Yale Scientists Enable Massively Parallel DNA Insertions Into Human Cells

Yale Cancer Center scientists have developed a novel technology that for the first time enables massively parallel DNA substitutions (known as “knock-ins”) into human cells by taking advantage of messenger RNA (mRNA), a platform now well-known from its use as the COVID vaccine vehicle. 

This new technology, named CLASH, allows scientists to generate tens of thousands of different variants of cells with different genetic modifications and pick the best one for desired purposes, such as using them in cell therapies for cancer treatment. The study is published Jan. 26 in the journal Nature Biotechnology.

“While there are different ways to modify the genome, inserting pieces of DNA has historically been more difficult than deleting pieces, and it’s even harder to insert many different pieces into various cells at the same time,” said senior author Sidi Chen, an associate professor of genetics at Yale School of Medicine and member of Yale Cancer Center. “We have been working hard over the past five years and came up with a solution: designing and creating large pools of adeno-associated virus [AAV] vectors and delivering them together with mRNA enabled us to achieve mass insertions instead of one-to-one gene substitutions.” 

Chen’s research team first applied this technology in human T cells to solve the challenge of cell therapy for cancer. Cell therapy such as chimeric antigen receptor T (CAR-T) therapy is successful and recently approved by the U.S. Food and Drug Administration to treat blood cancers but faces major hurdles in solid tumors such as breast cancer, lung cancer, and colon cancer. 

For the new study, the team used CLASH to modify the genome of T cells and created pools of new CAR-T variants, which they called “treasure islands.” They then tested these T cell variant pools against cancer, and let the best candidate emerge from selection. Researchers have found that a unique mutant can substantially enhance CAR-T against cancer in animals, and the effect is universal across different types of disease models.

“Although the proof-of-principle is in human T cells, the CLASH technology is in principle applicable to many different cell types, which allows us to rapidly create thousands of cell therapy candidates for diverse cancer treatment options,” said Chen. Future trials are needed before human application.

Chen is affiliated with the Systems Biology Institute and the Center for Cancer Systems Biology at Yale West Campus, the Yale Stem Cell Center, the Yale Center for Biomedical Data Science, and the Department of Genetics at Yale School of Medicine.

Yale’s Xiaoyun Dai is the first author of the paper. Co-first authors are Jonathan Park, a Yale student in the Medical Scientist Training Program; Yaying Du, a former postdoctoral associate in Chen’s lab; Zhenkun Na, a postdoctoral associate; and Stanley Lam, a Yale College student in Chen’s lab. Yale faculty members Hongyu Zhao and Sarah Slavoff are collaborators. Chen is the corresponding author.

The research was primarily funded by the National Institutes of Health and the U.S. Department of Defense.