Immune Responses to Capsid in AAV-Mediated Gene Transfer

项目1的总体目标是了解人类对AAV载体的免疫应答的性质。 这一研究路线是基于在患有严重血友病B的男性中向肝动脉施用AAV-因子IX（F.IX）的临床试验，其中我们记录了在载体注射后3-4周开始的F.IX表达丧失和短暂肝转氨酶升高的同时发生。在前一个资助期间，我们记录了：1）肝酶的上升和下降伴随着衣壳蛋白特异性CD 8 + T细胞群体的扩增和收缩，但没有对F.IX的T细胞应答; 2）如IFN-7 ELIspot所记录的，相当大比例的正常人受试者具有AAV衣壳蛋白特异性T细胞; 3）人肝细胞可以在转导细胞的表面上加工和呈递预先形成的衣壳抗原; 4）在初步研究中，临床上批准的蛋白酶体抑制剂硼替佐米减少转导细胞表面上的衣壳抗原呈递; 5）在非人灵长类动物和迄今为止的一名人受试者中，免疫抑制方案MMF/雷帕霉素可以与递送至肝动脉的AAV载体安全地共同施用。在下一个资助期内，我们提出了三个目标，以建立在这些观察结果的基础上：1）监测和表征在七个不同的AAV试验中经历AAV介导的基因转移的人类受试者对AAV衣壳的免疫应答。我们将使用ELISpot进行筛选和多功能T细胞分析，以更全面地评估T细胞应答;我们还将在所研究的受试者中获得随时间推移的完整血清细胞因子表达谱; 2）确定在表面暴露的酪氨酸残基中携带突变的替代AAV血清型和衣壳变体的衣壳抗原呈递的水平和生物学意义;这些实验将在完全人源化的体外系统中进行;和3）研究通过硼替佐米的药理学治疗或利用病毒免疫逃避的分子机制来阻断或减少抗原呈递的新策略。还将测试调节性T细胞作为衣壳T细胞的调节剂。目标1和2将涉及与项目2的广泛互动，目标3将涉及与项目3的广泛互动。

The overall goal of Project 1 is to understand the nature of the immune response to AAV vectors in humans. This line of investigation is based on a clinical trial of AAV-Factor IX (F.IX) administered to the hepatic artery in men with severe hemophilia B, in which we documented the simultaneous occurrence of loss of F.IX expression, and transient liver transaminase elevation, beginning 3-4 weeks after vector injection. In the previous funding period we documented: 1) that the rise and fall in liver enzymes was accompanied by expansion and contraction of a population of capsid-specific CD8+ T cells, but no T cell response to F.IX; 2) that a substantial proportion of normal human subjects harbor AAV capsid-specific T cells as documented by IFN-7 ELIspot; 3) that human hepatocytes can process and present preformed capsid antigen on the surface ofthe transduced cell; 4) in preliminary studies, that the clinically approved proteasome inhibitor bortezomib reduces capsid antigen presentation on the surface ofthe transduced cell; 5) in non-human primates, and one human subject thus far, that the immunosuppressive regimen MMF/rapamycin can be safely coadministered with AAV vector delivered to the hepatic artery. In the next funding period, we propose three aims to build on these observations by; 1) monitoring and characterizing the immune response to the AAV capsid in human subjects undergoing AAV-mediated gene transfer in seven different AAV trials. We will use ELISpot for screening and polyfunctional T cell analysis for more comprehensive assessment of T cell responses; we will also obtain a complete serum cytokine expression profile over time in the subjects studied; 2) determining the levels and the biological significance of capsid antigen presentation of alternate AAV serotypes and capsid variants carrying mutations in surface-exposed tyrosine residues; these experiments will be carried out in a fully-humanized in vitro system; and 3) investigating new strategies to block or reduce antigen presentation through a pharmacologic treatment with bortezomib or exploiting the molecular mechanisms of viral immune evasion. Regulatory T cells will also be tested as modulators of capsid T cells. Aims 1 and 2 will involve extensive interaction with Project 2, and Aim 3 with Project 3.