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.

Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.Adeno-associated virus (AAV) has evolved a biphasic life cycle to ensure persistence in its primate host. It needs an unrelated helper virus, adenovirus (Ad) or herpesvirus, for productive infection (1). In the absence of a helper, AAV-2 establishes latency by preferential integration into a specific site on human chromosome 19 (chr-19) (19q13.3-qter) called AAVS1 (10). The site specificity of AAV integration is mediated by the AAV Rep78 protein or by its C-terminally spliced variant Rep68 (1, 12). During productive AAV replication, Rep78 or Rep 68 is needed for AAV gene expression and DNA replication. The AAV origins of DNA replication reside in the 145-bp inverted terminal repeats (ITRs) that flank the 4.7-kb single-stranded AAV genome. Rep78 and/or Rep 68 bind to the Rep-binding site (RBS) on the AAV ITR (22) and nick and unwind the ITR at the terminal resolution site (trs) (8). The AAV ITRs also serve as cis elements for efficient chromosomal integration (1, 12). Binding of Rep to the ITR is required for site-specific integration (27), whereas nicking of the trs is not essential (31). DNA sequences homologous to RBS and the trs of the AAV ITR are found in the chr-19 preintegration site (9,21). In vitro studies demonstrated ternary complex formation of Rep68 with the AAV ITR and chr-19 AAVS1 (27). A 33-bp sequence spanning the chr-19 RBS and the trs homology element is sufficient to mediate site-specific AAV integration in vivo. Either of the elements and proper spacing between them are essential (13, 15).Mapped chr-19 integration sites have been derived from cloned cell lines generated with and without drug selection. The integration sites are highly variable within a range of a few hundred base pairs from the RBS of AAVS1 (9,16,17,21,24,29). Sequence rearrangements are prominent features. It is unclear whether these have evolved during clonal selection. The only system in which integration sequences have been analyzed early after AAV-2 infection is integration into AAVS1 carried by Epstein-...

Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats.

Seroepidemiology shows that infections with adeno-associated virus (AAV) are widespread, but diverse AAV serotypes isolated from humans or nonhuman primates have so far not been proven to be causes of human disease. In view of the increasing success of AAV-derived vectors in human gene therapy, definition of the in vivo sites of wild-type AAV persistence and the clinical consequences of its reactivation is becoming increasingly urgent. Here, we identify the presumed cell type for AAV persistence in the human host by highly sensitive AAV PCRs developed for the full spectrum of human AAV serotypes. In genomic-DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV positive, predominantly AAV type 2 (AAV2) (77%), AAV5 (19%), and additional serotypes. Roughly 11% of the blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, 76% of whom were AAV positive. Of these, at least 45% displayed mixed infections. Follow-up of single blood donors over 2 years allowed repeated detection of the initial and/or additional AAV serotypes, suggestive of fluctuating, persistent infection. Leukocyte separation revealed that AAV resided in CD3 ϩ T lymphocytes, perceived as the putative in vivo site of AAV persistence. Moreover, infectious AAVs of various serotypes could be rescued and propagated from numerous samples. The high prevalence and broad spectrum of human AAVs in leukocytes closely follow AAV seroepidemiology. Immunosuppression obviously enhances AAV replication in parallel with activation of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), reminiscent of herpesvirus-induced AAV activation.IMPORTANCE Adeno-associated virus is viewed as apathogenic and replication defective, requiring coinfection with adenovirus or herpesvirus for productive infection. In vivo persistence of a defective virus requires latency in specialized cell types to escape the host immune response until viral spread becomes possible. Reactivation from latency can be induced by diverse stimuli, including infections, typically induced upon host immunosuppression. We show for the first time that infectious AAV is highly prevalent in human leukocytes, specifically T lymphocytes, and that AAV is strongly amplified upon immunosuppression, along with reactivation of latent human herpesviruses. In the absence of an animal model to study the AAV life cycle, our findings in the human host will advance the understanding of AAV latency, reactivation, and in vivo pathogenesis.

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.Adeno-associated virus (AAV) has evolved a biphasic life cycle to ensure persistence in its primate host. It needs an unrelated helper virus, adenovirus (Ad), or herpesvirus for productive infection (16). In the absence of a helper, AAV establishes latency by preferential integration into a specific site on human chromosome 19 (chr.19), 19q13.3q-ter, called AAVS1 (10). AAV type 2 (AAV-2) contains a linear singlestranded DNA genome of 4.7 kb that covers the genes rep and cap, flanked by 145-bp inverted terminal repeats (ITRs) (27) that serve as origins of replication and as cis elements for chromosomal integration. The AAV regulatory proteins Rep78 and/or Rep68 (Rep78/68) are necessary for AAV DNA replication both in vivo and in vitro. Rep78/68 binds to the Rep-binding site (RBS) on the AAV-ITR (26) and nicks and unwinds the ITR at the terminal resolution site (trs) (7). Rep78/68 also mediates site-specific AAV integration at AAVS1 on chr.19 that comprises DNA sequences homologous to the RBS and the trs of the AAV-ITR (9, 23). In vitro studies showed ternary complex formation of Rep68 with the AAV-ITR and AAVS1 (31) and Rep68-mediated initiation of DNA replication starting from an origin sequence comprised of AAVS1 (29). A 33-bp sequence spanning the chr.19 RBS, a spacer sequence, and a trs homology element are sufficient to mediate site-specific AAV integration in vivo (12,14). chr.19 integration sites have been analyzed either in selected cell clones or in 293 cells carrying AAVS1 on an Epstein-Barr virus-based episome (1,2,9,17,20,23,28,32). To analyze integration into the authentic chr.19 preintegration locus early after AAV-2 infection, we have recently developed a sensitive and quantitative real-time PCR assay for AAV-ITR/AAVS1 junctions. Within 4 days postinfection (p.i.) site-specific integration frequency reached 10 to 20% of unselected HeLa cells.Further analysis showed that at least 1 in 1,000 infectious AAV-2 integrated site specifically (5, 6). MATERIALS AND METHODS Plasmids.The following plasmids have been described: pTAV2-0 (4), pDG (3), pAAVS1-TR (6), and psub201 (22). Plasmid psubgfpneo was generated by inserting the gfpneo cassette derived from pTR-UF5 (36) between the 19...

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus, AAVS1, on human chromosome 19 (chr19). To study the kinetics and frequency of chr19-specific integration, a rapid, sensitive and quantitative real-time PCR assay specific for AAV inverted terminal repeat (ITR)-chr19 junction sequences was developed. Since the assay only detected right-hand AAV ITR-specific integration events, the development of a complementary left-hand ITR-specific real-time PCR assay is described. The time-course of left-hand ITR-dependent AAV integration at AAVS1 of chr19 was determined in AAV-2-infected HeLa cells. Both the kinetics and frequencies of left-hand ITR-dependent integration were found to be similar to those of the right-hand ITR. In addition, left-hand ITR-specific fusion sequences and chromosomal breakpoints within AAVS1 were variable, yet were the same as those found in right-hand ITR-chr19 junction sequences. Thus, the AAV-2 genome integrates site-specifically into chr19 with similar efficiency in either orientation.

To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0–1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host’s chromosomes.

Recombinant adeno-associated virus (rAAV) has become the most widely used vector in the gene therapy field with hundreds of clinical trials ongoing and already several products on the market. AAV's physicochemical stability, and the various natural and engineered serotypes allow for targeting a broad range of cell types and tissue by diverse routes of administration. Progressing from early clinical studies to eventual market approval, many critical quality attributes have to be defined and reproducibly quantified, such as AAV stability, purity, aggregates, empty/full particles ratio, and rAAV genome titration. Droplet digital PCR (ddPCR) is becoming the tool of choice to perform absolute quantification of rAAV genomes. In the present study, we have identified critical parameters that could impact AAV titration and characterization accuracy, such as Poisson distribution confidence interval, primers/probe position, and potential aggregates. Our work presents how ddPCR can help to better characterize AAV vectors on the single particle level and highlights challenges that we are facing today in terms of AAV titration.