Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: Cell cycling does not influence hepatocyte transduction

Recombinant adeno-associated virus vectors (rAAV) show promise in preclinic al trials for the treatment of genetic diseases including hemophilia, Liver -directed gene transfer results in a slow rise in transgene expression, rea ching steady-state levels over a period of 5 weeks concomitant with the con version of the single-stranded rAAV molecules into high-molecular-weight co ncatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about similar to 5 % of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated, Whi le only similar to 5% of hepatocytes are stably transduced, in situ hybridi zation experiments demonstrated that the vast majority of the hepatocytes t ake up AAV-DNA genomes after portal vein infusion of the vector. Two differ ent vectors were infused together or staggered by 1, 3, or 5 weeks, and two -color fluorescent in situ hybridization and molecular analyses were perfor med 5 weeks after the infusion of the second vector, These experiments reve aled that a small but changing subpopulation of hepatocytes were permissive to stable transduction, Furthermore, in animals that received a single inf usion of two vectors, about one-third of the transduced cells contained het eroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through t he cell cycle was important for rAAV transduction, animals were continuousl y infused with 5'-bromo-2'-deoxyuridine (BrdU), starting at the time of adm inistration of a rAAV vector that expressed cytoplasmic beta-galactosidase, Colabeling for beta-galactosidase and BrdU revealed that there was no pref erence for transduction of cycling cells. This was further confirmed by dem onstrating no increase in rAAV transduction efficiencies in animals whose l ivers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes ta ke up vector, unknown cellular factors are required for stable transduction , and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.