| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Only recombinant adeno-associated virus (Raav) system may have the character for site specific integration into AAVS1 site on human chromosome 19q13.3-qter. Site-specific integration mechanism starts with the complex formation mediated by viral encoded Rep78/68 protein between AAV inverted terminal repeat (ITR) and AAVS1 sequences. Currently, it is considered, available rAAV vectors lacking the rep gene lost the ability of site specific integration. Detectable and long term expression of Rep 78/68 are known to be cytostatic to many cells and woeks as an inhibitor of sp1 promoters under stable expression. Therefore, transient presence of cytostatic Rep78/68 proteins is required only at AAV-vector integration for safety of the targeted cells. In this study, we examined the direct delivery of Rep Protein to 293 cells with AAV vector plasmid by lipofection and by HVJ-liposome, instead of cotransfection of Rep-expression plasmids. Rep78 protein (psub 201 ; 318-2227 nt) was synthesized as a
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fusion protein with maltose binding protein (MalE-REP) in E. coli. After digestion of MalE-REP by Factor X, REP fraction was prepared by passing through amylose resin for removal of Maltose binding protein. 293 cells were lipofected/infected with/containing Rep protein (MalE-REP or REP) and AAV vector plasmid (XF) containing the tk promoter driven neoR gene flanked by ITRs, and culture in the presence of G418. Specific-integration of AAV vector plasmid was detected by nested-PCR amplification from 3'-vector ITR to AAVs1, southern analysis by AAVS1 oligo, and direct sequencing of PCR bands. At least, five out of 50 G418-resistant cell lines extablished by lipofection, and one out of 23 G418 resistant clones by HVJ-liposome showed that AAV vector sequences were integrated into the AAVS1 locus. These results formally prove that Rep 78 and ITR are sufficient for AAVS1 specific integration. Direct transfer of Rep proteins by lipofection or HVJ-liposome should be available for site specific integration of the transgene into chromosome 19, although further studies are required for optimization of co-introduction of plasmid DNA and protein. Less
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