Validation of Clinical-Grade Electroporation Systems for CRISPR-Cas9-Mediated Gene Therapy in Primary Hepatocytes for the Correction of Inherited Metabolic Liver Disease.

Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah(-/-) graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver.