679. Specific Small Interfering RNAs (siRNAs) Targeted to CXCR4 and CCR5 Confer HIV-1 Resistance

GENE SILENCING carbohydrate and lipogenesis pathways in the liver, leading to de novo synthesis of fatty acids. We recently published that short hairpin RNA (shRNA) expressed from the U6 promoter to target fabp5 in a helper-dependent adenovirus vector leads to 75% gene silencing in the liver. Nevertheless, shRNA expression at the highest dose of virus (2x1011v.p) activated the interferon response. IFNs are part of the innate immune response against viral infection and up-regulate expression of multiple genes that contain interferon-stimulated response elements in their promoters, resulting in early blocking of viral transcription/translation and activation of apoptotic pathways. The presence of an interferon response was dose and shRNA sequence specific, and correlated with the presence of apoptotic cells and infiltrates. To study the potential to use the helper-dependent system for long-term gene silencing, we injected 1x1011 v.p to mice and collected liver weekly for 6 weeks after virus injection. Gene-specific knock-down was maintained over this period. However, the level of vector DNA present in liver was significantly reduced in animals sacrificed at week 6, compared to the week 1 and week 3 time points. Given that activation of the interferon response is dose-dependent, a possible explanation is that shRNA accumulate over time in liver, reaching the threshold that activates the interferon response. To prove this concept, the microRNA fraction was extracted and shRNA expression was quantified by Northern blot. We observed that the level of shRNA present in liver was higher at week 3 post-infection, compared to week 1, suggesting that shRNA accumulate over time. In conclusion, we have shown very efficient silencing of fabp5 and SREBP1 in the liver using helper dependent adenovirus vectors. One limitation is the presence of an interferon response that results in loss of transduced cells. Future studies will include the development of advanced helper dependent adenoviral vectors expressing shRNA under the control of inducible systems, so that the level of shRNA expressed can be modulated by administration of a drug, avoiding shRNA-induced non-specific effects.

679. Specific Small Interfering RNAs (siRNAs) Targeted to CXCR4 and CCR5 Confer HIV-1 Resistance

Su Jin Seo, In Young Oh, Tae Gyun Kim, Hyun Song, Min Jung Choi, Youn Ju Park, Kwang Soo Ahn. Gene Therapy Products Division, Biologics Bureau, Korea Food and Drug Administration, Seoul, Korea, Republic of. The recently discovered phenomenon of RNA interference(RNAi) offers another novel approach, which appears to be even more potent in targeted gene silencing and therefore can be potentially harnessed for HIV gene therapy. Infection of a susceptible cell by HIV-1 required viral envelope interactions with the primary cell surface receptor, CD4, and a coreceptor, either CXCR4 or CCR5. Therefore, a potentially promising strategy is to exploit siRNAs to prevent viral entry at the cell surface by down-regulating essential cell surface HIV-1 coreceptors. In our studies, we designed specific siRNA oligonucleotide and establishment of short hairpin RNA(shRNA) expressed plasmid constructs against HIV-1 cell surface coreceptors, CXCR4 and CCR5, to inhibit viral entry. These siRNA oligonucleotides prepared using synthetic RNA duplexes consisting of two unmodified 21mer oligonucleotides annealed together to from siRNAs for CXCR4 or CCR5. These plasmid constructs were designed to high-level express shRNA under the control of U6 promoter that employs RNA polymeraseIII(PolIII) in mammalian cells. shRNAs are synthesized from either CXCR4 or CCR5 DNA template. The resulting shRNAs are believed to be processed by Dicer to generate small RNA duplexes that resemble the active siRNA duplexes. HeLa-TZM cells transfected with siRNA oligonuceotide or shRNA expressed plasmid constructs showed significant down-regulation of their respective coreceptors.Also, we establishment of optimal condition for each siRNA oligonucleotide Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

and shRNA expressed plasmid construct transfection and confirmed effective reduction of CXCR4, CCR5 expression levels by each coreceptor specific siRNA. Real-time PCR and Western blot analysis showed the reduced levels of intracellular synthesis of CXCR4, CCR5 and FACS analysis confirmed marked down-regulation of CXCR4, CCR5 on the cell surface. Specially, effective reduced levels of CXCR4 and CCR5 expression at 5nM siRNA concentration and 48 hours after transfection. These results showed possible to introduce promising siRNA for in vivo gene therapeutic applications.

680. Exploration of Gene Therapies for Dominant Dystrophic Epidermolysis Bullosa

Clare P. Morgan, Danny Allen, Paul F. Kenna, Pete Humphries, Gwyneth J. Farrar. Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland. Epidermolysis Bullosa (EB) is a rare genetically determined blistering skin disorder, affecting approximately 1 in 17,000 of live births. The disease is classified into a number of different subtypes, one of which is dominantly inherited dystrophic EB (dDEB). The dominant dystrophic form of EB arises due to mutations of the COL7A1 gene which encodes type VII collagen, an important structural protein. The resulting mutant protein displays a dominant negative effect, causing the disease phenotype. A potential gene therapy approach for dDEB would likely require suppression of expression of the mutant COL7A1 gene: the method of RNA interference (RNAi) represents one such molecular tool which may be suitable for achieving such suppression. This study explores suppression of the wild-type COL7A1 gene in cultured human epidermal keratinocyte cells (HaCaTs) using an RNAi approach. Short hairpin RNA molecules (shRNAs) which have characteristics of the cells own endogenous microRNAs, cloned within artificial microRNA delivery plasmid vectors, were designed to target COL7A1. 14 such molecules along with a non-targeting negative control were tested in vitro and COL7A1 mRNA levels were measured by real time rtPCR. Stable cell lines expressing the most potent suppressors and non-targeting control were established, made possible by the presence of a blasticidin-resistance marker gene on the miRNA plasmid. Additionally, collagen type VII protein levels were evaluated by western blotting. Results demonstrated significant suppression of COL7A1 expression in HaCaT cells which endogenously express type VII collagen protein. These results obtained thus far represent an important step in the progression towards a suitable therapeutic approach for dDEB.

681. Multifunctional Nanoparticles for DNAzyme Delivery: Towards New Hepatitis C Drug

Soo-Ryoon Ryoo,1 Hong-Je Jang,1 Ki-Sun Kim,2 Dong-Eun Kim,2 Dal-Hee Min.1 1 Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, Republic of; 2Bioscience and Biotechnology, Konkuk University, Seoul, Korea, Republic of.

Gene therapy may be therapeutically useful in relieving symptoms and treating any kind of diseases which hybridize with their complementary target site in mRNA, blocking translation to protein expression of patho-physiologic genes. These strategies include DNAzymes, siRNA, antisense oligonucleotides, ribozymes, and aptamers. Among these molecules, deoxyribozymes (DNAzyme, Dz) have been paid attention as therapeutics both in cell-based assays and in preclinical models of diseases including cancer and viral infectious diseases. The “10–23” RNA cleaving DNAzyme has been shown to cleave any purine–pyrimidine junction under simulated physiological conditions, therefore efficiently inhibiting expression of target proteins in vitro and in vivo studies. These molecules S259