AAV1-CAG-Luc

The wild-type AAV genome consists of a linear single-stranded DNA (ssDNA) molecule of approximately 4.7 kilobases, which includes coding sequences for the Rep and Cap proteins, flanked by inverted terminal repeats (ITRs). Since the ITRs contain cis-acting sequences required for replication and packaging, the wild-type genome can be “de-removed” by removing all viral coding sequences and adapted for gene delivery by placing a heterologous DNA sequence of choice between the flanking ITRs. These heterologous sequences can encode conventional expression cassettes to express a selected transgene in target cells or encode templates for targeted genome editing by homologous recombination. Replication and assembly of recombinant AAV (rAAV) virions, either for gene addition or gene targeting, are then enabled by providing trans-acting AAV Rep and Cap proteins and the necessary adenoviral helper functions.

After molecular transduction into target cells, the single-stranded recombinant AAV genome must be converted to a double-stranded transcriptionally active form to facilitate transgene expression (functional transduction). Transcriptionally competent double-stranded DNA is formed by annealing of sense and antisense strands in cells transduced at high multiplicity, or by second strand synthesis. This process requires host cell activity and is the rate-limiting step for functional transduction in some cell types. In cells transduced by rAAV, vector genomes are maintained predominantly as episomal circular monomers and concatemers, but can also undergo random genomic integration. In actively replicating target cell populations, episomal vector genomes are rapidly lost leaving stable long-term expression dependent on the subset of genomes that have undergone genomic integration.