It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 x 10(11) particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9's delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

A subset of cellular DNA hairpins at double-strand breaks is processed by DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-and Artemis-associated endonuclease. DNA hairpin termini of adeno-associated virus (AAV) are processed by DNA repair machinery; however, how and what cellular factors are involved in the process remain elusive. Here, we show that DNA-PKcs and Artemis open AAV inverted terminal repeat (ITR) hairpin loops in a tissue-dependent manner. We investigated recombinant AAV (rAAV) genome metabolism in various tissues of DNA-PKcs-or Artemis-proficient or -deficient mice. In the absence of either factor, ITR hairpin opening was impaired, resulting in accumulation of double-stranded linear rAAV genomes capped with covalently closed hairpins at termini. The 5 end of 3-base hairpin loops of the ITR was the primary target for DNA-PKcs-and Artemis-mediated cleavage. In the muscle, heart, and kidney, DNA-PKcs-and Artemisdependent hairpin opening constituted a significant pathway, while in the liver, undefined alternative pathways effectively processed hairpins. In addition, our study revealed a Holliday junction resolvase-like activity in the liver that cleaved T-shaped ITR hairpin shoulders by making nicks at diametrically opposed sites. Thus, our approach furthers our understanding of not only rAAV biology but also fundamental DNA repair systems in various tissues of living animals.Adeno-associated virus (AAV) of various serotypes is a family of nonpathogenic replication-defective parvoviruses with a linear single-stranded DNA genome of approximately 5 kb. Recombinant AAV (rAAV) devoid of all the virally encoded genes is not only a promising viral vector for human gene therapy but also has recently emerged as a powerful tool for studying cellular biology, particularly DNA repair pathways (18). Upon infection in animals, rAAV genomes change their forms in infected cells by recombination through DNA hairpins at viral termini, named inverted terminal repeats (ITRs). DNA hairpins at AAV-ITR serve as a platform for all the rAAV genome recombinations, including self circularization (8), concatemerization (37), and integration (29). These processes are presumed to be mediated solely by cellular DNA repair machinery. Therefore, studying the interactions between viral DNA hairpins and host cellular DNA repair machinery will not only enhance our understanding of the biology of rAAV vectors, which is essential for successful human gene therapy, but also will provide important clues to elucidating fundamental cellular processes of DNA damage responses.DNA hairpin structures in mammalian cells are often a target for cellular DNA repair machinery. The best example is hairpin-coding ends that emerge during the V(D)J recombination, a physiological and programmed DNA double-strand break (DSB) that occurs in developing B and T lymphocytes (23,46). DNA hairpins at pathological DSBs caused by intrinsic and extrinsic sources such as reactive oxygen species and ionizing radiation also are processed by DNA repair machine...

No abstract

However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of տ20 bp (total length, տ40 bp) are a significant target for rAAV integration. Up to ϳ30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of տ20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.