Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4 + T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.

Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4 + T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.

IntroductionGenetic disease can potentially be cured by the introduction of a functional copy of the defective gene. Stable gene transfer results in a continuous supply of the functional gene product, and proof-of-principle for this concept has been achieved in humans with severe immune deficiencies and in animal models of several genetic diseases. 1,2 However, gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product. A particularly serious concern in treatments that rely on systemic delivery of the transgene product is the potential for formation of an antibody response to the therapeutic protein.A humoral immune response could not only neutralize the gene therapy but also interfere with conventional protein therapy. 3,4 Animal models of genetic disease have been used to study the risk of immune responses in gene therapy for lysosomal storage disorders and systemic protein deficiencies such as the X-linked bleeding disorder hemophilia.The goal of in vivo gene transfer for systemic protein delivery is to direct transgene expression to a particular tissue, thereby turning the target cells into factories that produce the therapeutic protein and secrete it into circulation. However, because of the genetic defect in the recipient of gene transfer, the functional protein may represent a neoantigen that can be subject to an adaptive immune response. Activation signals derived from the gene transfer process (ie, from the procedure or the vector) may trigger a local immune response that can lead to T-and B-cell activation in draining lymph nodes and subsequently in other lymphoid organs such as the spleen. 5,6 Nonetheless, sustained correction of lysosomal storage disease in mice and of canine and murine hemophilia B (deficiency in coagulation factor IX, FIX) has been achieved in juvenile and adult animals by in vivo hepatic gene transfer using adenoassociated viral (AAV) vectors. 7-10 Moreover, it has been demonstrated that the lack of immune responses in these experiments was the result of induction of immune tolerance by the hepatic route. 10,11 Experimental animals continued to express a human FIX (hFIX) transgene product without antibody formation even after challenge by administration of hFIX in adjuvant. 11 Lymphocytes from tolerized mice failed to proliferate and to secrete cytokines in response to in vitro restimulation with hFIX, indicating a lack of T helper cell responses. 11 While our laboratory has obtained evidence for induction of CD4 ϩ T-cell anergy and deletion using ovalbumin (ova) as a model antigen in hepatic AAV-mediated gene transfer, studies with hFIX also provided evidence for induction of CD4 ϩ regulatory T cells (T regs ). 11,12 These cells were found to suppress antibody and cytotoxic T-cell responses to hFIX, thereby allowing for secondary gene transfer with a more immunogenic adenoviral vector. 13 The subset of CD4 ϩ cells responsible for suppression had not yet been identified. Analysis of splenocytes showed an increase in CD4 ϩ CD...

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