[iStock/lvcandy]
[iStock/lvcandy]

The ability to precisely extricate and manipulate fragments of our genome in order to abrogate mutations or turn off overactive genes seems like the fantasy tales written about in science fiction novels and comic books. Yet, at present, researchers are becoming ever more successful at accomplishing those seemingly fictional techniques using the genome editing tool known as CRISPR/Cas9.

Now, investigators from the University of California San Francisco (UCSF) have developed a new strategy to precisely modify human T cells using CRISPR/Cas9—implications that could have sweeping impacts on AIDS, cancer, and various other autoimmune disorders.     

“Genome editing in human T cells has been a notable challenge for the field,” explained senior author Alexander Marson, Ph.D., a Sandler Fellow at UCSF. “So we spent the past year and a half trying to optimize 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.”

The findings from this study were published recently in PNAS through an article entitled “Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.”

The UCSF researchers were able to deactivate a protein on the surface of T cells called CXCR4, which is often exploited by the HIV virus to infect the T cell. Additionally, the scientists were able to nullify the PD-1 protein, which has made a big splash in the newly flourishing field of cancer immunotherapy, since blocking this protein with newly developed drugs has been shown to cause T cells to attack tumors.  

The CRISPR technique has captivated the imaginations of scientists over the past several years since it makes it possible to efficiently and inexpensively edit genetic information in virtually any organism. T cells are a high valued target for medical applications of the technology, as these cells not only stand at the epicenter of many disease processes, but can be easily gathered from patients, edited with CRISPR/Cas9, then returned to the body to exert therapeutic effects.

“It's been great to be part of this exciting collaboration, and I look forward to seeing the insights from this work used to help patients in the future,” said co-author Jennifer Doudna, Ph.D., professor of chemistry and of cell and molecular biology at UC Berkeley and a pioneering researcher on CRISPR/Cas9.

Specifically, Cas9, which cuts the target DNA and allows new genetic sequences to be inserted, has previously been introduced into cells using viruses or plasmids. Then, in a separate step, a genetic construct known as single-guide RNA, which steers Cas9 to the specific spots in DNA where cuts are desired, is also placed into the cells. However, this process has been inefficient and is generally not suitable for use within a clinical setting.

“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,” stated lead author Kathrin Schumann, Ph.D., a postdoctoral fellow in Dr. Marson's laboratory. “Using RNPs [ribonulceoproteins] made outside the cell, so that the cell is responsible for as little of the process as possible, has made a big difference.”

The UCSF team found that using an electroporation technique, where cells are briefly exposed to an electrical field that makes their membranes more permeable, was the most efficient method of delivering the genome editing tools inside the T cells.

The investigators were excited by their findings and are moving forward to develop standards in order to utilize their newly developed technique within a clinical setting.   

“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,” Dr. Marson noted. “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.”