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.

PROJECT NARRATIVE To overcome the scarcity of donor livers for transplantation, hepatocytes generated from human induced pluripotent stem cells (hiPSC) could provide an unlimited supply of cells to treat liver diseases with cell therapy; however, preparing hiPSC-derived hepatocyte-like cells (HLC) that can repopulate diseased livers remains a challenge and a major gap which the current proposal targets. Given the known immaturity and poor survival of HLCs in vivo in comparison to primary cells, we propose that engineering HLCs to express key regenerative and maturation factors represents a potential strategy to overcome the obstacles of HLC therapies. This approach includes HLCs engineered to express maturation factors and activate mitogen pathways in combination with the use of clinically safe nucleoside-modified mRNA technology, with the goal to develop successful HLC-based treatment for liver disease.