Characterization of Genome Integrity for Oversized Recombinant AAV Vector

Application of recombinant adeno-associated virus (rAAV) in gene therapy has been limited by its packaging capacity. Recent studies suggested that rAAV could achieve persistent transgene expression beyond 4.7-kb packaging limit. To clarify the mechanism leading to transgene expression from oversized...

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Published inMolecular therapy Vol. 18; no. 1; pp. 87 - 92
Main Authors Dong, Biao, Nakai, Hiroyuki, Xiao, Weidong
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 01.01.2010
Elsevier Limited
Nature Publishing Group
Subjects
Animals
Cell Line
Dependovirus - genetics
Gene expression
Gene therapy
Genetic Vectors - genetics
Genome, Viral - genetics
Genomes
HeLa Cells
Humans
Immunology
Infections
Mice
Mice, Inbred C57BL
Original
Plasmids
Thrombosis
Vectors (Biology)
United States > US
Pennsylvania
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Summary:Application of recombinant adeno-associated virus (rAAV) in gene therapy has been limited by its packaging capacity. Recent studies suggested that rAAV could achieve persistent transgene expression beyond 4.7-kb packaging limit. To clarify the mechanism leading to transgene expression from oversized rAAV vector, we constructed a series of rAAV vectors with genomes ranging from 2.9 to 7.2 kb. A plasmid replication origin and an ampicillin-resistant marker were included in the vector to facilitate the recovery of circularized, post-transduction AAV genome. Southern dot-blot analysis and silver staining confirmed that rAAVs could be produced at varying vector size. However, the vector yields decreased approximately tenfold for oversized vectors as compared to regular ones. Alkaline Southern blot hybridization suggested that the packaged genomes for oversized vectors were truncated. In the cells transduced by the above vectors, circularized rAAV monomers could be rescued at 24 hours after infection. Few recovered AAV genomes were >5 kb regardless of the initial vector size. In mice receiving the above vectors, larger circularized rAAV genomes could be recovered for oversized vectors at day 21 after vector administration. Our studies suggested that the partially packaged rAAV sequences may complement each other to restore full expression cassette.
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ISSN:1525-0016
1525-0024
DOI:10.1038/mt.2009.258