Enhance AAV Liver Transduction with Capsid Immune Evasion

 描述：腺相关病毒(AAV)载体已成功应用于肝靶向血友病B患者的I期临床试验。然而，这些研究表明，AAV衣壳特异性细胞毒性T淋巴细胞(CTL)具有清除AAV转导的肝细胞的潜力，从而导致治疗失败。我们先前的研究已经证明，AAV衣壳抗原呈递是剂量依赖的，并且需要衣壳泛素化来实现蛋白酶体介导的降解。AAV制剂中空病毒粒子的污染抑制了AAV载体全颗粒的转导，并潜在地增加了病毒衣壳抗原负载的风险。在这项建议中，我们将研究AAV空病毒粒子的衣壳抗原递呈，以及空粒子对完全病毒转导的抗原提呈的影响(目标1)。为了减少AAV转导细胞的抗原提呈，以避免衣壳特异性CTL介导的消除，有人建议通过修饰AAV衣壳表面或应用蛋白酶体抑制剂来增强AAV转导，同时降低有效剂量或逃避衣壳泛素化。我们将研究AAV突变体和蛋白酶体抑制剂对AAV衣壳抗原提呈的影响(目标2)。众所周知，AAV载体在小鼠模型中的转导并不总是翻译成人类。最后，我们将探索定向进化方法和合理的设计策略相结合的方法来分离具有人肝细胞特异性取向和人源化小鼠逃避衣壳特异性CTL反应能力的AAV载体(目标3)。阐明AAV空衣壳抗原在体内的呈递方式，并开发具有增强的人肝转导和CTL免疫逃避功能的AAV载体，将使我们能够设计出更安全、更有效的策略，以解决目前使用AAV进行人类肝脏基因治疗的临床并发症。

 DESCRIPTION: Adeno-associated virus (AAV) vector has been successfully applied in phase I clinical trials in hemophilia B patients with liver targeting. However, these studies have suggested that AAV capsid specific cytotoxic T lymphocytes (CTL) have the potential to eliminate AAV transduced hepatocytes and result in the therapeutic failure. Our prior studies have demonstrated that AAV capsid antigen presentation is dose-dependent and requires capsid ubiquitination for proteasome mediated degradation. The contamination of empty virions in AAV preparation inhibits transduction from full particles of AAV vectors and potentially increases the risk of virus capsid antigen load. In this proposal we will investigate capsid antige presentation from AAV empty virions and the effect of empty particles on antigen presentation from full virus transduction (Aim 1). To decrease antigen presentation on AAV transduced cells for avoiding capsid specific CTL-mediated elimination, it has been proposed to modify the AAV capsid surface or apply proteasome inhibitors to enhance AAV transduction while lowering the effective dose or to escape capsid ubiquitination. We will study the effect of AAV mutants and proteasome inhibitors on AAV capsid antigen presentation (Aim 2). It is well-known that the transduction of AAV vectors in mouse models does not always translate into the human. Finally, we will explore the directed evolution approach combined with a rational design strategy to isolate AAV vectors with human hepatocyte specific tropism and the ability to evade a capsid specific CTL response in humanized mice (Aim 3). Elucidation of AAV empty capsid antigen presentation in vivo and the development of an AAV vector with enhanced human liver transduction and CTL immune-evasion will allow us to design safer and more effective strategies that address the current clinical complications for human liver gene therapy using AAV.