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

摘要 α-1抗胰蛋白酶缺乏症(AATD)是一种遗传性疾病，最常见的原因是Z点突变 SERPINA1基因，导致Z-AAT蛋白在肝细胞内错误折叠聚合，细胞死亡，并经常 肝硬变。肝移植用野生型MM肝细胞替代ZZ是唯一有效的治疗方法 治疗AATD肝病。然而，供移植的器官供不应求，移植也有风险。 移植排斥反应。AATD患者诱导的多能干细胞(IPSCs)来源的肝细胞样细胞(HLC) 在对Z突变进行基因编辑后，可以提供无限的自体M-AAT产生细胞 无排斥反应负担的移植。然而，HLC嫁接不佳仍然是一个严重的差距 必须在基于IPSC的治疗可以用于患有AATD的患者之前解决 严重的肝病。 这项提案的目标是通过促进IPSC衍生的HLC的存活、增殖和 成熟是细胞植入的关键特征，用于治疗NSG-PIZ的AATD相关性肝病 转基因小鼠模型。为此，我们将使用经过基因编辑的AATD患者特定的IPSCs 从ZZ到MM。这些MM IPSCs将被设计成表达MM HLC的生理水平 2个已知的关键的肝细胞有丝分裂原受体、肝细胞生长因子受体、cMET和表皮 生长因子(EGF)受体、EGFR和3个已知对肝细胞至关重要的转录因子 成熟，ATF5，PROX1和CEBPA，使用及时控制的多西环素可诱导的猪Bac转座子 系统。受体cMET和EGFR将被相应的配体HGF和EGF激活， 通过静脉注射包裹在脂质纳米粒中的核苷修饰的信使核糖核酸(信使核苷-信使核糖核酸 LNP)，这是我们实验室最近建立的一种非一体化安全技术，用于治疗各种肝病。 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 多西环素诱导的iggyBac平台的转录单元，我们预计将完成平台和 (2)我们发现NSG中的病变肝细胞-- 将mRNA-LNPs有效地导入PIZ小鼠，验证了mRNA-LNP工具在PIZ小鼠体内传递有丝分裂原 (3)HGF+EGF mRNA-LNP处理提高了移植对照的原发能力 人肝细胞植入，也改善了HLC移植后的存活，尽管是暂时的 NSG-PIZ小鼠。这就留下了改进的空间，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 关键的肝细胞成熟因子ATF5、PROX1和CEBPA将导致AATD的HLC治疗成功 肝病。重要的是，该项目将率先使用信使核糖核酸，它已被广泛验证为 与最新的基于mRNA的疫苗一起安全，为AATD患者提供基于HLC的肝脏治疗。

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.