Gene therapy straightened red blood cells
Sickle cell anemia learned to treat with CRISPR
Alexander Ershov, N+1
Scientists from three American universities have tested a method of genetic therapy of sickle cell anemia using CRISPR/Cas9 technology on mice. The test results suggest that the changes made to the genome really persist for a long time. However, so far the effectiveness of making genetic "corrections" is quite low, and the safety of therapy has not even been investigated. The work was published in the journal Science Translation Medicine (DeWitt et al., Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells).
Sickle cell anemia is one of the most well-studied genetic diseases. It is caused by a single mutation in the sequence of one of the proteins that make up hemoglobin – beta-globin (ordinary hemoglobin is a tetramer of two alpha– and two beta-globins, each of which is equipped with a non-protein cofactor, heme). Replacing the amino acid glutamate with valine in the primary sequence of beta-globin leads to the fact that the resulting hemoglobin becomes "sticky" and begins to form aggregates inside red blood cells. This leads to the fact that red blood cells acquire an elongated shape, thanks to which the disease got its name.
A snapshot from the UC Berkeley Genome engineering press release paves the way for sickle cell cure - VM
Sickle-shaped red blood cells often accumulate in the places of branching of blood vessels and can form blood clots, leading to a stop of blood supply and even tissue death. In addition, such red blood cells are more often destroyed, which causes anemia and leads to problems with immunity. In general, sickle cell anemia reduces a person's life expectancy by about 30 years. Despite the fact that the pathological processes associated with the development of this disease are well studied, doctors now have almost no mechanisms for its actual therapy. The only effective method of intervention is the transplantation of hematopoietic cells from a donor together with bone marrow, but it is dangerous in itself for the immune system, not to mention the problems of finding a suitable donor.
Sickle cell anemia, however, is a convenient target for genetic therapy. The fact is that in this case, it is not necessary to replace the mutant sequence in all cells of the patient's body (as, for example, when trying to remove HIV from the genome). It is enough to have a targeted effect on stem hematopoietic cells, and not necessarily on all 100 percent of their population. Even if some of them begin to produce normal beta-globin, the red blood cells obtained from such cells, due to the increased time of their life, should stop the main symptoms of the disease.
Some research groups have already tried using genetic therapy to treat this disease. So, the first successes last year were announced by scientists in the journal Blood. However, then, to introduce the "correct" mutations, the authors of the article used the so-called zinc finger nucleases, or ZFN proteins (they are specially developed in silico for each mutation under study). And the efficiency of the process was low: only about one percent of the "correct" blood cells were preserved in the bone marrow of mice. In the new work, the researchers turned to a more modern CRISPR/Cas9 technology, which is now beginning to be actively tested in medicine.
Briefly, the experiment looked like this. Scientists took a line of cells resembling blood stem cells (hemoblastocysts) and injected a special drug into them using an electric current. It was a ribonucleoprotein: a complex of protein-nuclease Cas9, a guide RNA and the correct version of the gene. Penetrating into the cell, the nuclease cut the beta-globin gene near the pathogenic mutation, and then the cell's own enzymes healed the gap using a DNA sample from the drug. The efficiency of the editing process depends on many factors: not only the specific guide RNA and target, but also on the DNA sample. And a significant part of the described work was devoted just to the selection of conditions for the most effective introduction of mutations. As a result, the authors managed to ensure that about a third of the treated cells were "corrected" at the genetic level. However, the main part of the work was that these cells were injected into the bloodstream of mice and monitored how they behave and how long they can last (mice of a special line were used that do not produce an immune response to human cells).
The main positive result of the work is that hematopoietic cells with an altered genome were detected in the bone marrow of mice even a few months after therapy. For rodents that live no more than two years, this result can be considered very good. On the other hand, the "correct" hematopoietic cells accounted for only two percent of the total population. According to the authors, even this is enough to lead to a significant therapeutic effect (and this is twice as much as in previous work with ZFN nucleases). However, before the start of clinical trials, scientists plan to raise this share to at least five percent. After that, it is planned to investigate the side activity of nucleases, i.e. the possibility of introducing breaks in other parts of the genome. The danger of such ruptures in therapeutic applications of CRISPR/Cas9 has not yet been practically investigated. Only after the completion of these two stages will it be possible to talk about clinical trials of the technology.
Sickle cell anemia is just one example of the immediate goals of genetic therapy using CRISPR/Cas9. So, studies are already underway on the possibility of therapy for another hereditary disease, thalassemia (which is also associated with a mutation in beta-globin). It was this disease that was targeted when editing the first embryonic genome by a Chinese group at the beginning of last year. Another example is Duchene's myodystrophy, whose therapy has already been tested on mice. Bioengineers have high hopes for the combination of CRISPR/Cas9 technology and the method of chimeric antigen receptors – these studies were the first studies of this kind approved by the American FDA regulator. A more complex goal can be considered the use of CRISPR to remove HIV integrated into the host cell genome. This is likely to require significant progress in the effectiveness of genetic editing, but such experiments are already being conducted, including on laboratory animals.
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14.10.2016