101. Molecular Mechanism Responsible for High Level Transgene Expression in Murine and Nonhuman Primate Liver with the Second Generation of Novel AAV Serotype Vectors

Abstract

AAV vectors are being considered for in vivo application of gene therapy in the treatment of a variety of disorders. This study evaluates the biology of second generation vectors based on the novel serotypes AAV7 and 8 containing self-complimentary (sc) genomes in both murine nonhuman primate livers. Superior murine liver transduction by sc over the traditional single stranded (ss) vector genomes was confirmed for all serotypes examined. Stable levels of transgene expression were achieved in cynomolgus macaques with efficiencies at least 2 logs higher than what was achieved with AAV2 vectors using ss genomes. In an attempt to understand molecular mechanisms responsible for the higher level gene transfer, we performed a series of detailed molecular studies of the vector genomes. This included treatment with exonuclease with specific substrate preferences, single cutter restriction enzyme digestion mediated and locus specific hybridization based vector genome mapping, bacteriophage Phi29 DNA polymerase mediated double stranded circular template specific isothermal rolling circle linear amplification for rescue, cloning and characterization of persisted circular genomes, and differential quantification of different populations of vector genomes by real time PCR. Our data indicated that the higher level of transgene expression achieved with the sc vectors was due to both increased numbers of persisting genomes and increased activity of these genomes. Study of the molecular dynamics of vector genome processing in murine liver revealed that both ss and sc vectors persisted as episomal circular forms but sc vectors were more quickly and more efficiently converted into circular forms. The structure and formation of concatemers were similar with the types of genomes. The molecular structures of AAV genomes in macaques were more heterogeneous consisting of primarily circular monomer and concatemers in different configurations, and some unexpected molecular entities. More extensive rearrangements and deletions were observed in macaque than in mouse. It will be useful to further understand differences in the molecular processing of AAV genomes in mice and macaques as human applications are being considered.