Researchers at the University of California San Francisco created CRISPR guides that would cause green fluorescent protein to be expressed in only certain cellular locations and structures without using viruses. Photo courtesy of Alex Marson/UCSF
July 12 (UPI) -- Scientists have figured out a way to use genetically reprogram human immune cells without using viruses to insert DNA, as the CRISPR method of gene editing has traditionally required.
In a study published recently in Nature, researchers at the University of California San Francisco show a new CRISPR technique involving T cells could help develop new and safer treatments for cancer, autoimmunity and rare inherited disorders.
"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," senior author Dr. Alex Marson, associate professor of microbiology and immunology, said in a press release. "Now we're off to the races on all these fronts."
The researchers said they used a "cut and paste" method of rewriting genome sequences in human T cells using electroporation, which applies an electrical field to cells to make their membranes more permeable.
The researchers tested thousands of variables over one year. They found that mixing T cells, DNA and the CRISPR "scissors," and then exposing them to an appropriate electrical field, the T cells will take in these elements and integrate specified genetic sequences where CRISPR programmed the cut in the genome.
In the past, researchers have found cells have died when they tried to place long sequences of DNA into T cells.
The UCSF researchers attempted to repair a disease-causing genetic mutation in T cells from children with a rare genetic form of autoimmunity. They also customized T cells to seek out and kill human melanoma cells.
"There has been 30 years of work trying to get new genes into T cells," said first author Theo Roth, a doctoral student in UCSF's Medical Scientist Training Program. "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."
Roth found the correct ratios of T cell populations, DNA quantity and CRISPR abundance that, when combined with an electrical field delivered, would result in efficient and accurate editing of the T cells' genomes. Then he tested the ratios by sending CRISPR to label different T cell proteins with green fluorescent protein.
Further experiments showed how the method could be used to gather T cells against autoimmune disease or cancer.
The researchers generated large numbers of CRISPR-engineered cells reprogrammed to display the new T cell receptor with using viruses. Engineered human T cells went to the tumor site and showed anti-cancer activity when transferred into mice implanted with human melanoma tumors.
"This strategy of replacing the T cell receptor can be generalized to any T cell receptor," Marson said. "With this new technique we can cut and paste into a specified place, rewriting a specific page in the genome sequence."
The researchers say the method could lead to a multitude of advances with CRISPR and gene editing for disease treatment.