Presence of a trs -Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration

Abstract

Wild-type adeno-associated virus type 2 (wtAAV2) integration capabilities are unique as the virus exhibits a strong preference for integrating its genome in a defined locus on human chromosome 19, termed AAVS1 (1, 2). PCR-based technologies such as linear amplification-mediated PCR (LAM-PCR) (3-5) and linker selection-mediated PCR in combination with highthroughput sequencing now allow for the establishment of comprehensive integration profiles of integrating viruses, including AAV. Recent studies have shown a strong preference for wtAAV2 to integrate into AAVS1, although additional clusters of integration sites (ISs) were found throughout the cellular genome (6-8). AAVS1 is characterized by the presence of a Rep binding site (RBS), containing GAGC repeats and a terminal resolution site (trs), which together resemble the origin of replication of the virus and are necessary and sufficient for site-specific integration of AAV (9-12). The viral replication protein, Rep, targets the AAV genome for integration at AAVS1 by initiating replication at this chromosomal origin. This is achieved by binding of Rep to the RBS (11), after which it executes a strand-and site-specific nick after the first thymidine of the trs, which leaves a free 3= end from which replication can be initiated (13,14). The aforementioned large-scale integration studies uncovered that integration hot spots are strongly associated with RBS and prefer genomic regions with high chromosome accessibility. However, unlike AAVS1, no correlations of integration to the canonical trs (GGTTGG) or derivatives were found, which raised the question of whether all wtAAV2 integration events do only occur via the replicative mechanism described for integration into AAVS1. To address this, we performed integration analyses based on linear amplification-mediated PCR (LAM-PCR) and nonrestrictive LAM-PCR (nrLAM-PCR (3-5) on wtAAV2-infected HeLa cells and human dermal fibroblasts (HDFs) and analyzed the most abundant integration hot spots for Rep-mediated binding and target site nicking.Duplicates of HeLa cells (3 ϫ 10 7 cells total) and HDFs (7 ϫ 10 6 cells total) were infected with wtAAV2 at a multiplicity of infection of 10 4 genome-containing particles per cell and passaged 5 times to dilute out episomal concatemers. In the conventional LAM-PCR approach, 4 different restriction enzymes were used to retrieve ISs: MluCI, Taq ␣ I, MseI, and CviQI. PCRs were performed using linker-specific primers and primers binding to the 3= and 5= ends of the viral genome. Bioinformatic data mining of LAM-PCR and nrLAM-PCR sequences was performed using HISAP (15). We analyzed a total of 255,772 454 GS FLX (Roche Diagnostics) and 10,398,424 MiSeq (Illumina) sequencing reads derived from LAM-PCR products, thereby determining a total of 4,340 unique ISs (HeLa, 3,060 ISs; HDFs, 1,280 ISs) (Fig. 1A). By sequencing through the vector-genome junction, these ISs could be mapped with high nucleotide resolution. In addition to the canonical AAVS1 locus, we identified 275 other IS clusters, te...

Adeno-associated Vector Toxicity—To Be or Not to Be?

“…7ZR UHFHQW SXEOLFDWLRQV KDYH FRQ¿UPHG SUHIHUHQFH IRU $$9 to integrate into the AAVS1 region on chromosome 19, which is caused by the accumulation of Rep binding sites and terminal resolution site (trs)-like motifs within this region. 9,10 It is thus surprising that no AAV sequences were found within the AAVS1 region in either the HCCs or the matched nonmalignant tissue, 2 even though wild-type AAV has an active Rep integrase targeting this site.…”

Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface

“…This process is necessary for resolution of the ITRs and completion of the viral DNA replication cycle with the assistance of the host cell machinery ( 12 , 13 ). Similarly, the DNA binding and endonuclease activities are required for mediation of integration at chromosomal target loci that contain RBS/ trs , such as the integration hot spot AAVS1 ( 14 – 16 ). In addition, efficient nicking of the trs at both the viral and cellular origins requires ATP-dependent helicase activity for the generation of an optimal single-stranded substrate ( 7 , 17 ).…”

Reply to “Wild-type AAV Insertions in Hepatocellular Carcinoma Do Not Inform Debate Over Genotoxicity Risk of Vectorized AAV”

“…They differed with respect to the frequency of integration into AAVS1 that was determined (2.5% and 8.9%, respectively), but nevertheless spot. 13,14 Thus, although we acknowledge that one has to assume not only methodological variations in integration site retrieval but also cell-intrinsic factors that may bias integration site distribution of a single wtAAV integration in AAVS1.…”