Theodore E. Woodward Award. AAV-mediated gene transfer for hemophilia.

Our research efforts have been focussed on developing a gene transfer strategy for the treatment of the hemophilias. Hemophilia is an attractive target for studies in gene transfer because even small amounts of clotting factor can improve the clinical symptoms of the disease, the factor can be expressed in almost any tissue as long as it gains access to the circulation, and there are large and small animal models of the disease, so that promising approaches can be assessed for efficacy before moving into clinical studies (1). We have developed recombinant adeno-associated viral (AAV) vectors expressing blood coagulation Factor IX. AAV has a number of advantages as a gene transfer vector including: 1) the absence of viral coding sequences in the recombinant vector; 2) the ability to transduce a variety of non-dividing target cells, including liver, muscle and nervous system; 3) the ability to direct long-term expression of the transgene in immunocompetent animals. We have introduced AAV-F.IX vectors into skeletal muscle and liver, and shown long-term correction of the bleeding diatheses in both small and large animal models of hemophilia B (2-5). In the initial clinical trial, rAAV was introduced into skeletal muscle of subjects with severe hemophilia B. Results showed that the general characteristics of transduction were similar in mouse, canine and human muscle, and muscle biopsies of injected sites showed evidence of gene transfer and expression, but circulating levels of F.IX failed to reach the desired target of 3-10%. There were no serious adverse events associated with rAAV injection in skeletal muscle (6). Work has also proceeded on development of a liver-directed approach. Engineering of the expression cassette has resulted in better expression per particle, and circulating F.IX levels of 4-12% have now been achieved in hemophilia B dogs treated with vector doses lower than those already administered in the clinical study in skeletal muscle (5). After extensive safety studies in mice, rats, hemophilic dogs and non-human primates, a Phase I study of an AAV-mediated, liver-directed approach to treating hemophilia B has begun. There were no acute toxicities associated with administration of vector to the first two subjects, but subsequently a PCR assay on the subjects' semen was found to be positive for vector sequences. After a period of weeks, the positive signal disappeared. These findings were distinct from those seen in pre-clinical animal studies. To gain a clearer understanding of the biodistribution of vector to the gonads, we undertook additional studies in rabbits and mice. These showed that, following intravascular delivery of vector, there is hematogenous dissemination to the gonads and gradual washout of vector over time. Direct transduction of germ cells does not appear to occur (7). Based on these and other safety studies, the clinical trial has now resumed. A goal of this work will be to determine whether the therapeutic levels achieved in a large animal model of hemophilia can be realized in humans.