Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.     

Concatamerization of Adeno-Associated Virus Circular Genomes Occurs through Intermolecular Recombination

Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.     

Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue

Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a approximately 50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual trans-splicing lacZ vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of trans-splicing-mediated beta-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the trans-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.     

Host cell DNA repair pathways in adeno-associated viral genome processing

Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PK(CS), but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.     

Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle

Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression following administration to skeletal muscle. In rodent muscle, the vector genomes persist in the nucleus in concatemeric episomal forms. Here, we demonstrate with nonhuman primates that rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside predominantly as episomal monomeric and concatemeric circles. The episomal rAAV genomes assimilate into chromatin with a typical nucleosomal pattern. The persistence of the vector genomes and gene expression for years in quiescent tissues suggests that a bona fide chromatin structure is important for episomal maintenance and transgene expression. These findings were obtained from primate muscles transduced with rAAV1 and rAAV8 vectors for up to 22 months after intramuscular delivery of 5 × 10¹² viral genomes/kg. Because of this unique context, our data, which provide important insight into in situ vector biology, are highly relevant from a clinical standpoint.     

Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.     

Characterization of adeno-associated virus genomes isolated from human tissues

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.     

Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 x 10(10) to 1.8 x 10(12) particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in approximately 80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.     

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.     

A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy

Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.     

Role for highly regulated rep gene expression in adeno-associated virus vector production.

Recent success achieving long-term in vivo gene transfer without a significant immune response by using adeno-associated virus (AAV) vectors (X. Xiao, J. Li, and R. J. Samulski, J. Virol. 70:8098-8108, 1996) has encouraged further development of this vector for human gene therapy. Currently, studies focus on the generation of high-titer vectors by using the two-plasmid helper-vector system in adenovirus (Ad)-infected cells. To examine the effects of the AAV replication (rep) genes on recombinant AAV (rAAV) vector production, we have constructed a series of AAV helper plasmids that contain strong heterologous promoters in place of the endogenous p5 promoter. Although high-level rep gene expression was achieved, rAAV DNA failed to replicate in the absence of Ad infection. Moreover, unregulated overexpression of Rep78/68 led to substantially lower rAAV yields in the presence of Ad (10(4-5) versus 10(7-8)). In contrast, under similar conditions, reduced Rep78/68 expression resulted in much higher rAAV yields (10(9)). Molecular characterization showed that overexpression of the rep gene decreased rAAV DNA replication and severely inhibited capsid (cap) gene expression. Interestingly, a reduced rep level enhanced cap gene expression and supported normal rAAV DNA replication. These studies suggest a critical role for regulated rep gene expression in rAAV production and have facilitated the development of a new AAV helper plasmid that increases vector production eightfold over currently used constructs.     

A helper virus-free packaging system for recombinant adeno-associated virus vectors

Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected onto human 293 cells with a vector plasmid in the absence of Ad infection, the rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.     

The SSV-Seq 2.0 PCR-Free Method Improves the Sequencing of Adeno-Associated Viral Vector Genomes Containing GC-Rich Regions and Homopolymers

Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.     

Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.     

Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.     

Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome.

A model system using an episomal Epstein-Barr virus shuttle vector was recently developed to study the adeno-associated virus (AAV) site-specific integration event in chromosome 19q13.3-qter (C. Giraud, E. Winocour, and K.I. Berns, Proc. Natl. Acad. Sci. USA 91:10039-10043, 1994). In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (AAVS1 locus). In most of the recombinants, one end of the viral genome was joined to a portion of the AAVS1 DNA previously shown to be a minimum target for AAV integration. Within this AAVS1 segment, the AAV insertion points were strikingly clustered around a binding site for the AAV regulatory protein. In all cases, the second junction with AAV occurred with vector DNA outside of the AAVS1 segment. With respect to the viral genome, one junction with the shuttle vector DNA occurred either within the AAV inverted terminal repeat (itr), or near the P5 promoter, approximately 100 nucleotides distal to a modified itr. The modified itr in 5 of 11 recombinants involved a head-to-tail organization. In one such instance, the AAV insert contained slightly more than one genome equivalent arranged in a head-to-tail manner with a junction close to the P5 promoter; the AAV insert in this recombinant episome could be rescued by adenovirus infection and replicated to virus particles. The significance of the head-to-tail organization is discussed in terms of the possible circularization of AAV DNA before or during integration.     

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.     

Recombinant adeno-associated viral vectors are deficient in provoking a DNA damage response

Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UV-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis-acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.