CXCR4 is a receptor broadly expressed in cells of immune and the central nervous systems and can mediate migration of resting leukocytes and haematopoietic progenitors. It is also used by strains of HIV1 to gain entry into Helper T cells, and are typically detected at the later stages, associated with a rapid decline in CD4+ T cells and progression to AIDS.
They disabled a T-cell surface protein CXCR4, exploited by HIV when the virus infects T cells and causes AIDS. The group also successfully shut down PD-1, a protein that has attracted intense interest in the burgeoning field of cancer immunotherapy, as scientists have shown that using drugs to block PD-1 coaxes T cells to attack tumors.
T cells not only stand at the center of many disease processes, but could be easily gathered from patients, edited with CRISPR/Cas9, then returned to the body to exert therapeutic effects. But in practice, editing T cell genomes with CRISPR/Cas9 has proved surprisingly difficult.
Drugs have been used to block PD-1 to encourage T cells to attack tumors.
The new work was done under the Innovative Genomics Initiative (IGI), a joint UC Berkeley-UCSF program. Cas9, an enzyme in the CRISPR system that makes cuts in DNA and allows new genetic sequences to be inserted, has generally been introduced into cells using viruses or circular bits of DNA called 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.
Until recently, however, editing human T cells with CRISPR/Cas9 has been inefficient, with only a relatively small percentage of cells being successfully modified. In lab dishes, Schumann and Lin team assembled Cas9 ribonucleoproteins, or RNPs, which combine the Cas9 protein with single-guide RNA. They then used electroporation, in which cells are briefly exposed to an electrical field that makes their membranes more permeable, to quickly deliver these RNPs to the interior of the cells.
With these innovations, they successfully edited CXCR4 and PD-1, even knocking in new sequences to replace specific genetic “letters” in these proteins. The group was then able to sort the cells using markers expressed on the cell surface, to help pull out successfully edited cells for research, and eventually for therapeutic use. “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,” Schumann said. “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.”
Marson stressed that, while recent reports of CRISPR/Cas9 editing of human embryos have stirred up controversy, T cells are created anew in each individual, so modifications would not be passed on to future generations. He hopes that Cas9-based therapies for T cell-related disorders, which include autoimmune diseases as well as immunodeficiencies such as “bubble boy disease,” will enter the clinic in the future.
http://www.eurekalert.org/pub_releases/2015-07/uoc–ica072315.php
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