Research Overview

More than half of intractable diseases -- conditions with unclear onset mechanisms and no effective treatment -- are thought to involve genetic mutations. To develop novel therapies for these diseases, genetic repair technologies and patient-derived iPS cells serve as powerful tools. The Hotta laboratory is engaged in the development of a range of technologies with the intractable disease muscular dystrophy as its main target.

The emergence of CRISPR-Cas9 and other genome editing technologies has made it possible to partially rewrite target genes on the human genome. However, as different diseases and patients display different types of genetic mutations, repair is impossible without applying the appropriate genome editing technology in the right method. For Duchenne muscular dystrophy, we developed a genomic exon skipping method using CRISPR-Cas9 and demonstrated in patient-derived iPS cells that it can repair the genetic mutation. We are also focusing on the unique ability of the novel CRISPR-Cas3 system to delete a large genome sequence in one direction from the target site, and are aiming to use this feature to further expand the scope of genetic mutation repair by genome editing. Additionally, to deal with the risk of off-target mutations by genome editing, we have been exploring multiple perspectives for enhancing the safety of the CRISPR system.

After verification of the successful genetic repair in cultured cells in the laboratory, it is necessary to deliver CRISPR-Cas tools to the patient's body to repair genetic mutation. For this, we have developed a number of relevant technologies such as NanoMEDIC virus-like particle, which is able to deliver CRISPR-Cas9 protein into living skeletal muscle tissue with high efficiency and low toxicity, and lipid nanoparticle (LNP) to deliver CRISPR-Cas9 mRNA as joint research with Takeda Pharmaceutical Company.

In the later stage of muscular dystrophy, there is a marked reduction in skeletal muscle cells. Therapy based on genetic repair alone is therefore unlikely to be sufficient at sites lacking in muscle cells, where new skeletal muscle cells and other tissue must be supplemented. In addition to autologous cell therapy with genetically repaired patients' own iPS cells, allogeneic (isogeneic) cell therapy using iPS cells from healthy volunteers has several advantages in manufacturing. However, in the latter case, immune rejection due to the mismatch of HLA haplotypes is a major concern. To reduce the riks of immune rejection, we have developed a method of genome editing selective HLA genes of iPS cells.

The Hotta laboratory will continue to engage in developing unique technologies, utilizing CRISPR-Cas9, Cas3, and other genome editing technologies for genetic mutation repair at the molecular level; virus-like particles (VLP) and lipid nanoparticles (LNP) delivery technologies at the nanoparticle level; and HLA modification for immune escape at the cellular level. By combining the range of scientifically advanced technologies, we aim to uncover the close but still unknown world of the genome and develop innovative therapies for muscular dystrophy and beyond.