Vol 15, No 1 (2020)

Gene therapy is a group of methods for treatment of various groups of diseases by replacement of damaged, introducing new genes or changing their expression. This is a new and actively developing area in biomedicine. Approaches to regulation of gene therapy drugs at all stages - from production, preclinical and clinical trials, as well as registration and marketing approval are not fully developed and regularly updated. This article provides an overview of worldwide approaches to conducting studies of gene therapy drugs and the procedures for their "accelerated registration”. This article as well summarizes our experience of Lomonosov Moscow State University in development of new direction vectors - gene therapy for the delivery of several therapeutic proteins. In the near future, this approach can be used to increase the effectiveness of gene therapy for stimulating vascular and axon growth, and tissue regeneration.

More and more researches suggest that the nervous and immune systems actively interact. Investigation of the mechanisms that underlie the development of septic shock led to discovery of a qualitatively new type of neuro-immune interactions - the cholinergic anti-inflammatory pathway involving the vagus nerve. However, despite the electrical stimulation of the vagus nerve that is already actively used in order to relieve the symptoms of some autoimmune conditions, many molecular and cellular aspects of the cholinergic anti-inflammatory pathway remain unknown or controversial. This review discusses the mechanisms of the reception of inflammatory mediators by vagal afferent fibers and nervous cells in the area postrema, the nerve centers presumably involved in the processing of immune information, the efferent parasympathetic effects on the immune system, in particular, the release of pro-inflammatory cytokines by spleen macrophages, as well as the role of cholinergic anti-inflammatory pathway in maintaining homeostasis in the body.

Duchenne muscular dystrophy is an X-linked recessive muscular dystrophy associated with a mutations in the dystrophin protein gene. The most common laboratory model of Duchenne muscular dystrophy is mdx mice. The striated muscle fibers of mdx mice are characterized by the absence of dystrophin, the presence of centrally located nuclei, and the high level of renewal of the striated muscle fibers. In addition, mdx mice show a morphological aberrations at neuromuscular junctions, expressed in the breakdown of large clusters of acetylcholine receptors in the form of branches into small clusters in the form of islets. One approach to treating muscular dystrophy in mdx mice may be the nonmyeloablative transplantation of wild-type bone marrow cells after X-ray irradiation of mdx mice at a dose of 3 Gy. The aim of this work is to evaluate the effect of nonmyeloabla-tive transplantation of wild-type bone marrow cells on dystrophin synthesis and the structure of neuromuscular junctions of mdx mice. Mdx mice were irradiated with X-rays at a dose of 3 Gy, after 24 hours was performed intravenous transplantation of bone marrow cells of C57BL/6 mice. The m. quariceps femoris and diaphragm were examined 2, 4, 6, 9, 12 months after transplantation. Muscle studies were performed using immunohisto-chemical methods of study (immunohistochemical staining with antibodies to dystrophin). The neuromuscular junctions were stained with tetramethylrodamine-a-bungarotoxin. After intravenous bone marrow cells transplantation, the part of dystrophin-positive muscle fibers in the muscle quadriceps femoris was shown to increase to a 27,6±6,7% 6 months after transplantation. After 12 months, the part of dystrophin-positive muscle fibers decreased to 5,1±1,1%. There was also an increase in the proportion of striated muscle fibers without centrally located nuclei and a decrease in the part of dead striated muscle fibers. Similar changes were found in the striated muscle fibers of the diaphragm of mdx mice. In addition, transplantation of bone marrow cells after irradiation at a dose of 3 Gy increases the part of neuromuscular junctions with normal structure. Thus, nonmy-eloablative transplantation of wild-type bone marrow cells can be considered as one way to treat monogenic disease of striated muscle fibers muscular dystrophy of mdx mice.

Cell mosaicism is found in biological systems much more often than clinically identified forms of the disease, in some cases, "erased forms” or "normal variants” are phenotypic manifestations of mosaicism. Some diseases, difficult for a clinical diagnosis, such as ataxia-telangiectasia, are based on cell mosaicism. This work is aimed to study DNA repair disorders in the cell lines of dermal fibroblasts isolated from skin biopsies of 5 patients with a clinically diagnosed ataxia-telangiectasia. In the obtained cell lines, the method of indirect immunofluorescence was used to determine the number, intensity, focus area pATMSer1981 and 53BP1, as well as the number of cells with the active form of the ATM kinase. The mosaic pattern of malfunctioning of the active form of the ATM kinase, phospho-ATM Ser1981, was revealed at different time intervals after exposure to ionizing radiation at a dose of 2 Gy. Significant differences were found between the number of ATMSer1981 and 53BP1 foci, the fluorescence intensity and their area in the cells of patients with ataxia-telangiectasia and healthy donors. The results of this work can be used in the diagnosis of ataxia-telangiec-tasia and determining the degree of impairment of the functional activity of the ATM gene.

Gene-activated materials are getting translated to the clinical practice that is the result of increased research activity in this area. There is significant success in development of gene-activated materials for bone grafting procedures. In our study, we made the gene-activated bone substitute based on octacalcium phosphate and plasmid DNA with VEGFA gene using three-dimensional printing technologies (discs, size of 10x2 mm). During the subcutaneous test in rats, the dynamics of bioresorption of materials was evaluated in comparison with the control that was presented by the scaffold without gene constructs. 60-90 days after surgery, volumes of the implants twicely decreased while the diameter declined by 20% at 180 days, and there were no significant differences between the groups related to these parameters. According to a histological examination, no signs of pronounced inflammation were found in the operation zone, the materials were surrounded by a connective tissue capsule. Further studies are needed to evaluate the effect of gene-activated materials produced with the developed technology on reparative osteogenesis.

In the European Union and the United States, regulation of cell-based products for the placing on the market is rather extensive and multi-tiered. This is due to the complexities which emerge at the different steps of the process, e. g. when obtaining source materials (live human cells), further development, manufacturing and clinical investigation, as well as due to risks posed by cellular products for the patient and other people. In this paper we address foreign regulatory systems for products containing live cells as an active ingredient in order to establish the difference with the domestic system.