Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle

Abstract

Recombinant adeno-associated virus (rAAV) vectors are promising human gene transfer vectors, because they mediate long-term gene expression in vivo. The vector DNA form responsible for sustained gene expression has not been clearly defined, but it has been presumed that the vector integrates to some degree and persists in this manner. Using two independent methods, we were unable to identify rAAV integrants in mouse muscle. In the first approach, we were unable to recover host cell-vector DNA junctions from a lambda phage library generated using transduced mouse muscle DNA that contained a high vector copy number. Following this result, we devised a PCR assay based on the principle that integrated rAAV vector sequences could be amplified using primers specific for mouse interspersed repetitive sequences (B1 elements). Using this assay, we analyzed transduced mouse muscle DNA isolated from 6 to 57 weeks after injection and did not detect amplification above background levels. Based on the demonstrated sensitivity of the assay, these results suggested that >99.5% of vector DNA was not integrated. Additional analyses using a novel DNA exonuclease showed that the majority of the rAAV vector DNA in muscle persisted over time as transcriptionally active monomeric and concatameric episomes.

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice

“…For the lentiviral vector the mean number of integration events should be similar to the multiplicities of infection. For rAAV vectors, an approximate integration frequency of 0.1% has been reported in the literature, 30,33,45 which suggests 0.9 and 0.09 integrations per cell for rAAV.CMV.hrGFP and rAAV.hRPE65p.hRPE65, respectively. We detected no histological evidence of intraocular malignancy in any of the vector-injected p53-deficient mice.…”

“…Although the wild-type virus efficiently integrates in a specific location in the human genome (AAVS1 on chromosome 19), 29 integration of rAAV genomes is typically passive, of low frequency and largely random. [30][31][32] The vast majority of rAAV vector genomes remain as episomes in the host cell nucleus, with only an estimated 0.5% of genomes integrating into the host genome, 33 probably at pre-existing chromosomal breaks. 34 However, as doses ranging from 10 11 to 10 12 virus particles may be administered to the human eye, 15,35 this small fraction of integrating vector genomes potentially equates to a substantial number of integration events.…”

Long-term persistence of gene expression from adeno-associated virus serotype 5 in the mouse airways

“…With regard to the persistence of viral genome DNA, there is a growing acceptance that rAAV genomes have lost the ability of wild-type AAV genomes to efficiently integrate in a sitespecific manner, into host cell chromosomal DNA after in vivo gene transfer. 44 Instead, it appears that in mouse and non-human primate tissues, rAAV genomes tend to form extrachromosomal concatemers [45][46][47] and that the presence of head-to-tail ITRs may be one mechanism for persistence of these concatemers. It would be interesting to investigate whether a similar profile of head-to-tail concatemers is present in rAAV5/5-treated respiratory tract samples.…”

Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping

“…8,22 Among the rAAV serotypes analyzed to date, rAAV1 and rAAV8 are among the most efficient for muscle transduction. 8,[23][24][25][26] After IM administration, it was demonstrated that rAAV DNA resides as episomal circles [27][28][29] in a chromatin structure; 30 and from a biosafety perspective, the inefficient integration of rAAV into the host genome is an attractive feature for the legitimate use of this gene transfer system.…”