696. Pre-Clinical Evaluation of Allele-Specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides

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