1999;93:1875C1881. novel rabbit model, AAV-2 or AAV-8 sequences were detected in the semen of vasectomized animals that 4-IBP lack germ cells. Therefore, structures of the genitourinary (GU) tract, as well as the testis, contribute significantly to vector shedding in the semen. Collectively, data from these two models suggest that the risk of inadvertent germline transmission in males by AAV-8 vectors is low, similar to that of 4-IBP AAV-2, and that AAV dissemination to the semen is in part modulated by host-dependent factors. Introduction Early-phase clinical studies using recombinant adeno-associated viral (AAV) vectors are encouraging and provide the basis for the treatment of several genetic and acquired diseases. The therapeutic potential of Rabbit Polyclonal to GHRHR AAV serotype 2 (AAV-2) following local delivery to skeletal muscle or to the subretinal space is attested by the documented long-term local transgene expression and by evident improvement of the disease phenotype, respectively.1,2 However, the use of AAV for the treatment of some diseases will require intravascular delivery of the vector, which imposes additional safety concerns due to systemic vector dissemination. Following hepatic artery delivery of AAV-2 4-IBP for hemophilia B in humans, we demonstrated that the immune responses to the vector capsid and the risk of germline transmission are critical challenges to the safety of this strategy.3 The characterization of novel AAV vectors derived from alternate serotypes, the development of higher potency vectors derived from modifications in the AAV genomes, and the optimization of transgene expression or function4,5 support the likelihood of achieving efficient liver-directed gene expression by a simple peripheral intravenous injection. Thus, there is a fundamental interest in determining the risk of germline transmission as a key safety issue to support the use of these promising strategies in humans. In a previous work, we established a rabbit model to assess the risk of germline transmission by AAV-2 vector in males.6,7 We determined that the risk of vector dissemination to the semen was dependent on the route of vector administration; semen tested positive for vector sequences following intravascular delivery but not after intramuscular administration. Following intravascular delivery, vector sequences were transiently detected in semen and disappeared in dose- and time-dependent fashion. The kinetics of vector clearance was faster in the semen fractions enriched for motile sperm than in the total semen fractions. Long-term follow-up spanning hundreds of spermatogenesis cycles in 31 animals showed that there was no recurrence of detectable vector sequences in semen. Infectious vector particles were present only up to day 4 4-IBP postinjection, and were undetectable thereafter, which limited the risk of transmission of the vector. These data suggest that AAV-2 presents a low risk of germline transmission for humans. Moreover, the data agree with those of adult hemophilia male subjects enrolled in muscle- and liver-directed AAV-2 mediated human factor IX (= 8) or AAV-8 (= 17) vectors expressing hFIX under the control of a liver-specific promoter at two doses: 1 1012 vg/kg (low dose) or 1 1013 vg/kg (high dose). Injection 4-IBP of AAV vector was uneventful; there was no elevation of liver enzyme levels, which were monitored every week for 2 months after injection (data not shown). Circulating hFIX levels were initially detected at week 1, and reached plateau levels after week 10. In animals without inhibitory antibodies to the transgene, hFIX levels reached a therapeutic range of 6 and 12% (low dose), and 12 and 24% (high dose) of normal levels (5 g/ml) in the AAV-2 (= 7) and AAV-8 (= 13) cohorts, respectively (Figure 1). FIX levels in the AAV-8 injected rabbits were approximately twofold higher than those of AAV-2. However, because of the limited numbers of animals, this difference did not reach statistical significance. These data demonstrate that rabbit hepatocytes are efficiently transduced by both AAV-2 and AAV-8 vectors. Open in a separate window Figure 1 Plasma concentration of hFIX in experimental rabbits as a function of time after AAV injection. Rabbits received intravenous injection of AAV-2 or AAV-8 at doses of (a) 1 .
