Gene transfer into the CNS using recombinant adeno-associated virus: analysis of vector DNA forms resulting in sustained expression.

Recombinant adeno-associated virus (rAAV) vectors have shown significant promise as vehicles for in vivo gene transfer, particularly for transduction of organs composed primarily of non-dividing cells (i.e., muscle, CNS, and liver). However, the mechanistic basis for this desirable property remains unclear. To investigate the fate of rAAV genomes in mouse brain, we stereotactically injected an rAAV vector carrying the E. coli lacZ gene into the caudate of BALB/c mice and demonstrate efficient transduction of mouse brain cells that possess cellular morphology consistent with post-mitotic neurons. We observed a significant increase in beta-galactosidase expression from 5 to 56 days after injection that paralleled the disappearance of single-stranded DNA input genomes. Analysis of in vivo viral DNA forms over time out to 5 months after inoculation revealed that rAAV genomes associated with high molecular weight mouse chromosomal DNA by 14 days after injection and persisted for the length of this study. The pattern of Southern hybridization was consistent with random viral integration in predominantly head-to-tail concatameric arrays. Importantly, we also documented an additional DNA species that appears to be a monomeric episomal circular form based on nuclease sensitivity assays. These data are the first to document the existence of multiple vector DNA forms present within the adult murine brain following direct rAAV inoculation and therefore, provide insight into the molecular events that ultimately result in long-term rAAV mediated transgene expression.