Abstract
Top of pageAbstract We reported last year that double-stranded (ds) linear rAAV vector genomes with a |[ldquo]|dog bone|[rdquo]| structure (i.e., a dumbbell-shaped ds rAAV genome with both terminal hairpin loops closed) emerged in mouse hepatocytes in the absence of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs, i.e., scid phenotype) but not in the presence of DNA-PKcs (i.e., wild type) when a rAAV8 vector was injected via the tail vein at a very high dose, 7.2 |[times]| 10e12 vector genome (vg) per mouse. We and others have shown that such an altered fate of rAAV vector genomes in murine liver was not observed when rAAV2 vectors were injected at a regular dose. Based on this observation, we have hypothesized that 1) AAV-ITRs are recognized by cells as |[ldquo]|closed|[rdquo]| hairpins whether or not the ITRs have a 5' or 3' DNA end (i.e., |[ldquo]|open|[rdquo]| ITRs as opposed to the |[ldquo]|closed|[rdquo]|), and 2) hairpin opening of |[ldquo]|closed|[rdquo]| ITRs by a specific cellular endonuclease(s) is the very first step that triggers all vector genome recombinations toward the formation of ds circular monomer genomes and concatemers, and vector genome integration into chromosomal DNA in vivo. If AAV-ITR hairpins are not resolved, 5' and 3' DNA ends in vector genomes are ligated with each other intramolecularly by a cellular ligase activity, forming the |[ldquo]|dog bone|[rdquo]| structure, and further intermolecular recombinations through the ITRs would not occur. In the present study, we further investigated the |[ldquo]|dog bone|[rdquo]| genomes as a tool to understand the mechanisms of AAV-ITR hairpin opening and rAAV vector genome recombinations in mice. Consequently, we have elucidated the following: (1) |[ldquo]|dog bone|[rdquo]| genomes emerged in scid liver only when rAAV8 vectors were injected at doses of 1.8 |[times]| 10e12 or higher, at which vector copy numbers in the livers were > 800 ds-vg/dge (ds vector genomes per diploid genomic equivalent); (2) rAAV vectors delivered to scid liver by rAAV2 vectors never resulted in accumulation of |[ldquo]|dog bone|[rdquo]| genomes no matter how much vectors were injected; (3) in non-hepatic scid tissues such as heart, muscle and kidney, |[ldquo]|dog bone|[rdquo]| genomes accumulated at a much lower vector genome load (|[sim]|2 ds-vg/dge); (4) hepatocyte cell cycling recruited a supplementary AAV- ITR hairpin opening activity that did not exist in a quiescent state, facilitating vector genome recombinations through the AAV-ITRs. These observations demonstrate that AAV-ITR hairpin opening in non-hepatic tissues largely depends on the DNA-PKcs-associated cellular endonuclease activity, while in the liver, the pathways of AAV-ITR hairpin opening are redundant and cell cycle dependent. Based on the recent discovery of DNA-PKcs/Artemis pathway for DNA double-strand break repair together with our observations, we propose that a cellular endonuclease, presumably Artemis, plays a crucial role in AAV-ITR hairpin opening and subsequent vector genome recombinations in concert with DNA-PKcs. We are currently investigating the role of Artemis in this process using Artemis deficient cells.
Published Version
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