114. Revisiting rAAV Vector Integration in scid Mice: DNA-PKcs Deficiency Does Not Substantially Increase Integration Frequency in Hepatic and Non-Hepatic Tissues In Vivo

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Top of pageAbstract DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role in rAAV vector transduction. Recently, it has been reported that rAAV vector genome integration into chromosomes is substantially increased in scid mouse muscles and livers (PNAS 98: 4084, 2001; PNAS 101:2112, 2004). In scid livers, they observed that >40% of transgene expression and >50% of vector genomes remained after partial hepatectomy (PHx). However, we have not been able to confirm their observations. Rather we have found that liver regeneration is impaired and slowed in scid mice, particularly in older animals, resulting in insufficient dilution of episomal genomes after PHx. In order to revisit this subject, we have established a method that can directly quantify integrated vector genomes by Southern blot, and performed an experiment with a careful monitoring of liver regeneration after PHx. Since we found that animal age is a critical factor for liver regeneration in scid mice, we prepared relatively young age-matched 8-week-old C57BL/6 and C57BL/6 scid male mice. We injected these mice with AAV2 or 8-ISceI.AO3 vector as indicated in Table 1 (n=5|[ndash]|6 per group). The AAV-ISceI.AO3 vectors had an ISceI site in their vector genomes. With these vectors and ISceI digestion, we could clearly separate episomal and integrated vector genomes in transduced tissues, therefore quantify the amount of integrated vector genomes by Southern blot. 7 weeks after injection, 3 mice from each group underwent PHx, and all the mice were sacrificed 8 weeks after PHx. Careful liver weight measurement revealed that liver regeneration in scid mice was impaired by a factor of 0.88. Vector genome copy numbers at and 8 weeks after PHx are summarized in Table 1. The results demonstrate that vector genome copy numbers substantially dropped by 71|[ndash]|90% in scid mouse livers, and there was no significant difference in the reduction of vector genome copy numbers between B6 and scid mice. This sharply contrasts with the previous observations by Song et al. In addition, Southern blot analysis that can quantify integrated vector genomes revealed no detectable integrated vector genomes in any of the liver, skeletal muscle and kidney. Thus we conclude that scid phenotype does not substantially increase rAAV vector genome integration frequency at a detectable level in vivo.