Using human liver tissue equivalents to optimize AAV-mediated GT and better define age-related clinical risks

项目总结 使用AAV载体的基因治疗(GT)临床试验有望实现安全、负担得起的终生 纠正一次治疗后的出血性疾病。尽管如此，使用AAV载体治疗的临床试验 成人中的血友病A(HA)强调了障碍，例如存在预先存在的AAV抗体， 以及这些患者出现意想不到的肝毒性风险。重要的是，这种毒性在临床前动物身上看不到。 研究，强调了将数据从动物模型外推到人类的危险。因为下一步是 GT治疗重症HA将在儿童中实施这一方法，关键是要准确预测， 在这一人群中可能存在的、不可预见的风险。目前，尚不清楚这种意外的免疫/ 炎症反应是由于使用AAV作为递送媒介，或者它们是由强迫 FVIII在肝细胞中的表达，而肝细胞不是FVIII产生的天然部位。然而，由于类似的 AAV治疗血友病B的临床试验尚未发现毒性(肝细胞是FIX的天然部位 生产)，这是合理的假设，异位表达的FVIII可能发挥作用。此外，临床前数据已经 还表明，在使用高剂量时，AAV，长期以来被认为主要是上体的，可能表现出显著的 可能导致克隆性扩张和肝细胞癌的宿主基因组整合水平 (肝细胞癌)，其风险因肝细胞增殖而增加。这些都是安全的关键问题 将这些潜在治愈疗法的使用推广到儿科人群，在这些人群中， 肝脏的原始状态可能会增加这些风险。本提案的总体目标是 利用人类肝组织等效物(HLTE)平台回答这些问题并确定影响 受助人年龄对这些变量有影响。我们将使用hLTE来检验FVIII表达可以 得到改善，克服先前存在的对AAV的免疫力，并避免临床试验中看到的毒性，通过 优化FVIII转基因的密码子使用和/或序列以最小化未折叠的蛋白质反应 和内质网应激和/或通过靶向转导到肝内皮细胞，FVIII合成的天然部位。 具体地说，我们将使用生理上相关的hLTE平台来：1)定义AAV的年龄相关影响 转导与肝细胞靶向表达FVIII对人肝脏生物学和功能的影响及其触发的可能性 先天性免疫，以及优化FVIII转基因的密码子使用和序列含量是否可以 防止这种不良的免疫/炎症反应；2)检测是否靶向AAV转导到肝脏 内皮可以改善FVIII的表达，防止肝脏炎症/免疫，保护肝功能，以及 保护AAV免受现有的抗衣壳蛋白免疫；以及3)研究基因组整合频率是否会更高 在较年轻的时候，由于细胞周期增加，靶向肝内皮细胞是否会降低 潜在的遗传毒性。希望这些研究将确定使疗效和安全性最大化的方法。 将人肝靶向AAV GT用于HA，从而为其在儿科患者中的使用铺平了道路。

PROJECT SUMMARY Gene therapy (GT) clinical trials using AAV vectors are poised to fulfill the promise of a safe, affordable, lifelong correction of bleeding disorders following a single treatment. Still, clinical trials using AAV vectors to treat hemophilia A (HA) in adults have underscored the hurdles, such as the presence of pre-existing AAV antibodies, and unexpected risk of hepatoxicity in these patients. Importantly, this toxicity was not seen in preclinical animal studies, highlighting the dangers of extrapolating data from animal models to humans. Since the next step for GT to treat severe HA will be implementation of this approach in children, it is crucial to predict, as accurately as possible, unforeseen risks in this population. Currently, is unknown whether the unexpected immune/ inflammatory responses seen are due to the use of AAV as a delivery vehicle, or they are caused by the forced expression of FVIII within hepatocytes, which are not the native site of FVIII production. However, since similar toxicity has not been seen in AAV clinical trials for hemophilia B (hepatocytes are the natural site of FIX production), it is rational to posit that ectopic FVIII expression likely plays a role. In addition, preclinical data have also shown that, at the high doses used, AAV, long assumed to be largely episomal, may exhibit significant levels of host genome integration that could potentially drive clonal expansion and hepatocellular carcinoma (HCC), the risk of which increases as a result of hepatocyte proliferation. These are critical questions to safely extend the use of these potentially curative treatments to the pediatric population, in whom the higher proliferation and more primitive state of the liver may increase these risks. The overall goal of the present proposal is to utilize a human liver tissue equivalent (hLTE) platform to answer these questions and to determine the impact recipient age has on these variables. We will use hLTE to test the overall hypothesis that FVIII expression can be improved, the pre-existing immunity to AAV overcome, and the toxicity seen in clinical trials avoided, by optimizing the codon usage and/or sequence of the fVIII transgene to minimize the unfolded protein response and ER stress and/or by targeting transduction to hepatic endothelium, the native site of FVIII synthesis. Specifically, we will use a physiologically relevant hLTE platform to: 1) define age-dependent impact of AAV transduction vs. hepatocyte-targeted FVIII expression on human liver biology and function, the potential to trigger innate immunity, and whether optimizing the codon usage and sequence content of the fVIII transgene can prevent this undesired immune/inflammatory response; 2) test whether targeting AAV transduction to hepatic endothelium will improve FVIII expression, prevent hepatic inflammation/immunity, preserve liver function, and protect AAV from existing anti-capsid immunity; and 3) investigate if genomic integration frequency will be higher at younger ages, due to increased cell cycling, and whether targeting hepatic endothelial cells will decrease the potential for genotoxicity. It is hoped that these studies will identify the means to maximize the efficacy and safety of human liver-targeted AAV GT for HA and thereby pave the way for its use in pediatric patients.