Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors

We previously demonstrated that rAAV vectors carrying human and canine fact or IX (FIX) cDNA can infect, stably persist, and secrete functional human a nd canine FIX following direct intramuscular injection. In an attempt to im prove FIX protein secretion for eventual therapeutic use, we set out to det ermine if alteration of the AAV capsid would affect skeletal muscle transdu ction and factor IX secretion. Two reasons to pursue this question were (I) the persistence of high-titer neutralizing antibody (NAB) to AAV2 and (2) o ur previous study that supported a restricted tropism of muscle fiber types to AAV2 transduction. Using an identical CMV/canine factor IX (cFIX) expre ssion cassette, we cross-packaged this genome into virions generated from e ach of the five AAV serotypes. In a dose-response assay, equivalent amounts of rAAV/cFIX serotypes were tested in vitro and in vivo, In tissue culture cells, FIX antigen levels secreted into the supernatant varied depending o n the AAV serotype used; type 2 transduced maximally, with serotypes 3, 1, 5, and 4, respectively, expressing lower levels. However, when the same vir uses were tested in vivo using immunodeficient NOD/SCID animals, we obtaine d surprisingly different results. While the time to onset of detectable ser um levels appeared the same for all serotypes, types 1, 3, and 5 produced 1 00- to 1000-fold more cFIX than type 2. In fact, 12 weeks after transductio n, type 1 continued to express levels of cFIX on average at 80 mug/ml follo wed by type 5 (6.52 mug/ml), type 3 (3.27 mug/ml), type 4 (258 ng/ml), and finally type 2 (90 ng/ml). Coagulant activity of cFIX as measured by aPTT s upported the circulating levels measured by ELISA demonstrating the secrete d protein was functional, and RT-PCR of injected tissue correlated with the serotype-specific transduction data. In summary, we found significant diff erences in cFIX expression upon introducing various rAAV serotypes into mou se muscle. These data have direct bearing on the design of AAV gene therapy clinical trials for hemophilia and should also extend to most therapeutic transgenes.