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.

项目概要 使用 AAV 载体的基因治疗 (GT) 临床试验有望实现安全、实惠、终身的承诺 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 成人甲型血友病 (HA) 强调了这些障碍，例如预先存在的 AAV 抗体的存在， 这些患者存在意想不到的肝毒性风险。重要的是，在临床前动物中没有发现这种毒性 研究强调了将动物模型的数据外推到人类的危险。由于下一步为 GT 治疗严重 HA 将在儿童中实施这种方法，因此预测至关重要 该人群中可能存在不可预见的风险。目前尚不清楚是否会出现意外免疫/ 所看到的炎症反应是由于使用 AAV 作为递送载体，或者是由强制作用引起的 FVIII 在肝细胞内表达，肝细胞不是 FVIII 产生的天然位点。然而，由于类似 AAV 对 B 型血友病的临床试验中尚未发现毒性（肝细胞是 FIX 的天然位点） 产生），有理由认为异位 FVIII 表达可能发挥了作用。此外，临床前数据有 还表明，在使用高剂量时，长期以来被认为主要是附加型的 AAV 可能会表现出显着的 可能驱动克隆扩增和肝细胞癌的宿主基因组整合水平 （HCC），其风险因肝细胞增殖而增加。这些都是安全的关键问题 将这些潜在治疗方法的使用范围扩大到儿科人群，其中增殖率较高 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 利用人体肝脏组织等效 (hLTE) 平台来回答这些问题并确定影响 接受者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，克服了预先存在的对 AAV 的免疫力，并避免了临床试验中看到的毒性， 优化 fVIII 转基因的密码子使用和/或序列，以尽量减少未折叠蛋白反应 和 ER 应激和/或通过靶向转导至肝内皮（FVIII 合成的天然位点）。 具体来说，我们将使用生理相关的 hLTE 平台来：1）定义 AAV 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，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内容是否可以 防止这种不良的免疫/炎症反应； 2) 测试AAV是否靶向转导至肝脏 内皮细胞将改善 FVIII 表达，预防肝脏炎症/免疫，保护肝功能，以及 保护 AAV 免受现有抗衣壳免疫的影响； 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降低 潜在的遗传毒性。希望这些研究能够找到最大限度提高疗效和安全性的方法 人类肝脏靶向 AAV GT 用于 HA，从而为其在儿科患者中的使用铺平了道路。