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.