Gene therapy of beta-tallasemia
A safe method for correcting mutations of hematopoietic cells has been developed
Oleg Lischuk, N+1
Erythrocytes of mice with beta-thalassemia and healthy animals (Yale University)
American scientists have developed a method of genetic correction based on peptidonucleic acids, which in an experiment effectively eliminated the symptoms of beta-thalassemia in mice. The results of the work are published in the journal Nature Communications (Bahal et al., In vivo correction of anaemia in β-thalassemic mice by yPNA-mediated gene editing with nanoparticle delivery).
Beta-thalassemia is a hereditary disease in which the synthesis of the beta–subunit of hemoglobin (beta-globin) is disrupted. This leads to deformation and a small size of red blood cells and a decrease in their life expectancy, an increased number of reticulocytes (immature red blood cells) in the blood, anemia of varying severity and an increase in the spleen (sometimes tens of times compared to the norm).
Peptidonucleic acids (PNA) are synthetic analogues of nucleic acids in which a chain of sugar (ribose or deoxyribose) and phosphoric acid residues is replaced by a polyamide pseudopeptide sequence carrying nitrogenous bases. NNAs in the form of oligomers are able to complementarily bind to nucleic acids. In the case of double-stranded DNA (DNA/DNA), some PNAS can displace one of the DNA strands in the complementary region, forming a three-stranded helix (PNA/DNA/PNA). In a living cell, this leads to the launch of cellular mechanisms of DNA repair (repair). In the presence of a "healthy" DNA fragment, these mechanisms restore the genome in accordance with it, which allows correction of individual mutations. Unlike common genome editing methods, such as CRISPR/Cas9, this approach does not require the involvement of third-party nuclease enzymes, which reduces the risk of unwanted genome modifications.
Employees of the Universities of Yale, Massachusetts and Carnegie Mellon used in their work PNAS substituted in the gamma position with polyethylene glycol (gamma-PNA) for better binding to DNA. Gamma-PNAS complementary to the mutation-bearing region of the beta-globin gene, together with the corresponding normal single-stranded DNA fragments, were placed in polylactide-co-glycolide nanoparticles, which ensure their penetration into hematopoietic stem cells (CSCs).
The structure of DNA (RNA), PNA and gamma-PNA (from an article in Nature Communications)
At the first stage of the experiments, scientists were convinced that such a drug successfully corrects the CSC genome with a mutation leading to beta-thalassemia in vitro. Then they found out that the efficiency of DNA editing increases with the activation of cellular signaling pathways associated with the tyrosine kinase receptor c-Kit (CD117), which is present in hematopoietic cells.
After that, they tested the technique on transgenic mice with mutant human beta-globin (such animals have all the typical symptoms of the disease). First, the mice were injected with a stem cell factor (SCF) activating c-Kit. Three hours later, they were injected intravenously with nanoparticles with gamma-PNA and normal DNA. The procedure was repeated four times with an interval of two days.
After 35 days, the concentration of hemoglobin in the blood of animals reached normal values and the level of reticulocytes decreased. In addition, their sizes have decreased and the histological structure of the spleen has improved. DNA analysis showed that the level of genome correction reached seven percent, with side modifications occurring more than 1,200 times less frequently. Similar effects persisted for 140 days of observation.
The effectiveness and safety of the technique were also confirmed in the culture of human hematopoietic cells.
"The combination of nanoparticle delivery, next–generation NNA and SCF can become a minimally invasive and safe treatment for genetic blood diseases, which is carried out by simple intravenous administration," the researchers write. According to them, the developed technique is suitable for the treatment of not only beta-thalassemia, but also other hereditary hemoglobinopathies, such as sickle cell anemia.
Another promising approach to the genetic modification of hematopoietic cells for the treatment of blood diseases, some cancers and chronic viral infections is their gene therapy with the help of a viral vector carried out outside the body (a bone marrow sample is taken from the patient, a transgen is introduced into the specified cells and injected back). Recently, a compact semi-automatic system has been developed that allows such an intervention to be carried out in a medical facility without the need for expensive laboratories.
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