Adeno-associated virus type 2 as vector for human gene therapy: Characterization of virus-host interactions

Vectors based on adeno-associated virus type 2 (AAV) offer considerable promise for somatic gene therapy of various diseases (e.g. cystic fibrosis, hemophilia B, cancer). Limitations, however, still exist and require further improvement. The study presented here addresses two major problems that hamper a widespread use of AAV in human gene therapy: First, the loss of site-specific integration of recombinant AAV vectors (rAAV) due to the deletion of the rep gene, and secondly, the potential neutralization of AAV gene therapy vectors by preexisting antibodies. It could be demonstrated that site-specific integration of a transgene encoding rAAV vector can be efficiently restored by providing rep as plasmid DNA in trans. Based on these findings, a rAAV vector was developed where a plasmid coding for Rep was coupled as polylysine/DNA complex (PLL/DNA) directly to the capsid of the virion and co-transduction of such a PLL/DNA coupled to AAV was shown. Thus, providing rep-DNA as PLL/DNA should allow rAAV vectors to integrate specifically at chromosome 19. To characterize immunogenic domains of the AAV capsid and to develop strategies to circumvent neutralization by antibodies, AAV capsid mutants carrying peptide insertions in surface exposed loop regions were investigated in binding and neutralization assays. Three positions could be identified in the AAV capsid (at aa 534, 573, and 578 of the VP1 protein) where binding and neutralization of the virus by human serum samples was markedly reduced. These result suggest that capsid modifications could help to overcome binding and neutralization by human antisera in clinical gene therapy applications