564. Apolipoprotein B Knockdown by AAV-Delivered ShRNA Lowers Plasma Cholesterol in Mice

OLIGONUCLEOTIDE & RNAI THERAPEUTICS I 562. In Vivo Comparison of the Long-Term Efficacy of AAV Expressed ShRNA and MiRNA Targeting Apolipoprotein B100

Piotr Maczuga,1,2 Annemart Koornneef,1 Richard van Logtenstein,1,3 Florie Borel,1 Harald Petry,1 Sander van Deventer,1 Pavlina Konstantinova.1 1 Amsterdam Molecular Therapeutics, Amsterdam, Netherlands; 2 Department of Gastroenterology, Leiden University Medical Center, Leiden, Netherlands; 3Department of Hepatology, Academic Medical Center, Amsterdam, Netherlands. Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels, RNA interference was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). Highly effective siRNAs against conserved regions of human, mice and macaque ApoB were expressed from H1 Pol III promoter in the form of shRNA (shApoB10) or incorporated into artificial miRNA backbone (miApoB10) under the control of the CMV Pol II promoter. Both shApoB and miApoB inhibited endogenous ApoB with more than 80% in vitro in murine hepatocytes, despite the fact that miRNA constructs express 20 times less siRNA molecules per cell. Adeno-associated virus (AAV) was used for long-term transduction of murine liver with shApoB10 expressed from the H1 promoter or miApoB10 expressed from the liver specific LP1 promoter. In both cases strong knock- down of ApoB mRNA and protein levels were observed 4 weeks post injection. This correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. However, after 6 weeks the inhibitory effect of the shApoB10 started to wear off rapidly, while miApoB10 retained a more stable inhibitory profile. At 22 weeks post-injection, ApoB10 and plasma cholesterol were still decreased to 50% from baseline. Ongoing research aims to determine the mechanism underlying the differences seen between long-term AAV-shApoB and AAV-miApoB efficacy in murine livers. We hypothesize that the long-term stability of the miApoB is due to its lower toxicity and off-target properties compared to shApoB because expression of miApoB is specifically limited to hepatocytes. Our results demonstrate that the artificial miRNA have a greater intrinsic inhibitory activity than conventional shRNAs and are better approach for development of RNAi based gene therapy of hypercholesterolemia and associated cardiovascular disease. In the future we want to express artificial miRNA targeting ApoB from an inducible expression system. Regulated expression of sh/miRNA combined with the use of a tissue specific promoter and different AAV serotypes will contribute to better and safer gene therapy approach. Decrease in murine plasma cholesterol levels by AAV-shApoB10 and AAV-miApoB10.

563. Developing Use of Inosine-Containing siRNAs To Improve Hepatitis B Virus Silencing Specificity

Justin Hean,1 Carol Crowther,1 Abdullah Ely,1 Piet Herdewijn,2 Patrick Arbuthnot.1 1 Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa; 2Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Leuwen, Belgium. Demonstration that hepatitis B virus (HBV) replication can be effectively silenced by harnessing RNA interference (RNAi) has prompted development of synthetic and expressed anti HBV RNAi activators for therapeutic application. Synthetic siRNAs have advantages of being amenable to chemical modification, compatibility with delivery using non viral vectors and ‘drug-like’ properties that facilitate dose regulation. Our group has previously reported incorporation of altritol modifications into siRNAs that successfully diminish immunostimulation and improve stability of HBV silencing sequences in vivo. After systemic administration of altritol siRNA lipoplex formulations, effective suppression of viral replication was achieved in a stringent transgenic mouse model that simulates human persistent HBV infection. Despite these encouraging results, potentially toxic unintended translational suppressive effects on cellular mRNA, caused by seed sequence hybridization, remain a concern of developing RNAi-based therapeutics. To address this and improve specificity of siRNA-target interaction, effects of modifications to nitrogen bases within the seed region of altritol siRNAs were assessed. Inosines have been substituted for guanine residues at various positions within the guide strand seed region. In addition to attenuating off target hybridization, these modifications diminish thermodynamic stability of the siRNA duplex at the 5’ end of the antisense strands and further improve specificity by enabling intended guide strand selection. Modified siRNAs that target the conserved X open reading frame of HBV achieved highly efficient knockdown (>90%) of markers of viral replication in cultured cells. Position of the inosine residues within the seed region did not affect knockdown. Moreover, silencing efficacy was retained in siRNAs containing stabilizing altritol sugars. Efficacy was sustained without evidence of toxicity and induction of a non specific interferon response. These results indicate that incorporation of inosines into antiviral siRNA seeds, a novel modification, has potential for improving therapeutic application and is likely to have broad utility. To advance this approach, specificity of gene silencing and efficacy in murine models of HBV replication are currently being assessed.

564. Apolipoprotein B Knockdown by AAVDelivered ShRNA Lowers Plasma Cholesterol in Mice

Annemart Koornneef,1 Piotr Maczuga,1,2 Richard van Logtenstein,1 Florie Borel,1,3 Bas Blits,1 Tita Ritsema,1 Sander van Deventer,2 Harald Petry,1 Pavlina Konstantinova.1 1 R&D, Amsterdam Molecular Therapeutics, Amsterdam, Netherlands; 2Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, Netherlands; 3 Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, Netherlands. Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. S216

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

OLIGONUCLEOTIDE & RNAI THERAPEUTICS I Adeno-associated virus (AAV) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knock-down of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. No evidence was found for oversaturation of the endogenous RNAi pathway, since liver-specific microRNAs mir-122, mir29-a, and let7a were not significantly downregulated. In addition, mir-122 target genes AldoA, Bckdk, and Ndrg3 were unaffected. AAV-shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, while high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with AAV-shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dosedependent knock-down of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAibased gene therapy for the treatment of hypercholesterolemia.

565. Assessment of Zwitterionic Lipids as Vectors for Nucleic Acid Delivery

Juliane Nguyen,1 Colin L. Walsh,1 Francis C. Szoka.1 1 Depts. of Bioengineering, Therapeutic Science & Pharmaceutical Chemistry, UCSF, San Francisco, CA. Nonviral RNAi delivery technology provides great potential for the treatment of many human diseases which are not addressable by current medicine. Existing methods to deliver siRNA using cationic liposomes show high delivery efficiency but often cause unwanted toxic effects. Anionic liposomal systems display low cytotoxicity but their use in vivo is hampered by insufficient nucleic acid delivery, large sizes and low encapsulation efficiency. The objective of this research is to design a new class of lipids, which combine the advantages of both carrier systems into one vector: low cytotoxicity of anionic liposomes and high delivery efficiency of cationic liposomes. These lipids should display a neutral or negative surface charge at physiological pH and become positively charged at the lower pH of the endosomes. For this purpose we have synthesized a library of zwitterionic lipids that contain a cationic amine group and an anionic carboxylate group with pKa values of ∼ 5-6. These zwitterionic lipids were characterized for encapsulation of nucleic acids (pDNA and siRNA), size, zeta potential, and ability to mediate gene silencing of FVII in vivo.

566. Multidrug Resistance Transporters and miRNAs Expression: Changes in Hepatocellular Carcinoma

Florie Borel,1,2 Harald Petry,1 Peter Jansen,2 Sander van Deventer,1,3 Pavlina Konstantinova.1 1 Amsterdam Molecular Therapeutics, Amsterdam, Netherlands; 2 Department of Hepatology and Gastroenterology, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands; 3Department of Hepatology and Gastroenterology, Leiden University Medical Centre, Leiden, Netherlands. ATP-binding cassette (ABC) transporters family is involved in multidrug resistance by decreasing the intracellular concentration of toxic compounds. This is a phenotype caused by overexpression of these transporters in tumor cells. Overcoming increased drug extrusion could lead to significant improvement of chemotherapy effectiveness for hepatocellular carcinoma (HCC) patients. In the current study we determined changes in expression pattern of ABC genes in HCC. We established the expression profile of 15 ABC transporters in 19 non-treated HCC patients obtained from Collection des Carcinomes Hepatocellulaires (France). We show that 12 ABC genes are significantly up-regulated in HCC compared to adjacent healthy liver. We next wanted to determine if these changes in ABC expression are associated with miRNA dysregulation. The miRNA Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

profile of a subset of 10 patients reveals significant changes between HCC and healthy liver, for instance the let-7 family is 3 times downregulated in HCC. We have chosen a few down-regulated miRNAs which have bioinformatically predicted target in up-regulated ABC genes in order to validate them in vitro. ABC, sub-family C transporters Abcc1 and Abcc2, also known as multidrug resistance proteins MRP1 and MRP2, were up-regulated in HCC. Downregulation of MRP1/MRP2 could result in improved response of the tumors to chemotherapy. We designed short hairpin RNAs: 6 targeting murine Abcc1 and 7 targeting murine Abcc2. In vitro assays in murine HCC cells showed endogenous mRNA knock-down of more than 50% with shAbcc11, shAbcc17, and shAbcc22, shAbcc23, shAbcc27, shAbcc28. The knock-down effect was sequence-specific and not due to shRNA off-targeting or toxicity. scAAV8 transduces almost completely the murine liver. scAAV8 expressing shAbcc11, shAbcc17, shAbcc22, shAbcc28 were therefore produced and injected in BL/6 mice and knock-down of Abcc1/Abcc2 was determined 2 weeks post-injection. Knocking-down multidrug resistance proteins may improve the effectiveness of the current anti-cancer therapies.

567. Antisense Drug Development for Skipping Human Dystrophin Exons In Vitro and In Vivo Systematically

Bo Wu,1 Ehsan Benrashid,1 Peijuan Lu,1 Caryn Cloer,1 Allen Zillmer,1 Mona Shaban,1 QiLong Lu.1 1 McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, NC. Antisense therapy has recently been demonstrated with great potential for frame-restoring of dystrophin mRNA in human muscle cells and in local muscles of Duchenne muscular dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs), for targeted skipping of human dystrophin exons. Identification of AOs with highest efficiency and specificity is therefore crucial for AO drug development. In this study, we applied three cell culture systems, C2C12 myoblasts containing a GFP-reporter gene expressing human dystrophin exon 50, normal human myoblasts and a culture of DMD patient-derived skin fibroblasts, to screen AOs targeting human dystrophin exon 50. The GFP reporter system was most effective and sensitive for quantitative measurement of exon skipping efficiency. The efficacy of selected AOs was further investigated with Vivo-Morpholino chemistry in the hDMD transgenic mouse carrying the full-length human dystrophin gene. Our results suggest that a combination of in vitro cell culture based selection systems and a Vivo-Morpholino based evaluation in vivo systemically, provides stringent screening to identify effective AOs targeting human dystrophin exon for drug development to treat DMD patients.

568. Tracking Single Liposomes for Delivery of siRNA Matthew R. Tiffany,1 Francis C. Szoka.1 Bioengineering, Therapeutic Science & Pharmaceutical Chemistry, University of California, San Francisco, San Francsico, CA.

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The impetus for this work is based upon the observation that liposomes, and non-viral vectors as a whole, are inefficient at delivering their contents to the cytosol. For example, a single liposome encapsulates at least ∼10^2 siRNA molecules, and at the very least 10^3 liposomes are bound per cell in a typical transfection. Thus the potential for delivery starts at 10^5 siRNAs per cell. In contrast, current estimates indicate that the cell needs ∼10^3 siRNAs for effective silencing. The two log discrepancy between the amount dosed and the amount for a biological outcome suggests that either only a small fraction of molecules get delivered to the cytoplasm in S217