Inducing T_regs with hepatic gene therapy

The development of a humoral immune response to the transgene product is a potential complication of gene transfer in inherited disease, in which the transgene product may represent a neoantigen. The report of Cao and colleagues in this issue of Blood has documented how adeno-associated viral (AAV)–mediated gene transfer to the liver can suppress this antibody development through the generation of antigen-specific T regulatory cells (T_regs).

The success of gene therapy in the treatment of inherited diseases has been predictably complicated by responses from the host immune system.¹  Systemic delivery of therapeutic transgenes by viral vectors may result in humoral and cell-mediated responses to both the vector and the transgene product. These responses adversely influence the potential for vector readministration and the likelihood of achieving success of long-term expression of the transgene product. In addition, the development of antibodies to the transgene product also has the potential of neutralizing the therapeutic benefit derived from the exogenously administered protein. Examples of these immunologic scenarios have now been documented in many preclinical animal studies of gene-transfer replacement strategies, with the lysosomal storage diseases and hemophilias being prime examples.

The development of antibodies to the transgene product appears to be influenced by several factors, including the type of vector, the vector dose, the transgene promoter, and the site of transgene expression.²  In a previous study, this same group had already provided evidence indicating that hepatic gene transfer mediated by an AAV vector could generate tolerance to the factor IX transgene product in a murine model of hemophilia B.³  However, until the current report, the mechanism of this tolerance induction had been unclear.

In these new studies, the authors have first utilized a mouse model in which the animals are transgenic for a T-cell receptor (TCR) specific for a chicken ovalbumin peptide but lack endogenous TCRs due to a knockout of the RAG-2 locus. After delivery of an ova transgene by portal-vein injection of an AAV2 vector, the authors were able to demonstrate induction of ova-specific CD4⁺CD25⁺ T_regs (see figure). These cells were first detectable as early as 14 days after vector administration, and continued to increase in number in the thymus and secondary lymphoid organs during the 2 months after gene transfer. The hepatic gene-transfer–induced T_regs expressed FoxP3, GITR, and CTLA4, and suppressed CD4⁺CD25⁻ cells in a standard in vitro assay.

In subsequent studies of AAV-mediated hepatic factor IX transgene delivery to hemophilia B mice, the authors have used adoptive transfer of CD4⁺GITR⁺ cells to demonstrate that these cells can suppress anti–factor IX antibody generation after factor IX immunization. Finally, in vivo depletion of the CD4⁺CD25⁺ cells in hepatic gene-transfer–treated mice resulted in the development of an anti–factor IX response in 4 of 5 animals, demonstrating the requirement for T_regs for tolerance induction following this form of gene transfer.

These studies provide the first definitive evidence of antigen-specific T_reg generation following hepatic gene transfer. As such, they remind us that while gene-transfer approaches can mediate immunogenic responses in the host, they also have the potential of inducing tolerance to the transgene product.