Electroporation-based delivery for gene editing

Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder marked by severely elevated cholesterol and early atherosclerotic cardiovascular disease.¹ Liver transplantation is the only curative option, but it is limited by donor shortages, procedural risks, and lifelong immunosuppression.²

Over three decades ago, the first trial of ex vivo hepatocyte transplantation for HoFH failed to achieve sufficient lipid lowering effects.³ Although CRISPR-Cas9 now enables efficient gene editing, robust hepatocyte engraftment remains a major barrier for a cell therapy approach for HoFH.⁴

To overcome this challenge, our laboratory is developing nonviral strategies to perform gene editing in hepatocytes ex vivo to correct the HoFH phenotype and confer a selective advantage for their expansion in the liver. As a proof-of-concept of this therapeutic strategy, we used electroporation to deliver Cas9 ribonucleoproteins (RNP) to disrupt the gene encoding Cytochrome P450 reductase (Cypor) into healthy hepatocytes, followed by transplantation and transient acetaminophen (APAP) administration in a mouse model of HoFH.⁵

Gene-edited hepatocytes showed robust engraftment, underscoring the potential of our cell-therapy approach for the treatment of preclinical models of genetic diseases that affect the liver.^2,5

Genetic causes of FH

Familial hypercholesterolemia (FH) is the most common monogenic disorder and is characterized by markedly elevated plasma LDL-C levels from birth, up to 2-6 times higher than normal levels.⁶ Patients may also present with cutaneous or tendon xanthomas even during childhood, which are cholesterol deposits around eyelids, connective tissue and tendons.⁷ When left untreated, FH leads to accelerated, premature atherosclerotic cardiovascular disease.

FH has an autosomal semi-dominant inheritance pattern. Individuals with one copy of the pathogenic DNA variant have heterozygous FH, exhibiting an abnormal phenotype characterized by higher-than-normal cholesterol levels. Whereas individuals with two pathogenic DNA variants have HoH, which is characterized by extremely elevated LDL cholesterol levels, and more severe clinical features that can start in early childhood.⁸

Clinicians use genetic testing to characterize the pathogenic variants in the causative genes. Pathogenic variants causing FH occur primarily in three genes:⁸

• Low-density lipoprotein receptors (LDLR): the most common cause, occurring in 85-90% of patients; typically due to loss-of-function variants, resulting in defective or absent LDL receptors.⁹
• Apolipoprotein B (APOB): accounts for approximately 10% of patients; results in impaired LDL particle binding to the LDL receptor.
• Proprotein convertase subtilisin/kexin type 9 (PCSK9): occurring in less than 5% of patients; caused by gain-of-function variants that increase LDL receptor degradation.

However, despite advances in genetic diagnostics, FH is underdiagnosed and undertreated.

Current standard of care

After diagnosis, patients are typically started on statin therapy, the standard first-line treatment for FH. Statins inhibit hydroxymethylglutaryl-CoA (HMG-CoA) in the liver, reducing cholesterol synthesis and thereby upregulating LDL receptor expression.¹⁰

The efficacy of statins requires residual LDLR activity (>25%) and will not work at all in patients with very low LDLR activity or no functional LDLR. A major challenge associated with statins is risk for developing rhabdomyolysis which is a rare life-threatening condition where the muscle cells break down and are leaked into the bloodstream.¹¹ In most patients with HoFH, statins alone are largely inadequate and are therefore prescribed in combination with other lipid-lowering therapies.¹²

FH is also treated with PCSK9-directed therapies.¹³ Hepatocytes secrete PCSK9 to reduce the number of LDLR in the liver by targeting the LDLR to lysosomes for degradation. Two FDA-approved monoclonal antibodies, alirocumab and evolocumab, inhibit PCSK9 and lower LDL-C by ~30% on average (up to ~70%), depending on residual LDLR activity (>30%).¹⁴ Because LDL-C often remains above target, these agents are used in combination with other therapies and are ineffective in HoFH patients with null or severely impaired LDLR mutations.¹⁵

Another therapeutic approach for FH involves targeting angiopoietin-like 3 (ANGPTL3). ANGPTL3 is secreted by the liver and regulates lipid metabolism by inhibiting lipoprotein lipase and endothelial lipase enzymes responsible for hydrolyzing triglycerides and phospholipids. The FDA-approved monoclonal antibody Evinacumab binds and inhibits circulating ANGPTL3, thereby increasing lipoprotein and endothelial lipase activity. This promotes enhanced clearance of triglyceride-depleted LDL particles and lowers LDL cholesterol levels in an LDL receptor-independent manner.

Nucleic acid therapies represent an emerging approach for the treatment of FH. These approaches are based on RNA interference (RNAi) technology, which involves the delivery of small interfering RNAs (siRNAs) that are loaded into the RNA-induced silencing complex. When a target mRNA binds to the guide strand through sequence complementarity, the mRNA is cleaved and degraded. One such therapy is inclisiran, a siRNA that targets PCSK9 mRNA for degradation. When administered in combination with statin therapy, inclisiran has been shown to reduce LDL cholesterol levels by up to 50%. However, this therapy is effective in patients with heterozygous FH due to its dependence on residual LDL receptor activity and is not effective in patients with severe HoFH.

Non-pharmacological treatments, such as lipoprotein apheresis, remain a foundational therapy for patients with HoFH and are often combined with other lipid-lowering therapies. But lipoprotein apheresis has notable drawbacks, including rebound increases in LDL cholesterol after treatment and the need for frequent, often weekly, sessions, which create a significant time burden for patients.

Limited therapies for HoFH

Unfortunately, none of the FH therapies discussed so far are fully effective, particularly in patients with severe HoFH. These treatments are limited by both tolerability and efficacy issues, and patients with HoFH are often treated with multiple lipid-lowering medications, yet still fail to achieve target LDL cholesterol levels to reduce atherosclerotic cardiovascular disease risks.

Liver transplantation is the only curative therapy for FH but is reserved for a small subset of patients refractory to lipid-lowering treatments. Its use is limited by a severe donor organ shortage — approximately 9,424 patients were on the liver transplant waitlist in 2024 — and by substancial risks, including perioperative mortality and complications associated with lifelong immunosuppressive therapy. These limitations underscore the urgent need for novel FH therapies.

Novel therapeutic approaches

Gene editing to disrupt genes encoding for FH therapeutic targets in the liver, such as ANGPTL3 or PCSK9, represents a novel therapeutic approach for permanently treating FH.

The type II CRISPR system from Streptococcus pyogenes uses Cas9 to induce double-strand breaks at precise genomic locations adjacent to NGG protospacer-adjacent motif sequences that are complementary to the 20-nucleotide guide RNA.^16,17 The CRISPR-Cas9 system enables high levels of gene editing activity, and it is amenable to multiplex gene editing. However, a major drawback is the potential for off-target gene editing at sites with partial sequence complementarity to the guide RNA.

Gene editing using CRISPR-Cas9 relies on the induction of double-strand breaks at the target locus, which are repaired by two major DNA repair pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR).¹⁸ NHEJ is an error-prone pathway that introduces insertions or deletions at the break site, often resulting in frameshift mutations and premature stop codons that effectively knock out gene function. In some cases, NHEJ can also be harnessed to restore the reading frame.

HDR enables precise gene correction or insertion using a homologous donor template delivered alongside CRISPR-Cas9. However, a drawback of the HDR pathway is its dependence on the cell cycle, which occurs primarily during the late S and G2 phases. In contrast, the NHEJ pathway is active throughout the cell cycle, making it a more practical and promising pathway to exploit for gene editing in liver diseases.

Gene editing delivery challenges

The delivery of the CRISPR components into target cells remains a significant challenge in the gene editing field for liver diseases like FH, where the target cell type is hepatocytes.

Adeno-associated viral (AAV) vectors are the most commonly used delivery platform for introducing transgenes in animal models of liver disease and preclinical studies.¹⁹ However, AAVs present several limitations, including immunogenicity due to pre-existing immunity to the viral capsid and a restricted packaging capacity, which is particularly problematic for larger gene-editing systems such as base editors.^20,21 AAVs also carry a risk of insertional mutagenesis and can lead to prolonged Cas9 expression, increasing the likelihood of off-target editing and chromosomal translocation.²²

Lipid nanoparticles (LNPs) represent an alternative delivery strategy that enables transient expression of CRISPR components, such as Cas9 mRNA. Like AAVs, LNPs have been used clinically, including in a recent personalized gene-editing therapy for severe CPS1 deficiency at the CHOP. However, LNP-based delivery is limited by inefficient endosomal escape, often necessitating higher doses. While liver targeting is efficient, achieving cell-type-specific delivery in vivo remains difficult, with added risks of transfection-related toxicity and off-target organ exposure.

The first FDA-approved CRISPR therapy for sickle cell disease, Casgevy, uses an alternative delivery approach: electroporation.²³ Electroporation involves exposing cells to brief electrical pulses that transiently permeabilize the cell membrane, enabling highly efficient delivery of biomolecules, including CRISPR-Cas9 RNPs.

A key limitation of electroporation is that it is restricted to ex vivo gene therapies, where target cells can be isolated and manipulated outside the body and is therefore not feasible for in vivo gene editing. Diseases affecting inaccessible organs, such as the brain, heart, and skeletal muscle, electroporation is not feasible.

Therapeutic genetic editing approaches

There are two main therapeutic strategies for gene editing: in vivo and ex vivo approaches. In vivo gene editing involves delivering CRISPR components directly into the body using carriers such as viral vectors or LNPs. This strategy has already reached the clinic, including a personalized treatment for CPS1 deficiency at the CHOP and Intellia Therapeutics’ clinical trial for transthyretin amyloidosis.

The second strategy is an ex vivo approach, in which gene editing is performed outside the body in the target cell type. A prominent example is Casgevy, which involves isolating hematopoietic stem cells from the patient, editing them ex vivo using CRISPR-Cas9 delivered by electroporation, and reinfusing the modified cells back into the patient to engraft in the bone marrow.

Ex vivo gene editing has demonstrated strong clinical efficacy and offers important safety advantages over in vivo approaches. It avoids direct patient exposure to gene-editing reagents, thereby reducing pre-existing immunogenicity risks associated with Cas9. In addition, it enables delivery of transient gene-editing tools such as RNPs, allows edited cells to be expanded or functionally tested prior to transplantation, and provides a more controlled and potentially safer therapeutic pathway when feasible.

In vivo gene-editing approaches for FH have been explored in both preclinical and clinical studies. Preclinical studies using LNP delivery of base editors targeting ANGPTL3 have demonstrated reductions in LDL cholesterol levels in LDLR knockout mouse models and in non-human primates.^24,25

In LDLR-knockout primates, Verve Therapeutics reported a 46% reduction in LDL cholesterol levels. This strategy is currently being evaluated in clinical trials for patients refractory to existing PCSK9-directed therapies.²⁶ Verve is also separately evaluating PCSK9 base-editing approaches for the treatment of HoFH, with early clinical data showing a mean LDL cholesterol reduction of approximately 53%.

Our novel therapeutic strategy

Our lab’s therapeutic approach is inspired by early gene therapy trials conducted in the 1990s for FH and leverages modern gene-editing technologies. We aim to develop a curative, autologous therapy using hepatocytes isolated from the patient’s liver as the substrate.

In a clinical setting, the first step would involve resection of a small liver segment, preferably from the left lateral lobe, followed by perfusion with collagenases and mechanical dissociation to isolate hepatocytes. The second step would involve ex vivo CRISPR-Cas9–mediated gene editing to target therapeutic genes that both correct the disease phenotype and confer a selective advantage to the edited cells. The third step would involve transplantation of these cells back into the patient via the portal vein.

Finally, patients would be treated with a pharmacologic agent to promote the expansion of gene-edited hepatocytes and liver repopulation. Our approach leverages the liver’s high regenerative capacity to replace diseased hepatocytes with gene-modified ones that reduce LDL cholesterol.

Background on hepatocyte transplantation

Hepatocyte transplantation has been explored as an alternative to whole-organ liver transplantation and has been evaluated clinically for multiple indications, including FH and acute liver failure. In this approach, hepatocytes isolated from cadaveric donors — often from livers unsuitable for transplantation — are infused into recipients through the portal vein.

However, approximately 70-80% of transplanted cells are cleared by resident immune cells within three days, resulting in engraftment efficiencies below 1%.²⁷ Moreover, transplanted hepatocytes generally do not expand after engraftment, with the exception of a few rare inherited metabolic liver diseases.²⁸

Another drawback of hepatocyte transplantation is the need for immunosuppressive therapy to prevent rejection and dilution of engrafted cells in a growing liver, often necessitating eventual liver transplantation. As a result, hepatocyte transplantation has shown clinical benefit primarily as a temporary bridge to liver transplantation rather than a definitive therapy.

For most diseases, including FH, transplanted healthy hepatocytes lack a selective advantage to repopulate the liver.²⁸ Therefore, strategies that enable selective in vivo expansion of engrafted cells are critical to improving the efficacy and durability of cell-based therapies for liver disease.

Boosting hepatocyte engraftment in vivo

A major challenge in hepatocyte transplantation is achieving sufficient engraftment to correct liver disease. To address this, we are developing a novel strategy to enable in vivo clonal expansion of ex vivo gene-edited hepatocytes using CRISPR-Cas9–mediated disruption of Cypor, combined with transient administration of APAP.

At high doses, APAP is metabolized by cytochrome P450 enzymes into the toxic metabolite N-acetyl-p-benzoquinone imine, causing hepatocyte necrosis. Cypor is an essential cofactor for these enzymes, and its disruption renders hepatocytes resistant to APAP-induced toxicity. As a result, Cypor-deficient hepatocytes gain a selective advantage, clonally expanding as surrounding native hepatocytes undergo necrosis.²⁹

Proof of principle for this approach was demonstrated by our collaborator, Dr. Marcus Grompe, who showed that hydrodynamic delivery of CRISPR plasmids targeting Cypor, followed by repeated APAP administration, resulted in a >9-fold expansion of edited hepatocytes in vivo.

A nonviral ex vivo gene-editing platform

Building on this concept, our lab is developing a nonviral, ex vivo hepatocyte gene-editing platform. Our efforts focus on:^2,5,30,31

• Designing CRISPR guides targeting therapeutic FH target genes and conferring a selective advantage
• Delivering CRISPR-Cas9 RNPs by electroporation
• Assessing genomic safety, including off-target effects and translocations
• Evaluating engraftment and in vivo expansion following transplantation

Our overarching hypothesis is that hepatocytes gene-edited ex vivo using electroporation can clonally expand in vivo and repopulate the liver to treat inherited metabolic diseases.

We established robust hepatocyte isolation and electroporation protocols, transitioning from retrograde perfusion to Miltenyi Biotec’s gentleMACS Octo Dissociator, which improved reproducibility, yield and viability while reducing animal use. Using gentleMACS system, we routinely obtained >85% viability and high cell yields.Electroporation of CRISPR-Cas9 RNPs into freshly isolated hepatocytes achieved editing efficiencies approaching 80-90%, with preserved viability and plating capacity.

Validation in mouse models

To validate engraftment and functional correction, we used a mouse model of hereditary tyrosinemia type 1. The models have a loss-of-function mutation in the gene encoding fumarylacetoacetate hydrolase (Fah), a critical enzyme in the tyrosine metabolism pathway, leading to a toxic buildup of metabolites in the liver and elevated tyrosine levels.32 Hepatocytes were gene-edited ex vivo to disrupt the gene encoding 4-hydroxyphenylpyruvate dioxygenase (Hpd), an upstream enzyme in the pathway, then transplanted into Fah-/- recipients and subjected to NTBC cycling to promote selective expansion.^2,30

Gene-edited hepatocytes showed robust engraftment, restored NTBC independence in Fah-/- recipients, normalized amino acid levels, improved liver function markers, and prevented tumor formation ­— establishing a reliable preclinical platform for hepatocyte transplantation combined with ex vivo gene editing to treat inherited liver metabolic disease. In follow-up studies, electroporation using the FDA-approved MaxCyte GTX GMP-grade system achieved gene-editing efficiency, viability and engraftment comparable to our research-grade Lonza platform.²

We next applied this strategy to a mouse model of HoFH. Hepatocytes from wild-type donors were gene-edited ex vivo to disrupt Cypor, transplanted into Ldlr-/- mice, and subjected to repeated APAP administration.⁵ ALT kinetics confirmed selection of APAP-resistant cells, and genomic analysis demonstrated ~9% liver-wide indels, indicating successful engraftment and expansion of gene-edited hepatocytes. Immunofluorescence staining revealed large clonal patches of Cypor-deficient hepatocytes, with no evidence of hepatotoxicity.

Following selection, mice were placed on a Western diet to assess durability and metabolic impact. Engrafted cells persisted, and lipid analysis revealed ~18% reduction in LDL cholesterol and ~52% reduction in triglycerides.⁵ However, atherosclerotic plaque burden was unchanged. Histological analysis revealed lipid accumulation within Cypor-deficient hepatocytes, suggesting impaired cholesterol clearance due to disrupted P450-dependent pathways.

These findings indicate that while Cypor-based selection enables robust expansion, it negatively affects lipid handling, motivating follow-up studies targeting cytochrome P450 enzymes specifically responsible for metabolizing APAP to preserve cholesterol metabolic functions.

Translation and clinical relevance

In summary, we validated the use of the FDA-approved MaxCyte GTX GMP-grade electroporation system for gene editing in hepatocytes, demonstrating editing efficiency, viability and engraftment comparable to research-grade devices. In Fah-/- mice, this approach achieved near-complete liver repopulation and phenotypic correction, supporting translational feasibility.²

We demonstrated that hepatocytes gene-edited ex vivo by electroporation retain robust engraftment capacity when recovered in a defined cytokine medium. Using this platform, we established a proof of principle for a cell-based gene-editing therapy for hereditary tyrosinemia type 1, achieving complete rescue of the disease phenotype with CRISPR.

We further showed that APAP-mediated selection of Cypor-deficient hepatocytes gene-edited with Cas9 RNPs is feasible in a Ldlr-/- mouse model of FH. In addition, we demonstrate that the GentleMACS dissociation system provides a rapid, reproducible method for isolating viable primary hepatocytes for transplantation studies.⁵

Collectively, these findings establish the feasibility of combining nonviral ex vivo gene editing, hepatocyte transplantation, and in vivo selection, and support the clinical translation of cell-based gene-editing therapies for inherited metabolic liver diseases.

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