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

项目摘要 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，终身 在单次治疗后纠正出血性疾病。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）已经强调了障碍，例如预先存在的AAV抗体的存在， 和非预期的肝毒性风险。重要的是，在临床前动物中未观察到这种毒性 研究，强调从动物模型推断数据到人类的危险。由于下一步为 GT治疗重症HA将在儿童中实施这种方法，至关重要的是要预测， 潜在的不可预见的风险目前，尚不清楚是否意外免疫/ 所见的炎症反应是由于使用AAV作为递送载体，或者它们是由强迫的免疫应答引起的。 FVIII在肝细胞内的表达，肝细胞不是FVIII产生的天然位点。然而，由于类似 在血友病B的AAV临床试验中尚未观察到毒性（肝细胞是FIX的天然位点 生产），因此推断异位FVIII表达可能起作用是合理的。此外，临床前数据 还表明，在使用的高剂量下，长期以来被认为主要是游离型的AAV可以表现出显著的 宿主基因组整合水平可能潜在地驱动克隆扩增和肝细胞癌 (HCC)其风险由于肝细胞增殖而增加。这些都是关键问题， 将这些潜在的治愈性治疗扩展到儿科人群，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）平台来回答这些问题并确定其影响 接受者年龄对这些变量有影响。我们将使用hLTE来检验总体假设，即FVIII表达可以 通过以下方法，可以改善对AAV的免疫力，克服对AAV的预先存在的免疫力，并避免临床试验中观察到的毒性， 优化所述fVIII转基因的密码子使用和/或序列以使未折叠的蛋白质应答最小化 和ER应激和/或通过靶向转导至肝内皮（FVIII合成的天然位点）。 具体而言，我们将使用生理学相关的hLTE平台来：1）定义AAV对人外周血淋巴细胞的年龄依赖性影响。 转导与肝细胞靶向FVIII表达对人类肝脏生物学和功能的影响， 先天免疫，以及优化密码子使用和fVIII转基因的序列内容是否可以 预防这种不期望的免疫/炎症反应; 2）测试是否靶向AAV转导至肝细胞; 内皮将改善FVIII表达，预防肝脏炎症/免疫，保护肝功能， 保护AAV免受现有的抗衣壳免疫;和3）研究基因组整合频率是否会更高 在年轻时，由于细胞周期增加，以及靶向肝内皮细胞是否会减少 潜在遗传毒性。希望这些研究将确定最大限度地提高疗效和安全性的方法 人肝靶向的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.