Gene therapy of neurodegenerative diseases

Amyotrophic lateral sclerosis was cured at the cell level

Alexander Ershov, N+1

Molecular biologists from The University of California at San Diego (USA), based on CRISPR/Cas9 technology, has developed a method for eliminating transcripts of "parasitic" RNA. The accumulation of such RNA is associated with the development of many serious diseases, such as amyotrophic lateral sclerosis, myotonic muscle dystrophy, Huntington's disease, etc. Scientists have managed to test the new method so far only on individual cell lines, but the results of the research look quite promising from the point of view of the therapy of these still incurable diseases. The researchers' article was published in the journal Cell.

Microsatellites are sections of the genome that represent multiple repeats of short (less than 9 letters) nucleotide "phrases" like (CTG)n, (CCTG)n, etc. They make up a significant part of the entire human genome and are often considered as so-called junk, that is, DNA that does not perform any meaningful functions. Such repeats occur both outside and inside genes (primarily in non-coding regions, introns), they tend to change rapidly (increase or decrease) from generation to generation, which, for example, is used in genetics to establish kinship between close individuals.

The expansion of such sequences, if they are inside genes, is associated by doctors with the development of several serious diseases. Apparently, an increase in the number of repeats in DNA is harmful not in itself, but as a cause of improper maturation (splicing) of the matrix RNA of these genes. The development of the disease is usually accompanied by the accumulation of point aggregates of RNA in the nucleus with these "junk" sequences. Sometimes such RNA is even translated into proteins, which leads to the accumulation of insoluble protein aggregates and leads to cell death. At the level of the body, it looks like irreversible muscle dystrophy, the death of peripheral neurons, and so on.

The causal relationship between the accumulation of microsattelites in genes and the development of certain symptoms of the disease has so far been poorly investigated and is at the level of proven correlation for most of the diseases discussed. You can read more about one of the types of amyotrophic lateral sclerosis here, about myodystrophy – here and here, about Huntington's disease – here. Nevertheless, there is reason to believe that since each of the diseases is accompanied by the accumulation of RNA aggregates in the nuclei, the elimination of these aggregates can lead to the elimination of the symptoms of the disease. This is what guided the authors of the new article, who developed a method for eliminating aggregates and "cured" several cell cultures with its help.

Theoretically, it would be possible to eliminate the cause of the diseases under discussion with the help of point intervention in the genome: it is enough to remove repeats, bring the necessary genes to a "normal" state and the cause of the disease will be eliminated. The improvement of CRISPR/Cas9 technology has made this theoretical possibility a practical therapy option. In some cases, this approach has even reached the stage of clinical trials.

The problem is that genome editing involves cutting DNA, and this is fraught with a sharp increase in the number of mutations in cells. In addition, the use of even the most accurate "genomic scissors" inevitably leads to the appearance of unnecessary, off-target incisions in other places of the genome, which is also dangerous and may be accompanied by the development of malignant neoplasms. The discussion of the scale of such non-targeted gaps in genetic therapy is now in full swing, and, perhaps, in reality, the danger of CRISPR/Cas9 is greatly exaggerated. Anyway, the transition from DNA to RNA eliminates this problem completely: whatever side breaks occur in RNA, they will not affect the stability of the genome. Not for all diseases such a transition is possible, however, that is why the authors of the new article turned their attention to those diseases that are associated with the accumulation of microsattellite RNA.

The new method works as follows. A genetic construct is introduced into cells using a modified adenovirus, which contains a guide RNA and a specialized version of the Cas9 nuclease. The latter is not able to cut DNA, because the amino acids necessary for this have been artificially removed in its active center. However, the inactivated nuclease can bind to RNA molecules under the guidance of the corresponding guide RNA. A short RNase is also attached to the modified Cas9, called RCas9 by the authors, which destroys those RNAs to which the complex is attached.

In the course of experiments on cell lines, including those isolated from patients with myodystrophy, scientists have shown that RCas9 is really able to eliminate aggregates of disease-specific microsattellite RNA. In addition, the cells normalize the splicing altered during dystrophy (at the level of the entire genome, not a single gene with microsatellites) and the concentration of the number of peptides translated from microsatellite repeats drops sharply. As for side effects, the authors assessed the effect of the system on non-target RNAs as insignificant: according to transcriptome analysis, less than 10 different genes changed their expression level as a result of the use of RCas9. A separate part of the article is devoted to experiments to reduce the size of Cas9 in such a way that the resulting construct can be placed in a very limited host virus genome.

The CRISPR/Cas9 genome editing technology has been gradually coming into clinical practice lately. So far, most scientists do not risk using it to make permanent, inherited changes to the genome, although laboratory experiments in this direction are already being conducted not only in China, but also in the United States. Most therapies focus on making non–inherited changes in somatic cells or on an even milder version of the method - actions at the RNA level.

Portal "Eternal youth" http://vechnayamolodost.ru  29.08.2017