Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle.
Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.
Department of Genetics, Istituto di Ricerche di Biologia Molecolare, I.R.B.M.-Piero Angeletti, 00040 Pomezia, Rome, Italy.
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Lamartina
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Roscilli
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Rinaudo
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Delmastro
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Toniatti
D002411:Cation Exchange Resins; D002888:Chromosomes, Human, Pair 19; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000378:metabolism*; D004337:Drug Carriers; D006367:HeLa Cells; D006801:Humans; D008055:Lipids; D012091:Repetitive Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration
D002411:Cation Exchange Resins; D004268:DNA-Binding Proteins; D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2