Precise Modeling of Mitochondrial Diseases: Research Based on Optimized Mitobes

Academic Background

Mitochondrial diseases are a group of genetic disorders caused by mutations in mitochondrial DNA (mtDNA), which affect cellular energy metabolism and lead to dysfunction in multiple organs. Mitochondrial DNA mutations can be homoplasmic (affecting all mtDNA copies) or heteroplasmic (coexistence of mutant and wild-type mtDNA). These mutations are relatively rare in the population but can cause severe clinical symptoms, such as Leigh syndrome and Leber’s hereditary optic neuropathy (LHON). Due to the lack of suitable animal models, research and treatment progress in mitochondrial diseases have been limited. Therefore, developing animal models that can precisely mimic human mitochondrial diseases is crucial.

This study aims to optimize mitochondrial base editors (mitobes) to reduce off-target effects and improve editing efficiency and precision, thereby providing tools for the precise modeling of mitochondrial diseases. Through this technology, researchers can simulate human mitochondrial disease mutations in mouse models, laying the foundation for disease mechanism research and therapeutic strategy development.

Source of the Paper

This research was conducted by Xiaoxue Zhang, Xue Zhang, Jiwu Ren, Jiayi Li, Xiaoxu Wei, Ying Yu, Zongyi Yi, and Wensheng Wei. The research team is affiliated with Changping Laboratory, Peking University Genome Editing Research Center, and Peking-Tsinghua Center for Life Sciences. The paper was accepted by Nature on November 28, 2024, and published online in December 2024.

Research Process

1. Optimization and Development of Mitobes

The research team first optimized mitochondrial base editors (mitobes) to reduce their off-target effects on the transcriptome and mitochondrial genome. Mitobes are gene-editing tools that combine single-stranded DNA deaminase with a nickase, enabling C-to-T and A-to-G base editing in mitochondrial DNA. To further improve editing efficiency and precision, the researchers engineered the deaminases.

• Optimization of A-to-G Editors: Through saturation mutagenesis screening, the researchers found that the V28F mutation in the Tada8e-V106W protein significantly improved editing efficiency and reduced off-target effects at the transcriptome level. The optimized mitoABE v2 increased editing efficiency in mouse cells from 14% to 26%.

• Optimization of C-to-T Editors: The researchers tested 16 different cytosine deaminases and found that CBE6D performed best in terms of editing efficiency and off-target effects. The optimized mitoCBE v2 achieved 60% editing efficiency in mouse cells, with significantly reduced off-target effects.

2. Mitochondrial DNA Editing in Mouse Models

Using the optimized mitobes v2, the researchers targeted 70 mouse mitochondrial DNA sites analogous to human pathogenic mutations. By injecting circRNA-encoded mitobes v2 into mouse zygotes, the researchers successfully achieved up to 82% editing efficiency in mice, with no detectable off-target effects in the nuclear genome.

• Evaluation of Editing Efficiency: The researchers assessed editing efficiency in mouse embryos and F0 generation mice. The results showed that circRNA-encoded mitobes v2 had significantly higher editing efficiency in mouse embryos compared to mRNA-encoded versions, reaching up to 65%-82%.

• Persistence and Tissue Distribution of Editing: The researchers measured editing efficiency in 2-month-old and 6-month-old mice, finding that the editing effects persisted across multiple tissues and remained stable in different tissues.

3. Inheritance of Mitochondrial DNA Editing

By breeding edited female mice with wild-type male mice, the researchers studied the inheritance of mitochondrial DNA editing. The results showed that edited mitochondrial DNA could be maternally inherited, with some F1 generation mice exhibiting mutation loads as high as 100%. This indicates that mitobes v2 can produce mouse models with high mutation loads, providing an important tool for studying the genetic mechanisms of mitochondrial diseases.

4. Assessment of Disease Phenotypes

The researchers further evaluated the disease phenotypes of edited mice. Mice carrying the mt-ATP6 T8591C mutation exhibited reduced heart rate and left ventricular ejection fraction, consistent with symptoms of Leigh syndrome. In contrast, mice carrying the mt-ND5 A12784G mutation showed decreased visual acuity, similar to symptoms of Leber’s hereditary optic neuropathy (LHON).

Research Conclusions

By optimizing mitobes, the researchers successfully developed an efficient and precise mitochondrial DNA editing tool that can simulate human mitochondrial disease mutations in mouse models. This tool not only has high editing efficiency and low off-target effects but also allows the edited mitochondrial DNA to be maternally inherited by offspring. The results demonstrate that mitobes v2 can effectively mimic the phenotypes of mitochondrial diseases such as Leigh syndrome and LHON, providing an important tool for disease mechanism research and therapeutic strategy development.

Research Highlights

1. High Editing Efficiency and Low Off-Target Effects: The optimized mitobes v2 achieved up to 82% editing efficiency in mouse models, with no detectable off-target effects in the nuclear genome.

2. Persistence and Tissue Distribution: The edited mitochondrial DNA persisted across multiple tissues, with stable editing efficiency in different tissues.

3. Maternal Inheritance: The edited mitochondrial DNA could be maternally inherited, with some F1 generation mice exhibiting mutation loads as high as 100%.

4. Precise Simulation of Disease Phenotypes: The researchers successfully simulated the phenotypes of mitochondrial diseases such as Leigh syndrome and LHON, providing an important tool for disease mechanism research and therapeutic strategy development.

Research Significance

This study provides an important tool for the precise modeling of mitochondrial diseases, helping researchers better understand the genetic mechanisms of these diseases and laying the foundation for developing new therapeutic strategies. The high editing efficiency and low off-target effects of mitobes v2 make it a promising tool for future gene therapy applications.