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对肝动脉施用AAV-因子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F.IX缺失的同时发生， 表达，以及在载体注射后3-4周开始的短暂肝转氨酶升高。在 上一个资助期，我们记录了：1）肝酶的上升和下降伴随着 扩增和收缩的captain特异性CD 8 + T细胞的群体，但没有T细胞响应F.IX; 2） 相当大比例的正常人受试者具有AAV衣壳特异性T细胞，如由 IFN-7 ELIspot; 3）人肝细胞可以加工并呈递预先形成的衣壳抗原 4）在初步研究中，临床批准的蛋白酶体抑制剂硼替佐米 减少转导细胞表面的衣壳抗原呈递; 5）在非人灵长类动物中，和 迄今为止，一名人类受试者证实免疫抑制方案MMF/雷帕霉素可以安全地共同施用 用AAV载体递送至肝动脉。在下一个资助期内，我们建议 旨在通过以下方式建立这些观察结果：1）监测和表征对AAV的免疫应答 在七个不同的AAV试验中，在经历AAV介导的基因转移的人类受试者中的衣壳。我们将使用 ELISpot用于筛选和多功能T细胞分析，以更全面地评估T细胞 我们还将获得受试者体内完整的血清细胞因子表达谱 2）确定了交替感染病毒衣壳抗原提呈的水平及其生物学意义 在表面暴露的酪氨酸残基中携带突变的AAV血清型和衣壳变体;这些 实验将在完全人源化的体外系统中进行;和3）研究新的策略， 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。调节性T细胞也将被测试为调节剂， 衣壳T细胞。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 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.