AAV2-CMV-FLuc

Some genetic diseases are caused by relatively small numbers of nucleotide changes, insertions, or deletions. In these cases, approaches involving modification of a few nucleotides may be preferable to replacing or adding an entire gene. A number of in vitro experiments have achieved site-specific rAAV2 recombination in up to 1% of cultured cells, compared with an overall integration frequency of 10%. Preliminary experiments targeted selectable reporter genes inserted throughout the genome of different clones, each with a different type of mutation. In these studies, a rAAV vector genome was generated that was homologous to a portion of the target gene, differing only in the nucleotides to be modified, inserted, or deleted. Using this approach, mutation insertions of up to 14 bp, deletions of up to 4 bp, and substitutions of up to 2 bp were successfully corrected. Deletions appear to be corrected more efficiently than insertion mutations. In nearly all successful examples of gene targeting, the sequences flanking the corrected mutation matched the expected functional sequence. Deletions and insertions commonly seen at rAAV integration sites were not generally observed in these gene targeting events. In addition to corrections of approximately 15 bp, sequences up to 1.6 kb have also been successfully site-specifically inserted. The targeting rate increased with increasing homology to the target sequence, increasing vector dose, and increasing time point after infection. In addition, the targeting frequency was higher when the vector's proofreading nucleotide was closer to the center of the rAAV sequence than when the proofreading nucleotide was closer to the end of the vector genome.