Primary hepatocyte and engineered iPSC-derived hepatocyte-like cell transplantation to treat alpha-1 antitrypsin deficiencyassociated liver disease

原代肝细胞和工程 iPSC 衍生的肝细胞样细胞移植治疗 α-1 抗胰蛋白酶缺乏相关的肝病

• 批准号: 10607039
• 负责人: Anna R. Smith
• 金额: $ 4.77万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607039
• 关键词: Address; Autologous; Award; BMP4; Blood; CEBPA gene; COVID-19 vaccine; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Line; Cell Maturation; Cell Survival; Cell Therapy; Cell Transplantation; Cells; Cirrhosis; Clinical; Culture Media; Data; Disease; Disease model; Doxycycline; Encapsulated; Engineering; Engraftment; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Genes; Genetic Diseases; Genetic Transcription; Goals; Graft Rejection; Growth; Growth Factor; Growth Factor Receptors; HGF gene; Hepatic; Hepatocyte; Hepatocyte transplantation; Human; Human Engineering; Immune; In Vitro; Injections; Ligands; Liver; Liver Cirrhosis; Liver diseases; Lung; MET gene; Messenger RNA; Mitogen Receptors; Mitogens; Mus; Mutation; Nucleosides; Organ Transplantation; Pathway interactions; Patients; Peptide Hydrolases; Physiological; Point Mutation; Polymers; Production; Proliferating; Protease Inhibitor; Proteins; Pulmonary Emphysema; RNA vaccine; Risk; Serum; Specific qualifier value; System; Technology; Testing; Therapeutic; Toxic effect; Transcription Factor 3; Transfection; Transgenic Mice; Transplantation; alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; alternative treatment; cellular engineering; directed differentiation; hepatocyte engraftment; improved; in vivo; induced pluripotent stem cell; inducible gene expression; inhibitor; intravenous injection; lipid nanoparticle; liver function; liver transplantation; mouse model; polymerization; preconditioning; prevent; receptor; regenerative; stem cell therapy; tool; transcription factor

ABSTRACT Alpha-1 antitrypsin deficiency (AATD) is a genetic disease most commonly caused by the Z point mutation in the SERPINA1 gene, resulting in misfolded Z-AAT protein polymerization in hepatocytes, cell death, and often cirrhosis. Replacement of ZZ with wild type MM hepatocytes via liver transplantation is the only available cure for AATD liver disease. However, organs for transplant are in short supply and transplantation comes with risk of graft rejection. Hepatocyte-like cells (HLCs), derived from AATD patient induced pluripotent stem cells (iPSCs) after gene editing of the Z mutation, could provide an unlimited supply of autologous M-AAT producing cells for transplantation without the burden of rejection. Yet, poor engraftment of HLCs remains a critical gap that must be addressed before iPSC-based therapy can be made available to AATD patients suffering from severe liver disease. The goal of this proposal is to fill this gap by promoting iPSC-derived HLC survival, proliferation and maturation, key features for cell engraftment, to treat the AATD associated liver disease of the NSG-PiZ transgenic mouse model. To do so, we will employ AATD patient-specific iPSCs that have been gene edited from ZZ to MM. These MM iPSCs will be engineered to make MM HLCs that express physiological levels of the 2 key known hepatocyte mitogen receptors, hepatocyte growth factor (HGF) receptor, cMET, and epidermal growth factor (EGF) receptor, EGFR, as well as 3 transcription factors known to be critical for hepatocyte maturation, ATF5, PROX1, and CEBPA, using a timely controlled doxycycline inducible piggyBac transposon system. The receptors, cMET and EGFR, will be activated using the corresponding ligands, HGF and EGF, delivered via intravenous injection of nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA- LNP), a non-integrative and safe technology that our lab has recently established to treat various liver diseases. Our preliminary data support the feasibility of this project and are summarized as follows: (1) We have built the transcriptional units of the doxycycline-inducible piggyBac platform, and we expect to complete the platform and start engineering hiPSC lines when the award will be initiated; (2) We showed that diseased hepatocytes in NSG- PiZ mice are efficiently transfected with mRNA-LNPs, validating the mRNA-LNP tool to deliver mitogens in the liver of these mice; (3) We showed that HGF+EGF mRNA-LNP treatment enhances transplanted control primary human hepatocyte engraftment and also improves, albeit transiently, HLC survival after transplantation into NSG-PiZ mice. This leaves room for improvement, the goal of the present application. Thus, our central hypothesis is: Activation of the mitogen HGF/cMET and EGF/EGFR axes in combination with expression of 3 key hepatocyte maturation factors ATF5, PROX1, and CEBPA will lead to successful HLC therapy for AATD liver disease. Importantly, this project will pioneer the use of mRNA-LNPs, which have been widely validated as safe with the recent mRNA-based vaccines, to harness HLC-based liver therapy for AATD patients.

抽象的 Alpha-1 抗胰蛋白酶缺乏症 (AATD) 是一种遗传性疾病，最常见的原因是 Z 点突变 SERPINA1 基因，导致肝细胞中 Z-AAT 蛋白聚合错误折叠、细胞死亡，并且经常 肝硬化。通过肝移植用野生型 MM 肝细胞替代 ZZ 是唯一可行的治疗方法 用于 AATD 肝病。但移植器官供不应求，移植存在风险 移植物排斥反应。肝细胞样细胞 (HLC)，源自 AATD 患者诱导多能干细胞 (iPSC) Z突变基因编辑后，可以无限量供应自体M-AAT产生细胞 移植时无排斥反应。然而，HLC 的植入不良仍然是一个关键差距， 在向患有以下疾病的 AATD 患者提供基于 iPSC 的治疗之前必须解决这一问题： 严重的肝脏疾病。 该提案的目标是通过促进 iPSC 衍生的 HLC 存活、增殖和增殖来填补这一空白。 成熟，细胞移植的关键特征，用于治疗 NSG-PiZ 的 AATD 相关肝病 转基因小鼠模型。为此，我们将使用经过基因编辑的 AATD 患者特异性 iPSC 从ZZ到MM。这些 MM iPSC 将被设计成表达 MM HLC 的生理水平 2 个已知的关键肝细胞有丝分裂原受体、肝细胞生长因子 (HGF) 受体、cMET 和表皮 生长因子 (EGF) 受体、EGFR 以及已知对肝细胞至关重要的 3 种转录因子 成熟、ATF5、PROX1 和 CEBPA，使用及时控制的多西环素诱导性 piggyBac 转座子 系统。受体 cMET 和 EGFR 将使用相应的配体 HGF 和 EGF 激活， 通过静脉注射封装在脂质纳米颗粒中的核苷修饰的 mRNA（mRNA- LNP）是我们实验室最近建立的一种非整合且安全的技术，用于治疗各种肝脏疾病。 我们的初步数据支持了该项目的可行性，总结如下：（1）我们已经建成了 多西环素诱导的piggyBac平台的转录单元，我们期望完成该平台并 当奖项启动时开始设计 hiPSC 细胞系； (2) 我们发现 NSG- 中的患病肝细胞 PiZ 小鼠被 mRNA-LNP 有效转染，验证了 mRNA-LNP 工具能够在小鼠体内传递有丝分裂原。 这些小鼠的肝脏； (3) 我们表明 HGF+EGF mRNA-LNP 处理增强了移植对照的原代 人肝细胞植入，并且还改善（尽管是短暂的）移植后 HLC 存活率 NSG-PiZ 小鼠。这留下了改进的空间，这也是本申请的目标。因此，我们的中央 假设是：有丝分裂原 HGF/cMET 和 EGF/EGFR 轴的激活与 3 的表达相结合 关键肝细胞成熟因子 ATF5、PROX1 和 CEBPA 将导致 AATD 的 HLC 治疗成功 肝脏疾病。重要的是，该项目将率先使用 mRNA-LNP，该技术已被广泛验证为 使用最近的基于 mRNA 的疫苗是安全的，可以利用基于 HLC 的肝脏治疗来治疗 AATD 患者。