935. Brain Targeted Gene Therapy for Metachromatic Leukodystrophy: In Vitro Expression and Activity of a Melanotransferrin-Arylsulfatase A Fusion Protein

encoding an exogenous target luciferase gene was injected in mice by a hydrodynamics-based procedure. After 6 hours, the mice were injected with AeKG6!siRNA complexes via tail vein. Silencing occurred mainly in the liver. Then, after 18 hours, the livers were dissected from the mice and lucifcrasc activities were measured. The degree of silencing was dependent on the C!A ratio ofAeKG6 to siRNA , and maximal silencing was ca. 70%. In conclusion, we have succeeded in systemic siRNA delivery mediated by AeKG6. After modifying its surface and optimizing the mixing condition of the complex, the dendritic poly(L-lysine) could be expected to be a promising candidate that enables functional gene delivery in vivo.

934. Fully 2'-Deoxy-2'-Fluoro Substituted Nucleic Acids Induce RNA Interference in Mammalian Cell Culture

Richard A. Blidner, I Robert P. Hammer.' W. Todd Monroe" I Department ofBiological & Agricultural Engineering, Louisiana State University, Baton Rouge, LA; IDepartment ofChemistry, Louisiana State University; Baton Rouge, LA. RNA interference (RNAi) is a phenomenon in which RNA molecules elicit potent and sequence-specific post-transcriptional gene silencing. Various chemical modifications to nucleic acids have been incorporated in attempts to improve their pharmacological properties. Recent studies have shown that small interfering RNA (siRNA) containing pyrimidine 2'-fluoro modifications can elicit RNAi. In this study we demonstrate that 2 '-fluorinated nucleic acids (FNAs) with fluoro substitutions at all purines and pyrimidines can be generated for RNAi studies by either custom solid-phase synthesis or through in vitro transcription using a mutated T7 polymerase and fluorinated nucleoside triphosphates (NTPs). Single-stranded and hybridized FNAs were evaluated for their resistance to enzymatic digestion by a common ribonuclease and nuclease. Duplex siFNA and antisense FNA were evaluated for their ability to knockdown green fluorescent protein (GFP) transgene expression in mammalian cell culture. Results indicate that FNA can be successfully generated , and these FNA products show superior resistance to digestion over native RNA. A combination of melt curve analysis and terminal end-labeling with a cy5-ddUTP suggests that the reduced knockdown efficiency of transcribed FNAs may be attributable to base miscoding errors by the mutant polymerase. Phenotypic analysis in cell culture and quantitative PCR show that siFNA can induce RNAi ofthe reporter gene, albeit less efficiently than native siRNA. Silencing from antisense FNA was greatly reduced relative to the duplex form. This suggests that gene silencing from siFNA is likely an enzymatically driven process as opposed to simple translational arrest. In addition their effectively reducing gene expression through RNAi, the chemical stability ofsiFNAs may facilitate furtherchemical manipulation relative to unmodified RNA species.

935. Brain Targeted Gene Therapy for Metachromatic Leukodystrophy: In Vitro Expression and Activity of a MelanotransferrinArylsulfatase A Fusion Protein Donna Y.I-Iou,' Anna A. Li,' Murray A. Potter. I Pathology and Molecular Medicine, MeMaster University, Hamilton, ON, Canada. I

Metachromatic leukodystrophy (MLD) is an inherited neurodegenerative disorder stemming from the deficiency of the lysosomal enzyme arylsulfatase A (ARSA). Profound deficiency of ARSA leads to lysosomal accumulation ofthe substrate 3-0-sulfogalactosylccramide and results in progressive demyelination in the central and peripheral nervous systems. Currently, there is no treatment for MLD. The development of enzyme replacement therapy (ERT) and gene therapy of MLD is hampered by the presence of the S356

blood brain barrier (BBB) which impedes delivery of therapeutic products into the brain. The amount of enzyme required to clear sulfatide storage is relatively low, as evidenced by individuals with lower than normal levels of ARSA who appear clinically healthy. The challenge for gene therapy of MLD is to deliver ARSA across the BBB into the brain at the lower than normal levels sufficient to prevent sulfatide storage. Our strategy to overcome this hurdle is to develop a non-viral peripherally administered therapy to deliver therapeutic enzymes across the BBB. The ability of human melanotransferrin p97 (mtl) to be transcytosed across the BBB has been previously reported. Therefore we developed an expression vector fusing mtfto human ARSA to deliver ARSA activity across the BBB. We have also fused mtfto luciferase as a marker protein. Transfected MOCK and C2CI2 cells express and secrete intact fusion proteins, confirmed by western blotting using antibodies against mtf and ARSA. Enzyme activity assays show that the fusion with mtfdoes not reduce enzymatic activity ofeither luciferase or ARSA compared to un-fused luciferase and ARSA. In in vitro uptake assays , fibroblasts from three different MLD patients were able to take up the fusion protein (mtffused ARSA) from medium conditioned by transfeeted cells. The uptake occurs in a mannose six phosphate receptor independent manner. ARSA levels in two of the three treated MLD cells were significantly greater than untreated controls. ARSA levels in treated MLD cells reached 25% of the lower limit of the range of normal enzyme activity, levels comparable to those present in clinically normal heterozygotes. Aldan blue staining showed reduction in sulfatide storage in treated MLD fibroblasts as compared to controls. Whether the mtf fused ARSA is able to be delivered across the BBB and into the brain is now under investigation. This approach is applicable to a variety of delivery techniquies such as enzyme replacement therapy, viral or non-viral gene therapy or microencapsulated cell gene therapy. In microencapsulated cell gene therapy, cells expressing the therapeutic fusion protein are enclosed in an immuno-isolating, selectively permeable, biocompatible microcapsule. Implanted capsules secrete desired gene products while protecting the encapsulated cells from immune mediators. Our future goal is to deliver the fusion protein via non-autologous microencapsulated gene therapy in the mouse modelofMLD.

936. In Vivo Efficient Gene Expression by Freeze-Dried DNA Complexes Coated by Polyanions

Tomoko Ito,' Keiko Abe,' Naoko I. Tanaka,' Yoshiyuki Koyama.' I Department ofMaterials Science, Otsuma Women University Graduate School, Tokyo, Japan ; IDepartment ofFood Science, Otsuma Womens University Graduate School, Tokyo, Japan.

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Various polycations and cationic lipids which can spontaneously assemble with DNA have been widely explored as safer alternative to viral vector systems. The suspension of DNA eomplcxes with cationic molecules are quite unstable , and required to be quickly used after preparation, otherwise they would be immediately aggregated, and diminish activity. Freezing or lyophilizing thc complexes would also extremely reduce the transfcetion efficiency. We have found and reported that certain polyanions such as PEG derivative having carboxy lic acid (PEG-C) or hyaluronic acid could deposit and coated onto the DNA! polycation (or cationic lipid) complexes, and stabilize the dispersion. The polyanion-coating recharged the complexes to negative (Fig. I) and reduced the non-specific interaction with blood components. It also inproved the gene transcription efficiency probably owing to the moderate loosening of compacted DNA. Recently, we found that the polyanion-coated DNA complexes could be freeze-dried without decrease ofgene activity. In this study, we attempted efficient in vivo transfection by the freeze-dried DNA complexes coated by polyanions. Novel gene transfection methodMolecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C AmericanSocietyo f Gene Tllcr.lpr