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

Abstract

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.

Keywords: CRISPR-Cas9; clinical translation; electroporation; gene therapy; therapeutic liver repopulation.

Figure 1

Schematic of therapeutic procedure. Healthy hepatocytes were isolated from mTmG donor mice and electroporated with Hpd-aiming CRISPR-Cas9 RNPs. Following electroporation, donor cells were transplanted via splenic injection into Fah^-/- mice. Recipient mice were cycled off and on nitisinone until independence from the drug was achieved. Abbreviations: Hpd, hydroxyphenylpyruvate dioxygenase; RNP, ribonucleoprotein; Fah, fumarylacetoacetate hydrolase.

Figure 2

In vitro analyses of transplanted donor hepatocytes post-electroporation and recipient body weights post-transplantation. On-target gene editing efficiencies (a) measured in donor hepatocytes three days after electroporation (n = 1). Cell viability in hepatocytes (n = 1) was quantified by trypan-blue staining and cell counting and normalized to the viability of untreated hepatocytes (b). MTT assay viability measurements in plated hepatocytes after electroporation normalized to untreated controls (c) and albumin levels in conditioned media (d) at 24 h after plating. Bar graphs display individual values. Recipient body weights as a percentage of pre-operation weight over the entire selection period (e). For (e), the initial n = 4, there was one mortality in the untransfected recipients on day 9 and one in the 4D recipients on day 11. Abbreviations: ALB, albumin.

Figure 3

Liver repopulation and therapeutic efficacy in Fah^-/- recipients. Measures of donor cell engraftment by tdTomato fluorescence of isolated hepatocytes using flow cytometry (a) and quantification of Hpd indel mutations of homogenized liver tissue (b) (n = 3–4 for recipients and n = 2 for untreated Fah^-/- controls). Representative liver images subjected to anti-Fah IHC staining for recipients transplanted with (n = 4) GTx (c), (n = 3) 4D (d), and (n = 3) untransfected control (e) hepatocytes. IHC staining in untreated Fah^-/- mice on NTBC (n = 2) was shown as a negative control to confirm proper staining (f). Bar graphs display the median and biological replicates, and error bars display the range. Statistical significance was denoted as * for p < 0.05 and ** for p < 0.01. Abbreviations: Hpd, hydroxyphenylpyruvate dioxygenase; Fah, fumarylacetoacetate hydrolase; IHC, immunohistochemistry; NTBC, 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione.

Figure 4

Analyses of liver biochemical markers in Fah^-/- graft recipients. Blood serum concentrations of ALT (a), ALP (b), ALB (c), TBIL (d), CREA (e), and GLU (f) in recipient mice (n = 3–4) and no surgery controls (n = 3). Bar graphs display the median and biological replicates, and error bars display the range. All sample groups except for CREA levels were observed to take a normal distribution and were analyzed using the Brown–Forsythe and Welch one-way ANOVA with Dunnett’s T3 multiple comparisons significance test. The CREA groups followed a lognormal distribution and were analyzed using the Kruskal–Wallis one-way ANOVA with multiple comparisons significance test. Statistical significance was not observed. Abbreviations: ALT, alanine transaminase; ALP, alkaline phosphatase; ALB, albumin; TBIL, total bilirubin; CREA, creatinine; GLU, glucose.