Genome editing: new opportunities

In fact, they were able to use the CRISPR/Cas9 system, which could not be used before due to difficulties with the delivery of system components to lymphocytes.

The CRISPR/Cas9 system was recently discovered and has already become very popular due to the outstanding combination of efficiency and safety. Most of the noise in the media is caused by its potential use for editing the DNA of embryos, especially human ones. Editing embryonic genomes is an ethically ambiguous idea and, from a practical point of view, rather useless: almost all medical problems that editing could solve, such as the birth of a healthy child in a family with a disease transmitted from generation to generation, can be solved by embryo selection – it is both cheaper and safer. But because of the ethical ambiguity, this area is well known to everyone, and there are other areas of application of CRISPR/Cas9 that sometimes deserve more attention. Earlier, we wrote about the CRISPR/Cas9 system and its application in the treatment of various diseases in the essays "Genome Editing: pros and cons", "A new step in genome editing".

Genetic modification of blood cells could be useful for the treatment of many diseases, even not directly related to blood, because there are also cells of the immune system in the blood, for example, lymphocytes. One way or another, by changing the genome of blood cells, it would be possible to treat sickle cell anemia, beta-thalassemia, AIDS and other immunodeficiencies, for example, severe combined immunodeficiency, lymphomas, leukemias, probably some tumors of other organs, autoimmune diseases, in particular, diabetes.

For example, the HIV virus primarily penetrates into T-lymphocytes. Due to the fact that the virus multiplies in them, the cells die. A deficiency of T-cells makes a sick person defenseless against infections and oncological diseases, and he dies. The virus can store its copies in other cells, but a person gets sick and dies precisely because he has no T-cells left. The virus penetrates into T cells through receptors on their surface. He needs, first of all, the CD4 receptor, which all people have on the surface of some lymphocytes, and plays an important role in their vital activity. In addition to the CD4 receptor, the virus needs another, auxiliary, receptor. Some strains – CXCR4, some – CCR5, some will fit any. These are not as important receptors as CD4, and they can be removed from the surface of T-lymphocytes.  There are a certain number of people in the world who simply do not have a CCR5 receptor, and they are immune to CCR5-dependent HIV strains. The CXCR4 receptor is involved in the process of embryo implantation in the uterus and in the embryonic development of the nervous system, but adult T cells can do without it. T-cells and blood stem cells have a good proliferative potential, so if you take a few of these cells from a person, modify their genome and release them back into the bloodstream, a person, even if the virus persists in his body, will have enough cells into which he will not be able to penetrate, and a person will be able to behave normally to feel.

Recently, the direction of tumor immunotherapy seems to be increasingly promising. Normally, cells in the human body degenerate into cancerous cells from time to time, but the immune system calculates them and kills them. Problems begin when newly formed cancer cells acquire the ability to hide from T cells. The human immune system is calibrated to kill enemies, but not to touch their own. Cancer cells occupy an intermediate position, because they are much more similar to ordinary human cells than, say, a bacterium to an ordinary cell. At the same time, the immune system cannot treat the inspected cells too pickily all its life, otherwise an autoimmune disease will develop. But scientists have found a way to make T cells more evil cops when it's needed. To do this, turn off the PD-1 receptor, which is responsible for quenching the immune response. Antibodies to this receptor helped to achieve good results in the treatment of tumors, but cells devoid of PD-1 receptors would be more effective and would work much longer.

Genetically modified T-lymphocytes have already been successfully used for the treatment of B-cell leukemia (a story about this can be read in the essay "Gene therapy of leukemia: success again"). In this case, the cells were modified with specially created viruses so as to recognize and kill any B-cells, including malignant ones. After recovering, such patients should receive regular injections of antibodies due to the lack of B cells, but these are the only unpleasant consequences, and the method has already saved several lives of terminally ill people.

Non-viral modification of cells looks, however, preferable to viral. Viruses are usually embedded in a random place in the genome, can cause mutations that turn cells into malignant. Such episodes have taken place in the history of gene therapy. You don't have to think about them, saving people from certain death, but modern HIV-infected people, as a rule, can hope for a sufficiently long and high-quality life even without gene therapy.

The CRISPR/Cas9 system was borrowed from nature, or rather from bacteria. For them, it works as something like an acquired antiphage (antiviral) immunity. Having met with the virus once, the bacterium leaves fragments of the viral in its genome. RNA with the necessary signal sequences can be synthesized from these fragments. If the same virus enters the bacterial cell again, the RNA synthesized by the bacterium will interact with the viral genome according to the principle of complementarity. Then a special bacterial enzyme Cas9 comes into play, which is able to cut such interacting structures.

This mechanism is used in therapy. If you deliver RNA to the cell, a complementary gene that needs to be corrected, the Cas9 enzyme (can be in the form of DNA or RNA) and the correct copy of the gene, then they cope on their own.

The CRISPR/Cas9 system compares favorably with the rest in efficiency and safety. To modify the genome of a cell with its help, it is necessary that the system works only for a short time at the very beginning, and then, when the genome is edited, it is no longer necessary. Usually, RNA transfection is used for this. The necessary RNA molecules are introduced into the cell, the necessary proteins are synthesized from some of them, some are needed exactly as RNA, they do their job and degrade – RNA and proteins do not live in cells for long. Only the edited gene remains. Viruses embedded in the genome will remain in the random place in which they are embedded for the rest of the cell's life.

The only problem was that T-cells are poorly amenable to genetic modification, and the efficiency of CRISPR/Cas9 with them is generally low. The solution was found when scientists decided to deliver the Cas9 complex with a guide RNA using electroporation. Electroporation is a method of delivering different molecules to cells. Under the influence of short electrical impulses, pores appear in cell membranes through which molecules can penetrate inside. The pores soon close, and the cell continues to live its life.

The CRISPR/Cas9 system is able to make breaks in the specified place in DNA using guide RNA molecules. Cells have their own system for repairing breaks in DNA. If a break occurred only in one of the chains, then the break in it is repaired according to the principle of complementarity. The enzymes take the surviving chain as a sample, and complete the damaged one. If the gap occurs in both (and it does if CRISPR/Cas9 works effectively), then there is no sample, and the gap is patched with random nucleotides. In this way, you can make the gene inactive.

But not only the enzyme-nuclease and guide RNAs can be introduced into the cells, but also a DNA sample, checking with which the enzymes close the gap. Then you can not only inactivate a gene, but also edit it in the right way.

The analysis of the authors of the work showed that the cells in which the CXCR4 receptor simply lost activity were about 40% among the treated. In about 20% of the cells, the gene was not just inactivated, but replaced with the sequence that was additionally injected into the cells for this purpose. This is a very good result, which even viruses can envy.

CRISPR/Cas9 is a very promising technique for modifying any cells for any purpose. Due to its effectiveness and safety, it can significantly accelerate the development of gene therapy and solve some of its problems.

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27.07.2015