1. ¹Department of Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA
2. ²Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA

Abstract

rAAV8 vectors transduce various tissues with extremely high efficiency in neonates and infants. Many applications of rAAV8 vector injection into prenatal or neonatal mice have been proposed to treat various disorders. Intraperitoneal or intravascular injection of rAAV8 vector into neonatal mice results in widespread efficient transduction in heart and skeletal muscles, which can stably persist even at adult ages. In contrast, the liver is transduced efficiently with rAAV8 only in the neonatal period, and liver transduction substantially declines thereafter. Despite the limited efficacy in neonatal liver transduction, it is important to understand the biology of rAAV8 vectors in the liver following neonatal administration because 1) the neonatal liver is an ideal target organ for gene transfer in many instances; and 2) substantial vector genome dissemination to the liver is likely to occur in non-hepatic gene transfer. In order to substantially understand the biology of rAAV8 injected into neonates, we non-injected or injected intraperitoneally C57BL/6 and 44Bri mice with AAV8-EF1|[alpha]|-nlslacZ vector at birth, and followed up total 69 animals. 44Bri is an HBV transgenic strain that develops chronic liver injury at |[thksim]|3 months of age, causing continuous liver regeneration. At the age of 110|[ndash]|179 days, the mice received AAV9-LSP (liver-specific promoter)-hFIX vector via the tail vein. 17 days post rAAV9 injection, half of the mice underwent 2/3 partial hepatectomy (PHx), and all the mice were sacrificed 6 weeks after PHx. The rAAV9 vector served as an internal control for PHx in rAAV8-injected animals. Xgal staining of the liver sections revealed sparsely scattered single blue hepatocytes and occasional clusters comprising 2|[ndash]|20 blue hepatocytes. Non-parenchymal blue cell foci were also found in many liver sections analyzed. Several relatively large blue hepatocyte clusters comprising of supposedly 10e2|[sim]|10e4 hepatocytes were found in one wild type and two 44Bri mice after PHx, suggesting that rAAV8-transduced cells in these foci might have had growth advantage over surrounding non-transduced hepatocytes. hFIX expression from the rAAV9 and rAAV9 genome copy numbers in the liver substantially dropped (over 90%) by PHx, while rAAV8 vector copy number drops were significantly smaller than those of rAAV9 vector (27-92% vector copy number drops for wild type and 73% drop-869% increase for 44Bri mice). This indicates that, although a significant portion of rAAV8 vector genomes were extrachromosomal, vector genomes were apparently integrated into chromosomes, with frequency of at most 2.7 vector genome copy/cell in our study. Thus, rAAV8 injected at birth integrates into chromosomes in the liver, stably transducing both parenchymal and non-parenchymal hepatic cells sparsely. In parallel to this experiment, we are following up 69 non-injected and 117 rAAV8-injected (at birth) wild type and 44Bri male mice over 400 days to investigate procarcinogenic potential of rAAV8 in mice. A preliminary result in this study will be also presented.