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One Shock, Not One Cure: Electroporation Reveals Disease-Specific Constraints in Hepatocyte Gene Editing Therapy
by
, , , , , , , , , and
Callie Clark
1,
Menam Pokhrel
1,
Benjamin Arthur
1,
Pramita Suresh
1,
Ilayda Ates
1,
Justin Gibson
1,
Abishek Dhungana
1,
Ryan Mehlem
1,
Andrew Boysia
1,
Mugdha V. Padalkar
2,
Achala Pokhrel
3,
Jing Echesabal-Chen
3,
Anne Vonada
4,
Alexis Stamatikos
3,
Olga V. Savinova
2,
Markus Grompe
4 and
Renee N. Cottle
1,5,* 1
Department of Bioengineering, Clemson University, Clemson, SC 29634, USA
2
Department of Biomedical Sciences, New York Institute of Technology College of Osteopathic Medicine, Old Westbury, NY 115680, USA
3
Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC 29634, USA
4
Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR 97239, USA
5
Center for Human Genetics, Clemson University, Greenwood, SC 29646, USA
*
Author to whom correspondence should be addressed.
Biology 2025, 14(8), 1091; https://doi.org/10.3390/biology14081091
Submission received: 6 June 2025
/
Revised: 13 August 2025
/
Accepted: 16 August 2025
/
Published: 20 August 2025
Simple Summary
Liver transplantation remains the only definitive treatment for inherited metabolic liver diseases, but it carries significant drawbacks. In earlier research, we showed that we could use a gene-editing tool called CRISPR-Cas9 to engineer liver cells (called hepatocytes) and then help those edited hepatocytes to expand in the liver using short-term treatment with the common pain reliever acetaminophen (APAP). This cell therapy approach successfully cured a mouse model of phenylketonuria, a rare genetic disease of the liver. Here, we applied this approach to a different genetic liver disease, familial hypercholesterolemia, which is characterized by high cholesterol levels and increased risks of developing atherosclerotic cardiovascular disease. We gene-edited healthy liver hepatocytes and transplanted them into mice with the disease, then used APAP to select for the gene-edited hepatocytes in the liver. The gene-edited hepatocytes engrafted up to 13% of liver hepatocytes and the lipid levels decreased. However, disabling the gene (Cypor) using CRISPR-Cas9 for APAP selection also caused problems with how fats were processed in the liver. Collectively, our results suggest that while our cell-based gene editing approach can work well in some liver diseases, it might not be the best fit for all of them.
Abstract
We previously demonstrated lipid nanoparticle-mediated CRISPR-Cas9 gene editing to disrupt the gene encoding cytochrome P450 oxidoreductase (Cypor), combined with transient administration of acetaminophen (APAP), to repopulate the liver with healthy hepatocytes and rescue a phenylketonuria mouse model. This study aimed to investigate electroporation-mediated delivery of Cypor-targeting CRISPR-Cas9 ribonucleoproteins into wild-type hepatocytes, combined with liver engraftment under APAP treatment, as an in vivo selection approach in a mouse model of homozygous familial hypercholesterolemia (Ldlr−/−). Electroporation provides higher delivery efficiency compared to lipid nanoparticles. We observed engraftment levels up to 13% engraftment of electroporated Cypor-deficient hepatocytes with indels in the liver of Ldlr−/− mice after transient APAP administration, while negligible engraftment was observed in no-APAP controls (mean 9% and 2%, respectively, p = 0.0121). The engraftment of Cypor-deficient Ldlr+/+ hepatocytes was associated with reductions in LDL-cholesterol (18%) and triglycerides (52%) compared to the untransplanted control Ldlr−/− mice fed a Western diet for 5 weeks, but offered no protection from the development of diet-induced aortic root atherosclerosis or liver steatosis. While biochemical markers for liver damage normalized after discontinuation of APAP, we observed persistent lipid accumulation in the liver of Ldlr−/− mice grafted with Cypor-deficient Ldlr+/+ hepatocytes, likely stemming from the impact of Cypor deficiency on cholesterol clearance. Therefore, the combination of CRISPR-Cas9-mediated Cypor knockdown to induce clonal expansion of gene-edited hepatocytes using transient APAP administration is not a viable therapeutic strategy for familial hypercholesterolemia due to the essential role of Cypor in cholesterol metabolism. Unlike findings from phenylketonuria mouse model studies, the loss of Cypor function could not be compensated by unedited native hepatocytes in Ldlr−/− mice. Collectively, our results demonstrate that electroporation is a viable and informative approach for evaluating gene editing strategies for the treatment of inherited metabolic diseases that affect the liver. Our electroporation procedure revealed that a one-size-fits-all gene editing strategy may not be universally applicable for treating inherited metabolic liver disorders. Tailored gene editing and selection strategies may be needed for different liver disorders.
