**ER-targeted Intrabodies Mediating Specific** *In Vivo* **Knockdown of Transitory Proteins in Comparison to RNAi**

Oliver Backhaus and Thomas Böldicke

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62103

#### **Abstract**

In animals and mammalian cells, protein function can be analyzed by nucleotide se‐ quence-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, TALEN, zinc finger nucleases, or the RNAi technique. Alternatively, protein knock‐ down approaches are available based on direct interference of the target protein with the inhibitor.

Among protein knockdown techniques, the endoplasmic reticulum (ER) intrabodies are potent molecules for protein knockdown *in vitro* and *in vivo*. These molecules are in‐ creasingly used for protein knockdown in living cells and transgenic mice. ER intra‐ body knockdown technique is based on the retention of membrane proteins and secretory proteins inside the ER, mediated by recombinant antibody fragments. In con‐ trast to nucleotide sequence-based methods, the intrabody-mediated knockdown acts only on the posttranslational level.

In this review, the ER intrabody technology has been compared with the RNAi techni‐ que on the molecular level. The generation of intrabodies and RNAi has also been dis‐ cussed. Specificity and off-target effects (OTE) of these molecules as well as the therapeutic potential of ER intrabodies and RNAi have been compared.

**Keywords:** Knockdown techniques, intracellular antibodies, ER intrabodies, RNA inter‐ ference, off-target effects

#### **1. Introduction**

For the study of protein function in animals and mammalian cells, DNA-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, TALEN, zinc finger nucleases [1],

© 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.

as well as the RNAi technique [2] were proven and reliable tools. Besides the RNAi technique, approaches with miRNA are also very attractive [3]. Silencing of target mRNA can be achieved using siRNA, miRNA, or shRNA (Box 1).

#### **Box 1**

#### *siRNA*

Small interfering RNA (siRNA) are small pieces of double-stranded (ds) RNA, usually about 21 nt long, with 2-nt-long 3′ overhangs at each end. They can be applied for the interference with the protein translation by binding to the messenger RNA (mRNA), whereby promoting the degradation or destabi‐ lization of the mRNA.

#### *shRNA*

shRNAs form hairpin structures, which consist of a stem region of paired antisense and sense strands, connected by unpaired nucleotides building a loop. They are converted into siRNAs by the same RNAi machinery that processes miRNAs.

#### *miRNA*

MicroRNAs are small RNA molecules, encoded in the genome of plants and animals. These highly conserved, ~21-mer RNAs regulate the expression of genes by binding to the 3' untranslated regions (3'- UTR) of specific mRNAs.

Protein knockdown is possible with small molecule inhibitors including peptides, neutralizing and intracellular antibodies, and allosteric modulators [4–8]. In addition, aptamers and intramers, in general short single-stranded DNA or RNA oligonucleotides are also potent molecules for specific inhibition of small molecules, peptides, proteins, or even whole living cells [9].

Currently, RNAi is the most often used gene-silencing technique in functional genomics [2]. In this article, we described an emerging protein knockdown technology using intracellular antibodies (intrabodies) targeted to the ER and compared the advantages and disadvantages of this promising technique with the RNAi technology. We tried to make scientists, who are interested in protein research or have very specific protein-related questions, familiar with the ER intrabody technology [10]. The molecular mechanisms of both methods are different. RNAi-mediated knockdown is based on the interference of siRNA with mRNA (Figure 1), whereas the protein knockdown by ER intrabodies is exerted upon binding of a recombinant antibody fragment to its specific antigen inside the ER [10] (Figure 2).

Intrabodies are recombinant antibody fragments targeted to a cell expressing the specific antigen. Intracellular binding of the intrabody to the antigen results in inhibition of antigen function. Moreover, intrabodies can specifically be targeted to subcellular compartments such

ER-targeted Intrabodies Mediating Specific *In Vivo* Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103 139

as well as the RNAi technique [2] were proven and reliable tools. Besides the RNAi technique, approaches with miRNA are also very attractive [3]. Silencing of target mRNA can be achieved

Small interfering RNA (siRNA) are small pieces of double-stranded (ds) RNA, usually about 21 nt long, with 2-nt-long 3′ overhangs at each end. They can be applied for the interference with the protein translation by binding to the messenger RNA (mRNA), whereby promoting the degradation or destabi‐

shRNAs form hairpin structures, which consist of a stem region of paired antisense and sense strands, connected by unpaired nucleotides building a loop. They are converted into siRNAs by the same RNAi

MicroRNAs are small RNA molecules, encoded in the genome of plants and animals. These highly conserved, ~21-mer RNAs regulate the expression of genes by binding to the 3' untranslated regions (3'-

Protein knockdown is possible with small molecule inhibitors including peptides, neutralizing and intracellular antibodies, and allosteric modulators [4–8]. In addition, aptamers and intramers, in general short single-stranded DNA or RNA oligonucleotides are also potent molecules for specific inhibition of small molecules, peptides, proteins, or even whole living

Currently, RNAi is the most often used gene-silencing technique in functional genomics [2]. In this article, we described an emerging protein knockdown technology using intracellular antibodies (intrabodies) targeted to the ER and compared the advantages and disadvantages of this promising technique with the RNAi technology. We tried to make scientists, who are interested in protein research or have very specific protein-related questions, familiar with the ER intrabody technology [10]. The molecular mechanisms of both methods are different. RNAi-mediated knockdown is based on the interference of siRNA with mRNA (Figure 1), whereas the protein knockdown by ER intrabodies is exerted upon binding of a recombinant

Intrabodies are recombinant antibody fragments targeted to a cell expressing the specific antigen. Intracellular binding of the intrabody to the antigen results in inhibition of antigen function. Moreover, intrabodies can specifically be targeted to subcellular compartments such

antibody fragment to its specific antigen inside the ER [10] (Figure 2).

using siRNA, miRNA, or shRNA (Box 1).

**Box 1**

138 RNA Interference

*siRNA*

*shRNA*

*miRNA*

cells [9].

lization of the mRNA.

machinery that processes miRNAs.

UTR) of specific mRNAs.

**Figure 1. Principle of the knockdown of transitory proteins using the RNA interference technique.** For knockdown of the mRNA of transitory proteins, transfection with a specific shRNA-expressing plasmid is sufficient. Although by using the RNA interference technology all kinds of proteins could be targeted, only knockdown of transitory proteins is illustrated. (1) Specific shRNA is transcribed and processed by the RNase III Dicer-1 enzyme in mammalian cells in order to form the mature siRNA. (2) The Argonaute 2 protein (Ago2) is loaded with the siRNA and forms together with additional proteins the RNA-induced silencing complex (RISC), which is a multiprotein complex consisting of ef‐ fector (Argonaute proteins), accessory proteins, and si/miRNA. During the loading of the Argonaute protein, one strand of the siRNA duplex is discarded. Next, the RISC complex associates with its target mRNA via complementary base pairing of the siRNA and the target mRNA. In many cases, the recognition site comprises the 3′ untranslated re‐ gions (UTR) of the mRNA. Finally, target binding leads to mRNA degradation or translational inhibition [11]. mRNA degradation is mediated through the endonuclease activity of the Argonaute proteins. (3) As a result of mRNA knock‐ down, the target protein is not expressed on the cell surface [11].

**Figure 2. Principle of the specific knockdown of transitory proteins with endoplasmic reticulum (ER)-retained in‐ trabodies.** In wild-type cells, transitory proteins are transported through the ER and can be further processed (e.g., gly‐ cosylated) in the Golgi apparatus. These proteins could reside in the secretory cell compartments, secreted through the plasma membrane (PM), or become integrated in the PM as a membrane protein. For functional inhibition of these pro‐ teins, transfection with an ER intrabody expressing plasmid is sufficient. The intrabody construct consists of an N-ter‐ minal secretion sequence for the translocation in the ER (leader sequence) and the C-terminal retention signal (KDEL). (1) The intrabody inside of the ER binds to the target protein. This complex of antibody and target protein is further processed and transported through the secretory pathway. (2) In the cis-cisterna of the Golgi stack, the hERD2 receptor binds to the KDEL sequence and (3) initiates the retrograde transport back to the ER compartment. This continuous binding of the intrabody and retrograde transport prevents the target protein to reach its localization where it normal‐ ly acts. (4) The accumulated intrabody–antigen complex in the ER might be transported into the cytoplasm, where it is marked for degradation by the 26S proteasome [12, 13]. Böldicke and Burgdorf have shown that an anti-toll-like recep‐ tor 2 (TLR2) ER intrabody is degraded by the proteasome (unpublished data).

as the nucleus, cytoplasm, mitochondria, or ER [10] (Box 2). Currently, the most used and promising intrabodies are the ER intrabodies, because of the correct folding in the oxidative environment of the ER [14]. This contrasts with cytosolic intrabodies, in which disulfide bridges are not formed in the reducing environment of the cytoplasm [15, 16].

#### **Box 2**

**Figure 2. Principle of the specific knockdown of transitory proteins with endoplasmic reticulum (ER)-retained in‐ trabodies.** In wild-type cells, transitory proteins are transported through the ER and can be further processed (e.g., gly‐ cosylated) in the Golgi apparatus. These proteins could reside in the secretory cell compartments, secreted through the plasma membrane (PM), or become integrated in the PM as a membrane protein. For functional inhibition of these pro‐ teins, transfection with an ER intrabody expressing plasmid is sufficient. The intrabody construct consists of an N-ter‐ minal secretion sequence for the translocation in the ER (leader sequence) and the C-terminal retention signal (KDEL). (1) The intrabody inside of the ER binds to the target protein. This complex of antibody and target protein is further processed and transported through the secretory pathway. (2) In the cis-cisterna of the Golgi stack, the hERD2 receptor binds to the KDEL sequence and (3) initiates the retrograde transport back to the ER compartment. This continuous binding of the intrabody and retrograde transport prevents the target protein to reach its localization where it normal‐ ly acts. (4) The accumulated intrabody–antigen complex in the ER might be transported into the cytoplasm, where it is marked for degradation by the 26S proteasome [12, 13]. Böldicke and Burgdorf have shown that an anti-toll-like recep‐

tor 2 (TLR2) ER intrabody is degraded by the proteasome (unpublished data).

140 RNA Interference

*Intrabodies* are intracellularly expressed recombinant antibody fragments, which specifically inhibit the function of target proteins produced in the same cell [10].

*ER Intrabodies* retain their corresponding antigen inside the ER by inhibiting the translocation of the antigen to the cell compartment where it normally acts.

*Cytosolic Intrabodies* are expressed in the cytoplasm. They inactivate their targets or interfere with the binding of the target protein to its corresponding binding partner.

The effect of ER intrabodies is based on retention of proteins passing the secretory pathway. Secretory proteins, membrane proteins, and even Golgi or endosomal-located proteins can be targeted [17–19], which cannot be reached by classical antibodies, due to the extracellular presence. Successful functional knockdown was achieved for oncogenic receptors, viral proteins for preventing virus assembly, cellular virus receptors to block virus entry, and receptors of the immune system as well as of the nervous system [20–24].

The format of expressed intrabodies is, in general, the single-chain variable fragment (scFv) or less common the antigen-binding fragment (Fab) [25]. The only prerequisite of ER intrabodies is the efficient binding to the antigen, and the method to select and generate an ER intrabody is greatly simplified by phage display. On the contrary, functional cytoplasmic intrabodies have to inactivate the antigen or have to interfere with the binding of the target protein to its corresponding binding partner [10].

The starting material for construction of an ER intrabody is an scFv or Fab, which can be obtained by amplification of the variable domains from a hybridoma clone [26], or scFv fragments can be selected from phage or yeast display [27, 28].

Early attempts using the intrabody approach failed frequently due to the lack of reliable techniques for the identification of the correct functional antibody sequence from a hybridoma clone. The genes of the variable domains for construction of recombinant antibody fragments can be amplified from hybridoma clones using mixtures of consensus primers [29]. This approach was used in the beginning. As hybridoma cells could secrete several different antibodies, it was sometimes difficult to isolate the correct functional sequences of the variable domains. Presently, with reliable protein sequencing techniques, next generation of DNA sequencing and optimized consensus primer sequences, the functional antibody DNA can much better be identified. Furthermore, optimized strategies for amplification of the correct functional antibody sequence are available [30–32].

In the case of using *in vitro* display systems, like phage or yeast cell surface display, the selected scFv fragment has only to be cloned into the ER-targeting vector. For preliminary characteri‐ zation of the intrabody function, co-transfection of the intrabody expression plasmid with the corresponding antigen expression plasmid into HEK 293 cells is sufficient and followed by coimmunoprecipitation and immunofluorescence analysis [33].

In contrast to the ER intrabody technology, the advantage of the RNAi is that it can be applied for almost every mRNA and also non-coding RNAs. Here, we further compared the RNAi with the intrabody technology, regarding specificity, off-target effects, and therapeutic approaches.

#### **2. Intracellular intrabodies versus RNA interference**

#### **2.1. Generation of ER intrabodies**

The prerequisite for generating intrabodies is the availability of a hybridoma antibody clone or scFv/Fab fragments selected from *in vitro display* systems [10]. Starting from a hybrido‐ ma clone, the variable domains of the heavy and light chain are amplified by PCR from the cDNA. This can be achieved by (1) PCR amplification using consensus primer [29, 34–36], (2) rapid amplification of cDNA ends (RACE) [30], (3) PCR amplification using adaptorligated cDNA [31], or (4) inverse PCR with constant region heavy chain and light chain primer, amplifying the corresponding antibody sequence from circularized double-strand‐ ed cDNA [32] (Figure 3 A).

In most cases, using consensus primers is a fast and efficient approach for amplification of the correct functional antibody sequence from a hybridoma clone. However, less-common nonconsensus antibody sequences cannot be amplified and primer mismatching could be a problem. Approaches (no. 2–4 shown in Figure 3) for amplifying the variable antibody domains are more time-consuming; however, the correct functional antibody gene sequence can be obtained. The variable domains are compiled by assembly PCR, linking both variable domains together by a short flexible linker sequence, for example (Gly4Ser)3, resulting in the scFv fragment. Next, the scFv fragment will be cloned into the ER targeting vector, providing the ER signal sequence, an myc tag for detection of intrabody, and the KDEL retention sequence localized at the C-terminus of the intrabody gene [26].

Following the *in vitro* display pipeline, an scFv fragment or Fab fragment selected by phage or yeast cell surface display can directly be cloned into the ER targeting vector. Most recombi‐ nant antibody fragments in the scFv or Fab format are selected by phage display or also the frequently used yeast cell surface display [27, 28, 41]. Other *in vitro* display systems are bacterial, mammalian cell surface display, or ribosome display [42–44]. Cytoplasmic intra‐ bodies are generated from hybridoma clones or scFv/Fab fragments from *in vitro* display libraries in a similar way and cloned into an appropriate cytosolic targeting vector [10]. The main difference in comparison to ER intrabodies is that cytosolic intrabodies have to demon‐ strate neutralizing activity, and furthermore stable folding antibody fragments have to be selected [10].

ER-targeted Intrabodies Mediating Specific *In Vivo* Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103 143

In the case of using *in vitro* display systems, like phage or yeast cell surface display, the selected scFv fragment has only to be cloned into the ER-targeting vector. For preliminary characteri‐ zation of the intrabody function, co-transfection of the intrabody expression plasmid with the corresponding antigen expression plasmid into HEK 293 cells is sufficient and followed by co-

In contrast to the ER intrabody technology, the advantage of the RNAi is that it can be applied for almost every mRNA and also non-coding RNAs. Here, we further compared the RNAi with the intrabody technology, regarding specificity, off-target effects, and therapeutic

The prerequisite for generating intrabodies is the availability of a hybridoma antibody clone or scFv/Fab fragments selected from *in vitro display* systems [10]. Starting from a hybrido‐ ma clone, the variable domains of the heavy and light chain are amplified by PCR from the cDNA. This can be achieved by (1) PCR amplification using consensus primer [29, 34–36], (2) rapid amplification of cDNA ends (RACE) [30], (3) PCR amplification using adaptorligated cDNA [31], or (4) inverse PCR with constant region heavy chain and light chain primer, amplifying the corresponding antibody sequence from circularized double-strand‐

In most cases, using consensus primers is a fast and efficient approach for amplification of the correct functional antibody sequence from a hybridoma clone. However, less-common nonconsensus antibody sequences cannot be amplified and primer mismatching could be a problem. Approaches (no. 2–4 shown in Figure 3) for amplifying the variable antibody domains are more time-consuming; however, the correct functional antibody gene sequence can be obtained. The variable domains are compiled by assembly PCR, linking both variable domains together by a short flexible linker sequence, for example (Gly4Ser)3, resulting in the scFv fragment. Next, the scFv fragment will be cloned into the ER targeting vector, providing the ER signal sequence, an myc tag for detection of intrabody, and the KDEL retention sequence

Following the *in vitro* display pipeline, an scFv fragment or Fab fragment selected by phage or yeast cell surface display can directly be cloned into the ER targeting vector. Most recombi‐ nant antibody fragments in the scFv or Fab format are selected by phage display or also the frequently used yeast cell surface display [27, 28, 41]. Other *in vitro* display systems are bacterial, mammalian cell surface display, or ribosome display [42–44]. Cytoplasmic intra‐ bodies are generated from hybridoma clones or scFv/Fab fragments from *in vitro* display libraries in a similar way and cloned into an appropriate cytosolic targeting vector [10]. The main difference in comparison to ER intrabodies is that cytosolic intrabodies have to demon‐ strate neutralizing activity, and furthermore stable folding antibody fragments have to be

immunoprecipitation and immunofluorescence analysis [33].

**2. Intracellular intrabodies versus RNA interference**

localized at the C-terminus of the intrabody gene [26].

approaches.

142 RNA Interference

**2.1. Generation of ER intrabodies**

ed cDNA [32] (Figure 3 A).

selected [10].

**Figure 3. Generation of intrabody and RNA interference knockdown constructs.** (**A**) Generation of intrabody knock‐ down vectors. The scFv fragment could be either cloned from hybridoma cell lines or selected from huge human naive phage display libraries. The antibody variable domain of the light chain (VL) and heavy chain (VH) is amplified from cDNA using consensus primer mixtures (1), 5′ adapter-ligated PCR or rapid amplification of cDNA ends (RACE) (2) or with constant domain-specific primer from circularized cDNA (3). The antibody VL and VH genes are assembled as scFv by fusing both domains with a flexible (Gly4Ser)3-linker sequence and cloned into the ER targeting intrabody vec‐ tor. The scFvs are cloned between an upstream secretion signal and a downstream retention sequence (KDEL). Using the phage display system, selected scFvs can directly be cloned into the intrabody vector in one cloning step. Shown is an ER-targeting vector. (**B**) Generation of siRNA/shRNA/miRNA knockdown vectors. Rational *in silico* design of siR‐ NA, shRNA, or miRNA mimics using software algorithms like those mentioned in Ref. [37] or in Ref. [38], a recent publication, deduced from the target cDNA. The algorithms are designed to select appropriated sequences by means of empiric criteria. Main criteria are an siRNA length of 19–21 nucleotides (nt) in conjunction with 2 nt overhangs at

their 3′ ends, as well as thermodynamic properties of target mRNA hybridization. Rational design can be expanded by testing *in silico* the potential off-target effects of the designed sequences by using genome-wide enrichment of seed se‐ quence matches (GESS) [39] or Haystack [40]. Designed sequences are chemically synthesized and cloned into appro‐ priate mammalian or viral knockdown expression vectors. Alternatively, siRNA can be used for direct cell transfection. The siRNA/shRNA/miRNA sequences originated from rational design are screened for effective processing, specific knockdown capabilities, and potential off-target effects. Corresponding clones are selected, and for most applications 3–4 different targeting sequences were chosen and theses libraries are used for the RNA interference knockdown. ER: endoplasmic reticulum, CDS: coding DNA sequence, p:promoter.

#### **2.2. Generation of siRNA, miRNA, and shRNA**

In order to generate siRNAs for a specific target, only the mRNA information about the target sequence is needed [45] (Figure 3 B). siRNA-mediated mRNA knockdown can be performed in several ways. In general, cells can directly be transfected with siRNA, using transfection reagents like lipofectamine. Cells can also be transfected using siRNA/shRNA/miRNAexpressing plasmids or viral vectors. Long-lasting gene silencing can be achieved with shRNAs expressed from stably transfected plasmids or from integrated retro- or lentiviral vectors [46]. Several approaches exist for RNA interference-mediated knockdown, the principle workflow of *in silico* design, screening, and selection of siRNA/shRNA/miRNA, with best knockdown properties shown in Figure 3 B. Currently, software algorithms mentioned in Ref. [37] or [38] can help to find the appropriate knockdown sequences of 19–21 nt length siRNA by analysis of the optimal thermodynamic properties of mRNA hybridization. Potential off-target effects can be reduced by *in silico* optimization with GESS [39] or Haystack [40]. Resulting siRNA/ shRNA/miRNA sequences are tested for effective processing, specific knockdown capability, and low off-target effects.

#### **2.3. Stability**

Intrabodies are stably expressed inside the ER [14], whereas most cytosolic intrabodies are not correctly folded [15, 16]. On the other hand, siRNA can be cleaved by nucleases, present in the blood serum and cellular cytoplasm.

#### **2.4. Specificity and Off-Target Effects (OTE)**

For the knockdown of distinct target proteins, the specificity of the process is crucial. Other‐ wise, the resulting phenotypes of the induced knockdown experiment might be superimposed with off-target effects. The specificity of the RNAi and the ER intrabody knockdown technique is the main difference between them.

Intrabodies, which are also known as intracellular antibodies, are generated from monoclonal antibodies (mAbs) and phage or yeast antibody repertoires. Intrabodies are very specific to their targets due to antibody–antigen interactions.

The high specificity of ER intrabodies has been demonstrated for the specific knockdown of members of the TLRs. The knockdown of toll-like receptor 2 (TLR2) and TLR9, which functions as a part of the innate immunity and recognizes pathogen-associated molecular patterns (PAMP), did not influence the expression of other TLRs. The developed anti-TLR2 intrabody

did not inhibit TLR3-, TLR4-, and TLR9-driven signal transduction [33] and the anti-TLR9 intrabody did not inhibit TLR3-, TLR4-, TLR7-, and TLR8-driven signaling, respectively [18].

their 3′ ends, as well as thermodynamic properties of target mRNA hybridization. Rational design can be expanded by testing *in silico* the potential off-target effects of the designed sequences by using genome-wide enrichment of seed se‐ quence matches (GESS) [39] or Haystack [40]. Designed sequences are chemically synthesized and cloned into appro‐ priate mammalian or viral knockdown expression vectors. Alternatively, siRNA can be used for direct cell transfection. The siRNA/shRNA/miRNA sequences originated from rational design are screened for effective processing, specific knockdown capabilities, and potential off-target effects. Corresponding clones are selected, and for most applications 3–4 different targeting sequences were chosen and theses libraries are used for the RNA interference knockdown. ER:

In order to generate siRNAs for a specific target, only the mRNA information about the target sequence is needed [45] (Figure 3 B). siRNA-mediated mRNA knockdown can be performed in several ways. In general, cells can directly be transfected with siRNA, using transfection reagents like lipofectamine. Cells can also be transfected using siRNA/shRNA/miRNAexpressing plasmids or viral vectors. Long-lasting gene silencing can be achieved with shRNAs expressed from stably transfected plasmids or from integrated retro- or lentiviral vectors [46]. Several approaches exist for RNA interference-mediated knockdown, the principle workflow of *in silico* design, screening, and selection of siRNA/shRNA/miRNA, with best knockdown properties shown in Figure 3 B. Currently, software algorithms mentioned in Ref. [37] or [38] can help to find the appropriate knockdown sequences of 19–21 nt length siRNA by analysis of the optimal thermodynamic properties of mRNA hybridization. Potential off-target effects can be reduced by *in silico* optimization with GESS [39] or Haystack [40]. Resulting siRNA/ shRNA/miRNA sequences are tested for effective processing, specific knockdown capability,

Intrabodies are stably expressed inside the ER [14], whereas most cytosolic intrabodies are not correctly folded [15, 16]. On the other hand, siRNA can be cleaved by nucleases, present in the

For the knockdown of distinct target proteins, the specificity of the process is crucial. Other‐ wise, the resulting phenotypes of the induced knockdown experiment might be superimposed with off-target effects. The specificity of the RNAi and the ER intrabody knockdown technique

Intrabodies, which are also known as intracellular antibodies, are generated from monoclonal antibodies (mAbs) and phage or yeast antibody repertoires. Intrabodies are very specific to

The high specificity of ER intrabodies has been demonstrated for the specific knockdown of members of the TLRs. The knockdown of toll-like receptor 2 (TLR2) and TLR9, which functions as a part of the innate immunity and recognizes pathogen-associated molecular patterns (PAMP), did not influence the expression of other TLRs. The developed anti-TLR2 intrabody

endoplasmic reticulum, CDS: coding DNA sequence, p:promoter.

**2.2. Generation of siRNA, miRNA, and shRNA**

and low off-target effects.

blood serum and cellular cytoplasm.

is the main difference between them.

**2.4. Specificity and Off-Target Effects (OTE)**

their targets due to antibody–antigen interactions.

**2.3. Stability**

144 RNA Interference

Stress response induction in the endoplasmic reticulum (ER), due to the accumulation of retained and partially unfolded target proteins upon intrabody–antigen complex formation, was analyzed by measuring the unfolded protein response (UPR) for an overexpressed antip75NTR ER intrabody and could not be proven [24]. No off-target effects of expressed intrabodies are known yet, particularly any activation of the immune system.

On the other hand, unspecific silencing is a major problem using RNAi-mediated gene silencing, due to the expression of short-interfering RNA sequences, such as miRNA, siRNA, shRNA, or dsRNA [47]. The short seed region of these silencing RNAs recognizes and hybridizes with 2–8 nt to the target mRNA. Even with specific alignment software, it is practically impossible to exclude any possible transcript, which aligns with the target seed sequence, because statistically the chance is high to have the same sequence or secondary structure in other non-target mRNA transcripts too. However, at least software algorithms such as GESS [39] and Haystack [40] are able to predict potential off-targeted genes. By computer-aided optimization of the miRNA, siRNA, or shRNA, the OTEs can be reduced to a minimum.

siRNA can bind to TLR3, TLR7, and TLR8, resulting in secretion of type I interferon and proinflammatory cytokines [48–50]. Aberrant expression of up to more than 1000 genes has also been described [51].

Fortunately, some progress has been made in the repression of the RNAi-induced immune response. When siRNA is *in vitro* transcribed by the T7 polymerase, a 5′-triphosphate group is added. The 5′ triphosphate is recognized by the innate immunity, and it activates the type I interferon response. This can be prevented by chemical synthesis of siRNA, which misses the 5′-triphosphate group. Furthermore, the siRNA molecules can be modified by adding 2′-Omethyl groups, in order to reduce the recognition by toll-like receptors (TLRs) [52]. Interest‐ ingly, this modification additionally hampers degradation of the siRNA by RNases, leading to an increase in serum half-life [53]. Finally, strong destabilizing unlocked nucleic acids (UNAs), which were altered to have an acyclic ribose, also reduce the recognition by TLRs [54].

The specific suppression of one allele in heterozygous genes is of concern in dominantly inherited genetic disorders. Huntington's disease (HD) is caused by a dominant mutation of the huntingtin protein (Htt) and an excellent target for the examination of allele-specific knockdown of the mutated Htt, with high therapeutic potential. Huntington's disease is based on a long stretch of CAG triplets on one disease-caused allele [55]. Most of the patients are heterozygous for the *htt* gene mutation and 48% of the American and European HD patients are heterozygous at a single nucleotide polymorphism (SNP) site, making this genetic disease a *bono fide* target for specific protein knockdown. Approaches to inhibit the appearance of Huntington's disease is silencing of wild type and mutant Htt or silencing of only the diseasecausing allele.

Although it was found in HD mice that co-silencing of wild type and mutant Htt provides therapeutic benefit, nothing is known of such a long-term suppression of huntingtin [56]. Thus, the effect and safety over decades have yet to be proven in clinical trials. Therefore, there is still a need for high allele-specific inhibition of the mutant Htt protein, which is toxic due to an expanded polyglutamine (polyQ) motif (CAG motif). Targeting of the CAG motif is not selective for the mutant allele and affected both alleles. Genotyping of the Huntington's disease patients resulted in three single nucleotide polymorphisms (SNP) in huntingtin [57]. Therefore, an alternative strategy when using RNAi is targeting a single nucleotide mutation localized in the disease-caused sequence [58]. Furthermore, targeting of the mutant huntingtin SNPs or the expanded CAG motif by designed artificial miRNAs was recently demonstrated *in vitro*, using an allele-specific reporter system and *in vivo* in a transgenic mouse model [59].

For the RNAi technique, it is possible to discriminate between very similar targets with a specific reduction on the RNA and protein level [58, 60, 61]. However, there are still some concerns and limitations. The RNAi-mediated allele-specific knockdown may result in a broad off-targeting and therefore has to be further evaluated in appropriate preclinical model systems [62]. Using both target strategies, CAG motif and prevalent mutant SNPs, in the case of huntingtin, the wild-type allele is also affected by the knockdown, and the knockdown ratio between the wild type and mutant allele remains unsatisfactory. Furthermore, the shift to *in vivo* delivery systems can have a substantial impact on the specificity, as was demonstrated in the mouse model [59]. Next, a limited expression of the miRNA vectors is important to avoid saturation of the miRNA processing machinery, as the selectivity seems to be reduced when miRNAs are highly expressed *in vivo* [59].

Different alleles can also be targeted and discriminated using specific intracellular antibodies (intrabodies) and represent a valuable alternative to RNA interference. Intrabodies targeting, for example, huntingtin have to recognize an epitope common in most disease-associated huntingtin SNP forms, which also has to be different in the translated amino acid between the mutant and wild-type allele. Alternatively, they could target the expanded polyglutamine (polyQ) motif associated with misfolding and aggregation [63]. Furthermore, cytoplasmic intrabodies have been developed, which efficiently inhibited aggregation of mutant HD [64]. Interestingly, a disulfide bond-free single-domain intracellular antibody with high affinity was developed after affinity maturation [65] from a specific anti-HD scFv fragment, demonstrating the power of antibody engineering.

For the allele-specific knockdown, the intrabody technology utilizes the high specificity of monoclonal antibodies, with no or low concerns about off-target effects and activation of the immune system. In the case of huntingtin, no RNAi approach was able to discriminate effectively between the wild-type and mutant expanded polyglutamine stretch [59]. Here, intracellular antibodies could, in principle, recognize different conformational epitopes formed by polyglutamine and might be able to discriminate between the length of the polyQ motifs [63]. However, in the case of the cytoplasmic huntingtin protein, it is more difficult to generate and select cyto-intrabodies, due to the reducing environment of the cytoplasm. In general, the allele-specific knockdown strategy should be also applied with ER intrabodies.

The kind of mismatches introduced into siRNAs or artificial miRNAs, in order to increase allele specificity for preference of the mutant allele, can differ. Purine-to-purine mismatches, for example, are more effective than purine-to-pyrimidine mismatches. This limitation can be overcome by introduction of a second mismatch, preferentially into the seed or cleavage region of the siRNA/miRNA [59]. Using a set of SNP sites, common in disease-associated alleles, might enable reaching many patients [57], but it is hard to access the whole population. For those genotypes that could not be cured by using mutant SNP-targeting siRNA, intrabody-mediated protein knockdown, recognizing a prevalent mutant epitope could be superior. Whereby, in the case of SNPs due to the posttranslational targeting, the intrabody technology demonstrates one of its weaknesses. Discrimination between mutant and wild-type SNP could only be achieved when the mutant SNP induces a change of the encoded amino acid. In addition, mutant SNPs in introns and untranslated regions (UTR) cannot be addressed, as it is in the case of HD.


**Table 1.** Intrabodies versus siRNA

the effect and safety over decades have yet to be proven in clinical trials. Therefore, there is still a need for high allele-specific inhibition of the mutant Htt protein, which is toxic due to an expanded polyglutamine (polyQ) motif (CAG motif). Targeting of the CAG motif is not selective for the mutant allele and affected both alleles. Genotyping of the Huntington's disease patients resulted in three single nucleotide polymorphisms (SNP) in huntingtin [57]. Therefore, an alternative strategy when using RNAi is targeting a single nucleotide mutation localized in the disease-caused sequence [58]. Furthermore, targeting of the mutant huntingtin SNPs or the expanded CAG motif by designed artificial miRNAs was recently demonstrated *in vitro*,

using an allele-specific reporter system and *in vivo* in a transgenic mouse model [59].

miRNAs are highly expressed *in vivo* [59].

146 RNA Interference

the power of antibody engineering.

For the RNAi technique, it is possible to discriminate between very similar targets with a specific reduction on the RNA and protein level [58, 60, 61]. However, there are still some concerns and limitations. The RNAi-mediated allele-specific knockdown may result in a broad off-targeting and therefore has to be further evaluated in appropriate preclinical model systems [62]. Using both target strategies, CAG motif and prevalent mutant SNPs, in the case of huntingtin, the wild-type allele is also affected by the knockdown, and the knockdown ratio between the wild type and mutant allele remains unsatisfactory. Furthermore, the shift to *in vivo* delivery systems can have a substantial impact on the specificity, as was demonstrated in the mouse model [59]. Next, a limited expression of the miRNA vectors is important to avoid saturation of the miRNA processing machinery, as the selectivity seems to be reduced when

Different alleles can also be targeted and discriminated using specific intracellular antibodies (intrabodies) and represent a valuable alternative to RNA interference. Intrabodies targeting, for example, huntingtin have to recognize an epitope common in most disease-associated huntingtin SNP forms, which also has to be different in the translated amino acid between the mutant and wild-type allele. Alternatively, they could target the expanded polyglutamine (polyQ) motif associated with misfolding and aggregation [63]. Furthermore, cytoplasmic intrabodies have been developed, which efficiently inhibited aggregation of mutant HD [64]. Interestingly, a disulfide bond-free single-domain intracellular antibody with high affinity was developed after affinity maturation [65] from a specific anti-HD scFv fragment, demonstrating

For the allele-specific knockdown, the intrabody technology utilizes the high specificity of monoclonal antibodies, with no or low concerns about off-target effects and activation of the immune system. In the case of huntingtin, no RNAi approach was able to discriminate effectively between the wild-type and mutant expanded polyglutamine stretch [59]. Here, intracellular antibodies could, in principle, recognize different conformational epitopes formed by polyglutamine and might be able to discriminate between the length of the polyQ motifs [63]. However, in the case of the cytoplasmic huntingtin protein, it is more difficult to generate and select cyto-intrabodies, due to the reducing environment of the cytoplasm. In general, the allele-specific knockdown strategy should be also applied with ER intrabodies. The kind of mismatches introduced into siRNAs or artificial miRNAs, in order to increase allele specificity for preference of the mutant allele, can differ. Purine-to-purine mismatches, for example, are more effective than purine-to-pyrimidine mismatches. This limitation can be

#### **2.5. High-throughput screening**

Oligonucleotide and cDNA microarrays can be applied for simultaneous quantitative moni‐ toring of gene expression of thousands of genes [66]. A combination of cDNA microarrays and RNA interference was used to validate upregulated genes, playing an important role in cancer development [67]. In this case, a pre-screening with cDNA microarrays is performed followed by silencing of selected upregulated mRNAs using RNAi. This might also be possible with intrabodies.

Although high-throughput RNAi screening is very useful in order to validate new genes involved in cancer pathogenesis or infection processes [68, 69], such high-throughput screen‐ ing is not possible with intrabodies.

#### **2.6. Therapeutic potential of siRNA and ER intrabodies**

The therapeutic potential of siRNA and ER intrabodies has been shown in different mouse models [70–73]. It has been shown that siRNA protected mice from fulminant hepatitis [74], viral infection [75], sepsis [76], tumor growth [77], and macular degeneration [78]. In these mouse models, synthetic siRNA was delivered systemically, peritoneally, or subretinally.

Furthermore, in an Alzheimer's and spinocerebellar ataxia disease-related mouse model, RNAi suppresses the expression of amyloid-β peptide or ataxia, respectively [79, 80]. In these mouse models, target-specific RNAi was virally delivered using adeno-associated virus or Herpes simplex virus. Interestingly, the knockdown of angiopoietin-2 mRNA in a mouse model with pancreatic carcinoma and xenotransplantation suppresses metastasis and down‐ regulates metalloproteinase-2 [81].

Many ER intrabodies have shown therapeutic potential against relevant targets in cancer, infection, and brain diseases, for example, ErbB-2, EGFR, VEGFR-2, Tie-2, VEGFR-2 × Tie-2, metalloproteinases MMP-2, MMP-9, E7 oncoprotein of human papillomavirus, CCR5, TLR2, TLR9, and amyloid-β protein [18, 33, 82–90]. Nevertheless, only four of these antigens have been applied in xenograft tumor mouse models so far, using an anti-Tie intrabody [85], a bispecific VEGFR-2 × Tie-2 intrabody [86], an anti-amyloid-β protein intrabody in an Alz‐ heimer's disease mouse model [90], and an anti-E7 oncoprotein intrabody in a mouse infection model with human papillomavirus [89]. Intrabody delivery was performed via adenovirus, adeno-associated virus, and retrovirus, respectively.

#### *2.6.1. Transgenic mice*

Transgenic RNAi mouse against p120-Ras GTPase-activating protein [91] and cytokineactivated IκB kinase 1 (IKK1) has been established [92]. Furthermore, RNAi transgenic mice and non-germline genetically engineered RNAi cancer mouse models were established [93]. In contrast to constitutive RNAi transgenic mice, generation of conditional RNAi in mice is also possible [94].

Recently, two transgenic ER intrabody mice have been generated against VCAM and gelsolin [71, 72]. In addition, a transgenic mouse expressing an anti-EVH1 intrabody has been pub‐ lished [73]. However, the inhibitory results obtained with these mice have been criticized because the intrabody was directed to the secretory pathway, but confusingly recognized a cytosolic protein [95]. Interestingly, the transgenic VCAM intrabody mouse was viable in contrast to the lethal knockout mice generated by targeted homologous recombination [96]. The intrabody mice were deficient in VCAM-1 cell surface expression.

#### *2.6.2. Clinical approaches*

RNA interference was used to validate upregulated genes, playing an important role in cancer development [67]. In this case, a pre-screening with cDNA microarrays is performed followed by silencing of selected upregulated mRNAs using RNAi. This might also be possible with

Although high-throughput RNAi screening is very useful in order to validate new genes involved in cancer pathogenesis or infection processes [68, 69], such high-throughput screen‐

The therapeutic potential of siRNA and ER intrabodies has been shown in different mouse models [70–73]. It has been shown that siRNA protected mice from fulminant hepatitis [74], viral infection [75], sepsis [76], tumor growth [77], and macular degeneration [78]. In these mouse models, synthetic siRNA was delivered systemically, peritoneally, or subretinally.

Furthermore, in an Alzheimer's and spinocerebellar ataxia disease-related mouse model, RNAi suppresses the expression of amyloid-β peptide or ataxia, respectively [79, 80]. In these mouse models, target-specific RNAi was virally delivered using adeno-associated virus or Herpes simplex virus. Interestingly, the knockdown of angiopoietin-2 mRNA in a mouse model with pancreatic carcinoma and xenotransplantation suppresses metastasis and down‐

Many ER intrabodies have shown therapeutic potential against relevant targets in cancer, infection, and brain diseases, for example, ErbB-2, EGFR, VEGFR-2, Tie-2, VEGFR-2 × Tie-2, metalloproteinases MMP-2, MMP-9, E7 oncoprotein of human papillomavirus, CCR5, TLR2, TLR9, and amyloid-β protein [18, 33, 82–90]. Nevertheless, only four of these antigens have been applied in xenograft tumor mouse models so far, using an anti-Tie intrabody [85], a bispecific VEGFR-2 × Tie-2 intrabody [86], an anti-amyloid-β protein intrabody in an Alz‐ heimer's disease mouse model [90], and an anti-E7 oncoprotein intrabody in a mouse infection model with human papillomavirus [89]. Intrabody delivery was performed via adenovirus,

Transgenic RNAi mouse against p120-Ras GTPase-activating protein [91] and cytokineactivated IκB kinase 1 (IKK1) has been established [92]. Furthermore, RNAi transgenic mice and non-germline genetically engineered RNAi cancer mouse models were established [93]. In contrast to constitutive RNAi transgenic mice, generation of conditional RNAi in mice is

Recently, two transgenic ER intrabody mice have been generated against VCAM and gelsolin [71, 72]. In addition, a transgenic mouse expressing an anti-EVH1 intrabody has been pub‐ lished [73]. However, the inhibitory results obtained with these mice have been criticized because the intrabody was directed to the secretory pathway, but confusingly recognized a cytosolic protein [95]. Interestingly, the transgenic VCAM intrabody mouse was viable in

intrabodies.

148 RNA Interference

ing is not possible with intrabodies.

regulates metalloproteinase-2 [81].

*2.6.1. Transgenic mice*

also possible [94].

adeno-associated virus, and retrovirus, respectively.

**2.6. Therapeutic potential of siRNA and ER intrabodies**

Different clinical approaches have been performed with siRNA. RNAi-based clinical trials are ongoing (phase I–III) [62, 97]. For example, a Bevasiranib RNAi targeting VEGF has been applied to heal macular degeneration [98] and RNAi targeting the RSV nucleocapsid SPC3649 has shown significant anti-viral activity [99].

In comparison to the RNAi, only one example of an ER intrabody targeting erbB-2 has been applied in a clinical phase I study [82]. As demonstrated, none of the patients treated in this study exhibited a dramatic clinical benefit.

Both methods share the limitations of viral and non-viral delivery methods. Using integrating vectors, insertional mutagenesis is still the main problem [100]. Concerning non-viral delivery methods, lipid-based and peptide polymer-based delivery systems have been applied [101]. However, for some diseases like HD, the non-neurotropic feature of many delivery systems and the lack of passing the blood–brain barrier (BBB) remain problematic.

Cell- and tissue-specific targeting is also always a concern; however, transductional and transcriptional targeting is promising [102]. Tissue-specific carrier for siRNA includes aptamers, antibodies, peptides, proteins, and oligonucleotide agonists [101]. Referring to ER intrabodies, the use of mRNA in clinical approaches is promising [103].

#### **2.7. Other features**

Intrabodies are able to inhibit posttranslational modifications, such as phosphorylation sites [104, 105]. This is not possible using RNAi. Besides the high specificity of intrabodies, this is an important advantage of intrabodies over RNAi.

Recently, single-stranded siRNA was used to suppress the spliced variants of proteins [106]. This might also be possible with specific intrabodies (Table 1). In addition, targeting of specific protein domains and isomers of a protein might also be feasible. For example, miRNA suppresses specifically an oncogenic isoform [107]. Intriguingly, the suppression of different protein isoforms with only one intrabody or one siRNA, recognizing a common epitope within all isoforms, might be possible, for example, the knockdown of all interferon alpha isoforms (13 different subtypes in human).

#### **2.8. miRNA**

It is known that miRNA influences tumorigenesis [3], and therefore miRNA and combined miRNA/siRNA pharmacological approaches are attractive [108]. miRNA has been applied in cancer mouse models as for lymphoid malignancies [109]. Furthermore, important studies using miRNA has been performed for diagnosis, prognosis, and prediction of cancer [108]. One of the most developed microRNA-based candidates is MRX34, a miR-34 mimetic that restores the function of miR-34 in cancer cells [110], which is applied in an ongoing multicenter phase I clinical trial. The repression of expression of several potential miR-34 target oncogenes was demonstrated [111]. Finally, miRNAs can be used to reprogram somatic cells into pluripotent stem cells [112]. However, siRNA and miRNA share the same silencing machinery and microRNA causes also off-target effects [113].

#### **3. Conclusions and perspectives**

siRNA and ER intrabody technology are both efficient knockdown techniques. siRNA is acting on the mRNA level, whereas ER intrabodies are acting on the protein level. The strength of the RNAi technology results from the possibility that nearly all mRNAs of a cell can be targeted. Currently, the knockdown of proteins mediated by intrabodies is most promising with ER intrabodies, because they are correctly folded inside the ER and can be generated more easily than in the past. Because of the availability of many new scFv fragments, generated by research consortia, one cloning step is sufficient to convert selected scFv fragments into ER intrabodies.

Stable cytosolic intrabodies have to be selected with considerable effort. Two approaches are successful and reliable: the intracellular antibody capture technology, based on an antigendependent two-hybrid system [114] and single-domain antibodies [115], which are stably folded in a reducing environment for inhibition of cytoplasmic proteins. Single-domain antibodies comprise only one V region, the variable domain of the heavy or light chain. Most successfully applied are camelid single-domain antibodies (VHHs) [115–118]. Alternatively, human VL and VH domains are also potent molecules and their successful construction is ongoing [119, 120].

The number of ER intrabodies will increase due to the fact that international research consortia as the "Affinomics" initiative [121] in the European Union and similar initiatives in the United States have already generated several thousands of recombinant antibodies, including the Vregion genes, which can be used to build up a new repertoire of intrabodies. Using this pipeline, the duration for development of intrabodies is similar to that of siRNA/shRNA/miRNAs. In the future, scFvs against very valuable disease-related targets have to be provided.

The main advantage of intrabodies is their specificity, no off-target effects, and posttransla‐ tional modification inhibition. The specificity of an intrabody can be estimated by immuno‐ assays such as ELISA, flow cytometry, and immunoprecipitation. On the contrary, the specificity and off-target effects of RNAi are often more difficult to predict.

Conferring to *in vivo* application, RNAi has been currently applied predominantly in phase 1 and 2 studies [62, 97]. In the future, the success of clinical approaches using RNAi and ER intrabodies is dependent on the development of safe viral vectors and the development of nonviral vectors possessing high transfection efficiency [122].

Two attractive applications of RNAi, hardly to perform with ER intrabodies, are genome-wide screening [68, 69] and reprogramming of somatic cells into pluripotent stem cells [112].

Thus, the ER intrabody approach has demonstrated its huge potential for *in vitro* and *in vivo* analysis of protein function [10]. The ER intrabody technique can complement the RNAi technique in cases where siRNA, shRNA, and miRNA molecules demonstrate unwanted unspecificity and off-target effects.

#### **Acknowledgements**

phase I clinical trial. The repression of expression of several potential miR-34 target oncogenes was demonstrated [111]. Finally, miRNAs can be used to reprogram somatic cells into pluripotent stem cells [112]. However, siRNA and miRNA share the same silencing machinery

siRNA and ER intrabody technology are both efficient knockdown techniques. siRNA is acting on the mRNA level, whereas ER intrabodies are acting on the protein level. The strength of the RNAi technology results from the possibility that nearly all mRNAs of a cell can be targeted. Currently, the knockdown of proteins mediated by intrabodies is most promising with ER intrabodies, because they are correctly folded inside the ER and can be generated more easily than in the past. Because of the availability of many new scFv fragments, generated by research consortia, one cloning step is sufficient to convert selected scFv fragments into ER intrabodies. Stable cytosolic intrabodies have to be selected with considerable effort. Two approaches are successful and reliable: the intracellular antibody capture technology, based on an antigendependent two-hybrid system [114] and single-domain antibodies [115], which are stably folded in a reducing environment for inhibition of cytoplasmic proteins. Single-domain antibodies comprise only one V region, the variable domain of the heavy or light chain. Most successfully applied are camelid single-domain antibodies (VHHs) [115–118]. Alternatively, human VL and VH domains are also potent molecules and their successful construction is

The number of ER intrabodies will increase due to the fact that international research consortia as the "Affinomics" initiative [121] in the European Union and similar initiatives in the United States have already generated several thousands of recombinant antibodies, including the Vregion genes, which can be used to build up a new repertoire of intrabodies. Using this pipeline, the duration for development of intrabodies is similar to that of siRNA/shRNA/miRNAs. In

The main advantage of intrabodies is their specificity, no off-target effects, and posttransla‐ tional modification inhibition. The specificity of an intrabody can be estimated by immuno‐ assays such as ELISA, flow cytometry, and immunoprecipitation. On the contrary, the

Conferring to *in vivo* application, RNAi has been currently applied predominantly in phase 1 and 2 studies [62, 97]. In the future, the success of clinical approaches using RNAi and ER intrabodies is dependent on the development of safe viral vectors and the development of non-

Two attractive applications of RNAi, hardly to perform with ER intrabodies, are genome-wide screening [68, 69] and reprogramming of somatic cells into pluripotent stem cells [112].

Thus, the ER intrabody approach has demonstrated its huge potential for *in vitro* and *in vivo* analysis of protein function [10]. The ER intrabody technique can complement the RNAi

the future, scFvs against very valuable disease-related targets have to be provided.

specificity and off-target effects of RNAi are often more difficult to predict.

viral vectors possessing high transfection efficiency [122].

and microRNA causes also off-target effects [113].

**3. Conclusions and perspectives**

ongoing [119, 120].

150 RNA Interference

The authors thank Prof. Peter Müller for critical reading of the manuscript.

## **Author details**

Oliver Backhaus and Thomas Böldicke\*

\*Address all correspondence to: thomas.boeldicke@helmholtz-hzi.de

Helmholtz Centre for Infection Research, Department of Structural and Functional Protein Research, Braunschweig, Germany

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2010.e8


**RNA Interference for Disease Therapy**

## **RNAi Therapeutic Potentials and Prospects in CNS Disease**

Kyoung Joo Cho and Gyung Whan Kim

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62896

#### **Abstract**

Over the past 20 years, RNA interference (RNAi) technology has provided a new regula‐ tory paradigm in biology. This technique can efficiently suppress target genes of interest in mammalian cells. Small non-coding RNAs play important roles in gene regulation, in‐ cluding both in post-transcriptional and in translational regulation. For *in vivo* experi‐ ments, continuous development has resulted in successful new ways of designing, identifying, and delivering small interfering RNAs (siRNAs). Proof-of-principle studies *in vivo* have clearly demonstrated that both viral and non-viral delivery methods can pro‐ vide selective and potent target gene suppression without any clear toxic effects. There are also the persistent problems with off-target effects (OTEs), competition with cellular RNAi components, and effective delivery *in vivo*. Although recent researches and trials from a large number of animal model studies have confirmed that most OTEs are not dangerous, other important issues need to be addressed before RNAi-based drugs are ready for clinical use. Currently, RNAi may be harnessed as a new therapeutic modality for brain diseases. Finally, there are already several RNAi-based human clinical trials in progress. It is hoped that this technology will have also effective applications in human central nervous system (CNS)-related disease.

**Keywords:** RNAi therapy, brain, neurodegenerative disease, allele-specific, neurovascu‐ lar

#### **1. Introduction**

During developmental stage and in response to internal and external cellular stresses, small RNA molecules regulate gene expression [1]. Specialized ribonucleases and RNA-binding proteins govern the production and action of small regulatory RNAs [2]. In most eukaryotic cells, RNA interference (RNAi) is a regulatory mechanism using small double-stranded RNA

© 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.

(dsRNA) molecules to direct homology-dependent control of gene activity [3, 4]. Small size [20–30 nucleotide (nt)] non-coding RNAs and associated proteins regulate the expression of genetic information [5]. The discovery of RNAi phenomenon widened our understanding of gene regulation and revealed related pathways in small RNAs [6]. As it processes, RNAi has been finding widespread in plants [7] and animals [8]. Each small RNA associates with an Argonaute (AGO) family protein to form a sequence-specific complex. After then, genesilencing ribonucleo-protein complex with specificity conferred by base pairing between the small RNA (guide RNA) and its target mRNA [5]. The pathway is well known as the RNAinduced silencing complex (RISC), which gives a target mRNA silencing by degradation or transcriptional regression [2]. Small interfering RNAs (siRNAs) loaded into RISC are doublestranded, and AGO-2, which having an active catalytic domain in human, cleaves and releases the "passenger" strand. RISC is activated with a single-stranded "guide RNA" molecule to impose the specificity recognizing the target by intermolecular base pairing [9].

MicroRNAs (miRNAs) are other endogenous substrates for the RNAi machinery, but the cellular origins of miRNA and siRNA are distinct. miRNAs are derived from the genome, whereas siRNAs may be endogenous or arise through viral infection or other exogenous sources [2]. Typically, miRNAs are initially expressed in the nucleus with a transcript as long as primary miRNA (pri-miRNA), and the transcripts are at least over 1000 nt. Pri-miRNAs are processed by the microprocessor complex (histone deacetylase proteins) consisting in Drosha-DGCR8 [DiGeorge critical region 8 (a double cysteine-ligated Fe (III) heme protein)—DGCR8] in the nucleus [10, 11]. They are cleaved in the nucleus into 60–70 base pair (bp) hairpins, which are consisted in single-stranded 5′- and 3′-terminal overhangs and about 10-nt distal loops [12]. In cytoplasm, the loop is further processed by the RNAse III Dicer, and one strand is loaded onto RISC. The mature miRNAs bind to the 3′ UTR of target mRNAs and then degrade the target [13]. Despite their differing origins, these RNA processing pathways converge once either type of RNA assembles into the RISC.

With development of an efficient delivery system in various diseases, RNAi has been an emerging therapeutic approach for *in vivo* studies with specific synthetic siRNAs against each disease. It should be considered as novel and interesting therapeutic challenge with the major concern how to administer the siRNAs with specific, efficient, and targeted way. Despite some hurdles for applying to clinical challenges such as anatomical barriers, drug stability and availability, various delivery routes, and different genetic backgrounds, an application of siRNAs has become extremely attractive in development of new drugs. Currently, one of the important challenges in siRNA bioinformatics is target prediction, when there is still no proper tool with certain drug design grade. Besides specific challenges in siRNA therapeutics, an efficient delivery method, targeting a specific tissue or cell, is another fundamental challenge.

This chapter introduced two of main themes. The first is the possibilities of therapeutics using RNAi principles and technique. The second is the challenges with siRNAs or miRNAs specifically in the area of brain disease. In addition, this chapter provided some prospects of siRNAs or miRNAs on disease prognosis, progress, and therapeutics in the present and future.

### **2. Principles of RNAi therapy**

(dsRNA) molecules to direct homology-dependent control of gene activity [3, 4]. Small size [20–30 nucleotide (nt)] non-coding RNAs and associated proteins regulate the expression of genetic information [5]. The discovery of RNAi phenomenon widened our understanding of gene regulation and revealed related pathways in small RNAs [6]. As it processes, RNAi has been finding widespread in plants [7] and animals [8]. Each small RNA associates with an Argonaute (AGO) family protein to form a sequence-specific complex. After then, genesilencing ribonucleo-protein complex with specificity conferred by base pairing between the small RNA (guide RNA) and its target mRNA [5]. The pathway is well known as the RNAinduced silencing complex (RISC), which gives a target mRNA silencing by degradation or transcriptional regression [2]. Small interfering RNAs (siRNAs) loaded into RISC are doublestranded, and AGO-2, which having an active catalytic domain in human, cleaves and releases the "passenger" strand. RISC is activated with a single-stranded "guide RNA" molecule to

impose the specificity recognizing the target by intermolecular base pairing [9].

either type of RNA assembles into the RISC.

166 RNA Interference

MicroRNAs (miRNAs) are other endogenous substrates for the RNAi machinery, but the cellular origins of miRNA and siRNA are distinct. miRNAs are derived from the genome, whereas siRNAs may be endogenous or arise through viral infection or other exogenous sources [2]. Typically, miRNAs are initially expressed in the nucleus with a transcript as long as primary miRNA (pri-miRNA), and the transcripts are at least over 1000 nt. Pri-miRNAs are processed by the microprocessor complex (histone deacetylase proteins) consisting in Drosha-DGCR8 [DiGeorge critical region 8 (a double cysteine-ligated Fe (III) heme protein)—DGCR8] in the nucleus [10, 11]. They are cleaved in the nucleus into 60–70 base pair (bp) hairpins, which are consisted in single-stranded 5′- and 3′-terminal overhangs and about 10-nt distal loops [12]. In cytoplasm, the loop is further processed by the RNAse III Dicer, and one strand is loaded onto RISC. The mature miRNAs bind to the 3′ UTR of target mRNAs and then degrade the target [13]. Despite their differing origins, these RNA processing pathways converge once

With development of an efficient delivery system in various diseases, RNAi has been an emerging therapeutic approach for *in vivo* studies with specific synthetic siRNAs against each disease. It should be considered as novel and interesting therapeutic challenge with the major concern how to administer the siRNAs with specific, efficient, and targeted way. Despite some hurdles for applying to clinical challenges such as anatomical barriers, drug stability and availability, various delivery routes, and different genetic backgrounds, an application of siRNAs has become extremely attractive in development of new drugs. Currently, one of the important challenges in siRNA bioinformatics is target prediction, when there is still no proper tool with certain drug design grade. Besides specific challenges in siRNA therapeutics, an efficient delivery method, targeting a specific tissue or cell, is another fundamental challenge.

This chapter introduced two of main themes. The first is the possibilities of therapeutics using RNAi principles and technique. The second is the challenges with siRNAs or miRNAs specifically in the area of brain disease. In addition, this chapter provided some prospects of siRNAs or miRNAs on disease prognosis, progress, and therapeutics in the present and future. As far as it is true that siRNA has promising benefits, and, concomitantly, siRNA has still some of technological barriers to be widely used in clinical therapy, which generally due to the lack of efficient delivery tools. To success with siRNA therapies, an effective and safe carrier system is required that would overcome the inherent defects of siRNA and achieve maximum genesilencing effect. There are many approaches that are being developed to achieve the efficient delivery of siRNA. In that, non-viral vectors have advantages of reproducibility, low immu‐ nogenicity, and relatively low production cost [14]; therefore, non-viral vectors made siRNA to be a potential therapeutic and nucleic acid–based drugs, such as plasmid DNAs or antisense oligonucleotides (ASOs) [15].

#### **2.1. Advantages of RNAi**

Theoretically, all disease-associated genes could be amenable to antisense-mediated RNAi suppression. RNAi can be a strategy for silencing of virtually all annotated protein-encoding genes in the human genome in large scale. The high specificity of siRNA lets targeting of disease-specific alleles that differ from the normal allele by only one or few nucleotide substitutions. This high fidelity and specificity of siRNAs are useful for targeting for some oncogenes, too.

The first advantage is the powerfulness of RNAi when compared with other antisense strategies, such as antisense DNA oligonucleotides and ribozymes [16]. It is important fact that the effector molecules work at much lower concentration than any other antisense oligomers or ribozymes, suggesting that RNAi has higher potency. This is a critical point to set thera‐ peutics.

The second is efficacy. The efficacy is generally presented by the half level of maximal inhibition or the value of IC50 against target site. The efficacy level is crucial for determining thermodynamic stability [17], targeted gene accessibility [18], or structure [18] of designed siRNA. For designing siRNA, the most important thing is end stability that is different from each end and is also meaning asymmetry and consistent with selected miRNA [19]. However, to date, our knowledge of siRNA and the selection of targets are incomplete and being explored. The identification of "hyperfunctional" siRNAs, functioning at sub-nanomolar concentration, remains an elusive task.

#### **2.2. Basic strategies for targeting-specific molecules**

RNAi can be triggered by two different pathways: (1) a RNA-based approach, where the 21 nt long duplexed siRNA effectors are delivered to target cells, and (2) a DNA-based strategy, where the siRNA effectors are produced by intracellular processing of longer RNA hairpin transcripts [3]. DNA-based strategy is based on short hairpin RNA (shRNA) synthesis in nucleus and transportation to the cytoplasm through miRNA machinery, which subsequently is processed by Dicer. Although the direct use of siRNA effectors is simple and effective way for gene silencing, the effect is transient. Therefore, it is costly for clinical usage due to the need of multiple large-scale application. In contrast, DNA-based RNAi drugs have the potential and stably introduced for application in a gene therapy. In principle, DNA-based RNAi allows a single treatment of viral vector that delivers shRNA genes to the targeted cells/or tissues.

#### **2.3. Delivery routes for targeting**

The effective delivery of siRNAs acts to be significant step in accelerating RNAi-based treatments. The instability of RNA and the relatively inefficient encapsulation process of siRNA remain critical issues toward the clinical translation of RNAi as a therapeutic tool. There are several obstacles for extracellular introduction of siRNA to deliver the target. Under normal physiological condition, the introduced molecules ought to have a positive charge to diffuse to cell membrane [20]. It is the simplest way of naked nucleotides or transfecting siRNAs to deliver into cells [21]. Another technique is microinjection and electroporation for direct delivery, but it has higher level of cellular toxicity [22]. The delivery routes can be intraperitoneal, intra-vascular, intra-muscular, intra-splenic, intra-cranial, and intra-tumoral injection. In addition, siRNAs can be delivered through subretinal, subcutaneous, mucosal, topical application, and oral ingestion to improve delivery [22]. However, these transfection processes should be optimized for siRNA concentration, cell density, and ratio of transfection reagent to siRNA [23]. Carriers for delivery of siRNA with cationic environment surrounding of siRNAs can be liposomes and dendrimers. These carriers reduce the nuclease activity and improve siRNA delivery into cells [24].

Microsponge is one of the mediators for siRNA delivery. Carrier and cargo combine and selfassemble into nanoscale pleated sheets of hairpin RNA. Subsequently, this complex forms sponge-like microspheres [25]. The complex of siRNA and microsponges consists in cleavable RNA strands, and the stable hairpin RNA converts into working siRNA once cells uptake the complex. Therefore, it can provide a protection for siRNA during delivery and transport it to the cytoplasm. Single microsponge complex can deliver more than half a million copies of siRNA when uptaken into a cell [25].

#### **2.4. Stabilizing the siRNA delivery**

The stability of the siRNA complexes, penetrating into target cells without stimulating immune responses, is one of the limiting factors and the major bottleneck for developing siRNA therapeutic tools. It restricts the delivery of siRNA macromolecular complexes to the desired cell types, tissues, or organs. Usually, siRNAs do not easily penetrate the cellular membrane because of their negative charge and macromolecular size. Manipulation of nucleotide bases is needed to increase stability and protein interactions, which can harness to increase the structural improvement of siRNAs [26]. The delivery systems for siRNA consist of four main methods, namely naked, lipid-based, peptide-based, and polymer-based delivery [27]. Basically, polymer-based methods are similar to lipid-based methods in targeting, except some special triggers, such as temperature, pH, or pulse release [28].

Initial efforts to improve stability addressed above were focused on incorporating chemical modifications into the sugar backbone or bases of siRNA duplexes [29]. The modified siRNA molecules increased stability, which effectively lowered the dose to achieve measurable and reproducible gene silencing [30]. Several modifications were introduced. The thio (−SH), hydroxyl (−OH), or iodo (−I) can modify bases in specific sites or utilize the pseudouracil base in siRNA, which would augment potency of naked siRNA [31]. There are three most popular chemical modification sites on siRNA structure containing the phosphodiester backbone, ribose 2′-hydroxyl group (R-2′-OH), and ribose ring. Endogenous cellular endonucleases can easily digest phosphodiester bond in RNA backbone [32]. Alternative modification is oxygen bridges of RNA backbone that can be replaced with phosphorothioate, although it would increase toxicity and reduce silencing activity [33, 34]. Another alternative is boranophosphate linkages. These are more nuclease resistant and less toxic compared to phosphorothioate [35]. Phosphonoacetate linkages are other candidates [36]. The linkage is completely resistant to nuclease and is electrochemically neutral when they are esterified [36, 37]. Another modifica‐ tion is 2′-*O*-methoxyethyl (2′-*O*-MOE), 2′-*O*-alkyl, and other bulky groups. These modifica‐ tions can improve anti-nuclease shield of siRNA that simultaneously makes them less tolerable when they are positioned on 3′ overhangs [38]. Despite disturbing thermodynamic asymmetry of siRNA by addition of 2′-aminoethyl at 3′ end of passenger strand, this modification improves efficiency of target silencing [39].

On the other hand, alterations in sugar compartment of nucleotides reduced flexibility and nuclease sensitivity of siRNA structure [39, 40]. Binding of ribose 2′O into 1′C with methylene bridges, which finally produces oxetane, forms a locked conformation nucleic acid (locked nucleic acid—LNA) [41]. *In vivo* nuclease resistance of this structure is enhanced [42]. In contrast to LNA, derivatives of RNA without C2′–C3′ sugar bonds (unlocked nucleic acid— UNA) destabilize a sequence structure [43]. Substitution of pentose with hexose monosac‐ charides, such as cyclohexenyl, anitrol, and arabinose, was applied to develop CeNA, ANA, and 2′-F-ANA [44], subsequently resulting in enhanced stability of siRNA *in vivo* [45]. During systemic delivery, however, internal modifications failed to improve central nervous system (CNS) entry and uptake. Researchers put new efforts to move toward using liposomes, nanoparticles, and cell-penetrating peptides (CPPs), among others, to stabilize and navigate siRNAs into and throughout the brain [46].

#### *2.4.1. Liposomes*

of multiple large-scale application. In contrast, DNA-based RNAi drugs have the potential and stably introduced for application in a gene therapy. In principle, DNA-based RNAi allows a single treatment of viral vector that delivers shRNA genes to the targeted cells/or tissues.

The effective delivery of siRNAs acts to be significant step in accelerating RNAi-based treatments. The instability of RNA and the relatively inefficient encapsulation process of siRNA remain critical issues toward the clinical translation of RNAi as a therapeutic tool. There are several obstacles for extracellular introduction of siRNA to deliver the target. Under normal physiological condition, the introduced molecules ought to have a positive charge to diffuse to cell membrane [20]. It is the simplest way of naked nucleotides or transfecting siRNAs to deliver into cells [21]. Another technique is microinjection and electroporation for direct delivery, but it has higher level of cellular toxicity [22]. The delivery routes can be intraperitoneal, intra-vascular, intra-muscular, intra-splenic, intra-cranial, and intra-tumoral injection. In addition, siRNAs can be delivered through subretinal, subcutaneous, mucosal, topical application, and oral ingestion to improve delivery [22]. However, these transfection processes should be optimized for siRNA concentration, cell density, and ratio of transfection reagent to siRNA [23]. Carriers for delivery of siRNA with cationic environment surrounding of siRNAs can be liposomes and dendrimers. These carriers reduce the nuclease activity and

Microsponge is one of the mediators for siRNA delivery. Carrier and cargo combine and selfassemble into nanoscale pleated sheets of hairpin RNA. Subsequently, this complex forms sponge-like microspheres [25]. The complex of siRNA and microsponges consists in cleavable RNA strands, and the stable hairpin RNA converts into working siRNA once cells uptake the complex. Therefore, it can provide a protection for siRNA during delivery and transport it to the cytoplasm. Single microsponge complex can deliver more than half a million copies of

The stability of the siRNA complexes, penetrating into target cells without stimulating immune responses, is one of the limiting factors and the major bottleneck for developing siRNA therapeutic tools. It restricts the delivery of siRNA macromolecular complexes to the desired cell types, tissues, or organs. Usually, siRNAs do not easily penetrate the cellular membrane because of their negative charge and macromolecular size. Manipulation of nucleotide bases is needed to increase stability and protein interactions, which can harness to increase the structural improvement of siRNAs [26]. The delivery systems for siRNA consist of four main methods, namely naked, lipid-based, peptide-based, and polymer-based delivery [27]. Basically, polymer-based methods are similar to lipid-based methods in targeting, except some

Initial efforts to improve stability addressed above were focused on incorporating chemical modifications into the sugar backbone or bases of siRNA duplexes [29]. The modified siRNA

**2.3. Delivery routes for targeting**

168 RNA Interference

improve siRNA delivery into cells [24].

siRNA when uptaken into a cell [25].

**2.4. Stabilizing the siRNA delivery**

special triggers, such as temperature, pH, or pulse release [28].

Generally, liposomes are classified into three classes: multilmellar vesicle (0.5~20 μm), small unilamellar vesicles (25~100 nm), and large unilamellar vesicles (100~500 nm) [47]. Liposomes are developed for passive or active targeting mechanisms in different complexes of liposome and other interacting molecules, namely lipoplex (cationic liposome-pDNA complex), liposome polycationic DNA, mannose liposome, and so on [48, 49]. The siRNA with mannose (Man)-coated liposomes would be useful for treatment of some cancers, especially liver and brain cancers [50].

#### *2.4.2. Dendrimers*

Dendrimers are hyper-branched, tree-shaped, and 3-D structures [51]. Dendrimer can utilize broad spectrum, and the broad range of functional groups makes it possible to introduce dendrimers with extensive applications. There are different classes of cationic and anionic dendrimers, such as polyamidoamine (PAMAM), polypropylene imine (PPI), and polyethy‐ lene glycol (PEG)-grafted carbosilane [52]. Specific dendritic polymers like PAMAM have been widely utilized in *in vivo* drug delivery [53]. Conjugation of Tat peptide (GRKKRRQRRRPQ) with PAMAM-G5 can efficiently inhibit multi-drug resistance-1 (MDR-1) gene expression *in vitro* [51]. Capping poly-l-lysine (PLL) dendrimers with methotrexate enhances stability and decreases toxicity [54].

#### *2.4.3. Cationic polymers*

Cationic polymers include chitosan, gelatin, cationic dextran, cationic cellulose, and cationic cyclodextrin and some synthetic biocompatible polyethyleneimine (PEI), PLL, poly(amidoa‐ mine)s (PAAs), poly(amino-co-ester), and poly(2-*N*,*N*-dimethylaminoethylmethacrylate). Moreover, they are less immunogenic response because these polymers are natural biode‐ gradable [55].

#### *2.4.4. Cationic peptides*

CPPs are cationic peptides. CPPs interact covalently or non-covalently through disulfide or electrostatic–hydrogen interactions with siRNAs [56]. Viral protein (VP22) [57], MPG (a peptide vector) [58], amphipathic peptide [59], and poly-arginine [60] were reported the same abilities. In addition, small cationic polypeptides (poly His, Lys, and Arg) coat and neutralize siRNA helping to pass through membrane [61].

#### *2.4.5. Nanoparticles*

For systemic delivery, a targeted nanocarrier-siRNA complex has been used. There are some studies that have experimentally condensed DNA or RNA into cancer-targeted nanoparticles with PEI, PLL, and cyclodextrin-containing polymers [62]. PEI–PEG–arginine–glycine– aspartic acid (RGD) fusion was used to inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) expression [63]. Angiogenesis can be inhibited by downregulation or silencing of VEGFR-2 expression [64]. PEGylation of nanoparticles causes "muco-inert" properties, which enhances diffusion process through mucus and peptidoglycan barriers [65].

#### *2.4.6. Aptamer*

siRNAs can be coupled with aptamers or oligodeoxynucleotide through a disulfide bond. This releases actively into targeted cells siRNAs before cytosolic uptake. Conjugate of aptamer siRNA has suggested a novel therapeutics with widespread applications in medicine [66].

#### **2.5. Limitations**

#### *2.5.1. Competition with endogenous RNAs*

In human brain diseases and normal brain development, RNAi potentiates the important role in normal neuronal function, although it is underestimated. When exogenous shRNA is introduced into the neuron, it might be considered whether the RNAi machinery perturbs normal physiologic condition of the system. Bioactive drugs that rely on cellular processing to exert their action face the risk of saturating such pathways and hence perturb the natural system. Sometimes, ectopically introduced RNAi does not trigger the silencing process because siRNA/shRNA activity may depend on the endogenous miRNA to achieve efficient target silencing. Mice that received liver-directed associate adeovirus (AAV)-encoded shRNAs were damaged in liver with dose-dependent manner. Within 2 months, the mice were killed by introducing high doses of AAV-encoded shRNAs. It was interpreted that the liver-specific miRNA was unexpectedly down-regulated by introducing shRNA [67]. The enhanced expression of Exportin 5, the nuclear export component, increased RNAi efficacy, which was shown by competition assay [68].

#### *2.5.2. Stimulation of innate immune responses*

dendrimers with extensive applications. There are different classes of cationic and anionic dendrimers, such as polyamidoamine (PAMAM), polypropylene imine (PPI), and polyethy‐ lene glycol (PEG)-grafted carbosilane [52]. Specific dendritic polymers like PAMAM have been widely utilized in *in vivo* drug delivery [53]. Conjugation of Tat peptide (GRKKRRQRRRPQ) with PAMAM-G5 can efficiently inhibit multi-drug resistance-1 (MDR-1) gene expression *in vitro* [51]. Capping poly-l-lysine (PLL) dendrimers with methotrexate enhances stability and

Cationic polymers include chitosan, gelatin, cationic dextran, cationic cellulose, and cationic cyclodextrin and some synthetic biocompatible polyethyleneimine (PEI), PLL, poly(amidoa‐ mine)s (PAAs), poly(amino-co-ester), and poly(2-*N*,*N*-dimethylaminoethylmethacrylate). Moreover, they are less immunogenic response because these polymers are natural biode‐

CPPs are cationic peptides. CPPs interact covalently or non-covalently through disulfide or electrostatic–hydrogen interactions with siRNAs [56]. Viral protein (VP22) [57], MPG (a peptide vector) [58], amphipathic peptide [59], and poly-arginine [60] were reported the same abilities. In addition, small cationic polypeptides (poly His, Lys, and Arg) coat and neutralize

For systemic delivery, a targeted nanocarrier-siRNA complex has been used. There are some studies that have experimentally condensed DNA or RNA into cancer-targeted nanoparticles with PEI, PLL, and cyclodextrin-containing polymers [62]. PEI–PEG–arginine–glycine– aspartic acid (RGD) fusion was used to inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) expression [63]. Angiogenesis can be inhibited by downregulation or silencing of VEGFR-2 expression [64]. PEGylation of nanoparticles causes "muco-inert" properties, which

siRNAs can be coupled with aptamers or oligodeoxynucleotide through a disulfide bond. This releases actively into targeted cells siRNAs before cytosolic uptake. Conjugate of aptamer siRNA has suggested a novel therapeutics with widespread applications in medicine [66].

In human brain diseases and normal brain development, RNAi potentiates the important role in normal neuronal function, although it is underestimated. When exogenous shRNA is

enhances diffusion process through mucus and peptidoglycan barriers [65].

decreases toxicity [54].

170 RNA Interference

*2.4.3. Cationic polymers*

gradable [55].

*2.4.4. Cationic peptides*

*2.4.5. Nanoparticles*

*2.4.6. Aptamer*

**2.5. Limitations**

*2.5.1. Competition with endogenous RNAs*

siRNA helping to pass through membrane [61].

RNAi therapy is importantly considered because of its potential for generating an adverse immune response, particularly in neurodegenerative diseases with affected brain. It has been already known as "heightened state of alert" to start chronic pro-inflammatory signaling cascades [30]. All evolutionary conserved mechanisms aimed at combating against invading viral pathogens [69]. In general, innate immune responses to non-virally delivered siRNAs are mediated by members of the toll-like receptor (TLR) family or by the two different dsRNAsensing proteins: retinoic acid-inducible gene-1 and dsRNA-binding protein kinase [70]. Certain siRNA sequence motifs invoked TLR7-dependent immune stimulation [71]. The particular sequence motif (5′-GUCCUUCAA-3′) seems to be recognized by TLR7 in plasma‐ cytoid dendritic cells and activates immune responses. The GU-rich regions, so-called "danger motifs," stimulated innate immune responses and lead to secretion of inflammatory cytokines in a cell type and sequence-specific manner. As siRNA-mediated immune induction seems to rely on endosome-located TLR receptors (TLR7 and TLR8) [72], the delivery and compart‐ mentalization of the siRNA significantly influence the cellular responses [3]. These interactions can occur during endosomal or lysosomal compartments' internalization or intracellular release of the siRNA molecule. It has a manner of dose and sequence dependence. Importantly, the chemically modified or nanoparticle-encased siRNA complexes avoid stimulation of immune response.

#### *2.5.3. Suppression of off targets*

Harmfulness of RNAi is "OTE." Genome-wide sequencing analyses have clearly demonstrat‐ ed that siRNA-treated cells show off-target silencing of a large number of genes [73]. The research result suggests that siRNAs with a 2′-*O*-MOE modification at the second base can significantly reduce off target without compromising the degree of silencing target [74]. Experimentally, it has been verified that off targets have 6~7-nt long matching to the siRNA, and it is called "seed" region [75]. When the siRNA guide strand contains seed-sequence matching to mRNA 3′-UTR regions, the siRNA guide strand functions as a miRNA, which might lead to harmful OTEs by translational repression [76]. To avoid siRNA seed matching with mRNA 3′ UTRs, the use of online 3′-UTR search algorithms would potentially reduce the detrimental OTEs [75].

The OTEs can also derive from non-specific changes in gene expression due to the activation of the interferon response (IR) [77]. The OTEs can change another gene by binding either strand of the shRNA to partially complementary sequences rather than binding to the intended target gene [77]. In case of dsRNA, it can result in a signaling cascade that culminates with the activation of interferon responsive genes and global translational repression [78]. Neverthe‐ less, IR activation was variable among the siRNAs used for each of these studies, and one recent report did not detect IR activation by siRNAs [79]. In mice, injection of naked siRNA did not show detectable induction of an IR in one study while another study showed sequencedependent induction of innate immunity [79, 80].

#### **3. Applications of RNAi**

RNAi has been used to generate model systems to identify novel molecular targets [81], to study gene function [82], and to create a new niche for clinical therapeutics [83]. Many researchers reported that siRNAs have successfully been tested in various disease animal models. Recent reports reviewed the therapeutic potential of synthetic siRNAs in various human diseases and disorders [84].

#### **3.1. Application for therapy with RNAi** *in vivo*

Applications, such as gene function analysis, target identification and validation, and thera‐ peutic agents, are the main spots of this new technology [26]. Although RNAi is an efficient technique for *in vitro* studies, there are some challenges for *in vivo* applications. siRNAs have undesired characteristics, such as non-specific silencing of non-targeted genes and dosedependent immunogenic response [85]. In addition, it is extremely complicated to avoid the OTEs due to spatiotemporal gene expression pattern of these molecules [73]. Furthermore, age, sex, tissue, organ, tumor, and individual-specific specificity should be also considered as other variables [86]. Prediction of susceptible off-target domains that can influence silencing efficiency is the first step for applying *in vivo* therapy [73, 87]. Some studies recommend utilization of more sensitive alignment algorithms or siDirect instead of BLAST database [85, 88] to predict a target for siRNA matching without cross-reactivity [89].

The administration route for siRNA, such as oral or intravenous, is not feasible and not efficiently delivered the siRNA into target cells. A single injection of naked siRNA into the brain parenchyma failed to good efficacy [90]. A study reported that continuous infusion of siRNA into the ventricular CSF success with very high concentration [91]. To penetrate the blood–brain barrier (BBB) and reach the target cells in the interesting site, receptor-specific pegylated immunoliposome (PIG) is used. PIGs encapsulate the plasmid vector–encoding siRNA or shRNA and are administered with peripheral route to the brain. This tool has been tried in brain cancer animal model and successfully worked [92]. Another study showed effective and long-term knock down of endogenous tyrosine hydroxylase (TH) in rodent brain using shRNA-expressing adeno-associated virus (AAV) [93]. There have been many successful *in vivo* studies with using viral vector. They are included two models of autoimmune hepatitis [94], hepatitis B virus [95], respiratory viruses such as influenza virus [96], respiratory syncytial virus [97], parainfluenza virus, and sexually transmitted disease such as herpes simplex virus-2 [98]. Both non-viral and viral shRNA delivery systems have been trailed.

#### **3.2. Application for therapy with RNAi in brain diseases**

The OTEs can also derive from non-specific changes in gene expression due to the activation of the interferon response (IR) [77]. The OTEs can change another gene by binding either strand of the shRNA to partially complementary sequences rather than binding to the intended target gene [77]. In case of dsRNA, it can result in a signaling cascade that culminates with the activation of interferon responsive genes and global translational repression [78]. Neverthe‐ less, IR activation was variable among the siRNAs used for each of these studies, and one recent report did not detect IR activation by siRNAs [79]. In mice, injection of naked siRNA did not show detectable induction of an IR in one study while another study showed sequence-

RNAi has been used to generate model systems to identify novel molecular targets [81], to study gene function [82], and to create a new niche for clinical therapeutics [83]. Many researchers reported that siRNAs have successfully been tested in various disease animal models. Recent reports reviewed the therapeutic potential of synthetic siRNAs in various

Applications, such as gene function analysis, target identification and validation, and thera‐ peutic agents, are the main spots of this new technology [26]. Although RNAi is an efficient technique for *in vitro* studies, there are some challenges for *in vivo* applications. siRNAs have undesired characteristics, such as non-specific silencing of non-targeted genes and dosedependent immunogenic response [85]. In addition, it is extremely complicated to avoid the OTEs due to spatiotemporal gene expression pattern of these molecules [73]. Furthermore, age, sex, tissue, organ, tumor, and individual-specific specificity should be also considered as other variables [86]. Prediction of susceptible off-target domains that can influence silencing efficiency is the first step for applying *in vivo* therapy [73, 87]. Some studies recommend utilization of more sensitive alignment algorithms or siDirect instead of BLAST database [85,

The administration route for siRNA, such as oral or intravenous, is not feasible and not efficiently delivered the siRNA into target cells. A single injection of naked siRNA into the brain parenchyma failed to good efficacy [90]. A study reported that continuous infusion of siRNA into the ventricular CSF success with very high concentration [91]. To penetrate the blood–brain barrier (BBB) and reach the target cells in the interesting site, receptor-specific pegylated immunoliposome (PIG) is used. PIGs encapsulate the plasmid vector–encoding siRNA or shRNA and are administered with peripheral route to the brain. This tool has been tried in brain cancer animal model and successfully worked [92]. Another study showed effective and long-term knock down of endogenous tyrosine hydroxylase (TH) in rodent brain using shRNA-expressing adeno-associated virus (AAV) [93]. There have been many successful *in vivo* studies with using viral vector. They are included two models of autoimmune hepatitis

88] to predict a target for siRNA matching without cross-reactivity [89].

dependent induction of innate immunity [79, 80].

**3. Applications of RNAi**

172 RNA Interference

human diseases and disorders [84].

**3.1. Application for therapy with RNAi** *in vivo*

Many works using RNAi to suppress dominant disease genes have occurred primarily in cell culture models [99, 100]. Allele-specific silencing aims to suppress the disease gene without affecting any other normal genes. The possible therapeutic applications of RNAi for neuro‐ logical diseases are broad, ranging from acquired diseases, such as viral infections, to purely genetic disorders.

Particularly, one attractive group of candidate diseases for RNAi therapy is the dominantly inherited neurodegenerative diseases, including polyglutamine disorders such as Hunting‐ ton's disease (HD) [101], amyotrophic lateral sclerosis (ALS) [102], familial Alzheimer's disease (AD) [103], and frontotemporal dementia caused by tau mutations [104]. HD has been approaching with animal model mimicking the human disease to provide some therapeutic clues with various ways. In the new preclinical study, single injection of a cholesterol conju‐ gated-siRNA was targeting mutant Huntingtin (mhtt), and, subsequently, the pathologic symptoms containing behavioral dysfunction were improved [105].

The exciting recent works have taken place *in vivo* in mouse models of neurodegenerative brain disease. The best example of RNAi-mediated therapy to date is in spinocerebellar ataxia type-1 (SCA-1) [106]. As another case, RNAi-mediated therapy was tried on DYT1 dystonia with animal disease model. DYT1 dystonia is another inherited dystonia. DYT1 dystonia is caused by deletion of GAG that is coding TOR1A, which results in one of a pair of glutamic acid from the carboxyl terminal of the torsin A (TA) protein-coding region [107].

Prion disease is one of the brain diseases that is invariably fatal, and no therapy is available. Once serious damage to the brain has already occurred, clinical symptoms manifest after the untreatable brain damage. Causing this reason, prion disease treatments have aimed not to cure the disease but to slow disease progression [108]. Prion disease is caused by prions, in which a self-replicating, infectious protease resistant form of PrP (termed PrPSc), is the only essential component identified to date. PrPSc multiplies through conversion of the normal cellular PrP (PrPC) [109]. Some reviews are presenting that lentivector-mediated anti-PrPC shRNA expression effectively suppressed prion replication in a murine neuroblastoma cell line, and researchers created chimeric mice using embryonic stem cells, which were transfected with a lentiviral vector carrying an anti-PrPC shRNA. Results showed that the survival time after prion inoculation was markedly prolonged [110, 111].

#### **4. Prospects of RNA therapeutics in CNS disease**

The current phamaceuticals required more knowledge to decipher potentials of the RNAi in spite of flourishing future. It is crucial that each disease has not only a unique pattern but also the understanding for pathogenesis relating pathways and activating or inhibiting factors [112]. To introduce the DNA therapeutics into the CNS is much more complicated due to the BBB, which can be only permeable to lipophilic molecules of less than 400 Da [113]. Using human viruses, DNA delivery system has been extensively trailed for over three decades. However, the results have been not satisfactory. Therefore, a critical goal for clinical neuro‐ science is to develop the efficient RNAi therapy to prevent the neuronal damage [77]. We categorized the neurological disease containing cancers in below sections.

#### **4.1. Genetic neuronal disease-familial neurological disease**

The application of siRNA has been advanced in development of various incurable disease therapies, apart from the widespread usage of RNAi in fundamental biological application. Particularly, dominant inherited disorders are major application field. Among familial neurobiological diseases, HD has been tried to lots of therapies based on RNAi and may be beneficial effect from the therapy using siRNA. In the N171-82Q transgenic HD mouse model, a study using shRNA showed a 50–55% decrease in the N171-82Q mRNA when injected to striatum and a complete elimination of mHtt protein inclusions from the neuronal cells [114]. There was also a rescue of motor dysfunctions. siRNAs against the "R6/2 huntingtin (htt) mRNA" reduced brain atrophy and neuronal inclusions in the R6/2 transgenic mouse model [115]. With using a rAAV5 vector and administrating to the striatum, long-term expression of a mHtt-siRNA partially reduced in neuropathology condition [116].

Besides AAV, there is lentiviral vector that can be applied after onset of symptoms [117]. Using lentivirus vector decreased htt protein expression by up to 35% and altered htt-related pathways but did not reduce cellular viability for at least 9 months after treatment. To enhance cellular uptake of siRNA, cholesterol-conjugated duplexes (cc-siRNA) have been applied to target htt mRNA [118]. Allele-specific targeting of mhtt helped to overcome the side effects of RNAi where ASO or single nucleotide polymorphisms (SNPs) in the mHtt allele have been used to specifically target only the mutant gene product [119]. Intra-cellular antibody frag‐ ments bind to abnormal aggregations, and allele-specific siRNA disrupts mhtt gene [120, 121]. Targeting of just three SNPs with five siRNAs covered most of the HD patients in the population studied [122].

Tuberous sclerosis is a common, dominantly inherited disorder caused by mutations in the tumor suppressor complex-1 (TSC1) or tumor suppressor complex-2 (TSC2) genes [123]. The proteins hamartin (encoded by TSC1) and tuberin (encoded by TSC2) form a complex. This protein complex represses mTOR-S6K-4E-BP signaling pathway [123]. Mutated TSC1 and TSC2 lead to loss of activity resulting in unchecked cell growth and hamartoma formation in the CNS. Recent studies propose that the target may be the GTPase Rheb [124]. RNAi sup‐ pression of Rheb might respond the dysregulated cell proliferation in tuberous sclerosis.

Particularly, allele-specific silencing is apt for inherited neurological diseases. DYT1 is the most commonly inherited dystonia [125]. Although the pathogenesis of DYT1 is unclear, several facts make DYT1 a good candidate to explore the therapeutic potential of RNAi [77]. The three nucleotide difference between the wild type and the mutated gene has been enough to allow allele-specific silencing against mutant TA (the mutated protein in DYT1) in cultured cells using *in vitro* synthesized siRNA [107].

Allelic discrimination has also been demonstrated for superoxide dismutase (SOD) mutations responsible for familial ALS [100], and also a mutation in an acetylcholine receptor subunit causes congenital myasthenia [126]. In a tau mutation responsible for fronto-temporal dementia, siRNAs can act by discriminating between sequences differing by a single nucleo‐ tide [99].

An important role for RNAi in the brain is also presented for Fragile X syndrome (FXS) in human [127]. FXS is the one of the most common forms of inherited mental retardation caused by mutations in Fragile X Mental Retardation Protein (FMRP), a protein influencing synaptic plasticity [127]. FXS is stemmed from mutations in FMRP and is supported by the involvement of the RNAi process in human neurological disease [127]. Increasing evidences from different studies support the view that FMRP regulates protein translation by regulating RNAi in neurons [128, 129].

#### **4.2. Sporadic neurodegenerative diseases**

the understanding for pathogenesis relating pathways and activating or inhibiting factors [112]. To introduce the DNA therapeutics into the CNS is much more complicated due to the BBB, which can be only permeable to lipophilic molecules of less than 400 Da [113]. Using human viruses, DNA delivery system has been extensively trailed for over three decades. However, the results have been not satisfactory. Therefore, a critical goal for clinical neuro‐ science is to develop the efficient RNAi therapy to prevent the neuronal damage [77]. We

The application of siRNA has been advanced in development of various incurable disease therapies, apart from the widespread usage of RNAi in fundamental biological application. Particularly, dominant inherited disorders are major application field. Among familial neurobiological diseases, HD has been tried to lots of therapies based on RNAi and may be beneficial effect from the therapy using siRNA. In the N171-82Q transgenic HD mouse model, a study using shRNA showed a 50–55% decrease in the N171-82Q mRNA when injected to striatum and a complete elimination of mHtt protein inclusions from the neuronal cells [114]. There was also a rescue of motor dysfunctions. siRNAs against the "R6/2 huntingtin (htt) mRNA" reduced brain atrophy and neuronal inclusions in the R6/2 transgenic mouse model [115]. With using a rAAV5 vector and administrating to the striatum, long-term expression of

Besides AAV, there is lentiviral vector that can be applied after onset of symptoms [117]. Using lentivirus vector decreased htt protein expression by up to 35% and altered htt-related pathways but did not reduce cellular viability for at least 9 months after treatment. To enhance cellular uptake of siRNA, cholesterol-conjugated duplexes (cc-siRNA) have been applied to target htt mRNA [118]. Allele-specific targeting of mhtt helped to overcome the side effects of RNAi where ASO or single nucleotide polymorphisms (SNPs) in the mHtt allele have been used to specifically target only the mutant gene product [119]. Intra-cellular antibody frag‐ ments bind to abnormal aggregations, and allele-specific siRNA disrupts mhtt gene [120, 121]. Targeting of just three SNPs with five siRNAs covered most of the HD patients in the

Tuberous sclerosis is a common, dominantly inherited disorder caused by mutations in the tumor suppressor complex-1 (TSC1) or tumor suppressor complex-2 (TSC2) genes [123]. The proteins hamartin (encoded by TSC1) and tuberin (encoded by TSC2) form a complex. This protein complex represses mTOR-S6K-4E-BP signaling pathway [123]. Mutated TSC1 and TSC2 lead to loss of activity resulting in unchecked cell growth and hamartoma formation in the CNS. Recent studies propose that the target may be the GTPase Rheb [124]. RNAi sup‐ pression of Rheb might respond the dysregulated cell proliferation in tuberous sclerosis.

Particularly, allele-specific silencing is apt for inherited neurological diseases. DYT1 is the most commonly inherited dystonia [125]. Although the pathogenesis of DYT1 is unclear, several facts make DYT1 a good candidate to explore the therapeutic potential of RNAi [77]. The three nucleotide difference between the wild type and the mutated gene has been enough to allow

categorized the neurological disease containing cancers in below sections.

**4.1. Genetic neuronal disease-familial neurological disease**

a mHtt-siRNA partially reduced in neuropathology condition [116].

population studied [122].

174 RNA Interference

Neurodegenerative diseases are age dependent, and many of them are inherited. However, non-genetic neurological diseases, such as sporadic AD or migraine, are much more common than diseases due to single-gene mutations.

The most common sporadic neurodegenerative disease, AD, is also the best studied with siRNA therapy. Many studies of AD pathogenesis investigate an essential role for β-amyloid (Aβ) in familial and sporadic forms of AD [130]. Different RNAi strategies have been applied to regulate this pathogenic cascade. Researchers tried by directly silencing of amyloid precursor protein (APP) [131], by silencing of β-secretase (BACE1) that is one of two proteases required for Aβ production but not essential gene in mice [132], or by silencing of tau expres‐ sion that is a component of the neurofibrillary tangles of AD neurons. Therapeutic use of RNAi is now being tested in animal models of AD targeting these proteins.

Migraine, one of the most common neurological disorders, is caused by diminished production of calcitonin gene-related peptide (CGPR) in the trigeminal system. CGPR can protect from migraine attacks [133]. The CGPR-limited animals are normal, but the paroxysmal nature of this disorder necessitates to use promoters for CGPR. From the beginning of the pathogenic cascade, expression of the shRNA targeting CGPR can terminate the growing pain of this disease. This pain alleviating therapy for migraine is limited because of high threshold dose needed for RNAi [133].

#### **4.3. Motor dysfunction disease**

A viral delivery of shRNA was used to achieve a long-term RNAi in the CNS. In some reports, the delivery of shRNA-expressing lentivirus showed a rescue of spinal motor neurons with behavioral and histopathological phenotypes in a mouse model having dominant familial ALS [134].

Parkinson's disease (PD) is the second most common neurodegenerative disease. Patient brain of PD is often littered with Lewy body, which is abnormal protein aggregate primarily of alphasynuclein (α-syn) [135, 136]. parkinsonism is linked to hereditary to a single-point mutation in the α-syn as well as genetic duplication or triplication of the α-syn (SCNA) [104]. The studies targeting the α-syn expression revealed RNAi as a therapeutic approach to PD [30, 137]. To date, conflicting results were reported. Regarding the effectiveness and tolerability, there is a report that nigrostriatal degeneration was detected after depleting the α-syn level in the brain [138]. It can be inferred that RNAi approaches can be used to validate them in genetic and sporadic models of PD.

#### **4.4. Neurovascular disease**

RNAi can be applied to cardiovascular and cerebrovascular diseases. Cardiovascular disease results from the progressive occlusion of arteries, and it is most common in a process called atherosclerosis, which can ultimately culminate in a myocardial infarction or stroke [139]. It may be a trigger for the death of cardiac muscle cells or neurons [139]. Although some of the cells die rapidly by necrosis, many other cells die more slowly by apoptosis in such cardiac myocytes and brain neurons [140, 141]. RNAi technology may be used to intervene in athero‐ sclerosis or to reduce the damage of heart tissue and brain cells following a myocardial infarction or stroke [142].

Another vascular disease is an ocular disease. Representatively, there were two RNAi clinical trials. The trials performed direct intra-vitreal injection of siRNAs that are targeting VEGF or the VEGFR to test for the safety and efficacy in ocular diseases [143]. siRNAs, targeting VEGF and VEGFR1, are currently in the early stages of clinical trials. The direct injection approach can also prove its usefulness for the other ocular diseases.

#### **4.5. Cancer**

A chemo-resistance or radio resistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity have the potential to increase drug efficacy while reducing toxic effects on untargeted cells (144). Actually, oncogenes expressed at abnormally high levels are attractive targets for RNAi-based therapies against cancers [145], and such approaches have effectively inhibited tumor growth *in vivo* in mouse models.

In nasopharyngeal carcinoma, hyaluronan receptor (CD44) gene silencing resulted in pro‐ found reduction of malignant potential of the cells: tumorigenesis and metastasis of tumors in nude mice [105, 146]. It is also suggested a possible therapeutic effect of direct introduction of siRNA to CD44 into some human solid tumors with high expression of the CD44 gene [146]. Although the role of epidermal growth factor receptor (EGFR) in altering tumor chemosensi‐ tivity has not yet been fully elucidated, selectively targeting EGFR supplies the reversal possibility of chemoresistance in many tumor types [147]. Reduction of EGFR expression and increased chemosensitivity to docetaxel are emerging an effective strategy for the sensitization of cancer cells to taxane chemotherapy [147]. siRNA-PG-Amine polyplexes can be systemically delivered to tumors in mice [148], and siRNA-nanocarrier system can efficiently inhibit expression of a specific gene in tumor cells. Once the intact siRNA molecule moves to the target, the gene of interest gets silenced. The PG-amine-based delivery system actually combines both tumor passive targeting with the sequence selectivity of siRNA [148].

The limiting point of targeted therapy is alternative pathway compensation by gene amplifi‐ cation. The "synthetic lethality" is proposed idea to overcome the above problem [149]. This concept suggests that two genes may be considered to have a synthetically lethal relationship [150]. When a mutation is existed either of the two genes alone has no effect on cell survival, but when mutations in both genes cell death is triggered at the same time. By genome-wide RNAi library screening, some synthetic lethal molecules have been discovered. Anaphasepromoting complex/cyclosome (APC/C) and polo-like kinase (PLK) are synthetically lethal with the RAS oncogene in colorectal cancer [151]. The STK33 gene is also synthetic lethal interacting with a RAS mutation in multiple cancer cells from different tumor types [152]. Modified EGFR (amplification or truncation) and hyperactivation of AKT play a major role in the development of glioblastoma, one of the extreme malignancies [153]. There are approaches to develop the siRNA delivery efficiency such as the use of dsRNA-binding domain (DRBD) with a TAT peptide transduction domain (PTD) delivery peptide [154]. These facilities are stable and efficient delivery of siRNAs into cells [155].

#### **5. Conclusions**

Parkinson's disease (PD) is the second most common neurodegenerative disease. Patient brain of PD is often littered with Lewy body, which is abnormal protein aggregate primarily of alphasynuclein (α-syn) [135, 136]. parkinsonism is linked to hereditary to a single-point mutation in the α-syn as well as genetic duplication or triplication of the α-syn (SCNA) [104]. The studies targeting the α-syn expression revealed RNAi as a therapeutic approach to PD [30, 137]. To date, conflicting results were reported. Regarding the effectiveness and tolerability, there is a report that nigrostriatal degeneration was detected after depleting the α-syn level in the brain [138]. It can be inferred that RNAi approaches can be used to validate them in genetic and

RNAi can be applied to cardiovascular and cerebrovascular diseases. Cardiovascular disease results from the progressive occlusion of arteries, and it is most common in a process called atherosclerosis, which can ultimately culminate in a myocardial infarction or stroke [139]. It may be a trigger for the death of cardiac muscle cells or neurons [139]. Although some of the cells die rapidly by necrosis, many other cells die more slowly by apoptosis in such cardiac myocytes and brain neurons [140, 141]. RNAi technology may be used to intervene in athero‐ sclerosis or to reduce the damage of heart tissue and brain cells following a myocardial

Another vascular disease is an ocular disease. Representatively, there were two RNAi clinical trials. The trials performed direct intra-vitreal injection of siRNAs that are targeting VEGF or the VEGFR to test for the safety and efficacy in ocular diseases [143]. siRNAs, targeting VEGF and VEGFR1, are currently in the early stages of clinical trials. The direct injection approach

A chemo-resistance or radio resistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity have the potential to increase drug efficacy while reducing toxic effects on untargeted cells (144). Actually, oncogenes expressed at abnormally high levels are attractive targets for RNAi-based therapies against cancers [145], and such

In nasopharyngeal carcinoma, hyaluronan receptor (CD44) gene silencing resulted in pro‐ found reduction of malignant potential of the cells: tumorigenesis and metastasis of tumors in nude mice [105, 146]. It is also suggested a possible therapeutic effect of direct introduction of siRNA to CD44 into some human solid tumors with high expression of the CD44 gene [146]. Although the role of epidermal growth factor receptor (EGFR) in altering tumor chemosensi‐ tivity has not yet been fully elucidated, selectively targeting EGFR supplies the reversal possibility of chemoresistance in many tumor types [147]. Reduction of EGFR expression and increased chemosensitivity to docetaxel are emerging an effective strategy for the sensitization of cancer cells to taxane chemotherapy [147]. siRNA-PG-Amine polyplexes can be systemically

approaches have effectively inhibited tumor growth *in vivo* in mouse models.

sporadic models of PD.

176 RNA Interference

**4.4. Neurovascular disease**

infarction or stroke [142].

**4.5. Cancer**

can also prove its usefulness for the other ocular diseases.

Small RNAs and non-coding small RNAs were important discovery for molecular cell biology; these small RNAs have a vital role in gene regulation that can be controlled by RNA interfering technology. Presently, attempts to integrate gene expression profiling and protein interaction mapping are the main research objectives. The proof-of-principle studies *in vivo* have clearly demonstrated that both viral and non-viral delivery methods can provide selective and efficient target gene suppression without any clear toxic effects. Initial results have been very promising, and many pharmaceutical companies are already focusing on commercialization of various disease-specific RNAi drugs. Despite successful trials in a large number of animal model studies including brain diseases, to develop an efficient therapeutic application, there are numerous hurdles and concerns regarding targeted delivery of siRNAs into brain subre‐ gions that must be overcome before wide clinical application of RNAi as a new therapeutic solution. The OTEs, competition with endogenous cellular RNAi components, and effective delivery *in vivo* remain to be optimized. Although recent research has improved the safety and toxicity from the OTEs, it still remains a crucial issue and needs to be addressed before RNAibased drugs are ready for clinical use. Translational research using RNAi has taken place with an unprecedented speed, and already there are several RNAi-based human clinical trials in progress that will provide breakthrough therapeutic tools for effective treatment human CNSrelated disease.

#### **Author details**

Kyoung Joo Cho and Gyung Whan Kim\*

\*Address all correspondence to: gyungkim@yuhs.ac

Department of Neurology, Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea

#### **References**


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**Author details**

178 RNA Interference

Seoul, South Korea

1262504

**References**

Kyoung Joo Cho and Gyung Whan Kim\*

\*Address all correspondence to: gyungkim@yuhs.ac

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## **RNAi-based Gene Therapy for Blood Genetic Diseases**

Mengyu Hu, Qiankun Ni, Yuxia Yang and Jianyuan Luo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61644

#### **Abstract**

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Therapies for blood genetic diseases can be divided into different categories, including chemotherapy, radiotherapy, gene therapy, and hematopoietic stem cell transplantation. Among these treatments, gene targeting is progressively becoming a therapeutic alterna‐ tive that offers the possibility of a permanent cure for certain blood genetic diseases. In recent years, gene therapy has played a more important role in curing genetic blood dis‐ orders. RNA interference (RNAi) is one of the directions for gene therapy, which was in‐ tensively studied in the past decades for its potentials in the treatment of diseases. In order to provide useful references and prospective directions for further studies concern‐ ing RNAi-based gene therapy for blood genetic diseases, current RNAi-based gene thera‐ pies for several typical blood genetic diseases have been summarized and discussed in this chapter.

**Keywords:** RNA interference, gene therapy, mechanism, therapeutic strategy, blood ge‐ netic diseases

#### **1. Introduction**

Conceived in the 1960s, gene therapy did not produce any meaningful results until recent reports of success appeared in clinical studies. Gene therapy attempts to treat inherited diseases using normal copies of the defective genes. Insertion and expression of specific exogenous genetic materials via the transfer of nucleic acids directly *in vivo*, or through modified cells *in vitro*, correct a cellular dysfunction or provide a new cellular function. It has the potential to cure any genetic disease with long-lasting therapeutic benefits [1, 2]. Gene therapy can be classified into (i) germ line and (ii) somatic line gene therapy types. In the former, genomes of germ cells (sperms or eggs) are integrated by exogenous functional genes, which can be carried onto the patient's offsprings. In the latter, therapeutic genes are intro‐ duced into somatic cells and the effects will only be limited to the individual patient [3].

© 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.

According to the mechanisms, gene therapy may include three major categories: (a) direct modulation of the disease-causing gene, which can be applied to monogenic hereditary diseases; (b) indirect treatment through gene modulation, which can be applied to multifac‐ torial diseases; and (c) immunotherapy by gene modulation (DNA vaccines), which leads to the synthesis of the relevant antigen or an adjuvant [1].

RNA interference (RNAi) can be used for gene therapy. It was intensively studied in the past few decades for its potential in the treatment of blood genetic diseases. RNAi-based gene therapy possesses several therapeutic advantages such as high efficiency, sequence specificity, and potentially less immunogenicity. Less immunogenicity is largely due to the use of nonprotein-coding "gene products" to trigger RNAi, which makes gene therapy less likely to be potentially hampered by the host immune system [4, 5]. Compared to traditional small molecules and protein drugs, the target specificity and universal treatment spectrum make RNAi-based gene therapy an ideal treatment for blood genetic diseases. However, there are still obstacles that remain, such as barriers in the blood circulation system and the diseased tissues that block the actualization of the RNAi effects [6]. RNAi technology is a relatively new discovery, and it has already become a potent method for gene regulation. In order to provide useful references and prospective directions for further studies, current RNAi-based gene therapies for blood genetic diseases have been summarized and discussed in this chapter.

#### **2. The mechanism of RNAi-based gene therapy**

Being a well-described gene regulatory mechanism, RNAi not only suppresses transcription by transcriptional gene silencing (TGS) but also activates a homology-based mRNA degrada‐ tion process by post-transcriptional gene silencing (PTGS). Both silencing pathways resulted in the decrease of the coding transcript level (mRNA) [7]. We will focus on PTGS due to its important role in RNAi-based gene therapy. Two distinct mechanisms regulate PTGS. The first one is the repression and degradation of mRNAs with imperfect complementarity. Endoge‐ nous microRNAs (miRNAs) belong to this category. They induce translational repression and mRNA degradation when the guide (antisense) strand has limited complementarity to the target mRNA. The second one is the sequence-specific cleavage of perfectly complementary mRNAs. Exogenous small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) belong to this category. They have perfect or near-perfect base-pairings with the intended target mRNA. The miRNAs production and processing rely on host machinery that is guided by complementary miRNA strands to the target mRNA [8, 9]. During the process, doublestranded RNAs (dsRNA) of the target gene are produced and then processed into 21–24 noncoding small RNA duplexes with the help of RNaseIII enzyme dicer and its homologs. These siRNAs are then incorporated into a multi-subunit endonuclease silencing complex called RNA-induced silencing complex (RISC). siRNAs are associated with the defense against parasites, heterochromatin formation, transposon and transgene silencing, and PTGS. These siRNAs, loaded into the RISC, are used as the guide to recognize and degrade or suppress the complementary gene or mRNA utilizing the endonucleases activity of RISC. Gene silencing by RNAi can be used in different biological situations when sequence-specific knockdown of gene expression is required, thus providing a convenient tool for analysis of gene function as well as gene therapy [10, 11, 12].

Recent reports have shown that off-targeting can commonly occur during RNAi, despite the belief that initial gene silencing through RNAi was thought to be specific. Because nonspecific hybridization of siRNAs with non-target transcripts can induce undesired effects, it should be guaranteed that the dsRNA and corresponding siRNA sequences do not exert off-target effects that negatively influence host physiology. Off-target silencing is determined by the similarity of sequences between siRNA and non-target genes, or mRNA sequences not selected for RNAi, as well as the size of siRNA and transitive RNAi. Thus, off-targeting effects can be avoided by using highly specific sequences in siRNA expressed under specific and inducible promoters. In addition, it is important to examine the off-targeting effects of RNAi at multiple levels, since undesired effects on the host may occur through the silencing of genes associated with regulatory functions and multiple metabolic pathways, such as transcription factors or signaling molecules [10].

#### **3. RNAi-based gene therapy for blood genetic diseases**

#### **3.1. Blood diseases**

According to the mechanisms, gene therapy may include three major categories: (a) direct modulation of the disease-causing gene, which can be applied to monogenic hereditary diseases; (b) indirect treatment through gene modulation, which can be applied to multifac‐ torial diseases; and (c) immunotherapy by gene modulation (DNA vaccines), which leads to

RNA interference (RNAi) can be used for gene therapy. It was intensively studied in the past few decades for its potential in the treatment of blood genetic diseases. RNAi-based gene therapy possesses several therapeutic advantages such as high efficiency, sequence specificity, and potentially less immunogenicity. Less immunogenicity is largely due to the use of nonprotein-coding "gene products" to trigger RNAi, which makes gene therapy less likely to be potentially hampered by the host immune system [4, 5]. Compared to traditional small molecules and protein drugs, the target specificity and universal treatment spectrum make RNAi-based gene therapy an ideal treatment for blood genetic diseases. However, there are still obstacles that remain, such as barriers in the blood circulation system and the diseased tissues that block the actualization of the RNAi effects [6]. RNAi technology is a relatively new discovery, and it has already become a potent method for gene regulation. In order to provide useful references and prospective directions for further studies, current RNAi-based gene therapies for blood genetic diseases have been summarized and discussed in this chapter.

Being a well-described gene regulatory mechanism, RNAi not only suppresses transcription by transcriptional gene silencing (TGS) but also activates a homology-based mRNA degrada‐ tion process by post-transcriptional gene silencing (PTGS). Both silencing pathways resulted in the decrease of the coding transcript level (mRNA) [7]. We will focus on PTGS due to its important role in RNAi-based gene therapy. Two distinct mechanisms regulate PTGS. The first one is the repression and degradation of mRNAs with imperfect complementarity. Endoge‐ nous microRNAs (miRNAs) belong to this category. They induce translational repression and mRNA degradation when the guide (antisense) strand has limited complementarity to the target mRNA. The second one is the sequence-specific cleavage of perfectly complementary mRNAs. Exogenous small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) belong to this category. They have perfect or near-perfect base-pairings with the intended target mRNA. The miRNAs production and processing rely on host machinery that is guided by complementary miRNA strands to the target mRNA [8, 9]. During the process, doublestranded RNAs (dsRNA) of the target gene are produced and then processed into 21–24 noncoding small RNA duplexes with the help of RNaseIII enzyme dicer and its homologs. These siRNAs are then incorporated into a multi-subunit endonuclease silencing complex called RNA-induced silencing complex (RISC). siRNAs are associated with the defense against parasites, heterochromatin formation, transposon and transgene silencing, and PTGS. These siRNAs, loaded into the RISC, are used as the guide to recognize and degrade or suppress the complementary gene or mRNA utilizing the endonucleases activity of RISC. Gene silencing by RNAi can be used in different biological situations when sequence-specific knockdown of

the synthesis of the relevant antigen or an adjuvant [1].

192 RNA Interference

**2. The mechanism of RNAi-based gene therapy**

Blood diseases refer to the disorders in the hematopoietic system or plasma components. The development of blood disorders is always thought to be related with inheritance, the envi‐ ronment, drugs, and biological factors where the changes in chromosome and/or genes play a critical role in some specific hemopathies. Therapeutic approaches for blood diseases can be divided into different categories, including chemotherapy, radiotherapy, RNAi-based gene therapy, and hematopoietic stem cell transplantation. Among these, RNAi-based gene therapy is progressively becoming a therapeutic alternative which offers the possibility of a permanent cure for some blood diseases [13].

#### **3.2. Hemophilia**

Hemophilia A and B are X-linked monogenic bleeding disorders resulting from deficiencies of factor VIII and IX, respectively. Gene therapy, utilizing both viral and non-viral delivery vectors *in vivo* and *ex vivo*, has been attempted for the treatment of both hemophilia A and B [14, 15]. Given its recent clinical success, adeno-associated vector (AAV)-mediated hepatic gene transfer could be primarily used for the treatment of hemophilia B. However, a number of problems, such as current immunosuppressive regimen and pre-existing neutralizing antibodies, limit the broad applicability of this approach. For hemophilia A, while AAVmediated gene therapy has potential, a number of limitations reduce its desirability, such as packaging capacity and inefficient expression. While a number of transgene modifications have increased the expression levels, the vector doses require the corrective F.VIII expression to remain significantly higher than the F.IX. These expression limitations lead to further concerns about immune responses to both the capsid and, if expression levels are not sufficient, the transgene. As such, *ex vivo* gene transfer may be more effective for hemophilia A due to its ability to enhance expression through cellular division. While a number of promising gene therapies for hemophilia have been elucidated, there are clearly numerous problems that still need to be addressed to develop approved gene therapies, especially RNAi, for both hemo‐ philia A and B in humans [16]. RNAi-based gene therapy for hemophilia is still in its early stages of development.

#### **3.3. β-Thalassemia**

The globin chains have an extremely precise structure, ensuring their function of loading, delivering, and unloading oxygen. The globin chains are coded by genes in the chromosome 16 (α-gene) and 11(β-gene). The normal structure of globin is based on the balanced match between α-chains and β-chains. When the condition is not met, there will be a complete or partial defect in one or both allelic globin genes, such as β-thalassemia [17]. β-Thalassemia is a worldwide-distributed inherited hemoglobin disorder resulting in severe, chronic anemia [18, 19]. It is a heterozygous condition in which only a single β-globin gene is affected and results in the absence or reduced β-globin chain synthesis. The defects of β-globin synthesis lead to an excess of unmatched α-globin, which release free iron, non-heme iron, or hemi‐ chrome. These iron species promote a severe red cell membrane oxidative stress and lead to abnormal β-thalassemic red cell features. The abnormal red cells are finally removed by the macrophage system and results in anemia [20, 21].

Blood transfusion is a primary way to treat the most severe forms of β-thalassemia. Appro‐ priate goals and optimal safety of transfused blood are necessary for routine administration of red blood cells to patients. The problem is that the high frequency of blood transfusion can lead to iron overload. In its less severe form, chronic transfusions are not required, but iron overload may still develop due to the chronic suppression of the synthesis of the iron regula‐ tory hormone hepcidin by ineffective erythropoiesis. Untreated iron overload can be fatal, resulting in cardiac complications [3, 22]. Therefore, handling iron overload is a key factor for the successful treatment of this disease. TMPRSS6, a serine protease expressed predominantly in the liver, can inhibit an iron-responsive bone morphogenetic protein-mother against the decapentaplegic (BMP-SMAD) signaling pathway, resulting in the downregulation of hepcidin transcription. Researchers found that therapeutics with the lipid nanoparticle (LNP) formulated RNAi targeting of TMPRSS6, in conjunction with oral deferiprone therapy, is superior to monotherapy with dietary iron deficiency and iron chelate for reducing hepatic iron storage [22].

#### **3.4. B-lineage lymphoid malignancies**

B-precursor acute lymphoblastic leukemia (BPL) is the most common form of cancer in children and adolescents. A dysfunctional CD22 is expressed in BPL cells due to the deletion of Exon 12 (CD22ΔE12) resulting from a splicing defect related to homozygous intronic mutations. CD22 is a negative regulator of multiple signal transduction pathways critical for the proliferation and survival of B-lineage lymphoid cells, and CD22ΔE12 leads to uncontrol‐ led proliferation and the survival of B cells. Recently, researchers have found that CD22ΔE12 is especially associated with therapy-refractory clones in pediatric BPL, thus implicating the impact of the CD22ΔE12 genetic defect on the aggressive biology of relapsed or therapyrefractory pediatric BPL. At the same time, forced expression of CD22ΔE12 in transgenic mice causes fatal BPL, demonstrating that CD22ΔE12 alone is sufficient as an oncogenic driver lesion for malignant transformation and clonal expansion of B-cell precursors [23].

Recent studies have demonstrated that B-lineage lymphoid malignancies in children and adults are characterized by a high incidence of the CD22ΔE12 genetic alterations. Moreover, the relationship between CD22ΔE12 and aggressive biology of BPL cells is also reported through the demonstration that siRNA-mediated knockdown of CD22ΔE12 in primary BPL cells is associated with a remarkable inhibition of their carcinogenicity. A unique polypeptidebased nanoparticle formulation of CD22ΔE12-siRNA is described as a first-in-class RNAi therapeutic candidate targeting of CD22ΔE12. This formulation is capable of delivering siRNA cargo into the cytoplasm of leukemia cells, leading to a remarkable inhibition of leukemic cell growth [24]. It is expected that further development of this nanoparticle may promote an effective therapeutic RNAi strategy of aggressive or chemotherapy-resistant B-lineage lymphoid malignancies [23].

#### **3.5. Myeloid leukemia**

ability to enhance expression through cellular division. While a number of promising gene therapies for hemophilia have been elucidated, there are clearly numerous problems that still need to be addressed to develop approved gene therapies, especially RNAi, for both hemo‐ philia A and B in humans [16]. RNAi-based gene therapy for hemophilia is still in its early

The globin chains have an extremely precise structure, ensuring their function of loading, delivering, and unloading oxygen. The globin chains are coded by genes in the chromosome 16 (α-gene) and 11(β-gene). The normal structure of globin is based on the balanced match between α-chains and β-chains. When the condition is not met, there will be a complete or partial defect in one or both allelic globin genes, such as β-thalassemia [17]. β-Thalassemia is a worldwide-distributed inherited hemoglobin disorder resulting in severe, chronic anemia [18, 19]. It is a heterozygous condition in which only a single β-globin gene is affected and results in the absence or reduced β-globin chain synthesis. The defects of β-globin synthesis lead to an excess of unmatched α-globin, which release free iron, non-heme iron, or hemi‐ chrome. These iron species promote a severe red cell membrane oxidative stress and lead to abnormal β-thalassemic red cell features. The abnormal red cells are finally removed by the

Blood transfusion is a primary way to treat the most severe forms of β-thalassemia. Appro‐ priate goals and optimal safety of transfused blood are necessary for routine administration of red blood cells to patients. The problem is that the high frequency of blood transfusion can lead to iron overload. In its less severe form, chronic transfusions are not required, but iron overload may still develop due to the chronic suppression of the synthesis of the iron regula‐ tory hormone hepcidin by ineffective erythropoiesis. Untreated iron overload can be fatal, resulting in cardiac complications [3, 22]. Therefore, handling iron overload is a key factor for the successful treatment of this disease. TMPRSS6, a serine protease expressed predominantly in the liver, can inhibit an iron-responsive bone morphogenetic protein-mother against the decapentaplegic (BMP-SMAD) signaling pathway, resulting in the downregulation of hepcidin transcription. Researchers found that therapeutics with the lipid nanoparticle (LNP) formulated RNAi targeting of TMPRSS6, in conjunction with oral deferiprone therapy, is superior to monotherapy with dietary iron deficiency and iron chelate for reducing hepatic

B-precursor acute lymphoblastic leukemia (BPL) is the most common form of cancer in children and adolescents. A dysfunctional CD22 is expressed in BPL cells due to the deletion of Exon 12 (CD22ΔE12) resulting from a splicing defect related to homozygous intronic mutations. CD22 is a negative regulator of multiple signal transduction pathways critical for the proliferation and survival of B-lineage lymphoid cells, and CD22ΔE12 leads to uncontrol‐ led proliferation and the survival of B cells. Recently, researchers have found that CD22ΔE12 is especially associated with therapy-refractory clones in pediatric BPL, thus implicating the

stages of development.

macrophage system and results in anemia [20, 21].

**3.3. β-Thalassemia**

194 RNA Interference

iron storage [22].

**3.4. B-lineage lymphoid malignancies**

Leukemia arising from genetic alterations in normal hematopoietic stem or progenitor cells results in the impaired regulation of proliferation, differentiation, and apoptosis, as well as the survival of malignant cells. Overall, the relative 5-year survival rate for various leukemias is only around 50% [25]. Leukemia is still a worldwide health problem, although various therapies have been explored to cure the disease. Among them, chemotherapy is always considered to be a frontline treatment, mainly containing a broad spectrum of cytotoxic agents and therapeutic molecules. Although leukemic cells respond well to chemotherapy at the onset of treatment, over a period of 6–12 months the drugs might lose effectiveness in a considerable fraction of patients. Moreover, significant side effects of traditional cytotoxic agents are inevitable at efficacious doses, which limit its function with the progression of the disease. For example, in chronic myeloid leukemia (CML), resistance to current frontline therapy of imatinib and failure to a complete cytogenetic response may occur in 24% of patients within 18 months. With a better understanding of molecular changes in leukemia, the treatment targeting tumor-specific changes, such as RNAi, is expected to make a difference in the therapeutic effectiveness of the disease [26].

Researchers have explored the suitability of RNAi in suppressing the growth and proliferation of myeloid leukemia cell lines including HL-60, U937, THP-1, and K562, which express c-raf and bcl-2 genes. The results were exciting, as the siRNA duplexes succeeded in significantly decreasing the level of target proteins by eliminating the expression of c-raf and bcl-2 genes. This led to the inhibition of the differentiation and programmed cell death suppression of myeloid leukemia cells. These results demonstrated the possibility of RNAi as a novel therapeutic approach to myeloid leukemia [27]. In recent years, based on the molecular alteration of CML, two clinical trials of RNAi therapy have been conducted to target the aberrantly expressed isoforms of the BCR-ABL fusion protein. In one case, there are no publishable outcomes. In the other case, silencing aberrant proteins with RNAi have been found to be less prone to drug resistance [28]. In another *in vivo* application of targeted and non-virally delivered synthetic bcr-abl siRNA, a remarkable apoptosis of CML cells was found in a female patient with a recurrent Philadelphia chromosome with positive CML. The patient was resistant to imatinib and chemotherapy after hematopoietic stem cell transplantation. There were no clinically adverse events, implying feasibility and safety of the application of RNAi-based gene therapy for CML [29].

Furthermore, RNAi-based gene therapy also proves to be effective in acute promyelocytic leukemia (APL). APL is the M3 type of acute myeloid leukemia characterized by a clonal proliferation of abnormal promyelocytes in bone marrow and has a severe bleeding tendency. In 98% of APL patients, a balanced translocation between chromosomes 15 and 17 [t(15;17) (q22;q21)] was found, which leads to the formation and fusion of promelocytic leukemia protein (PML) and retinoic acid receptor alpha (RARα) [30, 31]. A variety of chromosomal aberrations have been identified in APL including t(11;17)(q23;q21), t(5;17)(q35;q12-21), t(11;17)(q13;q21), and der(17), in which the RARα gene is fused to the PLZF, NPM, NuMA, and STAT5b genes, respectively [32]. The differentiation of leukemic cells and complete remission of APL may occur after treatment with ATRA (all-trans retinoic acid). However, the alternative strategies of specific targeting of APL are required because of the ATRA resistance in patients with PLZF-RARα fusion mutation and a treatment of relapse. It has been found that the siRNA targeted knockdown of fused PML-RARα mRNA can induce differentiation and apoptosis of human APL cells; moreover, an injection of pretreated APL cells with anti-PML-RARα siRNA greatly inhibits the progression of APL in mice. Therefore, a targeted RNAi for PML-RARα fusion might be a promising treatment strategy for ATRA-resistant APL patients [33].

At present, several technological requirements and mechanistic challenges (i.e., targeted delivery of siRNAs) for efficient clinical trials have to be explored and overcome to make RNAibased gene therapy readily applicable for the treatment of myeloid leukemia [26].

#### **3.6. Multiple myeloma**

Multiple myeloma (MM) of clonal plasma cells in bone marrow can cause damage to multiple organs. MM is the second most common hematological malignancy. Chromosomal abnor‐ malities, oncogene activation, and growth factor dysregulation contribute to the development of MM. Like leukemia, chemotherapy is the most common treatment for MM right now. However, it is remarkably resistant to chemotherapy [34]. Molecular therapy became an alternative treatment, and the introduction of bortezomib has contributed to the improved survival of patients with MM. Resistance to this therapy inevitably occurs, and the clinical efficacy of bortezomib is significantly diminished. At present, RNAi is found to be helpful in sensitizing tumor cells to chemotherapy and radiation. Bmi-1, an oncogene, has been impli‐ cated in the pathogenesis of MM and might influence the response to bortezomib in MM patients. Bmi-1 has been silenced in two MM cell lines using shRNA targeting Bmi-1 (shBmi-1). The cell cycle progression and apoptosis of MM cells are evaluated. The prolonged G1 phase and enhanced apoptosis were observed that suggest RNAi-derived knockdown of Bmi-1 reducing the resistance to bortezomib. Therefore, Bmi-1-specific RNAi may serve as an important treatment strategy for MM [35].

#### **4. Conclusions and future perspectives**

found to be less prone to drug resistance [28]. In another *in vivo* application of targeted and non-virally delivered synthetic bcr-abl siRNA, a remarkable apoptosis of CML cells was found in a female patient with a recurrent Philadelphia chromosome with positive CML. The patient was resistant to imatinib and chemotherapy after hematopoietic stem cell transplantation. There were no clinically adverse events, implying feasibility and safety of the application of

Furthermore, RNAi-based gene therapy also proves to be effective in acute promyelocytic leukemia (APL). APL is the M3 type of acute myeloid leukemia characterized by a clonal proliferation of abnormal promyelocytes in bone marrow and has a severe bleeding tendency. In 98% of APL patients, a balanced translocation between chromosomes 15 and 17 [t(15;17) (q22;q21)] was found, which leads to the formation and fusion of promelocytic leukemia protein (PML) and retinoic acid receptor alpha (RARα) [30, 31]. A variety of chromosomal aberrations have been identified in APL including t(11;17)(q23;q21), t(5;17)(q35;q12-21), t(11;17)(q13;q21), and der(17), in which the RARα gene is fused to the PLZF, NPM, NuMA, and STAT5b genes, respectively [32]. The differentiation of leukemic cells and complete remission of APL may occur after treatment with ATRA (all-trans retinoic acid). However, the alternative strategies of specific targeting of APL are required because of the ATRA resistance in patients with PLZF-RARα fusion mutation and a treatment of relapse. It has been found that the siRNA targeted knockdown of fused PML-RARα mRNA can induce differentiation and apoptosis of human APL cells; moreover, an injection of pretreated APL cells with anti-PML-RARα siRNA greatly inhibits the progression of APL in mice. Therefore, a targeted RNAi for PML-RARα fusion might be a promising treatment strategy for ATRA-resistant APL

At present, several technological requirements and mechanistic challenges (i.e., targeted delivery of siRNAs) for efficient clinical trials have to be explored and overcome to make RNAi-

Multiple myeloma (MM) of clonal plasma cells in bone marrow can cause damage to multiple organs. MM is the second most common hematological malignancy. Chromosomal abnor‐ malities, oncogene activation, and growth factor dysregulation contribute to the development of MM. Like leukemia, chemotherapy is the most common treatment for MM right now. However, it is remarkably resistant to chemotherapy [34]. Molecular therapy became an alternative treatment, and the introduction of bortezomib has contributed to the improved survival of patients with MM. Resistance to this therapy inevitably occurs, and the clinical efficacy of bortezomib is significantly diminished. At present, RNAi is found to be helpful in sensitizing tumor cells to chemotherapy and radiation. Bmi-1, an oncogene, has been impli‐ cated in the pathogenesis of MM and might influence the response to bortezomib in MM patients. Bmi-1 has been silenced in two MM cell lines using shRNA targeting Bmi-1 (shBmi-1). The cell cycle progression and apoptosis of MM cells are evaluated. The prolonged G1 phase and enhanced apoptosis were observed that suggest RNAi-derived knockdown of Bmi-1 reducing the resistance to bortezomib. Therefore, Bmi-1-specific RNAi may serve as an

based gene therapy readily applicable for the treatment of myeloid leukemia [26].

RNAi-based gene therapy for CML [29].

patients [33].

196 RNA Interference

**3.6. Multiple myeloma**

important treatment strategy for MM [35].

As a powerful and novel treatment of blood genetic diseases, RNAi-based gene therapy has propelled the clinical testing of siRNAs for a variety of diseases at early stages. It is still too early to determine whether the RNAi-based therapeutics will be an efficient tool for the treatment of blood genetic diseases. Therefore, understanding the basic mechanisms of a targeted gene RNAi and its interconnection with genetic, biochemical, and physiological pathways, as well as "off-targets" and "side effects," are the most important factor for developing an efficient therapeutic RNAi strategy. It is highly possible that the RNAi-based gene therapy can be readily and efficiently used for clinical treatment in conjunction with other therapies. The power of sequence-specific suppression of gene expression without "offtargets" and undesired side effects makes RNAi-based gene therapy very promising. Although progress has been made [36], major obstacles, such as the development of methods for efficient targeted delivery of siRNAs in patients, still remain a critical task. In addition, uncovering target domains of genes related to blood genetic diseases and the discovery of more blood genetic disease-causing genes are also key research areas for developing RNAi-based gene therapy for important blood genetic diseases.

#### **Acknowledgements**

This study was supported by the Natural Science Foundation of Beijing [5122022].

#### **Author details**

Mengyu Hu1 #, Qiankun Ni1 #, Yuxia Yang1\* and Jianyuan Luo1,2\*

\*Address all correspondence to: JLuo@som.umaryland.edu; yangyx@bjmu.edu.cn

1 Department of Medical Genetics, Peking University Health Science Center, Beijing, China

2 Department of Medical & Research Technology, School of Medicine, University of Maryland, Baltimore, USA
