IMMUNE RESPONSES TO AAV IN GENE TRANSFER FOR HEMOPHILIA

Wistar Institute, Philadelphia PA

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Abstract

Hemophilia B is a bleeding diathesis due to a deficiency of blood coagulation Factor IX (F.IX). AAV-mediated, liver-directed gene transfer has yielded long-term (>5 years) expression of therapeutic levels of F.IX in the canine model of the disease. A clinical study based on these findings uncovered obstacles that had not been evident in pre-clinical studies. The first subject treated at a therapeutic dose initially demonstrated F.IX levels of approximately 10-12% for 4 weeks, but then F.IX levels gradually returned to the baseline level of <1%. The decline in F.IX levels was accompanied by a mild, self-limited transaminitis, that began 4 weeks after vector infusion and fully resolved several weeks later. Transient transaminitis was observed in a second subject and immunologic studies in this subject documented a T cell response to AAV capsid. The goal of this application is to understand, in terms of the cellular and humoral immune response, what happened to these subjects, and whether more detailed characterization of immune responses to AAV capsid will permit us to identify subjects likely to benefit from AAV-mediate, liver-directed gene transfer. In addition, we prepare to determine whether immunomodulatory therapies, or changes in the vector, can alter the outcome in favor of prolonged expression. To accomplish these goals, we shall pursue studies in murine and non-human primate animal models, and in normal and hemophilic subjects. In the first aim, we will examine T cell responses to AAV-2 capsid sequences in the normal human population, in hemophilic patients, and in hemophilic subjects who have been injected parenterally with AAV vectors. The second aim will determine how long AAV capsid proteins persist in an immunologically detectable form following introduction of vector into the livers of mice. In the third aim, we shall examine T cell responses to AAV-8 and to A modified Rhesus F.IX in non-human primates injected with an AAV-8 vector expressing F.IX. AAV-8 is a simian AAV;because NHP may be naturally infected prior to vector infusion, this may more accurately model the series of responses in humans infused with AAV-2 vectors. Lymphocytes from both liver and peripheral blood will be examined in these studies. These studies will be critical for developing an understanding of pre-existing immunity to AAV and its implications for AAV-mediated gene transfer.