Efficacy and Safety of AAV Gene Transfer for Hemophilia

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DESCRIPTION (provided by applicant) This career development award application outlines detailed plans for mentored training of Dr. Valder R. Arruda. The research plan details experiments related to AAV-mediated gene transfer for hemophilia B. In previous work we showed long-term expression of biologically active Factor IX in small as well as large animal models of hemophilia B following intramuscular injection of an AAV vector expressing human blood coagulation Factor IX. These data served as the basis for a clinical trial in which subjects with severe hemophilia B were treated by intramuscular injection with an AAV serotype 2-derived vector expressing human Factor IX. There has been no evidence of systemic or local toxicity in the treated subjects, but a dose has not yet been reached at which all subjects in a dose cohort achieve therapeutic levels of gene expression. The overall foal of the work proposed in this application is to evaluate several hypotheses that may lead to safer and more effective gene transfer. In the first aim, I will test the hypothesis that alternate serotypes of AAV, specifically AAV-1 and AAV-6, can direct higher levels of transgene expression in hemophilic doffs and human muscle cells. As shown in preliminary data, we have already shown in mice that these alternate serotypes can direct transgene expression at levels 10-20 fold higher than equivalent doses of an AAV-2 derived vector. However it is important to determine whether this effect is confined only to murine cells or is more widely observed. If higher levels of transgene expression are observed in human myotubes, we will be able to use this cell culture system to determine the mechanism(s) responsible for the higher expression. In the second aim, we propose to use a novel vector delivery technique to address a problem that plagues muscle-directed gene transfer, namely how to achieve transduction of large numbers of cells without Innumerable intramuscular injections. We hypothesize that we can achieve more extensive delivery to skeletal muscle tissue through the use of a technique in which vector is infused into the arterial circulation along with vasodilatory agents. This technique may allow more efficient delivery of vector and thus render muscle-directed gene transfer more feasible. Finally we propose to study molecular and cellular consequences of transduction of skeletal muscle by AAV vectors using microarray technology. This aim addresses one of the most difficult safety issues in gene transfer, i.e. long-term consequences of vector transduction. The data from these experiments will allow us to plan additional safety studies in relevant animal models to assess the long-term safety of treatment with AAV vectors. Other components of this application outline formal coursework, seminar participation, training in responsible conduct of research, and consultation with a formal committee that will enhance the applicant?s career development.