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.

摘要 α-1抗胰蛋白酶缺乏症（AATD）是一种遗传性疾病，最常见的是由Z点突变引起的。 SERPINA 1基因，导致肝细胞中错误折叠的Z-AAT蛋白聚合，细胞死亡， 肝硬化通过肝移植用野生型MM肝细胞替代ZZ是唯一可用的治疗方法 治疗AATD肝病然而，移植器官供应短缺，移植伴随着风险 移植排斥反应肝细胞样细胞（HLC），来源于AATD患者诱导的多能干细胞（iPSC） 在对Z突变进行基因编辑后，可以提供无限量的自体M-AAT生产细胞， 没有排斥负担的移植。然而，HLCs的植入不良仍然是一个关键的差距， 在基于iPSC的治疗可以提供给患有AATD的患者之前， 严重的肝病。 该提案的目标是通过促进iPSC衍生的HLC存活、增殖和分化来填补这一空白。 成熟，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，我们将采用经过基因编辑的AATD患者特异性iPSC 这些MM iPSC将被工程化以制造表达生理水平的MMPSC的MM HLC。 2种关键的已知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，ATF 5，PROX 1和CEBPA，使用及时控制的强力霉素诱导型piggyBac转座子 系统受体cMET和EGFR将被相应的配体HGF和EGF激活， 通过静脉内注射包封在脂质纳米颗粒中的核苷修饰的mRNA（mRNA- LNP），这是我们实验室最近建立的一种用于治疗各种肝脏疾病的非整合和安全技术。 我们的初步数据支持这个项目的可行性，并总结如下：（1）我们已经建立了 转录单位的doxycycline诱导piggyBac平台，我们希望完成该平台， 当该奖项将启动时，开始工程化hiPSC系;（2）我们表明，NSG- 用mRNA-LNP有效转染PiZ小鼠，验证了mRNA-LNP工具在小鼠中递送有丝分裂原。 （3）我们发现HGF+EGF mRNA-LNP治疗可以增强移植对照小鼠的原发性肝细胞癌。 人肝细胞移植后，HLC的存活率也得到改善，尽管是短暂的， NSG-PiZ小鼠。这留下了改进的空间，这是本申请的目标。因此，我们的中央 假设：促分裂原HGF/cMET和EGF/EGFR轴的激活与3 关键的肝细胞成熟因子ATF 5、PROX 1和CEBPA将导致AATD的成功HLC治疗 肝脏疾病重要的是，该项目将率先使用mRNA-LNP，这已被广泛验证， 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AATD患者。