Another success of gene therapy
Editing thalassemia
Alexandra Bruter, <url>
Gene therapy has taken a step towards the treatment of beta-thalassemia and diseases caused by a specific mutation in a single gene. The corresponding work was published in the journal Genome Research by a group of scientists from California (CRISPR corrects b-thalassaemia). Scientists obtained induced pluripotent stem cells from the patient's cells, edited their genome and forced them to differentiate into blood cells.
Beta-thalassemia is a genetic blood disease caused by mutations in the gene encoding one of the parts of hemoglobin. When mutations turn out to be in both copies of the gene, the disease can be very difficult. There is no treatment as such, only blood transfusions and bone marrow transplants, if a donor can be found. Drug treatment is aimed at reducing the toxicity of free iron, which in healthy people is bound in a hemoglobin molecule.
Patients with mutations survive, due to which hemoglobin synthesis completely stops, thanks to embryonic hemoglobin - this is another, less effective variety, which is encoded by another gene. In healthy people, its level of its synthesis drops rapidly after birth, although not to zero.
Since beta-thalassemia refers to monogenic diseases – those caused by a mutation in a single gene, and it seems to be a promising target for gene therapy. Turned on the right gene in the right place – and the patient is healthy. It's a small matter – to figure out how to turn it on.
The most effective method to date is viral delivery of the desired gene. A special virus is created that is unable to infect anyone more than once and instead of genes that would encode its own viral proteins, containing therapeutic genes. This virus infects cells. Now one person after treatment with such viruses lives with a genotype corresponding to a severe form of beta-thalassemia, but without blood transfusions and feels fine.
Viral delivery of therapeutic genes, however, is an effective but dangerous thing. First, the virus can embed its DNA in any place of the genome. This can disrupt the work of some gene needed by the cell and lead to undesirable consequences, up to the transformation of the cell into a cancerous one. In addition, the human body is accustomed to fight viruses, and when a large number of viral particles are introduced, an immune response occurs, sometimes quite severe. Therefore, the question arises whether it is possible to edit the genome in some safer way.
Restriction endonucleases are widely used in genetic engineering – enzymes that play the role of the immune system in bacteria. Bacteria have their own viruses, they are called bacteriophages. They are just like ordinary viruses, in order to multiply, they must infect a bacterium and embed their genome into the genome of a bacterial cell. Since it is deadly for bacteria, they defend themselves. They have enzymes that recognize small sequences of bacteriophage DNA (similar sequences in the bacteria themselves are prudently chemically modified and inaccessible to enzymes) and cut it. For some such enzymes, sequences ("recognition sites") strictly defined and, as a rule, consist of no more than 10 base pairs. This is very convenient for scientists who need to edit, stitch and glue small sections of DNA in the laboratory – there they manage to pick up enzymes whose recognition sites are found 1-2 times in the edited DNA. The whole genome, however, is much larger: the human genome is 3 billion base pairs, and laboratories most often deal with plasmids and viruses – it is, at most, several tens of thousands of base pairs. Probability theory suggests that a specific arbitrary "word" of eight letters will occur in the genome about 50 thousand times. If you edit the genome with such a tool, nothing good will come out of it, but only pieces will fly into the nooks and crannies, and the cell will quickly die, unable to repair all the gaps.
To the delight of scientists, bacteria have found another method of protection against bacteriophages – this is CRISPR/CAS9. This is also a kind of "immune system" of bacteria. It functions due to short sequences in the DNA of the bacterium corresponding to fragments from the DNA of bacteriophages. Actually, CRISPR means "Clustered Regularly Interspaced Short Palindromic Repeats" – short palindromic repeats regularly arranged in groups. From these fragments of bacteriophage DNA, the bacterium synthesizes RNA chains. RNA interacts on the principle of complementarity with the DNA of bacteriophages penetrating into the bacterial cell, and while they interact, special proteins make a break in the DNA at the site of interaction. In this particular case, this function is performed by the CAS9 protein. A similar method of gene inactivation exists in more complex organisms, up to humans. It's called RNA interference.
Scientists have learned to use this phenomenon to their advantage. It is possible to synthesize RNAs complementary to the place where you want to make the incision, and similar to RNAs from the CRISPR/CAS9 system and introduce them into cells together with the CAS9 protein. In fact, of course, DNA constructs encoding both are introduced into cells. The RNA will point to the place where you need to cut, and the protein will cut. The complementary part of such RNAs, as a rule, is about 20 bases, so it is easy to choose an RNA complementary to the only desired genome sequence.
The introduction of a break in the double–stranded genomic DNA greatly increases the likelihood of homologous recombination, a process in which homologous chromosomes can exchange homologous fragments. If a donor DNA carrying a normal hemoglobin gene is injected into a cell, and then a gap is inserted into the right place using the CRISPR/CAS9 system, then a chromosome with a normal gene is formed with a certain satisfied high probability.
The authors of the article did all this with iPS cells – pluripotent cells obtained from the somatic cells of the patient himself. Since the patient's own cells serve as the material, you don't have to worry about the immune response after the cells are transplanted back. Then the cells were differentiated into erythroblasts – the precursors of erythrocytes still containing the nucleus and the level of hemoglobin synthesis in them was evaluated. It turned out that it was increasing. However, the level of synthesis of the embryonic variety of hemoglobin increased even faster, due to which patients with beta-thalassemia survive.
The authors say that after finalizing the method of differentiation into adult erythroblasts, their method will be applicable to the treatment of people. In any case, another rather effective method of genome editing has been proposed, which may be useful for the treatment of a variety of diseases.
Portal "Eternal youth" http://vechnayamolodost.ru19.08.2014