Inadvertent germline transmission of AAV2 vector: findings in a rabbit model correlate with those in a human clinical trial.

The risk of germline transmission of vector sequences in humans is a major safety concern, because the enrollment of subjects of reproductive age in early-phase clinical trials of gene transfer continues to increase. In a study of adult men with hemophilia B, adeno-associated virus serotype 2 (AAV2) delivered to the liver via the hepatic artery resulted in unexpected transient vector dissemination to the semen. Here we report that intravenous AAV2 injection in rabbits proved a useful model to assess biologic parameters of vector dissemination to the semen. Detectable vector sequences in semen disappeared in a dose-dependent and time-dependent fashion. AAV infectious particles were present only as long as day 4 after injection and were undetectable thereafter. The kinetics of vector clearance was faster in the semen fractions enriched for motile sperm than in the total semen. In addition, increased frequency of semen sampling accelerated the clearance of vector sequences from semen. Long-term follow-up, spanning hundreds of spermatogenesis cycles, showed that there was no recurrence of detectable vector sequences in semen, thus reducing the probability of inadvertent transduction of early spermatogonia not committed to differentiation at the time of vector injection. We conclude that AAV2 presents minimal germline transmission risk for humans.

The inhibitory effects of anticoagulation on in vivo gene transfer by adeno-associated viral or adenoviral vectors.

Identifying factors that influence gene transfer efficacy is critical for a successful gene-based clinical study. Here we demonstrate that in vivo AAV-2-mediated gene transfer is efficiently inhibited by unfractionated heparin, but not by a heparin preparation containing mainly low-molecular-weight forms (LMWH). Surprisingly, inhibitors of thrombin or factor Xa (F.Xa) significantly reduced AAV-2 transduction in a dose-dependent manner. These effects were independent of the vector promoter, transgene, or strain of mice. Expression by alternate AAV serotypes 5 and 8 was not affected by anticoagulant drugs, which suggests an AAV-2-specific effect. Moreover, AAV-2-mediated gene expression was diminished in mice with deficiency in thrombin generation (factor IX deficiency) and enhanced in mice with procoagulant phenotype due to factor V Leiden. In addition, inhibitors of F.Xa diminished adenovirus-mediated gene expression. These results demonstrated that coagulation activity itself is critical to ensure optimal viral vector transduction. Since intravascular delivery of vectors often requires the use of anticoagulants, the use of LMWH appears to be safe. These observations are of relevance for approaches using AAV-2 or adenoviral vectors, especially in early phase studies designed to identify the minimum therapeutic doses.

Factor IX variants improve gene therapy efficacy for hemophilia B.

Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Novel hemophilia B mouse models exhibiting a range of mutations in the Factor IX gene.

Animal models have been critical to the development of novel therapeutics in hemophilia. A deficiency of current murine models of hemophilia B is that they are all due to gene deletions, a type of mutation that is relatively rare in the human hemophilia population. We generated mice with a range of mutations in the Factor IX (F.IX) gene; these more faithfully reflect the types of mutations that cause disease in the human population. Transgenic mice expressing either wild-type human F.IX (hF.IX), or F.IX variants with premature translation termination codons, or missense mutations, under the control of the murine transthyretin promoter, were generated and crossed with mice carrying a large deletion of the murine F.IX gene. Gene copy number, F.IX transcript levels in the liver, intrahepatocyte protein expression, and circulating levels of F.IX protein in the mice were determined and compared with data generated by transient transfection assays using the same F.IX variants. Mice were injected with a viral vector expressing hF.IX and displayed a range of immune responses to the transgene product, depending on the underlying mutation. These new mouse models faithfully mimic the mutations causing human disease, and will prove useful for testing novel therapies for hemophilia.

Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1.

Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10- to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected with AAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX.