Oakland, California, USA: In an achievement that has significant implications for research, medicine, and industry, 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 new method, described by UC San Francisco scientists 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 their 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.
“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.”
But just as important as the new technique’s speed and ease of use, said Marson, also scientific director of biomedicine at the Innovative Genomics Institute, is that the approach makes it possible to insert substantial 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.
Viruses cause infections by injecting their own genetic material through cell membranes, and 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.
T cells engineered with viruses are now approved by the U.S. Food and Drug Administration to combat certain types of leukemia and lymphoma. But creating viral vectors is a painstaking, expensive process, and a shortage of clinical-grade vectors has led to a manufacturing bottleneck for both gene therapies and cell-based therapies. Even when available, viral vectors are far from ideal, because they insert genes haphazardly into cellular genomes, which can damage existing healthy genes or leave newly introduced genes ungoverned by the regulatory mechanisms that ensure that cells function normally. These limitations, which could potentially lead to serious side effects, have been cause for concern in both gene therapy and cell therapies such as CAR-T-based immunotherapy.
“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, 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.
To validate these findings, Roth directed CRISPR to label an array of different T cell proteins with green fluorescent protein (GFP), and the outcome was highly specific, with very low levels of “off-target” effects: each subcellular structure Roth’s CRISPR templates had been designed to tag with GFP – and no others – glowed green under the microscope.
Search and Replace
In complementary experiments devised to serve as proof-of-principle of the new technique’s therapeutic promise, Roth, Marson, and colleagues showed how it could potentially be used to marshal T cells against either autoimmune disease or cancer.
In the first example, Roth and colleagues used T cells provided to the Marson lab by Yale School of Medicine’s Kevan Herold, MD. The cells came from three siblings with a rare, severe autoimmune disease that has so far been resistant to treatment. Genomic sequencing had shown that the T cells in these children carried mutations in a gene called IL2RA. This gene carries instructions for a cell-surface receptor essential for the development of regulatory T cells, or Tregs, which keep other immune cells in check and prevent autoimmunity.
With the non-viral CRISPR technique, the UCSF team was able to quickly repair the IL2RA defect in the children’s T cells, and to restore cellular signals that had been impaired by the mutations. In CAR-T therapy, T cells that have been removed from the body are engineered to enhance their cancer-fighting ability, and then returned to the body to target tumors. The researchers hope that a similar approach could be effective for treating autoimmune diseases in which Tregs malfunction, such as that seen in the three children with the IL2RA mutations.
In a second set of experiments conducted in collaboration with Cristina Puig-Saus, PhD, and Antoni Ribas, MD, PhD, of the Parker Institute for Cancer Immunotherapy at UCLA, the scientists completely replaced native T cell receptors in a population of normal human T cells with new receptors that had been specifically engineered to seek out a particular subtype of human melanoma cells. T cell receptors are the sensors the cells use to detect disease or infection, and in lab dishes the engineered cells efficiently homed in on the targeted melanoma cells while ignoring other cells, exhibiting the sort of specificity that is a major goal of precision cancer medicine.
Without using viruses, the researchers were able to generate large numbers of CRISPR-engineered cells reprogrammed to display the new T cell receptor. When transferred into mice implanted with human melanoma tumors, the engineered human T cells went to the tumor site and showed anti-cancer activity.
“This strategy of replacing the T cell receptor can be generalized to any T cell receptor,” said Marson, 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.”
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.”
Marson attributes the new method’s success with Roth’s “absolute perseverance” in the face of the widespread beliefs that viral vectors were necessary and that only small pieces of DNA could be tolerated by T cells. “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: the ratio of the CRISPR to the DNA; different ways of culturing the cells; different electrical currents. By optimizing each of these parameters and putting the best conditions together he was able to see this astounding result.”
Marson is a co-founder of Spotlight Therapeutics, and serves as an adviser to Juno Therapeutics and PACT Pharma. The Marson laboratory has received sponsored funding for Juno, Epinomics, and Sanofi, as well as a gift from Gilead Sciences. Roth, Puig-Saus, Eric Shifrut, PhD, Ribas, and Marson are inventors on new patent applications related to this research.
UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy, as well as UCSF Fresno, which is dedicated to improving health in the San Joaquin Valley; a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences; and a preeminent biomedical research enterprise. It also includes UCSF Health, which comprises three top-ranked hospitals – UCSF Medical Center and UCSF Benioff Children’s Hospitals in San Francisco and Oakland – as well as Langley Porter Psychiatric Hospital and Clinics, UCSF Benioff Children’s Physicians and the UCSF Faculty Practice. UCSF Health has affiliations with hospitals and health organizations throughout the Bay Area. UCSF faculty also provide all physician care at the public Zuckerberg San Francisco General Hospital and Trauma Center, and the SF VA Medical Center.
How CRISPR Tools are Unlocking New Ways to Fight Disease
Recent leaps in gene editing technology have brought ideas that just a decade ago seemed like science fiction to the cusp of reality.
The already famous CRISPR system allows scientists to edit faulty genes by cutting and replacing sections of DNA, but new and improved CRISPR techniques have expanded CRISPR’s scalpel into a Swiss Army knife. The new tools give researchers more flexible control of gene function without permanently altering an organism’s genetic code.
What is CRISPR?
CRISPR is an acronym for “clustered regularly interspaced short palindromic repeats.”
It is a system that bacteria use to defend themselves against viruses.
In bacteria, CRISPR acts like a vaccine, incorporating bits of genes from viruses. Bacteria can then reference this library of genes to recognize and attack viral invaders.
Scientists have learned how to use the CRISPR system to recognize specific genes in mammalian cells.
New research tools built on CRISPR can target genes, edit them, or turn them on or off.
These versatile tools are helping to untangle the complex genetics underlying diseases such as cancer and autoimmune disorders. They could identify new targets for drug development or point the way for gene therapies that could one day target genetic defects related to blindness or obesity.
The basis of these new techniques, known as CRISPR-dCas9, was invented in 2013 by UC San Francisco researchers Jonathan Weissman, PhD; Stanley Qi, PhD (now at Stanford University); and Wendell Lim, PhD.
They took the CRISPR system, well-known for its ability to target and snip out pieces of DNA, and mutated the gene-cutting protein Cas9 to create dCas9. Variants known as CRISPRa (for activation) and CRISPRi (for interference) still target genes, but can swap in different molecular tools to dial up or down gene expression.
Lim, who is chair of the Department of Cellular and Molecular Pharmacology, likens dCas9 to a universal adapter that can recognize any gene through DNA base pairing, and then modulate that gene with protein attachments.
“CRISPR-dCas9 is a very powerful way of recruiting anything you want to any part of the genome,” said Alexander Marson, MD, PhD, assistant professor of microbiology and immunology, one of many researchers who have adopted the new tools in their work.
Boosting Healthy Genes to Treat Obesity
In the lab of Nadav Ahituv, PhD, a School of Pharmacy professor in the Department of Bioengineering and Therapeutic Sciences, researchers have shown in mice that it’s possible to correct a genetic defect that causes severe obesity.
Healthy mice – and humans – have two copies of genes (one inherited from each parent). A defect in one copy that completely abolishes gene function, known as haploinsufficiency, can lead to disease. For example, mice with one defective copy of a particular gene involved in body weight tend to overeat and become obese. Ahituv realized that in this case, there was an opportunity for a potential therapeutic. “The trick here is you still have an existing copy that’s fine,” he said.
His team turned to CRISPRa, one of the CRISPR tools developed at UCSF, to target and boost the healthy copy of the gene, compensating for its broken twin. They packaged the CRISPRa system inside viruses that would deliver it into neuronal cells and injected the viruses into mice.
The treated mice stopped overeating and soon lost weight. When researchers examined the expression levels of the targeted gene, they found normal levels. Nine months later, the mice stayed at normal weight, and Ahituv says because neurons do not divide, the changes could last for the lifetime of the animal.
Haploinsufficiency for this obesity-related gene is responsible for a small percentage of cases of severe obesity in people. Considering the high rates of obesity in the U.S. population, even this small percentage represents quite a lot of people, says Ahituv.
His lab is now looking at five other haploinsufficient diseases, in particular at neurological and kidney diseases, as candidates for CRISPRa therapy, though much more work needs to be done before the technique can be tried in humans.
Researchers acknowledge they would have significant challenges to overcome in translating the new CRISPR tools into human therapies. A safe and precise delivery method would be key, as would a way to ensure the genetic changes are durable.
Revealing Hidden Switches
Even as these new CRISPR tools hint at new avenues for gene therapy, they’re already opening the field for other therapies by revealing the complex genetic interactions that underlie diseases.
The most immediate payoff may be in identifying new therapeutic targets, according to Bruce Conklin, MD, professor of medicine at UCSF and senior investigator at Gladstone Institutes, who works with CRISPRi and related techniques in his research.
“The power of these techniques is to test thousands of different drug targets in a single experiment. This has already helped with leads for potentially useful drugs,” he said.
It’s estimated that 98 percent of our DNA does not code for proteins, but instead act as a switchboard for the 2 percent that do. These so-called promotors and enhancers can be cryptic and time-consuming to study with conventional genetic techniques.
With CRISPRa, Marson’s lab can rapidly screen over 20,000 non-coding sites in the genome and study their function – essentially by flipping many switches and seeing which ones turn on the lights. “This is a critical step in identifying the functional significance of genetic elements,” said Marson.
In a recent CRISPRa screen, Marson’s lab, in collaboration with the lab of Jacob Corn, PhD, at UC Berkeley, identified several enhancers associated with inflammation and autoimmune disorders. In fact, one of the newly identified enhancers matched a common genetic variant known to increase the risk of irritable bowel disease, although its mechanism had previously been a mystery.
Lim has watched the CRISPR-dCas9 methods he helped pioneer disperse to research labs around the world in just a few years. From 2013 to the end of 2015, 49 papers included the term dCas9 in their title or abstract, according to PubMed. From 2016 through June 2018, 242 papers cited the same term – a nearly five-fold increase.
“It is amazing how quickly this technology has spread within just a few years.” said Lim. “It speaks to how flexible the simple core concept is.”
Gene Editing and CRISPR
Gene editing is already providing new therapies for rare diseases that just a generation ago were untreatable. New techniques with CRISPR hold promise for greatly improving both quality and length of life for those suffering from genetic conditions.