Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver

Abstract

Adeno-associated virus (AAV)-mediated gene transfer of factor IX (F.IX) to the liver results in long-term expression of transgene in experimental animals, but only short-term expression in humans. Loss of F.IX expression is likely due to a cytotoxic immune response to the AAV capsid, which results in clearance of transduced hepatocytes. We used a nonhuman primate model to assess the safety of AAV gene transfer coupled with an anti-T-cell regimen designed to block this immune response. Administration of a 3-drug regimen consisting of mycophenolate mofetil (MMF), sirolimus, and the anti-IL-2 receptor antibody daclizumab consistently resulted in formation of inhibitory antibodies to human F.IX following hepatic artery administration of an AAV-hF.IX vector, whereas a 2-drug regimen consisting only of MMF and sirolimus did not. Administration of daclizumab was accompanied by a dramatic drop in the population of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). We conclude that choice of immunosuppression (IS) regimen can modulate immune responses to the transgene product upon hepatic gene transfer in subjects not fully tolerant; and that induction of transgene tolerance may depend on a population of antigen-specific Tregs.

Figure 1

hF.IX expression levels and anti-hF.IX antibody formation after AAV2-hF.IX hepatic gene transfer in rhesus macaques receiving MMF, sirolimus, and daclizumab and in nonimmunosuppressed controls. Time 0 represents baseline samples; dotted vertical line shows the time IS was withdrawn. Each line is representative of one individual animal. All animals received 8 × 10¹² vg/kg of an AAV-2-hF.IX vector infused through the hepatic artery. Solid symbols indicate animals receiving a course of IS consisting of MMF, sirolimus, and daclizumab; open symbols, non-IS control animals. (A) Plasma human F.IX levels. (B) Plasma antihuman F.IX total IgG levels.

Figure 2

hF.IX expression levels and anti-hF.IX antibody formation after AAV2-hF.IX hepatic gene transfer in rhesus macaques receiving MMF and sirolimus. Time 0 represents baseline samples; dotted vertical line shows the time IS was withdrawn. Each line is representative of one individual animal. All animals received 8 × 10¹² vg/kg of an AAV-2-hF.IX vector infused through the hepatic artery. (A) Plasma human F.IX levels. (B) Plasma anti–human F.IX total IgG levels.

Figure 3

Anti–AAV-2 antibody formation following vector delivery. Time 0 represents the time of vector administration; dotted vertical line shows the time IS was withdrawn. All animals received 8 × 10¹² vg/kg of an AAV-2-hF.IX vector infused through the hepatic artery. Each line represents the average value (+ SD) of a group of 3 animals.

Figure 4

Staining for regulatory T cells on PBMCs isolated before and after AAV-2-hF.IX hepatic gene transfer. Each line represents one individual animal. y-axis shows the percent of CD4⁺CD25⁺FoxP3⁺ T cells. Cells were gated on CD4⁺CD25^hi and analyzed for FoxP3. (A) Animals receiving only the AAV vector with no IS. (B) Animals receiving both the vector and an IS regimen consisting of MMF, sirolimus, and daclizumab. (C) Animals receiving the vector and MMF and sirolimus.