Scientists have developed a new approach that produces enough cells for therapeutic applications
SAN FRANCISCO, August 25, 2022 /PRNewswire/ — A new variation of the CRISPR-Cas9 gene-editing system makes it easier to reengineer large numbers of cells for therapeutic applications. An approach developed at the Gladstone Institutes and UC San Francisco (UCSF), allows scientists to introduce particularly long DNA sequences into precise locations in cell genomes with remarkably high efficiency without the viral delivery systems traditionally used to deliver DNA into cells.
“One of our goals for many years has been to insert long DNA instructions into a targeted location in the genome in a way that does not depend on viral vectors,” he says Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology and lead author of the new study. “This is a huge step towards the next generation of safe and effective cell therapies.”
In a new paper published in the journal Natural biotechnologyMarson and his colleagues not only describe the technology, but show how it can be used to generate CAR-T cells with the potential to fight multiple myeloma, a blood cancer, as well as rewrite gene sequences where mutations can lead to rare inherited immunity. diseases.
“We have shown that we can engineer more than one billion cells in a single run, which is well above the number of cells we need to treat an individual patient,” says the first author Brian ShyMD, PhD, Clinical Fellow in the Marson Laboratory.
From double-stranded to single-stranded DNA
CRISPR-Cas9, a system that edits genes inside living cells, has been used as an essential research tool for the past decade. Many clinical scientists are increasingly excited about the potential of CRISPR-Cas9 to create living cell therapies.
Among other things, gene editing can be used to switch off, remove or replace a mutated gene causing a disease or to strengthen the activity of an immune cell in the fight against cancer. While the first therapeutic applications of CRISPR-Cas9 have recently entered clinical trials, the technology has still been limited by the challenge of safely producing large numbers of properly edited cells.
Traditionally, researchers have relied on viral vectors – envelopes of viruses without their disease-causing components – to deliver the DNA (called template DNA) used for gene therapy into cells. However, the production of large quantities of clinical-grade viral vectors has been a major obstacle in delivering cell therapies to patients. In addition, scientists cannot easily control where traditional viral vectors insert genes into the genome.
“Using viral vectors is expensive and resource intensive,” says Shy. “The main advantage of a non-viral approach to genetic engineering is that we are not as constrained by cost, manufacturing complexity and supply chain issues.”
In 2015, Marson’s group—in collaboration with the CRISPR pioneer’s lab Jennifer Doudna, PhD—first demonstrated that they could insert short DNA templates into immune cells without viral vectors by using an electric field to make the cells’ outer membranes more permeable. By 2018, they had developed a method for cutting and inserting longer DNA sequences into immune cells using CRISPR.
Then, in 2019, researchers discovered that by also using a modified version of DNA templates that can bind to the Cas9 enzyme—the same protein that acts as molecular scissors during CRISPR gene editing—they could deliver new sequences to the targeted genome. pages more efficiently.
However, more work was needed to improve the yield of successfully engineered immune cells and to make the process compatible with the production of future cell therapies. These goals motivated the team’s current study.
DNA can exist in single or double strands (like opposing pieces of Velcro), and Cas9 binds to double-stranded DNA. The researchers quickly discovered that high levels of double-stranded DNA template can be toxic to cells, so the method can only be used with low amounts of template DNA, resulting in low efficiency.
The team knew that single-stranded DNA was less toxic to cells, even at relatively high concentrations. So in the new paper, they describe a method of attaching a modified Cas9 enzyme to single-stranded template DNA by adding only a small overlap of double-stranded DNA to the ends.
“This gives us a balanced, best-of-both-worlds approach,” says Marson.
Single-stranded template DNA could more than double the efficiency of gene editing compared to the older double-stranded approach. And the molecules’ double-stranded ends allow researchers to use Cas9 to improve the delivery of nonviral vectors into cells.
“This technology has the potential to make new cell and gene therapies faster, better and cheaper,” he says Jonathan EsenstenMD, PhD, author of the new paper, who is an assistant professor of laboratory medicine at UCSF and a research associate at Gladstone.
The trip to the clinic
In the study, researchers used a new DNA template to create more than a billion CAR-T cells that target multiple myeloma. CAR-T cells are immune T cells genetically modified to effectively fight against specific cells or cancer. With the new single-stranded, Cas9-directed templates, approximately half of all T cells acquired the new gene and were consequently converted to CAR-T cells.
“We knew that targeting DNA templates to a specific location in the genome, called the TRAC site, would improve the antitumor efficacy of CAR-T cells,” says Justin Eyquem, PhD, co-author of the new paper. Assistant Professor of Medicine in the Department of Hematology and Oncology at UCSF and Associate Investigator at Gladstone. “This new non-viral approach allows us to achieve this targeting much more efficiently, which will accelerate the development of the next generation of CAR-T cell therapies.”
In addition, the researchers showed that their approach could, for the first time, completely replace two genes associated with rare genetic immune diseases, the so-called IL2RA and CTLA4 genes.
In the past, scientists have shown that they can replace small parts IL2RA the gene where specific patients have mutations. Now Marson’s team has proven they can replace the whole IL2RA and CTLA4 genes at once—a “one-size-fits-all” approach that could treat many patients with different mutations in those genes, rather than having to create personalized templates for each patient’s mutation. Almost 90 percent of cells treated with this genetic engineering approach acquired healthy versions of the genes.
The researchers are now seeking approval to advance clinical trials using the non-viral CRISPR technology in both CAR-T cell therapy and IL2RA-deficient treatment.
About the research project
The article “High-throughput genome engineering in primary cells using hybrid ssDNA repair template and small molecule cocktails” was published in the journal Natural biotechnology on August 25, 2022.
Other authors are Vivasvan S. Vykunta, Alvin Ha, Alexis Talbot, Theodore L. Roth, David N. Nguyen, Yan Yi ChienFranziska Blaeschke, Eric Shifrut, Shane Vedova, Murad R. Mammadov, Jing-Yi Chungand Ruby Yu from the Gladstone-UCSF Institute of Genomic Immunology; Wolfgang G. Pfeifer and Carlos E. Castro from The The Ohio State University; Hong Li and Lumeng Ye of GenScript Biotech; and David Wu, Jeffrey Wolfand Thomas G. Martin from UCSF.
This work was supported by the National Institute of Allergy and Infectious Diseases (P01AI138962 and P01AI155393), the UCSF Grand Multiple Myeloma Translational Initiative, the Weill Neurohub, the Larry L. Hillblom Foundation (2020-D-002-NET), and the Innovative Genomics Institute.
The researchers were also supported by the National Institutes of Health (L40AI140341, K08AI153767), the National Center for Advancing Translational Sciences (L30TR002983), the National Institute of Diabetes and Digestive and Kidney Diseases (F30DK120213), the Parker Simons Institute for Cancer Foundation, the Parker Simons Institute for Cancer Immunotherapy , Burroughs Wellcome Fund, Chan Zuckerberg Biohub, Cancer Research Institute, UCSF Herbert Perkins Cellular Therapy and Transfusion Medicine Fellowship, California Institute for Regenerative Medicine (INFR-10361), Care-for-Rare Foundation, German Research Foundation, UCSF Medical Scientist Training Program (T32GM007618), UCSF Endocrinology Training Grant (T32 DK007418), Mnemo Therapeutics, Takeda, Cytovia Therapeutics, Human Vaccines Project Michelson Prize, and National Science Foundation (1933344).
On the Gladstone Institutes
To ensure our work does the greatest good, Gladstone Institutes focuses on conditions with profound medical, economic and social impact – unaddressed diseases. Gladstone is an independent, not-for-profit life sciences research organization that uses visionary science and technology to overcome disease. Has an academic affiliation with University of California, San Francisco.
The University of California, San Francisco (UCSF) focuses exclusively on the health sciences and is dedicated to promoting health worldwide through advanced biomedical research, graduate education in the life sciences and health professions, and excellence in patient care. UCSF Health, which serves as UCSF’s primary academic medical center, includes top specialty hospitals and other clinical programs and has locations throughout the Bay Area. Learn more at ucsf.edu or view our fact sheet.
Gladstone Institutes: Julie Langelier | [email protected] | 415,734 2019
UCSF: Robin Marks | [email protected] | 628 399 0370
SOURCE Gladstone Institutes
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