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

使用人类肝脏组织等效物优化 AAV 介导的 GT 并更好地定义与年龄相关的临床风险

• 批准号: 10567919
• 负责人: Graca Duarte Almeida-Porada
• 金额: $ 39.78万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-15 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10567919
• 关键词: Adrenal Cortex Hormones; Adult; Affect; Age; Albumins; Animal Model; Animals; Antibodies; Biology; Blood Coagulation Disorders; Blood Vessels; Capsid; Cell Cycle; Cells; Cellular Stress; Child; Childhood; Clinical; Clinical Trials; Clonal Expansion; Codon Nucleotides; DNA; Data; Dose; Early treatment; Ectopic Expression; Endothelial Cells; Endothelium; Event; Exhibits; F8 gene; Fibrosis; Frequencies; Genes; Genetic Diseases; Genome; Genomics; Goals; Hemophilia A; Hemophilia B; Hepatic; Hepatocyte; Hepatotoxicity; Human; Immune; Immunity; Incidence; Inflammation; Inflammatory Response; Inherited; Innate Immune Response; Life; Liver; Mediating; Natural Immunity; Neonatal; Participant; Patients; Physiological; Play; Population; Primary carcinoma of the liver cells; Production; Proliferating; Proteins; Risk; Safety; Severities; Site; Specificity; System; Testing; Tissues; Toxic effect; Transaminases; Transgenes; Urea; adeno-associated viral vector; age related; angiogenesis; animal data; antibody conjugate; cell type; clinical risk; curative treatments; cytokine; delivery vehicle; drug metabolism; endoplasmic reticulum stress; gene therapy; gene therapy clinical trial; genomic locus; genotoxicity; improved; liver cell proliferation; liver function; liver inflammation; liver preservation; neonatal human; non-Native; novel; pediatric patients; pre-clinical; prevent; prophylactic; response; safety testing; transcriptomics; vasculogenesis; vector

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.

项目摘要 使用AAV载体的基因疗法（GT）临床试验有望实现安全，负担得起的终身的承诺 一次治疗后，纠正出血疾病。尽管如此，使用AAV矢量来治疗的临床试验 成年人中的血友病A（HA）强调了障碍，例如存在先前存在的AAV抗体， 这些患者的肝毒性风险。重要的是，这种毒性在临床前动物中没有看到 研究强调了将数据从动物模型推送到人类的危险。由于下一步 GT治疗严重的HA将是儿童中这种方法的实施，至关重要的是，准确地预测 在这个人群中可能会有无法预料的风险。目前，尚不清楚意外的免疫/是否 看到的炎症反应是由于使用AAV作为送货工具，或者它们是由强迫造成的 肝细胞中FVIII的表达，这不是FVIII产生的本地部位。但是，由于相似 在AAV临床试验中，毒性尚未看到Hemoblilia b（肝细胞是固定的自然部位 生产），认为异位FVIII表达可能起作用是合理的。此外，临床前数据具有 还表明，在使用的高剂量下，aav长期以来被认为是偶发性的，可能表现出显着的 宿主基因组整合的水平，可能驱动克隆膨胀和肝细胞癌 （HCC），由于肝细胞增殖而增加的风险。这些是要安全的关键问题 将这些潜在治愈性治疗的使用扩展到小儿种群，其中较高的增殖 肝脏的更原始状态可能会增加这些风险。本提案的总体目标是 利用人肝组织等效（HLTE）平台来回答这些问题并确定影响 接受者年龄在这些变量上具有。我们将使用HLTE测试FVIII表达可以的总体假设 得到改进，对AAV的先前免疫力克服，并且在临床试验中看到的毒性避免了 优化FVIII转基因的密码子使用率和/或序列，以最大程度地减少展开的蛋白质反应 以及通过靶向肝内皮（FVIII合成的本地部位）的转导向肝内皮的靶向和/或。 具体而言，我们将使用与生理相关的HLTE平台：1）定义AAV的年龄相关影响 在人肝生物学和功能上的转导与肝细胞靶向的FVIII表达，具有触发的潜力 先天免疫，以及优化FVIII转基因的密码子使用和序列含量是否可以 防止这种不需要的免疫/炎症反应； 2）测试是否靶向AAV转导向肝 内皮将改善FVIII表达，防止肝炎/免疫，保留肝功能，并 保护AAV免受现有的抗CAPSID免疫力； 3）研究基因组整合频率是否会更高 在年轻的年龄，由于细胞循环增加以及靶向肝内皮细胞是否会降低 遗传毒性的潜力。希望这些研究能够确定最大化功效和安全性的手段 HA的人体肝脏靶向AAV GT，从而为其在儿科患者中的使用铺平了道路。