Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues

Abstract

Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

DsAAV8-mediated expression of glucagon-like peptide-1 in pancreatic beta-cells ameliorates streptozotocin-induced diabetes

“…Furthermore, basal secretion of GLP-1 from beta-cells, as occurs with insulin, This targeted approach of GLP-1 production in beta-cells using AAV8 vector technology could potentially reduce the frequency of repeat administration required with current GLP-1-based therapies. While wild-type AAV is capable of integration into the human genome at a specific site on chromosome-19, designated AAVS1, 38,39,58 the vector used in this study was engineered to lack the Rep protein required for mediating this integration. Pancreatic beta-cells proliferate at a slow rate 59 and long-term episomal maintenance of transgenes delivered through AAV vectors has been demonstrated in slowly dividing cells.…”

Recombinant Adeno-Associated Viral Integration and Genotoxicity: Insights from Animal Models

“…8,9 This viral vector is suitable for both localized and systemic gene delivery and is capable of transducing a variety of cell types. To date, numerous naturally occurring AAV serotypes have been isolated from a number of different species, and novel capsids have been genetically engineered.…”

Existence of transient functional double-stranded DNA intermediates during recombinant AAV transduction

“…The circular intermediates of rAAV transduction have been studied extensively and were shown to be abundant after rAAV vector administration (24)(25)(26)(27)(28). Circular DNAs were eventually converted into high-molecular weight concatemers and were presumably stabilized.…”