Nonviral CRISPR Technology Developed for Faster, Cheaper T-Cell Engineering

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T cells engineered using nonviral CRISPR technology to express green fluorescent protein. [Alex Marson's Lab]


Researchers at the University of California, San Francisco (UCSF) have developed what they describe as a fast, simple CRISPR-based method for genetically reprogramming T-cell function, without using viral vectors. The technique, developed in the laboratory of associate professor of microbiology and immunology Alex Marson, M.D., Ph.D., uses electroporation to permeabilize T cells to the CRISPR gene editing components and DNA, and can be used to insert even large gene sequences at specified, CRISPR enzyme cut sites in the genomes of primary human T cells, without affecting cell viability or functionality.

In two initial proof-of-principle tests, the team used their approach to correct the gene defect and restore signaling function in T cells taken from children with a rare monogenic autoimmune disease, and to engineer T cells to recognize and mount antitumor responses against human melanomas in a mouse model. Research lead and student at UCSF Theo Roth, and colleagues, claim the technique could potentially be used to completely rewire signaling circuits in T cells for a wide range of human therapeutic applications.

“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,” says Dr. Marson, who is a member of the UCSF Helen Diller Family Comprehensive Cancer Center. “Now we're off to the races on all these fronts.” Dr. Marson is senior author of the team’s paper, which is published in Nature, and is titled, “Reprogramming human T cell function and specificity with non-viral genome targeting.”

Scientists have spent decades working to reprogram T cells for therapeutic purposes, using viral vectors to deliver stretches of DNA, or transgenes, that they want to insert. “There has been 30 years of work trying to get new genes into T cells,” comments Roth. However, the UCSF team continues, viral approaches don't target the new genetic elements to specific genomic sites. Effectively, they state, “the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive.” Another barrier to effective T-cell genome editing is the toxicity of DNA itself, they add. While inserting small, single-stranded DNA sequences into a T cell’s genome doesn’t result in any evident toxicity, large linear double-stranded DNA (dsDNA) templates are toxic at high concentrations. 

Roth and researchers in Dr. Marson’s lab spent a year manipulating the proportions of T cells, DNA, and CRISPR elements that, when subjected to the right electrical field, could allow the efficient, accurate site-specific editing of T-cell genomes, including the insertion of large genetic elements. Surprisingly, and “contrary to expectations,” they admit, they found that co-electroporation of the human primary T cells with the CRISPR elements and long (>1 kb) linear dsDNA templates acted to reduce the dsDNA template toxicity.

It was Roth’s “absolute perseverance” that eventually paid off, Dr. Marson states. “Theo was convinced that if we could figure out the right conditions we could overcome these perceived limitations, and he put in a Herculean effort to test thousands of different conditions, [including] the ratio of the CRISPR to the DNA, different ways of culturing the cells, [and] different electrical currents. By optimizing each of these parameters and putting the best conditions together he was able to see this astounding result.”

In a series of tests, the researchers first confirmed that the nonviral CRISPR system could be used to target sequences at different locations throughout the genome of human primary T cells. Further optimization then ensured that even rare occurrences of off-target effects could be avoided almost completely.

Having optimized the system in primary human T cells, the researchers then carried out a series of tests in two different clinically relevant settings. For the first, they identified a family with a monogenic autoimmune disease caused by mutations in the gene encoding the IL-2α receptor (IL2RA), which is essential for regulatory T cell (Treg) function. They used their nonviral genome-targeting technique to correct the identified mutations in cells taken from IL2RA-deficient children with the disorder, and demonstrated that the gene editing resulted in expression of the IL2RA on the cell surface and improved cell signal functioning.

The team then used the nonviral genome-editing system to replace an endogenous T-cell receptor sequence in human primary T cells with a new TCR that redirected the cells to recognize and react to a melanoma antigen. The primary T cells were collected from healthy human volunteers, engineered and expanded in vitro. When tested both against cultured melanoma cell lines and in human melanoma-bearing mice, the engineered human T cells recognized and mounted protective antitumor responses against the cancer cells, and reduced tumor burden in treated animals.

“This strategy of replacing the T-cell receptor can be generalized to any T-cell receptor,” says Dr. Marson, who is also a member of the Parker Institute for Cancer Immunotherapy at UCSF and a Chan Zuckerberg Biohub Investigator. “With this new technique we can cut and paste into a specified place, rewriting a specific page in the genome sequence.”

The researchers claim that avoiding the use of viral vectors should speed both research and clinical applications of genome editing technologies, keep costs down, and potentially improve safety. Projecting forward, they suggest that their process and materials could easily be adapted to pass regulatory requirements for clinical use. 

“Our therapeutic gene editing in human T cells is a process that takes only a short time from target selection to production of the genetically modified T-cell product,” they state. “In approximately one week, novel gRNAs [guide RNAs] and DNA repair templates can be designed, synthesized, and the DNA integrated into primary human T cells that remain viable, expandable, and functional.”

The studies reported in Nature were carried out in collaboration with academic researchers across the U.K. The speed with which Dr. Marson's lab can now make customized T cells has already transformed the research environment, making it possible to carry out work that would previously have been too difficult, time-consuming, or expensive. “We'll work on 20 'crazy' ideas,” Roth says, “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.”

Looking forward, the researchers project that technology could be used to “rewire” complex molecular circuits in human T cells. “Multiplexed integration of large functional sequences at endogenous loci should allow combinations of coding and noncoding elements to be corrected, inserted, modified, and rearranged,” they suggest. And although they acknowledge that a lot of work will be needed to improve our understanding of T-cell circuitry, the nonviral genome-editing technology should represent an approach that can rewrite T cell programs for “the next generation of immunotherapies.”








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