504. In Vivo Characterization of AAV Serotype 9 Vectors in Mice

Abstract

Recombinant AAV(rAAV) vectors are promising vehicles for achieving stable transduction in vivo. We have demonstrated that, in contrast to rAAV2 vectors, which have limited transduction efficiency in many tissues, rAAV8 vectors can transduce all the hepatocytes, cardiomyocytes and skeletal myofibers throughout the body, and a majority of pancreatic acinar cells by peripheral vein injection at a high dose, in mice. AAV serotype 9 has been recently isolated from non-human primate tissues and recombinant vectors based on this serotype have been shown to transduce various tissues with high efficiency. In order to characterize the behavior of rAAV9 vectors in mice, we injected mice with various rAAV9 vectors via several different routes and analyzed transgene expression and tissue distribution. First, we produced a liver specific promoter-driven human factor IX (hFIX)-expressing rAAV9 vector, AAV9-hFIX16, and injected adult C57BL/6 male and female mice via 4 different routes; i.e., portal vein (PV), tail vein (TV), intraperitoneal (IP) and subcutaneous (SC) injections at a dose of 8.0|[times]|10e10 vg/mouse. For comparison, we also injected mice with AAV8-hFIX16 at the same dose. Liver transduction efficiency was monitored by measuring hFIX levels in the plasma. Two weeks post-injection, PV injection with the rAAV9 vector (9PV), 9TV and 9IP achieved comparable hFIX levels of 350-400 |[mu]|g/ml, which was slightly higher than the mice injected with the rAAV8 vector via PV (8PV). SC injection achieved approximately half of the levels with both AAV9 and AAV8 vectors, but still expressed over 100 |[mu]|g/ml of hFIX in plasma at this dose. Liver transduction in females was lower than males by |[sim]|30% with both serotypes as seen with rAAV2 vectors in this mouse strain. Interestingly, TV and IP injections of the rAAV9 vector showed slower kinetics of hFIX expression compared to the PV injection, even though any of these routes achieved a comparable level of expression 2 weeks-post injection. Such distinct expression kinetics depending on the route of vector administration was not observed with rAAV8 vector. In the second experiment, we injected C57BL/6 mice with rAAV9 vectors expressing a lacZ marker gene; i.e., AAV9-EF1|[alpha]|-nlslacZ or AAV9-CMV-lacZ via TV at two different doses, 3.0|[times]|10e11 and 1.8|[times]|10e12 vg/mouse (n=2 per group, total 8 mice for 4 groups). Development of anti-|[acirc]| galactosidase antibody was monitored by ELISA. The antibody was not detected in any of the 8 mice 7 days post-injection, but detected in 3 of 4 mice injected with AAV9-CMV-lacZ 10 days post-injection, when all the 8 mice were sacrificed for tissue distribution analysis. Liver transduction efficiency with the CMV vector was 3% at 3.0|[times]|10e11 and reached 90% at 1.8|[times]|10e12 vg/mouse. The tissue distribution pattern with rAAV9 vectors was similar to that with rAAV8 vectors, in that, in addition to the liver, they transduced heart, skeletal muscle and pancreatic acinar cells with high efficiency. Of note is that rAAV9 had greater transduction efficiency in the heart than rAAV8, and 3.0|[times]|10e11 vg/mouse was enough to transduce all the cardiomyocytes via TV injection of rAAV9. Thus, rAAV9, as well as rAAV8, is a robust vector for gene therapy applications.