**5. Drug delivery**

414 Myocarditis

replication. In this regard, the CAR receptor which is shared by CVB3 and adenovirus is an attractive candidate since both CVB3 and adenovirus are considered as the common causal agents of myocarditis. To date, two studies have been reported to silence CAR expression with specific siRNAs. One study reported that transfection of HeLa cells with siRNAs, siCAR2 or siCAR9, almost completely silenced the expression of CAR and that further analysis by viral plaque assay revealed ~60% reduction of CVB3 particle formation (Werk et al., 2005). Another study using cardiac-derived HL-1 cell line and primary neonatal cardiomyocytes (PNCMs) demonstrated that treatment with recombinant adenoviruses expressing shRNAs against CAR resulted in almost completely silencing of CAR expression in both HL-1 cells and PNCMs. CAR knockout resulted in inhibition of CVB3 infections by up to 97% in HL-1 and up to 90% in PNCMs. Adenoviruses were inhibited by only 75% in

Another host gene, the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), has been suggested to be a potential target for siRNA to ameliorate CVB3-induced myocarditis. This suggestion is based on the investigation of Crocker and colleagues on a new role of TIMP-1 in exacerbating CVB-induced myocarditis (Crocker et al., 2007). They found that TIMP-1 expression was induced in the myocardium by CVB3 infection. Surprisingly, TIMP-1 knockout mice exhibited a profound attenuation of myocarditis, with increased survival. The amelioration of disease in TIMP-1 knockout mice was not attributable to either an altered T-cell response to the virus or to reduced viral replication. These data allowed the authors to propose and prove a novel function for TIMP-1: its highly localized up-regulation might arrest the matrix metalloproteinase (MMP)-dependent migration of inflammatory cells at the sites of infection thereby anatomically focusing the adaptive immune response. Finally, the benefits of TIMP-1 blockage in treating CVB myocarditis were confirmed by administration of siRNAs targeting TIMP-1, which diminished CVB3-induced myocarditis. However, this improvement of the disease is not due to the changes of viral titers, as

Recently, the active investigations on CVB3-induced signal transduction pathways have provided new avenues for the search of therapeutic targets for the treatment of myocarditis. Since CVB3, like other picornaviruses, requires the activation of certain signal pathways for initiating their life cycle, inactivation of some signal molecules in the signal cascade with specific siRNAs would block CVB3 replication. Such kind of studies that have been documented thus far include i) the knockdown of ubiquitin expression by siRNAs to downregulate the ubiquitination and subsequent alteration of protein function and/or degradation (Si et al., 2008); ii) silencing of proteosome activator REGγ to inhibit the REGγmediated degradation of several important intracellular proteins (Gao, 2010), such as cyclindependent kinase inhibitors p21, p16 and p19 and tumor suppressor p53; and iii) knockdown of genes critical important for autophagy formation, these genes include ATG7, Beeclin-1 and VPS34 (J. Wong et al., 2008). Although these target genes mentioned above have been tested *in vitro* using specific siRNAs in signal transduction studies and showed promising outcomes, their potential serving as a therapeutic target for treatment of CVB3

infection needs a further evaluation by pharmacological study in animal models.

miRNAs are a group of recently discovered new regulators of gene expression. These endogenous regulators control one third of human gene expression (Bartel, 2009; Q. Liu,

HL-1, but up to 92% in PNCMs (Fechner et al., 2007).

demonstrated by viral plaque assay (Crocker et al., 2007).

**4.4 Anti-CVB3 artificial miRNAs** 

NA-based agents are inefficiently taken up by mammalian cells and would therefore benefits from additional vehicles or modifications that facilitate drug delivery. Depending on transient delivery or long-term treatment, the delivery approaches can be divided into non-viral delivery of chemically synthesized agents and viral delivery of drug-expressing cassettes (Table 1). For the non-viral delivery measures, they can be further divided into unspecific and cell-type specific delivery. The former method has been widely used for delivery of many chemically synthesized ASONs, ribozymes and siRNAs as well as plasmids encoding shRNAs through transfection of tissue culture cells, hydrodynamic transduction/transfection of mice or intravenous (IV) injection of mice via tails using cationic lipoplexes or liposomes. The successful examples for inhibition of viral pathogens of myocarditis includes deliveries of i) ASONs targeting CVB3 IRES (Yuan et al., 2006) and both ends of the CVB3 genome (A. Wang et al., 2001), ii) siRNAs targeting CVB3 2A (Merl et al., 2005; Yuan et al., 2005) and 3D (Ahn et al., 2005; Schubert et al., 2005; Schubert et al., 2007), iii) plasmids expressing shRNAs targeting 3D and VP1 (J. Y. Kim et al., 2007) and vi) ribozymes targeting HCV RNA (Gonzalez-Carmona et al., 2006). For the cell-type specific method, proper modification and conjugation of 'naked' raw therapeutic molecules are required to achieve targeted delivery. The different chemical modifications described earlier for ASONs are applicable to all NA-based agents. The conjugation of these antivirals can be achieved by covalent linkage of a ligand to the molecules, which enables the drug internalization via specific interactions between the ligand and its receptor. The ligands can be an antibody, vitamin, short peptide, RNA aptamer, folic acid, etc. The details were reviewed elsewhere (X. Ye & Yang, 2009). By this strategy, we have specifically delivered siRNAs targeting CVB3 2A to HeLa (cancer) cells, a cell line susceptible to CVB3, through interactions between folate and its receptor highly expressed on the surface of all cancer cells but not the normal cells (Zhang et al., 2009). This study was carried out by covalent linkage of the siRNA targeting CVB3 2A to a bacterial phage-29 packaging RNA (pRNA). This small pRNA (~170 nts) can form dimer, trimer and hexamer by base pairing through its

infection.

Nucleic Acid-Based Strategies for the Treatment of Coxsackievirus-Induced Myocarditis 417

which guided the targeted delivery of siRNAs via ligand-receptor interactions and achieved strong inhibition of CVB3 replication (Zhang et al., 2009). The effectiveness of this strategy on targeted delivery of NA-based drugs was further solidified in targeted delivery of AmiRNAs to inhibit CVB3 replication (X. Ye, Liu, Z., Hemida, G. M., and Yang, D. C., 2011). Viral vector-mediated delivery of NA-based agents is another promising approach for treatment of persistent infection such as CVB3, HCV and many other viruses. This is because that the vector-encoded shRNA can produce relative long-term and continuous silencing. Most viral vectors are modified viruses, which can be applied to deliver a cargo sequence to cells. Currently the most commonly used viral vectors for the delivery of NAbased drugs are derived from the adenovirus, adeno-associated viruses (AAV) and lentiviruses. These vectors have been widely used and have achieved exciting promise (Fechner et al., 2008; Henry et al., 2006; Kuhlmann et al., 2008). Recent trends in further improvement of these vectors focus on modifications of their structure to increase the capability for targeted delivery. The efforts of this goal can be summarized into three categories (X. Ye & Yang, 2009): i) the genetic and chemical modifications of the vector to express a unique chimeric surface protein, such as adenovirus capsid proteins: fiber knob, penton and hexon. The common strategy is the insertion of a foreign peptide into fiber knob, which enables the vector to be capable of binding the specific cellular receptor (Koizumi et al., 2003; Mizuguchi& Hayakawa, 2002); ii) incorporation of heterologous protein from another virus with a restricted range of tissue tropism to the viral envelope; this approach is also called pseudotyping. An example of this strategy is the pseudotyping of lentivirus vector with the neurotropic rabies virus glycoprotein allows retro-axonal and trans-synoptic spread, thereby enhancing the transgene expression within the brain (L. F. Wong et al., 2004); and iii) the application of a tissue-specific promoter to express the vector-carried gene in a specific organ or cell type. An impressive finding has been reported regarding the utilization of cardiac myosin light chain 2v promoter and the hypoxia-response element by AAV vector to express vascular endothelial growth factor, an angiogenic factor, specifically in myocardium, leading to cardiac functional improvement (Su et al., 2004). Here we will briefly discuss the delivery of NA-based antivirals using these viral vectors for CVB3

Adenovirus is known to share the CAR receptor with CVB3. This receptor is highly expressed on the surface of cardiomyocytes. Thus, adenovirus-derived vector is an ideal carrier for delivery of NA-based antivirals to the heart. This vector has successfully delivered shRNAs targeting the CAR gene in a cardiac-derived HL-1 cell line and isolated PNCMs, resulting in the strong reduction of replication of both CVB3 and adenovirus (Fechner et al., 2007). Lentivirus vectors are derived from HIV. They have the ability to transduce quiescent as well as proliferating cells, thus increasing their therapeutic ranges. Particularly, after pseudotyping with G glycoprotein of vesicular stomatitis virus, they can transduce almost any cell type (Kurreck, 2009). Kim Y-J et al. constructed recombinant lentiviruses that express shRNAs targeting the CRE within CVB3 2C. Intraperitoneal injection of mice with these viruses clearly showed a protective effect against viral myocarditis by elimination of CVB3 infection and reducing pro-inflammatory cytokines, such as IL6 and INF-α. (Y. J. Kim et al., 2008). AAVs are attractive vectors for gene transfer since they efficiently transduce target cells and are nonpathogenic for humans. Fechner H et al. have employed a pseudotyped AAV2.9 vector, carrying the most cardiotropic AAV capsid currently known to successfully transduce PNCMs. This vector expressed siRNAs targeting CVB3 3D and reduced CVB3 replication by >3 log10 steps. Further evaluation by


Abbreviations: LNA: locked nucleic acid; RD: rhabdomyosarcoma; PBMC: peripheral blood mononuclear cell; HMF: human myocardial fibroblast; HCC: human conjunctive cell.

Table 1. NA-based agents for the treatment of CVB3 infection.

left- and right-hand loops (P. Guo, 2002) (Fig. 3). Thus, this pRNA multimer can carry multiple siRNAs and has the potential to overcome issues associated with drug resistance of viruses (P. Guo, 2005). In addition, this small pRNA vector has lower immunogenicity than big DNA vectors. Thus it is a safe vehicle for transportation of antiviral drugs (S. Guo et al., 2006). We labeled, by *in vitro* transcription, the 5'end of the pRNA with AMP-folic acid,

Abbreviations: LNA: locked nucleic acid; RD: rhabdomyosarcoma; PBMC: peripheral blood mononuclear cell; HMF: human myocardial fibroblast; HCC: human conjunctive cell.

left- and right-hand loops (P. Guo, 2002) (Fig. 3). Thus, this pRNA multimer can carry multiple siRNAs and has the potential to overcome issues associated with drug resistance of viruses (P. Guo, 2005). In addition, this small pRNA vector has lower immunogenicity than big DNA vectors. Thus it is a safe vehicle for transportation of antiviral drugs (S. Guo et al., 2006). We labeled, by *in vitro* transcription, the 5'end of the pRNA with AMP-folic acid,

Table 1. NA-based agents for the treatment of CVB3 infection.

which guided the targeted delivery of siRNAs via ligand-receptor interactions and achieved strong inhibition of CVB3 replication (Zhang et al., 2009). The effectiveness of this strategy on targeted delivery of NA-based drugs was further solidified in targeted delivery of AmiRNAs to inhibit CVB3 replication (X. Ye, Liu, Z., Hemida, G. M., and Yang, D. C., 2011). Viral vector-mediated delivery of NA-based agents is another promising approach for treatment of persistent infection such as CVB3, HCV and many other viruses. This is because that the vector-encoded shRNA can produce relative long-term and continuous silencing. Most viral vectors are modified viruses, which can be applied to deliver a cargo sequence to cells. Currently the most commonly used viral vectors for the delivery of NAbased drugs are derived from the adenovirus, adeno-associated viruses (AAV) and lentiviruses. These vectors have been widely used and have achieved exciting promise (Fechner et al., 2008; Henry et al., 2006; Kuhlmann et al., 2008). Recent trends in further improvement of these vectors focus on modifications of their structure to increase the capability for targeted delivery. The efforts of this goal can be summarized into three categories (X. Ye & Yang, 2009): i) the genetic and chemical modifications of the vector to express a unique chimeric surface protein, such as adenovirus capsid proteins: fiber knob, penton and hexon. The common strategy is the insertion of a foreign peptide into fiber knob, which enables the vector to be capable of binding the specific cellular receptor (Koizumi et al., 2003; Mizuguchi& Hayakawa, 2002); ii) incorporation of heterologous protein from another virus with a restricted range of tissue tropism to the viral envelope; this approach is also called pseudotyping. An example of this strategy is the pseudotyping of lentivirus vector with the neurotropic rabies virus glycoprotein allows retro-axonal and trans-synoptic spread, thereby enhancing the transgene expression within the brain (L. F. Wong et al., 2004); and iii) the application of a tissue-specific promoter to express the vector-carried gene in a specific organ or cell type. An impressive finding has been reported regarding the utilization of cardiac myosin light chain 2v promoter and the hypoxia-response element by AAV vector to express vascular endothelial growth factor, an angiogenic factor, specifically in myocardium, leading to cardiac functional improvement (Su et al., 2004). Here we will briefly discuss the delivery of NA-based antivirals using these viral vectors for CVB3 infection.

Adenovirus is known to share the CAR receptor with CVB3. This receptor is highly expressed on the surface of cardiomyocytes. Thus, adenovirus-derived vector is an ideal carrier for delivery of NA-based antivirals to the heart. This vector has successfully delivered shRNAs targeting the CAR gene in a cardiac-derived HL-1 cell line and isolated PNCMs, resulting in the strong reduction of replication of both CVB3 and adenovirus (Fechner et al., 2007). Lentivirus vectors are derived from HIV. They have the ability to transduce quiescent as well as proliferating cells, thus increasing their therapeutic ranges. Particularly, after pseudotyping with G glycoprotein of vesicular stomatitis virus, they can transduce almost any cell type (Kurreck, 2009). Kim Y-J et al. constructed recombinant lentiviruses that express shRNAs targeting the CRE within CVB3 2C. Intraperitoneal injection of mice with these viruses clearly showed a protective effect against viral myocarditis by elimination of CVB3 infection and reducing pro-inflammatory cytokines, such as IL6 and INF-α. (Y. J. Kim et al., 2008). AAVs are attractive vectors for gene transfer since they efficiently transduce target cells and are nonpathogenic for humans. Fechner H et al. have employed a pseudotyped AAV2.9 vector, carrying the most cardiotropic AAV capsid currently known to successfully transduce PNCMs. This vector expressed siRNAs targeting CVB3 3D and reduced CVB3 replication by >3 log10 steps. Further evaluation by

Nucleic Acid-Based Strategies for the Treatment of Coxsackievirus-Induced Myocarditis 419

system with which siRNAs can be delivered to their target cells. Despite great advances in the last years, further developments are still required to get systematically applied siRNAs to their required sites of action. Here, viral vectors systems for shRNA expression cassettes offer additional options for efficient and organ-specific delivery. However, this approach must be first overcome the reservations based on the negative experience with gene therapy. As discussed earlier, pRNA is a promising vehicle for targeted delivery of NA-based therapeutic molecules. For treatment of myocarditis, a myocardium-specific ligand such as peptides from the CVB3 antireceptor protein or RNA apatamers of cardiomyocytes should

Very recent advances in the understanding of miRNA biology and particularly their association with the molecular pathogenesis of a variety of diseases have served as a theoretical basis for drug development. On the one hand, miRNA, as one of the key factors for regulation of viral replication, tissue tropism and latency, are the ideal targets for inhibition. In this regard, construction of mRNAs that contain multiple tandem binding sites of a given miRNA may be useful to produce decoys or "miRNA sponges" to inhibit the function of a specific miRNA. In addition, chemically synthesized antisense RNA oligomers ('antagomirs') targeting a miRNA of interest could be also a promising approach to inhibit miRNA activity (Ebert et al., 2007; Krutzfeldt et al., 2005). Other strategies include i) overexpression of specific miRNAs using an expression vector to achieve a long-term effect of reversing the imbalance of miRNA expression caused by infections, and ii) introduction of pre-miRNA mimetics for transient replacement of a down-regulated miRNA. On the other hand, miRNA can serve as a useful tool for therapy. Since miRNA is tolerable to target mutation at its center region, application of multiple artificial miRNAs to target the 3'UTR and/or other regions of CVB3 RNA may improve the drug resistance. Given the immense interest in NA-based drug research and the rapid progress made in this field and other areas such as nano-biotechnology for drug delivery, the coming years are likely to see an increasing range of clinical applications, particularly for the RNAi-based drug candidates. The realization of the potential of NA-based therapies to address human viral pathogens

My special thanks go to my students, Postdoctoral Fellows and Research Associates, whose tireless efforts contributed to the advances of my research group. I also thank Paul Hanson for his critical reading of this manuscript and Xin Ye for his help on preparing the manuscript. The work was supported by grants from the Canada Institute of Health

Ahn, J., Jun, E. S., Lee, H. S., Yoon, S. Y., Kim, D., Joo, C. H., Kim, Y. K.& Lee, H. (2005). A

Amantana, A.& Iversen, P. L. (2005). Pharmacokinetics and biodistribution of

small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells

phosphorodiamidate morpholino antisense oligomers. *Current Opinion in* 

Research and the Heart and Stroke Foundation of British Columbia & Yukon.

from viral challenge. *The Journal of Virology*, 79(13), 8620-8624.

be identified, which will be used to replace folic acid on the pRNA vector.

suggests that this field has a very promising future.

*Pharmacology*, 5(5), 550-555.

**7. Acknowledgements** 

**8. References** 

intravenous injection of mice demonstrated significant reduction of virus titers and improvement of heart function compared to the control. This study showed for the first time that intravenously injected AAV2.9 has the potential to target RNAi to the heart and suggests AVV2.9-shRNA vectors as a novel therapeutic approach for cardiac disorders (Fechner et al., 2008).

Fig. 3. Structural schematic of packaging RNA (pRNA) multimers as a drug targeted delivery vehicle. (A) pRNA dimer forms through the base-paring between the loops of an A'b-pRNA-siRNA and a B'a-pRNA-ligand. The shaded areas on the loops indicate the basepair interactions between the monomers. siRNA is released when intracellular Dicer cleaves the dsRNA, which is indicated by an arrow. (B) pRNA trimers can be stoichiometrically formed by hand-in-hand loop interactions, which contains 1:1:1 of their linked conjugates. (C) hexamers allow for a customizable, combined therapy where multiple drugs may be added to the same complex.
