Next-generation human liver gene therapy

下一代人类肝脏基因疗法

• 批准号: 10326762
• 负责人: Holger Willenbring
• 金额: $ 78.56万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10326762
• 关键词: Animal Model; Bar Codes; Biliary; Capsid; Cells; Characteristics; Clinical; Clinical Trials; Collagen; DNA Sequence; DNA Shuffling; Development; Disease; Dose; Engineering; Fatty Liver; Gene Delivery; Hemophilia A; Hepatic Stellate Cell; Hepatocyte; Human; Libraries; Liver; Liver Fibrosis; Liver diseases; Monkeys; Mus; Myofibroblast; Pathogenicity; Performance; Perfusion; Population; Reaction; Reporting; Safety; Side; Source; Specificity; Therapeutic; Therapeutic Effect; Toxic effect; Treatment Efficacy; Virus; base; cell type; cholangiocyte; cholestatic liver disease; gene therapy; in vivo; next generation; screening; single-cell RNA sequencing; therapeutic gene; vector

Project Summary/Abstract Pioneering clinical trials of hepatocyte-targeted liver gene therapy in hemophilias have established the principal therapeutic efficacy of adenoassociated virus (AAV) vectors. Clinical trials have also revealed limitations of current AAV vectors, mainly lower than expected therapeutic efficacy and dose-dependent toxicity undermining both safety and efficacy. These clinical findings show that animal models, even monkeys or mice engrafted with human hepatocytes, fail to accurately predict the performance of AAV vectors in humans. The limitations of current AAV vectors need to be overcome for broad application of AAV liver gene therapy because most liver diseases require more hepatocytes to be transduced to achieve a therapeutic effect than hemophilias. To maximize the therapeutic effect that can be achieved within a safe AAV vector dose limit, we will identify AAV capsids that transduce hepatocytes in the human liver with maximum efficiency but no or limited off-targeting. For this, we will harness the near-clinical conditions provided by normothermic machine perfusion (NMP) of human livers. We will compare capsids currently used in clinical trials of liver gene therapy to engineered capsids reported to efficiently target mouse liver or human hepatocytes engrafted in mice. To compare capsids side by side, we have established analysis of AAV vector-expressed barcodes by single-cell RNA sequencing of comprehensive cell populations isolated from human livers after NMP. We will also target capsids de novo in human livers maintained by NMP by screening a library of 1 million chimeric capsids generated by shuffling of the DNA sequences of naturally occurring AAV capsids. In addition to achieving unprecedented levels of efficiency and specificity of transduction of hepatocytes, we aim to establish transduction of cholangiocytes, thereby facilitating the development of gene therapies for biliary diseases. We will also target activated hepatic stellate cells (myofibroblasts), the source of excessive collagen in liver fibrosis, and reactive cholangiocytes, which form the ductular reaction characteristic for cholestatic liver diseases. Efficient and specific in vivo gene delivery to these pathogenic and abundant cells will facilitate therapeutic strategies based on inactivation or repurposing, for example, reprogramming into hepatocytes. By identifying or generating capsids that transduce hepatocytes and other therapeutically relevant liver cell types with the highest level of efficiency and specificity our results will directly inform clinical trials that are at the planning stage and provide the basis for extending the reach of AAV liver gene therapy to common liver diseases like fatty liver disease and biliary diseases, including liver fibrosis as their common end stage.

项目摘要/摘要 血友病肝细胞靶向肝基因治疗的开创性临床试验确立了主要的 腺相关病毒载体的治疗效果。临床试验也揭示了这种药物的局限性。 目前的AAV载体，主要是治疗效果低于预期和剂量依赖毒性破坏 既安全又有效。这些临床发现表明，动物模型，即使是猴子或小鼠移植 对于人类肝细胞，无法准确预测AAV载体在人类体内的性能。其局限性 现有的AAV载体需要克服，才能广泛应用AAV肝脏基因治疗，因为大多数 与血友病相比，肝病需要更多的肝细胞来实现治疗效果。至 在安全的AAV载体剂量限制内最大化治疗效果，我们将识别AAV 在人类肝脏中以最高效率转导肝细胞的衣壳，但没有或有限地偏离靶点。 为此，我们将利用常温机器灌流(NMP)提供的近临床条件。 人体肝脏。我们将比较目前用于肝脏基因治疗临床试验的衣壳蛋白和基因工程衣壳蛋白。 据报道，衣壳蛋白可以有效地靶向移植到小鼠体内的小鼠肝脏或人肝细胞。要比较衣壳 同时，我们建立了AAV载体表达的条形码的单细胞RNA测序分析 从NMP后的人肝脏中分离出的综合细胞群。我们还将把目标锁定在 NMP通过筛选通过改组产生的100万个嵌合衣壳的文库来维持人类肝脏 自然产生的AAV衣壳的DNA序列。除了达到前所未有的水平， 肝细胞转导的效率和特异性，我们的目标是建立胆管细胞的转导， 从而促进胆道疾病基因疗法的发展。我们还将以激活的肝脏为目标 星状细胞(肌成纤维细胞)，肝纤维化中过量胶原的来源，以及反应性胆管细胞， 形成胆汁淤积性肝病的胆管反应特点。高效、特异的体内基因 对这些致病和丰富的细胞的输送将促进基于灭活或 重新调整用途，例如，重新编程进入肝细胞。通过识别或产生转导的衣壳 肝细胞和其他与治疗相关的肝细胞类型具有最高水平的效率和特异性 我们的结果将直接为处于计划阶段的临床试验提供信息，并为推广提供依据 AAV肝脏基因治疗对脂肪肝、胆道疾病等常见肝病的影响 包括肝纤维化作为其共同的终末期。