Breakthrough in Gene Therapy: Repair Drive Technology Enables In Vivo Expansion of Hepatocytes

Academic Background

Gene therapy has become a hot topic in medical research in recent years, especially for liver diseases. Due to the central role of the liver in metabolism, it has become a critical target for research. Although existing gene-editing technologies like CRISPR-Cas9 have made significant progress in gene knockout, most liver diseases require gene correction rather than disruption. However, the efficiency and precision of gene correction in terminally differentiated liver cells are severely limited, significantly restricting its clinical application. To address this issue, the research team developed a new technology called Repair Drive, which aims to selectively expand hepatocytes repaired via Homology-Directed Repair (HDR) by temporarily inhibiting an essential gene, thereby improving the efficiency of gene correction.

Source of the Paper

This paper was authored by Marco De Giorgi and his team, with members from renowned institutions such as Baylor College of Medicine, Rice University, and Alnylam Pharmaceuticals. The paper was published on February 12, 2025, in the journal Science Translational Medicine, titled “In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene.”

Research Process

1. Study Design

The core goal of the study was to develop a technology that could selectively expand HDR-repaired hepatocytes in vivo. The Repair Drive technology achieves this by temporarily inhibiting an essential gene, Fah (fumarylacetoacetate hydrolase), causing unrepaired hepatocytes to gradually die while allowing repaired hepatocytes to survive and proliferate.

2. Experimental Steps

a) Gene Editing and siRNA-Mediated Gene Suppression

The research team used adeno-associated virus (AAV) as a vector to deliver the CRISPR-Cas9 system and a repair template (donor plasmid) into the mouse liver. The repair template included the human Fah gene, which is resistant to siRNA, along with a therapeutic gene (such as human Factor IX, FIX). Mice were then injected with siRNA targeting the murine Fah gene to achieve transient suppression of Fah.

b) Liver Conditioning and Selective Expansion

Through multiple siRNA injections, the researchers simulated the liver conditioning process. With the Fah gene suppressed, unrepaired hepatocytes died due to the accumulation of toxic metabolites, while repaired hepatocytes survived and expanded. The team used fluorescent markers (e.g., tdTomato) to observe the expansion of repaired cells and employed digital PCR (ddPCR) and long-read sequencing to quantify gene editing events.

c) High-Protein Diet to Enhance Selection Pressure

To further increase selection pressure, the team conducted high-protein diet experiments, accelerating the death of unrepaired hepatocytes by enhancing tyrosine metabolism, thereby further promoting the selective expansion of repaired cells.

d) Long-Term Safety and Sustained Gene Expression

To evaluate the long-term safety and sustained gene expression of the Repair Drive technology, the team conducted a one-year follow-up, observing liver pathology, the persistence of gene expression, and potential long-term side effects such as tumorigenesis.

3. Data Analysis

The research team employed various advanced data analysis methods, including: - Digital PCR (ddPCR): To quantify the frequency of HDR and Non-Homologous End Joining (NHEJ) events. - Long-Read Sequencing: To analyze the detailed structure of gene editing events, distinguishing between different types of HDR and NHEJ events. - Single-Nucleus RNA Sequencing (snRNA-seq): To analyze the transcriptomic characteristics of repaired cells, revealing their distribution and function in different liver regions.

Key Results

1. Selective Expansion Efficacy

The study found that Repair Drive technology significantly increased the proportion of HDR-repaired hepatocytes. In healthy mice, the proportion of repaired cells reached 25%, and the expression of the therapeutic gene (e.g., FIX) increased fivefold. Additionally, with a high-protein diet, the proportion of repaired cells further increased to 24.6%.

2. Precision of Gene Editing Events

Through long-read sequencing, the team found that Repair Drive technology significantly reduced the occurrence of NHEJ events while increasing the frequency of HDR events. Although some unintended gene editing events (e.g., AAV genome insertion) persisted, these did not significantly affect the function of the repaired cells.

3. Long-Term Safety and Tolerability

In the one-year follow-up, Repair Drive technology demonstrated excellent safety and tolerability. Liver function and body weight in mice showed no significant changes, and there were no signs of tumorigenesis. Although a few mice developed localized proliferative lesions, these did not exhibit malignant characteristics.

Research Conclusion

Repair Drive technology, through the temporary inhibition of the essential gene Fah, achieved selective expansion of HDR-repaired hepatocytes in vivo, significantly improving the efficiency of gene therapy. This technology not only effectively increases the expression of therapeutic genes but also demonstrates excellent long-term safety and tolerability. The success of this technology provides new insights for the treatment of liver diseases, particularly for genetic disorders requiring gene correction rather than gene knockout.

Highlights of the Research

1. Innovative Technology: Repair Drive technology is the first to achieve selective expansion of repaired hepatocytes in vivo by temporarily inhibiting an essential gene, addressing the inefficiency of HDR in terminally differentiated tissues.
2. Efficiency and Precision: The technology significantly increases the frequency of HDR events while reducing unintended NHEJ events, ensuring the precision of gene correction.
3. Long-Term Safety: The one-year follow-up results indicate that Repair Drive technology has excellent safety and tolerability, with no significant long-term side effects observed.
4. Broad Application Potential: This technology can not only be used to treat coagulation disorders (e.g., hemophilia B) but can also be extended to other genetic metabolic diseases requiring gene correction.

Other Valuable Information

The research team also developed a technique called GISA-seq to detect off-target integration events of AAV vectors across the entire genome. This technology effectively identifies the integration sites of AAV genomes in the host genome, providing a new tool for assessing the safety of gene therapy.

The success of Repair Drive technology opens new avenues for the gene therapy of liver diseases and holds promise for widespread clinical application, offering hope to patients with hereditary liver disorders.