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Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 × 10 11 vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a β-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least ∼0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was ∼0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 10 3 hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.

Adeno-associated virus (AAV) is a small nonpathogenic, replication-defective DNA virus with a single-stranded DNA genome. Recombinant AAV (rAAV) is among the most promising gene delivery vectors for human gene therapy (15, 28). rAAV lacks machinery for virus genome insertion into host chromosomal DNA but does integrate into chromosomes at a low frequency (reviewed in reference 29). Murine leukemia virus preferentially integrates near transcription start sites (59), while human immunodeficiency virus favors transcribed regions for integration (49). As for rAAV, previous studies have shown that rAAV serotype 2 (rAAV2) vector integration occurs preferentially in genes and near gene regulatory sequences in mouse liver (36, 40) and in cultured human cells (32). A recent observation by Miller et al. has suggested that rAAV does not cause chromosomal breaks but integrates at preexisting chromosomal breakage sites (30). Although rAAV vector integration frequency is considered to be low, it is important to further understand the interactions between rAAV vector and host chromosomal DNA in various tissues in experimental animals. This is because a study has demonstrated increased incidence of liver cancer in rAAV2 vector-treated animals (5), the mechanisms for which remain elusive. In addition, the use of new serotype vectors with robust transduction efficiency, such as rAAV8, can increase vector genome loads in cells, which may pose an increased risk of undesirable genomic alterations in rAAV-transduced cells. Such robust serotype vectors now are widely used for many preclinical studies for gene therapy of various human diseases. Moreover, we have hypothesized that elucidation of the mechanisms of rAAV integration may help in understanding ongoing genomic instability in living animals, which is difficult to investigate with currently available strategies but is important for studies on carcinogenesis and aging. Furthermore, it has been shown that rAAV vectors serve as a powerful tool to study the mechanisms of fundamental biological processes in cells, such as DNA damage responses and DNA repair (11). To begin to further understand rAAV integration in various tissues of experimental animals, it is essential to establish a system by which rAAV integration sites can be identified on a large scale in nondividing cells with high efficiency and reliability without any cell manipulations. This minimizes possible technical biases and enables identification of rAAV integrations in nonhepatic tissues, for which selection is not easy to perform. However, currently available methods for high-throughput rAAV integration site analysis all rely on cell division either under a selective pressure (40) or without selection (32). Cell division is required for diluting out extrachromosomal rAAV vector genomes with high complexity, which are abundantly present in cells in a quiescent state and inhibit efficient isolation of rAAV integrants. In the present study, we have established a novel high-throughput method to identify rAAV integration sites in nondividing cells with high efficiency and high reliability independently of cell division, transgene expression, or selection. We have identified a thousand rAAV integration sites in quiescent somatic cells in mouse liver, skeletal muscle, and heart, and we discovered that DNA palindromes are a common target for rAAV integration. DNA palindromes are found prevalently in many organisms, including mammals (24, 58; S. M. Lewis, T. Zheng, S. Chen, T. Alleyne, J. Cheung, T. Chiang, and R. Richard, unpublished data). They have gained attention recently due to accumulating evidence that they have roles in promoting genomic instability in eukaryotes. This DNA motif has been shown to be involved in gene amplification (2, 7, 43, 46, 52-54, 60), nonrandom chromosomal translocations causing human diseases (6, 9, 12, 16-20, 55), genomic instability in animal models (1, 3, 4, 22, 23), retrovirus integration (14), and RAG protein-mediated transposition (45). Thus, the discovery that rAAV integrates preferentially at DNA palindromes not only provides further insights into the mechanisms of rAAV integration but also provides an unprecedented opportunity to study the biological impact and properties of naturally occurring DNA palindromes in tissues of living animals.