Scientists have genetically reprogrammed the human immune cells known as T cells without using viruses to insert DNA…
The researchers said they expect their technique—a rapid, versatile, and economical approach employing CRISPR gene-editing technology—to be widely adopted in the burgeoning field of cell therapy, accelerating the development of new and safer treatments for cancer, autoimmunity, and other diseases, including rare inherited disorders.
The achievement has significant implications for research, medicine, and industry, UC San Francisco scientists who conducted the work.
“This is a rapid, flexible method that can be used to alter, enhance, and reprogram T cells so we can give them the specificity we want to destroy cancer, recognize infections, or tamp down the excessive immune response seen in autoimmune disease,” said UCSF’s Alex Marson, MD, PhD, associate professor of microbiology and immunology, a member of the UCSF Helen Diller Family Comprehensive Cancer Center, and senior author of the new study.
“Now we’re off the races on all these fronts.”
The new method, which has been published in the journal Nature, offers a robust molecular “cut and paste” system to rewrite genome sequences in human T cells.
It relies on electroporation, a process in which an electrical field is applied to cells to make the T cell membranes temporarily more permeable. After experimenting with thousands of variables over the course of a year, the UCSF researchers found that when certain quantities of T cells, DNA, and the CRISPR “scissors” are mixed together and then exposed to an appropriate electrical field, the T cells will take in these elements and integrate specified genetic sequences precisely at the site of a CRISPR-programmed cut in the genome.
Just as important as the new technique’s speed and ease of use, said Marson.
The approach makes it possible to insert substantially long stretches of DNA into T cells, which can endow the cells with powerful new properties. Members of Marson’s lab have had some success using electroporation and CRISPR to insert bits of genetic material into T cells, but until now, numerous attempts by many researchers to place long sequences of DNA into T cells had caused the cells to die, leading most to believe that large DNA sequences are excessively toxic to T cells.
To demonstrate the new method’s versatility and power, the researchers used it to repair a disease-causing genetic mutation in T cells from children with a rare genetic form of autoimmunity, and also created customized T cells to seek and kill human melanoma cells.
This is a rapid, flexible method that can be used to alter, enhance, and reprogram T cells so we can give them the specificity we want to destroy cancer, recognize infections, or tamp down the excessive immune response seen in autoimmune disease.
Now we’re off the races on all these fronts
Alex Marson, MD, PhD
Viruses cause infections by injecting their own genetic material through cell membranes. Since the 1970s scientists have exploited this capability, stripping viruses of infectious features and using the resulting “viral vectors” to transport DNA into cells for research, gene therapy, and in a well-publicized recent example, to create the CAR-T cells used in cancer immunotherapy.
Marson’s method sidesteps the need for viruses.
“There has been thirty years of work trying to get new genes into T cells,” said first author Theo Roth, a student pursuing MD and PhD degrees in UCSF’s Medical Scientist Training Program who designed and led the new study in Marson’s lab. “Now there should no longer be a need to have six or seven people in a lab working with viruses just to engineer T cells, and if we begin to see hundreds of labs engineering these cells instead of just a few, and working with increasingly more complex DNA sequences, we’ll be trying so many more possibilities that it will significantly speed up the development of future generations of cell therapy.”
After nearly a year of trial-and-error, Roth determined the ratios of T cell populations, DNA quantity, and CRISPR abundance that, combined with an electrical field delivered with the proper parameters, would result in efficient and accurate editing of the T cells’ genomes.
“With this new technique we can cut and paste into a specified place, rewriting a specific page in the genome sequence,” said Marson.
Roth said that because the new technique makes it possible to create viable custom T cell lines in a little over a week, it has already transformed the research environment in Marson’s lab.
Ideas for experiments that were previously deemed too difficult or expensive because of the obstacles presented by viral vectors—are now ripe for investigation. “We’ll work on 20 ‘crazy’ ideas,” Roth said, “because we can create CRISPR templates very rapidly, and as soon as we have a template we can get it into T cells and grow them up quickly.”
