Nature Sub-Journal: A Breakthrough in Cell Engineering! High-yielding CRISPR without viral vectors

A new variant of the CRISPR-Cas9 gene-editing system makes it easier to redesign large numbers of cells for therapeutic applications

"One of our goals for years has been to put lengthy DNA instructions into the genome's target location in a way that doesn't rely on viral vectors," said Dr.

In a new paper published in the journal Nature Biotechnology, Marson and his colleagues not only describe the technique, but also show how it can be used to produce CAR-T cells that have the potential to fight multiple myeloma, a type of blood cancer, as well as rewriting the genetic sequence, mutations that can lead to rare inherited immune diseases

"We demonstrated that we can design more than 1 billion cells in a single run, which is far more than the number of cells we need to treat a single patient," said

From double-stranded to single-stranded DNA

CRISPR-Cas9, a system for editing genes within living cells, has been used as a basic research tool

Traditionally, researchers have relied on viral vectors — viral shells that don't contain pathogenic ingredients — to carry DNA used for gene therapy, called DNA templates, into cells

"Using viral vectors is expensive and resource-intensive," Shy said

In 2015, Marson's team collaborated with CRISPR pioneer Jennifer Doudna's lab to show for the first time that they could insert short DNA templates into immune cells without a viral vector, using an electric field to make the cell's outer membrane more permeable

DNA can exist in single- or double-stranded form (like a Velcro), while Cas9 is adsorbed on

The team knew that single-stranded DNA was less toxic to cells, even at relatively high concentrations

"It gives us a balanced, best-of-both-world approach," Marson said

Single-stranded template DNA can more than

"This technology has the potential to make new cell and gene therapies faster, better, and cheaper," said

The gateway to medical transformation

In this study, the researchers used new DNA templates to generate more than 1 billion CAR-T cells

"We know that localizing a DNA template to a specific location in the genome, called the TRAC site, will improve the anti-tumor ability of CAR-T cells," said Dr.
Justin Eyquem, ph.
D.
, co-author of the new paper, assistant professor of medicine in the UCSF's Department of Hematology and Oncology, and an affiliated researcher at
Gladstone.
"This new non-viral approach allows us to achieve targeting more effectively, which will accelerate the development of
the next generation of CAR-T cell therapies.
"

In addition, the researchers showed for the first time that their method could completely replace the two genes associated with rare genetic immune diseases, the IL2RA and CTLA4 genes.

In the past, scientists have shown that they can replace small parts of the IL2RA gene in the event of mutations in specific patients
.
Now, Marson's team has demonstrated that they can replace the entire IL2RA and CTLA4 genes at once, a "one-size-fits-all" approach that can treat many patients with different mutations in these genes without having to generate personalized templates
for each patient's mutation.
Nearly 90 percent of the cells treated with this genetic engineering method acquired a healthy version of the gene
.

Researchers are currently seeking approval to advance clinical trials
using non-viral CRISPR techniques in CAR-T cell therapy and treatment of IL2RA deficiency.