Developing nonviral gene delivery system in Primate

Genetic engineering in non-human primates has long been limited by the need for virus-based gene delivery methods. Recently, researchers in Japan successfully used a nonviral system to introduce a transgene—that is, a gene that has been artificially inserted into an organism—into cynomolgus monkeys, which is a species of primate closely related to humans.

Small animal models such as mice do not fully replicate the complexity of human diseases, particularly in areas like infectious disease and neuropsychiatric disorders. This limitation has made non-human primates an essential model for biomedical research. However, genetic modification of these primates has been challenging. For example, conventional virus-based methods require specialized containment facilities and are limited in terms of the size of transgenes that the viruses can carry. Also, these methods do not allow for precise selection of modified embryos before implantation.

To overcome these challenges, the research team sought an alternative to using viruses to carry transgenes, instead opting for a nonviral piggyBac transposon system. Transposons, which are sequences of DNA that can change positions within a genome, are valuable tools for gene transfer in genetic engineering as they can stably integrate genetic material into the host’s DNA. The piggyBac transposon system offers several advantages over traditional virus-based approaches, including greater flexibility in terms of the size of transgenes that can be carried and the ability to confirm successful modifications at the early embryo stage.  This allows for more efficient embryo screening before implantation, increasing the likelihood of producing genetically modified animals that carry the desired traits.

Using this approach, the team successfully generated transgenic cynomolgus monkeys, marking a major advancement in genetic engineering. In the resulting cynomolgus monkeys, there was widespread expression of fluorescent reporter genes (that is, the production of fluorescent reporter proteins based the genetic information). Red fluorescent protein was localized to cell membranes, and green fluorescent protein was localized to cell nuclei. Expression was confirmed across all tissues examined, including germ cells, demonstrating that the transgene was stably introduced. These findings suggest that the piggyBac transposon system has significant potential for creating genetically modified primates, which could be used to study human disease in ways that traditional rodent models cannot replicate.

While the transgene integration pattern was consistent across different tissues, expression levels varied. This variability underscores the need in future applications to carefully select promoters—the regulatory regions of DNA that turn on and off specific genes—based on the target tissue. For example, genes such as OCT3/4 and DDX4 play important roles in germ cell lineage differentiation, while SYN1 and THY1 are involved in Neuronal lineage differentiation. By selecting appropriate promoters for specific tissues, researchers can fine-tune gene expression to achieve the desired effects, an essential step in advancing genetic models for disease research.

“Our research represents a milestone in the field of genetic engineering,” explains Dr. Tomoyuki Tsukiyama who led this project. “Our method provides a practical and efficient way to introduce transgenes into non-human primates, which we hope will unlock new insights into complex human diseases.”

Looking ahead, the team plans to expand the applications of this system to include multiplex gene expression and precise transgene control, thereby allowing for more sophisticated genetic models. In addition, the researchers are exploring the potential for integrating epigenetic data about how genes are turned on and off into their work in order to better understand how gene expression is regulated at the molecular level. By refining these techniques, the researchers aim to explore disease mechanisms that remain inaccessible in rodent models and ultimately improve our understanding of complex health conditions in humans.

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About Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University

What key biological traits make us ‘human’, and how can knowing these lead us to better cures for disease? ASHBi investigates the core concepts of human biology with a particular focus on genome regulation and disease modeling, creating a foundation of knowledge for developing innovative and unique human-centric therapies.

About the World Premier International Research Center Initiative (WPI)

The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS).