Genome editing, which won the 2020 Nobel Prize in Chemistry, is seeing active movement toward industrial applications as a groundbreaking technology for rewriting the genetic information of organisms. "The Center of Innovation for Bio-Digital Transformation (Bio-DX)" is led by Professor Takashi Yamamoto, Director of the Genome Editing Innovation Center at Hiroshima University, and Professor Bono Hidemasa of the Graduate School of Integrated Sciences for Life at the same university, who specializes in genome analysis. The center is promoting research and development on the digitalization and programming of organisms. The aim is to implement the results it obtains in society and contribute to realizing a bioeconomic society.
Realization of a bioeconomic society: Participation of more than 50 universities and companies
The realization of a bioeconomic society that enables sustainable development "leaving no one behind" is the message championed by the research and development activities of the Center of Innovation for Bio-Digital Transformation (Bio-DX) (hereafter, "Bio-DX Center"), which has been selected for JST's Program on Open Innovation Platforms for Industry-Academia Co-Creation (COI-NEXT). Led by Hiroshima University, with participation from more than 30 universities and research institutions and more than 20 companies, the center aims to solve problems related to food, health, and energy by utilizing the functions of organisms, thereby contributing to achieving the SDGs (Sustainable Development Goals).
The characteristic feature of the Bio-DX Center lies in its integration of the "digitalization" and "programming" of organisms. The digitalization of organisms refers to decoding and analyzing their genetic information to enable computer simulation. Programming refers to using genome editing technology based on the knowledge obtained through digitalization to rewrite the base sequences of DNA-genetic information to create useful organisms.
Digitalization is led by Bono, who has specialized in "bioinformatics", the analysis of biological data with computers, for many years. The programming of organisms is handled by Yamamoto, who has a track record in research on genome editing and serves as the project leader. The strength of the center lies in its gathering of frontrunners from related fields, including Bono and Yamamoto.
Hiroshima University has positioned the Bio-DX Center as a development of the Genome Editing Consortium for Industry-Academia Co-Creation under JST's Program on Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA), which ended in 2021. "OPERA advanced research in an all-Japan system and obtained many achievements related to genome editing. The idea is to further accelerate social implementation based on those research results," says Yamamoto. The Bio-DX Center sets three targets (Figure 1) and establishes four research and development themes and six theme objectives (Figure 2) to realize "a bioeconomy that leads sustainable development through Bio-DX."
Reunion at alma mater leads to development of Platinum TALEN
Among the Bio-DX Center's projects, the development of "hypoallergenic eggs" that do not contain the causative substances of chicken egg allergy is closest to practical application and has attracted media attention. This is a theme that Professor Hiroyuki Horiuchi of the Graduate School of Integrated Sciences for Life at Hiroshima University has been working on for many years. Practical application of the research is finally approaching through the use of genome editing technology.
There are several substances that cause chicken egg allergies. Among them, a component called "ovomucoid (OVM)," a protein in egg white, is heat-resistant and causes allergies even after heat treatment. Horiuchi thought that eggs laid by chickens from which the gene that produces OVM has been eliminated would not cause chicken egg allergies. Initially, he used genetic recombination technology, but development was difficult. When genetic recombination was used, foreign genes intruded into places other than the target location, causing abnormalities in cells. With the methods at that time, about 90% of cells became abnormal. While Horiuchi was searching for a solution to this, he was reunited at his alma mater with Yamamoto, with whom he had researched side by side at the Hiroshima University Facility for Gene Science during his student days.
The two had contact through research even after their workplaces changed, and they decided to incorporate genome editing technology following this reunion. Currently, the most common genome editing tool is "CRISPR-Cas9," which won the Nobel Prize in 2020. However, there are restrictions on the DNA sequences that can be rewritten with this method, and there is a possibility that the OVM gene cannot be completely eliminated.
Therefore, Horiuchi and his colleagues improved a genome editing technology called TALEN, making use of a technology called "Platinum TALEN" developed independently by Yamamoto and his colleagues. "Platinum TALEN enables almost entirely free design and can completely remove genes. PtBio (Higashihiroshima City, Hiroshima Prefecture), a startup originating from Hiroshima University, manages the patents, and since the IP right ramifications are clear, the hurdle for industrial application is also lowered," he notes its superiority.
Creating OVM knockout eggs: Overseas expansion after successful clinical trials
In the research, primordial germ cells, which are cells that become sperm and eggs, were first genome-edited with Platinum TALEN to destroy (knockout) the gene that produces OVM. After that, fertilized eggs were created from sperm and eggs with destroyed genes and hatched (Figure 3). Horiuchi and his colleagues confirmed that eggs laid by hens born in this way did not contain OVM through protein-level analysis (Figure 4-A). Furthermore, they confirmed that mutant OVM, which is expected as a byproduct of genome editing, was not detected by immunoblotting, which detects proteins using specific interactions between antibodies and antigens (Figure 4-B).
One general risk in genome editing is an off-target effect that introduces mutations in sites other than the target gene. Therefore, Horiuchi and his colleagues conducted whole-genome analysis, which determines whole-genome DNA sequence information using next-generation sequencing technology, on chickens born from this genome editing. They showed that there were no off-target effects in which other genes were inserted, or mutations were introduced into other gene regions by Platinum TALEN.
Clinical trials are being conducted at the Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital. Hypoallergenic eggs created using this technique that do not contain OVM are heated, powdered, and then consumed by people with chicken egg allergies. Trials for 30 people have been completed to date, and trials for a total of about 50 people are scheduled to be completed by the end of this fiscal year. If safety is confirmed in these clinical trials, the product will be brought to market in two years, and expansion to Southeast Asia and India is also being considered. If this technology originating from Japan is realized, it will be a ray of hope for people with chicken egg allergies around the world.
Regarding its social implementation, Yamamoto says it is important to create things that people in the world desperately want. "I think hypoallergenic eggs are one of the things that people and their families who want to eat eggs but cannot, desperately want. On the other hand, some people may feel anxious if they do not understand what genome editing is. I think it is important for us to explain the technology carefully and have people understand it through dialogue."
Progress in elucidating "metabolic pathways": Toward social implementation through digital breeding
To create useful substances or clarify the causes of diseases by utilizing genome editing, the genome information of the organism to be genome-edited needs to be clarified. However, the genome information of most organisms has not been decoded. This is true for insects, which are cited as a final goal in the research and development challenges of Target 1 set by the Bio-DX Center.
Bono, who leads research on genome decoding and analysis at the center, previously worked on research on silkworms for about 15 years and analyzed mutants with characteristics similar to Parkinson's disease. He has a track record of elucidating the mechanism by which substances involved in pathological conditions are synthesized and decomposed in the body. Bono talks about the usefulness of genome decoding and analysis: "Silkworms have the property of producing large amounts of protein. If we can make them produce large amounts of pathogen proteins, there is also the possibility of using them as vaccines."
To ensure that organisms produce useful substances in large quantities, it is not sufficient to just analyze gene sequences. It is necessary to comprehensively analyze which genes are actually functioning and what substances are contained, and to clarify the "metabolic pathways"—the reactions through which those substances are produced.
Therefore, Bono reproduces metabolic pathways on a computer and simulates how the entire metabolic pathway changes when a certain gene is rewritten. "At the current stage, even if we want to create useful substances, in most cases the metabolic pathways are not known. An approach in which multiple pathways are selected as candidates through simulation and those that succeed in experiments are implemented in society is becoming important," he says.
This method of digitizing biological data using genome sequences and gene expression data obtained from experiments and already published data, executing simulations, and selecting target genes for genome editing is called "digital breeding." Bono and his colleagues are also conducting joint research with companies. The results of genome decoding of bed bugs resistant to pesticides, conducted with a pesticide manufacturer, are expected to lead to the development of effective pesticides in the future.
Domesticating first-generation genome editing and working on regional industry revitalization
Yamamoto, who is also working on the development of genome editing tools that can be applied industrially, emphasizes the importance of domestication and automation. Domestic tools are easy for domestic companies to handle, and if more effective ones can be selected from those automatically created at high speed, further cost reduction can be expected. Yamamoto's current focus is a genome editing technology called "zinc finger nuclease (ZFN)" (Figure 5), a first-generation genome editing technology that appeared in 1996. It takes time and money to create ZFN tools, and it has not been used much. After the creation of this technology, TALEN was announced in 2010, and CRISPR-Cas9, which appeared in 2012, is used in research laboratories around the world.
Because patents have been obtained for the latter two, it is necessary to pay expensive license fees, which can be a barrier when commercializing developed technologies. However, the patent for ZFN has already expired, and if the time and cost issues in tool development can be resolved, it could be a useful option. Yamamoto and his colleagues are also working on the development of a digital flow-type tool that automates the creation of ZFN tools, and he talks about his outlook: "We aim to make it usable in cultured cells three days after a technical assistant sets it up."
The Bio-DX Center is also advancing research on the mass production of biofuel and eicosapentaenoic acid (EPA), a functional component, using microscopic single-celled algae called microalgae. Furthermore, they are aiming to develop technology for the genome editing of bacteria and, as a long-term goal, to develop cedar that does not produce pollen.
The center also has an aspect of co-creation with regional industries. Yamamoto and his colleagues are enthusiastic about further accelerating cooperation with local companies, such as analyzing the genomes of organisms directly related to regional industries together with food manufacturers and sake brewers in their local Hiroshima Prefecture. Hiroshima University has also emphasized connecting on-site issues in graduate education and industry-academia collaboration and has advanced student education. Bono says that graduates who have obtained master's and doctoral degrees through these efforts are beginning to be active in the industrial and research worlds, and he is filled with anticipation: "I am looking forward to seeing what results young researchers will produce in the future."
(Article: Shosuke Shimada, Photography: Hiroshi Matsui)
