A new possibility has emerged for the development of gene therapy for mitochondrial genetic diseases. The Center for Genome Engineering at the Institute for Basic Science has developed a gene editing technique that can correct adenine bases in mitochondrial genes for the first time in the world. To fully understand this research, it is important to grasp the concepts and significance of the study, which the researchers will explain here.

Mitochondrial genetic diseases

Mitochondria exist in all eukaryotic cells, including humans. They serve as powerhouses generating chemical energy within cells, and due to their importance, problems with mitochondria can affect cell and tissue function. These issues typically arise when there are mutations in mitochondrial DNA, which can be passed down maternally.

Genetic diseases caused by various mutations in human mitochondrial DNA occur at a rate of 1 in 5,000 individuals. These diseases are not only associated with maternal inheritance but are also closely linked to conditions such as cancer, diabetes, and age-related diseases. Given their role in energy production, mitochondrial genetic diseases are highly pathogenic. For example, Leber's Hereditary Optic Neuropathy (LHON), one of the most well-known mitochondrial genetic conditions, primarily causes visual impairment and can lead to blindness. Other diseases include Mitochondrial Encephalopathy, which can cause brain degeneration, and Mitochondrial Myopathy, which affects muscle function. Most mitochondrial genetic diseases are highly pathogenic, often difficult to diagnose, and currently have no cure, with only palliative measures available to alleviate symptoms or delay progression.

[Figure 1] Mitochondrial DNA in Eukaryotic Cells (Source: Wikipedia; Mitochondrial DNA)

[Figure 2] Well-known YouTuber with Mitochondrial LHON Disease (Source: YouTube; OneShotHanSol)

Existing mitochondrial gene correction techniques

Mitochondria have their own DNA, separate from the nuclear genome DNA. Currently, there are a total of 95 clinically confirmed pathogenic mitochondrial DNA mutations. Among these, 90 (95%) are point mutations, which occur due to a single change in DNA base. Therefore, by using single-base correction techniques, the majority of pathogenic mitochondrial genetic diseases can be treated. However, despite the development of various gene correction techniques, correcting single bases in mitochondrial DNA was considered impossible until recently.

However, in 2020, a technique for correcting cytosine (C) bases in mitochondria (DdCBE) was published in Nature. Theoretically, this technique could correct 42 mutations, accounting for approximately 47% of all mitochondrial diseases. However, since the technique requires thymine (T) to be present before cytosine, the number of mutations that can be corrected is realistically limited to 9 out of the 95 mutations (10%). For research and treatment of mitochondrial genetic diseases, it was necessary to not only improve the existing technique of replacing cytosine with thymine but also develop a new technique to replace adenine (A) with guanine (G).

[Figure 3] Single nucleotide polymorphism (SNP) diversity of clinically confirmed mitochondrial genetic diseases. Nearly half involve changes from guanine to adenine and the other half from thymine to cytosine. Each can be theoretically corrected by (C to T) cytosine base correction technology, or (A to G) adenine base correction technology, respectively.

Gene Correction Technology Developed Domestically

In 2020, the cytosine base correction technology was initially developed by the Broad Institute in the United States, and it was also subsequently independently developed by the IBS Center for Genome Engineering. This base correction tool employs a different protein (using zinc finger proteins) to target a narrower range of the DNA, resulting in a more precise base correction technique. This achievement was published in Nature Communications.

Furthermore, the Center recently pioneered the world's first gene correction technology to correct adenine bases in mitochondrial DNA. This technology has the ability to correct 39 out of 90 point mutations, approximately 43%. Compared to the previous mitochondrial disease correction rate of only 10%, adding this new technique now can target over 50% of mutations. As a result of this research, various animal models related to mitochondrial diseases have been developed, and a new possibility of treating genetic diseases by correcting mitochondrial mutations has emerged.

[Figure 4] Schematic of Adenine Base Correction Technology. The adenine base correction technology, TALED, attaches to mitochondrial DNA and corrects A to G bases. MTS represents the mitochondrial targeting sequence, TALE repeats are the DNA-binding proteins, and both Split DddA and AD are thymine-deaminating enzymes. (Source: Cell)

Limitations and how to overcome them

While having a perfect technology would be ideal, there are currently two main limitations to mitochondrial gene correction technology. First is the limitation of cytosine base correction technology. As mentioned earlier, current cytosine base correction technology can only correct cytosines in TC sequences, making it unusable for gene sequences containing AC, GC, or CC. However, challenges are being undertaken worldwide to develop technologies that work well in sequences other than TC. Although a definitive technology has not yet emerged, various attempts are being made.

Second are the side effects. For gene correction technology to work, the editor must first attach to the gene. While ideally, it would attach to specific sequences (on-target sequences) and correct them, sometimes it may attach to unintended locations and cause undesired corrections. These unintended sequences are called off-target sequences. Since these off-target sequences generate unwanted mutations, they can cause unexpected side effects. For example, researchers have confirmed that adenine base correction technology can also act on RNA, not just DNA. When this technology attaches to RNA, it can cause a large number of off-target sequences due to nonspecific binding. In mouse models, these off-target sequences have been observed to affect embryonic development, leading to embryo death. To reduce these off-target sequences, researchers are redesigning the deaminase enzyme's DNA/RNA binding regions to reduce attachment to RNA off-target sequences while maintaining its affinity to on-target sequences

[Figure 5] 3D Structure of Adenine Deaminase Enzyme. The red image represents the target DNA, while the surrounding blue images depict the nearby amino acid residues.

This is how scientists develop new technologies and improve them by identifying and overcoming their limitations.

Taking a closer look at the once-neglected field of mitochondrial genetic diseases

As mentioned earlier, mitochondria serve as the powerhouses of cells, generating energy within living organisms including all animals and plants. Therefore, genetic disorders caused by mutations in mitochondrial DNA typically result in severe illnesses. Research outputs such as these in South Korea could offer solutions to mitochondrial genetic diseases that were previously considered impossible. Moreover, since the technology could potentially function in chloroplasts, which are organelles found in plants, it may have broader applications. This extends beyond molecular biology and genetics, potentially contributing to biopharmaceuticals, biotechnology, agriculture, forestry, fisheries, environmental industries, and more.

References

• ● Cho, S.I., Lee, S., Mok, Y.G., Lim, K., Lee, J., Lee, J.M., Chung, E., and Kim, J.S. (2022). Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell 185, 1764-1776 e1712. 10.1016/j.cell.2022.03.039.
• ● Lim, K., Cho, S.I., and Kim, J.S. (2022). Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases. Nat Commun 13, 366. 10.1038/s41467-022-27962-0..