- Email: [email protected]
697. New Insights Into the Mechanisms of Argonaute Protein Competition and Implications for RNAi Gene Therapies
Gene Inhibition directly to cRGD peptide or to internalizing RGD (iRGD), which is a variant RGD peptide that triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin. The cRGD- and iRGD-conjugated KRAS Adaptors were tested for efficacy against subcutaneous MiaPaca-2 xenografts, and tumor growth was inhibited to equal or greater extent as with the original cRGD-dendrimer system. iRGD may be of particular benefit for pancreatic adenocarcinomas, which have a densely fibrotic stroma that impedes drug delivery. Enhanced delivery of small- and large-molecule therapeutics into primary pancreatic adenocarcinomas in KPC mice has been achieved previously by conjugating or coinjecting iRGD peptide. We have shown that U1 Adaptors can successfully target human KRAS both in vitro and in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS in pancreatic cancer.
695. Splice-Correction of X-Linked Agammaglobulinemia in a Human BAC-Transgenic Mouse Model Using Oligonucleotides
Burcu Bestas,1 Pedro M.D. Moreno,1 Emelie K.M. Blomberg,1 Dara K. Mohammad,1 Karin E. Lundin,1 Robert Månsson,2 Anna Berglöf,1 Jesper Wengel,3 Edvard C.I. Smith.1 1 Laborattory Medicine, Karolinska Institutet, Huddinge/Stockholm, Sweden; 2Medicine, Karolinska Institutet, Huddinge/Stockholm, Sweden; 3Physics Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark. The inherited immunodeficiency, X-linked agammaglobulinemia (XLA), is caused by mutations in the BTK gene, and results in a B-lineage developmental block. We have recently assessed the treatment potential of splice-correcting oligonucleotides (SCOs) targeting a mutated BTK transcript, which contains a pseudo-exon (Bestas et al., J. Clin Invest 124: 4067, 2014). In order to study the potential of SCOs, we engineered a novel, Bacterial Artificial Chromosome (BAC)-transgenic mouse carrying a mutated human BTK gene, originally found in an XLA family. In order to avoid any influence of mouse endogenous Btk protein, we bred the BACtransgenic mice onto a Btk knockout background. In this model it was possible to correct the defect both in pro-B-cells in vitro, and also in mature B-cells, as demonstrated by the injection of SCOs in vivo. The corrected mRNA gave rise to a functional BTK protein. As a final proof-of-concept we were also able to correct the defect in primary patient cells. In this study we used different nucleotide chemistries, 2’-O-methyl, locked nucleic acid (LNA) and morpholino chemistries. We have now included also other nucleotide chemistries in order to make comparative studies. This is to our knowledge the first time that a lymphocyte defect, caused by abnormal splicing, has been corrected in vivo using a splice-correction approach.
696. Pre-Clinical Evaluation of Allele-Specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides
Amber L. Southwell,1 Niels H. Skotte,1 Nicholas Caron,1 Holly Kordasiewicz,2 Michael Oestergaard,2 Crystal N. Doty,1 Erika B. Villanueva,1 Yuanyun Xie,1 Boguslaw Felczak,1 Susan M. Freier,2 Eric E. Swayze,2 Punit P. Seth,2 C. Frank Bennet,2 Michael R. Hayden.1 1 Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC, Canada; 2ISIS Pharmaceuticals, Carlsbad, CA. Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than non-selective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. We have evaluated ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four welltolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 36 weeks or more after a single intracerebroventricular injection. We next conducted a pre-clinical therapeutic efficacy trial of lead ASOs and evaluated them for effect on the HD-like phenotypes of Hu97/18 mice. Treated mice underwent longitudinal behavioral and biochemical assessment followed by terminal neuropathology. Thus far we have determined that pre-symptomatic allele-specific muHTT silencing prevents onset of behavioral HD-like phenotypes. Evaluation of neuropathology and post-symptomatic intervention is ongoing. Contingent on findings from these studies and using delivery and dosing information gained from ongoing CNS ASO clinical trials, a primary SNP-targeted ASO drug could be fairly rapidly translated for human applications.
697. New Insights Into the Mechanisms of Argonaute Protein Competition and Implications for RNAi Gene Therapies
Mario Lederle,1 Kathrin Tegeler,1 Daniela Cerny,1 Patrick Zessin,1 Dirk Grimm.1 1 Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany. RNA interference (RNAi) has become an established tool for basic gene annotation and a promising option for therapeutic concepts that require specific inhibition of dysregulated or exogenous genes. However, we and others accumulated strong evidence that overexpression of short hairpin RNAs (shRNAs, an expressable and widely used form of RNAi trigger) can cause cytotoxicity leading up to organ damage and lethality in shRNA-treated animals. A meticulous dissection of the underlying mechanisms is critical as it will ultimately guide the design of safer RNAi therapies and thus foster their clinical translation. One informative finding was that co-expression of Ago2 (Slicer, as it cleaves targeted mRNA), a key component of the RNAinduced silencing complex RISC, increases shRNA efficiency in cells and animals, implying that Ago2 is a rate-limiting factor whose saturation may be involved in cytotoxicity. Moreover, over-expression of the three other mammalian Ago proteins - Ago1, Ago3 or Ago4 (all slicing-incompetent) - dampens RNAi potency, underscoring that a delicate balance of all four Ago variants is key to efficient and safe gene silencing. Here, we investigated three hypotheses that could explain how relative Ago1-4 levels may affect the functionality of shRNA-loaded RISC and hence the strength of target knockdown: over-abundance of non-Slicer Ago proteins could (i) dysregulate Ago2 expression (transcription and/or translation), (ii) re-localize intracellular Ago2 away from sites of RNAi activity, or (iii) quantitatively sequester shRNAs into slicing-incompetent RISC. Using codonoptimized Ago variants that are distinguishable by real-time PCR from the endogenous counterparts, as well as Western blotting, we could eliminate model (i). Likewise, wide-field and confocal S277
Gene Inhibition microscopy analyses revealed no evidence for Ago2 re-localization upon Ago1/3/4 over-expression and vice versa. We therefore focused on hypothesis (iii) and fused the Ago1-4 cDNAs with GFP or RFP tags that enabled their specific immunoprecipitation, alone or in combination, using the TRAP system. Indeed, we found that Slicer and non-Slicers compete for shRNA loading and mRNA knockdown in a dose-dependent manner, readily explaining all prior observations of RNAi enhancement by Ago2 over-expression, or shRNA inhibition by Ago1/3/4, respectively. Importantly, the combination of GFP/ RFP-Ago fusion constructs with the co-precipitation protocols newly established during this work should be widely useful to address other vital questions in the RNAi field, such as the selective processing of different cellular small RNAs by the four Ago proteins. Moreover, our notion that over-expression of non-Slicers has no adverse effect on endogenous Ago2 levels or localization paves the way for novel strategies to fine-tune therapeutic RNAi in a safe and potent manner. As a prototype, we present a new shRNA/Ago co-expression system whereby Ago1/2 levels can be regulated independently through exogenous macrolides, permitting a tight control of target knockdown efficiencies and providing an original avenue to reduce toxicity in future RNAi gene therapy applications.
698. Eliminating TLR9+ Prostate Cancer Stem Cells In Vivo Using NF-κB/RELA- or STAT3Targeting CpG-siRNA Conjugates
Dayson F. Moreira,1 Qifang Zhang,1 Dewan M. Hossain,1 Sergey Nechaev,1 Haiqing Li,1 Claudia M. Kowolik,1 Massimo D’Apuzzo,3 Stephen Forman,1 Jeremy Jones,1 Sumanta K. Pal,1 Marcin Kortylewski1.1 1 Beckman Research Institute at City of Hope, Duarte, CA.
Treatment of hormone-refractory and frequently reoccurring prostate cancers is a major clinical challenge. Here, we demonstrate that Toll-like Receptor 9 (TLR9) is commonly upregulated in latestage prostate cancers and provides a potential therapeutic target. As a sensor of immunogenic cell death, TLR9 bridges intra-prostatic inflammation to cancer stem cell phenotype. Our limited dilution/ serial transplantation experiments in vivo demonstrate that TLR9 is essential for prostate cancer cells’ potential to propagate and self-renew. In addition, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of prostate cancer cells. Genome-wide transcriptional analysis of cancer cells isolated from xenotransplanted prostate tumors revealed a unique TLR9-dependent gene expression signature. Further analysis of the TLR9 downstream signaling indicated that tumorigenic transcription factors NF-κB/RELA and STAT3 cooperate to orchestrate expression of stem cell-related genes. Both RELA and STAT3 bound and co-regulated promoters of NKX3.1 and KLF4 prostate cancer stem cell-related genes. We further demonstrated the feasibility of targeting prostate cancer-propagating potential in vivo by TLR9-targeted siRNA delivery using CpG-siRNA conjugates. Local administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates, inhibited tumor growth and cancer cell clonogenic potential in two xenotransplanted prostate cancer models. Selective elimination of tumor-propagating cells using TLR9-targeted blockade of NF-κB/RELA and STAT3 signaling has potential for clinical translation to benefit patients with late-stage prostate cancers.
S278
699. shRNA Sense Strand Neutralization Reduces Off-Targeting, Ameliorates Toxicity and Enhances Efficacy of RNAi-Based HBV Gene Therapy
Thomas Michler,1 Stefanie Grosse,2 Stefan Mockenhaupt,2 Ulrike Protzer,1 Dirk Grimm.2 1 Virology, Technical University, Munich, Germany; 2Virology, University of Heidelberg, Heidelberg, Germany. A decade after first reports of successful inhibition of hepatitis B virus (HBV) replication in vivo using shRNAs, only a single RNAi therapeutic is under phase II efficacy investigation in Hepatitis B patients. This slow clinical translation is puzzling, as current standard-of-care requires life-long and daily drug treatment which is often accompanied by side effects and occurrence of resistant HBV mutants. Also, while preventing the generation of infectious HBV virions, reverse-transcriptase inhibitors fail to suppress HBV surface and e-antigen, which enable viral persistence by modulating the host immune system and contribute to HBV-associated hepatocellular carcinoma. These problems may be solved using RNAi as anti-HBV therapeutic since (i) long-term suppression can be achieved by a single dose of an shRNA-encoding vector, and (ii) all four HBV transcripts share a common 3 end, allowing concurrent inhibition of HBV pre-genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis. Still, clinical translation remains hampered by safety concerns as shRNA over-expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss. One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity. Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute-2 (Ago2) ameliorates toxicity and enhances RNAi efficacy. Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off-target genes with partial complementarities to one of the two strands of the RNAi molecule. To alleviate such unintended gene silencing, we have developed a novel bi-cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called tough decoy or TuD. In cell culture, the TuD effectively sequestered and inactivated shRNA sense strands, and thereby improved RNAi specificity. These remarkable features translated well into an HBV-transgenic mouse model, where sense strand off-target activity was likewise prevented, as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity. Enhanced in vivo safety was also noted for two alternative AAV vectors, either co-expressing the shRNA and Ago2, or embedding the shRNA in a miR-122 backbone. Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over 3 months. We speculate that its enhanced antiviral efficacy results from increased loading of the desired antisense strand into RISC in absence of the sense strand, possibly also further amending toxicity by attenuating RISC saturation. The simple TuD design and the versatility of our new AAV vector pave the way for adaptation of our strategy to other applications and should facilitate clinical translation.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
695. Splice-Correction of X-Linked Agammaglobulinemia in a Human BAC-Transgenic Mouse Model Using Oligonucleotides
Burcu Bestas,1 Pedro M.D. Moreno,1 Emelie K.M. Blomberg,1 Dara K. Mohammad,1 Karin E. Lundin,1 Robert Månsson,2 Anna Berglöf,1 Jesper Wengel,3 Edvard C.I. Smith.1 1 Laborattory Medicine, Karolinska Institutet, Huddinge/Stockholm, Sweden; 2Medicine, Karolinska Institutet, Huddinge/Stockholm, Sweden; 3Physics Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark. The inherited immunodeficiency, X-linked agammaglobulinemia (XLA), is caused by mutations in the BTK gene, and results in a B-lineage developmental block. We have recently assessed the treatment potential of splice-correcting oligonucleotides (SCOs) targeting a mutated BTK transcript, which contains a pseudo-exon (Bestas et al., J. Clin Invest 124: 4067, 2014). In order to study the potential of SCOs, we engineered a novel, Bacterial Artificial Chromosome (BAC)-transgenic mouse carrying a mutated human BTK gene, originally found in an XLA family. In order to avoid any influence of mouse endogenous Btk protein, we bred the BACtransgenic mice onto a Btk knockout background. In this model it was possible to correct the defect both in pro-B-cells in vitro, and also in mature B-cells, as demonstrated by the injection of SCOs in vivo. The corrected mRNA gave rise to a functional BTK protein. As a final proof-of-concept we were also able to correct the defect in primary patient cells. In this study we used different nucleotide chemistries, 2’-O-methyl, locked nucleic acid (LNA) and morpholino chemistries. We have now included also other nucleotide chemistries in order to make comparative studies. This is to our knowledge the first time that a lymphocyte defect, caused by abnormal splicing, has been corrected in vivo using a splice-correction approach.
696. Pre-Clinical Evaluation of Allele-Specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides
Amber L. Southwell,1 Niels H. Skotte,1 Nicholas Caron,1 Holly Kordasiewicz,2 Michael Oestergaard,2 Crystal N. Doty,1 Erika B. Villanueva,1 Yuanyun Xie,1 Boguslaw Felczak,1 Susan M. Freier,2 Eric E. Swayze,2 Punit P. Seth,2 C. Frank Bennet,2 Michael R. Hayden.1 1 Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC, Canada; 2ISIS Pharmaceuticals, Carlsbad, CA. Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than non-selective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. We have evaluated ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four welltolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 36 weeks or more after a single intracerebroventricular injection. We next conducted a pre-clinical therapeutic efficacy trial of lead ASOs and evaluated them for effect on the HD-like phenotypes of Hu97/18 mice. Treated mice underwent longitudinal behavioral and biochemical assessment followed by terminal neuropathology. Thus far we have determined that pre-symptomatic allele-specific muHTT silencing prevents onset of behavioral HD-like phenotypes. Evaluation of neuropathology and post-symptomatic intervention is ongoing. Contingent on findings from these studies and using delivery and dosing information gained from ongoing CNS ASO clinical trials, a primary SNP-targeted ASO drug could be fairly rapidly translated for human applications.
697. New Insights Into the Mechanisms of Argonaute Protein Competition and Implications for RNAi Gene Therapies
Mario Lederle,1 Kathrin Tegeler,1 Daniela Cerny,1 Patrick Zessin,1 Dirk Grimm.1 1 Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany. RNA interference (RNAi) has become an established tool for basic gene annotation and a promising option for therapeutic concepts that require specific inhibition of dysregulated or exogenous genes. However, we and others accumulated strong evidence that overexpression of short hairpin RNAs (shRNAs, an expressable and widely used form of RNAi trigger) can cause cytotoxicity leading up to organ damage and lethality in shRNA-treated animals. A meticulous dissection of the underlying mechanisms is critical as it will ultimately guide the design of safer RNAi therapies and thus foster their clinical translation. One informative finding was that co-expression of Ago2 (Slicer, as it cleaves targeted mRNA), a key component of the RNAinduced silencing complex RISC, increases shRNA efficiency in cells and animals, implying that Ago2 is a rate-limiting factor whose saturation may be involved in cytotoxicity. Moreover, over-expression of the three other mammalian Ago proteins - Ago1, Ago3 or Ago4 (all slicing-incompetent) - dampens RNAi potency, underscoring that a delicate balance of all four Ago variants is key to efficient and safe gene silencing. Here, we investigated three hypotheses that could explain how relative Ago1-4 levels may affect the functionality of shRNA-loaded RISC and hence the strength of target knockdown: over-abundance of non-Slicer Ago proteins could (i) dysregulate Ago2 expression (transcription and/or translation), (ii) re-localize intracellular Ago2 away from sites of RNAi activity, or (iii) quantitatively sequester shRNAs into slicing-incompetent RISC. Using codonoptimized Ago variants that are distinguishable by real-time PCR from the endogenous counterparts, as well as Western blotting, we could eliminate model (i). Likewise, wide-field and confocal S277
Gene Inhibition microscopy analyses revealed no evidence for Ago2 re-localization upon Ago1/3/4 over-expression and vice versa. We therefore focused on hypothesis (iii) and fused the Ago1-4 cDNAs with GFP or RFP tags that enabled their specific immunoprecipitation, alone or in combination, using the TRAP system. Indeed, we found that Slicer and non-Slicers compete for shRNA loading and mRNA knockdown in a dose-dependent manner, readily explaining all prior observations of RNAi enhancement by Ago2 over-expression, or shRNA inhibition by Ago1/3/4, respectively. Importantly, the combination of GFP/ RFP-Ago fusion constructs with the co-precipitation protocols newly established during this work should be widely useful to address other vital questions in the RNAi field, such as the selective processing of different cellular small RNAs by the four Ago proteins. Moreover, our notion that over-expression of non-Slicers has no adverse effect on endogenous Ago2 levels or localization paves the way for novel strategies to fine-tune therapeutic RNAi in a safe and potent manner. As a prototype, we present a new shRNA/Ago co-expression system whereby Ago1/2 levels can be regulated independently through exogenous macrolides, permitting a tight control of target knockdown efficiencies and providing an original avenue to reduce toxicity in future RNAi gene therapy applications.
698. Eliminating TLR9+ Prostate Cancer Stem Cells In Vivo Using NF-κB/RELA- or STAT3Targeting CpG-siRNA Conjugates
Dayson F. Moreira,1 Qifang Zhang,1 Dewan M. Hossain,1 Sergey Nechaev,1 Haiqing Li,1 Claudia M. Kowolik,1 Massimo D’Apuzzo,3 Stephen Forman,1 Jeremy Jones,1 Sumanta K. Pal,1 Marcin Kortylewski1.1 1 Beckman Research Institute at City of Hope, Duarte, CA.
Treatment of hormone-refractory and frequently reoccurring prostate cancers is a major clinical challenge. Here, we demonstrate that Toll-like Receptor 9 (TLR9) is commonly upregulated in latestage prostate cancers and provides a potential therapeutic target. As a sensor of immunogenic cell death, TLR9 bridges intra-prostatic inflammation to cancer stem cell phenotype. Our limited dilution/ serial transplantation experiments in vivo demonstrate that TLR9 is essential for prostate cancer cells’ potential to propagate and self-renew. In addition, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of prostate cancer cells. Genome-wide transcriptional analysis of cancer cells isolated from xenotransplanted prostate tumors revealed a unique TLR9-dependent gene expression signature. Further analysis of the TLR9 downstream signaling indicated that tumorigenic transcription factors NF-κB/RELA and STAT3 cooperate to orchestrate expression of stem cell-related genes. Both RELA and STAT3 bound and co-regulated promoters of NKX3.1 and KLF4 prostate cancer stem cell-related genes. We further demonstrated the feasibility of targeting prostate cancer-propagating potential in vivo by TLR9-targeted siRNA delivery using CpG-siRNA conjugates. Local administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates, inhibited tumor growth and cancer cell clonogenic potential in two xenotransplanted prostate cancer models. Selective elimination of tumor-propagating cells using TLR9-targeted blockade of NF-κB/RELA and STAT3 signaling has potential for clinical translation to benefit patients with late-stage prostate cancers.
S278
699. shRNA Sense Strand Neutralization Reduces Off-Targeting, Ameliorates Toxicity and Enhances Efficacy of RNAi-Based HBV Gene Therapy
Thomas Michler,1 Stefanie Grosse,2 Stefan Mockenhaupt,2 Ulrike Protzer,1 Dirk Grimm.2 1 Virology, Technical University, Munich, Germany; 2Virology, University of Heidelberg, Heidelberg, Germany. A decade after first reports of successful inhibition of hepatitis B virus (HBV) replication in vivo using shRNAs, only a single RNAi therapeutic is under phase II efficacy investigation in Hepatitis B patients. This slow clinical translation is puzzling, as current standard-of-care requires life-long and daily drug treatment which is often accompanied by side effects and occurrence of resistant HBV mutants. Also, while preventing the generation of infectious HBV virions, reverse-transcriptase inhibitors fail to suppress HBV surface and e-antigen, which enable viral persistence by modulating the host immune system and contribute to HBV-associated hepatocellular carcinoma. These problems may be solved using RNAi as anti-HBV therapeutic since (i) long-term suppression can be achieved by a single dose of an shRNA-encoding vector, and (ii) all four HBV transcripts share a common 3 end, allowing concurrent inhibition of HBV pre-genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis. Still, clinical translation remains hampered by safety concerns as shRNA over-expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss. One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity. Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute-2 (Ago2) ameliorates toxicity and enhances RNAi efficacy. Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off-target genes with partial complementarities to one of the two strands of the RNAi molecule. To alleviate such unintended gene silencing, we have developed a novel bi-cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called tough decoy or TuD. In cell culture, the TuD effectively sequestered and inactivated shRNA sense strands, and thereby improved RNAi specificity. These remarkable features translated well into an HBV-transgenic mouse model, where sense strand off-target activity was likewise prevented, as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity. Enhanced in vivo safety was also noted for two alternative AAV vectors, either co-expressing the shRNA and Ago2, or embedding the shRNA in a miR-122 backbone. Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over 3 months. We speculate that its enhanced antiviral efficacy results from increased loading of the desired antisense strand into RISC in absence of the sense strand, possibly also further amending toxicity by attenuating RISC saturation. The simple TuD design and the versatility of our new AAV vector pave the way for adaptation of our strategy to other applications and should facilitate clinical translation.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy