These two authors contributed equally to the review.

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, Maria Victoria González#

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272 RNA Interference

#### **Chapter 13**

## **RNAi-Induced Immunity**

Wenyi Gu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61632

#### **Abstract**

RNA interference has a close relationship with the host defense system including adap‐ tive immunity. It is not only involved in regulating immune cells at different stages of the immune response but also directly induces or enhances antigen presentation and subse‐ quent immune responses. We have previously reported that a small hairpin RNA (shRNA) targeted the downstream site of a dominant cytotoxic T lymphocyte (CTL) epit‐ ope of human papillomavirus (HPV) type 16 oncogene E7 can stimulate an immune re‐ sponse against E7 expressing tumors in C57BL/6 mice. This results in the elimination of tumor growth *in vivo*, whereas an shRNA that targets the upstream site does not. Our re‐ cent data further confirm the long half-life of the 5'-mRNA fragment after shRNA degra‐ dation and its involvement in protein synthesis. This chapter summarizes these findings and provides some updated explanations for the findings.

**Keywords:** RNAi, shRNA, immune response, HPV E7, miRNA

#### **1. Introduction**

RNA interference (RNAi) is a conserved gene-regulation mechanism in all eukaryotic cells, where small RNAs including small interfering RNA (siRNA), small hairpin RNA (shRNA), and micro-RNA (miRNA, miR) interact with message RNAs (mRNAs) in a sequence-specif‐ ic manner and cause the cleavage or translational blockage of a gene [1, 2]. Because of its specificity and efficiency, it has been widely utilized as a routine tool for gene functional studies in biology laboratories worldwide. In addition, since RNAi blockage is very specific and at the transcriptional level, RNAi-based gene therapy (RNAi therapy) is thought to hold a great potential for treating many diseases, especially viral infections and genetic disorders. Synthesized siRNA is thus regarded as a specialized drug for gene therapies. So far, promis‐ ing results have been obtained with RNAi therapy in various diseases and many are being tested in clinical trials, including viral infections, cancers, and genetic or inflammatory dis‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

orders [3-7]. As cancers and other emerging diseases such as dementia and super-bug infec‐ tions become major public health issues, RNAi therapy can offer a new solution and has the additional ability to overcome drug resistances.

Beside the fundamental gene-silencing and gene-regulating roles of RNAi, which will also regulate the gene functions of immune cells and thus the immune responses, RNAi pathway itself has an additional function and involvement in inducing adaptive immunity. This func‐ tion has not been well-studied, and the mechanism is not clear. Its relationship with the im‐ mune system in terms of antigen reactivation and antigen presentation is still a new area to be investigated. Indeed, in plants and primitive species, RNAi is a part of the defense sys‐ tem against viral infections. However, in mammals, RNAi seems not directly involved in the immune system, probably due to the development of an advanced and sophisticated im‐ mune system. This chapter summarizes the evidence of RNAi-induced immunity against tu‐ mors and provides some updated possible explanations for the findings. The possible link between miRNA and its degraded products with the immune system has been also dis‐ cussed. Exploring the relationship between RNAi and the immune system may lead to new discoveries in RNAi biology and approaches for more effective cancer immunotherapy or treatment for viral and intracellular pathogen infections.

#### **2. The discovery of RNAi-induced adaptive immunity against tumors**

In 2009, we reported a discovery about RNAi-induced immunity [7]. We investigated two shRNAs encoded by lentiviral vectors on their ability to suppress tumor cell growth and stimulate antitumor immunity *in vivo*. One shRNA targeted the downstream site of a domi‐ nant cytotoxic T lymphocyte (CTL) epitope of the oncogene E7 of human papillomavirus (HPV) type 16 (termed downstream shRNA), while another shRNA targeted the upstream site of this epitope (termed upstream shRNA). Both shRNAs were equally effective at silenc‐ ing E7 gene expression (in mRNA and protein levels) and led to the inhibition of tumor cells growth *in vitro* and *in vivo* [7]. In spite of this, TC1 tumor cells (expressing HPV E6 and E7) treated with downstream shRNA stimulated an immune response against E7 in C57BL6 mice and resulted in elimination of tumor growth *in vivo*, whereas cells treated with the up‐ stream shRNA did not. When untreated TC1 tumor cells were injected to the same mice (challenging tumors), the group of downstream shRNA exhibited a total inhibition of chal‐ lenging tumor growth, whereas no inhibition was observed in the upstream shRNA group. This ability of downstream shRNA was absent in Rag-/- mice (lack of T- and B-cells), suggest‐ ing adaptive immune response or T-cell response was required. To prove that the immune response was antigen-specific, we carried out a same animal experiment by immunizing C57BL/6 mice with TC-1 cells treated with these shRNAs but challenged with another tumor cell line C2, which has the E7 expression and H-2b genetic background as C57BL/6 mice. Again, we observed that only mice immunized with downstream shRNA treated cells had a loss of tumor formation, indicating tumor clearance was specific to E7. Our data indicate that a more effective treatment can be developed for cervical cancer by combining RNAi treatment with immunotherapy. Our results also reveal that RNAi may be widely used as an antitumor immunity stimulator or enhancer (Fig 1).

orders [3-7]. As cancers and other emerging diseases such as dementia and super-bug infec‐ tions become major public health issues, RNAi therapy can offer a new solution and has the

Beside the fundamental gene-silencing and gene-regulating roles of RNAi, which will also regulate the gene functions of immune cells and thus the immune responses, RNAi pathway itself has an additional function and involvement in inducing adaptive immunity. This func‐ tion has not been well-studied, and the mechanism is not clear. Its relationship with the im‐ mune system in terms of antigen reactivation and antigen presentation is still a new area to be investigated. Indeed, in plants and primitive species, RNAi is a part of the defense sys‐ tem against viral infections. However, in mammals, RNAi seems not directly involved in the immune system, probably due to the development of an advanced and sophisticated im‐ mune system. This chapter summarizes the evidence of RNAi-induced immunity against tu‐ mors and provides some updated possible explanations for the findings. The possible link between miRNA and its degraded products with the immune system has been also dis‐ cussed. Exploring the relationship between RNAi and the immune system may lead to new discoveries in RNAi biology and approaches for more effective cancer immunotherapy or

**2. The discovery of RNAi-induced adaptive immunity against tumors**

In 2009, we reported a discovery about RNAi-induced immunity [7]. We investigated two shRNAs encoded by lentiviral vectors on their ability to suppress tumor cell growth and stimulate antitumor immunity *in vivo*. One shRNA targeted the downstream site of a domi‐ nant cytotoxic T lymphocyte (CTL) epitope of the oncogene E7 of human papillomavirus (HPV) type 16 (termed downstream shRNA), while another shRNA targeted the upstream site of this epitope (termed upstream shRNA). Both shRNAs were equally effective at silenc‐ ing E7 gene expression (in mRNA and protein levels) and led to the inhibition of tumor cells growth *in vitro* and *in vivo* [7]. In spite of this, TC1 tumor cells (expressing HPV E6 and E7) treated with downstream shRNA stimulated an immune response against E7 in C57BL6 mice and resulted in elimination of tumor growth *in vivo*, whereas cells treated with the up‐ stream shRNA did not. When untreated TC1 tumor cells were injected to the same mice (challenging tumors), the group of downstream shRNA exhibited a total inhibition of chal‐ lenging tumor growth, whereas no inhibition was observed in the upstream shRNA group. This ability of downstream shRNA was absent in Rag-/- mice (lack of T- and B-cells), suggest‐ ing adaptive immune response or T-cell response was required. To prove that the immune response was antigen-specific, we carried out a same animal experiment by immunizing C57BL/6 mice with TC-1 cells treated with these shRNAs but challenged with another tumor cell line C2, which has the E7 expression and H-2b genetic background as C57BL/6 mice. Again, we observed that only mice immunized with downstream shRNA treated cells had a loss of tumor formation, indicating tumor clearance was specific to E7. Our data indicate that a more effective treatment can be developed for cervical cancer by combining RNAi

additional ability to overcome drug resistances.

276 RNA Interference

treatment for viral and intracellular pathogen infections.

**Figure 1. Schematic diagram shows RNAi targeting site and 5' mRNA fragment.** It is known that in cervical cancer HPV E7 and mouse lymphoma EG7/OVA (ovalbumin) models, the shRNA targets the downstream site of the CTL epitope that produce the 5' fragment of mRNA. This makes immature proteins and further induces an immune re‐ sponse.

To prove the applicability of immune response to general tumor antigens, we tested a model antigen ovalbumin (OVA) expressed in EG7 cells that are a mouse thymoma cell line with C57BL6 genetic background. We chose the major CTL epitope of OVA, SIINFEKL, as the tar‐ get site and designed two upstream shRNAs OVA-1 and -2 and a downstream shRNA OVA-3. The shRNA-treated EG7 cells were used to immunize the mice, which were subse‐ quently challenged with untreated EG7 cells. We observed that only mice inoculated with OVA-3-shRNA treated cells had significantly reduced tumor formation but not with OVA-1 and -2 shRNAs.

#### **3. Confirmation of mRNA fragments and truncated proteins in treated cells**

To determine if the RNAi-induced immune response was actually from degraded products of RNAi, we designed a series of primers that would amplify RNA fragments inside and outside the targeting sites of upstream shRNA (E6-1) and downstream shRNA (E7-1, Fig 2A). If an inside fragment was present while the outside fragment was absent, it would indi‐ cate that the mRNA had been cleaved by shRNA at the target site. The cells were treated with E6-1 and E7-1 shRNAs and incubated for 2–4 days before real-time RT-PCR was car‐ ried out. As expected, the shortest PCR fragment, R3, was observed in all samples (Fig 2B). The full-length R1 PCR fragment was only observed in untreated TC1 cells or cells infected with the lentiviral vector control (PLL, Fig 2B). In cells treated with both E7-1 and E6-1, R1 was not found, indicating that shRNA-mediated cleavage was occurring. Of most interest was the R2 fragment which was found in all samples except cells treated with E6-1. These results suggest that R2 or R3 short fragments of E6-1 and E7-1 existed in the cells, at least temporarily at the time we isolated RNA. These short-form mRNAs may act as templates for short-form proteins (truncated proteins) and trigger antigen presentation to CD8+ T-cells and a CTL immune response to E7.

Apart from our data, a previous study reported that degradation of the 3' mRNA fragment resulting from siRNA-mediated cleavage was blocked for some mRNAs, leaving an mRNA fragment that could act as a template for cDNA synthesis. They suggested that this could give rise to false negative results and that this phenomenon may be avoided by the careful design of RT-qPCR primers for each individual siRNA experiment [8]. This report further confirms that mRNA fragments from RNAi do sometimes exist in the cells. In addition, it was noticed by researchers that un-degraded fragments of an siRNA-targeted mRNA may cause false positive effects of microarray analysis [9]. To avoid this, they developed a qRT-PCR protocol, which allowed for the determination of the optimal time point for mRNA analyses, indicating mRNA fragments after RNAi can be present in cell for a certain time.

What is the functional role of these mRNA fragments after RNAi? Our data demonstrated that they can be involved in translational machinery and produce truncated proteins. To ex‐ perimentally prove this, we utilized the OVA-expressing EG7 cell model again. The cells were treated with downstream and upstream shRNAs and further treated with the protease inhibitor MG132 to reduce protein degradation before immunoblotting was performed. The blots were probed with an antibody against the N terminus of OVA protein. The predicted size of a truncated protein produced by the cleavage of OVA-2 shRNA was 14.7 kDa. We observed a protein band about 15-kDa in cells treated with the OVA-2 shRNA but not in un‐ treated and OVA-1 or OVA-3 shRNA treated cells. It proves that truncated proteins can be produced in cells by the translation of mRNA fragment cleaved by shRNA. The predicted truncated product by shRNA-OVA3 was not observed due to cross-reacting proteins on the blot [7].

Our recent data (unpublished) showed that the cleaved 5' and 3' fragments of human papil‐ lomavirus type 16 (HPV-16) E6/7 mRNA after shRNA treatment were unevenly degraded. The 5' mRNA fragment was more abundant and displayed a greater stability than the corre‐ sponding 3' fragment in the treated cells. Further analysis revealed that the 5' fragment was polysome-associated, indicating its active translation, and this was further confirmed by us‐ ing tagged E7 protein to show that C-terminally truncated proteins were produced in treat‐ ed cells (Singhania et al. submitted).

outside the targeting sites of upstream shRNA (E6-1) and downstream shRNA (E7-1, Fig 2A). If an inside fragment was present while the outside fragment was absent, it would indi‐ cate that the mRNA had been cleaved by shRNA at the target site. The cells were treated with E6-1 and E7-1 shRNAs and incubated for 2–4 days before real-time RT-PCR was car‐ ried out. As expected, the shortest PCR fragment, R3, was observed in all samples (Fig 2B). The full-length R1 PCR fragment was only observed in untreated TC1 cells or cells infected with the lentiviral vector control (PLL, Fig 2B). In cells treated with both E7-1 and E6-1, R1 was not found, indicating that shRNA-mediated cleavage was occurring. Of most interest was the R2 fragment which was found in all samples except cells treated with E6-1. These results suggest that R2 or R3 short fragments of E6-1 and E7-1 existed in the cells, at least temporarily at the time we isolated RNA. These short-form mRNAs may act as templates for short-form proteins (truncated proteins) and trigger antigen presentation to CD8+ T-cells

Apart from our data, a previous study reported that degradation of the 3' mRNA fragment resulting from siRNA-mediated cleavage was blocked for some mRNAs, leaving an mRNA fragment that could act as a template for cDNA synthesis. They suggested that this could give rise to false negative results and that this phenomenon may be avoided by the careful design of RT-qPCR primers for each individual siRNA experiment [8]. This report further confirms that mRNA fragments from RNAi do sometimes exist in the cells. In addition, it was noticed by researchers that un-degraded fragments of an siRNA-targeted mRNA may cause false positive effects of microarray analysis [9]. To avoid this, they developed a qRT-PCR protocol, which allowed for the determination of the optimal time point for mRNA analyses, indicating mRNA fragments after RNAi can be present in cell for a certain time.

What is the functional role of these mRNA fragments after RNAi? Our data demonstrated that they can be involved in translational machinery and produce truncated proteins. To ex‐ perimentally prove this, we utilized the OVA-expressing EG7 cell model again. The cells were treated with downstream and upstream shRNAs and further treated with the protease inhibitor MG132 to reduce protein degradation before immunoblotting was performed. The blots were probed with an antibody against the N terminus of OVA protein. The predicted size of a truncated protein produced by the cleavage of OVA-2 shRNA was 14.7 kDa. We observed a protein band about 15-kDa in cells treated with the OVA-2 shRNA but not in un‐ treated and OVA-1 or OVA-3 shRNA treated cells. It proves that truncated proteins can be produced in cells by the translation of mRNA fragment cleaved by shRNA. The predicted truncated product by shRNA-OVA3 was not observed due to cross-reacting proteins on the

Our recent data (unpublished) showed that the cleaved 5' and 3' fragments of human papil‐ lomavirus type 16 (HPV-16) E6/7 mRNA after shRNA treatment were unevenly degraded. The 5' mRNA fragment was more abundant and displayed a greater stability than the corre‐ sponding 3' fragment in the treated cells. Further analysis revealed that the 5' fragment was polysome-associated, indicating its active translation, and this was further confirmed by us‐ ing tagged E7 protein to show that C-terminally truncated proteins were produced in treat‐

and a CTL immune response to E7.

278 RNA Interference

blot [7].

ed cells (Singhania et al. submitted).

**Figure 2. The shRNA targeting sites and primer design for HPV E6/E7 mRNA** (Adapted from Gu et al 2009). (A) The shRNA and primer sites on E6/E7 mRNA. (B) The PCR products on an agarose gel. Notes: TC1, the untreated cell con‐ trol. PLL, lentiviral vector control. \*: indicates the site of CTL epitope. F: forward primer, R: reverse primer.

#### **4. Possible models for explaining RNAi-induced immunity**

It is well established that miRNAs play an important role in regulating innate and adaptive immune responses as a part of their gene-regulating roles. Evidence has accumulated that miRNAs are involved in the adaptive immune responses by regulating T-cells, B-cells, and antigen-presenting cells (APCs). For example, miR-214 was reported to target phosphatase and tensin homolog (PTEN) and increase proliferation and the activation of T-cells [10], while miR-150, -155, and let-7 have been shown to be involved in the development of T-cells into memory cells [11]. In addition, miR-184 was shown to inhibit nuclear factor of activated T-cells-1 (NFAT-1) in the activation of CD4 T-cell in the early stage of adaptive immune re‐ sponses [12] and miR-181-a could promote CD4 and CD8 double positive T-cell develop‐ ment [13]. For B-cells, miR-155 is required for their normal function such as production of isotype-switched, high-affinity antibodies and for memory responses [14]. It has also been demonstrated that miR-155 is induced by B-cell receptor (BCR) [15]. However, overexpres‐ sion of miR-155 can immortalize B-cells and lead to transformation, for instance, EBV was shown to have induced miR-155 expression and transformed B-cells [16, 17]. In addition, miR-150 is important in B-cell development [18] and so is miR-17 [19, 20].

For dendritic cells (DCs), a recent review summarized the need of miRNAs in their lineage commitment from bone marrow progenitors and for the development of subsets such as plasmacytoid DCs and conventional DCs [21]. Liu et al. (2010) used software to predict and then conducted experiments to confirm that three members of the miR-148 family, miR-148a, miR-148b, and miR-152 are negative regulators of the innate immune response and antigen-presenting capacity of DCs. They showed that miR-148/152 expression was up‐ regulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. These miRNAs in turn inhibited the production of cytokines including IL-12, IL-6, TNF-al‐ pha, and IFN-beta and upregulation of MHC class II expression and DC-initiated antigenspecific T-cell proliferation by targeting Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) [22]. In addition, miR-150 and miR-223 has been shown to play an impor‐ tant regulatory role in Langerhans cells (LCs) by cross-presenting a soluble antigen to anti‐ gen-specific CD8(+) T-cells [23, 24]. Beside APCs such as DC and LC, miRNA is shown to be directly involved in antigen presentation. For example, Bartoszewski et al. (2011) demon‐ strated that the mRNA of human endoplasmic reticulum (ER) antigen peptide transporter 1 (TAP1) is a direct target of miR-346. They showed that the 3'-UTR (un-translational region) of TAP1 contains a 6-mer seeding region for miR-346 and the ER stress-associated reduction of TAP1 mRNA and protein levels could be reversed by inhibitory miRNA of miR-346 [25]. As TAP plays an important role in MHC class I-associated antigen presentation, their data provide an insight for miRNA-regulating MHC class-I-associated antigen presentation dur‐ ing ER stress.

The above-highlighted results clearly indicate miRNA's regulatory role in many aspects of adaptive immunity. However, is it possible that miRNA also takes part in host immunity through mRNA fragments produced after RNAi just like shRNA described in section 2 and 3? Normally, miRNAs target the 3' UTR and lead to the translation block or degradation of the targeted mRNAs. So when they degrade mRNAs, it is supposed to produce long 5' mRNA fragments and may also produce truncated proteins. If this is true, the translated de‐ fective proteins could be treated as truncated protein and be processed by proteasome. If there is a CTL epitope in the defective structure, it could be coupled with the MHC class I molecule and presented to T-cells by DCs through antigen cross-presentation, as described above with shRNA. This could be a link between RNAi pathway and antigen presentation or adaptive immune response.

In the shRNA case discussed above, the target should be at the downstream of a CTL epit‐ ope to induce immunity. When miRNAs target 3' UTR, a site certainly at the downstream site of any possible CTL epitopes, it is assumed to have the ability to produce truncated pro‐ teins and so to induce immune responses. Therefore, an important question for miRNA biol‐ ogy is whether miRNAs can routinely induce immune responses by degrading mRNA at 3' UTR and generating 5' mRNA fragments or truncated proteins that contain CTL epitopes? So far, there is no answer for this question. Another critical question is: what is the differ‐ ence between blocking and degrading mRNA by miRNAs at 3' UTR? Does this relate to an‐ tigen presentation of different proteins?

Because it has been shown that miRNA can act as siRNA and shRNA can be produced in the same pathway as miRNA [26], it is important and interesting to investigate if miRNA can induce the same immune response as shRNA. The systems of HPV 16 E7/TC1 and OVA/EG7 can be used as good models to investigate this. As miRNAs are routinely transcri‐ bed and involved in interacting with mRNA, this mechanism can be considered as a routine way in cells to generate CTL containing truncated proteins. However, because most mRNAs in cells are for self-proteins, their CTL epitopes will not be presented to T-cells. This leaves the question of whether this is a mechanism just for cells that express viral genes (such as HPV E7 in TC1 and C2 as above) or for cells expressing foreign genes/antigens (such as OVA in EG7)? The next question is: can this be generalized to any tumor antigens including self-antigens? This is an interesting subject to investigate and will facilitate our understand‐ ing of how RNAi pathways interact with and are involved in adaptive immune responses (antigen presentation) to utilize them for cancer immunotherapy.

Although some miRNA are highly conserved between lower animals and higher animals, mammals have far more miRNAs compared to nonmammals. This suggests that during evo‐ lution, as gene regulation became so complex and important in higher animals, miRNA or RNAi pathway gradually specialized into gene regulation. At the same time, as the adaptive immune system became well developed and highly specialized, these two systems got sepa‐ rated, but as described above, they still have some links. Future investigations leading to in‐ sight into these links will provide answers to the above questions.

#### **5. Conclusion**

plasmacytoid DCs and conventional DCs [21]. Liu et al. (2010) used software to predict and then conducted experiments to confirm that three members of the miR-148 family, miR-148a, miR-148b, and miR-152 are negative regulators of the innate immune response and antigen-presenting capacity of DCs. They showed that miR-148/152 expression was up‐ regulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. These miRNAs in turn inhibited the production of cytokines including IL-12, IL-6, TNF-al‐ pha, and IFN-beta and upregulation of MHC class II expression and DC-initiated antigenspecific T-cell proliferation by targeting Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) [22]. In addition, miR-150 and miR-223 has been shown to play an impor‐ tant regulatory role in Langerhans cells (LCs) by cross-presenting a soluble antigen to anti‐ gen-specific CD8(+) T-cells [23, 24]. Beside APCs such as DC and LC, miRNA is shown to be directly involved in antigen presentation. For example, Bartoszewski et al. (2011) demon‐ strated that the mRNA of human endoplasmic reticulum (ER) antigen peptide transporter 1 (TAP1) is a direct target of miR-346. They showed that the 3'-UTR (un-translational region) of TAP1 contains a 6-mer seeding region for miR-346 and the ER stress-associated reduction of TAP1 mRNA and protein levels could be reversed by inhibitory miRNA of miR-346 [25]. As TAP plays an important role in MHC class I-associated antigen presentation, their data provide an insight for miRNA-regulating MHC class-I-associated antigen presentation dur‐

The above-highlighted results clearly indicate miRNA's regulatory role in many aspects of adaptive immunity. However, is it possible that miRNA also takes part in host immunity through mRNA fragments produced after RNAi just like shRNA described in section 2 and 3? Normally, miRNAs target the 3' UTR and lead to the translation block or degradation of the targeted mRNAs. So when they degrade mRNAs, it is supposed to produce long 5' mRNA fragments and may also produce truncated proteins. If this is true, the translated de‐ fective proteins could be treated as truncated protein and be processed by proteasome. If there is a CTL epitope in the defective structure, it could be coupled with the MHC class I molecule and presented to T-cells by DCs through antigen cross-presentation, as described above with shRNA. This could be a link between RNAi pathway and antigen presentation

In the shRNA case discussed above, the target should be at the downstream of a CTL epit‐ ope to induce immunity. When miRNAs target 3' UTR, a site certainly at the downstream site of any possible CTL epitopes, it is assumed to have the ability to produce truncated pro‐ teins and so to induce immune responses. Therefore, an important question for miRNA biol‐ ogy is whether miRNAs can routinely induce immune responses by degrading mRNA at 3' UTR and generating 5' mRNA fragments or truncated proteins that contain CTL epitopes? So far, there is no answer for this question. Another critical question is: what is the differ‐ ence between blocking and degrading mRNA by miRNAs at 3' UTR? Does this relate to an‐

Because it has been shown that miRNA can act as siRNA and shRNA can be produced in the same pathway as miRNA [26], it is important and interesting to investigate if miRNA can induce the same immune response as shRNA. The systems of HPV 16 E7/TC1 and

ing ER stress.

280 RNA Interference

or adaptive immune response.

tigen presentation of different proteins?

In summary, RNAi-induced immunity opens a new perspective in which to explore the rela‐ tionship between RNAi pathways and the immune system, especially its involvement in an‐ tigen presentation in the adaptive immune response. For RNAi biology, it will provide an insight into the understanding of function roles of RNAi (including miRNA and siRNA) in host defense. In the field of gene therapy for cancers, RNAi can be used as an approach to silence oncogenes as well as a strategy to enhance immunity against cancer antigens (at least viral infection related cancers) and further explored as a novel cancer immunotherapy. Fi‐ nally, for intracellar pathogens, it can be used as a strategy for developing new vaccine through RNAi reactivating their antigens to the immune system.

#### **Author details**

Wenyi Gu\*

Address all correspondence to: w.gu@uq.edu.au

Australian Institute of Bioengineering and Nanotechnology, University of Queensland, QLD, Australia

#### **References**


[14] Calame, K., *MicroRNA-155 function in B Cells.* Immunity, 2007. 27(6): p. 825-7. doi: 10.1016/j.immuni.2007.11.010.

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282 RNA Interference

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[2] Elbashir, S.M., et al., *Duplexes of 21-nucleotide RNAs mediate RNA interference in cul‐ tured mammalian cells.* Nature, 2001. 411(6836): p. 494-8. doi:10.1038/35078107.

[3] Jacque, J.M., K. Triques, and M. Stevenson, *Modulation of HIV-1 replication by RNA in‐*

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[5] Putral, L.N., et al., *RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin.* Mol Pharmacol, 2005. 68(5): p.

[6] Bitko, V., et al., *Inhibition of respiratory viruses by nasally administered siRNA.* Nat Med,

[7] Gu, W., et al., *Both treated and untreated tumors are eliminated by short hairpin RNAbased induction of target-specific immune responses.* Proc Natl Acad Sci U S A, 2009.

[8] Holmes, K., et al., *Detection of siRNA induced mRNA silencing by RT-qPCR: considera‐ tions for experimental design.* BMC Res Notes, 2010. 3: p. 53. doi:

[9] Hahn, P., et al., *RNA interference: PCR strategies for the quantification of stable degrada‐ tion-fragments derived from siRNA-targeted mRNAs.* Biomol Eng, 2004. 21(3-5): p. 113-7.

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[11] Almanza, G., et al., *Selected microRNAs define cell fate determination of murine central memory CD8 T cells.* PLoS One, 2010. 5(6): p. e11243. doi: 10.1371/journal.pone.

[12] Weitzel, R.P., et al., *microRNA 184 regulates expression of NFAT1 in umbilical cord blood CD4+ T cells.* Blood, 2009. 113(26): p. 6648-57. doi: 10.1182/blood-2008-09-181156.

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*terference.* Nature, 2002. 418(6896): p. 435-8. doi:10.1038/nature00896.


## **Perspectives on RNA Interference in Immunopharmacology and Immunotherapy**

Zhaohua Hou, Qiuju Han, Cai Zhang and Jian Zhang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61575

#### **Abstract**

RNA interference (RNAi), mediated by short interfering RNA (siRNA), vector-derived short hairpin RNA (shRNA) and microRNA (miRNA), brings about revolutionary fea‐ tures to basic biomedical research and clinical application. New drugs based on RNAi have been developed for therapeutic applications. The family of RNAi molecules are effi‐ cient agents to modulate mammalian immune system, and many studies reported that these molecules could manipulate immune defence, surveillance and homeostasis. Both perfect match of siRNA/shRNA and non-perfect match of miRNA could be beneficial for designing RNAi-based drugs for treatment of tumour and viral infection. This chapter provides a view to control or utilize the immune regulation of various small RNAs that should help researchers to understand the successful clinical application of RNAi.

**Keywords:** RNA interference, siRNA, miRNA, immunopharmacology, immunotherapy

#### **1. Introduction**

RNA interference (RNAi) is a conserved mechanism against exogenous nucleic acid and transposon transcripts in plants and lower animals. No matter of transfected siRNA, vectordelivered shRNA or pre-miRNA (transcribed mainly by Pol II) , Dicer (DCLs) and Agronaute (AGO) family proteins efficiently process small RNAs into short double-stranded RNA(dsRNA). Further, dsRNAs assemble into the RNAi-induced silencing (protein) complex (RISC) to guide and cleave target mRNA, promote mRNA degradation or inhibit mRNA translation. The great potential of RNAi is to specifically repress the expression of diseasecausing genes while avoiding undesirable effects.

It is well accepted that siRNA can be recognized by endosomal pathways, Toll-like receptor 3 (TLR3), TLR7, TLR8 and cytoplasmic pathways, retinoic acid-inducible gene I (RIG-I),

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

melanoma differentiation-associated antigen 5 (MDA-5) and RNA-activated protein kinase (PKR), resulting in immune activation [1–5]. For example, it has been demonstrated that siRNA can cause activation of at least three key transcription factors, including NF-κB, interferon regulatory factor 3 (IRF-3) and IRF-7, and stimulate interferon (IFN) secretion. This activates T cells and dendritic cells (DCs) in the spleen in a TLR7-dependent manner [2,6]. Furthermore, 5′-triphosphate siRNA was demonstrated to activate RIG-I signal pathway, and then natural killer (NK) cells and DCs were activated [7,8]. In most circumstances, immune system stimulation is regarded as an unwanted side effect; therefore, siRNA-induced immune response should be controlled by using proper delivery system or chemical modification, although immune stimulation has been proved to be essential in cancer treatment and viral infection.

miRNAs are critical in regulating the development, differentiation, function and destiny of immune cells, including DCs, granulocytes, monocytes/macrophages, NK and natural killer T (NKT) cells and B and T lymphocytes. miRNAs influence both innate and adaptive immune defence and individual miRNAs may contribute their implications to various immunemediated diseases. Furthermore, pattern recognition receptors (PRRs), kinases, adaptors, inflammatory factors and IFN could all be targets of miRNAs. Extra effort has been made to develop miRNA-based oligos or vectors for anti-infection purpose by manipulating corre‐ sponding immune genes.

In addition to silencing of targeted genes in a sequence-specific manner, components of RNAi technology often induce immune response. Several strategies were reported to design RNAi molecules with gene silencing and immune regulatory properties. Bifunctional molecules rely on the activation of PRRs such as TLR7/8, TLR9 or RIG-I, or just rely on down-regulation of target gene. This chapter summarizes RNAi-involved immune responses in the past 10 years and discusses the anticipated therapeutic application.

#### **2. Chemically synthesized siRNA and vector-derived shRNA**

#### **2.1. RNAi drugs based on targeting specific immune genes**

Immune disorders, both autoimmune diseases and immune defective or deficiency, are always caused by high-level overexpression of certain immune genes. A variety of immune inhibitory genes can serve as targets for RNAi-mediated gene silencing. Targeting specific immune suppressor could re-balance immune network and subsets.

Elevated activity of signal transducer and activator of transcription 3 (STAT3) has been found in several kinds of human tumours. Use of RNAi to knockdown STAT3 expression and inhibit its activation would reduce the tumour cell growth such as pancreatic cancer, colorectal cancer, melanoma and hepatocarcinoma cells. STAT 3 knockdown could induce bystanders immune response in vitro and in vivo, where CD4+, CD8+ and NKT cells were activated as well as the secretion of interferon-γ (IFN-γ), interleukin-12 (IL-12) and tumour necrosis factor alpha (TNF-α) was increased significantly [9–11]. siRNA-STAT3, synthetical‐

ly linked to CpG (agonist of TLR9), was demonstrated to silence immune suppressor STAT3 gene in TLR9+ myeloid cells and B cells. This strategy of therapy leads to activation of various populations of immune cells, including DCs and macrophages, that ultimately induce potent anti-tumour immune responses [10,12]. Hossain DM et al. recently report‐ ed that CpG-siRNA-STAT3 conjugates could efficiently silence the target expression, and abrogate inhibition of CD8+ T cells in patients who received myeloid-derived suppressor cells (MDSCs) [13]. Researchers proved that immune-stimulation-inducing CpG(A)-STAT3 siRNA was non-toxic for normal human leukocytes [14]. In another experiment, Luo Z et al. [15] generated a nano-vaccine loaded with poly I:C (a TLR3 agonist) and STAT3 siRNA. Researchers found this kind of siRNA could promote the maturation of DC and reverse immunosuppression in the tumour micro-environment; the function of inhibitory cells in tumour-draining lymph nodes were inhibited; thus, anti-tumour immune responses were potently induced; and the survival were prolonged [15]. Therefore, STAT3 siRNAs are expected to be a promising immunomodulatory drugs to improve the treatment efficacy of cancer vaccines by abrogating tumour immunosuppression.

melanoma differentiation-associated antigen 5 (MDA-5) and RNA-activated protein kinase (PKR), resulting in immune activation [1–5]. For example, it has been demonstrated that siRNA can cause activation of at least three key transcription factors, including NF-κB, interferon regulatory factor 3 (IRF-3) and IRF-7, and stimulate interferon (IFN) secretion. This activates T cells and dendritic cells (DCs) in the spleen in a TLR7-dependent manner [2,6]. Furthermore, 5′-triphosphate siRNA was demonstrated to activate RIG-I signal pathway, and then natural killer (NK) cells and DCs were activated [7,8]. In most circumstances, immune system stimulation is regarded as an unwanted side effect; therefore, siRNA-induced immune response should be controlled by using proper delivery system or chemical modification, although immune stimulation has been proved to be essential in cancer treatment and viral

miRNAs are critical in regulating the development, differentiation, function and destiny of immune cells, including DCs, granulocytes, monocytes/macrophages, NK and natural killer T (NKT) cells and B and T lymphocytes. miRNAs influence both innate and adaptive immune defence and individual miRNAs may contribute their implications to various immunemediated diseases. Furthermore, pattern recognition receptors (PRRs), kinases, adaptors, inflammatory factors and IFN could all be targets of miRNAs. Extra effort has been made to develop miRNA-based oligos or vectors for anti-infection purpose by manipulating corre‐

In addition to silencing of targeted genes in a sequence-specific manner, components of RNAi technology often induce immune response. Several strategies were reported to design RNAi molecules with gene silencing and immune regulatory properties. Bifunctional molecules rely on the activation of PRRs such as TLR7/8, TLR9 or RIG-I, or just rely on down-regulation of target gene. This chapter summarizes RNAi-involved immune responses in the past 10 years

Immune disorders, both autoimmune diseases and immune defective or deficiency, are always caused by high-level overexpression of certain immune genes. A variety of immune inhibitory genes can serve as targets for RNAi-mediated gene silencing. Targeting specific immune

Elevated activity of signal transducer and activator of transcription 3 (STAT3) has been found in several kinds of human tumours. Use of RNAi to knockdown STAT3 expression and inhibit its activation would reduce the tumour cell growth such as pancreatic cancer, colorectal cancer, melanoma and hepatocarcinoma cells. STAT 3 knockdown could induce bystanders immune response in vitro and in vivo, where CD4+, CD8+ and NKT cells were activated as well as the secretion of interferon-γ (IFN-γ), interleukin-12 (IL-12) and tumour necrosis factor alpha (TNF-α) was increased significantly [9–11]. siRNA-STAT3, synthetical‐

**2. Chemically synthesized siRNA and vector-derived shRNA**

infection.

286 RNA Interference

sponding immune genes.

and discusses the anticipated therapeutic application.

**2.1. RNAi drugs based on targeting specific immune genes**

suppressor could re-balance immune network and subsets.

Suppressor of cytokine signalling 1 (SOCS1) is a negative regulator of antigen-presenting cell (APC)-based immune response. Silencing of SOCS1 gene expression by RNAi is essential for DCs to enhance Ag-specific anti-tumour immunity [16]. SOCS1-silenced bone marrow dendritic cells (BMDCs) were more potent in suppressing tumour growth [17]. When SOCS1 was silenced, maturation of DCs (i.e. expressions of CD80, CD40, CD86 and major histocom‐ patibility complex II [MHC II]) was significantly accelerated. As a result, SOCS1 inhibition upregulated the expression of IFN-γ and IL-12, and decreased IL-4 secretions, which induced Th1 cell differentiation and thereby affected the development of Th2 cell. The combined nanoparticle (NP) delivery, which can render both tumour antigen and siRNA-SOCS1 to BMDCs, simultaneously could enhance immunotherapeutic effects in BMDC-based cancer therapy [16,18]. DC-targeted delivery of SOCS1 siRNA has been shown to enhance antifungal immunity in response to *Candida albicans*in vitro and HIV-specific cytotoxic T cell in mice [16, 19]. This evidence suggests the use of SOCS1–siRNA, as a potent adjuvant to improve immune response.

A20 is usually regarded as an attractive target for siRNA-mediated gene knockdown in DCs because it is a negative feedback regulator of multiple pro-inflammatory signal transduction. Several reports demonstrated that RNAi-mediated A20 silencing in DCs enhanced expression of co-stimulatory molecules (CD80, CD86, CD40 and MHC class II) and pro-inflammatory cytokines (IL-6 and TNF-α). Tumour-infiltrating cytotoxic T lympho‐ cytes (CTLs), T helper cells that produced IL-6 and TNF-α were also activated by siA20- DC. A20 silencing in DCs can enhance the immune response against self-tumourassociated antigens [20,21]. Furthermore, A20-silenced DCs were proved to overcome CD4+CD25+regulatory T (Treg) cell suppression [21,22]. A20-silenced DCs could skew naive CD4+ T cells towards Th1 cell, but not Treg, Th2 or Th17 cells. Because a high amount of IL-10 was produced in A20-silenced DCs, simultaneous down-regulation of IL-10 and A20 resulted in enhanced T cell stimulatory capacity in DCs. A20 down-regulation resulted in enhanced CTLs immune response by the NF-κB and AP-1 pathways [20,23]. RNAi of A20 has enabled DCs to gain a potent ability to activate CTLs and Th cells, and inhibit Treg, providing a novel strategy to promote a tumour immune response.

Programmed death ligand (PD-L) is another exciting target on the surface of antigenpresenting cells (APCs); PD-L/PD-1 interactions were related to functional impairment and exhaustion of tumour antigen-specific CD8+ T cells. Although PD-L antibody exerts a potent anti-tumour effect, previous reports [24] have demonstrated that PD-L1-siRNA-PEI were preferentially and avidly engulfed by tumour-associated CD11c+PD-L1+ tolerogenic DCs at ovarian cancer locations. This kind of nanoparticle uptake stimulated multiple TLRs signalling, mainly via myeloid differentiation factor 88 (MyD88). Then, regulatory DCs activated into potent stimulators of CTLs that led to significant anti-tumour immunity in mouse models of ovarian cancer. Most importantly, PD-L knockdown DCs showed superior potential to expand minor histocompatibility antigen (MiHA)-specific CD8+ effector and memory T cells from leukaemia patients early after donor lymphocyte infusion and later during relapse. Combined PD-L1 and PD-L2 knockdown resulted in improved prolifera‐ tion of CD4+ T cells and enhanced cytokine production [25,26]. In addition, another report demonstrated the improved effector functions of tumour-specific CD4+ and CD8+ human T cells by siRNA-mediated silencing of PD-1 ligands, PD-L1 or PD-L2 [27]. These results suggest that siRNA-mediated knockdown of PD-L is a fascinating strategy to inhibit a negative regulatory mechanism of tumour-specific T cells.

siRNA-CD40, delivered by a novel delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-β-glucan, was captured and incorporated into DCs through its receptor, Dectin [28]. This strategy could induce antigenspecific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. CD40 knockdown significantly suppressed Th1-type cytokines and induced Th2-type cytokines in rats with myocarditis. Knockdown of CD40 in experimental autoimmune myocarditis (EAM) rats promoted Foxp3 gene expression and increased Treg cells [29].

In addition, when silencing of CD40 or CD80/CD86, DCs exhibited suppressed allostimulatory activity with impaired APC function. In the well-established collagen-induced arthritis (CIA) model, multigene-silenced DCs were capable of delaying onset of joint pathology. Therapeutic effects of gene-silenced DCs were mediated by the inhibition of collagen II-specific Ab production and suppression of T cell recall responses. Also, multigene-silenced DCs inhibited Th1 and Th17 response, demonstrating IFN-γ and IL-2 inhibition [30]. Thus, inhibition of specific co-stimulatory molecules of DCs reveals a promising approach of suppressing immune responses in autoimmunity. These findings highlight the potential of immunomo‐ dulation of siRNA-CD40, and have important implications for developing RNAi-based clinical therapy in the transplantation field.

It is well documented that tumours could secrete immunosuppressive molecules, including the cytokines transforming growth factor β (TGF-β) and IL-10. This creates an immunosup‐ pressive environment, which inhibits anti-tumour immunity. The suppression of Treg cell, induced by targeting TGF-β1 using siRNA, can enhance the efficacy of a DC vaccine against a poorly immunogenic tumour in mice [31]. Nanoparticle-delivered TGF-β siRNA enhances vaccination against advanced melanoma, and the tumour micro-environment was modified with increased levels of tumour-infiltrating CD8+ T cells and decreased level of regulatory T cells [32]. siRNA targeting IL-10 receptor α (siIL-10RA) initiated the significant antigen-specific CD8+ T cell immune responses. Concordantly, the combination of knockdown of IL-10RA and TGF-βR in DCs showed significant up-regulation of MHC I, enhancing co-stimulatory molecules CD40, CD80, CD86 and chemokine CCR7 after lipopolysaccharide (LPS) stimula‐ tion. It induced the strongest anti-tumour effects in the TC-1 P0 (a cervical cancer model expressing the human papillomavirus [HPV]-16 E7 antigen) tumour model, and even in the immune-resistant TC-1 (P3) ones [33]. These data revealed that siRNA co-targeting immuno‐ suppressive molecules enhance the potency of DC-based immunotherapeutics.

has enabled DCs to gain a potent ability to activate CTLs and Th cells, and inhibit Treg,

Programmed death ligand (PD-L) is another exciting target on the surface of antigenpresenting cells (APCs); PD-L/PD-1 interactions were related to functional impairment and exhaustion of tumour antigen-specific CD8+ T cells. Although PD-L antibody exerts a potent anti-tumour effect, previous reports [24] have demonstrated that PD-L1-siRNA-PEI were preferentially and avidly engulfed by tumour-associated CD11c+PD-L1+ tolerogenic DCs at ovarian cancer locations. This kind of nanoparticle uptake stimulated multiple TLRs signalling, mainly via myeloid differentiation factor 88 (MyD88). Then, regulatory DCs activated into potent stimulators of CTLs that led to significant anti-tumour immunity in mouse models of ovarian cancer. Most importantly, PD-L knockdown DCs showed superior potential to expand minor histocompatibility antigen (MiHA)-specific CD8+ effector and memory T cells from leukaemia patients early after donor lymphocyte infusion and later during relapse. Combined PD-L1 and PD-L2 knockdown resulted in improved prolifera‐ tion of CD4+ T cells and enhanced cytokine production [25,26]. In addition, another report demonstrated the improved effector functions of tumour-specific CD4+ and CD8+ human T cells by siRNA-mediated silencing of PD-1 ligands, PD-L1 or PD-L2 [27]. These results suggest that siRNA-mediated knockdown of PD-L is a fascinating strategy to inhibit a

siRNA-CD40, delivered by a novel delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-β-glucan, was captured and incorporated into DCs through its receptor, Dectin [28]. This strategy could induce antigenspecific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. CD40 knockdown significantly suppressed Th1-type cytokines and induced Th2-type cytokines in rats with myocarditis. Knockdown of CD40 in experimental autoimmune myocarditis (EAM)

In addition, when silencing of CD40 or CD80/CD86, DCs exhibited suppressed allostimulatory activity with impaired APC function. In the well-established collagen-induced arthritis (CIA) model, multigene-silenced DCs were capable of delaying onset of joint pathology. Therapeutic effects of gene-silenced DCs were mediated by the inhibition of collagen II-specific Ab production and suppression of T cell recall responses. Also, multigene-silenced DCs inhibited Th1 and Th17 response, demonstrating IFN-γ and IL-2 inhibition [30]. Thus, inhibition of specific co-stimulatory molecules of DCs reveals a promising approach of suppressing immune responses in autoimmunity. These findings highlight the potential of immunomo‐ dulation of siRNA-CD40, and have important implications for developing RNAi-based clinical

It is well documented that tumours could secrete immunosuppressive molecules, including the cytokines transforming growth factor β (TGF-β) and IL-10. This creates an immunosup‐ pressive environment, which inhibits anti-tumour immunity. The suppression of Treg cell, induced by targeting TGF-β1 using siRNA, can enhance the efficacy of a DC vaccine against a poorly immunogenic tumour in mice [31]. Nanoparticle-delivered TGF-β siRNA enhances

providing a novel strategy to promote a tumour immune response.

288 RNA Interference

negative regulatory mechanism of tumour-specific T cells.

rats promoted Foxp3 gene expression and increased Treg cells [29].

therapy in the transplantation field.

High-mobility group box 1 (HMGB1) is highly expressed in tumour cells and increased levels of HMGB1 in tumour cells are usually associated with a greater tumour angiogenesis, growth, invasion and metastasis. Knockdown of tumour cell-derived HMGB1 by shRNA did not affect tumour cell growth, while naturally acquired long-lasting tumour-specific IFN-γ- or TNF-αproducing CD8+ T cell responses were induced, and ability to induce Treg was attenuated. This led to naturally acquired CD8 T cell-dependent anti-tumour immunity [34].

Foxp3, a master gene that controls the development and function of Treg cells, contributes to pathogenesis of several different tumours. Owing to the intracellular localization of Foxp3, RNAi technology was employed to knockdown its activation to suppress Treg activity in vivo. Tsai et al. [35] performed a study targeting silencing Foxp3 gene expression by shRNAsmediated RNAi using a lentivirus vector in a murine model of leukaemia. The lentiviral vector was used to overcome poor transfection efficiency. Lentiviral-mediated Foxp3 RNAi showed suppressive effects on tumour growth and prolonged the survival of tumour-transplanted mice. Furthermore, Foxp3 knockdown mediated by siRNA increased the ratio of Th1/Th2 in chronic hepatitis B patients; transcription factors T-bet and GATA-3 may be partly involved in this progress [36]. This strategy provides a novel view about how to decrease the number of Treg cells and weaken its function.

Selective knockdown of CCL22 and CCL17 expression in monocyte-derived DC (MoDC) by siRNA decreased the ratios of CD4+ to CD8+ as well as lowered the frequency of Tregs recruited by MoDC. Furthermore, intratumoural injection of MoDC, which was transfected with siCCL22 and siCCL17, significantly reduced the number of Tregs while inducing CD8+ T cells infiltration in thymic nude mice with human tumour xenografts [37]. Using siRNA to selectively silence chemokines may lead to a new strategy for DC vaccine development to improve cancer biotherapy.

High expression of indoleamine 2, 3-dioxygenase (IDO) in DCs leads to the suppression of T cell responses. Gene silencing by siRNA or shRNA of IDO in DC would up-regulate IL-12 and IFN-γ and inhibit apoptosis in CD8 and CD4 T cells as well as Treg cells; IL-10 expression was significantly down-regulated, thus finally restraining tumour growth. DC-based vaccine with IDO silence was demonstrated to augment and enhance the anti-tumour response against breast cancer, melanoma, bladder tumour and liver cancer [38–40]. A novel APC-targeted siRNA delivery system using mannosed liposomes (Man-lipo) with encapsulated siRNA-IDO (Man-lipo-siIDO) was demonstrated to preferentially silence IDO in APCs and efficiently enhance anti-tumour immune response [41–43].

It was reported that natural killer group 2, member D (NKG2D) activation was involved in NK cell and CD8+ cell-mediated liver inflammation, and blockade of NKG2D by silencing of multiple NKG2D ligands on hepatocytes was considered efficient in liver disease intervention. Huang et al. [44] constructed a plasmid containing the three shRNA sequences (shRae1 shMult1-shH60). After hydrodynamic injection into mice, they found that the expression of all three NKG2D ligands on hepatocytes was down-regulated, and fulminant hepatitis mediated through NKG2D in NK cell was attenuated. Furthermore, simultaneous knockdown of multiple human NKG2D ligands (MHC class I polypeptide-related sequence B/A(MICA/B), UL16-binding protein 2 [ULBP2] and ULBP3) also significantly attenuated NK cell cytolysis. Simultaneous knockdown of multiple ligands of NKG2D is a potential therapeutic approach to treat liver diseases induced by NKG2D-expressing NK cells and CD8+ cells. Furthermore, inhibition of human leukocyte antigen-G (HLA-G) by siRNA boosted NK cell lytic function [45,46].

Among several molecules involved in immune response, the choice of targets should be carefully reviewed and validated comprehensively according to the emerging knowledge about their function.

#### **2.2. Advantage of non-target immune effect of siRNA/shRNA drugs**

siRNA/shRNA have the potential to recruit immune receptors specialized in RNA sensor, such as TLR3, TLR7/8 [2,3]. 5′-triphosphate siRNA (3p-siRNA) was demonstrated to be detected by RNA sensors RIG-I. These immunostimulatory siRNA or shRNA can non-specifically induce innate immune response, the so-called 'off-target' effects that have considerable implications for clinical application to cure cancer and infection disease.

IFN response is a common side effect of siRNAs and siRNAs with GU-rich sequences, which are very potent in inducing IFN-α response. A newly published report demonstrated that siRNA could induce IFN-α responses, and then induced the analgesic effects in the spinal cord. This off-target analgesia is dose- and sequence-dependent while non-GU-rich sequences also produced off-target analgesia at high doses, where pain relief by a designed siRNA may not be attributable to target gene knockdown but IFN response [47].

Early in 2004, Karikó et al. [48] demonstrated that siRNAs and shRNAs induce immune activation by signalling through TLR3 and activate sequence-independent inhibition of gene expression. Kleinman et al. [4] showed that non-targeted (against non-mammalian genes) and targeted (against vascular endothelial growth factor [VEGF] or VEGFR1) siRNAs suppressed choroidal neovascularization (CNV) via cell-surface TLR3 and its adaptor TIR-domaincontaining adaptor-inducing interferon-β (TRIF), leading to the induction of IFN-α and IL-12. The effect of non-targeted siRNA to suppress dermal neovascularization in mice was as effective as vascular endothelial growth factor (VEGF) siRNA. This finding showed that two investigational siRNAs in clinical trials owe their anti-angiogenic effect in mice, which was not due to target knockdown but due to TLR3 activation. The efficiency of RNAi by siRNA is believed to be comparable with anti-VEGF antibodies. Kleinman's group then concluded that a 21-nucleotide (nt) non-targeted siRNA suppresses both hemangiogenesis and lymphangio‐ genesis in mouse models of neovascularization, induced by corneal sutures or hindlimb ischemia, as efficiently as a 21-nt siRNA targeting VEGF-A [1].

(Man-lipo-siIDO) was demonstrated to preferentially silence IDO in APCs and efficiently

It was reported that natural killer group 2, member D (NKG2D) activation was involved in NK cell and CD8+ cell-mediated liver inflammation, and blockade of NKG2D by silencing of multiple NKG2D ligands on hepatocytes was considered efficient in liver disease intervention. Huang et al. [44] constructed a plasmid containing the three shRNA sequences (shRae1 shMult1-shH60). After hydrodynamic injection into mice, they found that the expression of all three NKG2D ligands on hepatocytes was down-regulated, and fulminant hepatitis mediated through NKG2D in NK cell was attenuated. Furthermore, simultaneous knockdown of multiple human NKG2D ligands (MHC class I polypeptide-related sequence B/A(MICA/B), UL16-binding protein 2 [ULBP2] and ULBP3) also significantly attenuated NK cell cytolysis. Simultaneous knockdown of multiple ligands of NKG2D is a potential therapeutic approach to treat liver diseases induced by NKG2D-expressing NK cells and CD8+ cells. Furthermore, inhibition of human leukocyte antigen-G (HLA-G) by siRNA boosted NK cell lytic function

Among several molecules involved in immune response, the choice of targets should be carefully reviewed and validated comprehensively according to the emerging knowledge

siRNA/shRNA have the potential to recruit immune receptors specialized in RNA sensor, such as TLR3, TLR7/8 [2,3]. 5′-triphosphate siRNA (3p-siRNA) was demonstrated to be detected by RNA sensors RIG-I. These immunostimulatory siRNA or shRNA can non-specifically induce innate immune response, the so-called 'off-target' effects that have considerable implications

IFN response is a common side effect of siRNAs and siRNAs with GU-rich sequences, which are very potent in inducing IFN-α response. A newly published report demonstrated that siRNA could induce IFN-α responses, and then induced the analgesic effects in the spinal cord. This off-target analgesia is dose- and sequence-dependent while non-GU-rich sequences also produced off-target analgesia at high doses, where pain relief by a designed siRNA may not

Early in 2004, Karikó et al. [48] demonstrated that siRNAs and shRNAs induce immune activation by signalling through TLR3 and activate sequence-independent inhibition of gene expression. Kleinman et al. [4] showed that non-targeted (against non-mammalian genes) and targeted (against vascular endothelial growth factor [VEGF] or VEGFR1) siRNAs suppressed choroidal neovascularization (CNV) via cell-surface TLR3 and its adaptor TIR-domaincontaining adaptor-inducing interferon-β (TRIF), leading to the induction of IFN-α and IL-12. The effect of non-targeted siRNA to suppress dermal neovascularization in mice was as effective as vascular endothelial growth factor (VEGF) siRNA. This finding showed that two investigational siRNAs in clinical trials owe their anti-angiogenic effect in mice, which was not due to target knockdown but due to TLR3 activation. The efficiency of RNAi by siRNA is

**2.2. Advantage of non-target immune effect of siRNA/shRNA drugs**

for clinical application to cure cancer and infection disease.

be attributable to target gene knockdown but IFN response [47].

enhance anti-tumour immune response [41–43].

[45,46].

290 RNA Interference

about their function.

Among 15 siRNAs, Khairuddin et al. [49] identified an extremely immunostimulatory siRNAs, targeting the HPV, which exerted potent anti-tumoural function. This bifunctional siRNA could reduce growth of established TC-1 tumours in C57BL/6 mice, and its effect was TLR7 dependent, where ablation of TLR7 recruitment via 2′O-methyl modification of the oligo backbone reduced these anti-tumour effects. Flatekval et al. [40] designed either monofunc‐ tional siRNAs devoid of immunostimulation or bifunctional siRNAs with IDO silencing and immunostimulatory activities. They showed that bifunctional siRNAs were able to knock‐ down IDO expression and induce cytokine production through either endosomal TLR7/8 or RIG-I.

In the past 10 years, several studies reported that bifunctional 3p-siRNA (Exp:targeting Bcl2/ TGF-β/Survivin/Glutaminase/IDO) with target silencing and an innate immunity stimulation via RIG-I activation could confer potent anti-tumour efficacy. This is illustrated for the first time by the work of Poeck et al. [8], who reported that bifunctional siRNAs, with 5′-triphos‐ phate targeting Bcl2 (3p-siRNA), led to better melanoma tumour reduction than OH-siRNA or 5′-triphosphate siRNAs containing target mismatches. Poeck and his colleagues revealed that siRNA with 5′-triphosphate ends could be recognized by RIG-I and activate an innate immune cells such as DC; then, expression of IFNs was directly induced, leading to apoptosis in tumour cells. These bifunctional 3p-siRNAs with RIG-I activation and RNAi-mediated Bcl2 silencing could provoke massive apoptosis of tumour cells in lung metastases in vivo*.* This was the first report demonstrating that 3p-siRNA represents a single molecule-based approach in which RIG-I function activates immune cell and gene silencing, leading to a key molecular event. Researchers subsequently found that 3p-TGFβ1-siRNA combining RIG-I activation with gene silencing of TGF-β1 induced profound tumour cell apoptosis and revealed potent antitumour efficacy in pancreatic cancer. This kind of 3p-siRNA induces a Th1 cytokine profile, demonstrating IFN-γ induction and IL-4 inhibition. High level of IFN and CXCL10 recruited more activated CD8+ T cells to the tumour. Frequency of immunosuppressive CD11b+ Gr-1+ myeloid cells was reduced after 3p-TGFβ1-siRNA treatment [50].

In addition, 3p-siRNA against survivin gene was designed and generated. This finding demonstrated that 3p-survivin-siRNA inhibited lung cancer cell proliferation and induced a RIG-I-dependent type-I interferon response [7]. Recently, 5′-triphosphate siRNA combining glutaminase (GLS) silencing with RIG-I activation was demonstrated to induce more promi‐ nent anti-tumour responses than RIG-I ligand or GLS silencing capability alone. 3p-siRNA-GLS effectively induced intrinsic proapoptotic signalling, and GLS silence sensitized malignant cells to apoptosis induced by RIG-I activation. Moreover, cytotoxicity was en‐ hanced, resulting from disturbed glutaminolysis induced by GLS silencing. Finally, RIG-I activation by 3p-siRNA-GLS blocked autophagic degradation, leading to dysfunction of mitochondria, whereas GLS silencing severely impaired reactive oxygen species (ROS) scavenging systems, leading to a vicious circle of ROS-mediated cytotoxicity [51]. Immature monocyte-derived DCs had been transfected with siRNA-bearing 5′-triphosphate-activated T cells [40].

In addition, 3p-siRNA can inhibit hepatitis B virus (HBV), Influenza A Virus and Coxsackie‐ virus, by gene silencing and RIG-I activation. RNAi provides a promising approach for the specific treatment of HBV infection. Our laboratory has previously demonstrated that 3p-HBxsiRNA and shRNA-HBx not only directly inhibit HBV replication but also stimulate innate immunity against HBV, which are both beneficial for the inversion of HBV-induced immune tolerance [52]. In HBV-positive hepatoma HepG2.2.15 cells, 3p-HBx-siRNA combining RIG-I activation with HBx gene silencing induce stronger type I IFN response than non-target 3pscramble-siRNA, indicating that a potent immunostimulatory effect may partly contribute to the reversal of immune tolerance through decreasing HBV load; 3p-HBx-siRNA more strongly inhibited HBV replication and promoted IFN production than HBx-siRNA in primary HBV(+) hepatocytes, and this effect was mediated by RIG-I activation [52]. This was consistent of the other two reports [53,54]. Our dually functional vector containing both an immunostimulatory single-stranded RNA (ssRNA) and an HBx-silencing shRNA could reverse HBV-induced hepatocyte-intrinsic immune tolerance; TLR7 signalling pathway was attributed to this progress [55].

Lin et al. [56] designed and tested a 3p-mNP1496-siRNA against influenza virus. They found that 3p-mNP1496-siRNA could activate the RIG-I-mediated IFN-β pathway and significantly reduce virus load and virus-induced pathogenesis. The inhibition effect was in an siRNA- and RIG-I-dependent manner, demonstrating siRNA playing dual antiviral roles: viral genespecific silencing and non-gene-specific RIG-I activation. This strategy was also proved to elicit potent antiviral effects in coxsackievirus myocarditis, and virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA and non-target 3p-siRNA in inhibiting viral replication and subsequent cytotoxicity [57].

In the attempt to inhibit the expression of woodchuck hepatitis virus (WHV), Meng et al. [58] found that innate immune responses could be enhanced by RNAi through the PKR- and TLRdependent signalling pathways in primary hepatocytes. The immunostimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.

Furthermore, siRNA can also synergistically enhance DNA-mediated type III IFN (a newly characterized antiviral interferon) response in non-immune or primary immune cells. This enhancement is mediated by crosstalk signalling pathway between RIG-I (RNA sensor) and IFI16 (DNA sensor) [59].

Designing with GU sequences, addition of triphosphate motifs to siRNA, co-treatment with CpG oligos are believed to activate innate immunity when siRNA was applied in vitro and in vivo. Accumulating evidence suggests these bifunctional siRNAs could activate NK cells and CD8+ T cells in different models. Thus, specific clinical applications of RNAi can benefit from a concurrent activation of the immune system.

### **3. miRNA**

monocyte-derived DCs had been transfected with siRNA-bearing 5′-triphosphate-activated T

In addition, 3p-siRNA can inhibit hepatitis B virus (HBV), Influenza A Virus and Coxsackie‐ virus, by gene silencing and RIG-I activation. RNAi provides a promising approach for the specific treatment of HBV infection. Our laboratory has previously demonstrated that 3p-HBxsiRNA and shRNA-HBx not only directly inhibit HBV replication but also stimulate innate immunity against HBV, which are both beneficial for the inversion of HBV-induced immune tolerance [52]. In HBV-positive hepatoma HepG2.2.15 cells, 3p-HBx-siRNA combining RIG-I activation with HBx gene silencing induce stronger type I IFN response than non-target 3pscramble-siRNA, indicating that a potent immunostimulatory effect may partly contribute to the reversal of immune tolerance through decreasing HBV load; 3p-HBx-siRNA more strongly inhibited HBV replication and promoted IFN production than HBx-siRNA in primary HBV(+) hepatocytes, and this effect was mediated by RIG-I activation [52]. This was consistent of the other two reports [53,54]. Our dually functional vector containing both an immunostimulatory single-stranded RNA (ssRNA) and an HBx-silencing shRNA could reverse HBV-induced hepatocyte-intrinsic immune tolerance; TLR7 signalling pathway was attributed to this

Lin et al. [56] designed and tested a 3p-mNP1496-siRNA against influenza virus. They found that 3p-mNP1496-siRNA could activate the RIG-I-mediated IFN-β pathway and significantly reduce virus load and virus-induced pathogenesis. The inhibition effect was in an siRNA- and RIG-I-dependent manner, demonstrating siRNA playing dual antiviral roles: viral genespecific silencing and non-gene-specific RIG-I activation. This strategy was also proved to elicit potent antiviral effects in coxsackievirus myocarditis, and virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA and non-target 3p-siRNA in inhibiting viral

In the attempt to inhibit the expression of woodchuck hepatitis virus (WHV), Meng et al. [58] found that innate immune responses could be enhanced by RNAi through the PKR- and TLRdependent signalling pathways in primary hepatocytes. The immunostimulation by RNAi

Furthermore, siRNA can also synergistically enhance DNA-mediated type III IFN (a newly characterized antiviral interferon) response in non-immune or primary immune cells. This enhancement is mediated by crosstalk signalling pathway between RIG-I (RNA sensor) and

Designing with GU sequences, addition of triphosphate motifs to siRNA, co-treatment with CpG oligos are believed to activate innate immunity when siRNA was applied in vitro and in vivo. Accumulating evidence suggests these bifunctional siRNAs could activate NK cells and CD8+ T cells in different models. Thus, specific clinical applications of RNAi can benefit from

cells [40].

292 RNA Interference

progress [55].

replication and subsequent cytotoxicity [57].

a concurrent activation of the immune system.

IFI16 (DNA sensor) [59].

may contribute to the antiviral activity of siRNAs in vivo.

It has been well discussed how miRNAs regulate signalling pathways, and the dynamics of the immune response, tolerance and homeostasis. Here we summarize and explore updated achievements of special miRNAs in immunopharmacology.

#### **3.1. miRNAs as intrinsic targets in antiviral immunity**

In addition to the conventional innate and adaptive immune responses, even in the earlier phase after virus invasion, the host cell suppresses viral replication by evolving the profile of special and constitutively expressed genes. These cell-intrinsic antiviral approaches based on host restriction factors may be no less important than in considerations of conventional immunity. At the same time, viruses also gain some countermeasures or adapt the unique phenotype of their hosts substantially to survive. Moreover, miRNAs may also be involved in the inextricably intertwined relationship between viruses and their hosts.

In 2005, a liver-specific miRNA, miR122, which is involved in cholesterol and lipid metabolism [60], was illustrated to be necessary for hepatitis C virus (HCV) accumulation in cultured liver cells [61]. Researchers found that miR122 directly binds to two close sites in the 5′ non-coding region of the HCV genome and promote HCV translation [62–64]. This miRNA kept conserved among all HCV subtypes [65,66]. Even in non-hepatic cell, miR122 could boost HCV replication [67]. Moreover, miR122 was further proved to be significantly reduced after IFN-β treatment, and miR122 mimics neutralized IFN-induced anti-HCV effect [68]. Epidemiological and genomic researches further suggested that the level of miR122 in individuals with HCV might be an 'indicator' for IFN therapy, and only those patients with high levels of miR122 responded well to IFN therapy [69,70]. Therefore, miR122 antagonist would also be called as IFN 'sensi‐ tizer' in HCV immune treatment.

Santaris Pharma designed and synthesized an LNA-based miR122 inhibitor, named Miravirs‐ en (or SPC3649), to eradicate HCV. The product was first evaluated in preclinical studies in mice [71], cynomolgus monkeys [72], green African monkeys and chimpanzees [73,74]. Here the key concern is that whether miR122 inhibitor can effectively lower the level of free miR122 and inhibit HCV replication without disturbing normal cholesterol and lipid metabolism or without any potential chemical toxicity. Interestingly, although there was a reduction of cholesterol levels in plasma by nearly 40%, Miravirsen caused a dose-dependent reduction of miR122 and maintained ∼5-week-long half-life in the liver of monkeys and chimpanzees [73, 74]. Moreover, in the high-dose treatment group, Miravirsen decreased HCV subtype 1a or 1b more than 2 orders of magnitude compared to control group. In all animal species, Miravirsen was reported to be safe, without serious adverse effects or dose-related toxicities in rats, monkeys and human [75,76].

In May 2008, Miravirsen was put into human clinical trials as the first miRNA-based drug (https://clinicaltrials.gov/ct2/show/NCT00979927). There was a significant, dose-dependent reduction and sustained decrease of HCV viremia after drug administration in human subjects, and several patients became even HCV undetectable during the study. At the same time, only infrequent and moderately adverse effects were caused to some volunteers and did not influence the trial process [77]. Because miR122 is only liver enriched in physiological condi‐ tions and there is high amount of miR122 in adult human liver, it may be an ideal target to design highly specific anti-HCV drugs with good resistance to HCV infected person, particu‐ larly to those who have no tolerance to traditional treatments. In the following years, miR196 [78], let-7b family [79] and some other miRNAs were then proved to influence HCV life cycle, providing new target to restrict hepatitis C infection and avoid chronic infection.

Besides HCV, some other kind of viruses also encode miRNAs or regulate the miRNAs expression in host cells to disturb the expression of many immune-associated genes directly and/or indirectly, so that they can be critical regulators for viral life cycle. For example, in HEK293T cell, prototype foamy virus I (PFV-1) encodes Tas protein to counteract cell-encoded miR32, which could inhibit PFV-1 gene expression and accumulation [80]. Kaposi's sarcomaassociated herpesvirus (KSHV)-induced miR132 could silence p300 expression, which is critical for the transcription initialization of many antiviral genes, to help themselves maintain long-time latency [81]. The hematopoietic-cell-specific miR142-3p potently restricts the replication of eastern equine encephalitis virus in myeloid-lineage cells by binding to the 3′ untranslated region (UTR) of viral genome [82]. Even Drosha, the enzyme that processes miRNA biogenesis and maturation, was an independent factor for limiting RNA virus replication along with canonical type I IFN system in particular cell type [83]. Above of all, it is much likely that miRNA mimics (for viral inhibitory miRNAs) or antagonists (for viral beneficial miRNAs) can be effective antiviral strategies as intrinsic immune drugs.

#### **3.2. miRNA regulation antimicrobial and anti-tumour immunity**

#### *3.2.1. miRNA in antimicrobial innate immunity*

Of the known PRRs, TLRs and RIG-like receptors (RLRs) have been well studied in mediating antimicrobial and inflammatory responses during infections, which may be targets of patho‐ gens or host-encoded miRNAs.

The first PRR targeting miRNA let7i was reported in 2007 [84], which targeted TLR4 mRNA in a MyD88/NF-κB-dependent way during *Cryptosporidium parvum* infection, controlling the production of inflammatory factors. During *Bacillus Calmette-Guérin* (BCG) infection, miR124 exerts its function by targeting multiple components of the TLR signalling pathway, including TLR6, MyD88, TNF receptor-associated factor 6 (TRAF6) and TNF-α in mouse lung cell [85]. After HCV infection, miR373 was induced and negatively regulated the type I IFN signalling pathway by suppressing Janus kinase 1(JAK1) and IRF9 in hepatocytes [86]. Experimental evaluation using miR124 inhibitors or miR373 knockout up-regulated BCG-induced pro-inflammatory factors or type I IFN and so as to inhibit BCG or HCV more efficiently. Besides using host miRNAs, human cytomegalovirus (HCMV) targeted TLR2 by encoding its own miRNA, miR-UL112-3p, and reduced the expression of IL-1β, IL-6 and IL-8 upon stimulation with a TLR2 agonist [87]. Neutralizing this miRNA might recover normal cytokines production.

Besides immune inhibitory miRNAs, dengue virus (DENV)-induced miR30e\* up-regulated IFN-β and the downstream IFN-stimulated genes (ISGs) by suppressing IkBα and promoting NF-κB-dependent IFN production [88]. The transfection of miR30e\* would increase the expression of 2′-5′-oligoadenylate synthetase 1(OAS-1), myxovirus resistance A (MxA) and interferon-induced transmembrane protein (IFITM). In 2014, miR526 [89] was proved to enhance RIG-I-induced viral replication by suppression of the expression of cylindromatosis (CYLD), which suppresses RIG-I K-63-linked polyubiquitin. Moreover, Enterovirus 71(EV71) inhibited miR526 transcription in an IRF-dependent way and so as to attenuate virus-triggered type I interferon production. These studies suggested that recruitment or increase of miR30e\* or miR526 would stimulate type I IFN expression and inhibit virus more quickly.

#### *3.2.2. 'Immune miRs' as immunopharmaceutic agents*

infrequent and moderately adverse effects were caused to some volunteers and did not influence the trial process [77]. Because miR122 is only liver enriched in physiological condi‐ tions and there is high amount of miR122 in adult human liver, it may be an ideal target to design highly specific anti-HCV drugs with good resistance to HCV infected person, particu‐ larly to those who have no tolerance to traditional treatments. In the following years, miR196 [78], let-7b family [79] and some other miRNAs were then proved to influence HCV life cycle,

Besides HCV, some other kind of viruses also encode miRNAs or regulate the miRNAs expression in host cells to disturb the expression of many immune-associated genes directly and/or indirectly, so that they can be critical regulators for viral life cycle. For example, in HEK293T cell, prototype foamy virus I (PFV-1) encodes Tas protein to counteract cell-encoded miR32, which could inhibit PFV-1 gene expression and accumulation [80]. Kaposi's sarcomaassociated herpesvirus (KSHV)-induced miR132 could silence p300 expression, which is critical for the transcription initialization of many antiviral genes, to help themselves maintain long-time latency [81]. The hematopoietic-cell-specific miR142-3p potently restricts the replication of eastern equine encephalitis virus in myeloid-lineage cells by binding to the 3′ untranslated region (UTR) of viral genome [82]. Even Drosha, the enzyme that processes miRNA biogenesis and maturation, was an independent factor for limiting RNA virus replication along with canonical type I IFN system in particular cell type [83]. Above of all, it is much likely that miRNA mimics (for viral inhibitory miRNAs) or antagonists (for viral

providing new target to restrict hepatitis C infection and avoid chronic infection.

beneficial miRNAs) can be effective antiviral strategies as intrinsic immune drugs.

Of the known PRRs, TLRs and RIG-like receptors (RLRs) have been well studied in mediating antimicrobial and inflammatory responses during infections, which may be targets of patho‐

The first PRR targeting miRNA let7i was reported in 2007 [84], which targeted TLR4 mRNA in a MyD88/NF-κB-dependent way during *Cryptosporidium parvum* infection, controlling the production of inflammatory factors. During *Bacillus Calmette-Guérin* (BCG) infection, miR124 exerts its function by targeting multiple components of the TLR signalling pathway, including TLR6, MyD88, TNF receptor-associated factor 6 (TRAF6) and TNF-α in mouse lung cell [85]. After HCV infection, miR373 was induced and negatively regulated the type I IFN signalling pathway by suppressing Janus kinase 1(JAK1) and IRF9 in hepatocytes [86]. Experimental evaluation using miR124 inhibitors or miR373 knockout up-regulated BCG-induced pro-inflammatory factors or type I IFN and so as to inhibit BCG or HCV more efficiently. Besides using host miRNAs, human cytomegalovirus (HCMV) targeted TLR2 by encoding its own miRNA, miR-UL112-3p, and reduced the expression of IL-1β, IL-6 and IL-8 upon stimulation with a TLR2 agonist [87]. Neutralizing this miRNA might

**3.2. miRNA regulation antimicrobial and anti-tumour immunity**

*3.2.1. miRNA in antimicrobial innate immunity*

gens or host-encoded miRNAs.

294 RNA Interference

recover normal cytokines production.

With the general knowledge of immunologically relevant miRNAs established in the past 10 years, many miRNAs have been intensively investigated using gain- and loss-of-function methods, showing how this novel class of small non-coding RNA participates in mammalian immunity. And individual immune miRNA might contribute its implications to various immune-mediated diseases.

The role of miR125b in immune signalling may be paradoxical. After stimulation with LPS, miR125b was down-regulated and TNF-α, one of miR125b targets, was overexpressed in RAW264.7, which is essential for antimicrobial activity. Moreover, during *M. tuberculosis* infection, the overexpression of miR125a significantly attenuated the antimicrobial effects in macrophages through targeting UV radiation resistance–associated gene (UVRAG) [90]. Nevertheless, in diffuse large B cell lymphoma (DLBCL), miR125a and miR125b directly target a negative NF-κB regulator tumour necrosis factor alpha-induced protein 3(TNFAIP3) and present a positive self-regulatory property to maintain prolonged NF-κB activity. Taken together, whether overexpression or inhibition of miR125b in an anti-infection therapeutic study depends on concrete circumstances.

miR146a also acts as a negative regulator in immune sensing. Both in mouse and human, miR146a was always exploited by virus to attenuate innate and adaptive antiviral immunity mainly in DC [91], lymphocyte [92] and hepatocytes by inhibiting interleukin-1 receptorassociated kinase 1(IRAK1), TRAF6 [93], son of sevenless homolog 1 (SOS1) [94] and STAT1 [95]. Silencing of miR146a via the delivery of sponge or antagomiR could restore the expression of inflammatory factors, augment type I IFN production and promote clearance of vesicular stomatitis virus (VSV) [96], dengue virus [97], enterovirus 71 (EV71) [94,98] and HBV [95]. Because miR146a was also abnormally expressed in hepatocellular carcinoma (HCC) and exerted negative anti-tumour effects by up-regulation of immunoinhibitory cytokines such as TGF-β, IL-17, VEGF, miR146a may also be a novel immunotherapeutic target for HCC [99].

Unlike miR146a, miR155 always promotes immune signal transduction, enhances immune function or speeds lymphocyte proliferation. Mice lacking miR155 have impaired CTL cell responses to infections with lymphocytic choriomeningitis virus and the intracellular bacteria *Listeria monocytogenes* because of insufficient activation of Akt pathway after TCR cross-linking [100]. miR155 knockout mice died soon after Erdman (a variant from severe acute respiratory syndrome [SARS]) infection and held higher level of colony-forming units (CFU) in lungs than wild-type mice [101]. During HIV infection, miR155 inhibited the HIV-activating effects of tripartite motif-containing protein 32 (TRIM32), and therefore, it might promote a return to latency in CD4+ reservoir cells [102]. In addition, in NK cells, miR155 might regulate T cell immunoglobulin-3 (Tim-3)/T-bet/STAT-5-signalling axis, and following cytokine expression that balanced antiviral response and immune injury during chronic HCV infection [103]. A remarkably ectopic up-expression of miR155 can be observed by delivering hepatotropic adeno-associated virus 8 (AAV8) vectors to the liver of mice, and then high level of miR155 enhanced GAP's protective capacity against parasite [104]. These studies imply miR155 as an immune-augmenting adjuvant in improving the antigenicity of vaccination.

miR223 was already proved to be of importance in myeloid progenitor cells proliferation and responsiveness to pathogenic stimuli in neutrophils by targeting myocyte-specific enhancer factor 2C (MEF2C), acting as a fine-tune regulator both in normal granulocytes generation and in preventing aberrant expansion and over-activated inflammatory responses. In recent years, miR223 was involved in inflammasome response by targeting NLR family pyrin domain containing 3 (NLRP3) in human [105]. Moreover, Epstein–Barr virus (EBV) encoded a mimic of hsa-miR223, called miR-BART15, targeting the same site within the NLRP3 3'-UTR to repress inflammasome activation. Furthermore, this miRNA can be secreted from EBV-infected B cells into exosomes to rheostat NLRP3 inflammasome activity in non-infected cells [106]. miR223 sponge would balance the amount of NLRP3 and 'absorb' EBV-miR-BART15 in macrophages and DCs.

It is noteworthy that two groups of miRNAs, which shaped NK-mediated cytotoxicity, have potent value for developing antiviral and anti-tumour biodrugs. First, NKG2D–NKG2D-L interaction plays a predominant role in 'NK cell–abnormal cell' recognition. MICB/A, ULBPs targeting miRNAs, are not only encoded by human genome as stress regulators but also synthesized by some virus (e.g. HCMV-miR-UL112 [107], EBV-miRBART2 [108], KSHV-miR-K12-7 [108] and BK virus (BKV)-miR-B1-3p, JC virus (JCV)-miR-J1-3p[109]), to escape from NK cell killing. Meanwhile, viral infected cell and tumour cell always express low MICA/B because of up-regulated MICB/MICA, targeting miRNAs such as miR20a, 93, 103, 106b [110] to maintain a compromised micro-environment. Furthermore, non-classical human leukocyte antigen G (HLA-G) is known as an inhibitory ligand, which suppresses the cytotoxic activity of T and NK cells. Studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR148a, miR148b and miR152, and lower expression of these miRNAs in renal carcinoma [111] and placental choriocarcinoma cells [112]. Stable manipulation of these activating and inhibitory miRNAs may enhance NK and LAK cell-mediated cytotoxicity against infected and tumour cells. Therefore, it could be concluded that modulating the expression or inhibition of specific miRNAs could boost immune response during viral infections or against cancers.

#### **3.3. miRNA in maintaining immune homeostasis**

Because several miRNAs participate in immune cell development and differentiation, abnormal expression of miRNA may cause a disturbance of homeostasis by changing the ratio of helper and regulatory cell subsets, or perturb the functionality and survival of effectmemory cells that lead to lymphoproliferative disease. Utilization of miRNA interference techniques may recover regular immune balance.

#### *3.3.1. miR17-92, miR146a and miR155 in Systemic Lupus Erythematosus (SLE)*

In 2007, a unique mouse strain, 'sanroque', presented a pattern of lupus pathology, revealing the core role of T follicular helper (Tfh) in systemic autoimmunity [113]. miR17-92 was found to regulate Tfh cell differentiation, which is essential for maintenance of the germinal centre formation and sustained antibody responses. Overexpression of this miRNA in T cells would enhance Tfh cell proliferation and survive an autoantibody production [112]. Similarly, miR155 increased IL-21-mediated STAT3 signalling in T cell [114], which might accelerate Tfh differ‐ entiation and maturation as well. Moreover, miR155 deficiency ameliorates autoimmune inflammation of SLE by targeting s1pr1 in mice [115]. Therefore, miR17-92 and miR155 might be a new target to restrain aggressive autoimmune response in SLE.

#### *3.3.2. miR29 and miR146 in type 1 diabetes*

syndrome [SARS]) infection and held higher level of colony-forming units (CFU) in lungs than wild-type mice [101]. During HIV infection, miR155 inhibited the HIV-activating effects of tripartite motif-containing protein 32 (TRIM32), and therefore, it might promote a return to latency in CD4+ reservoir cells [102]. In addition, in NK cells, miR155 might regulate T cell immunoglobulin-3 (Tim-3)/T-bet/STAT-5-signalling axis, and following cytokine expression that balanced antiviral response and immune injury during chronic HCV infection [103]. A remarkably ectopic up-expression of miR155 can be observed by delivering hepatotropic adeno-associated virus 8 (AAV8) vectors to the liver of mice, and then high level of miR155 enhanced GAP's protective capacity against parasite [104]. These studies imply miR155 as an

miR223 was already proved to be of importance in myeloid progenitor cells proliferation and responsiveness to pathogenic stimuli in neutrophils by targeting myocyte-specific enhancer factor 2C (MEF2C), acting as a fine-tune regulator both in normal granulocytes generation and in preventing aberrant expansion and over-activated inflammatory responses. In recent years, miR223 was involved in inflammasome response by targeting NLR family pyrin domain containing 3 (NLRP3) in human [105]. Moreover, Epstein–Barr virus (EBV) encoded a mimic of hsa-miR223, called miR-BART15, targeting the same site within the NLRP3 3'-UTR to repress inflammasome activation. Furthermore, this miRNA can be secreted from EBV-infected B cells into exosomes to rheostat NLRP3 inflammasome activity in non-infected cells [106]. miR223 sponge would balance the amount of NLRP3 and 'absorb' EBV-miR-BART15 in macrophages

It is noteworthy that two groups of miRNAs, which shaped NK-mediated cytotoxicity, have potent value for developing antiviral and anti-tumour biodrugs. First, NKG2D–NKG2D-L interaction plays a predominant role in 'NK cell–abnormal cell' recognition. MICB/A, ULBPs targeting miRNAs, are not only encoded by human genome as stress regulators but also synthesized by some virus (e.g. HCMV-miR-UL112 [107], EBV-miRBART2 [108], KSHV-miR-K12-7 [108] and BK virus (BKV)-miR-B1-3p, JC virus (JCV)-miR-J1-3p[109]), to escape from NK cell killing. Meanwhile, viral infected cell and tumour cell always express low MICA/B because of up-regulated MICB/MICA, targeting miRNAs such as miR20a, 93, 103, 106b [110] to maintain a compromised micro-environment. Furthermore, non-classical human leukocyte antigen G (HLA-G) is known as an inhibitory ligand, which suppresses the cytotoxic activity of T and NK cells. Studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR148a, miR148b and miR152, and lower expression of these miRNAs in renal carcinoma [111] and placental choriocarcinoma cells [112]. Stable manipulation of these activating and inhibitory miRNAs may enhance NK and LAK cell-mediated cytotoxicity against infected and tumour cells. Therefore, it could be concluded that modulating the expression or inhibition of specific miRNAs could boost immune response during viral

Because several miRNAs participate in immune cell development and differentiation, abnormal expression of miRNA may cause a disturbance of homeostasis by changing the ratio of helper and regulatory cell subsets, or perturb the functionality and survival of effect-

immune-augmenting adjuvant in improving the antigenicity of vaccination.

and DCs.

296 RNA Interference

infections or against cancers.

**3.3. miRNA in maintaining immune homeostasis**

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from the persisting destruction of pancreatic β-cells by autoreactive CD8+ T cell and Th1 cytokines. Dicer 1 deletion in β-cell would disrupt normal β-cell development and survival, lead to impairment of insulin secretion and diabetes development [116], apparently suggesting that miRNAs network is necessary for normal glycometabolism. Recently, endogenous miR29b released from pancre‐ atic β-cells within exosomes stimulated TNF-α secretion in spleen cells isolated from diabetesprone non-obese diabetic (NOD) mice. Delivery of miR29b to mice activated myeloid cell and pDCs to induce IFN-α, TNF-α and IL-6 production [117]. Abnormal expression of miR146 is associated with high serum titers of glutamic acid decarboxylase antibody in T1D patients, indicating the involvement of miR146 in the sustained immune imbalance during T1D progress [118]. These findings raised the possibility of developing a new clue for T1D immu‐ notherapy using miRNA-based agents.

#### *3.3.3. miR146a and miR155 in rheumatoid arthritis*

The role of miR146a in controlling Treg-mediated decrease of Th1 responses has been dem‐ onstrated [119]. In contrast, miR155 promoted Th1 and Th17 differentiation and cell formation and lowered T cell sensitivity to IFN-γ-driven proliferation by targeting C-MAF and IFNγRα [120]. Therefore, imbalance of miR146a and miR155 may be an epigenetic phenotype for autoimmune response. In rheumatoid arthritis (RA), decreased expression of miR146a contributes to an abnormal Treg phenotype and allows Th1/Th17 skewing while low level of miR155 failed to support effective Th2 immunity [121]. Systemic administration of miR146a has potential therapeutic intervention for preventing bone destruction by inhibited Th1 and Th17 cells, as well as IL-1β, IL-6 and TNF-α [122].

#### *3.3.4. miR15 and miR326 in multiple sclerosis*

Multiple sclerosis (MS) is manifested by chronic and progressive inflammatory demyelination of the central nervous system and is one of the main causes of regressive neurological diseases. Study on MS animal model illustrated that mice with fewer Th17 cells were less susceptible to experimental autoimmune encephalomyelitis (EAE) [123]. Therefore, Th17-targeting biother‐ apeutic approaches may be a promising way to cure multiple sclerosis. Gang Pei' s laboratory [124] found that miR326 promoted Th17 differentiation by targeting Ets-1 (a negative regulator of Th17 polarization) and antagonizing miR326 by sponge vector that resulted in fewer Th17 cells and Th17 cytokines and remitting EAE symptom. Inversely, increased miR155 in primary human microglia up-regulated pro-inflammatory cytokine secretion and co-stimulatory surface marker expression suggested that miR155 inhibition in myeloid cell might be useful to suppress allogeneic T cell responses [125]. In conclusion, reverse pathological expressed miRNAs and re-balance dysregulated immune genes are of consideration to treat multiple sclerosis.

#### **4. Conclusion**

RNAi technology holds promise for treating various human diseases. It is becoming apparent that clinical outcome of cancer immunotherapy and infectious diseases can be improved by targeted strategies to abrogate tumour-induced immunosuppression. Anti-tumour strategies using siRNA/shRNA/miRNA for both silencing of oncogenes and recruiting of innate recep‐ tors were designed. The present researches highlighted the potential therapeutic applications of this new generation of siRNAs in immunotherapy.

Additionally, but importantly, siRNA/shRNA or miRNA drugs with regard to pharmacody‐ namic difficulties and unwanted side effects are even more complicated compared to low molecular weight drugs and hard to be delivered into immune cells. This requires more extensive procedure than any other traditional drugs. Considering clinical challenges for RNAbased nucleic acid drugs, including barriers and RNases, the advanced tissue-directed delivery systems with safety, high efficiency and specificity, long-term function and controllability are required. Although the exploration of such tiny regulators causally bring pharmacists a considerable effort to draw up individualized and tailor-made strategies, we believe that immunoregulation triggered by siRNA/shRNA/miRNA can be used to regulate the host immunity against cancers or viruses. The development of multifunctional RNAi molecules will greatly contribute to the future arsenal of tools to combat not only microbial pathogens but also hard-to-treat cancer.

#### **Acknowledgements**

This work was supported by the Natural Science Foundation of China (81172789, 81373222, 31200651), National Basic Research Program of China (No. 2013CB531503) and National Mega Project on Major Infectious Diseases Prevention and Treatment (2012ZX10002006), The Special Foundation of Taishan Overseas Distinguished Experts and Scholars, The Priority Research Program of Shandong Academy of Sciences, Natural Science Foundation of Shandong Province (No.BS2015YY023), Natural Science Foundation of Shandong Academy of Sciences (No. 2014QN004) and Science and Technology Development Foundation of Shandong Analysis and Test Center.

#### **Author details**

Study on MS animal model illustrated that mice with fewer Th17 cells were less susceptible to experimental autoimmune encephalomyelitis (EAE) [123]. Therefore, Th17-targeting biother‐ apeutic approaches may be a promising way to cure multiple sclerosis. Gang Pei' s laboratory [124] found that miR326 promoted Th17 differentiation by targeting Ets-1 (a negative regulator of Th17 polarization) and antagonizing miR326 by sponge vector that resulted in fewer Th17 cells and Th17 cytokines and remitting EAE symptom. Inversely, increased miR155 in primary human microglia up-regulated pro-inflammatory cytokine secretion and co-stimulatory surface marker expression suggested that miR155 inhibition in myeloid cell might be useful to suppress allogeneic T cell responses [125]. In conclusion, reverse pathological expressed miRNAs and re-balance dysregulated immune genes are of consideration to treat multiple

RNAi technology holds promise for treating various human diseases. It is becoming apparent that clinical outcome of cancer immunotherapy and infectious diseases can be improved by targeted strategies to abrogate tumour-induced immunosuppression. Anti-tumour strategies using siRNA/shRNA/miRNA for both silencing of oncogenes and recruiting of innate recep‐ tors were designed. The present researches highlighted the potential therapeutic applications

Additionally, but importantly, siRNA/shRNA or miRNA drugs with regard to pharmacody‐ namic difficulties and unwanted side effects are even more complicated compared to low molecular weight drugs and hard to be delivered into immune cells. This requires more extensive procedure than any other traditional drugs. Considering clinical challenges for RNAbased nucleic acid drugs, including barriers and RNases, the advanced tissue-directed delivery systems with safety, high efficiency and specificity, long-term function and controllability are required. Although the exploration of such tiny regulators causally bring pharmacists a considerable effort to draw up individualized and tailor-made strategies, we believe that immunoregulation triggered by siRNA/shRNA/miRNA can be used to regulate the host immunity against cancers or viruses. The development of multifunctional RNAi molecules will greatly contribute to the future arsenal of tools to combat not only microbial pathogens

This work was supported by the Natural Science Foundation of China (81172789, 81373222, 31200651), National Basic Research Program of China (No. 2013CB531503) and National Mega Project on Major Infectious Diseases Prevention and Treatment (2012ZX10002006), The Special Foundation of Taishan Overseas Distinguished Experts and Scholars, The Priority Research Program of Shandong Academy of Sciences, Natural Science Foundation of Shandong

sclerosis.

298 RNA Interference

**4. Conclusion**

but also hard-to-treat cancer.

**Acknowledgements**

of this new generation of siRNAs in immunotherapy.

Zhaohua Hou1 , Qiuju Han2\*, Cai Zhang2 and Jian Zhang2

\*Address all correspondence to: hanqiuju@sdu.edu.cn

1 Laboratory of Immunology for Environment and Health, Shandong Analysis and Test Center, Shandong Academy of Sciences, Jinan, China

2 Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, Jinan, China

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## **RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies**

Constanca Figueiredo and Rainer Blasczyk

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61829

#### **Abstract**

Remarkable progress in the experimental and clinical applications of cell-based therapies has identified stem cells and their derived products as potential candidates for regenera‐ tive therapies for many disorders. The use of autologous stem cells as source for regener‐ ative therapeutic products is strongly limited by their low availability. Therefore, the future applications of *in vitro* pharmed therapeutic cell products will most likely occur in an allogeneic manner. However, the high variability of the human leukocyte antigen (HLA) represents a major obstacle to the application of off-the-shelf products. We have developed a strategy to decrease the immunogenicity of *in vitro* generated cell products by silencing HLA expression using RNAi. HLA expression was permanently silenced in CD34+ hematopoietic stem and progenitor cells and induced the pluripotent stem cells to generate HLA-universal cells sources, which were then used for the differentiation of low immunogenic cell products. In this chapter, we will provide an overview about an RNAibased strategy to reduce the immunogenicity of cell-based therapies, and in particular in the generation of HLA-universal platelets and tissues.

**Keywords:** HLA, immunogenicity, transplant rejection, blood pharming

#### **1. Introduction**

The high variability of the human leukocyte antigen (HLA) constitutes a major hurdle in allogeneic transplantation and to the application of off-the-shelf cell products in regenerative medicine. Recently, remarkable progresses in the field of stem cell biology, cell pharming, and tissue engineering have made feasible the differentiation of cells and tissues that might serve as a bridging strategy or even an alternative to the very scarce donated tissues and organs. However, HLA incompatibility may pose a threat to the applicability of such *in vitro* generated cell products by increasing the risk of immune rejection after the transplantation.[1] To

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

overcome this major hurdle in the fields of transplantation, cell and tissue engineering, we have developed an RNA interference (RNAi)-based approach to reduce the immunogenicity of cells and tissues, allowing their application in an universal manner. A lentiviral vector encoding for specific short hairpins RNA (shRNA), targeting HLA transcripts were used to achieve a permanent silencing of HLA expression. As HLA residual expression is crucial to prevent the natural killer (NK) cell activity, RNAi appears as a superior tool in comparison to gene editing technologies that cause a complete gene deletion such as the transcription activator-like effector nuclease (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems.[2-4] By decreasing the immunogenicity of cells and tissues, the need for immunosuppressive regimens might be reduced after HLA-mismatched trans‐ plantation. Furthermore, as HLA plays a pivotal role in the recognition of virus-infected cells and cancer cells, the combination of conditional promoter systems allows the re-expression of HLA and constitutes a safety mechanism. The generation of low immunogenic cells and tissues would bring enormous benefits to the patients and open novel horizons in the field of transplantation and regenerative medicine.

#### **2. The HLA system**

Evolution conferred highly refined mechanisms to all animals from sponges to mammals to distinguish self from non-self, and thereby allowing an immune response against potential pathogens. In humans, a tight interplay between adaptive and innate immune systems allows their defense against virtually all pathogens and cancer cells.[5, 6] Nevertheless, those sophisticated surveillance mechanisms pose a major hurdle to allogeneic transplantation. The alloimmune responses are mainly based on the recognition of mismatched major histocom‐ patibility complex (MHC) antigens by antibodies and T-cells. In humans, the MHC is known as HLA and it comprises a group of linked genes. MHC class I and II regulate the immune response through the presentation of peptides to T-cells. Allogeneic MHC in the graft's antigen-presenting cells (APCs) is recognized after transplantation through a direct pathway. Afterwards, own patient APCs process and present the allogeneic antigens to T-cells by an indirect alloantigen recognition pathway.[7, 8] HLA loci are encoded on the short arm of human chromosome 6. Based on their structure, HLA molecules are grouped into class I and class II (Figure 1). HLA class I classical genes comprise the A, B, and C loci, and are expressed in the majority of cells. HLA class II genes include DR, DQ, and DP and are constitutively expressed only in professional APCs.[9] The non-self recognition mediated by the engagement of the T-cell receptor with the donor HLA is the basis for the allogeneic immune response.

#### **2.1. HLA incompatibility increases the risk of rejection**

Despite the progresses in the field of transplantation, graft rejection remains the major concern regarding the application of off-the-shelf products. HLA comprises the most polymorphic loci of the entire human genome. The probability to find a complete HLA-matched donor for a specific patient is very low and, therefore, in most of the cases, patients will be treated with partially HLA-mismatched tissues and organs. In addition, even in fully HLA donor/recipient

overcome this major hurdle in the fields of transplantation, cell and tissue engineering, we have developed an RNA interference (RNAi)-based approach to reduce the immunogenicity of cells and tissues, allowing their application in an universal manner. A lentiviral vector encoding for specific short hairpins RNA (shRNA), targeting HLA transcripts were used to achieve a permanent silencing of HLA expression. As HLA residual expression is crucial to prevent the natural killer (NK) cell activity, RNAi appears as a superior tool in comparison to gene editing technologies that cause a complete gene deletion such as the transcription activator-like effector nuclease (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems.[2-4] By decreasing the immunogenicity of cells and tissues, the need for immunosuppressive regimens might be reduced after HLA-mismatched trans‐ plantation. Furthermore, as HLA plays a pivotal role in the recognition of virus-infected cells and cancer cells, the combination of conditional promoter systems allows the re-expression of HLA and constitutes a safety mechanism. The generation of low immunogenic cells and tissues would bring enormous benefits to the patients and open novel horizons in the field of

Evolution conferred highly refined mechanisms to all animals from sponges to mammals to distinguish self from non-self, and thereby allowing an immune response against potential pathogens. In humans, a tight interplay between adaptive and innate immune systems allows their defense against virtually all pathogens and cancer cells.[5, 6] Nevertheless, those sophisticated surveillance mechanisms pose a major hurdle to allogeneic transplantation. The alloimmune responses are mainly based on the recognition of mismatched major histocom‐ patibility complex (MHC) antigens by antibodies and T-cells. In humans, the MHC is known as HLA and it comprises a group of linked genes. MHC class I and II regulate the immune response through the presentation of peptides to T-cells. Allogeneic MHC in the graft's antigen-presenting cells (APCs) is recognized after transplantation through a direct pathway. Afterwards, own patient APCs process and present the allogeneic antigens to T-cells by an indirect alloantigen recognition pathway.[7, 8] HLA loci are encoded on the short arm of human chromosome 6. Based on their structure, HLA molecules are grouped into class I and class II (Figure 1). HLA class I classical genes comprise the A, B, and C loci, and are expressed in the majority of cells. HLA class II genes include DR, DQ, and DP and are constitutively expressed only in professional APCs.[9] The non-self recognition mediated by the engagement of the T-cell receptor with the donor HLA is the basis for the allogeneic immune response.

Despite the progresses in the field of transplantation, graft rejection remains the major concern regarding the application of off-the-shelf products. HLA comprises the most polymorphic loci of the entire human genome. The probability to find a complete HLA-matched donor for a specific patient is very low and, therefore, in most of the cases, patients will be treated with partially HLA-mismatched tissues and organs. In addition, even in fully HLA donor/recipient

transplantation and regenerative medicine.

**2.1. HLA incompatibility increases the risk of rejection**

**2. The HLA system**

312 RNA Interference

Figure 1. Structure representation of (A) HLA class I (HLA‐B) and (B) HLA class II (HLA‐DQ) molecules. The structures were designed using the software http://www.mh‐hannover.de/institute/transfusion/histocheck/. **Figure 1.** Structure representation of (A) HLA class I (HLA-B) and (B) HLA class II (HLA-DQ) molecules. The struc‐ tures were designed using the software http://www.mh-hannover.de/institute/transfusion/histocheck/.

pairs, disparities at the minor histocompatibility antigens (mHAg) derived from other polymorphic proteins and presented at the HLA complexes are capable of triggering antigenspecific immune responses that cause graft rejection. Improved use of post-transplant immu‐ nosuppression to prevent acute and chronic rejection allowed allogeneic transplantation as a widespread, successful therapy.[10-13] However, graft rejection remains a major concern in the field of transplantation. The registry of the International Society for Heart and Lung Transplantation (ISHLT) reported that within the first year after lung transplantation, up to 55% patients need to be treated for acute rejection and only 50% are alive after five years.[14] Also, the number of HLA mismatches between a donor/recipient pair is associated with stronger immunosuppressive regimens. In particular, the number of HLA-DR mismatches and the number of HLA-A and -B mismatches as well as rejection treatment showed significant associations with the dose of maintenance steroids. Although immunosuppression allows the acceptance of the allogeneic graft, it has severe side effects that may contribute to death even with a functioning graft.[15] The occurrence of post-transplant complications related to the immunosuppressive therapy such as cancer, opportunistic infection, toxicity, and hip fractures have indicated the necessity to develop alternative strategies to allow the therapeutical use of off-the-shelf HLA and mHAg-mismatched cell-based products.[16] Furthermore, due to the shortage of organs and tissues for transplantation, there is a high demand regarding the development of *in vitro* pharmed cell products, and engineered tissues or organs. Progresses in the fields of stem cell biology and tissue engineering have demonstrated the feasibility to generate *in vitro* potential alternative cell-based products that might serve as an alternative or overcome the need for using donated tissues. Nevertheless, the future use of such products will occur in an allogeneic manner, and therefore, it will be required that those products will be able to escape an allogeneic immune response. **2.1. HLA incompatibility increases the risk of rejection** Despite the progresses in the field of transplantation, graft rejection remains the major concern regarding the application of off‐the‐shelf products. HLA comprises the most polymorphic loci of the entire human genome. The probability to find a complete HLA‐matched donor for a specific patient is very low and, therefore, in most of the cases, patients will be treated with partially HLA‐mismatched tissues and organs. In addition, even in fully HLA donor/recipient pairs, disparities at the minor histocompatibility antigens (mHAg) derived from other polymorphic proteins and presented at the HLA complexes are capable of triggering antigen‐specific immune responses that cause graft rejection. Improved use of post‐transplant immunosuppression to prevent acute and chronic rejection allowed allogeneic transplantation as a widespread, successful therapy.10–13 However, graft rejection remains a major concern in the field of transplantation. The registry of the International Society for Heart and Lung Transplantation (ISHLT) reported that within the first year after lung transplantation, up to 55% patients need to be treated for acute rejection and only 50% are alive after five years.14 Also, the number of HLA mismatches between a donor/recipient pair is associated with stronger immunosuppressive regimens. In particular, the number of HLA‐DR mismatches and the number of HLA‐A and ‐B mismatches as well as rejection treatment showed significant associations with the dose of maintenance steroids. Although immunosuppression allows the acceptance of the allogeneic graft, it has severe side effects that may contribute to death even with a functioning graft.15 The occurrence of post‐transplant complications related to the immunosuppressive therapy such as cancer, opportunistic infection, toxicity, and hip fractures have indicated the necessity to develop alternative strategies to allow the therapeutical use of off‐the‐shelf HLA and mHAg‐mismatched cell‐based

3

products.16 Furthermore, due to the shortage of organs and tissues for transplantation, there is a high demand

#### **2.2. RNAi-mediated HLA targeting as a strategy to decrease the cell immunogenicity**

Rejection of allogeneic grafts is based on the recognition of the HLA complexes by the specific pre-formed complement-binding anti-HLA antibodies or by the engagement of the T-cell receptor, which leads to T-cell activation and the initiation of the immune response.[17, 18] RNA interference is an invaluable technique in cell biology and regenerative strategies to silence the target gene expression. Our studies have focused on the downregulation of HLA class I and class II expression on the graft cells. So far, several strategies have been developed to induce the acceptance of the allogeneic graft. Similar to the immunosuppression, those strategies involve the modulation of immune responses and aim the induction of tolerance to the graft. In our studies, we genetically modify the graft to silence its HLA expression to prevent the recognition of the allogeneic graft as non-self by the recipient's immune system. In this approach, we do not induce tolerance toward the allogeneic graft, but we generate a condition of immunological blindness in which the recipient's immune system is not able to recognize the allogeneic cells (due to the missing HLA) but is fully capable of defending the patient against common clinical conditions associated with the use of immunosuppressive drugs such as opportunistic infections and leukemia. To prevent the recognition of the grafted cells as off the shelf, we have downregulated the expression of HLA class I and class II antigens using RNAi. We have constructed lentiviral vectors encoding for short-hairpin RNA sequences targeting β2-microglobulin (shβ2m) or the alpha-chain of HLA-DR (shDRA) to silence the expression of HLA class I and class II antigens, respectively. Our studies demonstrated the feasibility to stably downregulate HLA class I and II expression in several cell lines (e.g., B-LCL, MonoMac-6, HeLa) as well as in primary cells (e.g., endothelial cells, CD34+ progenitor, induced pluripotent stem cells). Cell transduction for the delivery of shRNAs targeting specific HLA transcripts resulted in a decrease by up to 90% of β2m or HLA-DRA transcript levels and HLA class I expression. *In vitro* assays have shown that HLA class I-silenced cells were protected against the antibody-mediated complement-dependent cytotoxicity. Furthermore, in T-cell cytotoxicity assays, significantly lower cell lysis rates were observed when HLAsilenced cells were used as targets in comparison to fully HLA-expressing cells. In addition, HLA-silenced cells demonstrated to induce significantly lower T-cell proliferation, proinflammatory cytokine secretion, and degranulation. The residual HLA class I expression showed to be sufficient to prevent NK cell cytotoxicity. Altogether, HLA-silenced cells showed a protective effect against the humoral and cellular allogeneic immune response.[2, 3, 19]

#### **2.3. MHC-silenced cells survive after fully HLA-incompatible transplantation**

Despite the widespread use of immunosuppressive regimens to prevent graft rejection, their therapeutic window is very narrow. Immunosuppressive drugs frequently cause adverse effects including thrombocytopenia, leukopenia, hypercholesterolemia, stomatitis, nephro‐ toxicity, and diarrhea, and they lead to an increased risk for infections and cancer.[20, 21] Silencing HLA expression using RNAi may represent an alternative to immunosuppression; hence it has the potential to offer many benefits for the patients. In addition, silencing HLA expression may allow the future application of HLA-mismatched off-the-shelf products in a universal manner independently of the genetic background of the donor and recipient. In an allogeneic transplantation rat model, we have confirmed the improved capacity of MHCsilenced cells to survive in allogeneic environment upon transplantation and even in the absence of immunosuppression. A lentiviral vector encoding for a shRNA sequence targeting rat MHC class I (RT1-A) and the sequence for firefly luciferase as a reporter gene was used to silence MHC class I Lewis rat-derived fibroblasts. In contrast to nonmodified fibroblasts, MHC class I-silenced fibroblasts were able to survive after subcutaneous transplantation in a complete MHC-mismatched setting. MHC class I-silenced fibroblasts were able to engraft and were detectable during the entire monitoring period (8 weeks). Nonmodified cells were rejected in all animals.[22] This study showed the superior performance of MHC-silenced cells after MHC-incompatible transplantation.

#### **2.4. Generation of HLA-silenced platelets**

**2.2. RNAi-mediated HLA targeting as a strategy to decrease the cell immunogenicity**

314 RNA Interference

**2.3. MHC-silenced cells survive after fully HLA-incompatible transplantation**

Despite the widespread use of immunosuppressive regimens to prevent graft rejection, their therapeutic window is very narrow. Immunosuppressive drugs frequently cause adverse effects including thrombocytopenia, leukopenia, hypercholesterolemia, stomatitis, nephro‐ toxicity, and diarrhea, and they lead to an increased risk for infections and cancer.[20, 21] Silencing HLA expression using RNAi may represent an alternative to immunosuppression; hence it has the potential to offer many benefits for the patients. In addition, silencing HLA expression may allow the future application of HLA-mismatched off-the-shelf products in a universal manner independently of the genetic background of the donor and recipient. In an

Rejection of allogeneic grafts is based on the recognition of the HLA complexes by the specific pre-formed complement-binding anti-HLA antibodies or by the engagement of the T-cell receptor, which leads to T-cell activation and the initiation of the immune response.[17, 18] RNA interference is an invaluable technique in cell biology and regenerative strategies to silence the target gene expression. Our studies have focused on the downregulation of HLA class I and class II expression on the graft cells. So far, several strategies have been developed to induce the acceptance of the allogeneic graft. Similar to the immunosuppression, those strategies involve the modulation of immune responses and aim the induction of tolerance to the graft. In our studies, we genetically modify the graft to silence its HLA expression to prevent the recognition of the allogeneic graft as non-self by the recipient's immune system. In this approach, we do not induce tolerance toward the allogeneic graft, but we generate a condition of immunological blindness in which the recipient's immune system is not able to recognize the allogeneic cells (due to the missing HLA) but is fully capable of defending the patient against common clinical conditions associated with the use of immunosuppressive drugs such as opportunistic infections and leukemia. To prevent the recognition of the grafted cells as off the shelf, we have downregulated the expression of HLA class I and class II antigens using RNAi. We have constructed lentiviral vectors encoding for short-hairpin RNA sequences targeting β2-microglobulin (shβ2m) or the alpha-chain of HLA-DR (shDRA) to silence the expression of HLA class I and class II antigens, respectively. Our studies demonstrated the feasibility to stably downregulate HLA class I and II expression in several cell lines (e.g., B-LCL, MonoMac-6, HeLa) as well as in primary cells (e.g., endothelial cells, CD34+ progenitor, induced pluripotent stem cells). Cell transduction for the delivery of shRNAs targeting specific HLA transcripts resulted in a decrease by up to 90% of β2m or HLA-DRA transcript levels and HLA class I expression. *In vitro* assays have shown that HLA class I-silenced cells were protected against the antibody-mediated complement-dependent cytotoxicity. Furthermore, in T-cell cytotoxicity assays, significantly lower cell lysis rates were observed when HLAsilenced cells were used as targets in comparison to fully HLA-expressing cells. In addition, HLA-silenced cells demonstrated to induce significantly lower T-cell proliferation, proinflammatory cytokine secretion, and degranulation. The residual HLA class I expression showed to be sufficient to prevent NK cell cytotoxicity. Altogether, HLA-silenced cells showed a protective effect against the humoral and cellular allogeneic immune response.[2, 3, 19]

Since the 1950s, blood transfusion therapy has become routine clinical practice; however, the concept of blood pharming – ex vivo production of mature blood cells – is quite new. In humans, platelet production is sustained by a well-regulated process known as thrombopoi‐ esis. In the bone marrow, CD34+ progenitor cells differentiate into polyploid megakaryocytes (the precursor of platelets). Megakaryocytes lack the expression of CD34, but express several glycoproteins essential for the platelet function.[23, 24] In general, platelet numbers in blood range from 150 x 109 to 400 x 109 per liter, and an estimated 1 x 1011 platelets are produced each day in the adult human. Thrombocytopenia and severe thrombocytopenia characterized as platelet counts less than 50 x 109 and 10 x 109 per liter, respectively, increase the risk of spontaneous bleeding and represent a threat for the patient's life.25 Platelet transfusion has been widely used to prevent and treat life-threatening thrombocytopenia; however, prepara‐ tion of a unit of concentrated platelets for transfusion requires at least 4–6 units of whole blood, thereby significantly increasing the risk of blood-borne infections and adverse immunologic reactions. Furthermore, platelet transfusion refractoriness – lack of adequate post-transfusion platelet counts – remains a major complication often observed in patients receiving multiple transfusions. This condition is frequently caused by the development of antibodies specific to HLA. Currently, platelet transfusion relies on volunteer blood donation; however, the demand for blood products in particular of platelets often exceeds their availability.[25]

The potential of multipotent progenitor and stem cells in regenerative medicine has been recognized.[26, 27] Platelet transfusion refractoriness due to the presence of anti-HLA antibodies constitutes a life-threatening risk for many patients suffering from hematological disorders, and hence require multiple platelet transfusions. Thus, it would be highly desirable to produce HLA-deficient platelets to facilitate the management of severe alloimmunized thrombocytopenic patients. In our studies, we have combined the concept of blood pharming with RNAi as a strategy to downregulate HLA gene expression. The ultimate goal of this approach is the large-scale production of platelets *in vitro* that may be used as an alternative to the conventional donated blood platelets. In addition, we aim for the production of genetically modified platelets with the capacity to survive even under platelet transfusion refractoriness.

In our studies, CD34+ hematopoietic progenitor cells and induced pluripotent stem cells (iPSCs) were used to produce HLA-silenced platelets *in vitro*. CD34+ cells or iPSCs were transduced with a lentiviral vector encoding for the shRNA sequence targeting β2m which induces HLA class I silencing. We have demonstrated the feasibility to generate HLA-universal CD34+ cells and iPSCs which might be used for the differentiation of HLA-silenced cellproducts. In our studies, we have differentiated HLA-silenced megakaryocytes and platelets from both cell sources. In our previous studies, we have demonstrated the possibility to generate HLA-silenced platelets with comparable function to blood-derived platelets. How‐ ever, in contrast to blood-derived platelets, *in vitro* generated HLA-silenced platelets were able to escape antibody-mediated complement-dependent cytotoxicity as well as cellular-depend‐ ent cytotoxicity. Also, in a platelet transfusion refractoriness mouse model, HLA-silenced platelets showed the capacity to survive and were even detectable 10 days after transfusion. [28] The limited availability of CD34+ cells derived from G-CSF mobilized donors is a major obstacle to the large-scale production of *in vitro* pharmed platelets. The breakthrough Nobel Prize–winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. Human iPSCs can be used for studying embryo‐ genesis, disease modeling, drug testing, and regenerative medicine.[29] In contrast to CD34+ cells, iPSCs represent an unlimited cell source for the *in vitro* production of a variety of cellbased products including platelets. Therefore, we have recently established an efficient protocol for the differentiation of megakaryocytes and platelets from iPSCs (Figure 2). First, we have generated an HLA-universal iPSC line. Then, the lentiviral vector containing the shRNA targeting β2-microglobulin was used to silence HLA expression on iPSCs. A significant and durable reduction of HLA expression was observed even after passaging. Nevertheless, the HLA-silenced iPSC line showed comparable expression of pluripotency markers (such as SSEA-4 and Tra-1-60) as the original HLA class I-expressing iPSC line (Figure 3). The data indicate that silencing HLA expression does not affect the pluripotency potential of iPSCs.

**Figure 2.** Schematic representation of the differentiation of HLA-silenced platelets from iPSCs. A lentiviral vector en‐ coding for an HLA-specific shRNA is used to transduce the iPSCs. Afterward, HLA-silenced iPSCs will be differentiat‐ ed using a cytokine cocktail containing thrombopoietin (TPO) until the release of platelets.

**Proplatelet producing megakaryocyte**

1

**Platelets**

**Megakaryocyte**

**Endomitosis**

Figure 2. Schematic representation of the differentiation of HLA-silenced platelets from iPSCs. A lentiviral vector encoding for an HLA-specific shRNA is used to transduce the iPSCs. Afterward, HLA-silenced iPSCs will be

**CFU-MK Promegakaryoblast**

**iPSC**

Lentiviral vector encoding the shRNA

**Cell division**

In our studies, CD34+ hematopoietic progenitor cells and induced pluripotent stem cells (iPSCs) were used to produce HLA-silenced platelets *in vitro*. CD34+ cells or iPSCs were transduced with a lentiviral vector encoding for the shRNA sequence targeting β2m which induces HLA class I silencing. We have demonstrated the feasibility to generate HLA-universal CD34+ cells and iPSCs which might be used for the differentiation of HLA-silenced cellproducts. In our studies, we have differentiated HLA-silenced megakaryocytes and platelets from both cell sources. In our previous studies, we have demonstrated the possibility to generate HLA-silenced platelets with comparable function to blood-derived platelets. How‐ ever, in contrast to blood-derived platelets, *in vitro* generated HLA-silenced platelets were able to escape antibody-mediated complement-dependent cytotoxicity as well as cellular-depend‐ ent cytotoxicity. Also, in a platelet transfusion refractoriness mouse model, HLA-silenced platelets showed the capacity to survive and were even detectable 10 days after transfusion. [28] The limited availability of CD34+ cells derived from G-CSF mobilized donors is a major obstacle to the large-scale production of *in vitro* pharmed platelets. The breakthrough Nobel Prize–winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. Human iPSCs can be used for studying embryo‐ genesis, disease modeling, drug testing, and regenerative medicine.[29] In contrast to CD34+ cells, iPSCs represent an unlimited cell source for the *in vitro* production of a variety of cellbased products including platelets. Therefore, we have recently established an efficient protocol for the differentiation of megakaryocytes and platelets from iPSCs (Figure 2). First, we have generated an HLA-universal iPSC line. Then, the lentiviral vector containing the shRNA targeting β2-microglobulin was used to silence HLA expression on iPSCs. A significant and durable reduction of HLA expression was observed even after passaging. Nevertheless, the HLA-silenced iPSC line showed comparable expression of pluripotency markers (such as SSEA-4 and Tra-1-60) as the original HLA class I-expressing iPSC line (Figure 3). The data indicate that silencing HLA expression does not affect the pluripotency potential of iPSCs.

**CFU-MK Promegakaryoblast**

ed using a cytokine cocktail containing thrombopoietin (TPO) until the release of platelets.

**iPSC**

Lentiviral vector encoding the shRNA

316 RNA Interference

**Cell division**

**Megakaryocyte**

**Endomitosis**

**Figure 2.** Schematic representation of the differentiation of HLA-silenced platelets from iPSCs. A lentiviral vector en‐ coding for an HLA-specific shRNA is used to transduce the iPSCs. Afterward, HLA-silenced iPSCs will be differentiat‐

**Proplatelet producing megakaryocyte**

1

**Platelets**

Figure 3. Generation of an HLA-universal iPSC line. (A) iPSCs were adapted to monolayer culture conditions and transduced with a lentiviral vector encoding for a nonspecific shRNA (shNS) or a β2-microglobulin-specific shRNA to silence the expression of HLA class I; (B) expression of SSEA4 and Tra-1-60 on nonmodified and shβ2m-expressing iPSCs; (C) expression of HLA class I of a HLA-silenced shNS-expressing iPSC lines. Cells were stained with an HLA class I antibody (W6/32) and the expression of HLA was measured by flow cytometry after **Figure 3.** Generation of an HLA-universal iPSC line. (A) iPSCs were adapted to monolayer culture conditions and transduced with a lentiviral vector encoding for a nonspecific shRNA (shNS) or a β2-microglobulin-specific shRNA to silence the expression of HLA class I; (B) expression of SSEA4 and Tra-1-60 on nonmodified and shβ2m-expressing iPSCs; (C) expression of HLA class I of a HLA-silenced shNS-expressing iPSC lines. Cells were stained with an HLA class I antibody (W6/32) and the expression of HLA was measured by flow cytometry after different number of passag‐ es. Mean fluorescence intensities (MFI) detected on HLA-silenced iPSCs were normalized to the MFI measured on shNS-expressing iPSCs at the same passage.

7 different number of passages. Mean fluorescence intensities (MFI) detected on HLA-silenced iPSCs were normalized to the MFI measured on shNS-expressing iPSCs at the same passage. In addition, the future application of iPSCs will occur most likely in an allogeneic manner to facilitate their availability during the time of need and standardization of the protocols. Hence, the use of HLA-silenced iPSCs may facilitate the application of HLA-mismatched iPSCderived cell products. For megakaryocyte differentiation, HLA-silenced iPSCs were cultured in monolayer in the presence of vascular endothelial growth factor (VEGF) and BMP-4 for mesoderm induction and afterward in the presence of TPO. During ontogeny, definitive hematopoietic cells are generated de novo from a specialized subset of endothelium, known as hemogenic endothelium. Endothelial-to-hematopoietic transition during embryogenesis provides the first long-term hematopoietic stem and progenitor cells in the embryo.[30] In our differentiation cultures of iPSCs into megakaryocytes, structures resembling the hemogenic endothelium were observed (Figure 4). In suspension, megakaryocytes could be detected by an increase in DNA content higher than 4n and the expression of typical megakaryocytic markers such as CD41 and CD42a. In addition, the megakaryocytes showed the capability to build pro-platelets. Moreover, the shRNA-mediated silencing effect was maintained during the entire differentiation. Importantly, iPSC-derived megakaryocytes and pro-platelets showed a significant reduction of β2-microglobulin and HLA class I antigens in comparison to those differentiated from iPSC expressing a nonspecific shRNA (control) (Figure 5). Differentiation rates of iPSC into megakaryocytes by up to 82% were observed (Figure 6).

observed (Figure 6).

In addition, the future application of iPSCs will occur most likely in an allogeneic manner to facilitate their availability during the time of need and standardization of the protocols. Hence, the use of HLA‐silenced iPSCs may facilitate the application of HLA‐mismatched iPSC‐derived cell products. For megakaryocyte differentiation, HLA‐silenced iPSCs were cultured in monolayer in the presence of vascular endothelial growth factor (VEGF) and BMP‐4 for mesoderm induction and afterward in the presence of TPO. During ontogeny, definitive hematopoietic cells are generated de novo from a specialized subset of endothelium, known as hemogenic endothelium. Endothelial‐to‐hematopoietic transition during embryogenesis provides the first long‐term hematopoietic stem and progenitor cells in the embryo.30 In our differentiation cultures of iPSCs into megakaryocytes, structures resembling the hemogenic endothelium were observed (Figure 4). In suspension, megakaryocytes could be detected by an increase in DNA content higher than 4n and the expression of typical megakaryocytic markers such as CD41 and CD42a. In addition, the megakaryocytes showed the capability to build pro‐platelets. Moreover, the shRNA‐mediated silencing effect was maintained during the entire differentiation. Importantly, iPSC‐derived megakaryocytes and pro‐platelets showed a significant reduction of β2‐microglobulin and HLA class I antigens in comparison to those differentiated from iPSC expressing a

Figure 4. Formation of hemogenic‐like endothelium during the differentiation of iPSCs into megakaryocytes. The **Figure 4.** Formation of hemogenic-like endothelium during the differentiation of iPSCs into megakaryocytes. The pho‐ tos display an island of hemogenic-like endothelium at a magnification of (A) 10x; (B) 20x.

photos display an island of hemogenic‐like endothelium at a magnification of (A) 10x; (B) 20x.

**Figure 5.** Differentiation of HLA-universal megakaryocytes from HLA-silenced iPSC. (A) The histogram represents the flow cytometric analysis of HLA-silenced iPSC-derived megakaryocytes after staining with propidium iodide; (B) light microscopic and (C) fluorescence microscopic analysis of an iPSC-derived megakaryocyte after staining with 4′,6-Dia‐ midin-2-phenylindol (DAPI, blue); (D) light microscopic analysis of pro-platelets indicated with white arrows; (E) realtime PCR analysis of β2-microglobulin levels in megakaryocytes derived from iPSCs transduced with a lentiviral vector encoding the shRNA targeting β2-microglobulin (shβ2m) or a nonspecific shRNA (shNS) as a control.

**Figure 6.** Phenotypic analysis of HLA-universal megakaryocytes derived from iPSCs. The expression of the megakar‐ yocyte markers CD41 and CD42a was measured by flow cytometry in the cells harvested from the iPSC differentiation cultures at different time points. The gates in the dotplots indicate megakaryocytes characterized by the double expres‐ sion of CD41 and CD42a.

8

**0.0**

**Figure 5.** Differentiation of HLA-universal megakaryocytes from HLA-silenced iPSC. (A) The histogram represents the flow cytometric analysis of HLA-silenced iPSC-derived megakaryocytes after staining with propidium iodide; (B) light microscopic and (C) fluorescence microscopic analysis of an iPSC-derived megakaryocyte after staining with 4′,6-Dia‐ midin-2-phenylindol (DAPI, blue); (D) light microscopic analysis of pro-platelets indicated with white arrows; (E) realtime PCR analysis of β2-microglobulin levels in megakaryocytes derived from iPSCs transduced with a lentiviral

vector encoding the shRNA targeting β2-microglobulin (shβ2m) or a nonspecific shRNA (shNS) as a control.

**0.5**

**RQ**

**2m: GAPDH**

**1.0**

**iPSC**

**d19**

sh2m

**1.5** shNS

C

Figure 4. Formation of hemogenic‐like endothelium during the differentiation of iPSCs into megakaryocytes. The

**Figure 4.** Formation of hemogenic-like endothelium during the differentiation of iPSCs into megakaryocytes. The pho‐

photos display an island of hemogenic‐like endothelium at a magnification of (A) 10x; (B) 20x.

tos display an island of hemogenic-like endothelium at a magnification of (A) 10x; (B) 20x.

2n

Counts

PI

4n

8n

D E

A B

In addition, the future application of iPSCs will occur most likely in an allogeneic manner to facilitate their availability during the time of need and standardization of the protocols. Hence, the use of HLA‐silenced iPSCs may facilitate the application of HLA‐mismatched iPSC‐derived cell products. For megakaryocyte differentiation, HLA‐silenced iPSCs were cultured in monolayer in the presence of vascular endothelial growth factor (VEGF) and BMP‐4 for mesoderm induction and afterward in the presence of TPO. During ontogeny, definitive hematopoietic cells are generated de novo from a specialized subset of endothelium, known as hemogenic endothelium. Endothelial‐to‐hematopoietic transition during embryogenesis provides the first long‐term hematopoietic stem and progenitor cells in the embryo.30 In our differentiation cultures of iPSCs into megakaryocytes, structures resembling the hemogenic endothelium were observed (Figure 4). In suspension, megakaryocytes could be detected by an increase in DNA content higher than 4n and the expression of typical megakaryocytic markers such as CD41 and CD42a. In addition, the megakaryocytes showed the capability to build pro‐platelets. Moreover, the shRNA‐mediated silencing effect was maintained during the entire differentiation. Importantly, iPSC‐derived megakaryocytes and pro‐platelets showed a significant reduction of β2‐microglobulin and HLA class I antigens in comparison to those differentiated from iPSC expressing a nonspecific shRNA (control) (Figure 5). Differentiation rates of iPSC into megakaryocytes by up to 82% were

A B

observed (Figure 6).

318 RNA Interference

The complement-dependent cytotoxic (CDC) crossmatch is an informative assay that detects alloantibodies in pre- and post-transplant patients, which may guide the most appropriate clinical management of transplant patients.[31] The capacity of *in vitro* generated HLAsilenced megakaryocytes and platelets to escape antibody-mediated complement-dependent cytotoxicity was evaluated in CDC tests. HLA-silenced megakaryocytes incubated with specific HLA antibodies and complement showed comparable cell lyses rates to the megakar‐ yocytes incubated with nonspecific HLA antibodies. In contrast, significantly higher cell lysis rates were observed when HLA-expressing megakaryocytes were incubated with specific anti-HLA antibodies (Figure 7). These data suggest that HLA-universal iPSC-derived megakaryo‐ cytes are protected from anti-HLA antibody-mediated complement-dependent cytotoxicity and have the potential to survive under refractoriness conditions.

Figure 7. HLA-silenced megakaryocytes are protected from antibody-mediated complement-dependent cytotoxicity. HLA-expressing (shNS) or HLA-silenced (shβ2m) iPSC-derived megakaryocytes were incubated with a nonspecific antibody (NC) or an HLA-specific antibody and complement. Cell lysis was detected by flow **Figure 7.** HLA-silenced megakaryocytes are protected from antibody-mediated complement-dependent cytotoxicity. HLA-expressing (shNS) or HLA-silenced (shβ2m) iPSC-derived megakaryocytes were incubated with a nonspecific antibody (NC) or an HLA-specific antibody and complement. Cell lysis was detected by flow cytometric analysis upon staining with propidium iodide. The bar graph represents means and standard deviations of three independent experi‐ ments. Statistical significance was calculated using Student's *t*-test (\**p* < 0.05, \*\**p* < 0.01; \*\*\**p* < 0.001).

cytometric analysis upon staining with propidium iodide. The bar graph represents means and standard

#### deviations of three independent experiments. Statistical significance was calculated using Student's *t*-test (\**p*< **2.5. Generation of HLA-silenced tissues**

0.05, \*\**p*< 0.01; \*\*\**p*< 0.001). **2.5. Generation of HLA-silenced tissues**  Due to the high variability of the HLA loci and the shortage of organs and tissues for transplantation, it is very difficult to find a complete HLA-matched graft for a given recipient. The number of HLA mismatches between the graft and the recipient are associated with a higher risk for graft rejection and even morbidity and mortality due to immunosuppression-related side effects. Thus, it would be desirable to engineer the grafts in order to decrease their immunogenicity by silencing HLA expression. Worldwide, the demand for organs and tissues for transplantation is very high and it is not possible to satisfy all needs. This discrepancy is even accentuated in the Middle East and countries in the East such as India and China. According to the World Health Organization (WHO), there are over 10 million people in the world who are blind in one or both eyes due to corneal injury or Due to the high variability of the HLA loci and the shortage of organs and tissues for trans‐ plantation, it is very difficult to find a complete HLA-matched graft for a given recipient. The number of HLA mismatches between the graft and the recipient are associated with a higher risk for graft rejection and even morbidity and mortality due to immunosuppression-related side effects. Thus, it would be desirable to engineer the grafts in order to decrease their immunogenicity by silencing HLA expression. Worldwide, the demand for organs and tissues for transplantation is very high and it is not possible to satisfy all needs. This discrepancy is even accentuated in the Middle East and countries in the East such as India and China. According to the World Health Organization (WHO), there are over 10 million people in the world who are blind in one or both eyes due to corneal injury or disease and up to 45 million people could benefit from corneal transplants. However, according to data from eye banks and health agencies, less than 150,000 corneal transplants are done annually worldwide due to the shortage of human cadaver corneas. Furthermore, during the first five years after penetrating keratoplasty, rejection is responsible for 28–35% of total corneal graft loss. High risk-corneal recipients even showed increased rejection rates (30–56%).[32, 33]

11 disease and up to 45 million people could benefit from corneal transplants. However, according to data from eye banks and health agencies, less than 150,000 corneal transplants are done annually worldwide due to the shortage of human cadaver corneas. Furthermore, during the first five years after penetrating keratoplasty, rejection is The cornea presents a simple anatomical structure, in which the endothelium is easily accessible to the shRNA-encoding viral vector containing supernatant (Figure 8). The integrity of the endothelial cell layer is crucial for the transparency of the cornea and it is the major target for rejection. Therefore, silencing HLA expression on the corneal endothelium may improve cornea survival in high-risk patients after allogeneic keratoplasty.

responsible for 28–35% of total corneal graft loss. High risk-corneal recipients even showed increased rejection

 The cornea presents a simple anatomical structure, in which the endothelium is easily accessible to the shRNAencoding viral vector containing supernatant (Figure 8). The integrity of the endothelial cell layer is crucial for the transparency of the cornea and it is the major target for rejection. Therefore, silencing HLA expression on the

**Figure 8.** Anatomy of the cornea. Optical coherence tomography of a mouse cornea showing the three main layers.

We have silenced HLA expression in human and mice corneas. The tissue was transduced for 8 h with the lentiviral vector encoding for the MHC-specific shRNAs as described above. We were able to silence the MHC expression on the cornea endothelium, which is the major target during graft rejection. In this study, we demonstrated the feasibility to generate HLA universal tissues in their original 3D structure (Figure 9). Silencing HLA expression on tissues is expected to significantly improve graft survival rates in high-risk keratoplasty patients. We have silenced HLA expression in human and mice corneas. The tissue was transduced for 8 h with the lentiviral vector encoding for the MHC-specific shRNAs as described above. We were able to silence the MHC expression on the cornea endothelium, which is the major target during graft rejection. In this study, we demonstrated the feasibility to generate HLA universal tissues in their original 3D structure (Figure 9). Silencing HLA expression on tissues is expected to significantly improve graft survival rates in high-risk keratoplasty

12 **Figure 9.** Generation of MHC-silenced corneas. A mouse cornea was explanted and transduced with a lentiviral vector encoding the shβ2m to cause a downregulation of MHC class I antigens and the expression of green fluorescence pro‐ tein (GFP) sequences. (A) Light microscopic and (B) fluorescence microscopic analyses of a transduced mouse cornea.

#### **2.6. Conclusion**

patients.

rates (30–56%).32,33

11

The cornea presents a simple anatomical structure, in which the endothelium is easily accessible to the shRNA-encoding viral vector containing supernatant (Figure 8). The integrity of the endothelial cell layer is crucial for the transparency of the cornea and it is the major target for rejection. Therefore, silencing HLA expression on the corneal endothelium may

Figure 7. HLA-silenced megakaryocytes are protected from antibody-mediated complement-dependent cytotoxicity. HLA-expressing (shNS) or HLA-silenced (shβ2m) iPSC-derived megakaryocytes were incubated with a nonspecific antibody (NC) or an HLA-specific antibody and complement. Cell lysis was detected by flow cytometric analysis upon staining with propidium iodide. The bar graph represents means and standard deviations of three independent experiments. Statistical significance was calculated using Student's *t*-test (\**p*<

**Figure 7.** HLA-silenced megakaryocytes are protected from antibody-mediated complement-dependent cytotoxicity. HLA-expressing (shNS) or HLA-silenced (shβ2m) iPSC-derived megakaryocytes were incubated with a nonspecific antibody (NC) or an HLA-specific antibody and complement. Cell lysis was detected by flow cytometric analysis upon staining with propidium iodide. The bar graph represents means and standard deviations of three independent experi‐

Due to the high variability of the HLA loci and the shortage of organs and tissues for trans‐ plantation, it is very difficult to find a complete HLA-matched graft for a given recipient. The number of HLA mismatches between the graft and the recipient are associated with a higher risk for graft rejection and even morbidity and mortality due to immunosuppression-related side effects. Thus, it would be desirable to engineer the grafts in order to decrease their immunogenicity by silencing HLA expression. Worldwide, the demand for organs and tissues for transplantation is very high and it is not possible to satisfy all needs. This discrepancy is even accentuated in the Middle East and countries in the East such as India and China. According to the World Health Organization (WHO), there are over 10 million people in the world who are blind in one or both eyes due to corneal injury or disease and up to 45 million people could benefit from corneal transplants. However, according to data from eye banks and health agencies, less than 150,000 corneal transplants are done annually worldwide due to the shortage of human cadaver corneas. Furthermore, during the first five years after penetrating keratoplasty, rejection is responsible for 28–35% of total corneal graft loss. High risk-corneal

ments. Statistical significance was calculated using Student's *t*-test (\**p* < 0.05, \*\**p* < 0.01; \*\*\**p* < 0.001).

**anti-HLA-A\*02**

 **Ab**

\*\*\*

shNS sh2m

Due to the high variability of the HLA loci and the shortage of organs and tissues for transplantation, it is very difficult to find a complete HLA-matched graft for a given recipient. The number of HLA mismatches between the graft and the recipient are associated with a higher risk for graft rejection and even morbidity and mortality due to immunosuppression-related side effects. Thus, it would be desirable to engineer the grafts in order to decrease their immunogenicity by silencing HLA expression. Worldwide, the demand for organs and tissues for transplantation is very high and it is not possible to satisfy all needs. This discrepancy is even accentuated in the Middle East and countries in the East such as India and China. According to the World Health Organization (WHO), there are over 10 million people in the world who are blind in one or both eyes due to corneal injury or disease and up to 45 million people could benefit from corneal transplants. However, according to data from eye banks and health agencies, less than 150,000 corneal transplants are done annually worldwide due to the shortage of human cadaver corneas. Furthermore, during the first five years after penetrating keratoplasty, rejection is

0.05, \*\**p*< 0.01; \*\*\**p*< 0.001).

**2.5. Generation of HLA-silenced tissues**

**2.5. Generation of HLA-silenced tissues** 

recipients even showed increased rejection rates (30–56%).[32, 33]

improve cornea survival in high-risk patients after allogeneic keratoplasty.

**NS**

**0**

**5**

**10**

**% Cell Lysis**

320 RNA Interference

**15**

**Ab (NC)**

Recently, gene regulation or editing strategies have emerged as powerful tools to improve the design and efficacy of personalized cell-based therapies. In the field of histocompatibility and transplantation, RNAi seems to be a favorite approach to reduce the immunogenicity of allogeneic and *in vitro* generated cells and tissues. The lentiviral vector-mediated delivery of shRNAs targeting HLA transcripts prevents the activation of cellular and humoral allogeneic immune responses that cause the rejection of the foreign cells. However, this RNAi-based strategy permits the residual expression of HLA class I antigens which are crucial to inhibit NK cell cytotoxicity. With the establishment of iPSCs, the concept of cell pharming came one step closer to reality as iPSCs may serve as unlimited cell sources for different cell products such as platelets. The combination of RNAi-mediated HLA silencing and the capacity to generate platelets *in vitro* may represent a novel therapeutic approach for the management of alloimmunized thrombocytopenic patients with an increased risk to develop refractoriness to platelet transfusion. Furthermore, our results also indicate the feasibility to reduce MHC expression in the 3D original structure of tissues. Abrogating the histocompatibility barrier between donors and recipients may improve therapeutic efficacy, reduce the adverse events associated with strong immunosuppressive regimens, and improve transplant patient life's quality. In conclusion, RNAi-mediated silencing of HLA expression may open new avenues in tissue engineering and transplantation.

#### **Author details**

Constanca Figueiredo\* and Rainer Blasczyk

\*Address all correspondence to: figueiredo.constanca@mh-hannover.de

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany

#### **References**


[6] Buchmann K. Evolution of innate immunity: clues from Invertebrates via fish to mammals. *Front Immunol*. 2014;5:459.

allogeneic and *in vitro* generated cells and tissues. The lentiviral vector-mediated delivery of shRNAs targeting HLA transcripts prevents the activation of cellular and humoral allogeneic immune responses that cause the rejection of the foreign cells. However, this RNAi-based strategy permits the residual expression of HLA class I antigens which are crucial to inhibit NK cell cytotoxicity. With the establishment of iPSCs, the concept of cell pharming came one step closer to reality as iPSCs may serve as unlimited cell sources for different cell products such as platelets. The combination of RNAi-mediated HLA silencing and the capacity to generate platelets *in vitro* may represent a novel therapeutic approach for the management of alloimmunized thrombocytopenic patients with an increased risk to develop refractoriness to platelet transfusion. Furthermore, our results also indicate the feasibility to reduce MHC expression in the 3D original structure of tissues. Abrogating the histocompatibility barrier between donors and recipients may improve therapeutic efficacy, reduce the adverse events associated with strong immunosuppressive regimens, and improve transplant patient life's quality. In conclusion, RNAi-mediated silencing of HLA expression may open new avenues

in tissue engineering and transplantation.

and Rainer Blasczyk

solutions. *Curr Stem Cell Rep*. 2015;1(2):110–117.

\*Address all correspondence to: figueiredo.constanca@mh-hannover.de

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany

by lentiviral shRNA delivery. *J Mol Med (Berl)*. 2006;84(5):425–437.

[1] Barry J, Hyllner J, Stacey G, Taylor CJ, Turner M. Setting up a Haplobank: issues and

[2] Figueiredo C, Seltsam A, Blasczyk R. Class-, gene-, and group-specific HLA silencing

[3] Jaimes Y, Seltsam A, Eiz-Vesper B, Blasczyk R, Figueiredo C. Regulation of HLA class II expression prevents allogeneic T-cell responses. *Tissue Antigens*. 2011;77(1):

[4] Wiegmann B, Figueiredo C, Gras C, et al. Prevention of rejection of allogeneic endo‐ thelial cells in a biohybrid lung by silencing HLA-class I expression. *Biomaterials*.

[5] Hirano M, Das S, Guo P, Cooper MD. The evolution of adaptive immunity in verte‐

**Author details**

322 RNA Interference

**References**

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2014;35(28):8123–8133.

brates. *Adv Immunol*. 2011;109:125–157.

Constanca Figueiredo\*


## **Utility of Potent Anti-viral MicroRNAs in Emerging Infectious Diseases**

Zhabiz Golkar, Donald G. Pace and Omar Bagasra

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61687

#### **Abstract**

[21] Khan S, Khan S, Baboota S, Ali J. Immunosuppressive drug therapy - biopharmaceut‐ ical challenges and remedies. *Expert Opin Drug Deliv*. 2015;12(8):1333–1349.

[22] Figueiredo C, Wedekind D, Muller T, et al. MHC universal cells survive in an alloge‐ neic environment after incompatible transplantation. *Biomed Res Int*.

[24] Patel-Hett S, Wang H, Begonja AJ, et al. The spectrin-based membrane skeleton sta‐ bilizes mouse megakaryocyte membrane systems and is essential for proplatelet and

[25] Stanworth SJ, Navarrete C, Estcourt L, Marsh J. Platelet refractoriness - practical ap‐ proaches and ongoing dilemmas in patient management. *Br J Haematol*. 2015;171(3):

[26] Crane AM, Kramer P, Bui JH, et al. Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells. *Stem Cell Reports*.

[27] Tong Z, Solanki A, Hamilos A, et al. Application of biomaterials to advance induced

[28] Gras C, Schulze K, Goudeva L, Guzman CA, Blasczyk R, Figueiredo C. HLA-univer‐ sal platelet transfusions prevent platelet refractoriness in a mouse model. *Hum Gene*

[29] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐ ic and adult fibroblast cultures by defined factors. *Cell*. 2006;126(4):663–676.

[30] Zape JP, Zovein AC. Hemogenic endothelium: origins, regulation, and implications

[31] Lawrence C, Willicombe M, Brookes PA, et al. Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal

[32] Stuart PM, Yin X, Plambeck S, Pan F, Ferguson TA. The role of Fas ligand as an effec‐ tor molecule in corneal graft rejection. *Eur J Immunol*. 2005;35(9):2591–2597.

[33] Yin XT, Zobell S, Jarosz JG, Stuart PM. Anti-IL-17 therapy restricts and reverses late-

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term corneal allorejection. *J Immunol*. 2015;194(8):4029–4038.

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pluripotent stem cell research and therapy. *EMBO J*. 2015;34(8):987–1008.

[23] Thon JN, Italiano JE. Platelet formation. *Semin Hematol*. 2010;47(3):220–226.

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2013;2013:796046.

324 RNA Interference

297-305

2015;4(4):569–577.

*Ther*. 2013;24(12):1018–1028.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that have emerged as im‐ portant posttranscriptional regulators of gene expression. miRNA provides intracellular immune defense when the body is faced with challenges from transgenes, viruses, trans‐ posons, and aberrant mRNAs. miRNA molecules trigger gene silencing in eukaryotic cells. To date, more than 3,000 different human miRNAs (*hsa-miRs*) have been identified, and it is generally agreed that cellular gene regulation is significantly impacted by the presence of miRNAs. A single miRNA has the complex capacity to target multiple genes simultaneously. In a viral infection context, miRNAs have been connected with the inter‐ play between host and pathogen, and occupy a major role in the host–parasite interaction and pathogenesis. While numerous viral miRNAs from DNA viruses have been identi‐ fied, characterization of functional RNA virus-encoded miRNAs and their potential tar‐ gets is still ongoing. Here, we describe an in silico approach to analyze the most recent Ebola virus (EBOV) genome sequences causing West African epidemics. We identified numerous "candidate" miRNAs that can be utilized to quell the Ebola virus. Future ap‐ proaches will focus on experimental validation of these miRNAs during quelling the Ebo‐ la target transcripts for further elucidating their biological functions in primates and other animal models.

**Keywords:** Ebola virus, gene alignment, miRNA, prevention, vaccine

#### **1. Introduction**

#### **1.1. Inhibition of Ebola virus by anti-Ebola miRNAs In silico**

Since the HIV-1 pandemic of the 1980s and more recent outbreaks of bird flu, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome-Corona Virus (MER-CoV), it has been widely believed that there would be new pandemics of highly pathogenic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

viruses [1]. Fortunately, until recently, many of the new emerging pathogenic agents, such as Ebola virus (EBOV) and Marburg virus (MARV), have failed to demonstrate the transmissi‐ bility or animal reservoirs required to become true pandemic threats [2, 3]. Few, if any, antivirals can claim to be specific enough to halt the epidemic. Recently, a whole EBOV replication-defective vaccine—EBOVdVP30—has been found to be very effective in nonhu‐ man primates, while two others are in Phase II trials [4].

The testing of the recently developed replication-defective recombinant chimpanzee adeno‐ virus type 3–vectored ebolavirus (cAd3-EBO) vaccine is based on a demonstration of efficacy in a nonhuman primate model [5]. However, a curious finding has puzzled the investigators —preexisting neutralizing antibodies against cAd6 and cAd68 in human serum samples were found in ~40% of Ugandans and in 15% of the US and European volunteers. The increased prevalence of neutralizing antibodies against chimpanzee adenoviruses in sub-Saharan Africa may indicate cross-species transmission of these viruses from chimpanzees to humans. The possibility of fairly high levels of neutralizing antibodies against cAd3 may complicate the evaluation of the effectiveness of Ebola vaccines currently underway.

#### **1.2. Virology of Ebola virus**

Filoviruses are taxonomically classified within the order *Mononegavirales*, a large group of enveloped viruses whose genomes are composed of a nonsegmented, single-stranded minus [2, 3] RNA molecule. Following their discovery, filoviruses were originally grouped with rhabdoviruses, as the appearance of these virus particles appeared similar [3]. However, subsequent filamentous morphology and extensive genetic, physiochemical, and virologic studies of Marburg virus (MARV) and Ebola virus (EBOV) revealed distinctive characteristics, and these viruses were placed into a separate family, the *Filoviridae*[3]. Further characterization of these agents demonstrated that EBOV and MARV represent divergent lineages of filovi‐ ruses, and that their variances were significant enough to warrant the formation of the two genera, MARV and EBOV [4]. Subsequent to the International Committee on Taxonomy of Viruses recommendation, the MARV genus contains a single species, the Lake Victoria Marburg virus, as this strain exhibits only limited genetic variation.

EBOV is the causative agent of Ebola virus disease (EVD) [6]. The mortality rate can vary from 40% to 93% depending on the strains [6]. The viral life cycle begins with host cell entry through a mechanism that is still poorly understood. The incubation period for EVD is 2–21 days, and typical early symptoms include fever, chills, malaise, and myalgia (all of which could be misdiagnosed as malaria, which is highly prevalent in West African nations), followed by the onset of symptoms indicative of multi-organ stress and subsequent failure, sometimes followed by hemorrhagic episodes that can easily be misdiagnosed as Lassa fever.

EBOV is a negative, single-stranded RNA virus with an unusual, variable-length, filamentous, branched morphology whose helical capsid is enclosed inside a membrane. The mechanism of attachment and entry into the cell is still not completely defined (see discussion below). Once inside, the viral RNA polymerase (L protein) begins to copy the negative strand (–ve) RNA to make the positive strand (+ve) transcripts that mimic the structure of mRNA and are translated by host ribosomes. Replication is thought to occur in the cytoplasm. An unusual feature of the transcription and translation of the Ebola genes is the fact that the glycoproteins (GP) are only expressed through transcriptional editing. The genome of the Zaire Ebola virus (EBOV), the most pathogenic among all species of EBOV, is 18,959 nucleotides (nts) in length and contains seven transcriptional units that guide synthesis of at least nine distinct primary translation products: the nucleoprotein (NP), virion protein (VP) 35, VP40, glycoprotein (GP), soluble glycoprotein (sGP), small soluble glycoprotein (ssGP), VP30, VP24, and the large (L) protein. L is the catalytic subunit of the viral polymerase complex (Figure 1). Similar to other nonsegmented negative-sense (NNS) RNA viruses, EBOVs encode a multiprotein complex to carry out replication and transcription. In the case of EBOV, viral RNA synthesis requires the viral NP, VP35, VP30, and L proteins. Transcription of filovirus mRNAs is presumed to occur as in other NNS viruses, where there is a gradient of viral mRNAs with the abundance of each mRNA transcript decreasing as the polymerase transcribes toward the 5′ end of the template [7]. Each EBOV mRNA is presumed to be efficiently modified with a 5′–7'-methylguanosine (m7 G) cap and a 3′p (A) tail [8].

viruses [1]. Fortunately, until recently, many of the new emerging pathogenic agents, such as Ebola virus (EBOV) and Marburg virus (MARV), have failed to demonstrate the transmissi‐ bility or animal reservoirs required to become true pandemic threats [2, 3]. Few, if any, antivirals can claim to be specific enough to halt the epidemic. Recently, a whole EBOV replication-defective vaccine—EBOVdVP30—has been found to be very effective in nonhu‐

The testing of the recently developed replication-defective recombinant chimpanzee adeno‐ virus type 3–vectored ebolavirus (cAd3-EBO) vaccine is based on a demonstration of efficacy in a nonhuman primate model [5]. However, a curious finding has puzzled the investigators —preexisting neutralizing antibodies against cAd6 and cAd68 in human serum samples were found in ~40% of Ugandans and in 15% of the US and European volunteers. The increased prevalence of neutralizing antibodies against chimpanzee adenoviruses in sub-Saharan Africa may indicate cross-species transmission of these viruses from chimpanzees to humans. The possibility of fairly high levels of neutralizing antibodies against cAd3 may complicate the

Filoviruses are taxonomically classified within the order *Mononegavirales*, a large group of enveloped viruses whose genomes are composed of a nonsegmented, single-stranded minus [2, 3] RNA molecule. Following their discovery, filoviruses were originally grouped with rhabdoviruses, as the appearance of these virus particles appeared similar [3]. However, subsequent filamentous morphology and extensive genetic, physiochemical, and virologic studies of Marburg virus (MARV) and Ebola virus (EBOV) revealed distinctive characteristics, and these viruses were placed into a separate family, the *Filoviridae*[3]. Further characterization of these agents demonstrated that EBOV and MARV represent divergent lineages of filovi‐ ruses, and that their variances were significant enough to warrant the formation of the two genera, MARV and EBOV [4]. Subsequent to the International Committee on Taxonomy of Viruses recommendation, the MARV genus contains a single species, the Lake Victoria

EBOV is the causative agent of Ebola virus disease (EVD) [6]. The mortality rate can vary from 40% to 93% depending on the strains [6]. The viral life cycle begins with host cell entry through a mechanism that is still poorly understood. The incubation period for EVD is 2–21 days, and typical early symptoms include fever, chills, malaise, and myalgia (all of which could be misdiagnosed as malaria, which is highly prevalent in West African nations), followed by the onset of symptoms indicative of multi-organ stress and subsequent failure, sometimes

EBOV is a negative, single-stranded RNA virus with an unusual, variable-length, filamentous, branched morphology whose helical capsid is enclosed inside a membrane. The mechanism of attachment and entry into the cell is still not completely defined (see discussion below). Once inside, the viral RNA polymerase (L protein) begins to copy the negative strand (–ve) RNA to make the positive strand (+ve) transcripts that mimic the structure of mRNA and are translated by host ribosomes. Replication is thought to occur in the cytoplasm. An unusual

followed by hemorrhagic episodes that can easily be misdiagnosed as Lassa fever.

man primates, while two others are in Phase II trials [4].

**1.2. Virology of Ebola virus**

326 RNA Interference

evaluation of the effectiveness of Ebola vaccines currently underway.

Marburg virus, as this strain exhibits only limited genetic variation.

**Figure 1.** The genome of EBOV, 18 9 kb in length, has the following gene order: 3′ leader nucleoprotein (NP), virion proteins (VP) VP35-VP40, membrane glycoprotein (GP), viral polymerase (VP) VP30-VP24, viral polymerase L protein, and 5′ trailer.

The Ebola virus genus possesses greater diversity, and four viral species have been recognized: Zaire Ebola, Sudan Ebola, Reston Ebola, and Ivory Coast Ebola (EBOV-Z, EBOV-S, EBOV-R, and EBOV-IC, respectively). Each of the EBOV species has a different degree of pathogenicity and mortality rate [9]. Therefore, EBOV-S and EBOV-Z, which are the predominant EBOVs associated with known outbreaks, are more pathogenic than EBOV-R and EBOV-IC [10]. EBOV-IC has only caused a single nonfatal human infection, but EBOV-R has caused fatal infection in nonhuman primates [8]. However, EBOV-S, EBOV-Z, and EBOV-B often cause severe hemorrhagic diseases with markedly high case fatality rates (40–90%) [10]. The EBOV genome is 18.9 kb in length with the following gene order: 3′ leader nucleoprotein (NP), virion protein (VP) 35-VP40, glycoprotein (GP), VP30, VP24, polymerase (L), and 5′ trailer. The GP differences between any two species range from 37% to 41% at the nucleotide level and from 34% to 43% at the amino acid level [40]. However, variations within EBOV-Z species are very low (∼2–3%) [11]. Thus, GP nucleotides are usually used in the phylogenetic analysis of EBOV (Figure 2).

**Figure 2.** The Ebola Pandemic Map depicts the history of Ebola in Africa. The sporadic cases of Ebola were common in the central African countries such as the DRC (988 cases with 767 fatalities), Uganda (606 cases with 283 fatalities), South Sudan (335 cases with 180 fatalities), Gabon (214 cases with 150 fatalities), the Republic of the Congo (248 cases with 210 fatalities), and South Africa (2 cases with 1 fatality).

Because of their high mortality rate, which can vary from 40% to 93%, and their potential for person-to-person transmission and lack of an approved vaccine or antiviral therapy, MARV and EBOV are classified as biosafety level 4 (BSL-4) viruses by World Health Organization [12, 13]

#### **1.3. History of MARV and EBOV**

34% to 43% at the amino acid level [40]. However, variations within EBOV-Z species are very low (∼2–3%) [11]. Thus, GP nucleotides are usually used in the phylogenetic analysis of EBOV

**Figure 2.** The Ebola Pandemic Map depicts the history of Ebola in Africa. The sporadic cases of Ebola were common in the central African countries such as the DRC (988 cases with 767 fatalities), Uganda (606 cases with 283 fatalities), South Sudan (335 cases with 180 fatalities), Gabon (214 cases with 150 fatalities), the Republic of the Congo (248 cases

Because of their high mortality rate, which can vary from 40% to 93%, and their potential for person-to-person transmission and lack of an approved vaccine or antiviral therapy, MARV and EBOV are classified as biosafety level 4 (BSL-4) viruses by World Health

with 210 fatalities), and South Africa (2 cases with 1 fatality).

Organization [12, 13]

(Figure 2).

328 RNA Interference

Viruses are obligate intracellular parasites and essentially rely on host cells for raw materials, replication, transcription, and translations of their genetic codes. Until a few years ago, we assumed that the major intracellular defenses against viral pathogens were interferons [13]. Since the discovery of RNA interference (RNAi) and miRNAs, we know that one of the fundamental functions of miRNAs is to prevent replication of foreign viruses by pre- and posttranscriptions and suppressions of viral expression [12]. Therefore, besides endogenous gene regulation, miRNAs are the primary intracellular immune defense system [13]. Viruses have also evolved to counter the antiviral effects of miRNAs by viral miRNAs (vmiRNAs).

Recently, Li and Chen [14] have conducted molecular epidemiologic analyses of presently extant Ebola viral genomes to ascertain their evolutionary viral history. Of considerable potential importance are interpretations derived from a dataset that is between 1,000 and 2,100 years old and includes four Ebola species (EBOV-Z, EBOV-S, EBOV-TF, EBOV-R) [15]. Logically, one could assume that over the past 2,000 years, humans have evolved counter‐ measures to the Ebola virus via innate, adaptive, and miRNA-based immunity. The identification in a human database of 71 miRNAs capable of potentially quelling EBOV strongly suggests that *Homo sapiens* already have developed primary intracellular defens‐ es to quell EBOV infection [16]. This raises a question: Why have EBOVs been circulating for about 2,000 years, and yet they seem to have emerged only recently? The earliest known cases of Ebola date to the 1970s. One theory proposes that EBOV-Z experienced a recent genetic bottleneck [17]. Before Ebola viral strains were introduced to primates, they had already been circulating among small mammals, including bats, rodents, marsupials, shrews, and so on [16]. Although these bats and other animals were infected [15, 16], no evidence demonstrated that such infections were fatal to them [18]. This indicates that a natural balance had been achieved between the viruses' pathogenicity and the host's immune system, especially at the intracellular levels where miRNAs provide immunologi‐ cal protection [19]. This homeostasis, this balance, apparently was broken in 1900, when EBOV genetic diversity experienced a dramatic drop [16]. Accordingly, most lineages of the various EBOV species became extinct because of such influences as threatening human activities, climate change, and a steep decline in the number of animals to serve as a reservoir for viral replication. Probably due to altered patterns of positive selection in the glycoprotein (GP), which diversified substantially and was found to be part of fusion and receptor binding within cellular membranes, infection patterns through direct exposure were changing. Therefore, by about 1970, few lineages that possessed broader tropism and enhanced fitness had the capacity to infect primates via direct exposure [16]. Similar examples can be seen in the emergence of HIV-1, which appeared to have surfaced in the 1950s through a zoonotic event that involved common infections among chimpanzees (i.e., SIV) and then accidentally jumped to humans [16–18]. Due to the paucity of significant differences in EBOV genetic diversity since 1970, the decreased number of surviving viruses may have become the only circulating lineages in primates and viral reservoirs. EBOV-Z has the ability to traverse a long distance through bats, which serve as a migratory reservoir. Outbreaks with their epicenter in Congo have been caused by the EBOV-Z species [20–23]. Through analysis of miRNA numbers that demonstrate high homologies in seed sequences and that show high identity to EBOV species, we have deduced that the genetic variations at the GP may serve as a type of Achilles' heel. After all, only one miRNA showed identity to GP, while eight proved capable of blocking polymerase steps. This indicates that minor variations within the GP amino acid sequence could allow for viral entrance into host target cells in humans. The subsequent transcription of negative-stranded RNA viruses into positive RNA strands occurs amid a struggle to overcome the miRNAs with quelling potential that can halt this process. It is possible that at the time of exposure to EBOV, all of the protective miRNAs may not be present in the target cells, or may be present, but not in sufficient quantities to block early EBOV replication [24].

**Figure 3.** The illustration depicts a simplified structure of Ebola virus. The functions of various viral proteins are de‐ scribed in the text. GP, glycoprotein; NP, nucleoprotein; VP40, matrix protein; VP30, transcription factor; and polymer‐ ase enzyme.

Figure 3 shows the VP24, VP30, VP35, VP40, and L nucleoproteins that constitute the nucleo‐ capsid, which is crucial in both the transcription and viral replication processes [25, 26]. The glycoprotein is located in the lipid membrane of the Ebola virus; this is also the place in the host target cells where receptors that facilitate viral entry are embedded [27]. Viral matrix proteins VP40 and VP24 are essential to viral budding, stability, and structure. VP40 is the primary matrix protein, and is the viral protein that is expressed most abundantly. It plays a central role in the process of Ebola budding from the plasma membrane. For example, in mammalian cells, the mere expression of VP40 is sufficient to create virus-like particles (VLPs) with morphological characteristics that are similar to those of the actual Ebola virus [28, 29]. Given VP40's absence, studies have found that the nucleocapsid was not transported effec‐ tively into the plasma membrane, and as this membrane is the site of assembly, budding, and incorporation into the virions, considerable attention should be given to the role of this matrix protein [30]. The utilization of miRNAs that specifically target VP40 mRNA degradation is important to our understanding of just how VP40 functions and what potential roles it might play in the regulation of VLP assembly in both in vitro and live cell settings. *hsa-miR-4692* and *hsa-miR-548-az* effectively target VP40; therefore, the overexpression of these particular miRNAs within host cells could totally disrupt the viral life cycle and may have a decisive impact in the categorization of therapeutic targets (data unpublished). The tendency of Ebola VP40 to assemble virus-like particles (VLPs) presents an appealing model for analysis of the Ebola viral assembly at biosafety level 2 made possible by the noninfectious nature of geneti‐ cally engineered VLPs [31].

Through analysis of miRNA numbers that demonstrate high homologies in seed sequences and that show high identity to EBOV species, we have deduced that the genetic variations at the GP may serve as a type of Achilles' heel. After all, only one miRNA showed identity to GP, while eight proved capable of blocking polymerase steps. This indicates that minor variations within the GP amino acid sequence could allow for viral entrance into host target cells in humans. The subsequent transcription of negative-stranded RNA viruses into positive RNA strands occurs amid a struggle to overcome the miRNAs with quelling potential that can halt this process. It is possible that at the time of exposure to EBOV, all of the protective miRNAs may not be present in the target cells, or may be present, but not in sufficient quantities to block

**Figure 3.** The illustration depicts a simplified structure of Ebola virus. The functions of various viral proteins are de‐ scribed in the text. GP, glycoprotein; NP, nucleoprotein; VP40, matrix protein; VP30, transcription factor; and polymer‐

Figure 3 shows the VP24, VP30, VP35, VP40, and L nucleoproteins that constitute the nucleo‐ capsid, which is crucial in both the transcription and viral replication processes [25, 26]. The glycoprotein is located in the lipid membrane of the Ebola virus; this is also the place in the host target cells where receptors that facilitate viral entry are embedded [27]. Viral matrix proteins VP40 and VP24 are essential to viral budding, stability, and structure. VP40 is the primary matrix protein, and is the viral protein that is expressed most abundantly. It plays a central role in the process of Ebola budding from the plasma membrane. For example, in mammalian cells, the mere expression of VP40 is sufficient to create virus-like particles (VLPs) with morphological characteristics that are similar to those of the actual Ebola virus [28, 29]. Given VP40's absence, studies have found that the nucleocapsid was not transported effec‐ tively into the plasma membrane, and as this membrane is the site of assembly, budding, and

early EBOV replication [24].

330 RNA Interference

ase enzyme.

VP40's association with the plasma membrane is of fundamental importance [30]; it is here that assembly is initiated as well as oligomerization [31], and nucleoprotein recruitment. Besides membrane association, VP40 also associates or otherwise interacts with host cell factors, including the endosomal sorting complex that supports transport (ESCRT) machinery [27, 29], the vesicle coat II proteins (COPII) [24], as well as the protein actin [25, 30]; these host cell factors, respectively, have been shown to enable VP40 budding, transport, and movement. Moreover, host cell protein kinases could contribute to Ebola infectivity as c-Abl1 can phos‐ phorylate Tyr13 in VP40 [32, 33]. Still we have an inadequate understanding of how VP40 actually assembles on the plasma membrane before virion release occurs. Localization of VP40 in the plasma membrane is believed to be important as studies give evidence that hydrophobic residues located within the C-terminal domain, including Leu213, are essential in the localiza‐ tion and budding processes [32]. Detection of VP40 oligomers in VLPs and UV-inactivated virions has occurred [34, 35]; they have been detected mainly in filamentous structures stemming from the plasma membrane [36]. Therefore, VP40 oligomerization apparently occurs on the same plasma membrane in which oligomers selectively have found to reside [37]. In terms of structure, VP40 has predominantly been found to oligomerize into either hexamers or octamers [38, 39]. These share a comparable monomer–monomer (or intradimeric) antipar‐ allel interface. However, the detection of oligomeric structures in live cells suggests that these structures, too, could exert a critical influence on both viral assembly and egress [40]. We discovered that *hsa-miR-4692* and *hsa-miR-548-az* both target VP40 (data unpublished).

The formation of virus-like particles (VLPs) requires VP40 oligomers; these are associated with membranes that are resistant to detergent [41], which underscores the active part that the plasma membrane may play in VP40 oligomerization. Moreover, on the plasma membrane, matrix protein oligomerization may function as a scaffold in host protein recruitment, and also supply the force needed to effect the formation of virus particles and the deformation of membranes. A comprehension of VP40 plasma membrane association thus becomes crucial to our understanding of how the formation of protein buds occurs on the plasma membrane. Gupta K [42] recently investigated the role that the VP40 C-terminal domain plays in mem‐ brane association as well as in membrane penetration. These investigators utilized the monolayer penetration methodology to conduct in vitro research into the molecular basis of the penetration of the VP40 membrane. To study VP40 assembly and its associated egress in cells, they employed a multipronged methodology that blended cellular imaging, number and brightness (N&B) analysis, analysis of the egress of virus-like particles, site-directed muta‐ genesis, and total internal reflection (TIRF) microscopy. N&B analysis permitted them to ascertain the average number of molecules and also the brightness within each pixel within a fluorescence microscopy image. This permitted them to detect the oligomeric status of proteins that are labeled fluorescently. They concluded that within the VP40 C-terminal domain, a hydrophobic interface actually penetrates the plasma membrane, which plays a key role in the oligomerization of VP40. The knocking out of plasma membrane penetration by hydrophobic mutants also substantially reduces the egress of VLPs [38, 39]. Therefore, degradation of VP40 mRNA by a two-pronged attack from *hsa-miR-4692* and *hsa-miR-548-az* can stop Ebola.

A distinguishing characteristic of filovirus genomes is their 3′- and 5′-UTRs that are long related to other RNA viruses of the nonsegmented negative-strand (NNS) variety [40]. Of particular note, Kochetov AV [41] concentrated on the 5′-UTRs in the mRNA of seven EBOV viruses, due to the critical importance of the 5′-UTRs in translation initiation. In four of these seven mRNAs, small alternate upstream open reading frames (uORFs) were identified, but their significance is yet to be fully characterized. In cellular mRNAs, uORFs are known to be a common feature; they are critical in modulating translation of primary ORFs (pORFs), which was accomplish by reducing the efficiency and quantity of the scanning ribosomes associated with the reinitiation that occurs at the start codon of pORFs [42]. At a uAUG, rather than a pAUG, translation initiation frequency is affected by a variety of factors, including the strength of the Kozak consensus sequence that surrounds the uAUG. Moreover, between the pAUG and the upstream open reading frame (uORF) is an intercistronic space that, combined with the phosphorylation status of and the eIF-2α [43, 44], controls whether translation takes place at the principal protein initiation site (pAUG) or at the termination codon (uAUG).

When eIF-2α∼P is absent, cap-dependent translation has been found to be efficient, which permits higher ribosome initiation rates at the uORF [45]. When eIF2α∼P is enhanced, impairment of translation initiation occurs, which causes a ribosome to continue scanning beyond the uAUG; in this case, initiation occurs at the pAUG. In short, when cell stress occurs, eIF2α∼P facilitates translation initiation of select mRNAs that possess uORFs at the primary open ready frame (pORF) [46].

They characterized how the EBOV 5′-UTRs modulate translation. Mutating any of the four uAUGs present in the EBOV genome enhances translation at the corresponding pORF. The most dramatic effect was with the L gene where the L uAUG can potently suppress pORF translation; however, in response to eIF2α∼P, the L uAUG maintains L translation. Modulat‐ ing viral polymerase levels is biologically significant as ablating the L uORF in a recombinant EBOV reduces viral titers 10- to 100-fold in cell culture, severely impairs viral RNA synthesis, and functions to maintain virus titers in cells treated with stress-inducing agents. These data suggest that a uORF in the EBOV L mRNA regulates polymerase expression in response to the status of the cellular innate immune response and is required for optimal virus replication.

It would be relatively easy to incorporate a combination of relevant miRNAs in a miRNAexpression vector to test the utility of these miRNAs in genetically engineered VLP cell models in vitro that can be performed in a BSL-2 facility, and then to extend these studies in animal models utilizing safe vectors in a BSL-4 environment.

Currently, there are several genetically engineered vaccines containing genes for surface proteins (GP) that are in clinical trial. The first among these is a vaccine that Ebola GP genes stitched into a weakened chimpanzee adenovirus that serves as a vector. The second vaccine contains the Ebola surface protein gene inside a weakened version of vesicular stomatitis virus (VSV), which commonly infects farm animals. The potential dangers of employing of VSV are obvious: it can save men but potentially harm livestock in West Africa. The chimpanzee adenovirus will be a zoonotic event itself, and its potential danger cannot be underestimated [47, 48].

The third vaccine uses a vector known as MVA, a modified version of the smallpox vaccine virus, and involves protection from an Ebola virus "challenge" 10 months after the last vaccination.

We noted that none of these three approaches mentioned a simple and well-tested method of human and animal vaccination. What happened to the simple, whole formalin-killed or UV-killed, less pathogenic EBOV vaccines that have been tried in so many viral vaccina‐ tions [49, 50]?

With viruses like the major Ebola strands, where the mortality rate is over 50%, it will be difficult to find a reasonable and ethical way to carry out an unbiased clinical trial. However, if one can prepare a "dead Ebola virus" with antigenicity intact, it would be easy to immunize "high risk groups" without utilizing unusual vectors as exemplified by "harmless" chimpan‐ zee adenovirus, VSV or MVA (modified smallpox virus), each with unknown long-term risk factors and accompanied by immediate concerns of viral vector-induced antigenic competition that may potentially quell proper immune responses to the Ebola antigens [51]. We believe that a dead vaccine may induce the protective miRNAs and quell the pandemic. Increasingly, miRNA-induced intracellular immunity is being better understood, and several clinical trials are under way to treat viral diseases and cancers [52–55]. The cost of each of these vaccines would run into millions of dollars and would be prohibitively expensive to any of the individuals who are predicted to be infected with the virus in West African nations. In contrast to the proposed recombinant vaccines, each of the more traditional "killed vaccines" has been very inexpensive to produce and has benefited billions of humans [56].

#### **2. Conclusions**

brightness (N&B) analysis, analysis of the egress of virus-like particles, site-directed muta‐ genesis, and total internal reflection (TIRF) microscopy. N&B analysis permitted them to ascertain the average number of molecules and also the brightness within each pixel within a fluorescence microscopy image. This permitted them to detect the oligomeric status of proteins that are labeled fluorescently. They concluded that within the VP40 C-terminal domain, a hydrophobic interface actually penetrates the plasma membrane, which plays a key role in the oligomerization of VP40. The knocking out of plasma membrane penetration by hydrophobic mutants also substantially reduces the egress of VLPs [38, 39]. Therefore, degradation of VP40 mRNA by a two-pronged attack from *hsa-miR-4692* and *hsa-miR-548-az* can stop Ebola.

A distinguishing characteristic of filovirus genomes is their 3′- and 5′-UTRs that are long related to other RNA viruses of the nonsegmented negative-strand (NNS) variety [40]. Of particular note, Kochetov AV [41] concentrated on the 5′-UTRs in the mRNA of seven EBOV viruses, due to the critical importance of the 5′-UTRs in translation initiation. In four of these seven mRNAs, small alternate upstream open reading frames (uORFs) were identified, but their significance is yet to be fully characterized. In cellular mRNAs, uORFs are known to be a common feature; they are critical in modulating translation of primary ORFs (pORFs), which was accomplish by reducing the efficiency and quantity of the scanning ribosomes associated with the reinitiation that occurs at the start codon of pORFs [42]. At a uAUG, rather than a pAUG, translation initiation frequency is affected by a variety of factors, including the strength of the Kozak consensus sequence that surrounds the uAUG. Moreover, between the pAUG and the upstream open reading frame (uORF) is an intercistronic space that, combined with the phosphorylation status of and the eIF-2α [43, 44], controls whether translation takes place

at the principal protein initiation site (pAUG) or at the termination codon (uAUG).

open ready frame (pORF) [46].

332 RNA Interference

models utilizing safe vectors in a BSL-4 environment.

When eIF-2α∼P is absent, cap-dependent translation has been found to be efficient, which permits higher ribosome initiation rates at the uORF [45]. When eIF2α∼P is enhanced, impairment of translation initiation occurs, which causes a ribosome to continue scanning beyond the uAUG; in this case, initiation occurs at the pAUG. In short, when cell stress occurs, eIF2α∼P facilitates translation initiation of select mRNAs that possess uORFs at the primary

They characterized how the EBOV 5′-UTRs modulate translation. Mutating any of the four uAUGs present in the EBOV genome enhances translation at the corresponding pORF. The most dramatic effect was with the L gene where the L uAUG can potently suppress pORF translation; however, in response to eIF2α∼P, the L uAUG maintains L translation. Modulat‐ ing viral polymerase levels is biologically significant as ablating the L uORF in a recombinant EBOV reduces viral titers 10- to 100-fold in cell culture, severely impairs viral RNA synthesis, and functions to maintain virus titers in cells treated with stress-inducing agents. These data suggest that a uORF in the EBOV L mRNA regulates polymerase expression in response to the status of the cellular innate immune response and is required for optimal virus replication. It would be relatively easy to incorporate a combination of relevant miRNAs in a miRNAexpression vector to test the utility of these miRNAs in genetically engineered VLP cell models in vitro that can be performed in a BSL-2 facility, and then to extend these studies in animal

The current ongoing Ebola outbreaks in West Africa that began almost three years ago in March 2013 have already claimed 11,000 lives and over 27,000 cases. The rapid spread of the infection demands the need for rapid prevention methods. Currently, there are several vaccines that are in different phases of clinical trials. In this report, we highlight an alternative to the standard vaccine for Ebola prevention. We show that a preventive method based on miRNAs could be utilized and tested in nonhuman primates. Some of the lessons that we have learned from the recent West Africa Ebola outbreaks is to test the vaccine and other preventive methods that are currently available against Ebola before the major outbreaks occur. Therefore, we recom‐ mend that vaccines and preventive methods must be developed to the point that the measures correlate for human protection (Phase I level), so when the outbreaks occur, the vaccine and other measures can be rolled out quickly to prevent the spread of the disease.

#### **Acknowledgements**

We would like to thank Dr. Donald Gene Pace for his editorial assistance.

#### **Author details**

Zhabiz Golkar1 , Donald G. Pace2 and Omar Bagasra3\*

\*Address all correspondence to: obagasra@claflin.edu

1 Department of Biology, School of Health and Natural Science, Voorhees College, Denmark, SC, USA

2 School of Humanities & Social Science, Claflin University, Orangeburg, SC, USA

3 School of Natural Science, Claflin University, Orangeburg, SC, USA

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other measures can be rolled out quickly to prevent the spread of the disease.

We would like to thank Dr. Donald Gene Pace for his editorial assistance.

and Omar Bagasra3\*

1 Department of Biology, School of Health and Natural Science, Voorhees College,

2 School of Humanities & Social Science, Claflin University, Orangeburg, SC, USA

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338 RNA Interference

## **RNAi — Implications in Entomological Research and Pest Control**

Nidhi Thakur, Jaspreet Kaur Mundey and Santosh Kumar Upadhyay

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61814

#### **Abstract**

RNA interference (RNAi) has progressed swiftly in the past decade to become a conven‐ ient and dominant genetic tool that has immense utility in diverse fields. The entomologi‐ cal research, ranging from functional genomics to agriculture, has gained enormous momentum due to this technology. RNAi tool helped to discover the functions of new genes and study the complicated genetic networks, thus providing an evolutionary in‐ sight into various processes. RNAi is also becoming a method of choice for controlling in‐ sect pest populations. It is envisaged as tailor-made insecticide, which is highly species specific. However, the efficiency of this mechanism is limited by various factors such as the stability of the trigger molecule, the candidate gene selection, delivery system adopt‐ ed and, most importantly, the choice of the target species. Apart from the successful im‐ plication in diverse areas, there are certain drawbacks of this technology such as 'offtarget' effects, lack of sensitivity of various species, etc. Further research would relieve these limitations and support the manifestation of this genetic tool with much more relia‐ bility.

**Keywords:** RNAi, insects, pest management, efficiency of RNAi

#### **1. Introduction**

RNA interference (RNAi) is a highly conserved, sequence-specific mechanism of gene silencing which is triggered by the presence of double-stranded RNA (dsRNA). Since its discovery in 1998, RNAi has attained the status of a powerful genetic tool [1]. This reverse genetics technique is now immensely used in biomedical research, functional genetics and many other areas of biological research. Broadly, all the reactions that take place for RNA silencing are initiated when a long dsRNA is processed into small dsRNAs of about 21 to 24 bp by the RNaseIII enzyme, called Dicer. These small dsRNAs are called small interfering RNA (siRNA),

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

which when unwound using ATP-dependent activity are incorporated into the multi-subunit RNA-induced silencing complex (RISC). Here, the siRNA guides the RISC complex to degrade cellular RNA molecules that are complementary to its sequence [2]. Earlier this process was described in the experimental RNAi studies, and now it is the most accepted tool for gene knockdown studies.

The advent of RNAi also revolutionised the entomological research, as novel gene functions were efficiently discovered. In 1998, Kennerdell and Carthew were the first to use RNAi *in vivo* to study the genes *Frizzled* and *Frizzled*-*2* in *Drosophila melanogaster*[3]. The tremendous success of RNAi in model organisms has prompted its use for research in other insect species as well. In genomics and post-genomics era with the availability of a large amount of sequence information, RNAi further provides an opportunity to investigate the vital functions and crucial interactions that are of importance in both biomedical research and agriculture [4–6]. RNAi studies on insects of economic importance would provide new insights into unravelling the molecular interactions between various disease vectors and ultimately helping in the discovery of novel vaccine and drug targets. Disease vectors such as mosquito, ticks, mites, lice and others were studied extensively using RNAi [7]. These insects cause many serious diseases in humans and animals.

Among other applications, this genetic tool is also gaining popularity as a promising technol‐ ogy in controlling a wide array of agricultural pests. There is a substantial amount of literature available which documents the success of RNAi as a feasible and sustainable strategy in managing the agricultural pests [8–11]. The core RNAi machinery being present in all the insects makes it possible to silence a wide variety of target genes to produce diverse physio‐ logical, developmental and reproductive restrains. The sequence specificity of endogenous RNAi pathway allows the targeted suppression of genes essential for insect survival and thus offers the development of a specific, logical and sustainable strategy to combat against insect pests.

Agricultural pests are notorious and they cannot be efficiently managed by employing a single control agent or technique. Most commonly, the integrated pest management (IPM) strategies are utilised for combating the diversity of insect pests in the agro-ecosystem [12]. Along with mechanical, cultural, biological and chemical methods, the transgenic technology should also be embraced in the IPM regime. In this regard, RNAi can play an important role along with the available insecticidal molecules. Among various transgenic approaches to manage insect pests, *Bacillus thuringiensis* (Bt) toxin has shown spectacular success [13]; however, many important insect pests (primarily sap-sucking pests) are not amenable to Bt protection [14]. RNAi can be harnessed to defend crops against insect pests. Successful application of RNAi technology in agricultural pest management requires (i) suitable candidate gene where its silencing can cause mortality of the insect and (ii) an effective method of dsRNA delivery. Nevertheless, prior to field application, many aspects of this multi-faceted technology including safety and possible risks to environment need to be evaluated in detail.

In this chapter, we discussed the potential of this technology in gene silencing experiments to study the gene function as well as on opportunity to combat against agricultural pests and other disease vectors. Further, the factors responsible for a successful RNAi experiment, the link with immune response and viral infections have been discussed, highlighting the possible shortcomings of this strategy.

#### **2. RNAi in insects**

which when unwound using ATP-dependent activity are incorporated into the multi-subunit RNA-induced silencing complex (RISC). Here, the siRNA guides the RISC complex to degrade cellular RNA molecules that are complementary to its sequence [2]. Earlier this process was described in the experimental RNAi studies, and now it is the most accepted tool for gene

The advent of RNAi also revolutionised the entomological research, as novel gene functions were efficiently discovered. In 1998, Kennerdell and Carthew were the first to use RNAi *in vivo* to study the genes *Frizzled* and *Frizzled*-*2* in *Drosophila melanogaster*[3]. The tremendous success of RNAi in model organisms has prompted its use for research in other insect species as well. In genomics and post-genomics era with the availability of a large amount of sequence information, RNAi further provides an opportunity to investigate the vital functions and crucial interactions that are of importance in both biomedical research and agriculture [4–6]. RNAi studies on insects of economic importance would provide new insights into unravelling the molecular interactions between various disease vectors and ultimately helping in the discovery of novel vaccine and drug targets. Disease vectors such as mosquito, ticks, mites, lice and others were studied extensively using RNAi [7]. These insects cause many serious

Among other applications, this genetic tool is also gaining popularity as a promising technol‐ ogy in controlling a wide array of agricultural pests. There is a substantial amount of literature available which documents the success of RNAi as a feasible and sustainable strategy in managing the agricultural pests [8–11]. The core RNAi machinery being present in all the insects makes it possible to silence a wide variety of target genes to produce diverse physio‐ logical, developmental and reproductive restrains. The sequence specificity of endogenous RNAi pathway allows the targeted suppression of genes essential for insect survival and thus offers the development of a specific, logical and sustainable strategy to combat against insect

Agricultural pests are notorious and they cannot be efficiently managed by employing a single control agent or technique. Most commonly, the integrated pest management (IPM) strategies are utilised for combating the diversity of insect pests in the agro-ecosystem [12]. Along with mechanical, cultural, biological and chemical methods, the transgenic technology should also be embraced in the IPM regime. In this regard, RNAi can play an important role along with the available insecticidal molecules. Among various transgenic approaches to manage insect pests, *Bacillus thuringiensis* (Bt) toxin has shown spectacular success [13]; however, many important insect pests (primarily sap-sucking pests) are not amenable to Bt protection [14]. RNAi can be harnessed to defend crops against insect pests. Successful application of RNAi technology in agricultural pest management requires (i) suitable candidate gene where its silencing can cause mortality of the insect and (ii) an effective method of dsRNA delivery. Nevertheless, prior to field application, many aspects of this multi-faceted technology

including safety and possible risks to environment need to be evaluated in detail.

In this chapter, we discussed the potential of this technology in gene silencing experiments to study the gene function as well as on opportunity to combat against agricultural pests and other disease vectors. Further, the factors responsible for a successful RNAi experiment, the

knockdown studies.

342 RNA Interference

diseases in humans and animals.

pests.

RNAi offers species-specific molecules that can be flexibly manipulated and used in under‐ standing various complicated biochemical pathways. The research application of RNAi in entomology has elucidated the functions of several genes. Decrease in the mRNA levels of a candidate gene due to introduction of a complementary dsRNA fragment, and the study of the corresponding phenotype, illuminates a gene function. RNAi has been used to study various mechanisms related to insect development (embryonic and post-embryonic), repro‐ duction, behaviour and other complicated biosynthetic pathways [15].

Various insect orders have demonstrated amenability to RNAi-mediated silencing. Species of orders Coleoptera, Lepidoptera, Diptera, Hemiptera, Orthoptera, Blattodea and Hymenoptera have been studied for various aspects using RNAi technique [15]. The silencing efficiency ranges from 0% to 100% in different insects. A large majority of the target genes were gutspecific genes; however, genes from salivary glands, brain and antennae have also been targeted [16]. RNAi-based studies can be carried out by either *in vivo* or *in vitro* studies. The former method is much easier and involves incubating the cells with the dsRNA added to the medium. However, the *in vivo* approach is more useful in the field of functional genomics, especially in case of non-model organisms. Here, the dsRNA dosage and developmental stage of insect can be specified. In addition, RNAi can be very helpful in identifying the mutant genes that are fatal to the organism,. The significance and compilation of various categories of RNAi experiments in entomology are summarised in Table 1 [17–45].




**Table 1.** RNAi in the study of gene function in insects.

**Experiments Insect Gene and function**

*Schistocerca*

RNAi)

*Rhodnius prolixus*

*Americana* (Nymphal RNAi)

*Blattella germanica* (nymphal

*T. castaneum* (larval RNAi) *Laccase* 2 [28]

**Larval/nymphal/pupal**

**Regeneration-dependent**

**RNAi in behavioural**

**RNAi**

**biology**

**RNAi**

344 RNA Interference

*N. vitripennis* Role of *bicoid* gene in the structural pattern of the anterior

and thorax formation [24] *Oplegnathus. fasciatus Hox* genes and genes involved in segmentation and segment

specification [25]

*Bombyx mori* (pupal RNAi) Fatty acid transport protein (Bm'FATP) [29]

*S. Americana* Importance of early retinal genes *eyes*

(20E) [32] *G. bimaculatus* Mechanisms of leg regeneration [33]

rhythm [34]

*G. bimaculatus* Insect leg regeneration [36]

*T. castaneum* (larval RNAi) To study the molecular basis of adult morphological diversity in various organs [26]

*T. castaneum* (larval RNAi) *Ubx/Utx* during hindwing/elytron development [27]

suppression of ommochrome formation in the eye [30]

RXR/USP, along with EcR, of the

*G. bimaculatus period* (*per*)gene for circadian-dependent locomotor activity

*G. bimaculatus* Genes responsible for certain human disorders: *fragile X*

*G. bimaculatus* Orthologs of *Drosophila hedgehog* (*Gb'hh*), *wingless* (*Gb'wg*)

*Anopheles gambiae* Apyrase AgApy in the salivary glands shows important role in host probing behaviour [39]

*D. melanogaster* 3-Hydroxy-3-methylglutaryl CoA reductase has been identified for the control of

*receptor* (*DopR*) [35]

Nitrophorin 2

*mental retardation 1* (*fmr1*) and *Dopamine*

and *decapentaplegic* (*Gb'dpp*) are expressed

a decrease of anticoagulant activity and less efficient feeding behaviour [38]

during leg regeneration and play essential roles in the establishment of the proximal–distal axis [37]

body region. Also in the absence of *bicoid* gene, *orthodenticle*, *hunchback* and *giant* genes are responsible for proper head

The eye colour gene of first-instar nymphs triggered a

*absent (eya)* or *sine oculis (so)* in eye development [31]

heterodimeric nuclear receptor of 20-hydroxyecdysone

Apart from deciphering the function of genes involved in various metabolic pathways, RNAi also finds relevance in other aspects of insect science. It is quite beneficial in maintaining the beneficial insects and saving them from various parasites and pathogens. Certainly, this is useful in case of those parasites and pathogens, which have operative RNAi machinery. A successful study in this regard shows the control of honey bee parasite *Nosema ceranae*. When the gene related to energy metabolism was silenced, it was observed that the honey bee population had reduced infestation of *Nosema*, and lower mortality [46]. In another study, multiple genes of an ectoparasite of honey bee *Varroa destructor* were targeted [47]. It poses a great threat to the health of bees, and its control is of utmost importance for the rearing industry. It is a blood-sucking parasite, so the bees were fed on a meal containing dsRNA against the genes of *Varroa*. The RNAi-mediated control decreased the mite population by 50%, causing no evident damage to the bees. RNAi has also been useful in elucidating the impor‐ tance of various immunological pathways in *D. melanogaster* [48]*.* Host–parasite relationships such as that of *Anopheles–Plasmodium* have also been studied by using RNAi. Early research was conducted on *defensin* and it was shown to be important for protecting mosquitoes against infections of Gram-positive bacteria [49]. Later, the same group demonstrated how the development of Plasmodium is affected by *Anopheles gambiae* immune genes [50]. Similarly, in *Manduca sexta* haemocytes, knockdown of haemolin (a bacterial recognition protein) decreased the ability of insects to clear *Escherichia coli* from the haemolymph. This eventually reduced their ability to engulf bacteria and highlighted the role of haemolin in the *M. sexta* immune response [51].

#### **3. RNAi in pest control**

Plants are damaged by a plethora of insect pests. The losses due to these pests and expenditure on the chemical pesticides amount to billions of dollars. In an attempt to reduce these losses, many reports have been published which demonstrate the successful application of RNAi technique in crop protection. Huvenne and Smagghe [52] have summarised the reports on insects in which RNAi has been applied through feeding, and they discussed several factors that influence the success of RNAi on target insects, such as the concentration of dsRNA, the nucleotide sequence, the length of the dsRNA fragment and the life stage of the target insects. The downregulation of expression of critical genes, caused by dsRNA/siRNA, eventually leads to death/growth retardation of the insect and forms the basis of pest control. In this view, the efficient delivery and uptake of the dsRNA trigger is of prime importance. In contrast to the study of gene function, pest control strategies cannot depend on injection of the molecule. The deployment of pest controlling siRNA/miRNA molecules would involve oral exposure, either as transgenic plants or as sprays. Most of the nutrients from gut lumen are absorbed in the midgut tissue of the insect; therefore, this tissue is an attractive target for RNAi. After ingestion, the dsRNA enters into the gut lumen of the insect. Insect gut is divided into foregut, midgut and hindgut. While both foregut and hindgut are covered with chitin, it is only the midgut that has exposed cell surfaces. It is the site of nutrient exchange between the haemolymph and the gut contents. Therefore, midgut epithelium is an attractive target as it is the primary tissue exposed to dsRNA in the gut lumen. The stability of dsRNA molecule and the efficiency of the silencing process (discussed in section 5) is determined by the gut pH and nucleases.

A breakthrough research in RNAi-mediated pest control was published in 2007 on the western corn rootworm, *Diabrotica virgifera virgifera* (WCRW) [53], and cotton bollworm *Helicoverpa armigera* (CBW) [54]. In the former study, a candidate gene was screened based on the complete cDNA library. Out of a total of 290 dsRNAs, *Vacoular ATPase (V-ATPase)* subunit A was finally selected for the development of transgenic corn plants. The larvae reared on transformed plants caused much less damage to the roots and also showed the reduced expression of the target gene. The other study of Mao et al. [9] targeted the pesticide detoxifying gene *Cytochrome P450* (*CYP6AE14*), which provides gossypol tolerance to the insect. Transgenic plants express‐ ing dsRNA corresponding to *CYP6AE14* levels of this transcript in insect body was decreased and the larval growth was retarded. Following these two studies, many research groups started to consider RNAi as a feasible technique which could be employed in the transgenic ap‐ proaches to manage insects. As we know from the various studies conducted, the Bt (*Bacillus thuringiensis*) toxins are effective on lepidopteran and coleopteran pests but fail to work against hemipteran pests like aphids, whiteflies, phyllids, etc. [55–58].

RNAi-engineered plants can be more useful in case of these phloem-feeding hemipteran pests, which are notorious not only because of feeding damages but also because of their ability to transmit plant viruses [59]. The midgut genes of *Nilaparvata lugens* were downregulated by feeding on transgenic rice expressing dsRNA against three separate genes, but no lethal phenotype was detected in this case [60]. Pitino et al. [61] demonstrated RNAi in aphid *Myzus persicae* directed towards the receptor of activated kinase (*Rack-1*) gene by transgenic expres‐ sion in *Arabidopsis thaliana*. Another important pest, the whitefly, is also now amenable to RNAi [62]. Its control was demonstrated both by feeding on artificial diet and by feeding on trans‐ genic tobacco plants expressing dsRNA of *V-ATPase A* gene [11, 63]. RNAi experiments conducted on agricultural pests as well as on insect vectors of several human diseases are summarised in Table 2 [53–118].

**3. RNAi in pest control**

346 RNA Interference

Plants are damaged by a plethora of insect pests. The losses due to these pests and expenditure on the chemical pesticides amount to billions of dollars. In an attempt to reduce these losses, many reports have been published which demonstrate the successful application of RNAi technique in crop protection. Huvenne and Smagghe [52] have summarised the reports on insects in which RNAi has been applied through feeding, and they discussed several factors that influence the success of RNAi on target insects, such as the concentration of dsRNA, the nucleotide sequence, the length of the dsRNA fragment and the life stage of the target insects. The downregulation of expression of critical genes, caused by dsRNA/siRNA, eventually leads to death/growth retardation of the insect and forms the basis of pest control. In this view, the efficient delivery and uptake of the dsRNA trigger is of prime importance. In contrast to the study of gene function, pest control strategies cannot depend on injection of the molecule. The deployment of pest controlling siRNA/miRNA molecules would involve oral exposure, either as transgenic plants or as sprays. Most of the nutrients from gut lumen are absorbed in the midgut tissue of the insect; therefore, this tissue is an attractive target for RNAi. After ingestion, the dsRNA enters into the gut lumen of the insect. Insect gut is divided into foregut, midgut and hindgut. While both foregut and hindgut are covered with chitin, it is only the midgut that has exposed cell surfaces. It is the site of nutrient exchange between the haemolymph and the gut contents. Therefore, midgut epithelium is an attractive target as it is the primary tissue exposed to dsRNA in the gut lumen. The stability of dsRNA molecule and the efficiency of the

silencing process (discussed in section 5) is determined by the gut pH and nucleases.

hemipteran pests like aphids, whiteflies, phyllids, etc. [55–58].

A breakthrough research in RNAi-mediated pest control was published in 2007 on the western corn rootworm, *Diabrotica virgifera virgifera* (WCRW) [53], and cotton bollworm *Helicoverpa armigera* (CBW) [54]. In the former study, a candidate gene was screened based on the complete cDNA library. Out of a total of 290 dsRNAs, *Vacoular ATPase (V-ATPase)* subunit A was finally selected for the development of transgenic corn plants. The larvae reared on transformed plants caused much less damage to the roots and also showed the reduced expression of the target gene. The other study of Mao et al. [9] targeted the pesticide detoxifying gene *Cytochrome P450* (*CYP6AE14*), which provides gossypol tolerance to the insect. Transgenic plants express‐ ing dsRNA corresponding to *CYP6AE14* levels of this transcript in insect body was decreased and the larval growth was retarded. Following these two studies, many research groups started to consider RNAi as a feasible technique which could be employed in the transgenic ap‐ proaches to manage insects. As we know from the various studies conducted, the Bt (*Bacillus thuringiensis*) toxins are effective on lepidopteran and coleopteran pests but fail to work against

RNAi-engineered plants can be more useful in case of these phloem-feeding hemipteran pests, which are notorious not only because of feeding damages but also because of their ability to transmit plant viruses [59]. The midgut genes of *Nilaparvata lugens* were downregulated by feeding on transgenic rice expressing dsRNA against three separate genes, but no lethal phenotype was detected in this case [60]. Pitino et al. [61] demonstrated RNAi in aphid *Myzus persicae* directed towards the receptor of activated kinase (*Rack-1*) gene by transgenic expres‐




**Table 2.** Different insect pests targeted by RNAi.

**Insect pests Mode of delivery Gene target**

In planta

Injection

*Oncopeltus fasciatus* Injection Hunchback [89]

Injection and feeding

Feeding Feeding and injection

*Myzus persicae* Transgenic plant *MpC002* and *Rack-1* [96]

Artificial diet Transgenic plant

*Bactericerca cockerelli* Injection/feeding Actin, V-ATPase [97]

*Ostrinia furnacalis* Spraying LIM protein 1; myosin 3 light chain;

*Ostrinia nubilalis* Spraying Chitinase (*OnCht*); chitin synthase

*Epiphyas postvittana* Feeding Carboxylesterase gene (*EposCXE1*);

*Athalia rosae* Injection *Ar white gene*

Catalase [85] CbE E4 [86]

protease [92]

V-ATPase [63]

**Order Hymenoptera**

**Order Isoptera** *Reticulitermes flavipes* Feeding Endogenous digestive cellulase enzyme, hexamerin storage protein [98] **Order Lepidoptera**

V-ATPase subunit A [11]

Inhibitor of apoptosis gene (IAP) [87] Polygalacturonase (PG) [88]

Gland nitrophorin 2 (*NP2*) [91]

ATP synthase subunit [93] Trehalose phosphate synthase [94]

Snap, Chickadee, CG5885, GATAd [62]

chymotrypsin-like serine protease; chymotrypsin-like protease C1; chymortypsin-like serine protease C3; hydroxybutyrate dehydrogenase; Kazal-type serine proteinase inhibitor 1; fatty acid binding protein 1; unknown;

pheromone binding protein [101]

caboxypeptidse 4 [99]

(*OnCHS2*) [100]

Circadian clock gene, mammalian-type cryptochrome; Bla [90]

Hexose transporter; carboxypeptidase; trypsin-like serine

Cathepsin B-like protease; nicotinic acetylcholine receptors [95]

Actin; ADP/ATP translocase; *α*-tubulin; ribosomal protein L9;

*Sitobion avenae* Feeding

348 RNA Interference

*Lygus lineolaris* Injection

*Riptortus pedestris* Injection

*Bemisia tabaci* Injection

*Nilaparvata lugens* Feeding and transgenic

The choice of a suitable target gene is central to pest control strategy. The gene selection approaches can be based on choosing the gene with known function such as detoxification enzymes, cell synthesis, nutrition, metabolism and cytoskeleton structure. These types of genes can be selected as insect pest control targets. For this, expressed sequence tag (EST) library of European corn borer (*Ostrinia nubilalis*) was screened to find out that a *chitinase* gene (*OnCht*) and a *chitin synthase* gene (*OnCHS2*), which are very important in regulating the growth and development of this insect [119]. Likewise, the EST library of *Bemisia tabaci* was also used to screen out few important genes for RNAi-mediated control [63]. The cDNA library screening approach was also used. Mao et al. [9] constructed a cDNA library from RNAs expressed in the midgut of fifth-instar larvae exposed to gossypol. Several cDNA libraries of WCR (*D. virgifera virgifera*) were prepared and considered upon the underlying principle that genes encoding proteins with essential functions would be the best RNAi targets for causing lethality [8].

As an extension of the cDNA library screening approach, the next-generation sequencing (NGS) technologies have led to novel opportunities for expression profiling in organisms lacking any genome or transcriptome sequence information. It enables the direct sequencing of cDNA generated from mRNA (RNA-seq) [120, 121]. Hence, it provides the *de novo* genera‐ tion of the transcriptome for a non-model organism, including various pests. Wang et al. [99] adopted Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) to screen optimal RNAi targets from Asian corn borer (ACB; *Ostrinia furnacalis*). The same technique has been used for the grain aphid, *Sitobion avenae* and *Spodoptera litura* [122, 123]. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost-effective and easy way to select for candidate target genes for RNAi, which may not be only limited to the midgut tissue but can also be selected from the whole insect.

The convenience of RNAi to target specific pests while not harming other species is a perfect method of pest management. However, target gene selection and efficient delivery methods are the two major cornerstones of pest management by RNAi. The candidate gene for RNAi can be tailored to be species specific or they can also have a broad spectrum. Specificity can be achieved by designing dsRNAs that target the more variable regions of genes, such as untranslated regions (UTRs). It was first demonstrated in *Drosophila* where UTR of the *gammatubulin* gene was targeted and even closely related species could be targeted selectively [75]. However, it can also be possible to target multiple organisms with a single gene. One such example is of *V-ATPase* gene, which is an effective target in *B. tabaci*, *D. virgifera* and *B. dorsalis*[8, 63, 124]; all of these insects belong to different orders. In this case, either a conserved region can be selected which could affect closely related species or a mixture of dsRNA fragments from different genes belonging to different species can be selected.

Delivery methods that ensure continuous supply of dsRNA/siRNA will be applicable in the fields. A more reliable and verified method would be transgenic plants as the dsRNA can be applied as bait, sprays, or supplied through irrigation systems [125, 126]. The application approach by spray could be quite practical like the spray of chemical pesticides. Gan et al. [127] have also demonstrated the control of viral infection using dsRNA spraying. Similar results were obtained with Asian corn borer, *Ostrinia furnacalis*. This study showed that larval lethality or developmental disorders can be achieved by gene-specific RNAi, and spraying can be an efficient method for continuous supply of dsRNA [99]. The coating of dsRNA molecule with liposomes is used for delivering siRNA to mammalian cells, specific tissues and some insects [128, 75]. This coating prevents the degradation of the molecule and enhances its uptake ability; spraying may also be explored for such particles. Zhang et al. [129] used the chitosan nano‐ particle based RNAi technology to suppress the expression of two chitin synthase genes (*AgCHS1* and *AgCHS2*) in African malaria mosquito (*A*. *gambiae*) larvae. Bacterial expression or chemical synthesis allows large-scale production of dsRNA at efficient costs [75, 130, 131].

#### **4. RNAi: Link with immunity and viral infections**

The RNAi mechanisms evolved primarily as a defence mechanism against viruses and transposons [132]. Research has established that RNAi pathway also contributes to the innate immunity of the insects against the viruses having either dsRNA genome or such replicative intermediates. It was demonstrated that the *Drosophila* S2 cells utilise the endocytosis-mediated pathway involving the pattern recognition scavenger receptors for the uptake of dsRNA from the surroundings. These receptors are key players in the innate immune responses of the cell [133, 134]. Saleh et al. [135] demonstrated the strong link of dsRNA uptake pathway and the activation of the immune response in the infected cells. The normal cells used dsRNA uptake pathway to internalise viral dsRNA and subsequently showed manifestation of antiviral response in these cells. On the contrary, the mutant cells (defective genes used for dsRNA uptake) did not show activation of any antiviral response. Mutants in the core siRNA compo‐ nents Dicer-2, AGO-2 and R2D2 are more susceptible to viral infections [140].

Further, it was also reported that receptors such as Sr-CI and Eater, which contribute to majority of dsRNA uptake in the *Drosophila* S2 cells, were significantly down-regulated after pathogenic virus treatments, and significant changes in phagocytic activity were observed. The role of RNAi in antiviral defence has also been firmly established in mosquitoes [136]. Viruses can also affect the availability of RNAi machinery for other candidate dsRNA molecules. They can saturate the RNAi machinery and affect the efficiency of RNAi mecha‐ nism. Many viruses are known to produce viral suppressors of RNA silencing (VSRs), which bind to the key elements of the RNAi pathway rendering it unavailable. Many of the viral proteins (viz. B2 protein from Flockhouse virus, 1A proteins from *Drosophila* C virus and cricket paralysis virus) are known to interfere in the siRNA pathway of RNA-mediated silencing [137– 140]. These viral proteins may affect the biogenesis of the trigger molecule by binding to important enzymes such as dicer, which generate the siRNA from long dsRNA or affect the target cleavage by binding to RISC. The viral proteins may also sequester the dsRNA signal molecule or form complexes with the replicative intermediates of the siRNA pathway.

Viruses also produce large amount of RNAs and small RNAs that accumulate in the infected cells. It has also been hypothesised that the occurrence of alternative and effective antiviral pathway may become important in controlling the viral infections and may supersede the RNAi pathway. Few of these possible pathways have been worked upon. Goic et al. [141] have reported the potential interaction of nucleic acid-based acquired immunity with the core RNAi machinery in the study of persistent infection of S2 cells by Flock House Virus (FHV). The insects also protect themselves from foreign nucleic acids by becoming refractory to RNAi. In the oriental fruit fly, *Bactrocera dorsalis*, orally administered dsRNA-targeting endogenous genes, resistance to RNAi was seen due to a blockade in the dsRNA uptake pathway. A very interesting hypothesis is presented by Swevers et al. [142] about the possible impact of persistent viral infection in the insects. In their work, the authors have analysed various factors that determine the response to exogenous dsRNA in the background of viral infection.

#### **5. Efficiency of RNAi**

As an extension of the cDNA library screening approach, the next-generation sequencing (NGS) technologies have led to novel opportunities for expression profiling in organisms lacking any genome or transcriptome sequence information. It enables the direct sequencing of cDNA generated from mRNA (RNA-seq) [120, 121]. Hence, it provides the *de novo* genera‐ tion of the transcriptome for a non-model organism, including various pests. Wang et al. [99] adopted Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) to screen optimal RNAi targets from Asian corn borer (ACB; *Ostrinia furnacalis*). The same technique has been used for the grain aphid, *Sitobion avenae* and *Spodoptera litura* [122, 123]. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost-effective and easy way to select for candidate target genes for RNAi, which may not be only limited to the

The convenience of RNAi to target specific pests while not harming other species is a perfect method of pest management. However, target gene selection and efficient delivery methods are the two major cornerstones of pest management by RNAi. The candidate gene for RNAi can be tailored to be species specific or they can also have a broad spectrum. Specificity can be achieved by designing dsRNAs that target the more variable regions of genes, such as untranslated regions (UTRs). It was first demonstrated in *Drosophila* where UTR of the *gammatubulin* gene was targeted and even closely related species could be targeted selectively [75]. However, it can also be possible to target multiple organisms with a single gene. One such example is of *V-ATPase* gene, which is an effective target in *B. tabaci*, *D. virgifera* and *B. dorsalis*[8, 63, 124]; all of these insects belong to different orders. In this case, either a conserved region can be selected which could affect closely related species or a mixture of dsRNA

Delivery methods that ensure continuous supply of dsRNA/siRNA will be applicable in the fields. A more reliable and verified method would be transgenic plants as the dsRNA can be applied as bait, sprays, or supplied through irrigation systems [125, 126]. The application approach by spray could be quite practical like the spray of chemical pesticides. Gan et al. [127] have also demonstrated the control of viral infection using dsRNA spraying. Similar results were obtained with Asian corn borer, *Ostrinia furnacalis*. This study showed that larval lethality or developmental disorders can be achieved by gene-specific RNAi, and spraying can be an efficient method for continuous supply of dsRNA [99]. The coating of dsRNA molecule with liposomes is used for delivering siRNA to mammalian cells, specific tissues and some insects [128, 75]. This coating prevents the degradation of the molecule and enhances its uptake ability; spraying may also be explored for such particles. Zhang et al. [129] used the chitosan nano‐ particle based RNAi technology to suppress the expression of two chitin synthase genes (*AgCHS1* and *AgCHS2*) in African malaria mosquito (*A*. *gambiae*) larvae. Bacterial expression or chemical synthesis allows large-scale production of dsRNA at efficient costs [75, 130, 131].

The RNAi mechanisms evolved primarily as a defence mechanism against viruses and transposons [132]. Research has established that RNAi pathway also contributes to the innate

fragments from different genes belonging to different species can be selected.

**4. RNAi: Link with immunity and viral infections**

midgut tissue but can also be selected from the whole insect.

350 RNA Interference

Though RNAi is a conserved mechanism in eukaryotes, its efficiency is governed by various factors. The response of different insect species towards this mechanism of gene silencing is imperative for successful implementation in the study of gene function and more importantly in the pest management programs. Also, the efficacy is governed by many factors which are not intrinsic to the organism such as the delivery, dosage and choice of the candidate gene. Comprehending these factors will provide a better insight into designing the experiments for successful application of RNAi. The available reports indicate that the lower *Insecta* species such as that of *Blatella* show a much robust and persistent RNAi response, while higher *Insecta* species belonging to the orders Lepidoptera and Diptera are non-compliant [15]. The sensi‐ tivity could vary within and among the orders. For instance, many Lepidopteran insects are resistant to RNAi. Terenius et al. [143] have reviewed various factors that may be contributing to the poor responsiveness of these insects. The efficiency can vary with insect species, target gene, developmental stage of the organism, expression of RNAi machinery, method of delivery, stability of dsRNA, etc. A few factors that may be crucial in determining the efficiency of RNAi are discussed below.

#### **5.1. The RNAi machinery**

RNAi evolved in organisms as a defence mechanism against viral infections at the cellular level [144, 145]. The differences in the expression of core RNAi machinery can be a prime reason affecting the adequacy of RNAi mechanism. The systemic RNA-interference-deficient 1 (sid-1) protein forms a gated channel which is selective for dsRNA molecule. Its role is well established in the systemic spread of the RNAi signal in the model organism *Caenorhabditis elegans* [146]. The presence of *SID-1*gene orthologs in insects varies with the insect orders [52]. The dipterans lack this gene completely. The mosquito *Culex quinquefasciatus* also lacks sid-1 ortholog but shows the systemic spread of the dsRNA trigger [147]. On the one hand, honey bee (*Apis mellifera*) showed an increase in the expression of *SID-1* during the RNAi experiments, indicating its role in the uptake pathway [148]. On the other hand, in *Tribolium castaneum*, the silencing of all three orthologs of *SID-1* casted no influence on the efficiency of RNAi [149].

In *Bombyx mori*, three orthologs are present but no significant success has been found in this lepidopteran, while mosquitoes show systemic RNAi despite the absence of *sid-1* in several species [149–152]. R2D2, a cofactor of dicer-2 enzyme which cleaves long dsRNA into siRNA for loading into the RISC, is absent in *B. mori* making the insect very insensitive to RNAi. Another important enzyme RNA-dependent RNA polymerase (RdRP), which amplifies the primary siRNA signal in *C. elegans*, is entirely not reported in the insects [149]. *T. castaneum* showed a robust systemic RNAi, but a wide survey of RNAi related genes did not show any traces of RdRP [149]. However, RdRP-like activity was substituted in *Drosophila* cell lines by certain other enzymatic pathways. In many cases, the absence of certain well-known genes of the RNAi pathway is directly responsible for the poor response of the organism while in several other examples, the absence is compensated by other genes/pathways which play key roles of their counterparts.

#### **5.2. The RNAi molecule**

The exogenous dsRNA molecule is the trigger for initiating the RNAi pathway. These molecules are delivered in the form of dsRNA, siRNA or hairpin RNA. Apart from sequence specificity, other parameters are also crucial in determining the efficiency of RNAi experi‐ ments. The study on the administration of dsRNA (feeding by means of artificial diet, natural diet, droplet method, blood meal, transgenic plants, etc.) in insects has used varied length of dsRNA molecule. The nucleotide length used in these reports ranges from 134 to 1842 bp, while most of the studies used 300–500 bp as the optimal length [153]. However, silencing effects have also been observed in the case of single siRNA synthesised chemically (administration in *H. armigera*) or a cocktail of siRNA (obtained by using dicer enzyme to chop the dsRNA molecule) [154]. In the cell line experiments done on the *Drosophila* S2 cell line, 211 bp was found to be the optimum length of dsRNA that could be absorbed by the cells [135]. Not only the length but also the specificity of the RNAi molecule is a concern. It can be understood by the reports on *Drosophila* that feeding specific sequence of *V-ATPase* dsRNA caused no silencing in non-target species [75], which implies the specificity of the process. On the contrary, various other studies report non-specific silencing. Off-target effects were reported in *Rhodnius prolixus* [81] and Colorado potato beetle (*Leptinotarsa decemlineata*) [8]. Single mismatches are known to impair the RNAi effect in the mammalian cell lines [155]. Further studies will clarify whether single mismatches show similar impact in insects as well. In case of pest management programs, long dsRNAs (>200 bp) are generally used which generate many probable siRNA maximising the RNAi response [153]. The dosage of the dsRNA molecule also plays an important role in determining the efficiency of the process. Higher doses are required in case of feeding experiments as compared to injection. The silencing effect in *R. prolixus* was enhanced by multiple doses [38]. Therefore, it follows that doses and types of administration (oral or injectable) also need to be optimised according to the life stage of the organism and the target tissue.

#### **5.3. Delivery of the molecule/uptake of silencing signal**

imperative for successful implementation in the study of gene function and more importantly in the pest management programs. Also, the efficacy is governed by many factors which are not intrinsic to the organism such as the delivery, dosage and choice of the candidate gene. Comprehending these factors will provide a better insight into designing the experiments for successful application of RNAi. The available reports indicate that the lower *Insecta* species such as that of *Blatella* show a much robust and persistent RNAi response, while higher *Insecta* species belonging to the orders Lepidoptera and Diptera are non-compliant [15]. The sensi‐ tivity could vary within and among the orders. For instance, many Lepidopteran insects are resistant to RNAi. Terenius et al. [143] have reviewed various factors that may be contributing to the poor responsiveness of these insects. The efficiency can vary with insect species, target gene, developmental stage of the organism, expression of RNAi machinery, method of delivery, stability of dsRNA, etc. A few factors that may be crucial in determining the efficiency

RNAi evolved in organisms as a defence mechanism against viral infections at the cellular level [144, 145]. The differences in the expression of core RNAi machinery can be a prime reason affecting the adequacy of RNAi mechanism. The systemic RNA-interference-deficient 1 (sid-1) protein forms a gated channel which is selective for dsRNA molecule. Its role is well established in the systemic spread of the RNAi signal in the model organism *Caenorhabditis elegans* [146]. The presence of *SID-1*gene orthologs in insects varies with the insect orders [52]. The dipterans lack this gene completely. The mosquito *Culex quinquefasciatus* also lacks sid-1 ortholog but shows the systemic spread of the dsRNA trigger [147]. On the one hand, honey bee (*Apis mellifera*) showed an increase in the expression of *SID-1* during the RNAi experiments, indicating its role in the uptake pathway [148]. On the other hand, in *Tribolium castaneum*, the silencing of all three orthologs of *SID-1* casted no influence on the efficiency of RNAi [149].

In *Bombyx mori*, three orthologs are present but no significant success has been found in this lepidopteran, while mosquitoes show systemic RNAi despite the absence of *sid-1* in several species [149–152]. R2D2, a cofactor of dicer-2 enzyme which cleaves long dsRNA into siRNA for loading into the RISC, is absent in *B. mori* making the insect very insensitive to RNAi. Another important enzyme RNA-dependent RNA polymerase (RdRP), which amplifies the primary siRNA signal in *C. elegans*, is entirely not reported in the insects [149]. *T. castaneum* showed a robust systemic RNAi, but a wide survey of RNAi related genes did not show any traces of RdRP [149]. However, RdRP-like activity was substituted in *Drosophila* cell lines by certain other enzymatic pathways. In many cases, the absence of certain well-known genes of the RNAi pathway is directly responsible for the poor response of the organism while in several other examples, the absence is compensated by other genes/pathways which play key roles of

The exogenous dsRNA molecule is the trigger for initiating the RNAi pathway. These molecules are delivered in the form of dsRNA, siRNA or hairpin RNA. Apart from sequence

of RNAi are discussed below.

**5.1. The RNAi machinery**

352 RNA Interference

their counterparts.

**5.2. The RNAi molecule**

One of the most decisive factors for inducing RNAi is the efficient delivery of the dsRNA molecule. The common methods of delivery are by microinjection, soaking, oral delivery and transgenic technique [156]. Microinjection-based delivery is the most commonly used techni‐ que in studying gene functions. It has proven to work well for *Tribolium, Drosophila* and many other lepidopteran insects. Although it works well for larger insects, success with smaller insects is limited due to the invasive nature of this technique. The survival of aphids after microinjection procedure is highly dependent on the injected volume [157].

Further, factors such as needle choice, optimal volume and place of injection are very crucial considerations and tend to vary with organisms and laboratories. Feeding-based experiments involve either *in vitro* synthesised or bacterially expressed dsRNA molecules. The success of oral delivery methods indicates the possible employment of RNAi technique for target pest control. However, the stability of the molecule will always be a concern in the gut lumen. Artificial diet mixed with dsRNA could not induce RNAi in *Drosophila* spp. Ingested dsRNA against a gut-specific *aminopeptidase N* gene also failed to develop RNAi response in *Spodoptera litura* [43]. Therefore, it can be suggested that oral delivery is not equally suitable for all species. Another convenient method of delivery is soaking. Nevertheless, this method is more appli‐ cable for cell line experiments rather than whole insects.

After the delivery of the molecules, the next step is the uptake of the molecules by insects. Huvenne and Smagghe [52] have elaborately reviewed the basic mechanisms involved in the uptake of dsRNA in the insects. The spreading of the RNAi signal, i.e., systemic RNAi, is an important determinant of the efficiency of RNAi. In cases of functional genetics, cell autono‐ mous RNAi has been successfully employed to study the function of genes; however, for implementation in the pest control programs, non-cell autonomous RNAi is important. The systemic spread of the silencing signal is absent in the most studied model insect *Drosophila*. In contrast, the most studied insect *Tribolium* shows a powerful systemic silencing effect [149].

#### **5.4. Potency of the silencing signal**

The manifestation of the RNAi effect also depends on the stability and persistence of the dsRNA molecule. In *Acyrthosiphon pisum*, the silencing effect on the aquaporin gene began to reduce after five days [157]. The early stability of dsRNA molecule may be disrupted by the non-specific nucleases as reported in many of the lepidopteran insects [143]. These are extracellular enzymes different from dicer and digest the trigger molecule, thereby preventing the RNAi cascade. In certain cases, the activity of dsRNA degrading enzymes have been studied and their levels were measured in different stages, which was found related to the developmental stage. The dsRNase activity is also found in the digestive juices of *Bombyx mori*, saliva of *Linus lineolaris* and in haemolymph of *Manduca sexta* [87, 158, 159]. The existence/ stability/mode of action of these enzymes are not sufficiently studied and future research in this direction needs to be carried out to comprehend the stability of dsRNA molecule in the *in vivo* studies. The choice of gene can also decrease the strength of the silencing signal. Ideally, the protein whose function is to be silenced should have a short half-life, whilst the mRNA turnover number should be high. The stability of protein explained the weak RNAi response in both *D. melanogaster* and *T. casteneum* [160]. However, such studies have not been conducted for the majority of the genes and therefore it can be concluded that expression of RNAi is limited by many uncovered phenomena.

#### **6. Conclusions**

The advent of RNA interference has been a crucial phase of the modern day science. The wide array of applications in the entomological research has led to many momentous findings. The functionality of many genes has been understood by this technique. Its implication in func‐ tional genomics is not only restricted to the study of a given set of genes but is also used to unveil the interaction of different genes in a particular metabolic pathway. The rapid pace of RNAi-based research suggests that it would soon facilitate better understanding of evolution, circadian rhythms, behavioural pattern, reproductive biology and interaction between host and parasites/pathogens. However, successful manifestation of RNAi is dependent on several factors. The insect species might lack the basic RNAi machinery [161] or may rapidly degrade alien dsRNA. Such factors could be intrinsic to the concerned tissue or gene. The gene might have high transcription rate and could evade the effect of RNAi or the target mRNA may be too transient.

As happens with every phenomenon, this mechanism can also undergo selection pressure. Viruliferous insects that also have RNAi suppressors would be able to thrive on RNAiprotected crops. Furthermore, single nucleotide polymorphisms (SNPs) that result in lower effectiveness of the RNAi could potentially be selected for and lead to the evolution of resistance [153]. Genetic variations among insect species are already a challenge for RNAi. Therefore, parallel research must be carried out to develop strategies, which would minimise the resistance development and selective pressures.

RNAi has proved its utility as a futuristic tool of insect pest management. However, there are several issues that need to be addressed before the implementation of this technology in fields. The knowledge gaps underlying large-scale implications of pesticidal RNAi-based crops on the environment should be identified and bridged. The off-target gene silencing is a serious concern where unintended organisms are adversely affected [162]. The non-target effects can be categorised as off-target gene silencing, silencing the target gene in non-target organisms, immune stimulation and saturation of the RNAi machinery [163]. A balanced approach should be taken with maximum effects on the target pests with minimal effects on non-target organisms.

#### **Acknowledgements**

After the delivery of the molecules, the next step is the uptake of the molecules by insects. Huvenne and Smagghe [52] have elaborately reviewed the basic mechanisms involved in the uptake of dsRNA in the insects. The spreading of the RNAi signal, i.e., systemic RNAi, is an important determinant of the efficiency of RNAi. In cases of functional genetics, cell autono‐ mous RNAi has been successfully employed to study the function of genes; however, for implementation in the pest control programs, non-cell autonomous RNAi is important. The systemic spread of the silencing signal is absent in the most studied model insect *Drosophila*. In contrast, the most studied insect *Tribolium* shows a powerful systemic silencing effect [149].

The manifestation of the RNAi effect also depends on the stability and persistence of the dsRNA molecule. In *Acyrthosiphon pisum*, the silencing effect on the aquaporin gene began to reduce after five days [157]. The early stability of dsRNA molecule may be disrupted by the non-specific nucleases as reported in many of the lepidopteran insects [143]. These are extracellular enzymes different from dicer and digest the trigger molecule, thereby preventing the RNAi cascade. In certain cases, the activity of dsRNA degrading enzymes have been studied and their levels were measured in different stages, which was found related to the developmental stage. The dsRNase activity is also found in the digestive juices of *Bombyx mori*, saliva of *Linus lineolaris* and in haemolymph of *Manduca sexta* [87, 158, 159]. The existence/ stability/mode of action of these enzymes are not sufficiently studied and future research in this direction needs to be carried out to comprehend the stability of dsRNA molecule in the *in vivo* studies. The choice of gene can also decrease the strength of the silencing signal. Ideally, the protein whose function is to be silenced should have a short half-life, whilst the mRNA turnover number should be high. The stability of protein explained the weak RNAi response in both *D. melanogaster* and *T. casteneum* [160]. However, such studies have not been conducted for the majority of the genes and therefore it can be concluded that expression of RNAi is

The advent of RNA interference has been a crucial phase of the modern day science. The wide array of applications in the entomological research has led to many momentous findings. The functionality of many genes has been understood by this technique. Its implication in func‐ tional genomics is not only restricted to the study of a given set of genes but is also used to unveil the interaction of different genes in a particular metabolic pathway. The rapid pace of RNAi-based research suggests that it would soon facilitate better understanding of evolution, circadian rhythms, behavioural pattern, reproductive biology and interaction between host and parasites/pathogens. However, successful manifestation of RNAi is dependent on several factors. The insect species might lack the basic RNAi machinery [161] or may rapidly degrade alien dsRNA. Such factors could be intrinsic to the concerned tissue or gene. The gene might have high transcription rate and could evade the effect of RNAi or the target mRNA may be

**5.4. Potency of the silencing signal**

354 RNA Interference

limited by many uncovered phenomena.

**6. Conclusions**

too transient.

SKU is thankful to the Department of Science and Technology, India, for DST-INSPIRE faculty fellowship and Panjab University for facility.

#### **Author details**

Nidhi Thakur1,2, Jaspreet Kaur Mundey3 and Santosh Kumar Upadhyay3\*

\*Address all correspondence to: skupadhyay@pu.ac.in

1 Plant Molecular Biology Lab, CSIR-National Botanical Research Institute, Lucknow, India

2 Academy of Scientific and Innovative Research, Anusandhan Bhawan, New Delhi, India

3 Department of Botany, Panjab University, Chandigarh, India

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## **Management of Insect Pest by RNAi — A New Tool for Crop Protection**

Thais Barros Rodrigues and Antonio Figueira

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61807

#### **Abstract**

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2004.08.006.

The fast-growing human population requires the development of new agricultural tech‐ nologies to meet consumers´ demand, while minimizing environmental impacts. Insect pests are one of the main causes for losses in agriculture production, and current control technologies based on pesticide application or the use of transgenic crops expressing *Ba‐ cillus thuringiensis* toxin proteins are facing efficacy challenges. Novel approaches to con‐ trol pests are urgently necessary. RNA interference (RNAi) is a gene silencing mechanism triggered by providing double-stranded RNA (dsRNA), that when ingested into insects can lead to death or affect the viability of the target pest. Transgenic plants expressing dsRNA version of insect specific target genes are the new generation of resistant plants. However, the RNAi mechanism is not conserved among insect orders, and its elucidation is the key to develop commercial RNAi crops. In this chapter, we review the core RNAi pathway in insects and the dsRNA uptake, amplification, and spread of systemic silenc‐ ing signals in some key insect species. We also highlight some of the experimental steps before developing an insect-pest-resistant "RNAi plant". Lastly, we review some of the most recent development studies to control agricultural insect pests by RNAi transgenic plants.

**Keywords:** Biotechnology, dsRNA, Entomology, Gene silencing, Insect control, RNA in‐ terference

#### **1. Introduction**

Agriculture has to continually adapt to rising environmental concerns in conjunction with meeting the increasing consumers´ demand. The fast-growing human population creates the need for the sustainable intensification of agriculture throughout the world which can be acomplished by adopting mechanization and new technologies to close yield gaps while

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

minimizing environmental impacts. In the past few decades, insect pest control has been mainly conducted by the application of chemical pesticides because of the low cost and efficacy; but their indiscriminate use has caused escalating problems with the evolution of insect resistance to the pesticides together with secondary pest outbreaks. The development of new biotechnological approaches, with the introduction of transgenic crops expressing *Bacillus thuringiensis* (Bt) Cry toxin proteins, also known as insect-resistant transgenic Bt-plants, decreased pesticide utilization in certain key crops, such as cotton and maize, and brought economical and environmental benefits [1-3]. But once again, insect resistance has arisen, now against the Bt toxins, and outbreaks of nontarget pests have emerged [1, 4], which makes necessary the development of novel approaches to control selected agriculture pests.

RNA interference (RNAi) is a gene silencing mechanism at the cellular level triggered by double-stranded RNA (dsRNA) and is likely to be the new approach underlying the next generation of insect-resistant transgenic plants. In some studies, successful delivery of dsRNA molecules to insects by ingestion resulted in the expected essential gene target silencing [5, 6], which led to death or affected the viability of the target insect, resulting in control of the pest.

In general, long dsRNAs are processed by species-specific RNase-III-like enzymes, resulting in smaller double-stranded molecules. These shorter RNAs are loaded into RNA complexes as a guide for finding target mRNAs that are either cleaved or blocked for translation in posttranscriptional silencing, or inducing histone modifications when involved in transcrip‐ tional silencing response [7, 8]. However, the RNAi systemic spreading mechanism is not conserved across organisms, and its elucidation is an essential step in developing an efficient method to control agricultural pests by RNAi technology.

In this chapter, we review how the RNAi mechanism occurs in insects, highlighting the core RNAi pathway and components, and new developments regarding dsRNA uptake, amplifi‐ cation, and the spread of systemic silencing signals in some key insect species. We also discuss the critical experimental steps before developing an "RNAi plant" protected against a specific insect pest, with consideration to application. Lastly, we review some of the most recent published studies to control agriculturally important insect pests based on RNAi transgenic plants.

#### **2. RNAi mechanism**

RNAi is an important and natural antiviral defense mechanism, protecting organisms from RNA viruses, or even avoiding the random integration of transposons [9]. Over time, with the discovery of some aspects of the mechanism, RNAi has become a widely used tool to knock down and analyze the function of genes. Most of the RNAi pathways have dsRNA as the precursor triggering molecule that vary in length and origin [7, 8]. In addition, the RNAi pathways differ not only in the RNA precursor molecule, but also in genes, enzymes, and effector complexes involved throughout the process. However, some key steps are conserved. Briefly, RNA duplexes are processed into short RNA duplexes, which are then used to guide the recognition of their target, either to cleave a complementary mRNA, or to repress their target translation at a posttranscriptional silencing level, or to modify the chromatin structure at the transcriptional level [7, 8].

minimizing environmental impacts. In the past few decades, insect pest control has been mainly conducted by the application of chemical pesticides because of the low cost and efficacy; but their indiscriminate use has caused escalating problems with the evolution of insect resistance to the pesticides together with secondary pest outbreaks. The development of new biotechnological approaches, with the introduction of transgenic crops expressing *Bacillus thuringiensis* (Bt) Cry toxin proteins, also known as insect-resistant transgenic Bt-plants, decreased pesticide utilization in certain key crops, such as cotton and maize, and brought economical and environmental benefits [1-3]. But once again, insect resistance has arisen, now against the Bt toxins, and outbreaks of nontarget pests have emerged [1, 4], which makes

necessary the development of novel approaches to control selected agriculture pests.

method to control agricultural pests by RNAi technology.

plants.

372 RNA Interference

**2. RNAi mechanism**

RNA interference (RNAi) is a gene silencing mechanism at the cellular level triggered by double-stranded RNA (dsRNA) and is likely to be the new approach underlying the next generation of insect-resistant transgenic plants. In some studies, successful delivery of dsRNA molecules to insects by ingestion resulted in the expected essential gene target silencing [5, 6], which led to death or affected the viability of the target insect, resulting in control of the pest. In general, long dsRNAs are processed by species-specific RNase-III-like enzymes, resulting in smaller double-stranded molecules. These shorter RNAs are loaded into RNA complexes as a guide for finding target mRNAs that are either cleaved or blocked for translation in posttranscriptional silencing, or inducing histone modifications when involved in transcrip‐ tional silencing response [7, 8]. However, the RNAi systemic spreading mechanism is not conserved across organisms, and its elucidation is an essential step in developing an efficient

In this chapter, we review how the RNAi mechanism occurs in insects, highlighting the core RNAi pathway and components, and new developments regarding dsRNA uptake, amplifi‐ cation, and the spread of systemic silencing signals in some key insect species. We also discuss the critical experimental steps before developing an "RNAi plant" protected against a specific insect pest, with consideration to application. Lastly, we review some of the most recent published studies to control agriculturally important insect pests based on RNAi transgenic

RNAi is an important and natural antiviral defense mechanism, protecting organisms from RNA viruses, or even avoiding the random integration of transposons [9]. Over time, with the discovery of some aspects of the mechanism, RNAi has become a widely used tool to knock down and analyze the function of genes. Most of the RNAi pathways have dsRNA as the precursor triggering molecule that vary in length and origin [7, 8]. In addition, the RNAi pathways differ not only in the RNA precursor molecule, but also in genes, enzymes, and effector complexes involved throughout the process. However, some key steps are conserved. Briefly, RNA duplexes are processed into short RNA duplexes, which are then used to guide the recognition of their target, either to cleave a complementary mRNA, or to repress their

The RNA precursor molecules from the RNAi pathways, all of which are already identified in insects, are small RNAs, categorized into three classes [8, 10]: the first two classes are small interfering RNAs (siRNAs; 20–25 nucleotides) and microRNAs (miRNAs; 21–24 nucleotides) [7] (Figure 1). Both miRNAs and siRNAs share a common RNase-III processing enzyme, Dicer, and closely related effector complexes and both can regulate gene expression at the posttran‐ scriptional level [8, 10, 11]. Conversely, the third class of small RNAs, the PIWI-interacting RNAs (piRNAs; 24–30 nucleotides), are generated independent of the Dicer activity [12]. piRNAs have been reported to play an essential role in germ-line development, stem cell renewal, transposon silencing, and epigenetic regulation [13-16]. These piRNAs originate from a diversity of sequences, including repetitive DNA and transposons, and they seem to act both at the posttranscriptional and chromatin levels [10]. The mechanism that generates and amplifies piRNAs is not well-understood, but involves slicer activities (Argonaute proteins associated with cleaving activity) [8, 10, 17]. Considered a specialized subclass of piRNAs, **r**epeat-**a**ssociated **si**RNAs (rasiRNAs; 25-29 nucleotides) were identified in the *Drosophila* genome [18], and suggested by Meister and Tuschl [7] to be involved in guiding chromatin modification in this insect species (Figure 1).

The most recognized RNAi pathways are the siRNA and miRNA; despite being triggered by different molecules, both precursors are long double-stranded RNAs (dsRNAs). Naturally in a cell, long dsRNAs can derive from RNA virus replication, from the transcription of conver‐ gent cellular genes or mobile genetic elements, or from self-annealing cellular transcripts [8]. In the siRNA pathway, these long dsRNA are processed by Dicer into siRNA duplexes. By contrast, in the miRNA pathway, miRNAs are generated from endogenous transcripts (primary miRNAs; pri-miRNAs) that form stem–loop structures [19, 20]. In the nucleus, these hairpin regions are recognized and cleaved into precursor mirRNAs (pre-miRNAs) by Drosha, another RNase-III-family enzyme, and the pre-miRNAs are transported to the cytoplasm through the nuclear export receptor Exportin-5 (Expo-5) [19, 20]. Subsequently, the premiRNA undergoes another endonucleolytic cleavage, now catalyzed by Dicer, generating an miRNA duplex [19, 20] (Figure 1).

The siRNA and miRNAs duplex containing ribonucleoprotein particles (RNPs) are subse‐ quently rearranged into effector complexes. Although it is difficult to assign distinct functional labels, an siRNA-containing effector complex is referred to as an "RNA-induced silencing complex" (RISC), and an miRNA-containing effector complex is referred to as an miRNP [7]. In these complexes, the regulation is at a posttranscriptional level and every RISC or miRNP contains a member of the Argonaute (Ago) protein family [7]. For the regulation at the transcriptional level as guided by rasiRNAs, a specialized nuclear Argonaute-containing complex, known as the RNA-Induced Transcriptional Silencing complex (RITS) mediates gene silencing [10]. In general, one strand of the short-RNA duplex (the guide strand) is loaded onto an Argonaute protein at the core of the effector complexes. During loading, the nonguide strand is cleaved by an Argonaute protein and ejected. The Argonaute protein then uses the guide RNA to associate with target RNAs that contain a perfectly complementary sequence

**Figure 1.** RNA-mediated gene silencing pathways. In the nucleus, primary miRNA transcripts (pri-miRNAs) are proc‐ essed to mirRNA precursors (pre-miRNA) by the RNase-III-like enzyme Drosha. The pre-miRNA is exported through the export receptor Exportin-5 (Exp-5) to the cytoplasm, and then processed by Dicer to microRNA (miRNA). These miRNAs are unwound and assembled into miRNP (miRNA containing effector complex of RiboNucleo Protein parti‐ cles) or RISC (RNA Induced Silencing Complex), triggering silencing responses by translational repression of target mRNAs or mRNA-target degradation, respectively. In the cytoplasm, long dsRNA are processed to siRNA (small in‐ terfering RNA) by the RNase-III-like enzyme Dicer. The short dsRNAs are unwound and assembled into RISC or RITS (RNA-Induced Transcriptional Silencing) complexes. The siRNA guides the RNA cleavage by the RISC complex, while rasiRNA (repeat-associated short interfering RNA) guides the condensation of heterochromatin by the RITS complex. 7mG: 7-methyl guanine; AAAA: poly-adenosine tail; Me: methyl group; P: 5'- phosphate. (Adapted from [7])

and then catalyzes the slicing of these targets, either to be cleaved by RISC, to be blocked for translation in miRNP or by inducing histone modifications in RITS [7] (Figure 1). The mecha‐ nism of miRNA-guided translational regulation is not as well-understood in the case of targetRNA cleavage, and to make things more complicated, miRNAs can act as siRNAs, and siRNAs can act as miRNAs [7].

Dicer is one of the enzymes involved in RNAi mechanism that is encoded by a variable number of genes and presents distinct specificity among organisms [21]. For instance, mammals and *Caenorhabditis elegans*, the best characterized animal for RNAi, have each a single Dicer responsible for functions in both siRNA and miRNA pathways [12], while *Drosophila mela‐ nogaster* has two paralogues: Dicer-1 (Dcr-1) that preferentially processes miRNA precursors, and Dicer-2 (Dcr-2) required to process long dsRNA into siRNAs [22]. However, in *Tribolium castaneum*, a model organism among insects for systemic silencing by RNAi, Dcr-2 showed an important role at the RNAi pathway, whereas Dcr-1 is suggested to have an involvement in wing development, most likely through the miRNA pathway [23].

Another gene family involved in RNAi pathways is the Argonaute proteins (Ago). Ago is a central protein component of silencing complexes (RISC, RITS, miRNP) that acts in mediating target recognition and silencing [24]. Argonaute proteins contain two domains: a PAZ domain involved in dsRNA binding, and a PIWI-domain responsible for RNase activity [23]. In *Drosophila*, Ago-1 is involved in the miRNA pathway; Ago-2 in the siRNA pathway; while Piwi, Aubergine (Aub), and Ago-3 are associated in transcriptional silencing [13, 25-27]. In *Tribolium*, a single class of Argonaute was identified (Tc-Ago-1) in the miRNA pathway, while two classes of Ago-2 paralogues (Tc-Ago-2a and Tc-Ago-2b) were found in the siRNA pathway, probably deriving from gene duplication in the beetle lineage [23]. This duplicated Tc-Ago-2 might lead to higher amounts of Ago-2 protein, potentially with the enhancement of the RNAi response [23]. In the silkmoth *Bombyx mori*, a Lepidoptera species in which the RNAi response is considered much less robust [28, 29], *AGO* genes from all three main RNAi pathways were identified (*BmAGO-1* – miRNA; *BmAGO-2* – siRNA; *BmAGO-3* – piRNA), which were shown to be involved in the RNAi response in Bm5 cells [30]. Taken together, these findings, and other reviews [29], support the idea of a function overlap of the three main RNAi pathways in *B. mori* [29].

#### **2.1. Systemic RNAi**

Systemic RNAi is described as a silencing signal transmitted widely throughout a treated organism [5, 31]. The knowledge about the systemic RNAi mechanism in insects is important as it may affect the approaches adopted to develop "RNAi-mediated pest control" because the systemic mechanism is not conserved among those organisms. Systemic RNAi has two important steps to be considered: the uptake of dsRNA by the cells and the systemic spreading of the signals. Some of the main genes involved in systemic RNAi are presented below and discussed for the model organisms.

#### *2.1.1. dsRNA uptake*

and then catalyzes the slicing of these targets, either to be cleaved by RISC, to be blocked for translation in miRNP or by inducing histone modifications in RITS [7] (Figure 1). The mecha‐ nism of miRNA-guided translational regulation is not as well-understood in the case of target-

**Figure 1.** RNA-mediated gene silencing pathways. In the nucleus, primary miRNA transcripts (pri-miRNAs) are proc‐ essed to mirRNA precursors (pre-miRNA) by the RNase-III-like enzyme Drosha. The pre-miRNA is exported through the export receptor Exportin-5 (Exp-5) to the cytoplasm, and then processed by Dicer to microRNA (miRNA). These miRNAs are unwound and assembled into miRNP (miRNA containing effector complex of RiboNucleo Protein parti‐ cles) or RISC (RNA Induced Silencing Complex), triggering silencing responses by translational repression of target mRNAs or mRNA-target degradation, respectively. In the cytoplasm, long dsRNA are processed to siRNA (small in‐ terfering RNA) by the RNase-III-like enzyme Dicer. The short dsRNAs are unwound and assembled into RISC or RITS (RNA-Induced Transcriptional Silencing) complexes. The siRNA guides the RNA cleavage by the RISC complex, while rasiRNA (repeat-associated short interfering RNA) guides the condensation of heterochromatin by the RITS complex. 7mG: 7-methyl guanine; AAAA: poly-adenosine tail; Me: methyl group; P: 5'- phosphate. (Adapted from [7])

374 RNA Interference

In insects, two types of dsRNA uptake mechanisms have been identified [32]. The first one involves a multi-transmembrane domain protein, Systemic Interference Defective (Sid). In *C. elegans*, Sid-1 is essential and sufficient to mediate uptake and systemic spread of RNAi signal in both somatic and germ-line cells [33, 34]; conversely, in insects, Sid-1-like (Sil) proteins appear to be variable across orders [32]. For instance, Diptera do not present *SIL* genes, while *Tribolium* and *B. mori* presented three *SIL* homologues [23, 35]. However, these *Tribolium* genes share more identity with another *C. elegans* gene, *TAG-130*, not required for systemic RNAi in *C. elegans* compared to that with *SID-1* [23]. The second dsRNA uptake mechanism involves endocytosis, specific for dsRNAs acquired from the environment, known as environmental RNAi [5, 31]. First discovered in *Drosophila* S2 cells and later in *C. elegans*, this uptake of dsRNA by endocytosis appears to be evolutionary-conserved [36-38]. However, we should be cautious to conclude that all organisms have a dsRNA uptake mechanism based on endocytosis. For instance, Ulvila and colleagues [38] working with *Drosophila* S2 cells, which are hemocyte-like, described high rates of endocytosis as compared to the majority of other cell types in this species [39].

Other important proteins for systemic RNAi were identified in *C. elegans* but are specific only to germ-line cells, such as Rsd-2, Rsd-3, and Rsd-6 [40]. The Rsd-2 protein contains no particular known motifs but interacts with Rsd-6 that has a Tudor domain, suggesting that these two proteins act together [40]. The *RSD-3* gene encodes a protein that contains an epsin aminoterminal homology (ENTH) domain, found in proteins involved in vesicle trafficking, sug‐ gesting the involvement of endocytosis in systemic RNAi [40]. In *Tribolium*, a homologue for *RSD-3* (*Tc-RSD3*) has been found, but in *Drosophila*, which does not exhibit a systemic silencing response, a homologous Rsd-3 protein (Epsin-like) was identified [23]. So, the presence of Rsd-3 does not seems to determine whether or not systemic RNAi occurs in insects, and it is possible that the expression level and/or tissue specificity of this gene may affect the degree of RNAi efficiency and the dsRNA uptake from the environment [23].

#### *2.1.2. RNAi systemic spreading: amplification and maintenance of dsRNA*

Once the dsRNA overcomes all the uptake barriers, the silencing signal should be transported from treated cell to other cells, and spread to other tissues. Further, dsRNA should be con‐ stantly produced, e.g., either by the amplification of dsRNA by an RNA-dependent RNA polymerase (RdRP), and/or constantly acquired for the maintenance of the silencing responses.

In *C. elegans*, primary siRNAs processed by Dicer are used as a template for an RdRP activity to produce secondary dsRNAs [41]. The RdRP activity is key for the RNAi signal amplification. However, so far in insects, no RdRP-related protein has been found [21, 23], suggesting that strong RNAi response in insects does not rely on amplification of the trigger dsRNA, and it must be based on a different mechanism yet to be identified. Alternatively, constant supply of dsRNA may be provided by RNAi-plants to provide continuous effects.

The presence of the main genes involved in systemic RNAi and amplification in *Drosophila* and *Tribolium* fail to explain the respective absence and presence of systemic RNAi [23]. Never‐ theless, the several differences in the number of these core component genes found between species suggests an interesting avenue for further investigation [23]. For instance, Ago protein that have already been shown to determine RNAi efficiency [42], was found to be duplicated in the *Tribolium* genome, while *Drosophila* carries only a copy of *AGO-2* gene, suggesting a relationship between number of *AGO* copies and RNAi response [23].

Another component that might affect RNAi efficiency in different insects are the proteins containing the dsRNA-binding motif (dsRBM), which help small molecules to properly load inside of the silencing complexes [23]. These proteins act together with Dicer, and seem to be responsible for determining Dicer specificity in *Drosophila* [23]. In the *T. castaneum* genome, two *R2D2-like* genes (a particular dsRBM) were found (*TcR2D2* and *TcC3PO*), which might help *Tribolium* to be hypersensitive to dsRNA molecule uptake by the cells [23]. However, in the *Anthonomus grandis* transcriptome, only an *R2D2* contig was identified [21]. The presence of an additional R2D2-like protein in *Tribolium* might also allow a longer-lasting RNAi effect, once dsRBM proteins are known to bind to dsRNAs, and might be involved in the maintenance of dsRNA in cells [23].

### **3. Factors affecting the silencing effect and RNAi efficiency as an insect control method**

The RNAi approach to control insect pests had been considered for many years, but application of this technology was just realized after it was shown that ingestion of dsRNA would trigger RNAi. The concept of RNAi-plant mediated pest control was demonstrated in 2007 by the development of transgenic plants producing dsRNAs against specific insect genes, with the consequent effect on the target species [43, 44]. The main prerequisites to generate successful RNAi insect-resistant transgenic plants are: (i) identification of a specific gene with an essential function in the insect that can cause developmental deformities and/or larval lethality when knocked down or knocked out; and (ii) dsRNA delivery by oral ingestion that must be uptaken by the insect cells, and spread systemically.

The insect must uptake the dsRNA version of a target gene region by feeding. To silence the target gene, this specific dsRNA must be taken up from the gut lumen into the gut cells as what is considered as "environmental RNAi." If the target gene is expressed in a tissue distinct from the digestive system, the silencing signal should successfully spread via cells and tissues as a systemic RNAi. Both environmental and systemic RNAi are considered noncell-autonomous RNAi, which means that the interfering effect takes places in tissues/cells different from the location of application or production of the dsRNA. Conversely, in the cell-autonomous RNAi, the silencing process is limited to the cell in which the dsRNA is introduced [5]. However, the mechanism of ingested dsRNA uptake and systemic spreading of the silencing signal in the insect have yet to be fully characterized and understood.

Some factors can affect the efficiency of the dsRNA uptake and systemic silencing spread in different insects. Here, we highlight important points that must be considered in developing an RNAi approach against insect pests.

#### **3.1. Target gene**

in both somatic and germ-line cells [33, 34]; conversely, in insects, Sid-1-like (Sil) proteins appear to be variable across orders [32]. For instance, Diptera do not present *SIL* genes, while *Tribolium* and *B. mori* presented three *SIL* homologues [23, 35]. However, these *Tribolium* genes share more identity with another *C. elegans* gene, *TAG-130*, not required for systemic RNAi in *C. elegans* compared to that with *SID-1* [23]. The second dsRNA uptake mechanism involves endocytosis, specific for dsRNAs acquired from the environment, known as environmental RNAi [5, 31]. First discovered in *Drosophila* S2 cells and later in *C. elegans*, this uptake of dsRNA by endocytosis appears to be evolutionary-conserved [36-38]. However, we should be cautious to conclude that all organisms have a dsRNA uptake mechanism based on endocytosis. For instance, Ulvila and colleagues [38] working with *Drosophila* S2 cells, which are hemocyte-like, described high rates of endocytosis as compared to the majority of other cell types in this

Other important proteins for systemic RNAi were identified in *C. elegans* but are specific only to germ-line cells, such as Rsd-2, Rsd-3, and Rsd-6 [40]. The Rsd-2 protein contains no particular known motifs but interacts with Rsd-6 that has a Tudor domain, suggesting that these two proteins act together [40]. The *RSD-3* gene encodes a protein that contains an epsin aminoterminal homology (ENTH) domain, found in proteins involved in vesicle trafficking, sug‐ gesting the involvement of endocytosis in systemic RNAi [40]. In *Tribolium*, a homologue for *RSD-3* (*Tc-RSD3*) has been found, but in *Drosophila*, which does not exhibit a systemic silencing response, a homologous Rsd-3 protein (Epsin-like) was identified [23]. So, the presence of Rsd-3 does not seems to determine whether or not systemic RNAi occurs in insects, and it is possible that the expression level and/or tissue specificity of this gene may affect the degree

Once the dsRNA overcomes all the uptake barriers, the silencing signal should be transported from treated cell to other cells, and spread to other tissues. Further, dsRNA should be con‐ stantly produced, e.g., either by the amplification of dsRNA by an RNA-dependent RNA polymerase (RdRP), and/or constantly acquired for the maintenance of the silencing responses. In *C. elegans*, primary siRNAs processed by Dicer are used as a template for an RdRP activity to produce secondary dsRNAs [41]. The RdRP activity is key for the RNAi signal amplification. However, so far in insects, no RdRP-related protein has been found [21, 23], suggesting that strong RNAi response in insects does not rely on amplification of the trigger dsRNA, and it must be based on a different mechanism yet to be identified. Alternatively, constant supply of

The presence of the main genes involved in systemic RNAi and amplification in *Drosophila* and *Tribolium* fail to explain the respective absence and presence of systemic RNAi [23]. Never‐ theless, the several differences in the number of these core component genes found between species suggests an interesting avenue for further investigation [23]. For instance, Ago protein that have already been shown to determine RNAi efficiency [42], was found to be duplicated in the *Tribolium* genome, while *Drosophila* carries only a copy of *AGO-2* gene, suggesting a

of RNAi efficiency and the dsRNA uptake from the environment [23].

*2.1.2. RNAi systemic spreading: amplification and maintenance of dsRNA*

dsRNA may be provided by RNAi-plants to provide continuous effects.

relationship between number of *AGO* copies and RNAi response [23].

species [39].

376 RNA Interference

The choice of the target gene should be carefully considered. Each gene requires particular effort to be silenced. Terenius and colleagues [28] reviewed more than 150 RNAi experimental results from RNAi of lepidopterans involving 130 genes, from which only 38% were silenced at a satisfactory level, while 48% failed to be silenced, and 14% were silenced at insufficient levels. Among the target genes, those involved in immunity were more effectively silenced, and, in contrast, genes expressed in epidermal tissues seem to be most difficult. Differences for RNAi sensitivity among genes in the same tissue was described in [28].

#### **3.2. dsRNA design**

The design of the dsRNA determines the one particular target gene to be silenced, but offtarget effects can occur if siRNAs have some sequence similarity with unintended genes. Tobacco plants expressing *Helicoverpa armigera* ecdysone receptor (*EcR*) dsRNA improved resistance to another insect, *Spodoptera frugiperda*, due to the high identity shared between the nucleotide sequence of *HaEcR* and *SeEcR g*enes [45]. Although this result implies that an RNAiplant can control two or even more lepidopteran pests, this can also affect nontarget insects, becoming a biosafety issue.

#### **3.3. dsRNA length**

The length of the dsRNA fragments plays an essential role in the effectiveness of molecular uptake in insects, which is directly involved in the success of the target gene silencing. In most of the RNAi experiments, the insects are fed with long dsRNAs [5]. Some experiments showed that long dsRNAs are more efficiently uptaken than siRNAs [37, 46]. This may be due to the fact that a long dsRNA, with 100% match of the target mRNA, after processing into siRNA will provide a greater diversity of siRNAs available to cause specific suppression of target gene and increase the desired effect, and, additionally, reduce the likelihood of developing resist‐ ance [47]. In contrast, other studies reported suppression of genes in different insects via incorporation of siRNA in diet instead of dsRNA [48, 49].

#### **3.4. dsRNA concentration**

Optimal concentration of dsRNA delivered to the insect is required to induce sufficient gene target silencing. It is noteworthy to mention that exceeding the optimal dsRNA concentration may not result in more silencing [50, 51]. However, higher concentration of dsRNA decreased the duration of dsRNA exposure to reach 50% mortality of *Diabrotica virgifera virgifera* [46], suggesting an inverse relationship between dsRNA concentration and duration of exposure. In cricket (*Gryllus bimaculatus*), the highest concentrations of dsRNA yielded more efficient gene knockdown [52]. The amount of dsRNA sufficient to significantly reduce mRNA levels of *PER* and *CLK* genes were one and two μM, respectively. These concentrations reduced the expression level of the targets, but a higher concentration (20 μM) for the C*YC* gene was required, suggesting that the sensitivity to dsRNAs also depends specifically on the gene [52].

#### **3.5. Controls**

Empty vector, empty cassette, buffer only, irrelevant or nonspecific control (such as dsGFP – Green Fluorescent Protein gene region), or any other kind of negative control are essential to discriminate specific gene silencing from the simple induction of siRNA processing machinery by exposure to a dsRNA. Mainly, a negative control should demonstrate the specificity of the dsRNA designed for a target, not interfering in specific target expression, and even unspecific effects. Also, any control should have similar size and concentration of the used dsRNA [53].

#### **3.6. Molecular silencing confirmation**

results from RNAi of lepidopterans involving 130 genes, from which only 38% were silenced at a satisfactory level, while 48% failed to be silenced, and 14% were silenced at insufficient levels. Among the target genes, those involved in immunity were more effectively silenced, and, in contrast, genes expressed in epidermal tissues seem to be most difficult. Differences

The design of the dsRNA determines the one particular target gene to be silenced, but offtarget effects can occur if siRNAs have some sequence similarity with unintended genes. Tobacco plants expressing *Helicoverpa armigera* ecdysone receptor (*EcR*) dsRNA improved resistance to another insect, *Spodoptera frugiperda*, due to the high identity shared between the nucleotide sequence of *HaEcR* and *SeEcR g*enes [45]. Although this result implies that an RNAiplant can control two or even more lepidopteran pests, this can also affect nontarget insects,

The length of the dsRNA fragments plays an essential role in the effectiveness of molecular uptake in insects, which is directly involved in the success of the target gene silencing. In most of the RNAi experiments, the insects are fed with long dsRNAs [5]. Some experiments showed that long dsRNAs are more efficiently uptaken than siRNAs [37, 46]. This may be due to the fact that a long dsRNA, with 100% match of the target mRNA, after processing into siRNA will provide a greater diversity of siRNAs available to cause specific suppression of target gene and increase the desired effect, and, additionally, reduce the likelihood of developing resist‐ ance [47]. In contrast, other studies reported suppression of genes in different insects via

Optimal concentration of dsRNA delivered to the insect is required to induce sufficient gene target silencing. It is noteworthy to mention that exceeding the optimal dsRNA concentration may not result in more silencing [50, 51]. However, higher concentration of dsRNA decreased the duration of dsRNA exposure to reach 50% mortality of *Diabrotica virgifera virgifera* [46], suggesting an inverse relationship between dsRNA concentration and duration of exposure. In cricket (*Gryllus bimaculatus*), the highest concentrations of dsRNA yielded more efficient gene knockdown [52]. The amount of dsRNA sufficient to significantly reduce mRNA levels of *PER* and *CLK* genes were one and two μM, respectively. These concentrations reduced the expression level of the targets, but a higher concentration (20 μM) for the C*YC* gene was required, suggesting that the sensitivity to dsRNAs also depends specifically on the gene [52].

Empty vector, empty cassette, buffer only, irrelevant or nonspecific control (such as dsGFP – Green Fluorescent Protein gene region), or any other kind of negative control are essential to

for RNAi sensitivity among genes in the same tissue was described in [28].

incorporation of siRNA in diet instead of dsRNA [48, 49].

**3.2. dsRNA design**

378 RNA Interference

becoming a biosafety issue.

**3.4. dsRNA concentration**

**3.5. Controls**

**3.3. dsRNA length**

An efficient molecular confirmation of the RNAi silencing should be conducted, which includes target RNA expression, and analyses of protein amount and/or enzyme activity. In RNA analysis, additional care should be taken for expression analysis. The method of choice for RNA expression analysis is the quantitative amplification of reversed transcripts or RTqPCR, considered a very sensitive and accurate method. To provide precision in RT-qPCR, some essential care is required, such as the choice of appropriate stable reference genes and primer pair design with sufficient amplification efficiency. The reference genes should exhibit stable expression among experimental conditions, providing reliable estimate of gene expres‐ sion results [54]. Additionally, primers should be designed flanking the region used to design the dsRNA to ensure that the initial cleavage of the mRNA could be detected, thus avoiding false-positives [55]. Conventional care of RT–qPCR reactions defined by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines must be strictly followed [56].

#### **3.7. Protein stability and phenotype analysis**

Proteins can exhibit a long half-life and interfere with the phenotypic changes. However, phenotype changes are not totally related to small decrease of protein levels; haplo-sufficient genes produce proteins capable of performing the biological processes normally, even at half of the protein levels [53]. Phenotype changes could be more difficult to be observed in RNAi responses if the protein product of the target gene has a long half-life. For example, the reduction of *Da6* (nicotinic acetylcholine receptor subunit) expression in both *D. melanogaster* and *T. castaneum* exhibited weak phenotype responses [57], which may be explained based on the long stability of the nicotinic acetylcholine receptors (nAChRs) [58]. Nevertheless, for most of the genes, mRNA turnover and protein half-life are unknown and this lack of information presents one of the principal challenges for the RNAi experiments [39].

#### **3.8. Insect issues, life stage, nucleases, and gut pH**

Some insect characteristics should also be considered before starting an RNAi experiment including the developmental stage of insects. Although handling advanced developmental stages of insects is more efficient, silencing effects are more prominent in earlier stages. For instance, in second instar larvae of *Rhodnius prolixus*, the gene *nitropin 2* was knocked down for 42%, but no silencing was observed in the fourth instar individuals, even though both larval stages were treated with the same concentration of dsRNA [59]. In *Spodoptera frugiperda*, fifth instar larvae presented higher gene silencing as compared to adult moths [60].

Another consideration that can affect the RNAi silencing efficiency is the presence of insect nucleases and gut pH. For instance, feeding assays with *Lygus lineolaris* showed no mortality effects because the saliva of *L. lineolaris* contains dsRNA-degrading activity. Thus, dsRNA ingested did not result in siRNA fragments, rather were completely digested to monomers [61]. Also, dsRNA degradation is reduced during molting period and further reduced by starvation in some insects [62]. The stability of the dsRNA in the midgut could be affected not only by enzymatic but also by chemical hydrolysis [63]. In both cases, gut pH is an important factor; particularly, it is quite variable among insect orders, with variation even among gut regions [64]. For example, in general, lepidopterans exhibit a strong alkaline gut (up to pH 10.5 in some species), which provides a highly hostile environment for dsRNA [64]; therefore, this order is particularly recalcitrant to gene silencing by RNAi.

#### **4. Overview on the use of RNAi to control insects by transgenic plants**

Most of the current transgenic crops with specific control against insect pests are based on *Bacillus thuringiensis* (Bt) toxins, which act in gut epithelial cell membrane in susceptible insects. Bt toxins are highly specific against certain orders of insects, where the most successful use was achieved against Lepidoptera and Coleoptera. However, continuous exposure of those insects to Bt crops evolved field-resistance, affecting the efficiency in controlling those pests [4, 65]. Also, there are limitations of Bt transgenics to manage some other important agricultural pests, such as the sap-sucking insects (Hemiptera). This encouraged the development of new strategies to help in controlling agricultural pests.

In 2007, two studies demonstrated the concept of plants expressing dsRNAs derived from hairpin vectors that directed dsRNAs to target gene regions of economically important agricultural pests: the cotton bollworm (*Helicoverpa armigera*; Lepidoptera; [44]) and the Western corn rootworm (WCR; *Diabrotica virgifera virgifera*; Coleoptera; [43]). After the demonstration of plants resistant to insects, the application of RNAi by transgenic plants became a potential new approach to control important agricultural pests, which led to the flourishing of a new field of research. So far, searches on the publication database "Web of Science" from Thomson Reuters (August 2015), identified 543 published studies based on the combination of the topics "RNAi," "plant" and "insect," and only one quarter was published before 2008.

To implement RNAi in agricultural pest control, the target insect should uptake the dsRNA autonomously, e.g., from transgenic plants expressing dsRNA. This feeding should be continuous, since insects lack an amplification mechanism based on RdRP, such as *C. elegans*. Many of the main agricultural pest species have already been targeted by RNAi technology using various genes and delivery methods [66]. However, three orders have been the major focus of the development of transgenic plants expressing target gene regions for RNAi: Coleoptera, Hemiptera, and Lepidoptera. Here, we list some of the most recently published RNAi transgenic plants studies performed against those insect orders (Table 1).


**Table 1.** Overview of the recently published studies on the use of plant-RNAi against different insect pests

#### **4.1. Coleoptera**

Another consideration that can affect the RNAi silencing efficiency is the presence of insect nucleases and gut pH. For instance, feeding assays with *Lygus lineolaris* showed no mortality effects because the saliva of *L. lineolaris* contains dsRNA-degrading activity. Thus, dsRNA ingested did not result in siRNA fragments, rather were completely digested to monomers [61]. Also, dsRNA degradation is reduced during molting period and further reduced by starvation in some insects [62]. The stability of the dsRNA in the midgut could be affected not only by enzymatic but also by chemical hydrolysis [63]. In both cases, gut pH is an important factor; particularly, it is quite variable among insect orders, with variation even among gut regions [64]. For example, in general, lepidopterans exhibit a strong alkaline gut (up to pH 10.5 in some species), which provides a highly hostile environment for dsRNA [64]; therefore, this

**4. Overview on the use of RNAi to control insects by transgenic plants**

Most of the current transgenic crops with specific control against insect pests are based on *Bacillus thuringiensis* (Bt) toxins, which act in gut epithelial cell membrane in susceptible insects. Bt toxins are highly specific against certain orders of insects, where the most successful use was achieved against Lepidoptera and Coleoptera. However, continuous exposure of those insects to Bt crops evolved field-resistance, affecting the efficiency in controlling those pests [4, 65]. Also, there are limitations of Bt transgenics to manage some other important agricultural pests, such as the sap-sucking insects (Hemiptera). This encouraged the development of new

In 2007, two studies demonstrated the concept of plants expressing dsRNAs derived from hairpin vectors that directed dsRNAs to target gene regions of economically important agricultural pests: the cotton bollworm (*Helicoverpa armigera*; Lepidoptera; [44]) and the Western corn rootworm (WCR; *Diabrotica virgifera virgifera*; Coleoptera; [43]). After the demonstration of plants resistant to insects, the application of RNAi by transgenic plants became a potential new approach to control important agricultural pests, which led to the flourishing of a new field of research. So far, searches on the publication database "Web of Science" from Thomson Reuters (August 2015), identified 543 published studies based on the combination of the topics "RNAi," "plant" and "insect," and only one quarter was published

To implement RNAi in agricultural pest control, the target insect should uptake the dsRNA autonomously, e.g., from transgenic plants expressing dsRNA. This feeding should be continuous, since insects lack an amplification mechanism based on RdRP, such as *C. elegans*. Many of the main agricultural pest species have already been targeted by RNAi technology using various genes and delivery methods [66]. However, three orders have been the major focus of the development of transgenic plants expressing target gene regions for RNAi: Coleoptera, Hemiptera, and Lepidoptera. Here, we list some of the most recently published

RNAi transgenic plants studies performed against those insect orders (Table 1).

order is particularly recalcitrant to gene silencing by RNAi.

strategies to help in controlling agricultural pests.

before 2008.

380 RNA Interference

The coleopterans are likely to be the first target to be controlled by the new generation of transgenics, the "RNAi-plants." *Diabrotica virgifera virgifera (*Western corn rootworm, WCR) is one of the most important agricultural pests, and this species, along with other coleopterans, requires little effort to have genes silenced by RNAi, independent of the delivery method and gene target. The significant breakthrough was demonstrated when WCR presented significant larval stunting and mortality, causing less injury to maize roots that express a hairpin version of *V-ATPase A* [43]. Since then, many studies have been conducted using various target genes, while not focusing only on pest control, but also to characterize the mechanism of action of the RNAi [46]. A recent study with WCR demonstrated that long dsRNAs of *Dv V-ATPase C* expressed in maize provides highly efficient root protection, but the siRNA population generated in the transgenic plant does not lead to lethal RNAi responses when consumed by the insect [67].

Studies have also been performed in other species, such as the important potato pest *Leptino‐ tarsa decemlineata* (Colorado potato beetle, CPB). So far, this is the first study to compare efficacy at controlling pest insects by dsRNAs expressed either in the chloroplast or in the cytoplasm. Transgenic potato plants expressing hairpin versions of *β-actin* and *Shrub* genes (both insectidal dsRNAs) in the chloroplasts (transplastomic plants) conferred the most potent insecticidal activity (insects died after 5 days), while the conventional expression from nuclear transgenics did not affect the beetles [68].

An explanation for this result is that since choroplasts do not have cellular RNAi machinery [69], the dsRNAs produced inside these organelles are not cleaved by a plant Dicer and the beetles ingest almost entirely long dsRNA. In contrast, beetles fed on nuclear-transformed plants consumed mostly siRNAs; previous dsRNA-feeding studies already indicated that ingested long dsRNAs were much more effective than ingested siRNAs. It should be high‐ lighted that all the other studies have been based on an efficient cytoplasm-derived dsRNA in various crops, indicating that possibly potato plants process long dsRNA more effective than the plants from those other studies (Table 1 [69]).

#### **4.2. Lepidoptera**

Plants producing *Bt* proteins were the first generation of transgenic plants to control insects, and most of the lepidopterans were successfully managed by Bt crops for years. However, the durability of Bt technology appears to be unsure. The number of pest species that evolved Bt resistance in the field, reducing transgenic efficacy, increased from one in 2005 to five in 2010 [4]. Among those five species, four are lepidopterans [4]. The lepidopterans could be the first and main targets for RNAi crops if they were not as recalcitrant to gene silencing, without any definite explanation for the limited and unstable RNAi responses [28, 29]. For instance, the concentration of dsRNA necessary to knock down a specific gene by feeding *Diatraea saccha‐ ralis* (Lepidoptera) neonate larvae is much higher than the one required for the same larval stage of *D. v. virgifera* (Coleoptera).

The first successful RNAi plant protected against a lepidopteran (*Helicoverpa armigera*) was demonstrated by silencing the *CYP6AE14* gene, necessary for detoxifying gossypol from cotton (*Gossypum hirsutum*) [44]. Studies have been conducted to explore alternative target genes to control and understand the RNAi mechanism in lepiopterans. Larval lethality and molting defects were detected in *H. armigera* fed with transgenic tobacco plants expressing dsRNA targeted to the nuclear ecdysone receptor complex (*HaEcR*), absolutely required for insect development [45]. The transgenic tobacco expressing dsRNA of *HaEcR* had an improved resistance to another lepidopteran pest, *Spodoptera exigua*. This cross-species effect might indicate a risk to affect nontarget insects, highlighting the importance of biosafety studies that should be carefully conducted [45].

The expression of dsRNA in both *Escherichia coli* and transgenic tobacco plants to silence a molt-regulating transcription factor gene (*HR3*) of *H. armigera* resulted in developmental deformity and larval lethality [70]. Transgenic *Arabidopsis thaliana* plants expressing dsRNA targeted to *arginine kinase* (*AK*) of *H. armigera* (*HaAK*) led to a 55% mortality rate in first instar larvae, while retarding growth in surviving larvae [71]. However, no lethal phenotypes were observed for the third instar larvae, although transcript levels of *HaAK* were distinctly suppressed [71].

#### **4.3. Hemiptera**

Studies have also been performed in other species, such as the important potato pest *Leptino‐ tarsa decemlineata* (Colorado potato beetle, CPB). So far, this is the first study to compare efficacy at controlling pest insects by dsRNAs expressed either in the chloroplast or in the cytoplasm. Transgenic potato plants expressing hairpin versions of *β-actin* and *Shrub* genes (both insectidal dsRNAs) in the chloroplasts (transplastomic plants) conferred the most potent insecticidal activity (insects died after 5 days), while the conventional expression from nuclear transgenics

An explanation for this result is that since choroplasts do not have cellular RNAi machinery [69], the dsRNAs produced inside these organelles are not cleaved by a plant Dicer and the beetles ingest almost entirely long dsRNA. In contrast, beetles fed on nuclear-transformed plants consumed mostly siRNAs; previous dsRNA-feeding studies already indicated that ingested long dsRNAs were much more effective than ingested siRNAs. It should be high‐ lighted that all the other studies have been based on an efficient cytoplasm-derived dsRNA in various crops, indicating that possibly potato plants process long dsRNA more effective than

Plants producing *Bt* proteins were the first generation of transgenic plants to control insects, and most of the lepidopterans were successfully managed by Bt crops for years. However, the durability of Bt technology appears to be unsure. The number of pest species that evolved Bt resistance in the field, reducing transgenic efficacy, increased from one in 2005 to five in 2010 [4]. Among those five species, four are lepidopterans [4]. The lepidopterans could be the first and main targets for RNAi crops if they were not as recalcitrant to gene silencing, without any definite explanation for the limited and unstable RNAi responses [28, 29]. For instance, the concentration of dsRNA necessary to knock down a specific gene by feeding *Diatraea saccha‐ ralis* (Lepidoptera) neonate larvae is much higher than the one required for the same larval

The first successful RNAi plant protected against a lepidopteran (*Helicoverpa armigera*) was demonstrated by silencing the *CYP6AE14* gene, necessary for detoxifying gossypol from cotton (*Gossypum hirsutum*) [44]. Studies have been conducted to explore alternative target genes to control and understand the RNAi mechanism in lepiopterans. Larval lethality and molting defects were detected in *H. armigera* fed with transgenic tobacco plants expressing dsRNA targeted to the nuclear ecdysone receptor complex (*HaEcR*), absolutely required for insect development [45]. The transgenic tobacco expressing dsRNA of *HaEcR* had an improved resistance to another lepidopteran pest, *Spodoptera exigua*. This cross-species effect might indicate a risk to affect nontarget insects, highlighting the importance of biosafety studies that

The expression of dsRNA in both *Escherichia coli* and transgenic tobacco plants to silence a molt-regulating transcription factor gene (*HR3*) of *H. armigera* resulted in developmental deformity and larval lethality [70]. Transgenic *Arabidopsis thaliana* plants expressing dsRNA targeted to *arginine kinase* (*AK*) of *H. armigera* (*HaAK*) led to a 55% mortality rate in first instar larvae, while retarding growth in surviving larvae [71]. However, no lethal phenotypes were

did not affect the beetles [68].

382 RNA Interference

**4.2. Lepidoptera**

the plants from those other studies (Table 1 [69]).

stage of *D. v. virgifera* (Coleoptera).

should be carefully conducted [45].

Hemipterans are characterized as piercing/sucking insects, representing major agricultural pests that inflict direct damages by sucking sap, or indirectly by acting as a vector of several viruses and bacterial infections. Since hemipterans feed through sucking the phloem, only systemic chemical insecticides are effective against these insects, resulting in high residual toxicity. The problem is further aggravated as no Bt toxin has been identified as exhibiting adequate insecticidal effects against hemipterans. Transgenic crops based on RNAi offer a large potential to control hemipteran, requiring expression of target gene dsRNAs on the phloem. One first report was published in 2011 about developing transgenic *Nicotiana benthamiana* and *A. thaliana* expressing dsRNA targeting genes expressed in *Myzus persicae* gut (*RACK1*) and salivary glands (*MpC002*) [72]. A reduction of the expression level of the target genes and a decrease in *M. persicae* fecundity were observed, but no lethal effects [72].

The same phenotype was observed when *M. persicae* fed on *A. thaliana* expressing dsRNA of a serine protease gene (*MySP*), with no lethal effects, but reduced fecundity [73]. Once again, reduced reproduction, but no lethal phenotype was observed when *M. persicae* was fed on tobacco plants expressing dsRNA targeting the *hunchback* gene [74]. A possible explanation for not achieving the expected phenotypes (mortality) after target gene depletion is that the dsRNA level produced by RNAi transgenic plants could not be sufficient for an efficient uptake of dsRNA or siRNA by sap-sucking insects [74]. However, more recently, mortality rates were observed in *Bemisia tabaci* fed in tobacco plants expressing dsRNA of *v-ATPaseA* [75]. This result provides a proof-of-concept that plants expressing dsRNA, at an efficient level and targeting crucial genes, could resist the attack of Hemipteran pests [75].

#### **5. Conclusion and future perspectives**

Since the concept of a transgenic plant expressing dsRNA targeted to a specific essential gene in the insect that affects its viability was first demonstrated in 2007, the technology has been extended to a large number of insect species from various orders. Elucidating the various mechanisms and components of the RNA interference pathway has progressed, but many aspects remain to be clarified. Many differences in components and mechanisms among insect orders and between insects and other organisms still need to be worked out. Some of these differences (e.g., genes involved, gene number, and level of expression) may explain variation in recalcitrance among insect species and need to be further investigated. Of particular interest are the mechanisms of dsRNA uptake, signal amplification, and systemic spread in the major pest species. Additional insect- or order-specific characteristics, such as gut pH, presence of dsRNA-degrading activity in digestive system, among others that could be associated with differences in recalcitrance to RNAi need to be dissected and clarified.

Due to the variety of RNAi response to RNAi in insects, no single protocol is suitable for all species. Issues related to the choice of effective target genes, including determining the size of optimal dsRNA length and ideal gene region. Assuming that the method of choice to deliver dsRNA is transgenic plants, a major question still to be addressed is the impact of plant dsRNA processing in the effective RNAi-induced silencing. There is still a need for investigation in this area. The choice of a suitable inducible promoter for expressing the dsRNA construct is another point barely explored.

Based on the recent publications reviewed in this chapter, the progress in developing "RNAiplants" to control important insect pests widely demonstrated the potential of this technology to complement or replace Bt crops, providing resistance against a broad variety of insect pests. However, to be applied on a commercial level, several issues related to the RNAi mechanism and biosafety still need to be addressed. As a new technology, risk assessments and govern‐ ment regulations still have to be developed. However, RNAi transgenic crops are expected to have wider acceptance and reduced biosafety requirements for RNAi traits, in comparison to a protein incorporated into a plant, such as a Bt transgenic [39]. Thus, RNAi-mediated pest control will open a new paradigm in insect pest management.

#### **Author details**

Thais Barros Rodrigues\* and Antonio Figueira

\*Address all correspondence to: thaisbarros.bio@gmail.com

Centro de Energia Nuclear na Agricultura (CENA), Universidade de São Paulo, Piracicaba, SP, Brazil

#### **References**


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Due to the variety of RNAi response to RNAi in insects, no single protocol is suitable for all species. Issues related to the choice of effective target genes, including determining the size of optimal dsRNA length and ideal gene region. Assuming that the method of choice to deliver dsRNA is transgenic plants, a major question still to be addressed is the impact of plant dsRNA processing in the effective RNAi-induced silencing. There is still a need for investigation in this area. The choice of a suitable inducible promoter for expressing the dsRNA construct is

Based on the recent publications reviewed in this chapter, the progress in developing "RNAiplants" to control important insect pests widely demonstrated the potential of this technology to complement or replace Bt crops, providing resistance against a broad variety of insect pests. However, to be applied on a commercial level, several issues related to the RNAi mechanism and biosafety still need to be addressed. As a new technology, risk assessments and govern‐ ment regulations still have to be developed. However, RNAi transgenic crops are expected to have wider acceptance and reduced biosafety requirements for RNAi traits, in comparison to a protein incorporated into a plant, such as a Bt transgenic [39]. Thus, RNAi-mediated pest

Centro de Energia Nuclear na Agricultura (CENA), Universidade de São Paulo, Piracicaba,

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## **RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC)**

Eduardo C. de Andrade and Wayne B. Hunter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61612

#### **Abstract**

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390 RNA Interference

nal.pone.0025709

RNAi technologies are more environmentally friendly, as the technology provides great‐ er specificity in pest targeting, while reducing the potential negative effects on ecosys‐ tems and leaving beneficial insects and other organisms unharmed in crop ecosystems. Consequently, the increase in native fauna improves the efficacy of biological control agents against pests and pathogens. A growing understanding of the ubiquitous nature of RNAi, along with evidence for efficient, non-transgenic, topical applications has al‐ ready begun to garner support among organic and industry producers. Designing solu‐ tions to agricultural problems based upon the same mechanisms used in nature provides newer, safer solutions to pests and pathogens for all agricultural industries.

**Keywords:** Future, Crops, Organic, Non-transgenic, RNAi

#### **1. Introduction**

A phenomenon initially reported in plants [1] called the attention of the scientific community, leading to the discovery of a sophisticated mechanism of gene regulation and protection against invasive nucleic acids [2–4]. Furthermore, the mechanism described in plants, referred to as post-transcriptional gene silencing (PTGS), or virus-induced gene silencing (VIGS), had been described in the 1990s [5] and was often referred to as pathogen-derived resistance [6].

RNAi is a natural process of gene regulation and antiviral defense system of eukaryotic cells. RNAi is a mechanism that functions as a "gene silencer" by targeting specific RNA sequences. RNAi results in degradation, and in some situations, translation inhibition, resulting in a reduction or complete elimination of the expression of a targeted RNA [7]. RNAi is also linked to suppressing gene expression at transcriptional level by directing epigenetic alterations on chromatin [8].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The basic RNAi process consists of the trigger molecule, a long endogenous or exogenous double-strand RNA (dsRNA) molecule that is expressed in, or introduced into, the cell, which is processed by Dicer, a ribonuclease III (RNase III) enzyme into small RNA duplexes of 21– 23 nucleotides in length. These duplexes are separated with one strand (the guide strand), producing a protein complex known as the RNA-induced silencing complex (RISC). The RISC complex uses the specific sequence of the guide strand to determine potential target messenger RNAs (mRNA). Once bound to the mRNA, the guide strand directs a RISC-bound endonu‐ clease [called "slicer", an Argonaute (AGO) protein] to cleave the mRNA which has homology to the guide strand [9]. Thus, the RISC complex can target the messenger RNA (mRNA), an invasive virus RNA, or a transposable element transcript (Please note that RNAi cannot eliminate transposons itself, but its transcripts.) [10]. These components appear to be cosmo‐ politan in their distribution across the RNAi spectrum of the eukaryotic phyla. This implies that a common ancestor had a functional RNAi pathway [11], estimated to have occurred over a billion years or more ago [12].

The generation of virus-resistant transgenic plants, by expressing fragments of viral genomes, was the first demonstration of the beneficial use of the RNAi [13,14]. However, with the demonstration that ingestion of dsRNAs can robustly silence genes in *Caenorhabditis elegans*[7], RNAi became not only an important tool in genetic studies to identify gene function but also opened a new field of application in plant protection against insects, arthropods, and patho‐ gens.

In this chapter, we outlined some aspects on the development of RNAi-based strategies to control insects, presenting some considerations and research steps that are important to be addressed.

#### **2. RNAi-based products for agricultural management of insect pest**

Advances in genomics and initiatives to sequence genomes of agriculturally important organisms created big breakthroughs within the entomology fields of study, including taxonomy, insect physiology, toxicology, immunology, pest management, and microbe–host interactions. This new paradigm affected how research could be conducted, to make discov‐ eries and increase the speed by which these could be accomplished. With these breakthroughs, entomologists, pathologists, and biologists are rapidly advancing toward better, safer, and more specific pest and pathogen management. Development of gene-based methods was dependent upon having the knowledge to understand how the cells in a living organism respond to the threats from viral pathogens, how they regulate their own gene expression, and how they maintain these natural complex systems throughout their lives.

The initial genomes which were sequenced and annotated to elucidate these very complicated interactions from: the nematode – *C. elegans*, the fruit fly – *Drosophila melanogaster*, the red flour beetle – *Tribolium castaneum*, the silkworm – *Bombyx mori*, the pea aphid – *Acyrthosiphon pisum*, the honey bee – *Apis mellifera*, to bumble bee – *Bombus terrestris* and including a few more examples from the world of agriculture [15]. Current genome initiatives, like the i5K Arthropod initiative [15] and the Beijing Institute of Genomics, BGI, China, plan to sequence thousands of arthropod and plant genomes. The enormous amount of new information produced will also increase the understanding of the genetic basis of the mechanisms nature uses to solve problems at the cellular and organismal levels.

The basic RNAi process consists of the trigger molecule, a long endogenous or exogenous double-strand RNA (dsRNA) molecule that is expressed in, or introduced into, the cell, which is processed by Dicer, a ribonuclease III (RNase III) enzyme into small RNA duplexes of 21– 23 nucleotides in length. These duplexes are separated with one strand (the guide strand), producing a protein complex known as the RNA-induced silencing complex (RISC). The RISC complex uses the specific sequence of the guide strand to determine potential target messenger RNAs (mRNA). Once bound to the mRNA, the guide strand directs a RISC-bound endonu‐ clease [called "slicer", an Argonaute (AGO) protein] to cleave the mRNA which has homology to the guide strand [9]. Thus, the RISC complex can target the messenger RNA (mRNA), an invasive virus RNA, or a transposable element transcript (Please note that RNAi cannot eliminate transposons itself, but its transcripts.) [10]. These components appear to be cosmo‐ politan in their distribution across the RNAi spectrum of the eukaryotic phyla. This implies that a common ancestor had a functional RNAi pathway [11], estimated to have occurred over

The generation of virus-resistant transgenic plants, by expressing fragments of viral genomes, was the first demonstration of the beneficial use of the RNAi [13,14]. However, with the demonstration that ingestion of dsRNAs can robustly silence genes in *Caenorhabditis elegans*[7], RNAi became not only an important tool in genetic studies to identify gene function but also opened a new field of application in plant protection against insects, arthropods, and patho‐

In this chapter, we outlined some aspects on the development of RNAi-based strategies to control insects, presenting some considerations and research steps that are important to be

Advances in genomics and initiatives to sequence genomes of agriculturally important organisms created big breakthroughs within the entomology fields of study, including taxonomy, insect physiology, toxicology, immunology, pest management, and microbe–host interactions. This new paradigm affected how research could be conducted, to make discov‐ eries and increase the speed by which these could be accomplished. With these breakthroughs, entomologists, pathologists, and biologists are rapidly advancing toward better, safer, and more specific pest and pathogen management. Development of gene-based methods was dependent upon having the knowledge to understand how the cells in a living organism respond to the threats from viral pathogens, how they regulate their own gene expression, and

The initial genomes which were sequenced and annotated to elucidate these very complicated interactions from: the nematode – *C. elegans*, the fruit fly – *Drosophila melanogaster*, the red flour beetle – *Tribolium castaneum*, the silkworm – *Bombyx mori*, the pea aphid – *Acyrthosiphon pisum*, the honey bee – *Apis mellifera*, to bumble bee – *Bombus terrestris* and including a few more examples from the world of agriculture [15]. Current genome initiatives, like the i5K

**2. RNAi-based products for agricultural management of insect pest**

how they maintain these natural complex systems throughout their lives.

a billion years or more ago [12].

gens.

392 RNA Interference

addressed.

The number of arthropod species which have had successful RNAi reports continues to increase, and this trend will carry agriculture into the future, covering five Classes, in four Subphyla of the Arthropoda phylum, which includes eight insect orders and over 30 insect species [16–17].

## **3. Pitfalls and solutions – Relevant considerations for development of RNAi in insects**

Though the use of RNAi strategies to control a desired insect pest seems to be very straight‐ forward; however, some issues should be taken into considerations: (1) for oral treatments the dsRNA must survive ingestion, then be absorbed by the epithelial cells, and depending upon the target translocated through the hemolymph to reach other tissues. In insects, the dsRNA is mainly introduced through feeding, but dermal application has already been reported to possibly bypass gut issues [18–21]; (2) once inside the body, the dsRNA must enter into the cell to activate the RNAi mechanism. In insects, cell uptake of dsRNA varies widely between species, because there are different mechanisms of systemic absorption and translocation of dsRNAs within and between cells, respectively, leading to differences in response and influencing the efficiency in silencing the target gene [17,22]; (3) once the RNAi mechanisms are triggered in a group of cells, it has been demonstrated that a systemic spread of silencing may also occur, so that other tissues / cells are also affected, which may increase the RNA effect. Successful studies have shown that dsRNA can circulate in the hemolymph, and cause a suppression of genes in tissues distant from initial entry sites in the insect gut, affecting cuticle formation, the nervous system, or ovaries[23–26]. However, in insects results can be highly variable and research efforts continue to elucidate the effects of systemic signaling.

In theory, any cellular mRNA can be inactivated in a precise and controlled manner. With this in mind, the use of the RNAi mechanism to manage an insect pest relies on the capacity to design the dsRNA. The sequence of the dsRNA provides specificity and the researcher must determine the active concentration needed to obtain the desired RNAi outcome; thus, proper design and evaluation of the dsRNA becomes critical.

Identification of vitally important (i.e., with high mortality) target genes of a particular insect is a crucial step toward development of RNAi-based control strategy. Thanks to world science development and increasing efforts of the research community, the identification of an essential target can be achieved by an extended literature search and analyses of available DNA/RNA sequence databases [27]. Once identified, "candidate" target sequences are used to design potent "RNAi causing structures". Then, the dsRNAs must be experimentally validated for functionality, specificity, and stability, toward the specific RNAi target of interest. Furthermore, the development of an efficient delivery system for "RNAi causing structures" is another key step. There are several methods available that include, but are not limited to: microinjection [28], soaking (for mosquito larvae, and nematodes) [29,30], and feeding (chewing and piercing-sucking insects) [17,17,27,31,32].

One approach to identify potential target genes which will function under field conditions is to perform bioassays that closely mimic the conditions in the field, for example, using a bioassay that mimics the feeding of a hemipteran insect acquiring the dsRNA during the natural feeding process performed on the crop plant. One problem with delivering dsRNA through feeding is that, depending on the bioassay, it may be difficult to measure the dose of dsRNA ingested, from the dose absorbed by the gut cells and the target cells [33].

Oral delivery of dsRNA through feeding can be performed by using artificial diet, detached plant parts (leaves, buds, roots), or intact plants [17,24,34,35]. Delivering dsRNA through the diet provides an easy procedure to screen large numbers of dsRNAs in insect larvae and adults [23,24,34,37]. In addition, it allows addressing different issues, such as effective length of dsRNA, determine regions of the gene to be target which may provide better suppression, and to determine the effective lethal concentration (LC50) [23,38].

Although oral feeding provides a more natural screening system, it is important to take into consideration that for some insect species from across all taxonomic orders, they may not provide an effective RNAi response when conducting oral feeding bioassays regardless of the dosage of dsRNA, as the dsRNA may not enter, or be detected in the insect's body [22]. In contrast, in these same insects when dsRNA was injected directly into the insect's body, a potent RNAi response was observed [23,39,40]. Indeed, lack of positive results using feeding bioassays does not necessarily indicate that the insect is insensitive to RNAi, but in a majority of the situations, insects have nucleases in the saliva, in the midgut, or even in the hemolymph that degrades dsRNA before it can be absorbed by the cells [40–42].

Wynant et al. [40] discussed the interactions of enzymes and RNAi-causing dsRNAs in the alimentary tract and hemolymph of insects and other arthropods using oral delivery. The elucidation of the roles of microbes and host enzymes on RNAi efficacy across arthropod species continues to be a challenging field of research [43].

Where information about absorption of dsRNA or presence of nucleases is not available for a particular insect species, the use of reporter dsRNAs molecules are useful to clarify possible issues. It is essential that the dsRNAs sequence should not match with any insect's transcript sequence. The dsRNA "movement" can be monitored (detected), or quantified in the insect's body by RT-qPCR, showing that the insect has acquired the potent, fully functional, systemi‐ cally spreading dsRNA during feeding (plant, diet, drop of water, etc.) (Figure 1).

When conducting a RT-PCR detection of reporter dsRNA after insect feeding, it is important to sample a tissue other than the gut such as the hemolymph, fat body, or ovary. Careful collection of tissues which are not in direct contact with the gut provides evidence that the dsRNA was truly absorbed by the cells, and you are not just detecting dsRNA just in the digestive tract.

With small insects, as the Asian Citrus Psyllid (ACP) *Diaphorina citri* (whitefly or aphids), collection of material can be difficult without bringing in gut tissues, so another option is to

is another key step. There are several methods available that include, but are not limited to: microinjection [28], soaking (for mosquito larvae, and nematodes) [29,30], and feeding

One approach to identify potential target genes which will function under field conditions is to perform bioassays that closely mimic the conditions in the field, for example, using a bioassay that mimics the feeding of a hemipteran insect acquiring the dsRNA during the natural feeding process performed on the crop plant. One problem with delivering dsRNA through feeding is that, depending on the bioassay, it may be difficult to measure the dose of

Oral delivery of dsRNA through feeding can be performed by using artificial diet, detached plant parts (leaves, buds, roots), or intact plants [17,24,34,35]. Delivering dsRNA through the diet provides an easy procedure to screen large numbers of dsRNAs in insect larvae and adults [23,24,34,37]. In addition, it allows addressing different issues, such as effective length of dsRNA, determine regions of the gene to be target which may provide better suppression, and

Although oral feeding provides a more natural screening system, it is important to take into consideration that for some insect species from across all taxonomic orders, they may not provide an effective RNAi response when conducting oral feeding bioassays regardless of the dosage of dsRNA, as the dsRNA may not enter, or be detected in the insect's body [22]. In contrast, in these same insects when dsRNA was injected directly into the insect's body, a potent RNAi response was observed [23,39,40]. Indeed, lack of positive results using feeding bioassays does not necessarily indicate that the insect is insensitive to RNAi, but in a majority of the situations, insects have nucleases in the saliva, in the midgut, or even in the hemolymph

Wynant et al. [40] discussed the interactions of enzymes and RNAi-causing dsRNAs in the alimentary tract and hemolymph of insects and other arthropods using oral delivery. The elucidation of the roles of microbes and host enzymes on RNAi efficacy across arthropod

Where information about absorption of dsRNA or presence of nucleases is not available for a particular insect species, the use of reporter dsRNAs molecules are useful to clarify possible issues. It is essential that the dsRNAs sequence should not match with any insect's transcript sequence. The dsRNA "movement" can be monitored (detected), or quantified in the insect's body by RT-qPCR, showing that the insect has acquired the potent, fully functional, systemi‐

When conducting a RT-PCR detection of reporter dsRNA after insect feeding, it is important to sample a tissue other than the gut such as the hemolymph, fat body, or ovary. Careful collection of tissues which are not in direct contact with the gut provides evidence that the dsRNA was truly absorbed by the cells, and you are not just detecting dsRNA just in the

With small insects, as the Asian Citrus Psyllid (ACP) *Diaphorina citri* (whitefly or aphids), collection of material can be difficult without bringing in gut tissues, so another option is to

cally spreading dsRNA during feeding (plant, diet, drop of water, etc.) (Figure 1).

dsRNA ingested, from the dose absorbed by the gut cells and the target cells [33].

(chewing and piercing-sucking insects) [17,17,27,31,32].

394 RNA Interference

to determine the effective lethal concentration (LC50) [23,38].

that degrades dsRNA before it can be absorbed by the cells [40–42].

species continues to be a challenging field of research [43].

digestive tract.

**Figure 1.** Topically applied RNA, provides long dsRNAs for insect ingestion. If absorbed into the plant, then long dsRNA persists in plant vascular tissues, xylem, and phloem, for several weeks due to lower metabolic activity, fewer enzymes, and microbes. Once the dsRNA enters a plant or insect cell, the RNAi mechanism is triggered, and the dsRNA processed [Image: Hunter, W.B., USDA-ARS, 2015].

let the insects feed on the source of the dsRNA (plant, diet, etc.) for a period of 24–48 h, then transfer them to an untreated food source. After feeding for 36 h, or more, the insect should excrete any food residue from the treated food source, which contains dsRNA. After this period, proceed with sample collection for dsRNA detection.

The reporter dsRNA is designed so that the sequence does not match with any known mRNA transcript in your insect. This is to avoid off target of other transcripts in the insect. Some commonly used dsRNAs which are used as negative controls in RNAi experiments are: green fluorescent protein (GFP), β-glucuronidase (GUS), and enhanced yellow fluorescent protein (EYFP) [44].

When designing RNAi experiments, important questions arise regarding the design of dsRNA, including: the length of the molecule and the region targeted within the mRNA. The minimal required length to achieve an RNAi effect will vary depending on insect species [45]. For example, in *Tribolium castaneum*, analysis showed that the dsRNA length had a strong influence on the efficacy of the RNAi response, with longer dsRNA proven to be more effective on curtailing gene expression [46]. In *T. castaneum* dsRNA, it was observed that length was crucial for cellular uptake; a minimum of 70 nucleotides were necessary to achieve the desired interference. However, other studies in the potato/tomato psyllid, *Bactericera cockerelli* [47], the pea aphid *Acyrthosiphon pisum* [48], and the lepidopteran *Manduca sexta* [49] have reported gene suppression using shorter dsRNAs, between 21 and 27 nucleotides in length. These molecules are called small interfering RNAs (siRNA). Overall, the majority of studies dem‐ onstrate success with dsRNA ranging from 140 to 520 nucleotides in length. Interestingly, Huvenne and Smagghe [31] reported success using a dsRNA 1,842 nucleotides in length. dsRNAs longer than 200 nucleotides provide the advantage of resulting in many siRNAs, postcleavage against the targeted mRNA, thus maximizing the RNAi response and preventing the development of individuals with "resistance" due to the natural genetic variation.

There is no consensus on the mRNA region that the dsRNA should match to (e.g., 5′ or 3′). For example, in the pea aphid, *A. pisum*, no difference in mortality was observed in groups of insects that received dsRNA matching the 5′ or 3′ end of the *hunchback* (hb) gene [27]. In the mosquito, *Aedes aegypti* greater RNAi effects were achieved when insect larvae ingested dsRNA targeting the 3′ end versus 5′ end of an apoptosis gene, *AaeIAP1* [18]. These different results highlight the need to screen several dsRNA molecules across the entire mRNA [50].

In the context of field applications of RNAi for insect management, dsRNAs can be designed to be highly specific to both the target gene and the insect species. If desired, the RNAs can be designed to have a broader spectrum to affect several pest species. For example, RNAi strategies can be designed to remove one aphid species from a cropping system, or be designed to remove multiple aphid species from that same ecosystem [24,38].

#### **4. Bioassays for dsRNA screening**

For RNAi research attempting the development of a viable pest management product. It is of utmost importance to identify the best delivery mechanisms (i.e., topical sprays, baits, or transgenic plants) as early as possible; this will expedite the entire process and can cut years off of the development and commercialization timeline.

The example outlined below highlights RNAi bioassays directed toward two citrus insect pests, each one with different feeding behaviors: piercing-sucking plant-feeding (the Asian citrus psyllid *Diaphorina citri*, Hemiptera) and a chewing beetle pest (the weevil *Diaprepes abbreviatus,* Coleoptera). In both situations, the bioassays were designed to evaluate the efficacy of oral ingestion of dsRNAs under "natural feeding conditions" which mimic conditions the insects will encounter in the field.

#### **4.1. Bioassays for piercing-sucking insects**

The artificial feeding bioassay is being widely used for studies on insect nutrition, pathogen acquisition, toxicity, and RNAi (Figure 2A) [51–53].

It is notable that liquid feeding bioassays (dsRNAs mixed in a liquid diet or a sucrose solution) frequently result in high mortality levels in the controls, and increased degradation of dsRNA in the solution due to bacterial or fungal contaminations [42,57]. In addition, these bioassays require significantly high dsRNA concentrations to achieve insect mortality. Concentrations up to 1μg/μL [58–60] cannot be reproduced inside plant vascular tissues.

Hemipteran pests in citrus (psyllids, leafhoppers, aphids, whiteflies) have piercing-sucking mouthparts that are inserted into the plant vascular system to feed. The development of an

onstrate success with dsRNA ranging from 140 to 520 nucleotides in length. Interestingly, Huvenne and Smagghe [31] reported success using a dsRNA 1,842 nucleotides in length. dsRNAs longer than 200 nucleotides provide the advantage of resulting in many siRNAs, postcleavage against the targeted mRNA, thus maximizing the RNAi response and preventing the

There is no consensus on the mRNA region that the dsRNA should match to (e.g., 5′ or 3′). For example, in the pea aphid, *A. pisum*, no difference in mortality was observed in groups of insects that received dsRNA matching the 5′ or 3′ end of the *hunchback* (hb) gene [27]. In the mosquito, *Aedes aegypti* greater RNAi effects were achieved when insect larvae ingested dsRNA targeting the 3′ end versus 5′ end of an apoptosis gene, *AaeIAP1* [18]. These different results highlight the need to screen several dsRNA molecules across the entire mRNA [50]. In the context of field applications of RNAi for insect management, dsRNAs can be designed to be highly specific to both the target gene and the insect species. If desired, the RNAs can be designed to have a broader spectrum to affect several pest species. For example, RNAi strategies can be designed to remove one aphid species from a cropping system, or be designed

For RNAi research attempting the development of a viable pest management product. It is of utmost importance to identify the best delivery mechanisms (i.e., topical sprays, baits, or transgenic plants) as early as possible; this will expedite the entire process and can cut years

The example outlined below highlights RNAi bioassays directed toward two citrus insect pests, each one with different feeding behaviors: piercing-sucking plant-feeding (the Asian citrus psyllid *Diaphorina citri*, Hemiptera) and a chewing beetle pest (the weevil *Diaprepes abbreviatus,* Coleoptera). In both situations, the bioassays were designed to evaluate the efficacy of oral ingestion of dsRNAs under "natural feeding conditions" which mimic conditions the

The artificial feeding bioassay is being widely used for studies on insect nutrition, pathogen

It is notable that liquid feeding bioassays (dsRNAs mixed in a liquid diet or a sucrose solution) frequently result in high mortality levels in the controls, and increased degradation of dsRNA in the solution due to bacterial or fungal contaminations [42,57]. In addition, these bioassays require significantly high dsRNA concentrations to achieve insect mortality. Concentrations

Hemipteran pests in citrus (psyllids, leafhoppers, aphids, whiteflies) have piercing-sucking mouthparts that are inserted into the plant vascular system to feed. The development of an

up to 1μg/μL [58–60] cannot be reproduced inside plant vascular tissues.

development of individuals with "resistance" due to the natural genetic variation.

to remove multiple aphid species from that same ecosystem [24,38].

**4. Bioassays for dsRNA screening**

396 RNA Interference

insects will encounter in the field.

**4.1. Bioassays for piercing-sucking insects**

acquisition, toxicity, and RNAi (Figure 2A) [51–53].

off of the development and commercialization timeline.

**Figure 2.** RNAi feeding assays used at the USDA lab, ARS, Ft. Pierce, FL (2007-2014), to successfully screen dsRNA constructs rapidly and under controlled conditions: (A) sucrose solutions within artificial membranes (5 ng dsRNA/μL) [54], (B) rooted okra plant cuttings, (C) citrus flush (5 ng to 100 ng dsRNA/ 0.25 g plant tissue). Plant cut‐ tings absorb dsRNA directly providing systemic movement of dsRNA, (D) insects are given a feeding access period to ingest the dsRNA across 10 days to observe mortality (Hunter and Andrade, USDA-ARS 2015) [54–56].

RNAi control strategy against these insects relies on effective delivery of the dsRNA through the vascular tissues.

Demonstration of the first dsRNA delivery into full-sized citrus trees and grapevines, without a delivery vector, expression vector, or transformation event was performed in 2008 [56]. These results showed that two hemipteran insects, the xylem-feeding leafhopper (*H. vitripennis*), and the phloem-feeding Asian citrus psyllid (*D. citri*), tested positive for ingested dsRNA after feeding from host plants treated with dsRNA as either a foliar spray or root drench. These results along with other studies that demonstrated successful RNAi through oral ingestion in insects [24,34,36,61,62] support the potential for exogenously delivered RNAi control strat‐ egies.

Use of cut plant feeding bioassays for hemipteran pests enables the screening of a large number of dsRNAs molecules at a reduced cost of materials and time. The bioassay, can use leaf disks, whole leaf, new growth leaves and stem, or rooted cuttings, to absorb and deliver dsRNAs. In citrus, the "flush", which are new growth foliar shoots, are collected from potted citrus seedlings grown in a glasshouse (USDA-ARS, Fort Pierce, FL). The leaves and stem material are about 7–8 cm long. The plant material is washed in 0.2% bleach water, for 10 min. Then the base of each stem is cut at a 45 degree angle while submerged in filtered water. The material is then placed into a 1.5 mL tube containing 0.5 mL water (Figure 2B and C). The dsRNA solution, 300 μL, is added to the water, the tube top is wrapped with plastic or Parafilm™ and placed under artificial lighting to stimulate absorption of dsRNA solution. The next day the tube is filled with water using a 26 gauge syringe needle and syringed filtered (0.45 μ). The treated cuttings are then placed into a cage and adult insects provided feeding access for 10 days (Figure 2D). The plant material can remain viable for up to 40 days on average. While most bioassays may terminate after eight to 10 days of observations for mortality, having a longer feeding access time enables observations on insect oviposition, egg viability, or nymph development.

Each dsRNA molecules has an optimal concentration. So each dsRNA molecule is evaluated across a range of total concentrations (i.e., 5, 20, 50, 100 nanograms/ tissue). The bioassay permits screening for synergistic effects of multiple dsRNAs and to screen a single dsRNA against multiple insect species. For example, the assay using citrus flush permits screening of dsRNAs designed against psyllids for off target effects in the citrus aphid (*Toxoptera citrici‐ dus*) and glassy-winged sharpshooter leafhopper (*Homalodisca vitripennis*(Germar), two closely related hemipterans, which also use citrus trees as a host plant.

#### **4.2. Bioassays for chewing insects**

For insects which are foliage feeders, the delivery of dsRNA can be achieved as a foliar topical spray. In this scenario, the dsRNAs are evaluated similarly as topical insecticides. The dsRNA solution is sprayed on leaves, and then fed to the insects. An example of the effectiveness of this approach was reported by Bolognesi [45] working with the coleopteran *Diabrotica virgifera*, in which dsRNA administered through feeding, silenced genes in tissues far from the gut epithelium.

Similar results were obtained while developing an RNAi strategy against the Diaprepes root weevil (DRW), *Diaprepes abbreviatus* L., (Curculionidae: Coleoptera) (Andrade and Hunter). The adults feed and oviposition on mature citrus leaves. Topically applied dsRNA was sprayed on a bouquet of citrus leaves for delivery to DRW adults (Figure 3A). RNA's have been shown to move through the plant xylem and phloem [6].

**Figure 3.** Citrus "*leaf bouquet"* feeding bioassay for *Diaprepes* root weevil. **(A)** Fresh stems with leaves are washed in 0.2% bleach water, rinsed with Nanopure™ filtered water, then the stems are freshly cut while submerged in water. The leaf bouquets are placed in water-filled containers and placed under artificial lighting for 24 h prior to use. **(B)** Topical foliar treatment: the dsRNA is mixed with water and applied using a low-volume aerosol sprayer. **(C)** After the leaves dry, the "bouquets" are caged with adult insects.

A test spray using only water established the volume needed to provide full coverage of leaf bouquets without excessive run off (Figure 3B). After the leaves have dried, they are caged with adult insects (Figure 3C). Freshly treated citrus leaf bouquets replaced previous bouquets every five to seven days for a 5-week period. The total amount of dsRNA to be sprayed over the leaf bouquet was determined by evaluating a range of concentrations in a pretest experi‐ ment for efficacy. The effects from RNAi in insects usually start to appear within 4 to 5 days post-ingestion, which suggests there may be a dose response [63]. Since foliage feeding insects tend to eat a lot of leaf material each day, a low-dose spray may be able to deliver a significant amount of dsRNA.

#### **5. Final considerations on RNAi applied to agriculture**

longer feeding access time enables observations on insect oviposition, egg viability, or nymph

Each dsRNA molecules has an optimal concentration. So each dsRNA molecule is evaluated across a range of total concentrations (i.e., 5, 20, 50, 100 nanograms/ tissue). The bioassay permits screening for synergistic effects of multiple dsRNAs and to screen a single dsRNA against multiple insect species. For example, the assay using citrus flush permits screening of dsRNAs designed against psyllids for off target effects in the citrus aphid (*Toxoptera citrici‐ dus*) and glassy-winged sharpshooter leafhopper (*Homalodisca vitripennis*(Germar), two closely

For insects which are foliage feeders, the delivery of dsRNA can be achieved as a foliar topical spray. In this scenario, the dsRNAs are evaluated similarly as topical insecticides. The dsRNA solution is sprayed on leaves, and then fed to the insects. An example of the effectiveness of this approach was reported by Bolognesi [45] working with the coleopteran *Diabrotica virgifera*, in which dsRNA administered through feeding, silenced genes in tissues far from the

Similar results were obtained while developing an RNAi strategy against the Diaprepes root weevil (DRW), *Diaprepes abbreviatus* L., (Curculionidae: Coleoptera) (Andrade and Hunter). The adults feed and oviposition on mature citrus leaves. Topically applied dsRNA was sprayed on a bouquet of citrus leaves for delivery to DRW adults (Figure 3A). RNA's have been shown

**Figure 3.** Citrus "*leaf bouquet"* feeding bioassay for *Diaprepes* root weevil. **(A)** Fresh stems with leaves are washed in 0.2% bleach water, rinsed with Nanopure™ filtered water, then the stems are freshly cut while submerged in water. The leaf bouquets are placed in water-filled containers and placed under artificial lighting for 24 h prior to use. **(B)** Topical foliar treatment: the dsRNA is mixed with water and applied using a low-volume aerosol sprayer. **(C)** After

related hemipterans, which also use citrus trees as a host plant.

**4.2. Bioassays for chewing insects**

to move through the plant xylem and phloem [6].

the leaves dry, the "bouquets" are caged with adult insects.

development.

398 RNA Interference

gut epithelium.

Efficient delivery and increased stability of dsRNA need to be developed if non-transgenic, topically delivered, RNAi strategies are to be established. Increased stability and superior delivery into some insects can be achieved using nanoparticle-mediated RNAi [63–65], traditional crop improvement strategies, in which plants express hairpin dsRNAs, will continue to be a mainstay of agricultural approaches [63,65,66].

Transgenic plants have successfully used RNAi strategies to produce crops with improved virus resistance, increased nutrition and fiber content [67]; biotechnology companies are trying to move towards a faster, more natural process of topically applied RNAi. dsRNA molecules are part of naturally occurring processes in all living organisms. They exist in our foods, and our bodies [66]. The short persistence time of dsRNA in the environment is demonstrated by the fact that analyses of soils and plant debris, treated with dsRNA have consistently shown rapid breakdown of dsRNAs within 2–3 days [68], also means less concerns about unintended contamination of water supplies, soils, or adverse air quality effects. Furthermore, since all living things have evolved to break down dsRNA and use the nucleic acids as cellular nutrients, this technology will be safer than conventional chemistries for those who apply RNAi products, or eat the produce [66–69].

RNAi technologies have greater specificity in pest targeting, which reduces negative impacts on crop ecosystems by leaving more insects and other organisms unharmed in the field. The increased fauna consequently improves the efficacy of pollination, and biological control agents that help suppress a broad range of pests. The increased understanding of the ubiqui‐ tous nature of RNAi, along with evidence of efficient topical application, has already begun to garner support for this technology among members of the organic grower's communities, which desperately need a truly natural, innovative breakthrough, to manage many of the pests and pathogens which plague the organic industries.

#### **5.1. Cost-effective methods for the mass production and formulation of dsRNA**

Cost-efficient methods for mass production of vast amounts of dsRNA are being developed, and include bacterial, plant, and synthetic production [65]. While small amounts of dsRNA can be easily produced in the laboratory for research purposes, commercially available kits are not a viable, cost-effective method for the production of large quantities of dsRNA [65]. The costs associated with the commercialization and implementation of RNAi products are decreasing rapidly. The costs of dsRNA production have dropped from \$500,000 USD for 40 g in 2008 to less than \$4,000 USD for 40 g today. *For example, see* [70]. As interests in commer‐ cialization of RNAi-based products increase, better production systems will be developed to meet the predicted demands of these growing markets [17,65,71]. One of the most cost-effective methods for production is in bacteria, since for most countries bacteria-produced dsRNA would provide an affordable production system which could advance RNAi as, "*The* novel biological insecticide of the future!" Most agricultural companies interested in the future of RNAi are working on developing their own technologies that will further reduce production costs predicted to be near \$4 USD per one gram by the end of 2015. *For example, see* [72].

#### **5.2. Other applications**

Future applications of RNAi and other gene-based targeting biotechnologies will add value to existing beneficial insects (pollinators, predators, parasitoids). A real-world example is a study conducted over several years in which an RNAi product designed to reduce Israeli acute paralysis virus replication was fed to honey bees. The treated bees had significantly greater survival and produced significantly more honey [73]. RNAi strategies have also reduced honey bee parasites, like *Varroa* mites [74,75], and internal microsporidian parasites [76] without deleterious effects to the bees. The highly specific nature of RNAi approaches can be exploited to reduce pests with no harmful effects on non-target species. The use of RNAi in combination with beneficial pollinators and natural enemies has the potential to raise the level of all pest management efforts [17].

Biotechnology has demonstrated the safe production of plants which are more nutritious, less toxic, more resistant to drought, and more efficient for biofuel production [66, 67]. RNAi has already been successfully used to produce crops which are virus- and drought-resistant [66]. However, plants expressing dsRNAs while stable and safe take years to develop and millions of dollars to commercialize [65]. Development of topically applied RNAi, which is a nontransgenic approach improves crop traits and provides a major step forward for environmen‐ tally sound crop management [66, 67].

#### **6. Conclusions**

As more insects and mites develop chemical resistance to one or more insecticides, now estimated to be over 500 species with resistance to one or more products [77], it is imperative that new types of pest control are developed. The public would like the world to be filled with environmentally friendly technologies, safe for human and animal consumption, technologies which are safe for use around animals and beneficial insects, safe for each type of ecosystem, forest, field, crop, or backyard, a technology that will not endanger water or food quality, a more natural solution, with a natural approach toward problem-solving. So enters RNA interference!

RNA interference, or gene silencing, is a way to reduce specific mRNAs so that a particu‐ lar protein is either not made or it is reduced. The RNAi mechanism is a natural one which occurs in the cells of humans, animals, insects, and plants, and appears to have evolved as a primary defense system against virus replication [78]. Andrew Fire and Craig Mello, won the Nobel Prize in 2006 [79] for explaining how the RNAi mechanism is triggered, when a cell encounters double-stranded RNA, and how this could be used to benefit humanity. Humanity's greatest discoveries have come from observing the natural world; while RNAi will not solve every problem, it certainly can help improve plant health, reduce insect pests and pathogens [17, 66, 67, 80]. Some of the benefits from developing RNAi as topically applied products are: 1) The rapid degradation of the molecules ensures low environmen‐ tal risks. All cells have the capacity to degrade dsRNA, and the salvage pathways to recycle these bases and nucleosides to form new nucleotides. Thus, cells constantly breaking down DNA and RNA into recycled nucleotides [81]. 2) Topical RNAi applications do not insert genes, so do not produce proteins. RNAi reduces the expression of the targeted proteins, a modulation effect of the natural system. 3) RNAi can be designed and tested faster (in about 2–3 years) than producing transgenic crops, which can take 10 to 20 years and cost hundreds of millions of dollars [65]. 4) Finally, RNAi strategies as topical sprays would, for the first time, be able to remove one or two closely related insect species, while leaving all the other insects unharmed [38]. The ability to design RNAi as highly specific pest control will finally provide relief to biological control agents and beneficial insects [17, 44, 73], significantly improving integrated pest management programs.

The advantages and promises from RNAi technology sound amazing. However, serious efforts in outreach and education are needed to better inform the different stake holders including the general public, and agricultural industry, leaders as well as decision makers in the regulatory and political communities to help expedite the release and adoption of RNAi products and technology.

#### **7. Disclaimer**

not a viable, cost-effective method for the production of large quantities of dsRNA [65]. The costs associated with the commercialization and implementation of RNAi products are decreasing rapidly. The costs of dsRNA production have dropped from \$500,000 USD for 40 g in 2008 to less than \$4,000 USD for 40 g today. *For example, see* [70]. As interests in commer‐ cialization of RNAi-based products increase, better production systems will be developed to meet the predicted demands of these growing markets [17,65,71]. One of the most cost-effective methods for production is in bacteria, since for most countries bacteria-produced dsRNA would provide an affordable production system which could advance RNAi as, "*The* novel biological insecticide of the future!" Most agricultural companies interested in the future of RNAi are working on developing their own technologies that will further reduce production costs predicted to be near \$4 USD per one gram by the end of 2015. *For example, see* [72].

Future applications of RNAi and other gene-based targeting biotechnologies will add value to existing beneficial insects (pollinators, predators, parasitoids). A real-world example is a study conducted over several years in which an RNAi product designed to reduce Israeli acute paralysis virus replication was fed to honey bees. The treated bees had significantly greater survival and produced significantly more honey [73]. RNAi strategies have also reduced honey bee parasites, like *Varroa* mites [74,75], and internal microsporidian parasites [76] without deleterious effects to the bees. The highly specific nature of RNAi approaches can be exploited to reduce pests with no harmful effects on non-target species. The use of RNAi in combination with beneficial pollinators and natural enemies has the potential to raise the level of all pest

Biotechnology has demonstrated the safe production of plants which are more nutritious, less toxic, more resistant to drought, and more efficient for biofuel production [66, 67]. RNAi has already been successfully used to produce crops which are virus- and drought-resistant [66]. However, plants expressing dsRNAs while stable and safe take years to develop and millions of dollars to commercialize [65]. Development of topically applied RNAi, which is a nontransgenic approach improves crop traits and provides a major step forward for environmen‐

As more insects and mites develop chemical resistance to one or more insecticides, now estimated to be over 500 species with resistance to one or more products [77], it is imperative that new types of pest control are developed. The public would like the world to be filled with environmentally friendly technologies, safe for human and animal consumption, technologies which are safe for use around animals and beneficial insects, safe for each type of ecosystem, forest, field, crop, or backyard, a technology that will not endanger water or food quality, a more natural solution, with a natural approach toward problem-solving. So enters RNA

**5.2. Other applications**

400 RNA Interference

management efforts [17].

**6. Conclusions**

interference!

tally sound crop management [66, 67].

Mention of trade names or commercial products herein is solely for the purpose of providing specific information and does not imply recommendation or endorsement, to the exclusion of other similar products or services by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

#### **Acknowledgements**

We thank Dr. Xiomara Sinisterra-Hunter, AgTec, LLC, Plant Biotechnology Consultant, Port St. Lucie, FL, for critical reviews of the manuscript, and Maria Gonzalez, Biological science technician, USDA, Fort Pierce, FL, for technical assistance.

#### **Author details**

Eduardo C. de Andrade1 and Wayne B. Hunter2


#### **References**


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**Author details**

402 RNA Interference

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## **RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control**

Remil Linggatong Galay, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki and Tetsuya Tanaka

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61577

#### **Abstract**

Ticks (Acari: Ixodida) are blood-sucking arthropods globally recognized as vectors of nu‐ merous diseases. They are primarily responsible for the transmission of various patho‐ gens, including viruses, rickettsiae, and blood parasites of animals. Ticks are second to mosquitoes in terms of disease transmission to humans. The continuous emergence of tick-borne diseases and acaricide resistance of ticks necessitates the development of new and more effective control agents and strategies; therefore, understanding of different as‐ pects of tick biology and their interaction with pathogens is very crucial in developing effective control strategies. RNA interference (RNAi) has been widely used in the area of tick research as a versatile reverse genetic tool to elucidate the functions of various tick proteins. During the past decade, numerous studies on ticks utilized RNAi to evaluate potentially key tick proteins involved in blood feeding, reproduction, evasion of host im‐ mune response, interaction with pathogens, and pathogen transmission that may be tar‐ geted for tick and pathogen control. This chapter reviewed the application of RNAi in tick research over the past decade, focusing on the impact of this technique in the ad‐ vancement of knowledge on tick and pathogen biology.

**Keywords:** Acari, ticks, Ixodidae, RNA interference, tick-borne diseases

#### **1. Introduction**

Ticks belong to the class of Arachnida together with spiders, scorpions, and mites. To date, there are about 900 species of ticks, majority of which are hard ticks belonging to the Ixodidae family, as well as about 200 species are soft ticks belonging to the Argasidae family, and a single species belonging to the Nuttalliellidae family [1]. Most of the ticks of medical and veterinary importance are hard ticks. Through their blood-feeding behavior, ticks can directly

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

affect their host by causing anemia, irritation, and allergic reactions particularly in heavy infestation. The saliva of some tick species may also contain neurotoxic substances that may cause the condition termed "tick paralysis" [2]. Additionally, the transmission of pathogens including viruses, bacteria, and parasitic protozoa also occurs during blood feeding [1]. Ticks are considered second to mosquitoes in terms of their impact on public health, but they are the most important vectors of different pathogens in both domestic and wild animals [3]. Tick infestation and tick-borne diseases (TBDs) continue to have great economic impact on livestock production, particularly on cattle and small ruminants, in several continents [2]. The annual loss in cattle production worldwide due to ticks and TBDs has been estimated to be worth billions of USD [4].

The complete dependence of ticks to host blood for the completion of their life cycle and generation of offspring is the reason for their notoriety as vectors of several diseases. Depend‐ ing on the species, a tick may utilize one to three hosts during their life cycle. Most of the pathogens they transmit can be carried on throughout their life cycle through transstadial (from one stage to the next) transmission and to the next generation through transovarial (from adults to eggs) transmission [5]. A single tick may carry multiple pathogens [6], thereby having the potential of infecting a host with a cocktail of pathogens. Most tick-borne infections are zoonotic in nature, and more of these are being recognized in recent years [1, 7]. Among the TBDs that are well-known in the veterinary and medical field are anaplasmosis, borreliosis, rickettsiosis, ehrlichiosis, babesiosis, theileriosis, and tick-borne encephalitis.

The significant impact of ticks and TBDs underscores the importance of tick control. For several decades, the application of chemical acaricides has been the primary tick control method, and acaricides were used extensively in livestock production. However, the continuous emergence of resistant tick strains makes most chemical acaricides ineffective [8]. Moreover, the increasing concerns for animal product and environmental contamination set limitations for this control method. To search for new and more effective means of controlling ticks and TBDs, researchers have actively expanded the understanding on tick biology.

RNA interference (RNAi) is a reverse genetic approach for manipulation of genes that commonly utilizes double-stranded RNA (dsRNA) to induce post-transcriptional genespecific silencing [9]. RNAi has been extensively employed in many studies on tick biology and pathogen interaction since the first report of RNAi application in the hard tick *Amblyomma americanum* [10]. In fact, it is evident that a number of laboratories in different countries working on tick research are routinely performing RNAi, as shown by an increasing number of recent publications utilizing this technique. Typically, functional studies using RNAi involve gene knockdown with subsequent infestation and evaluation of phenotypes, such as blood feeding and reproduction success (Figure 1). Indeed, RNAi has been particularly useful in searching for tick proteins that can be targeted for control of tick development and TBDs [11].

This chapter aims to show the extent of RNAi application in tick research, emphasizing the progress of advanced knowledge on tick biology and tick-pathogen interaction. We first discussed so far known RNAi mechanisms and the current RNAi inducing methods in ticks; then, briefly described the studies on tick physiology, immunity, and pathogen interaction that employed RNAi, highlighting the prospects of applications of RNAi in tick research.

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577 413

**Galay et al, Fig.1 Figure 1.** A typical RNAi experiment in the hard tick *Haemaphysalis longicornis*. The double-stranded RNA (dsRNA) is introduced to adult ticks by microinjection. Unfed adult ticks, placed on a double adhesive tape attached on a glass slide (A), are injected with dsRNA using a pointed microcapillary glass attached to a microinjector (B) through the membrane of the fourth coxa under a stereomicroscope (C). Successful silencing, as shown by the absence of a band for the target gene, such as *Hlfer1*, is confirmed around 4 d post-injection through RT-PCR after adjusting the cDNA level using an internal control, such as actin (D). Ticks were infested on a host and allowed to feed to repletion (E) and after dropping, parameters such as engorged body weight (F), survival, egg laying (G, H), and hatch were compared.

#### **2. RNAi pathway in ticks**

affect their host by causing anemia, irritation, and allergic reactions particularly in heavy infestation. The saliva of some tick species may also contain neurotoxic substances that may cause the condition termed "tick paralysis" [2]. Additionally, the transmission of pathogens including viruses, bacteria, and parasitic protozoa also occurs during blood feeding [1]. Ticks are considered second to mosquitoes in terms of their impact on public health, but they are the most important vectors of different pathogens in both domestic and wild animals [3]. Tick infestation and tick-borne diseases (TBDs) continue to have great economic impact on livestock production, particularly on cattle and small ruminants, in several continents [2]. The annual loss in cattle production worldwide due to ticks and TBDs has been estimated to be worth

The complete dependence of ticks to host blood for the completion of their life cycle and generation of offspring is the reason for their notoriety as vectors of several diseases. Depend‐ ing on the species, a tick may utilize one to three hosts during their life cycle. Most of the pathogens they transmit can be carried on throughout their life cycle through transstadial (from one stage to the next) transmission and to the next generation through transovarial (from adults to eggs) transmission [5]. A single tick may carry multiple pathogens [6], thereby having the potential of infecting a host with a cocktail of pathogens. Most tick-borne infections are zoonotic in nature, and more of these are being recognized in recent years [1, 7]. Among the TBDs that are well-known in the veterinary and medical field are anaplasmosis, borreliosis,

The significant impact of ticks and TBDs underscores the importance of tick control. For several decades, the application of chemical acaricides has been the primary tick control method, and acaricides were used extensively in livestock production. However, the continuous emergence of resistant tick strains makes most chemical acaricides ineffective [8]. Moreover, the increasing concerns for animal product and environmental contamination set limitations for this control method. To search for new and more effective means of controlling ticks and TBDs, researchers

RNA interference (RNAi) is a reverse genetic approach for manipulation of genes that commonly utilizes double-stranded RNA (dsRNA) to induce post-transcriptional genespecific silencing [9]. RNAi has been extensively employed in many studies on tick biology and pathogen interaction since the first report of RNAi application in the hard tick *Amblyomma americanum* [10]. In fact, it is evident that a number of laboratories in different countries working on tick research are routinely performing RNAi, as shown by an increasing number of recent publications utilizing this technique. Typically, functional studies using RNAi involve gene knockdown with subsequent infestation and evaluation of phenotypes, such as blood feeding and reproduction success (Figure 1). Indeed, RNAi has been particularly useful in searching for tick proteins that can be targeted for control of tick development and TBDs [11]. This chapter aims to show the extent of RNAi application in tick research, emphasizing the progress of advanced knowledge on tick biology and tick-pathogen interaction. We first discussed so far known RNAi mechanisms and the current RNAi inducing methods in ticks; then, briefly described the studies on tick physiology, immunity, and pathogen interaction that

employed RNAi, highlighting the prospects of applications of RNAi in tick research.

rickettsiosis, ehrlichiosis, babesiosis, theileriosis, and tick-borne encephalitis.

have actively expanded the understanding on tick biology.

billions of USD [4].

412 RNA Interference

The mechanism of RNAi has been well studied in the nematode *Caenorhabditis elegans* and the fruit fly *Drosophila melanogaster* [9, 12]. RNAi begins with the uptake of dsRNA by the cell, followed by its cleavage to produce small interfering RNAs (siRNAs). Cleavage of dsRNA is accomplished by an RNAse III enzyme called Dicer. The siRNAs are then incorporated into RNA-induced silencing complex (RISC), which then drives the degradation or translational inhibition of the target mRNA that results to gene silencing. This silencing signal may spread among the cells and different tissues, leading to systemic gene silencing in the whole organism [13]. The mechanism of RNAi in ticks has not been fully elucidated, but the study of Kurscheid et al. [14] revealed that several components of RNAi machinery in other invertebrates are also present in the ticks, and they proposed a putative tick RNAi pathway. Here, we briefly describe the available knowledge on key components of RNAi machinery in ticks, in comparison of other invertebrates.

#### **2.1. dsRNA uptake**

There are two recognized dsRNA uptake mechanisms in invertebrates: a transmembrane channel-mediated uptake through systemic RNA interference defective (SID) transmembrane proteins described in *C. elegans*, and an endocytosis-mediated uptake described in most arthropods [15, 16]. Several SIDs identified in *C. elegans* have been shown to be involved in the spread of RNAi [17]. SID-1, SID-3, and SID-5, which have wide tissue distribution, are involved in the systemic spread of RNAi [18–20], whereas SID-2, localized mainly in the gut, is involved primarily in the intestinal uptake of ingested dsRNA [21]. The multi-domain SID-1 along the plasma membrane facilitates the traffic of dsRNA into and out of the cells. Homologs of SID-1 are present in some arthropods and vertebrates [18]. Both SID-3, a conserved tyrosine kinase, and SID-5 have intracytoplasmic localization, the latter being associated with late endosomes [19, 20]. SID-2 has luminal localization in the intestinal cells, and it was also found in the lower levels of excretory duct cells [21]. In addition to SIDs, endocytosis has also been also implicated as a dsRNA uptake mechanism in *C. elegans* through a protein containing an epsin N-terminal homology (ENTH) domain [22]. In *D. melanogaster*, dsRNA uptake in cells is facilitated mainly by scavenger receptor-mediated endocytosis [23]. Two scavenger receptors, Eater and Sr-CI, have been identified to be responsible for the majority of dsRNA uptake. These scavenger receptors are mainly expressed in the plasmatocytes and have a primary role in the phagocy‐ tosis of bacterial pathogens [24, 25].

SID homologues have not been identified in ticks. However, a homologue of ENTH, Epn-I, has been identified in the hard ticks *Rhiphicephalus* (*Boophilus*) *microplus* and *Ixodes scapularis* [14]. A class B scavenger receptor identified in *Haemaphysalis longicornis* (HlSRB) has been demonstrated to mediate systemic RNAi in this tick [26, 27]. Combined injection of dsRNA against *HlSRB* and other target genes, *Vitellogenin-*1 (*HlVg-1*) and *Vitellogenin Receptor* (*HlVgR*) effectively silenced these genes. However, silencing *HlSRB* prior to injection of dsRNA against *HlVg-1* and *HlVgR* inhibited the silencing of the latter two genes, suggesting that the uptake of the injected dsRNA is dependent on HlSRB in ticks. Similar to *D. melanogaster* scavenger receptors, HlSRB is also involved in the phagocytosis of bacteria [28], but it is expressed not only in the hemocytes but also in the other organs such as midguts, salivary glands, and ovary [26]. The presence of ENTH homologue and scavenger receptor indicates that the uptake of dsRNA in ticks is through endocytosis. Additionally, the presence of scavenger receptor in different tick tissues strongly implies its involvement in systemic RNAi, particularly after dsRNA injection. Introduction of dsRNA into the hemocoel of ticks directly exposes the different tick organs to dsRNA, and the scavenger receptor in these organs most likely mediates the entry of dsRNA into the cells.

#### **2.2. dsRNA processing and RISC assembly**

accomplished by an RNAse III enzyme called Dicer. The siRNAs are then incorporated into RNA-induced silencing complex (RISC), which then drives the degradation or translational inhibition of the target mRNA that results to gene silencing. This silencing signal may spread among the cells and different tissues, leading to systemic gene silencing in the whole organism [13]. The mechanism of RNAi in ticks has not been fully elucidated, but the study of Kurscheid et al. [14] revealed that several components of RNAi machinery in other invertebrates are also present in the ticks, and they proposed a putative tick RNAi pathway. Here, we briefly describe the available knowledge on key components of RNAi machinery in ticks, in comparison of

There are two recognized dsRNA uptake mechanisms in invertebrates: a transmembrane channel-mediated uptake through systemic RNA interference defective (SID) transmembrane proteins described in *C. elegans*, and an endocytosis-mediated uptake described in most arthropods [15, 16]. Several SIDs identified in *C. elegans* have been shown to be involved in the spread of RNAi [17]. SID-1, SID-3, and SID-5, which have wide tissue distribution, are involved in the systemic spread of RNAi [18–20], whereas SID-2, localized mainly in the gut, is involved primarily in the intestinal uptake of ingested dsRNA [21]. The multi-domain SID-1 along the plasma membrane facilitates the traffic of dsRNA into and out of the cells. Homologs of SID-1 are present in some arthropods and vertebrates [18]. Both SID-3, a conserved tyrosine kinase, and SID-5 have intracytoplasmic localization, the latter being associated with late endosomes [19, 20]. SID-2 has luminal localization in the intestinal cells, and it was also found in the lower levels of excretory duct cells [21]. In addition to SIDs, endocytosis has also been also implicated as a dsRNA uptake mechanism in *C. elegans* through a protein containing an epsin N-terminal homology (ENTH) domain [22]. In *D. melanogaster*, dsRNA uptake in cells is facilitated mainly by scavenger receptor-mediated endocytosis [23]. Two scavenger receptors, Eater and Sr-CI, have been identified to be responsible for the majority of dsRNA uptake. These scavenger receptors are mainly expressed in the plasmatocytes and have a primary role in the phagocy‐

SID homologues have not been identified in ticks. However, a homologue of ENTH, Epn-I, has been identified in the hard ticks *Rhiphicephalus* (*Boophilus*) *microplus* and *Ixodes scapularis* [14]. A class B scavenger receptor identified in *Haemaphysalis longicornis* (HlSRB) has been demonstrated to mediate systemic RNAi in this tick [26, 27]. Combined injection of dsRNA against *HlSRB* and other target genes, *Vitellogenin-*1 (*HlVg-1*) and *Vitellogenin Receptor* (*HlVgR*) effectively silenced these genes. However, silencing *HlSRB* prior to injection of dsRNA against *HlVg-1* and *HlVgR* inhibited the silencing of the latter two genes, suggesting that the uptake of the injected dsRNA is dependent on HlSRB in ticks. Similar to *D. melanogaster* scavenger receptors, HlSRB is also involved in the phagocytosis of bacteria [28], but it is expressed not only in the hemocytes but also in the other organs such as midguts, salivary glands, and ovary [26]. The presence of ENTH homologue and scavenger receptor indicates that the uptake of dsRNA in ticks is through endocytosis. Additionally, the presence of scavenger receptor in different tick tissues strongly implies its involvement in systemic RNAi, particularly after

other invertebrates.

414 RNA Interference

**2.1. dsRNA uptake**

tosis of bacterial pathogens [24, 25].

The recommended length of dsRNA to effectively induce silencing of the target gene in nonmammalian systems is more than 200 bp [15]. A study in *R.* (*B.*) *microplus* showed, however, that short dsRNAs between 100 and 200 bp were also effective in inducing silencing of *Ubiquitin-63E* homologue, with minimal off-target effects, but short hairpin dsRNAs were not able to induce silencing effects [29]. After cellular uptake, dsRNAs are cleaved into 21–25 nt siRNAs by an RNAse III enzyme called Dicer. In contrast to *C. elegans* and mammals that have only oneDicer,*D.melanogaster* andmosquitoeshave twoDicers [15].Dicer-2 is the one involved in the generation of siRNA, whereas Dicer-1 acts on stem loop RNA precursors to generate micro RNA (miRNA). Both, however, are required for siRNA-induced gene silencing due to their distinct roles in siRISC assembly [30]. Only a single putative Dicer has been identified so far in the hard tick *I. scapularis*, which is more similar to mammalian Dicer-1 [14].

The RNAi inhibition of a target mRNA is accomplished by RISC formed by siRNAs and Argonaute (AGO) proteins. AGO proteins are highly conserved between species, encoded by multiple genes in most organisms. All AGO proteins are characterized by two domains: the PAZ domain and the PIWI domain [31]. Upon ATP activation, AGO mediates RISC recognition of mRNA target that are homologous to siRNAs, subsequently leading to the cleavage of the mRNA target [9]. In most insects, including *D. melanogaster* and mosquitoes, five AGO genes have been identified [15, 31]. In ticks, a homologue of AGO-1 has been identified in *I. scapu‐ laris* and *R.* (*B.*) *microplus,* and a homologue of AGO-2 has been identified in *I. scapularis* [14]. However, the functions of these tick AGOs remains to be confirmed.

#### **2.3. Amplification of RNAi signal**

The ability to spread throughout the whole organism, inducing total systemic silencing of the target gene in spite of introducing only a relatively small amount of dsRNA, is an important aspect of RNAi observed in plants and invertebrates. This systemic RNAi-induced gene silencing in both plants and *C. elegans* involves RNA-directed RNA polymerase (RdRP) that amplifies the RNAi signal [9]. RdRP function in RNAi has not been found in arthropods, but a putative homologue of RdRP EGO-1 protein of *C. elegans* has been identified in the hard tick *I. scapularis*, and a partial sequence was also identified from *R.* (*B.*) *microplus* [14].

#### **3. Methods of introducing dsRNA in ticks**

#### **3.1. Injection**

Direct injection is the most widely used technique for introducing dsRNA for in vivo gene silencing, not only in ticks but also in insects [11, 32]. Through this method, dsRNA is usually

introduced directly into the hemocoel of ticks allowing the dsRNA to circulate within the hemolymph. In most reports, a high concentration of at least 1 μg dsRNA per tick has been shown to be effective in inducing gene silencing [33], but in some reports, lower concentration has been found to be similarly effective [34–36]. Injection has been accomplished using a 33– 36-gauge needle attached to a Hamilton syringe particularly in large tick species, such as *Amblyomma americanum*[37], *Dermacentor variabilis*, and *D. marginatus*[38], while microinjection using a microcapillary drawn to a fine point needle and inserted to a micromanipulator has been commonly employed in smaller tick species, including *Ixodes* [39], and *Haemaphysalis*[26] ticks. Different injection sites include the lower right quadrant of the ventral surface of the exoskeleton [40], the groove between the basis capituli and the scutum [37], the ventral torso of the idiosoma, away from the anal opening [39], and the coxal membrane in the fourth coxae [26, 41]. In some reports, dsRNA was injected through the spiracle [29, 42, 43] and anal pore; the latter inducing midgut-specific silencing of the target gene [44]. Injection of dsRNA has been commonly performed in unfed adult ticks, subsequently allowed to recover for at least 24 h before infestation or use in succeeding experiments. Exceptionally, dsRNA has been also injected in engorged *R.* (*B*.) *microplus* [42, 45-47], *A. americanum, I. scapularis*, and *D. variabilis* adults [43], which produced significant effects on the eggs and larvae, and microinjection has also been accomplished in *I. scapularis* [48-52] and engorged *O. moubata* nymphs [53].

#### **3.2. Soaking**

Soaking in dsRNA has been previously employed to study RNAi in the cell lines of *D. melanogaster* [23, 54]. In tick research, this method has been applied to induce in vitro RNAi not only in cell cultures [55-57], but also in some organs including whole salivary glands [36, 58-60] and midguts [61]. Soaking live *Varroa destructor*[62] and *Dermanyssus gallinae*[63] mites, as well as *Aedes aegypti* larvae [64] in a solution of dsRNA has been already demonstrated in producing effective silencing of the target genes in these organisms. However, soaking whole ticks in a solution of dsRNA has not been commonly performed. Soaking *Haemaphysalis longicornis* nymphs in a solution of dsRNA for 24 h resulted to significant transcript reduction of the target gene, although the effect on the phenotype was not observed in all the nymphs [65]. In our laboratory, we have attempted to soak *H. longicornis* larvae, nymphs, and adults in a dsRNA solution overnight, which resulted to a significant decrease in the mRNA level of a targeted gene (Galay et al., unpublished results). Soaking offers a simpler and less invasive method of introducing dsRNA without injuring the ticks and is applicable to immature tick stages. Furthermore, it does not require injection equipment; therefore, it is less laborious.

#### **3.3. Electroporation**

Electroporation is a technique that employs electric impulses to promote DNA uptake of cells and has been primarily used with in vitro cell transfection [66]. In tick research, this technique has been first applied to facilitate the introduction of dsRNA in *I. scapularis* eggs and nymphs [67]. After electroporation, fluorescein-labeled dsRNA was visualized all over the nymph's body and eggs, indicating the successful entry of dsRNA. In a more recent report, the wax coating of the eggs was first removed using heptane and hypochlorite prior to electroporation [68]. Using heptane alone did not significantly decrease the hatching rate. Thus, heptane may be more helpful in evaluating the effect of a particular dsRNA to egg hatching. This technique also offers a less invasive method of dsRNA introduction that can be applied to immature tick stages and eggs.

#### **3.4. Feeding**

introduced directly into the hemocoel of ticks allowing the dsRNA to circulate within the hemolymph. In most reports, a high concentration of at least 1 μg dsRNA per tick has been shown to be effective in inducing gene silencing [33], but in some reports, lower concentration has been found to be similarly effective [34–36]. Injection has been accomplished using a 33– 36-gauge needle attached to a Hamilton syringe particularly in large tick species, such as *Amblyomma americanum*[37], *Dermacentor variabilis*, and *D. marginatus*[38], while microinjection using a microcapillary drawn to a fine point needle and inserted to a micromanipulator has been commonly employed in smaller tick species, including *Ixodes* [39], and *Haemaphysalis*[26] ticks. Different injection sites include the lower right quadrant of the ventral surface of the exoskeleton [40], the groove between the basis capituli and the scutum [37], the ventral torso of the idiosoma, away from the anal opening [39], and the coxal membrane in the fourth coxae [26, 41]. In some reports, dsRNA was injected through the spiracle [29, 42, 43] and anal pore; the latter inducing midgut-specific silencing of the target gene [44]. Injection of dsRNA has been commonly performed in unfed adult ticks, subsequently allowed to recover for at least 24 h before infestation or use in succeeding experiments. Exceptionally, dsRNA has been also injected in engorged *R.* (*B*.) *microplus* [42, 45-47], *A. americanum, I. scapularis*, and *D. variabilis* adults [43], which produced significant effects on the eggs and larvae, and microinjection has

also been accomplished in *I. scapularis* [48-52] and engorged *O. moubata* nymphs [53].

Soaking in dsRNA has been previously employed to study RNAi in the cell lines of *D. melanogaster* [23, 54]. In tick research, this method has been applied to induce in vitro RNAi not only in cell cultures [55-57], but also in some organs including whole salivary glands [36, 58-60] and midguts [61]. Soaking live *Varroa destructor*[62] and *Dermanyssus gallinae*[63] mites, as well as *Aedes aegypti* larvae [64] in a solution of dsRNA has been already demonstrated in producing effective silencing of the target genes in these organisms. However, soaking whole ticks in a solution of dsRNA has not been commonly performed. Soaking *Haemaphysalis longicornis* nymphs in a solution of dsRNA for 24 h resulted to significant transcript reduction of the target gene, although the effect on the phenotype was not observed in all the nymphs [65]. In our laboratory, we have attempted to soak *H. longicornis* larvae, nymphs, and adults in a dsRNA solution overnight, which resulted to a significant decrease in the mRNA level of a targeted gene (Galay et al., unpublished results). Soaking offers a simpler and less invasive method of introducing dsRNA without injuring the ticks and is applicable to immature tick stages. Furthermore, it does not require injection equipment; therefore, it is less laborious.

Electroporation is a technique that employs electric impulses to promote DNA uptake of cells and has been primarily used with in vitro cell transfection [66]. In tick research, this technique has been first applied to facilitate the introduction of dsRNA in *I. scapularis* eggs and nymphs [67]. After electroporation, fluorescein-labeled dsRNA was visualized all over the nymph's body and eggs, indicating the successful entry of dsRNA. In a more recent report, the wax coating of the eggs was first removed using heptane and hypochlorite prior to electroporation

**3.2. Soaking**

416 RNA Interference

**3.3. Electroporation**

Feeding dsRNA in insects has been achieved in different species using diets mixed with dsRNA, liposome-embedded or lipophilic siRNAs, and bacteria and transgenic plants that can synthesize dsRNA [32, 69]. Although in vitro feeding assays have been shown to be useful in studying different tick molecules and tick-pathogen interaction [70], its application in RNAi study in ticks has been limited. A study on the Lyme disease vector *I. scapularis* employed capillary feeding of dsRNA to nymphs to suppress anticomplement gene *isac* [71]. In another study, adult *R.* (*B.*) *microplus* ticks were capillary fed with *ubiquitin* dsRNA mixed in whole blood or *Bm86* dsRNA mixed in bovine serum [72]. In both cases, ticks were pre-fed in an animal host before capillary feeding was performed. While this method may be advantageous over injection due to very minimal injury, drawbacks may arise from the uncertainty whether an individual tick will ingest the amount of dsRNA that will effectively induce silencing, and the possibility of variation in the amount of dsRNA ingested by the ticks within a treatment group. Furthermore, capillary feeding is difficult to perform and may not be applicable in ticks with short hypostome.

#### **4. RNAi and study of tick physiology**

#### **4.1. Genes related to salivary functions**

The saliva is an important arsenal of ticks containing hundreds of pharmacologically potent substances that facilitate attachment to their hosts and blood-sucking [73]. Different salivary proteins have redundant functions in counteracting the hemostatic [74], inflammatory, and immune mechanisms [75] of the host. Aside from its function in tick feeding, the salivary glands are also involved in osmoregulation and transmission of pathogens [76].

Many studies on characterization of salivary proteins in the recent years employed RNAi (Table 1). In fact, the first report on the application of RNAi in tick research described a tick inhibitor of inflammatory mediator, a salivary histamine-binding protein, wherein researchers induced in vitro RNAi by soaking salivary glands in dsRNA [10, 58]. Soluble N-ethylmalei‐ mide-sensitive factor attachment receptors (SNARE) complex proteins, which mediate exocytosis in secretory pathways of the salivary glands, have been characterized in *Amblyom‐ ma* ticks. These include N-ethylmaleimide-sensitive fusion (NSF) protein, Synaptosomal Associated Protein of 25 kDa (SNAP-25) [77], Ykt6 [65], and vesicle transport through inter‐ action with t-SNAREs (Vti) [78]. Silencing various genes such as Salp14, Salp9pac [39], Neuronal isoform munc18-1 (nSec1) [59], and synaptobrevin [36] affected the secretion of anticoagulant or the anticoagulant activity of salivary gland extracts, indicating that these genes are important in tick anti-hemostatic mechanism.

Longistatin [79] and acidic chitinases [80] have been found to be important in the formation of blood pool and tick cement cone, respectively. The attachment site of *longistatin*-silenced *H. longicornis* ticks did not show pathological changes, such as hemorrhagic lesions correspond‐ ing to the blood pool, while the attachment site of ticks simultaneously silenced acidic chitinases exhibited blood leakage and these ticks can be easily removed. Various protease inhibitors that have roles in anti-hemostatic, anti-inflammatory, and immunomodulatory mechanism have been also characterized using RNAi, including a cystatin, sialostatine L [81], a Kunitz type protease inhibitor, rhipilin [82], and serine protease inhibitors (serpin) [83, 84].

Other salivary proteins with immunomodulatory function, such as the anti-complement protein, isac [71], and two proteins that can inhibit neutrophil function, ISL 929 and 1373 [85], have also been knockdowned in *I. scapularis*. Silencing of *isac* in nymphs, induced by capillary feeding of dsRNA, not only reduced blood feeding, but also decreased the load of the spiro‐ chete *Borrelia burgdorferi* in the tick. Meanwhile, the saliva of ticks devoid of ISL 929 and 1373 had reduced ability in inhibiting host integrin. An osmoregulatory protein aquaporin, characterized in *I. ricinus* through RNAi, showed that suppression of this protein impaired the concentration of blood meal due to failure in removing water [86].

#### **4.2. Genes related to digestion and midgut function**

The midgut of ticks houses various kinds of enzymes that act on a large amount of ingested host blood, which contains great quantities of hemoglobin [151]. Functional studies on these enzymes and other midgut proteins using RNAi have expanded the understanding of tick digestive physiology (Table 1). Silencing hemoglobinolytic enzymes, such as leucine amino‐ peptidase [91, 92], longipain [95], and cathepsin L [96, 97] had negative impact on tick feeding. Moreover, the longipain of *H. longicornis* was found to have a protective role in *Babesia* infection through its babesiacidal activity [95]. Other proteins important in tick digestion that have been characterized using RNAi are thrombin inhibitors that prevent blood coagulation and serine proteinase, which induce erythrocyte degradation. Silencing of thrombin inhibitor hemalin from *H. longicornis* [93] and boophilin from *R.* (*B*.) *microplus* [47] prolonged the blood feeding period and decreased the oviposition of these ticks, respectively. Silencing serine protease reduced the weight of ticks after blood feeding due to impaired erythrocyte degradation [94].

#### **4.3. Genes related to reproductive function**

Ticks are known for their high fecundity, laying hundreds of eggs per batch in the case of soft ticks and up to thousands in the case of hard ticks. A series of physiological events takes place in female ticks during and after blood feeding that initiate ovarian maturation and subsequent oviposition. Vitellogenesis, the synthesis and oocyte deposition of the yolk pro‐ tein precursor (vitellogenin), is a key process for ovarian development and oocyte matura‐ tion induced by blood meal in ticks [152]. Three genes encoding vitellogenin have been identified and characterized in *H. longicornis* [102].


anticoagulant or the anticoagulant activity of salivary gland extracts, indicating that these

Longistatin [79] and acidic chitinases [80] have been found to be important in the formation of blood pool and tick cement cone, respectively. The attachment site of *longistatin*-silenced *H. longicornis* ticks did not show pathological changes, such as hemorrhagic lesions correspond‐ ing to the blood pool, while the attachment site of ticks simultaneously silenced acidic chitinases exhibited blood leakage and these ticks can be easily removed. Various protease inhibitors that have roles in anti-hemostatic, anti-inflammatory, and immunomodulatory mechanism have been also characterized using RNAi, including a cystatin, sialostatine L [81], a Kunitz type protease inhibitor, rhipilin [82], and serine protease inhibitors (serpin) [83, 84].

Other salivary proteins with immunomodulatory function, such as the anti-complement protein, isac [71], and two proteins that can inhibit neutrophil function, ISL 929 and 1373 [85], have also been knockdowned in *I. scapularis*. Silencing of *isac* in nymphs, induced by capillary feeding of dsRNA, not only reduced blood feeding, but also decreased the load of the spiro‐ chete *Borrelia burgdorferi* in the tick. Meanwhile, the saliva of ticks devoid of ISL 929 and 1373 had reduced ability in inhibiting host integrin. An osmoregulatory protein aquaporin, characterized in *I. ricinus* through RNAi, showed that suppression of this protein impaired the

The midgut of ticks houses various kinds of enzymes that act on a large amount of ingested host blood, which contains great quantities of hemoglobin [151]. Functional studies on these enzymes and other midgut proteins using RNAi have expanded the understanding of tick digestive physiology (Table 1). Silencing hemoglobinolytic enzymes, such as leucine amino‐ peptidase [91, 92], longipain [95], and cathepsin L [96, 97] had negative impact on tick feeding. Moreover, the longipain of *H. longicornis* was found to have a protective role in *Babesia* infection through its babesiacidal activity [95]. Other proteins important in tick digestion that have been characterized using RNAi are thrombin inhibitors that prevent blood coagulation and serine proteinase, which induce erythrocyte degradation. Silencing of thrombin inhibitor hemalin from *H. longicornis* [93] and boophilin from *R.* (*B*.) *microplus* [47] prolonged the blood feeding period and decreased the oviposition of these ticks, respectively. Silencing serine protease reduced the weight of ticks after blood feeding due to impaired erythrocyte degradation [94].

Ticks are known for their high fecundity, laying hundreds of eggs per batch in the case of soft ticks and up to thousands in the case of hard ticks. A series of physiological events takes place in female ticks during and after blood feeding that initiate ovarian maturation and subsequent oviposition. Vitellogenesis, the synthesis and oocyte deposition of the yolk pro‐ tein precursor (vitellogenin), is a key process for ovarian development and oocyte matura‐ tion induced by blood meal in ticks [152]. Three genes encoding vitellogenin have been

genes are important in tick anti-hemostatic mechanism.

418 RNA Interference

concentration of blood meal due to failure in removing water [86].

**4.2. Genes related to digestion and midgut function**

**4.3. Genes related to reproductive function**

identified and characterized in *H. longicornis* [102].



**Target gene Tick species RNAi Effect Refs**

Vti (SNARE) *A. americanum*, *A. maculatum*Decreased post-blood meal weight and

Glutaminyl cyclase (QC) *A. maculatum*, *I. scapularis* Decreased post-blood meal weight, egg weight

AV422 *A. americanum* Decreased post-blood meal weight [88] Acidic chitinase (Ach) *A. americanum* Leakage of blood from the mouthparts in late

Longepsin *H. longicornis* No effects reported [90] Leucine aminopeptidase *H. longicornis* Extended pre-oviposition period, decreased egg

Serine proteinase *H. longicornis* Suppressed erythrocyte degradation; decreased

Longipain *H. longicornis* Impaired blood feeding, decreased post-blood

Cathepsin L *I. ricinus* Decreased weight gain [96]

Astacin *R.* (*B.*) *microplus* Decreased egg weight and egg conversion ratio [84]

Vitellogenin receptor (VgR) *Dermacentor variabilis* Failure of Vg uptake by oocytes; failed

*A. maculatum* Decreased post-blood meal weight, decreased

and hatch

skin

*H. longicornis* Longer blood feeding period, failure to

egg weight, failure in hatching

survival, failed oviposition

abnormalities in the oocytes

*R.* (*B.*) *microplus* Decreased oviposition [47]

post-blood meal weight

*H. longicornis* Decreased post-blood meal weight [97]

*H. longicornis* Decreased egg conversion ratio [98]

oviposition

*H. longicornis* Suppressed oocyte maturation and failed

transmission

and transovarial transmission

feeding phase, loose attachment in the host's

weight and egg conversion ratio, morphological

meal weight, increased *B. gibsoni* infection level

oviposition, failure of *B. gibsoni* transovarial

engorge, decreased inhibitory activity of fibrinogen clot formation in the midgut

[85]

[86]

[87]

[89]

[91, 92]

[93]

[94]

[95]

[34]

[99]

Synaptosomal Associated Protein of 25 kDa (SNAP-25)

420 RNA Interference

**Digestive activity**

Hemalin (thrombin

Boophilin (thrombin

**Tick reproduction**

(FRP)

Follistatin-related protein

inhibitor)

inhibitor)



**Target gene Tick species RNAi Effect Refs**

Ubiquitin *R.* (*B.*) *microplus* Shorter post-blood meal survival, decreased or

CD147 receptor *A. americanum* Inhibited feeding, low post-blood meal weight

oviposition

*H. longicornis* Longer blood feeding period, decreased post-

after LKR silencing

*R. annulatus* High mortality [45]

*R.* (*B.*) *microplus* Decreased oviposition and hatching [115]

*A. americanum* Decreased post-blood meal weight [117]

tender cuticle

*A. americanum* High mortality and very low post-blood meal

*A. americanum* High mortality and very low post-blood meal

*A. americanum* High mortality and very low post-blood meal

*A. americanum* High mortality and very low post-blood meal

*A. americanum* High mortality and very low post-blood meal

*H. longicornis* Mortality after attachment, retarded blood

granulocytes

*A. americanum* 100% mortality [118]

and egg conversion ratio

feeding and longer feeding period, decreased post-blood meal weight, decreased egg weight

after engorgement, decreased oviposition and hatch; inhibited bacterial phagocytosis of

weight

weight

weight

weight

weight

Scavenger receptor *H. longicornis* Decreased post-blood meal weight, mortality

High post-blood meal mortality, decreased post-blood meal weight and failure of

blood meal weight, longer pre-oviposition period, decreased oviposition and hatch after SDH silencing; higher volume of hemolymph

absence of egg output, impaired embryogenesis

[45]

[114]

[14, 29, 45, 72];

[116]

[118]

[118]

[118]

[118]

[118]

[119]

[26, 28]

Elongation factor 1-α *R. annulatus, R.* (*B.*)

Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH)

422 RNA Interference

Glycogen synthase kinase-3

Insulin-like growth factor binding protein-related

Putative 5.8S, ITS2 and 28S

Putative 2B7 60S ribosomal

(GSK-3)

proteins

rRNA

protein L13e

synthetase

protein L13a

rRNA

(HlChI)

Putative interphase cytoplasm foci protein 45

Putative threonyl-tRNA

Putative 60S ribosomal

Putative mitochondrial 12S

Chymotrypsin inhibitor

*microplus*



**Target gene Tick species RNAi Effect Refs**

Midgut protein Hl86 *H. longicornis* Decreased post-blood meal weight [128]

Midgut protein Ree86 *R. evertsi evertsi* No significant effect [130] Midgut protein ReeATAQ *R. evertsi evertsi* No significant effect [130] Longicin *H. longicornis* Decreased post-blood meal weight, increased *B.*

α2-macroglobulin proteins *I. ricinus* Decreased phagocytic action of hemocytes [132, 133]

Dual oxidase (Duox) *I. scapularis* Decreased level of *B. burgdorferi* [136] Peroxidase ISCW017368 *I. scapularis* Decreased level of *B. burgdorferi* [136] Glutathione S-transferase *R.* (*B.*) *microplus* Decreased tick attachment and post-blood meal

Selenoprotein W *R.* (*B.*) *microplus* Decreased tick attachment and post-blood meal

Thioredoxin reductase *A. maculatum* Decreased native microbial load in midguts and

Subolesin *D. variabilis* Inhibited *Anaplasma marginale* infection in

Selenoprotein K *A. maculatum* Decreased oviposition [138] Selenoprotein M *A. maculatum* Decreased oviposition [138, 139]

weight

weight

salivary glands

salivary glands

*R.* (*B.*) *microplus* Increased resistance to bacteria [140]

Macrophage migration inhibitory factor

424 RNA Interference

Janus kinase ( JAK)– signaling transducer activator of transcription (STAT) pathway

Rmcystatin3 (cysteine protease inhibitor)

**Pathogen acquisition/ transmission**

Midgut protein Bm86 *R.* (*B.*) *microplus* Decreased number of engorging ticks, lower

oviposition

weight

post-blood meal body weight and survival after feeding in *B. bovis-*infected host, decreased egg

*gibsoni* infection in the midgut and ovary, and

transmission in the eggs

*A. americanum* No effect on phenotypes [134]

*I. scapularis* Increased *A. phagocytophilum* infection level [135]

*R.sanguineus* Increased susceptibility to permethrin [137]

[125]

[129]

[131]

[45]

[45]

[139]

[141, 142]

Midgut protein Rs86 *R. sanguineus* Decreased post-blood meal weight and


**Table 1.** Genes functionally characterized through RNAi in different tick species.

Silencing these genes through RNAi greatly reduced the reproductive capacity of female ticks, which showed immature and light-colored oocytes. The uptake of vitellogenin in the oocytes is facilitated by vitellogenin receptor, which has been characterized in *D. variabilis* [34], *H.longicornis* [99], and *A. hebraeum* [100]. Aside from the negative impact in oviposition consistently induced by RNAi in all these studies, silencing of *H. longicornis* vitellogenin receptor also reportedly inhibited the transovarial transmission of *Babesia gibsoni*. Three factors involved in the initiation of vitellogenesis, the GATA factor, S6 kinase [103], and target of rapamycin (TOR) pathway [104], have been also characterized in *H. longicornis* ticks using RNAi. The significance of other proteins to reproduction, such as a tick homologue of the human follistatin-related protein [98] and the engorgement protein voraxin [101] from the male gonad, has been also demonstrated using RNAi.

#### **4.4. Genes related to structural and metabolic functions**

Various gene encoding proteins important in cellular structure and metabolism have been characterized using RNAi. Due to their wide distribution and systemic function, knockdown of these proteins caused detrimental effects on different tick physiological functions and some even proved to be lethal (Table 1). Among these proteins is the multifunctional ubiquitin, which has been first targeted based on a homologous gene of *D. melanogaster* in a study investigating the components of tick RNAi pathway [14]. Ubiquitin knockdown in *R.* (*B.*) *microplus* shortened the post-blood meal survival of ticks and impaired egg viability and hatch. In separate studies, ubiquitin has also been the subject in examining off-target effects of RNAi [29] and the feasibility of dsRNA feeding in *R.* (*B.*) *microplus* [72]. RNAi-mediated silencing of ribosomal proteins in *A. americanum* [118], and ubiquitin, elongation factor-1 alpha and several other proteins in *R.* (*B*.) *microplus* and *R. annulatus* [45] has been employed to screen potential antigens for tick control.

**Target gene Tick species RNAi Effect Refs**

*annulatus*

*R. annulatus*

acquisition or transmission

*phagocytophilum* infection level

*I. scapularis* Increased *A. phagocytophilum* infection [149]

*R.* (*B.*) *microplus* Failure in transmission of *A. marginale* [150]

*I. scapularis* No significant effect on *B. burgdorferi*

Silencing these genes through RNAi greatly reduced the reproductive capacity of female ticks, which showed immature and light-colored oocytes. The uptake of vitellogenin in the oocytes is facilitated by vitellogenin receptor, which has been characterized in *D. variabilis* [34], *H.longicornis* [99], and *A. hebraeum* [100]. Aside from the negative impact in oviposition consistently induced by RNAi in all these studies, silencing of *H. longicornis* vitellogenin receptor also reportedly inhibited the transovarial transmission of *Babesia gibsoni*. Three factors involved in the initiation of vitellogenesis, the GATA factor, S6 kinase [103], and target of rapamycin (TOR) pathway [104], have been also characterized in *H. longicornis* ticks using RNAi. The significance of other proteins to reproduction, such as a tick homologue of the human follistatin-related protein [98] and the engorgement protein voraxin [101] from the male

Various gene encoding proteins important in cellular structure and metabolism have been characterized using RNAi. Due to their wide distribution and systemic function, knockdown of these proteins caused detrimental effects on different tick physiological functions and some even proved to be lethal (Table 1). Among these proteins is the multifunctional ubiquitin, which has been first targeted based on a homologous gene of *D. melanogaster* in a study investigating the components of tick RNAi pathway [14]. Ubiquitin knockdown in *R.* (*B.*)

Antifreeze glycoprotein *I. scapularis* Decreased survival and mobility of ticks in

**Table 1.** Genes functionally characterized through RNAi in different tick species.

gonad, has been also demonstrated using RNAi.

**4.4. Genes related to structural and metabolic functions**

Decreased *B. bigemina* infection level [126]

[126]

[126]

[50]

[148]

Decreased post-blood meal weight in *R.*

Decreased *B. bigemina* infection level in *R. microplus*; decreased post-blood meal weight in

extremely cold temperature; decreased *A.*

Serum amyloid A *R. annulatus*, *R.* (*B.*)

Ricinusin *R. annulatus*, *R.* (*B.*)

Calreticulin *R. annulatus*, *R.* (*B.*)

Chitin deacetylase-like protein (IsCDA)

inhibitor of apoptosis protein (E3 ubiquitin ligase,

Cytochrome c oxidase

x-linked

426 RNA Interference

XIAP)

subunit III

*microplus*

*microplus*

*microplus*

In the hard tick *H. longicornis*, individual knockdown of glutamine:fructose-6-phosphate aminotransferase [105], cyclophilin A [108], the ribosomal protein P0 [109], protein disulphide isomerases [110], and tropomyosin [123] resulted to decreased survival of ticks after engorge‐ ment. Two proteins with apparent roles in withstanding long starvation period, lysineketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) [114] and 4E-BP [120], have also been characterized in *H. longicornis*. LKR/SDH mRNA expression is higher in starved ticks than in unfed ticks and knockdown of LKR resulted to high volume of hemolymph after blood feeding, suggesting its role in osmoregulation. Meanwhile, 4E-BP knockdown led to decreased lipid storage in the midguts and fat bodies of ticks during longer starvation period. An interesting report on the application of RNAi in studying tick neurobiology targetedβ-actin and Na+ -K+ -ATPase of *I. scapularis* using fluorescently labeled dsRNAs to monitor the uptake in tick synganglia [106].

The significance of proteins involved in iron metabolism to tick feeding and reproduction has been also demonstrated using RNAi in two hard tick species, *I. ricinus* [111] and *H. longicor‐ nis* [112]. Silencing two types of the iron storage protein ferritin greatly reduced the ticks' capacity to engorge and produce eggs, also affecting post-blood meal survival due to occur‐ rence of iron-mediated oxidative stress [113]. An iron regulatory protein responsible for translation of iron binding proteins was characterized in *I. scapularis*, with its knockdown greatly reducing egg hatchability [111]. Two enzymes, spook and shade, were characterized in the soft tick *O. moubata* and were shown to be important in ecdysteroidogenesis through RNAi [53]. Silencing spook protein in nymphs caused arrested development and molting, whereas silencing shade delayed molting and led to abnormal ecdysis.

#### **5. RNAi studies on tick protective antigens and immunity**

The immune system of ticks has a vital role of protecting them from harmful substances in the blood, including components of their host's immune system, and from various pathogens that they acquire in their blood feeding activity. Tick protective antigens, therefore, gain wide interest due to their potential as target for tick control. The highly conserved tick protective antigen subolesin, previously known as 4D8, was first identified from *I. scapularis* through cDNA expression library immunization (ELI) [153], after which, it has been also identified in other hard tick species, and using RNAi, was found to be important in the success of blood feeding and reproduction [124]. An ortholog of subolesin has also been characterized in two soft tick species and RNAi demonstrated that subolesin is also important in the reproduction of soft ticks [127]. The function of subolesin is unclear, but a report showed that subolesin knockdown affected the expression of several genes involved in multiple cellular pathways, suggesting a role in gene expression by interacting with regulatory proteins [154]. Aside from being reported as a promising anti-tick vaccine antigen candidate in many studies, it has been also proposed that subolesin may be targeted in ticks that subsequently will be released for sterile acarine technique (SAT) [38].

The membrane-bound glycoprotein Bm86 expressed mainly in the midgut of *R.* (*B.*) *micro‐ plus* [155] is the first, and until recently, the only tick antigen that is commercially available as an anti-tick vaccine in some countries. The exact function of Bm86, however, remains unclear yet. RNAi has been employed to knockdown *Bm86* and its homologues in other tick species, including *R. sanguineus*, *H. longicornis*, and *R. evertsi evertsi*, which in most cases affected the blood feeding and reproduction of adult ticks, except in *R. evertsi evertsi* wherein knockdown of two homologues did not yield significant effects [130]. A study in *R.* (*B.*) *microplus* also showed that knockdown of *Bm86* decreased the blood feeding capacity and survival of ticks after feeding on a *B. bovis-*infected host, suggesting that Bm86 may have a critical role in the fitness of ticks after feeding from an acutely *B. bovis-*infected host [129].

The function of some components of immunity, such as α2-macroglobulin proteins [132, 133], antimicrobial peptides [131], Janus kinase (JAK)-signaling transducer activator of transcription (STAT) pathway [135], dityrosine network [136], and cysteine protease inhibitor in the hemocytes [140] have been analyzed using RNAi. The α2-macroglobulin proteins of *I. ricinus*, related to vertebrate complement system, were shown to be involved in the phagocytic activity of hemocytes against Gram-negative bacteria [132, 133]. In contrast, a cysteine protease Rmcystatin3 identified in *R.* (*B.*) *microplus* was implicated as a negative modulator of tick immune response after its silencing greatly reduced the number of bacterial load in the ticks [140]. The role of a defensin from *H. longicornis*, longicin, in ticks' immune defense against *Babesia* parasites was demonstrated through RNAi, as exhibited by a higher load of *B. gibsoni* in the midgut and ovary of *longicin*-silenced ticks after infestation in an infected host [131]. Meanwhile, JAK-STAT pathway was shown to be important in *Anaplasma phagocytophilum* infection in ticks after its knockdown increased the infection in the salivary glands of nymphs that fed on infected mice [135]. A dual oxidase and a peroxidase, ISCW017368, which together forms a dityrosine network, were separately silenced in *I. scapularis*, both resulting to reduced *Borrelia burgdorferi* persistence in ticks [136].

The obligatory blood feeding lifestyle of ticks exposes them to high levels of pro-oxidants that may trigger oxidative stress. Antioxidant enzymes function to protect them from the harmful effects of oxidative stress. Furthermore, these antioxidant enzymes provide detoxification mechanisms to counteract toxins that they encounter in the environment, such as chemical acaricides. RNAi has been very useful in evaluating the function of these antioxidants. Silencing a selenoprotein in *R.*(*B.*) *microplus* reduced the engorged body weight and egg output [45]. In contrast, a study on *A. maculatum* showed that silencing two selenoproteins did not alter blood feeding, although the egg output was reduced. Interestingly, the total antioxidant capacities of the saliva from knockdowned ticks were higher, indicating that other antioxidant enzymes may have compensated for the absence of selenoproteins [138]. In another study, silencing thioredoxin reductase, another selenoprotein, in *A. maculatum* did not have a negative impact on blood feeding and reproduction. Likewise, variations in transcriptional expression of some antioxidant enzymes were also observed, suggesting compensatory mechanism in the absence of thioredoxin reductase [139]. However, the more interesting finding in that study was the decreased microbiota population following thioredoxin reductase knockdown, possibly because of disturbed redox homeostasis balance. Meanwhile, silencing a glutathione S-transferase (GST) gene affected the attachment of ticks and reduced the post-blood meal bodyweight of *R.* (*B.*) *microplus* [45]. It also made *R. sanguineus* ticks more susceptible to permethrin, although no significant effects on tick attachment, feeding and reproductive capacity were observed [137].

#### **6. Understanding tick-pathogen interaction through RNAi**

knockdown affected the expression of several genes involved in multiple cellular pathways, suggesting a role in gene expression by interacting with regulatory proteins [154]. Aside from being reported as a promising anti-tick vaccine antigen candidate in many studies, it has been also proposed that subolesin may be targeted in ticks that subsequently will be released for

The membrane-bound glycoprotein Bm86 expressed mainly in the midgut of *R.* (*B.*) *micro‐ plus* [155] is the first, and until recently, the only tick antigen that is commercially available as an anti-tick vaccine in some countries. The exact function of Bm86, however, remains unclear yet. RNAi has been employed to knockdown *Bm86* and its homologues in other tick species, including *R. sanguineus*, *H. longicornis*, and *R. evertsi evertsi*, which in most cases affected the blood feeding and reproduction of adult ticks, except in *R. evertsi evertsi* wherein knockdown of two homologues did not yield significant effects [130]. A study in *R.* (*B.*) *microplus* also showed that knockdown of *Bm86* decreased the blood feeding capacity and survival of ticks after feeding on a *B. bovis-*infected host, suggesting that Bm86 may have a critical role in the

The function of some components of immunity, such as α2-macroglobulin proteins [132, 133], antimicrobial peptides [131], Janus kinase (JAK)-signaling transducer activator of transcription (STAT) pathway [135], dityrosine network [136], and cysteine protease inhibitor in the hemocytes [140] have been analyzed using RNAi. The α2-macroglobulin proteins of *I. ricinus*, related to vertebrate complement system, were shown to be involved in the phagocytic activity of hemocytes against Gram-negative bacteria [132, 133]. In contrast, a cysteine protease Rmcystatin3 identified in *R.* (*B.*) *microplus* was implicated as a negative modulator of tick immune response after its silencing greatly reduced the number of bacterial load in the ticks [140]. The role of a defensin from *H. longicornis*, longicin, in ticks' immune defense against *Babesia* parasites was demonstrated through RNAi, as exhibited by a higher load of *B. gibsoni* in the midgut and ovary of *longicin*-silenced ticks after infestation in an infected host [131]. Meanwhile, JAK-STAT pathway was shown to be important in *Anaplasma phagocytophilum* infection in ticks after its knockdown increased the infection in the salivary glands of nymphs that fed on infected mice [135]. A dual oxidase and a peroxidase, ISCW017368, which together forms a dityrosine network, were separately silenced in *I. scapularis*, both resulting to reduced

The obligatory blood feeding lifestyle of ticks exposes them to high levels of pro-oxidants that may trigger oxidative stress. Antioxidant enzymes function to protect them from the harmful effects of oxidative stress. Furthermore, these antioxidant enzymes provide detoxification mechanisms to counteract toxins that they encounter in the environment, such as chemical acaricides. RNAi has been very useful in evaluating the function of these antioxidants. Silencing a selenoprotein in *R.*(*B.*) *microplus* reduced the engorged body weight and egg output [45]. In contrast, a study on *A. maculatum* showed that silencing two selenoproteins did not alter blood feeding, although the egg output was reduced. Interestingly, the total antioxidant capacities of the saliva from knockdowned ticks were higher, indicating that other antioxidant enzymes may have compensated for the absence of selenoproteins [138]. In another study, silencing thioredoxin reductase, another selenoprotein, in *A. maculatum* did not have a negative

fitness of ticks after feeding from an acutely *B. bovis-*infected host [129].

sterile acarine technique (SAT) [38].

428 RNA Interference

*Borrelia burgdorferi* persistence in ticks [136].

RNAi has undoubtedly paved a way to better understand the different aspects of ticks' association with various pathogens. Numerous tick proteins with different functions have been found to be involved in the acquisition, establishment, and transmission of pathogens. Several proteins have been studied through RNAi to determine their importance in the development cycle of different pathogens. The knockdown of subolesin [142, 156], GST, vATPase, and selenoprotein M [142] in *D. variabilis*, and putative von Willebrand factor, flagelliform silk protein and subolesin in *R.* (*B.*) *microplus* [143] decreased the infection level of *A. marginale*in these hard ticks, implying that these proteins are significant in the establish‐ ment of infection of this rickettsia.

RNAi also demonstrated that the Lyme disease agent *B. burgdorferi* can utilize several proteins of *I. scapularis* to facilitate its transmission to the host. These include salivary proteins such as tick histamine release factor [147], Salp15 [52], and the lectin complement pathway inhibitor (TSLPI) [145]; the latter two provide protection for *B. burgdorferi* against components of the host immune system. Salivary proteins Salp14 [49], Salp16 [48], and Salp25D [44] have been examined for their function in acquiring *A. phagocytophilum* or *B. burgdorferi* through RNAi. Knockdown of Salp14 did not affect the acquisition of either rickettsiae, whereas the knock‐ down of Salp16 and Salp25D decreased the infection level of *A. phagocytophilum* and *B. burgdorferi* in the tick, respectively.

An interesting study on *I. scapularis* showed that *A. phagocytophilum* promotes cold tolerance through an antifreeze glycoprotein [148]. In the absence of this antifreeze glycoprotein, the survival rate of ticks after exposure to extremely cold temperature and the infection level of *A. phagocytophilum* following exposure was reduced. Tick defensins, varisin from *D. variabi‐ lis* [144], and ricinusin from *Rhipicephalus* ticks [126] have been silenced to examine their functions in pathogen establishment; the former reduced *A. marginale*infection level, while the latter did not have an effect on *B. bigemina* infection.

Several reports also demonstrated the interaction of *Babesia* parasites and tick proteins through RNAi. Knockdown of the immunophilin gene in *R.* (*B.*) *microplus* had negative impact on the reproductive performance of the tick and also increased the infection rate of *B. bovis* in larval progeny [41], while knockdown of TROSPA, serum amyloid A, and calreticulin reduced the infection level of *B. bigemina* in *Rhipicephalus* ticks [126]. Silencing a Kunitz-type serine protease inhibitor from *D. variabilis* increased the rickettsial infection in the midgut [146], whereas in *R.* (*B.*) *microplus*, silencing a Kunitz-type serine protease inhibitor, Spi, tended to increase the infection rate of *B. bovis* in larval progeny [41], but silencing of another Kunitz-type protease inhibitor 5 (KTPI) did not have any effect on *Babesia* infection [126].

#### **7. Future directions in tick research and application in tick control**

Indeed, great progress in understanding tick biology has been already accomplished in the past. However, many aspects of tick physiology and host-tick-pathogen interaction need to be unraveled yet. Moreover, several optimizations can still be done to improve RNAi in tick research. While being the most widely used method of introducing dsRNA, the injection method (particularly microinjection) that requires elaborate equipment may not be accessible to all laboratories. Moreover, injection is mostly applicable to adult and sometimes nymphal stages, and may be injurious to the ticks, especially when performed by an inexperienced researcher. The soaking method is simpler, less invasive, and less laborious. Electroporation has been recently shown to be effective in introducing dsRNA in eggs [66] and may be useful in studying the function of genes that are involved in embryogenesis and physiology of immature tick stages.

RNAi may also prove to be a promising tick control method and not just a research tool. In pest insect management, the possibility of using RNAi as a novel tool of pest control is already being explored by feeding liposome-coated dsRNA or dsRNA expressed in transgenic plants or bacteria [32]. RNAi targeting several genes have been accomplished by feeding plants expressing dsRNA in several species of economically important crop pests [16]. Feeding dsRNA to ticks is still an underdeveloped approach, which has been yet accomplished only by artificial feeding. Coating dsRNAs with liposomes or nanocarriers may increase dsRNA stability that may make it feasible for administration to the host. Genes that are highly conserved across different tick species, and are of importance in tick survival are good candidate targets. These include proteins with structural and metabolic functions, such as ubiquitin, tropomyosin, and ferritin. However, the specificity of dsRNA to the tick gene should be highly considered. Additional consideration would be the establishment of a minimum effective dose, since the synthesis of dsRNA is costly.

Additionally, RNAi has been proposed as an alternative method for the sterile insect technique in blood-sucking mosquitoes that will produce sterile males by feeding dsRNA in mosquito larvae [64]. Quite similarly, the application of RNAi for tick control was also proposed in a single report on *D. variabilis*, wherein the highly conserved subolesin was targeted leading to reproductive incapacity [38]. In conclusion, the authors suggested that RNAi may be used to massively produce sterile ticks (SAT) that may be released in the field. Releasing subolesinsilenced ticks may also aid in the control of *A. marginale*, since it has been reported that subolesin knockdown reduced the infection level of this pathogen [141–143]. Introducing dsRNA to eggs through electroporation described above may be a more convenient way of producing knockdowned ticks.

#### **8. Summary**

infection level of *B. bigemina* in *Rhipicephalus* ticks [126]. Silencing a Kunitz-type serine protease inhibitor from *D. variabilis* increased the rickettsial infection in the midgut [146], whereas in *R.* (*B.*) *microplus*, silencing a Kunitz-type serine protease inhibitor, Spi, tended to increase the infection rate of *B. bovis* in larval progeny [41], but silencing of another Kunitz-type protease

Indeed, great progress in understanding tick biology has been already accomplished in the past. However, many aspects of tick physiology and host-tick-pathogen interaction need to be unraveled yet. Moreover, several optimizations can still be done to improve RNAi in tick research. While being the most widely used method of introducing dsRNA, the injection method (particularly microinjection) that requires elaborate equipment may not be accessible to all laboratories. Moreover, injection is mostly applicable to adult and sometimes nymphal stages, and may be injurious to the ticks, especially when performed by an inexperienced researcher. The soaking method is simpler, less invasive, and less laborious. Electroporation has been recently shown to be effective in introducing dsRNA in eggs [66] and may be useful in studying the function of genes that are involved in embryogenesis and physiology of

RNAi may also prove to be a promising tick control method and not just a research tool. In pest insect management, the possibility of using RNAi as a novel tool of pest control is already being explored by feeding liposome-coated dsRNA or dsRNA expressed in transgenic plants or bacteria [32]. RNAi targeting several genes have been accomplished by feeding plants expressing dsRNA in several species of economically important crop pests [16]. Feeding dsRNA to ticks is still an underdeveloped approach, which has been yet accomplished only by artificial feeding. Coating dsRNAs with liposomes or nanocarriers may increase dsRNA stability that may make it feasible for administration to the host. Genes that are highly conserved across different tick species, and are of importance in tick survival are good candidate targets. These include proteins with structural and metabolic functions, such as ubiquitin, tropomyosin, and ferritin. However, the specificity of dsRNA to the tick gene should be highly considered. Additional consideration would be the establishment of a minimum

Additionally, RNAi has been proposed as an alternative method for the sterile insect technique in blood-sucking mosquitoes that will produce sterile males by feeding dsRNA in mosquito larvae [64]. Quite similarly, the application of RNAi for tick control was also proposed in a single report on *D. variabilis*, wherein the highly conserved subolesin was targeted leading to reproductive incapacity [38]. In conclusion, the authors suggested that RNAi may be used to massively produce sterile ticks (SAT) that may be released in the field. Releasing subolesinsilenced ticks may also aid in the control of *A. marginale*, since it has been reported that subolesin knockdown reduced the infection level of this pathogen [141–143]. Introducing dsRNA to eggs through electroporation described above may be a more convenient way of

**7. Future directions in tick research and application in tick control**

inhibitor 5 (KTPI) did not have any effect on *Babesia* infection [126].

immature tick stages.

430 RNA Interference

effective dose, since the synthesis of dsRNA is costly.

producing knockdowned ticks.

In this chapter, we have reviewed the application of RNAi in tick research and described the significant contribution of RNAi in advancing our knowledge on tick biology and tickpathogen interaction. RNAi has revolutionized the advancement of our understanding of various aspects of tick blood feeding and digestion, reproduction, metabolism, and immunity. As a functional analysis tool, RNAi has become very handy in elucidating the functions of different proteins from more than 10 hard tick species and a few soft tick species. It has been particularly helpful in screening potential target antigens for anti-tick and tick-borne pathogen vaccine development [157]. Several methods of introducing dsRNA in ticks have been employed but injection has remained to be the most widely used technique. The number of published research on ticks that involves the application of RNAi has been continuously increasing through the years, and it is expected to continue doing so. A great majority of the published reports focused on hard ticks, but due to some physiological differences, more research using RNAi on soft ticks should be conducted. Finally, with numerous potential target genes already identified, the application of RNAi as a tick control method should be investi‐ gated in the future, starting with optimization of dsRNA delivery method for practical use.

#### **Acknowledgements**

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 22580335, 25292173, and 26660229. We also thank our colleague, Melbourne R. Talactac, at the Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, for his assistance in writing this chapter.

#### **Author details**

Remil Linggatong Galay1,2, Rika Umemiya-Shirafuji3 , Masami Mochizuki1 , Kozo Fujisaki4 and Tetsuya Tanaka2\*

\*Address all correspondence to: tetsuya@ms.kagoshima-u.ac.jp

1 Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, Los Baños, Philippines

2 Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan

3 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan

4 National Agricultural and Food Research Organization, Tsukuba, Ibaraki, Japan

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## *Edited by Ibrokhim Y. Abdurakhmonov*

RNA interference (RNAi), a hallmark of all biological sciences of twenty-first century, is an evolutionarily conserved and double-stranded RNA-dependent eukaryotic cell defense process. Opportunity to utilize an organisms own gene and to systematically induce and trigger RNAi for any desired sequence made RNAi an efficient approach for functional genomics, providing a solution for conventional longstanding obstacles in life sciences. RNAi research and application have significantly advanced during past two decades. This book RNA interference provides an updated knowledge and progress on RNAi in various organisms, explaining basic principles, types, and property of inducers, structural modifications, delivery systems/methodologies, and various successful bench-to-field or clinic applications and disease therapies with some aspects of limitations, alternative tools, safety, and risk assessment.

Photo by bluebay2014 / DollarPhotoClub

RNA Interference

RNA Interference

*Edited by Ibrokhim Y. Abdurakhmonov*