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Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (T(reg))-deficient model (Rag-2(-/-) mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4(+)CD25(+) T(regs) by in vivo gene transfer. Hepatic gene transfer-induced T(regs) express FoxP3, GITR, and CTLA4, and suppress CD4(+)CD25(-) T cells. T(regs) are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4(+)CD25(+)GITR(+) that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4(+)CD25(+) T(regs) leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4(+)CD25(+) T(regs) in preventing immune responses to the transgene product in gene transfer.

Adeno-associated viral (AAV) vectors have been successfully used for therapeutic expression of systemic transgene products (such as factor IX or erythropoietin) following in vivo administration to skeletal muscle of animal models of inherited hematologic disorders. However, an immune response may be initiated if the transgene product represents a neoantigen. Here, we use ovalbumin (OVA) as a model antigen and demonstrate immune-mediated elimination of expression on muscle-directed AAV-2 gene transfer. Administration to immune competent mice resulted in transient systemic OVA expression. Within 10 days, OVA-specific T-helper cells had been activated in draining lymph nodes, an inflammatory immune response ensued, and OVA-expressing muscle fibers were destroyed by a cytotoxic CD8(+) T-cell response. Use of a muscle-specific promoter did not prevent this immune response. Adoptively transferred CD4(+) cells transgenic for a T-cell receptor specific to OVA peptide-major histocompatibility complex class II showed antigen-specific, vector dose-dependent proliferation confined to the draining lymph nodes of AAV-OVA-transduced muscle within 5 days after gene transfer and subsequently participated in lymphocytic infiltration of transduced muscle. This study documents that a local immune response limits sustained expression of a secreted protein in muscle gene transfer, a finding that may have consequences for design of clinical protocols.

Immune responses to the therapeutic gene product are a potentially serious complication in treatment of genetic disease by gene therapy. Induction and maintenance of immunologic hypo-responsiveness to the therapeutic antigen is therefore critical to the success of gene-based treatment of inherited protein deficiency. Here, we demonstrate induction of antigen-specific CD4+ T-cell tolerance to a secreted transgene product (ovalbumin, ova) in ova-specific T-cell receptor (TCR) transgenic mice by hepatic adeno-associated virus (AAV)-mediated gene transfer. Transduced mice maintained stable circulating ova levels without evidence of an immune response. Lymph node cells and splenocytes were hypo-responsive to ova as early as day 10 after gene transfer. Numbers of TCR+CD4+ cells were reduced in secondary lymphoid organs and in the thymus by 1 to 2 months after vector administration. The remaining TCR+CD4+ cell population was anergic to ova antigen in vitro and enriched for CD25+ cells. These data provide direct evidence that transgene expression following in vivo viral gene transfer can induce CD4+ T-cell tolerance to the transgene product, involving anergy and deletion mechanisms.