Adeno-associated virus vector integration junctions

Abstract

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.

Infectious Clones and Vectors Derived from Adeno-Associated Virus (AAV) Serotypes Other Than AAV Type 2

“…Wild-type virus stocks of AAV1, AAV3, and AAV6 used for cloning and sequencing were prepared by infecting 293 cells with the AAV isolate and adenovirus, freeze-thawing infected cell lysates 3 days later, and pelleting the cell debris by centrifugation at 5,800 ϫ g for 30 min. The crude lysates were concentrated by pelleting through sucrose, and the AAV6 stock was further purified on a cesium chloride gradient as described previously (48). AAV1 (ATCC VR-645) and AAV3 (ATCC VR-681) were obtained from ATCC, and AAV6 was obtained from a laboratory adenovirus type 5 stock after wild-type AAV was identified in vector stocks prepared with this adenovirus helper.…”

“…AAV vector stocks were prepared from 293 or 293T cells and purified on CsCl gradients as described previously (48). pA2LAPSN with pRepCap2, pA3LAPSN with pRepCap3, and pA6LAPSN with pRepCap6 were used to produce AAV2-LAPSN, AAV3-LAPSN, and AAV6-LAPSN, respectively.…”

RAD52: Viral Friend or Foe?

“…This model is also consistent with the known mechanism of rAAV random integration. Because the viral ITRs are extensively altered with deletions and there is frequent microhomology at the chromosomal junctions, the insertion of the viral dsDNA replication intermediate into the genome is widely believed to occur through A-NHEJ [60,61]. Figure 2, RAD52 may facilitate, via a BIR-like mechanism, the replication of single-stranded AAV DNA into dsDNA.…”

Strategies for cancer gene therapy

“…They are capable of infecting dividing and non-dividing cells. AAV vector integration into the host genome can be site specific or random [70] and results in prolonged transgene expression [71]. In contrast to Ad vectors, AAV vectors do not cause a significant host inflammatory response [72].…”