Science and Technology Platform Program for Advanced Biological Medicine


Establishment of ultra-high-precision gene correction method for treatment of hereditary intractable diseases

<Project Leader> Shinichiro Nakada

Institute for Advanced Co-Creation Studies, Osaka University

Shinichiro Nakada

To date, many researchers have considered correcting mutations in hereditary diseases. Recently, gene editing efficiency has been dramatically improved by the development of the CRISPR/Cas system as a new genome editing technology. Now, genetic correction for hereditary intractable diseases can be expected as a practical treatment.

Whether ex vivo gene correction, which returns cells that have undergone gene modification outside the body, or in vivo gene correction, where gene modification takes place in the body, new risks from genetic corrections should be avoided as much as possible. When using cells with unintended mutations in medicine, patients are at unpredictable risk. Unfortunately, it has been pointed out that genome editing using CRISPR/Cas generates many unintended mutations. For the practical use of genome editing medicine, improving the safety of genome editing methods, namely, reducing unintended mutations is considered an important research topic. Sources of unintended mutations caused by genome editing are DNA double-strand breaks generated by CRISPR/Cas and exogenous DNA used for gene correction. DNA double-strand breaks frequently induce insertion and deletion mutations (indels), and exogenous DNA generates mutations by random integration into the genome. Most recently, Base Editor and Prime Editor have been developed as genome editing technologies that eliminate these risks and improve genome editing efficiency. However, enzymes such as deaminase and reverse transcriptase used in these technologies may cause unintended mutations. We have been developing a high-precision gene modification method (SNGD method) that eliminates the risk of unintended mutations caused by DNA double-strand breaks (Nakajima et al. Genome Res. 2018, Japanese Patent Application 2016-131382). This method achieves higher gene correction efficiency for point mutations and single nucleotide insertion mutations, and remarkable suppression of indel generation at the on-target site compared to the conventional method using DNA double-strand breaks. In addition, several researchers have demonstrated that accurate gene editing by the SNGD method has been achieved efficiently in iPS cells. In this research and development project, we aim to minimize the occurrence of unintended mutations in cells that have successfully corrected the gene. The new gene correction technology we are developing meets the following requirements. (1) No indel derived from DNA double-strand breaks at the off-targets, (2) no exogenous DNA random integration, (3) no DNA/RNA mutation induced by enzymes other than Cas9, (4) And the genome editing tool does not remain in the cell. If this technology makes it possible to correct genes accurately in cells with differentiating potential, it is expected that this technology will contribute to the development of genome editing medicine.

Figure 1 Figure 1: Development of a genetic correction method for hereditary intractable diseases.
The long-term goal of this research project is to correct genes in patient-derived cells in a highly safe manner. The cells are then used to treat the patient with hereditary intractable disease, leading to cure of the disease or reduction of symptoms. During this research and development period, we will establish accurate gene correction technology and demonstrate safety.
Figure 2 Figure 2: Weaknesses of existing genome editing methods (problems that this research group aims to overcome)
The conventional method has a problem that insertion and deletion mutations occur frequently. Newly developed genome editing methods such as BE and PE have weaknesses such as off-target mutations caused by newly used enzymes. In this study, we aim to overcome these weaknesses.
Figure 3 Figure 3: Cells that have been genetically corrected by the method under development.
Gene correction was performed on the thymidine kinase gene mutant cells. It can be confirmed that cells that have succeeded in gene correction restore DNA synthesis by the salvage pathway and proliferate. Cells are growing in the wells where the culture has turned yellow.
Figure 4 Figure 4: Our team
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