Cancer immunotherapy has become even more effective
Genomic editing turned lymphocytes into anti-cancer "universal soldiers"
Oleg Lischuk, N+1
American scientists have improved the effectiveness of cancer therapy with T-lymphocytes with chimeric antigen receptors (CAR-T), using the CRISPR/Cas9 system to obtain them. The results of the work are published in the journal Nature (Eyquem et al., Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection), and related editorial material is also devoted to them (Marcela V. Maus, Immunology: T-cell tweaks to target tumours).
CAR-T technology is the most promising experimental cancer treatment method to date. It consists in the fact that the lymphocytes of the patient or donor are genetically modified so that they synthesize an artificial receptor, which consists of an antibody fragment that recognizes tumor cells and an intracellular part that activates the lymphocyte. During the CAR-T experiments, some forms of cancer were successfully cured, primarily hematological: the most effective were cells that recognize the CD19 B-lymphocyte antigen. However, in the treatment of most dense tumors, their effectiveness was insufficient.
Most modern methods of CAR-T production do not affect the main T-lymphocyte receptor (TCR). Thus, the modified cells express both artificial and "native" receptors. At the same time, lymphocytes with TCR, which are specific to the body's own antigens, are destroyed relatively quickly naturally in order to prevent the development of autoimmune diseases. This also weakens their attack on tumors, since some of the antigens of cancer cells coincide with the antigens of healthy cells, and leads to insufficient effectiveness of CAR-T. Experiments have shown that "switching off" the DNA sequence encoding the permanent section of the alpha chain TCR (TRAC) not only increases the effectiveness of CAR-T, but also allows the use of pre-prepared donor lymphocytes for treatment without the risk of rejection reaction.
Taking into account these data, the staff of the Sloan-Kettering Institute improved the method of obtaining CAR-T recognizing CD19. Using the CRISPR/Cas9 genome editing system, they embedded the chimeric antigen receptor gene directly into the sequence encoding TRAC, thereby interrupting it and stopping the expression of TCR. That is, they solved two tasks at once with one point effect: they provided CAR synthesis and deprived lymphocytes of "native" receptors.
A scheme for obtaining CAR-T using CRISPR/Cas9 (from an article in Nature).
In the search for promoters (regions of genes triggering their expression) that ensure uniform and long-term synthesis of CAR, scientists tested four different promoter sequences on mice with a leukemia model, as well as CAR genes devoid of promoters embedded in two different regions of the genome.
It turned out that embedding the CAR gene into the TRAC sequence allows it to use a natural promoter of T-cell receptors, which ensures the most stable expression of CAR and maximum antitumor efficacy.
Thus, the developed technique makes it possible to increase the effectiveness of CAR-T therapy, use donor cells, which makes the technology more versatile and reduces its cost. In addition, targeted insertion of the CAR gene by the CRISPR/Cas9 system is associated with a lower risk of side effects compared to embedding in a random part of the genome, which occurs when using a viral vector.
Earlier, the US Federal Commission on Biosafety and Ethics approved the first experiment on editing the human genome using the CRISPR/Cas9 system. During trials at three clinical centers, it is planned to use this technology to remove two CAR-T-lymphocyte genes. One of them encodes TCR, and the other is the PD–1 protein that inactivates T cells. If the trials are approved by the US Food and Drug Administration (FDA) and clinics, 15 patients with multiple myeloma, melanoma and sarcoma will receive experimental therapy.
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28.02.2017