1:51 PM
 | 
Jul 12, 2018
 |  BC Extra  |  Preclinical News

Virus-free CRISPR T cell editing

A team led by University of California San Francisco researchers developed a non-viral method to engineer T cell genomes in about one week using CRISPR/Cas9 DNA editing. The method could reduce the time and cost needed to generate T cell therapies, and increase the size of genetic constructs that can be incorporated into the cells' DNA.

Genome editing using viral vectors can take months to complete, and is generally limited to delivery of DNA around 1 kilobase or smaller. Moreover, previous studies have shown viral vectors can cause non-specific gene integration.

In a Nature paper, the team used an optimized electroporation-based method to deliver a homology-directed repair DNA template and CRISPR-associated protein 9 (Cas9) complexed to a guide RNA to primary human T cells. Through systematic screening, the team identified optimal cell culture conditions, concentrations of DNA and RNA/Cas9 complexes, and handling protocols to support efficient genomic editing in T cells.

The researchers first used the method to insert a reporter protein into six loci in T cells with editing rates between 16% and 57.6%. The method increased T cell viability compared with electroporation of DNA alone, and led to off-target integration rates around 1%.

The team next edited T cells derived from a family of patients with autoimmune disease to correct loss-of-function mutations in interleukin-2 receptor alpha chain (CD25). CRISPR editing of the T cells induced expression of CD25 in up to 25% of the T cells, compared with CD25 expression in less than 1% of unelectroporated cells.

Finally, the CRISPR method was used to reprogram the antigen specificity of T cells by replacing endogenous TCR sequences with a TCR targeting cancer/testis antigen 1B (NY-ESO-1; CTAG1B) -- a 1.5 kb sequence -- with editing efficiencies around 10%. In mice with tumors derived from melanoma cells expressing NY-ESO-1, the CRISPR-edited T cells reduced tumor size compared with T cells engineered to express the TCR using a lentiviral vector.

The authors suggested that the method could be used to generate CAR T cells or create synthetic gene circuits in T cells.

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