cMOAT)

GENE REGULATION: TARGETS in structure or CG content diminished their potential, testing molecules with only one loop, with CG motifs only in one loop, with a smaller size and a linear PO-ODN. Interestingly, dSLIM30L1 significantly increased IL-12p40 production by B-cells, and IFN-γ production by NK-cells. Using total PBMCs, we found induction of a specific cytokine pattern by dSLIM-30L1 with levels of IFN-γ, IFN-α and IL-12p40 which is distinct from the pattern induced by the linear, PS-based lin-30L1. Furthermore, the PObased dSLIM had significantly less prolonged blood clotting time compared to PS-based lin-30L1. In conclusion, we present a new class of immunomodulators (dSLIM) with multiple immune properties which are distinct to PSbased CpG-ODNs of the same sequence, while exhibiting reduced PS-dependent side-effects. Some authors (AS, JK, SAKM, BW, MS) are employees of Mologen AG

647. AP20817-Inducible Activation of a Chimeric Insulin Receptor Mimics Insulin Action in Hepatocytes Transduced with AAV Alberto Auricchio,1 Gabriella Cotugno,1 Pietro Formisano,2 Francesco Beguinot,2 Enrico M. Surace,1 Roy Pollock.3 1 Telethon Institute of Genetics and Medicine (TIGEM), Napoli, Italy; 2Molecular and Cellular Biology and Pathology, “Federico II” University, Napoli, Italy; 3ARIAD Pharmaceuticals, Inc., Cambridge, MA. Insulin gene replacement therapies for type I diabetes, that result in ectopic insulin production from tissues engineered with viral vectors, lack the precise temporal control associated with natural hormone secretion. A potential solution to this problem is to direct gene transfer to insulin target cells and introduce constructs that activate the insulin signaling pathway in response to an exogenously administered drug. Insulin regulates metabolism and glucose homeostasis through binding to a specific membrane receptor (IR) that displays tyrosine kinase activity. We constructed a chimeric insulin receptor (Fv2IRE) whose dimerization and subsequent activation can be induced by a small molecule dimerizer drug, AP20187. This system should activate the IR signaling pathway in insulin target cells with kinetics similar to those of the natural hormone. The Fv2IRE chimeric receptor, which binds AP20187, was introduced in human HepG2 hepatocytes and primary human fibroblasts using AAV2/1 vectors. AP20187 is able to induce Fv2IRE dimerization and transphosphorylation in a dose dependent way. This activation leads to AP20187 dose dependent tyrosine phosphorylation of the canonic IR substrate IRS1. This occurs independently of the activation of the endogenous IR expressed in HepG2 cells. AP20187 activation of Fv2IRE induces glycogen synthase activation and proliferation in AAV transduced- cells. In conclusion, the AP20187-Fv2IRE system represents a potential therapeutic and research tool to be exploited in vivo in animal models of type I diabetes or of other genetic defects of insulin action.

648. In-Vivo Adenovirus Mediated RNA Interference of Multidrug Resistance Protein-2 (MRP-2/ABCC2/cMOAT) Iñigo Narvaiza,1 María Vera,1 Nerea Razquin,1 Mikel Zaratiegui,1 Jesús Prieto,1 Puri Fortes.1 1 Division of Gene Therapy and Hepatology, Foundation for Applied Medical Research. University of Navarra, Pamplona, Navarra, Spain. RNA interference (RNAi) is a natural process, originally described in Caenorhabditis elegans and plants, initiated by the cleavage of long double stranded RNA (dsRNAs) by the ribonuclease III Dicer generating short interfering RNAs (siRNAs). siRNAs Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

induce selective degradation of mRNAs that are homologous to their sequence. In mammalian cells, RNAi interference can be achieved by transfection of chemically synthesized siRNAs or short-hairpin RNA (shRNAs) precursor expressing plasmids. In-vivo gene silencing of endogenous and exogenous genes has been proven by hydrodynamics injection of both shRNAs and synthetic siRNAs, or by using shRNA expressing viral vectors such as lentivirus or adenoviruses. We have constructed first generation recombinant adenoviruses encoding different shRNAs against murine multidrug resistance protein 2 (MRP2/ABCC2/cMOAT) (AdshMRPs). Mutations in MRP2 gene are responsible for Dubin-Johnson syndrome, a hereditary disease characterized by hyperbilirubinemia. AdshMRP infected murine hepatocellular carcinoma cells (Hepa 1-6 cell line) have decreased MRP2 mRNA levels as quantified by quantitativePCR. In addition, we are able to detect siRNAs targeting MRP2 mRNA by primer extension experiments in AdshMRP2 infected Hepa 1-6 cells. In-vivo experiments in C57BL/6 mice revealed the presence of antisense siRNAs against MRP2 in liver extracts 7, 12, 24 and 150 days after AdshMRP intra-venous (i.v.) injection. Furthermore, AdshMRP i.v. administration in mice resulted not only in basal serum hyperbilirubinemia, but also in significant impairment of synthetic bilirubin uptake after bilirubin i.v. administration for up to three weeks. Our results demonstrate that adenoviral vectors are efficient tools for shRNA delivery into the liver, allowing the targeted silencing of an endogenous mRNA that result in transient function inhibition.

649. Repression of Vascular Endothelial Growth Factor-A Over-Expression in Glioblastoma Cells Using Engineered Zinc-Finger Transcription Factors Andrew W. Snowden, Lei Zhang, Fyodor Urnov, Carolyn Dent, Andrew R. McNamara, Yann Jouvenot, Steve H. Zhang, Edward J. Rebar, Andrew C. Jamieson, Casey C. Case, Philip D. Gregory. 1 Sangamo Biosciences Inc, 501 Canal Blvd, Richmond, CA. Angiogenesis is of critical importance to tumor progression and therefore is an attractive target in the treatment of cancer. The proangiogenic Vascular Endothelial Growth Factor A (VEGF-A) a potent effector of angiogenesis, is over-expressed in a variety of human cancers, including highly invasive brain tumors. In this study, we have applied engineered zinc-finger protein transcription factors (ZFP TFs) to the regulation of VEGF-A expression in human cancer cell lines. We show that ZFP TFs containing either the ligand-binding domain of thyroid hormone receptor or its viral relative vErbA are potent transcriptional repressors of VEGF-A expression. The engineered ZFP chimera specifically binds its target site within the VEGF-A promoter in vitro and in vivo and represses in a HDAC dependant manner via targeted deacetylation of promoter nucleosomes. The therapeutic relevance of ZFP-driven VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant over-expression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of this gene by >20-fold. Furthermore, the levels of VEGF-A following ZFP TF-mediated repression were comparable to those of a non-angiogenic cancer line, U251MG, suggesting that this degree of repression may be sufficient to suppress tumor angiogenesis. The use of ZFP-mediated recruitment of alternative repression domains and activities which may confer long term, epigenetic repression; such as DNA methylation, histone H3 lysine 9 methylation and HP1 recruitment in the repression of VEGF-A transcription will also be discussed.

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