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Recent studies have shown that recombinant adeno-associated virus (rAAV) can persist in episomal form; however, factors affecting rAAV persistence are poorly understood. DNA-dependent PK (DNA-PK) is a DNA repair enzyme, which we previously found played an important role in determining the molecular fate of the rAAV genome in mouse skeletal muscle. In the present study, we tested the effect of DNA-PK on AAV serotype 2 integration in vitro and in vivo in mouse liver. In an in vitro integration system, addition of DNA-PK decreased AAV integration, whereas antibody against DNA-PKcs increased integration. In vivo, matched doses of a recombinant AAV serotype 2 vector were injected into the portal vein of either C57BL͞6 (DNA-PKcs ؉/؉ ) or severe combined immunodeficient (DNA-PKcs ؊/؊ ) mice. After partial hepatectomy to stimulate hepatocyte proliferation, retention of vector genomes and of transgene expression was substantially higher in severe combined immunodeficient mice, indicating that in the absence of DNA-PKcs, a greater proportion of genomes integrated into the cellular genome. In summary, we have provided evidence that DNA-PK inhibits AAV integration both in vitro and in vivo.R ecombinant adeno-associated virus (rAAV) vectors have been increasingly used for gene therapy because they are relatively nontoxic and can mediate long-term transgene expression. Because this vector is used more frequently in clinical trials, it has become crucial to attain an improved understanding of its potential for insertional mutagenesis. Recent studies have shown that the majority of rAAV genomes persist in episomal form after in vivo delivery and that the hallmark of these episomal forms is the production of vector-to-vector junctions, resulting in either circular episomes or high molecular weight concatamers (1-3). Little is known about the cellular factors required for the maturation of rAAV DNA into these stable episomal forms. We previously demonstrated that in skeletal muscle of severe combined immunodeficient (SCID) [DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-negative] mice, some rAAV serotype 2 (rAAV2) genomes persist as linear episomes and then gradually integrate into the cellular genome, whereas in C57BL͞6 (DNA-PKcs-positive) mice, they form circular episomes (2). Most recently, Duan et al. (4) also have shown that SCID skeletal muscle retains both circular and linear forms of rAAV genomes, whereas C57BL͞6 muscle retains only circular forms of rAAV.The DNA-PK is composed of a DNA-binding Ku70͞Ku80 heterodimer and a large catalytic subunit (DNA-PKcs) and functions as a nuclear serine͞threonine protein kinase (5). The Ku protein was first identified as an autoantigen in patients with lupus. It is a heterodimer composed of two tightly associated subunits, Ku70 and Ku80, and is the most abundant DNA end-binding protein in mammalian cells. It recognizes a variety of DNA structures (blunt, overhanging, or hairpin) and binds with high affinity in a DNA sequence-independent manner. In the present studies, we sho...

A fundamental question about microbial pathogens is how they elicit host-specific symptoms. We report here that expression of a single gene from a plant-pathogenic bacterium in plant cells elicits host-specific symptoms diagnostic of the disease caused by the pathogen. Expression of pthA from Xanthomonas citri in citrus cells is sufficient to cause division, enlargement, and death of host cells. Since elicitation of these symptoms depends on a functional type III protein secretion system in X. citri, we deduce that the PthA protein is a specific plant signal, its site of action is inside the plant cell, and it is a major determinant of host range.

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