A newly developed CRISPR system, using single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating Cas9 target sequences achieved two- to threefold better knock-in efficiency and yield relative to dsDNA. In addition, the approach, which does not rely on viral vectors, allows scientists to introduce especially long DNA sequences to precise locations in the genomes of cells.

This work is published in Nature Biotechnology in the paper, “High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.”

“One of our goals for many years has been to put lengthy DNA instructions into a targeted site in the genome in a way that doesn’t depend on viral vectors,” said Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology. “This is a huge step toward the next generation of safe and effective cell therapies.”

Marson 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 to rewrite gene sequences where mutations can lead to rare inherited immune diseases.

“We showed 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,” said Brian Shy, MD, PhD, a clinical fellow in Marson’s lab.

“Using viral vectors is expensive and resource intensive,” said Shy. “A major benefit of a nonviral approach to gene engineering is that we’re not as limited by cost, manufacturing complexity, and supply chain challenges.”

Enhancing CRISPR-mediated insertion efficiency using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences can be toxic to primary cells. Single-stranded DNA is less toxic to cells, even at relatively high concentrations. In the new method, the researchers describe a method to attach the modified Cas9 enzyme to a single-stranded template DNA, by adding just a small overhang of double-stranded DNA at the ends.

“This gives us a balanced, best-of-both-worlds approach,” said Marson.

Single-stranded template DNA could more than double the efficiency of gene editing compared to the older, double-stranded approach. And the double-stranded ends of the molecules let researchers use Cas9 to enhance the delivery of nonviral vectors into cells.

“This technology has the potential to make new cell and gene therapies faster, better, and less expensive,” said Jonathan Esensten, MD, PhD, an assistant professor of laboratory medicine at UCSF and an affiliate investigator at Gladstone.

In the study, researchers used the new DNA template to generate more than a billion CAR-T cells that target multiple myeloma. Approximately half of all T cells gained the new gene and, as a result, were converted to CAR-T cells.

“We knew that targeting the DNA templates to a specific location in the genome, called the TRAC site, would improve the anti-tumor potency of CAR-T cells,” said Justin Eyquem, PhD, assistant professor of medicine in the division of hematology and oncology at UCSF, and affiliate investigator at Gladstone. “This new nonviral approach enables us to achieve that 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, replace in their entirety two genes associated with rare genetic immune diseases, the IL2RA and the CTLA4 genes.

In the past, scientists had shown they could replace small sections of the IL2RA gene where particular patients have mutations. Now, Marson’s team proved that they can replace whole IL2RA and CTLA4 genes at once—a “one size fits all” approach that could treat many patients with different mutations in these genes, rather than having to generate personalized templates for each patient’s mutation. Nearly 90% of the cells treated with this gene engineering approach gained the healthy versions of the genes.

The researchers are now seeking approval to advance clinical trials using nonviral CRISPR technology in both CAR-T cell therapy and the treatment of IL2RA deficiency.

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