Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy S37 STEM CELL BIOLOGY In recent work we showed that reduction of mutant htt in a mouse model of HD, using a viral vector expressing short hairpin RNAs (shRNAs), protected the animal from the onset of behavioral and neuropathological hallmarks of the disease. Here, we tested if delivery of synthetic siRNAs directly to the brain by nonviral methods could be similarly effective. This approach has many advantages, most important of which is that if problems arise, the therapy can be stopped. Major hurdles for nonviral delivery, however, include formulating the inhibitory nucleic acids for delivery to brain, and stabilizing the siRNAs for improved longevity in vivo. We tested chemically modified siRNAs specific for human and mouse huntingtin, encapsulated in various lipid nanoparticles (LNP). The prepared LNP formulations were screened for their ability to silence full-length htt in vitro, followed by testing in vivo. Using Alzet® osmotic pumps, siRNAs encapsulated in LNPs were infused into the lateral ventrical or striatum for 7 or 14 days, respectively, at concentrations ranging from 0.1 to 1 μg/μl (total dose ranging from 8.4 to 84 μg). We noted an impressive 80% reduction in htt mRNA levels by QPCR compared to scrambled control sequences, or naïve brain. This level of reduction is similar to that achieved with adeno- associated virus vector expressed shRNAs, and was accomplished at doses 18-fold lower than those previously reported as required for target gene reduction in adult rodent brain using other formulations. In summary, we show for the first time, reduction of target htt in vivo in adult mouse striatum, a major site of neurodegeneration in HD. Ongoing studies will determine the safety and feasibility of the synthetic siRNA approach for HD therapy. Collaborative effort between Sirna Therapeutics and the University of Iowa. 89. Gene Targeting of β-Globin IVS2 Using Sequence-Specific Peptide Nucleic Acids Jean Y. Kuan, 1 Ryszard Kole, 2 Peter E. Nielsen, 3 Peter M. Glazer. 1 1 Genetics and Therapeutic Radiology, Yale University School of Medicine, New Haven, CT; 2 Pharmacology, University of North Carolina, Chapel Hill, Chapel Hill, NC; 3 Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Copenhagen, Denmark. Many of the mutations that lead to the blood disease β-thalassemia are the result of single base mutations that result in a decrease or lack of β-globin protein. The reversion of these mutations back to the wild-type sequence in genomic DNA would result in permanent restoration of globin protein expression. One mutation, G>A change which occurs at the first nucleotide of intron 2 in the β-globin gene (designated IVS2-1 G>A), destroys the 5’ donor splice site at the end of exon 2 and abolishes correct splicing of pre-mRNA. Because triplex formation via peptide nucleic acids (PNAs) has been shown to provoke repair and recombination upon binding to duplex DNA, it is believed that PNA binding can be utilized to stimulate gene conversion events. Several potential PNA target sites have been identified near the IVS2-1 G>A mutation including a polypurine run beginning 24 nt downstream from IVS2-1, a binding site which we have designated IVS2-24. Using a reporter system consisting of GFP interrupted by β-globin IVS2, we have demonstrated restoration of reporter gene expression when cell lines containing the inactivating mutation were treated with PNAs and short donor molecules comprised of the wild-type sequence. Reporter gene expression has been confirmed by both FACS and fluorescence microscopy and was observed over a 6 week time period after a single treatment. This demonstrates a potentially powerful tool in the gene-targeting of diseases caused by single base mutations. 90. miRNA Shuttles Improve Therapeutic RNAi Ryan L. Boudreau, Beverly L. Davidson. 1 Internal Medicine, University of Iowa, Iowa City, IA; 2 Physiology & Biophysics, University of Iowa, Iowa City, IA. Huntington’s disease (HD) and Spinocerebellar ataxia type I (SCA1) are dominant neurodegenerative diseases caused by polyglutamine (polyQ) expansion. The mutations produce toxic proteins, with polyQ length predictive of disease onset and severity. There is no therapy for HD or SCA1, supporting the investigation of novel strategies. In other work, we show that gene silencing by RNA interference (RNAi) can be achieved in vitro and in vivo by expressing short hairpin RNAs (shRNAs) which are subsequently processed into small interfering RNAs (siRNAs) specific for ataxin- 1 or huntingtin mRNA. Viral delivery of RNAi to disease mouse models resulted in partial recovery of morphological and behavioral phenotypes, suggesting improvements could be explored. To improve our RNAi strategy, we have previously developed an inducible RNAi expression system which exploits microRNAs (miRNAs) as siRNA shuttles. Although previous studies suggest that miRNA shuttles are processed by RNAi machinery more efficiently than shRNAs, the silencing efficiencies of miRNA shuttles and shRNAs have not been adequately compared. To address this, we generated RNAi constructs which express siRNAs specific for unique targets (ataxin- 1, huntingtin or eGFP) in the context of either miRNA shuttles or shRNAs. shRNAs were driven by the polIII-based U6 or H1 promoters, while miRNA shuttles were driven by U6 or the polII- based CMV promoter. We assessed the ability of the RNAi constructs to silence Renilla luciferase with 3’UTRs containing target sequences corresponding to either ataxin-1, huntingtin or eGFP. Furthermore, sense and antisense sequences were targeted to determine which RNAi strands (guide or passenger) enter RISC. RNAi and luciferase target constructs were co-transfected into 293 cells and luciferase assays performed 48h later. Preliminary results show that the RNAi constructs direct sequence-specific silencing of the appropriate luciferase target. Furthermore, silencing was more efficient with miRNA shuttles than shRNAs, independent of the targeted sequence. These results suggest that miRNA shuttles may be used to increase the therapeutic efficacy of RNAi in SCA1 and HD mouse models. STEM CELL BIOLOGY 91. Retroviral Vector Integrations Relate to Hematopoietic Stem Cell Gene Expression Patterns Martijn H. Brugman, 1 Karin Pike-Overzet, 2 Carla Oerlemans- Bergs, 1 Sigrid Swagemakers, 3 Dick de Ridder, 4 Frank J. T. Staal, 2 Monique M. A. Verstegen, 1 Gerard Wagemaker. 1 1 Department of Hematology, Erasmus MC, Rotterdam, Netherlands; 2 Department of Immunology, Erasmus MC, Rotterdam, Netherlands; 3 Department of Bioinformatics, Erasmus MC, Rotterdam, Netherlands; 4 Information & Communication Theory Group, Delft University of Technology, Delft, Netherlands. The oncogenic risk of retrovirus mediated gene transfer has received major attention and is a considerable safety concern. To analyze the mechanisms involved we have identified integration sites in life-long monitored primary and secondary recipient mice of bone marrow cells transduced with an MLV retroviral vector. In these mice, clones with integrants by definition originate from long- term repopulating stem cells. Vector integrations were identified by LAM-PCR. To determine gene expression levels in the target population for transduction, we performed microarray analyses of