High-precision Base Editing Technology in Primates and Human Retina

Research Background

Stargardt disease is a currently untreatable, inherited neurodegenerative disease that leads to macular degeneration and blindness due to loss-of-function mutations in the ABCA4 gene. The protein encoded by the ABCA4 gene is a membrane lipid flippase localized in photoreceptors and retinal pigment epithelial cells (RPE cells), responsible for preventing the accumulation of toxic retinoids in the retina. The most common mutation in Stargardt disease is the c.5882G>A (p.Gly1961Glu) point mutation in the ABCA4 gene, which causes a loss of protein function, thereby triggering the disease.

Although there have been some studies on base editing in cell lines and mouse models, achieving efficient gene editing in human and non-human primate (NHP) neural tissues remains a challenge. This study aims to develop an adenine base editing strategy based on adeno-associated virus (AAV) to correct the c.5882G>A mutation in the ABCA4 gene and verify its efficiency and precision in human and non-human primate models.

Source of the Paper

This paper was collaboratively completed by scientists including Alissa Muller, Jack Sullivan, and Wibke Schwarzer from multiple research institutions, with primary authors from the Institute of Molecular and Clinical Ophthalmology Basel, Beam Therapeutics, and others. The paper was published in February 2025 in the journal Nature Medicine, titled “High-efficiency base editing in the retina in primates and human tissues.”

Research Workflow and Detailed Methods

1. Design and Optimization of the Base Editing Strategy

The research team first designed a dual AAV vector encoding a split-intein adenine base editor (ABE) to correct the c.5882G>A mutation in the ABCA4 gene. To optimize editing efficiency, the research team conducted ABCA4 base editing optimization in human models, including retinal organoids, induced pluripotent stem cell (iPS cell)-derived RPE cells, adult retinal explants, and RPE/choroid explants.

2. Validation of Base Editing in In Vitro Models

The research team tested different base editors and guide RNAs (gRNAs) in HEK293T cell lines carrying the ABCA4 c.5882G>A mutation and screened out the most efficient gRNA. Subsequently, the research team validated the efficiency of base editing in human retinal organoids, retinal explants, and RPE/choroid explants. The results showed that the editing efficiency of the ABCA4 gene reached high levels in in vitro models.

3. Validation of Base Editing in Mouse Models

The research team tested the AAV9-PHP.eB vector in mouse models and delivered the base editor to the retina via subretinal injection. The results showed that the editing efficiency of the ABCA4 gene in the mouse retina and RPE/choroid tissue was relatively high, with no detectable off-target editing.

4. Validation of Base Editing in Non-Human Primate Models

The research team further validated the efficiency of base editing in non-human primate models (NHPs). By subretinal injection, the research team delivered the AAV5-SABE1 vector to the NHP retina and evaluated the editing efficiency. The results showed that the editing efficiency of the ABCA4 gene in the NHP retina and RPE/choroid tissue reached high levels, with significant editing effects observed in cone cells and RPE cells.

5. Optimization of the Base Editor and Dose Effect

The research team further optimized the packaging efficiency of the AAV vector and tested the editing efficiency of different doses of the AAV5-SABE1 vector in NHPs. The results showed that the optimized AAV5-V2-SABE1 vector could still achieve efficient gene editing at lower doses, with significantly improved editing efficiency in cone cells and RPE cells.

Main Research Results

1. Efficient Base Editing in In Vitro Models: In human retinal organoids, retinal explants, and RPE/choroid explants, the editing efficiency of the ABCA4 gene reached high levels, with no detectable off-target editing.
2. Efficient Base Editing in Mouse Models: In the mouse retina and RPE/choroid tissue, the editing efficiency of the ABCA4 gene was relatively high, with no detectable off-target editing.
3. Efficient Base Editing in Non-Human Primate Models: In the NHP retina and RPE/choroid tissue, the editing efficiency of the ABCA4 gene reached high levels, with significant editing effects observed in cone cells and RPE cells.
4. Optimization of the Base Editor and Dose Effect: The optimized AAV5-V2-SABE1 vector could still achieve efficient gene editing at lower doses, with significantly improved editing efficiency in cone cells and RPE cells.

Research Conclusions and Significance

This study demonstrates the feasibility of achieving efficient and precise base editing in human and non-human primate retinas, providing a new strategy for gene therapy of Stargardt disease. The results show that AAV-based adenine base editing technology can efficiently correct the c.5882G>A mutation in the ABCA4 gene, with significant editing effects observed in cone cells and RPE cells. Additionally, by optimizing the packaging efficiency of the AAV vector, the research team further improved the efficiency of base editing and reduced the required vector dose.

Research Highlights

1. Efficient Base Editing: This study achieved efficient base editing in human and non-human primate retinas, providing a new strategy for gene therapy of Stargardt disease.
2. Precision: The results show that base editing exhibits high precision at the target site, with no detectable off-target editing.
3. Optimized AAV Vector: By optimizing the packaging efficiency of the AAV vector, the research team further improved the efficiency of base editing and reduced the required vector dose.
4. Validation in Non-Human Primate Models: The efficiency of base editing was validated in non-human primate models, providing important references for clinical trials.

Other Valuable Information

The research team also analyzed the off-target effects of base editing through whole-genome screening. The results showed no detectable off-target editing in human explants, further demonstrating the precision of base editing. Additionally, the research team evaluated the editing efficiency of base editing in other tissues outside the retina, showing that editing was limited to the retina and RPE/choroid tissue.

The success of this study provides important references for the treatment of other hereditary retinal diseases targetable by base editing, showcasing the enormous potential of base editing technology in the treatment of genetic diseases.