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 α-1抗胰蛋白酶缺乏症（AATD）是一种遗传疾病，最常见于Z点突变引起 SERPINA1基因，导致肝细胞中的Z-AAT蛋白聚合错误折叠，细胞死亡，并且通常 肝硬化。用肝移植用野生型MM肝细胞代替ZZ是唯一可用的治疗方法 用于AATD肝病。但是，用于移植的器官供应不足，移植有风险 移植排斥。源自AATD患者诱导多能干细胞（IPSC）的肝细胞样细胞（HLC）（HLC） 基因编辑Z突变后，可以为自体M-AAT产生细胞的无限供应 移植而没有拒绝负担。然而，HLC的不良植入仍然是一个关键的差距 必须对基于IPSC的疗法进行治疗的AATD患者 严重的肝病。 该建议的目的是通过促进IPSC衍生的HLC生存，扩散和 成熟，是细胞植入的关键特征，以治疗NSG-PIZ的AATD相关肝病 转基因小鼠模型。为此，我们将采用已编辑基因的AATD患者特异性IPSC 从ZZ到MM。这些MM IPSC将经过设计，以使MM HLC表达生理水平的MM HLC 2个关键已知的肝细胞促分裂原受体，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个转录因子 成熟，ATF5，Prox1和Cebpa，使用及时控制的强力霉素诱导型Piggybac Transon 系统。 CMET和EGFR的受体将使用相应的配体HGF和EGF激活。 通过静脉注射的核苷修饰的mRNA封装在脂质纳米颗粒（mRNA-- LNP），这是一种非综合和安全的技术，我们的实验室最近已建立以治疗各种肝病。 我们的初步数据支持该项目的可行性，并总结如下：（1）我们已经建立了 强力霉素诱导的Piggybac平台的转录单元，我们希望完成平台和 启动该奖励时，开始工程HIPSC线路； （2）我们表明NSG-中患病的肝细胞 PIZ小鼠用mRNA-LNP有效地转染，验证mRNA-LNP工具以在 这些小鼠的肝脏； （3）我们表明HGF+EGF mRNA-LNP治疗增强了移植对照的初级 人类肝细胞植入植物，尽管在移植到 NSG-PIZ小鼠。这留给了改进的空间，即本应用的目标。因此，我们的中心 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 关键的肝细胞成熟因子ATF5，PROX1和CEBPA将导致AATD成功的HLC治疗 肝病。重要的是，该项目将开拓MRNA-LNP的使用，该项目已被广泛验证为 可用于最近基于mRNA的疫苗，以利用HLC基于AATD患者的肝脏疗法。