Host and Vector-dependent Effects on the Risk of Germline Transmission of AAV Vectors

Abstract

The assessment of the risk of germline transmission of vector-coded sequences is critical for clinical translation of gene transfer strategies. We used rabbit models to analyze the risk of germline transmission of adeno-associated viral (AAV) vectors. Intravenous injection of AAV-2 or AAV-8 resulted in liver-mediated, long-term expression of therapeutic levels of human factor IX (hFIX) in a dose-dependent manner. In high-dose cohorts, AAV-8 resulted in twofold higher levels of circulating hFIX and of vector DNA in liver compared to AAV-2. Vector sequences were found in the semen of all rabbits. The kinetics of vector clearance from semen was dose- and time-dependent but serotype-independent. No late recurrence of AAV-8 sequences was found in the semen over several consecutive cycles of spermatogenesis. In a novel rabbit model, AAV-2 or AAV-8 sequences were detected in the semen of vasectomized animals that 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 AAV-2, and that AAV dissemination to the semen is in part modulated by host-dependent factors.

Safety of Liver Gene Transfer Following Peripheral Intravascular Delivery of Adeno-Associated Virus (AAV)-5 and AAV-6 in a Large Animal Model

“…This is consistent with the observation that AAV-6 gcn values were higher in the liver than those of AAV-5. The efficacy of the AAV-6 vectors is comparable and potentially superior (in the high-dose cohort) to AAV-8 vector, as has been previously reported using this model (Favaro et al, 2009).…”

“…Previous data demonstrated that the duration of detectable vector sequences in the semen of rabbits injected with AAV-2 and AAV-8 was vector dose-dependent (Favaro et al, 2009). Here we documented similar findings for AAV-6.…”

“…The primers (forward, 5'-ttcgatctacaaagttcaccatctataac-3'; reverse, 5'-aaactggtcccttccacttcag-3') and the fluorescein aminohexylamidite-labeled probe (5'-aatctctacctccttcatg gaagccagca-3') were designed to detect the AAV-hFIX16 vector sequences. The lowest sensitivity of the quantitative PCR was 25 copies per 1 mg of genomic DNA (Favaro et al, 2009).…”

“…Bethesda assays were performed using citrated plasma samples following heat inactivation at 568C for 1 hr to eliminate the endogenous rabbit FIX. Serial dilutions of heat-inactivated rabbit plasma were then incubated with human plasma for 2 hr at 378C (Favaro et al, 2009). Residual hFIX clotting activity was determined by one-stage activated partial thromboplastin time and compared with a standard curve.…”

The gene therapy journey for hemophilia: are we there yet?

“…15 These data suggested that the presence of vector DNA in the semen was transient and that the risk of observing this was dose-related. 16,17 The trial was therefore modified to include a recommendation to bank sperm in advance of the procedure for those considering fathering additional children, and a recommendation that participants use barrier birth control until 2 sequential semen specimens were negative by PCR for vector sequences (Table 1). Subsequent clinical studies have confirmed the animal data, and there have been no cases of semen being persistently positive for vector sequences after administration of either AAV2 or AAV8 vectors.…”