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Journal of Virology, April 2009, p. 3919-3929, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02466-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Reducing the Risk of Adeno-Associated Virus (AAV) Vector Mobilization with AAV Type 5 Vectors{triangledown}

F. Curtis Hewitt,1,2,§ Chengwen Li,1,§ Steven J. Gray,1 Shelley Cockrell,4 Michael Washburn,2 and R. Jude Samulski1,2,3*

Gene Therapy Center, University of North Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, North Carolina 27599-7352,1 Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,2 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,3 Interdisciplinary Biomedical Graduate Program, University of Pittsburgh, Pittsburgh, Pennsylvania 152614

Received 1 December 2008/ Accepted 1 February 2009

Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

* Corresponding author. Mailing address: Gene Therapy Center, University of North Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, NC 27599-7352. Phone: (919) 962-3285. Fax: (919) 966-0907. E-mail: rjs{at}med.unc.edu

{triangledown} Published ahead of print on 11 February 2009.

§ These authors contributed equally to this work.

Journal of Virology, April 2009, p. 3919-3929, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02466-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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