A new method has been developed, primarily by University of California San Francisco (UCSF) researcher Theo Roth of Alex Marson’s laboratory, to introduce gene editing CRISPR technology into T-cells without the use of viral vectors. The method was published in the July 11, 2018 issue of the journal Nature.
The method is based on electroporation, the use of electricity to briefly punch small holes in the membranes of cells so that DNA and other molecules can enter. Roth spent the best part of a year optimising the ratios and concentrations of T-cells, DNA, and CRISPR enzymes, and other conditions needed in order for electroporation to permit efficient entry of DNA and CRISPR enzymes into the T-cells. The technique has allowed the insertion of longer stretches of DNA, into the T-cell genome, than was previously thought to be possible. Custom T-cells can be created in just over a week.
From a cell therapy manufacturing perspective, this is a major step forward as, to date, FDA approved CAR-T cell therapies require the costly manufacture of clinical-grade viral vectors in order to reprogram a patient’s T cells. It should also speed up basic T-cell disease and therapy research as specialized knowledge, designated equipment and areas for virus production will no longer be required for a laboratory to make meaningful contributions.
In order to demonstrate the capabilities of the system, the researchers restored IL2RA gene function to T-cells extracted from three children with a rare autoimmune disease, in which their regulatory T-cells are impaired by IL2RA mutation.
In a second demonstration the researchers developed a CAR-T to specifically target melanoma cancer cells. In a mouse model, these T-cells, engineered with CRISPR without viral vectors, were able to target xenografts of melanoma tumors and demonstrated anti-tumor activity.
As an indication of the interest from industry, the Marson lab has received financial support from Juno, Epinomics, Sanofi, and Gilead Sciences. Marson is also a co-founder of Spotlight Therapeutics.