846. Expanded CAG Repeats Enhance the Production of a Pathogenic Cav2.1 Splice Variant That Can Be Selectively Targeted Using RNAi

OLIGONUCLEOTIDE & RNAI THERAPEUTICS II 846. Expanded CAG Repeats Enhance the Production of a Pathogenic Cav2.1 Splice Variant That Can Be Selectively Targeted Using RNAi

Wei-Ling Tsou,3 Bing-Weng Soong,3 Henry L. Paulson,2 Edgardo Rodriguez-Lebron.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Neurology, University of Michigan, Ann Arbor, MI; 3Life Sciences-Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan. Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion near the C-terminus of the Cav2.1 voltage-gated calcium channel subunit (CACNA1A). SCA6 is characterized by marked cerebellar dysfunction and atrophy resulting from the preferential loss of cerebellar Purkinje neurons. There is currently no treatment for this debilitating disorder, and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting the progression of behavioral, histological and molecular abnormalities in several models of Spinocerebellar ataxia (SCA), including SCA type 1 and SCA type 3. However, RNAi-based strategies that selectively target mutant alleles in SCA are currently lacking. In SCA6, alternative splicing near the 3’end of the Cav2.1 transcript generates two mRNA isoforms encoding functional Cav2.1 channel subunits, only one of which contains the pathogenic polyQ domain. Taking advantage of a 5 base pair sequence difference between the two isoforms, we designed splice-isoform-specific (SIS)-RNAi molecules to selectively suppress expression of the polyQ-encoding splice variant in SCA6. Significant and selective suppression of the polyQ-encoding Cav2.1 splice variant was achieved in a variety of cell-based models. A miR124-based microRNA shuttle used for the intracellular production of SIS-RNAi molecules was efficiently recognized, processed and modified by the cellular RNAi machinery. Intriguingly, we find that pathogenic CAG expansions in Cav2.1 enhance splicing activity at the 3’end of the transcript and lead to an increase in the levels of polyQ-encoding Cav2.1 mRNA isoform and polyQ-containing Cav2.1 protein. Current experiments aim to assess the activity, specificity and safety of rAAV-SIS-RNAi mediated inhibition of polyQ-containing Cav2.1 channel subunits in the adult mouse cerebellum. Our results support the preclinical development of SIS-RNAi as a molecular therapy for SCA6 and provide the basis for the development of similar SIS-RNAi-based strategies in other dominantly inherited diseases.

847. Apolipoprotein B Knock-Down by AAVDelivered sh/miRNA Lowers Plasma Cholesterol in Mice

Piotr Maczuga,1,2 Annemart Koornneef,1 Richard van Logtenstein,1 Florie Borel,1,3 Bas Blits,1 Sander van Deventer,2 Harald Petry,1 Pavlina Konstantinova.1 1 Research & Development, Amsterdam Molecular Therapeutics, Amsterdam, Netherlands; 2Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, Netherlands; 3Department 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 initially 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, shApoB10, for in vivo inhibition studies. To compare the intrinsic inhibitory properties and long-term efficacy of shRNA and miRNA, we expressed shApoB10 from a miRNA scaffold (miApoB) using the liver-specific S324

LP1 promoter. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB and miApoB. Expression of the AAV-shApoB and AAV-miApoB resulted in 90% ApoB protein knock-down, associated with 80% cholesterol decrease in murine plasma for the first 6 weeks. However, after 6 weeks the inhibitory effect of the shApoB started to wear off, while miApoB retained a more stable inhibitory profile. At 22 weeks post-injection, ApoB and plasma cholesterol were still decreased to 50% from baseline. Furthermore, 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 shApoB, a known phenotypic side effect of lowering ApoB levels. 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 longterm 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 a robust long-term knock-down of ApoB by AAV-delivered miApoB in murine liver, thus providing an excellent candidate for development of RNAibased gene therapy for the treatment of hypercholesterolemia and associated cardiovascular disease.

848. Double-Stranded Let-7 Mimics, Potential Candidates for Cancer Gene Therapy

Qizhao Wang,1 Chao Lu,1 Weidong Xiao,1,2 Ruian Xu.1 1 Engineering Research Center of Molecular Medicine & Institute of Molecular Medicine, Huaqiao University, Ministry of Education, Quanzhou, Fujian, China; 2Department of Microbiology, Temple University, Philadelphia, PA.

MicroRNAs (miRNAs), a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The ability of one single miRNA regulating multiple functionally related mRNAs makes it a new potential candidate for cancer gene therpay. Let-7s miRNAs have been demonstrated as tumor suppressive genes in a variety types of cancers, providing one choice of gene therapy by replenishing this miRNA. Here we showed that introduction of double-stranded mimics form of Let-7s inhibited the growth and migration, and induced the cell cycle arrest of lung cancer cell lines in vitro. Reporter assay verified that Let-7 mimics could silence the expression of K-ras and Hmga2 genes through binding to the 3’UTR of targeting mRNAs, and mutation of seed sequence significantly depressed the gene silencing activity. Both Let-7 mimics and K-ras siRNAs loaded into RISC within 3 h and A 3hs-interval between the introduction of funcional Let-7 mimics and K-ras siRNA enhance the anti-cancer activities of Let-7s. By using competition and RISC loading assays, it was found that the action Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy