Next-generation human liver gene therapy

项目总结/摘要 血友病肝细胞靶向肝基因治疗的开创性临床试验已经确立了原则， 腺相关病毒（AAV）载体的治疗功效。临床试验也揭示了 目前的AAV载体，主要是低于预期的治疗效果和剂量依赖性毒性破坏 安全性和有效性。这些临床发现表明，动物模型，即使是猴子或小鼠移植， 与人肝细胞的关系，不能准确预测AAV载体在人体中的性能。的限制 目前的AAV载体需要克服广泛应用的AAV肝基因治疗，因为大多数 肝病比血友病需要更多的肝细胞被转导以达到治疗效果。到 为了最大化在安全的AAV载体剂量限度内可以实现的治疗效果，我们将鉴定AAV 衣壳，其以最大效率在人肝脏中包裹肝细胞，但没有或有限的脱靶。 为此，我们将利用常温机器灌注（NMP）提供的近临床条件， 人类的肝脏我们将比较目前用于肝脏基因治疗临床试验的衣壳和工程改造的衣壳。 据报道，衣壳有效地靶向小鼠肝脏或移植到小鼠中的人肝细胞。为了比较衣壳 同时，我们已经通过单细胞RNA测序建立了AAV载体表达条形码的分析 从NMP后的人类肝脏中分离的综合细胞群。我们还将目标衣壳从头在 通过筛选通过以下改组产生的100万个嵌合衣壳的文库， 天然存在的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治疗的发展。我们还将针对激活的肝脏 星状细胞（肌成纤维细胞），肝纤维化中过量胶原的来源，和反应性胆管细胞， 其形成胆汁淤积性肝病的特征性小管反应。高效特异的体内基因 递送到这些致病的和丰富的细胞将促进基于灭活或 再利用，例如，重编程为肝细胞。通过识别或产生衣壳， 肝细胞和其他治疗相关的肝细胞类型，具有最高水平的效率和特异性 我们的研究结果将直接为处于计划阶段的临床试验提供信息，并为扩展 AAV肝基因治疗对脂肪肝、胆道疾病等常见肝病的治疗范围， 包括肝纤维化作为其共同的终末期。

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.