Human T cells play an essential role in a wide array of diseases, from cancer to AIDS. A research team lead by scientists in San Francisco has pioneered a technique allowing for precise genomic editing of the cells to be performed, carving a path for future CRISPR/Cas9-based therapies, which could target a host of deadly diseases.
Since its discovery, the CRISPR/Cas9 gene editing system has been seen as a breakthrough for modern biomedical research, allowing accurate, but inexpensive editing of genetic information in nearly any organism. As T cells are critical for immune system function, they were immediately seen as an ideal candidate for medical applications using the new technology. Their location within the bloodstream would also allow for simple acquisition from patients, before being edited, and subsequently returned to the body for treatment.
Although this system was immediately recognised as having far reaching medical applications, the editing of T cells by CRISPR/Cas9 proved problematic, as explained by Dr Alexander Marson, the leader of the study, published in Proceedings of the National Academy of Sciences. “Genome editing in human T cells has been a notable challenge for the field, so we spent the past year and a half trying to optimise editing in functional T cells. There are a lot of potential therapeutic applications, and we want to make sure we’re driving this as hard as we can.”
Until now, delivery of CRISPR/Cas9 into human T cells has been inefficient, and only a relatively small number of cells could be transfected successfully. This problem was resolved by creating Cas9 ribonucleoproteins (RNPs), which combined the Cas9 protein with a single strand of RNA. These RNPs were then transfected into the T cells using electroporation, where an electrical field is used to permeablise the membranes for molecule delivery. “We tried for a long time to introduce Cas9 with plasmids or lentiviruses, and then to express separately the single-guide RNA in the cell. Using RNPs made outside the cell, so that the cell is responsible for as little of the process as possible, has made a big difference,” explained Doctor Kathrin Schumann, a postdoctoral fellow in Marson’s group.
Using this technology, the team were able to disable two genes which encode the proteins CXCR4 and PD-1. CXCR4, a protein expressed on the T cell surface membrane is exploited by HIV to infect cells, where as PD-1 is a protein which has recently garnered interest from the cancer immunotherapy field, as its inhibition causes the T cells to attack tumours.
Although recent advances in CRISPR/Cas9 technology have sparked controversy due to reports of embryos editing, T cells are generated anew in each person so modifications would not be passed down to future generations. Overall Marson hopes that Cas9-based treatments for T cell-related disorders will enter the clinic in the not too distant future.
“There’s actually well-trodden ground putting modified T cells into patients. There are companies out there already doing it and figuring out the safety profile, so there’s increasing clinical infrastructure that we could potentially piggyback on as we work out more details of genome editing,” Marson commented. “I think CRISPR-edited T cells will eventually go into patients, and it would be wrong not to think about the steps we need to take to get there safely and effectively.”
Sources: In CRISPR Advance, Scientists Successfully Edit Human T Cells, http://www.ucsf.edu/news/2015/07/131146/crispr-advance-scientists-successfully-edit-human-t-cells.
Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. K Schumanna, S Linc, E Boyer et al. PNAS July 27, 2015 DOI: doi: 10.1073/pnas.1512503112