Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

Abstract

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

“…Studies originally demonstrated targeted integration through Southern blot analysis, fluorescence in situ hybridization (FISH), and AAVS1-specific PCR (2,9,10). Two studies have used low-throughput genomic approaches, involving enzyme digestion, ligation-mediated PCR, and cloning, to investigate AAV integration (28,29). One study was unable to detect any integrants in AAVS1 (28).…”

“…One study was unable to detect any integrants in AAVS1 (28). The other study found that AAVS1 integrations in exon 1 of PPP1R12C represented less than one percent of events, while integration in the general vicinity (within 100 kb) of AAVS1 accounted for less than 10 percent (29). Efforts to apply computational techniques to AAV integration have been limited by the small and biased data pools, which preclude thorough bioinformatics (29).…”

Intégration des vecteurs AAV et mutagenèse insertionnelle

“…En effet, l'intégration de l'AAV sauvage peut avoir lieu dans une zone très étendue, en amont et en aval du site AAVS1, et entraîner des modifications profondes du génome cellulaire (délétions, duplications) ainsi que des délétions des extrémi-tés du génome viral. Il est ainsi impossible de connaître, a priori, les séquences virales présentes au niveau des jonctions avec l'ADN Figure 3A) [20][21][22]. Cela s'explique par la présence dans le génome cellulaire de nombreux sites RBS reconnus par les protéines Rep de l'AAV.…”

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1

“…The RBS is made up of multiple repeats of the tetranucleotide sequence 5Ј-GCTC-3Ј or small variations of this sequence. Interestingly, most of the AAV serotypes have three contiguous repeats flanked by pseudo-repeats, a feature that is shared with AAVS1 (18). The site-specific integration process is contingent on the presence of the large regulatory proteins Rep78/Rep68, the AAVS1 site, and a cis-acting viral DNA sequence (1,17,19,20).…”