Immune Responses to Capsid in AAV-Mediated Gene Transfer

项目1的总体目标是了解人类对AAV媒介的免疫反应的性质。 这项研究是基于对肝动脉注射AAV-IX(F.IX)的临床试验。 在患有严重血友病B的男性中，我们记录了同时发生F.IX丢失的情况 表达和一过性转氨酶升高，开始于载体注射后3-4周。在 在之前的资助期间，我们记录了：1)肝酶的上升和下降伴随着 大量衣壳特异性CD8‘T细胞的扩张和收缩，但T细胞对F.IX没有反应；2) 很大一部分正常人体内有AAV衣壳特异性T细胞，这是由 干扰素-7 ELISPOT；3)人肝细胞能处理和呈递表面预形成的衣壳抗原 4)在初步研究中，临床批准的蛋白酶体抑制剂Bortezomib 减少衣壳抗原在转导细胞表面的呈递；5)在非人类灵长类动物中，以及 到目前为止，一名人类受试者认为，免疫抑制方案MMF/雷帕霉素可以安全地联合使用 将AAV载体送入肝动脉。在下一个资助期，我们提出三项建议 目的是通过以下方式建立这些观察：1)监测和表征对AAV的免疫反应 在七个不同的AAV试验中，接受AAV介导的基因转移的人类受试者的衣壳。我们将使用 ELISPOT用于筛查和多功能T细胞分析以更全面地评估T细胞 我们还将通过FIME获得受试者完整的血清细胞因子表达谱 2)轮状病毒衣壳抗原提呈水平的测定及其生物学意义 AAV血清型和衣壳变异体携带表面暴露的酪氨酸残基突变；这些 实验将在完全人性化的体外系统中进行；以及3)研究新的策略 通过用硼替佐米进行药物治疗或利用 病毒免疫逃避的分子机制。调节性T细胞也将作为调节器进行测试 衣壳T细胞。目标1和目标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 investigafion 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 elevafion, beginning 3-4 weeks after vector injecfion. In the previous funding period we documented: 1) that the rise and fall in liver enzymes was accompanied by expansion and contraction of a populafion 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 polyfuncfional T cell analysis for more comprehensive assessment of T cell responses; we will also obtain a complete serum cytokine expression profile over fime 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 interacfion with Project 2, and Aim 3 with Project 3.