Vol 12, No 4 (2017)

Exosomes are extracellular vesicles with the diameter of 30-120 nm, originating from early endosomes. Exosomes have been actively studied in the last decade, and a great amount of data has appeared on their nature and role in the intercellular transport and signaling both in the normal and pathological conditions. A particular interest to exosomes in the clinical practice emerged after the separation of their circulating fraction from the blood and the study of tumor genetic markers in them became possible (so called “liquid biopsy”). The objective of this review is to familiarize clinical specialists with the fundamentals of exosomes' biology and physiology and with the main achievements on their practical application in the medicine, as a natural drug delivery system, as well as for high-precision, early non-invasive differential diagnostics of diseases.

The HTT gene (Huntingtin, IT-15) was described in 1993 as highly expressed in various parts of the brain and other human and rodent tissues. The interest to this gene is due to the fact that the expansion of trinucleotide repeats in the first exon leads to the Huntington's disease. However, the causes of selective death of striata neurons in the course of the disease development are still unknown. Studying the HTT expression pattern in different tissues allows us to understand the role of HTT isoforms in different human tissues and organs. We studied the expression and alternative splicing of HTT in different parts of the brain and other human tissues in healthy people and Huntington's disease patients. No aberrant HTT forms were found in striatal neurons. This confirms the important role of the HTT gene for this type of neurons.

Bone substitutes with osteoinductive and (or) osteogenic capacities are highly needed in clinical practice for treatment of patients with skeletal bone pathology. Gene-activated bone substitutes consisting of a scaffold and gene constructs belong to such materials. In this study, we made two proto-types of gene-activated bone substitutes based on octacalcium phosphate (OCP) granules and two variants of plasmid DNA - the system delivering single gene encoding vascular endothelial growth factor A-165 (VEGFA-165, pl-VEGFA) and the other plasmid carrying simultaneously VEGFA and gene of stromal-derived factor 1a (pl-VEGFA-SDF). All the materials were implanted to rabbits into the full-thickness parietal bone defects with diameter 10 mm, OCP without plasmid DNA we used as a control. Both gene-activated materials showed pronounced osteoinduction providing new bone formation in the central part of the defects and complete parietal bone repair by 90 days after surgery. In addition, we found newly formed bone level to be higher in pl-VEGFA-SDF group comparing with pl-VEGFA (p<0.05), while there were the opposite results at 60 days' time point (p<0.05). Thus, gene-activated bone substitutes we developed had osteoinductive effect, dynamic and features of which were different and depended on biological action of the factors delivered by transgenes.

Today we have an inadequate set of methods for treating spinal cord injuries. Gene therapy (direct or cell-mediated) is one of the most promising approache for successfully solving this problem. The present study focused on evaluating the therapeutic efficacy of genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and the neuronal cell adhesion molecule (NCAM) in the model of contusion injury in rats. The therapeutic genes in two combinations (VEGF + GDNF + NCAM and VEGF + ANG + NCAM) either were administered intrathecally, with the help of adenoviral vectors, or on cellular carriers - genetically modified mononuclear cells of human umbilical cord blood. On 30 day after a spinal cord injury, the safety of the myelin fibers of the white matter and the kinematics of the left hindlimb joints in experimental animals were analyzed. Both therapeutic combinations of genes have shown a positive effect on the conduction pathways and kinematics of the joints.

The success of cell therapy depends on an effective method of cell delivery and engraftment. The use of transgenic cells carrying a reporter system based on the luciferase gene allows to perform the quantitative evaluation of the transplantation efficiency in dynamics using biochemical methods. The purpose of this work was to monitor the persistence of rat cardiosphere-derived cells (CDC) after allogeneic transplantation into the periinfarction zone. Transplantation was performed by intramyocardial injection of a cell suspension in a culture medium or in platelet rich plasma (PRP). When injected into the myocardium PRP forms fibrin clots which serves as a matrix for the transplanted cells. The cells were modified by the luciferase enzyme gene by transduction with lentiviruses (CDC-Luc). The activity of luciferase was determined in protein extracts of the myocardium at different time points after the transplantation. It was shown that in the first hour after injections, CDC-Luc is quantitatively detected in the peri-infarction zone irrespective of the use of platelet gel or medium, and their amount does not decrease within 48 hours. During this period, we found a positive effect of the fibrin matrix on the cells - the luminescence of CDC-Luc protein extracts in the platelet gel composition was significantly higher. We suggested that the platelet gel promotes a more favorable microenvironment for CDC-Luc and facilitates the adaptation of cells after transplantation, what reflected in the recovery of the level of luciferase production in cells. Further, we found negative dynamics: CDC-Luc injected in the culture medium is retained in the myocardium for 5 days and on the seventh day their presence is not determined, CDC-Luc in the fibrin matrix is retained in the myocardium for 10 days after transplantation. Thus, despite the successful transplantation of CDC, the integration of cells into the myocardium does not occur. Nevertheless, the use of platelet gel prolongates the time of CDC persistence in the tissue and enhances of their paracrine effect. The use of fibrin matrix can be useful for long-lived cells, such as cardiomyocytes, in particular, to improve the efficiency of transplantation of the tissue engineering biological pacemaker. A luciferase reportering system can be effective for in vitro and in vivo monitoring of cell fate, both in biotechnological stages of cultivation and assembly of the tissue engineering biopee maker, and after myocardial transplantation. In the future, the developed methodological approach will be used to study of tissue-engineering biopacemakers in experimental animals.

Osteopetrosis is a group of rare hereditary diseases, the general structural manifestation of which is the excessive volume of bone tissue due to violation of osteoclastic resorption. The only way to treat this group of patients so far remains transplantation of hematopoietic stem cells, but the degree of its effectiveness largely depends on the severity of morphological changes in the hematopoietic microenvironment in the bone marrow. In this regard, a comprehensive clinical and morphological analysis, in conjunction with the results of transplantation can help in determining the prognosis of the disease depending on the genetic type of osteopetrosis. The material for the studies were biopsies and smears of bone marrow of patients with osteopetrosis, who were received at the R. Gorbacheva Memorial Research Institute for Pediatric Oncology, Hematology and Transplantation from the republics of Chuvashia and Mari El for carrying out transplantation of hematopoietic stem cells. The histological preparations were stained with hematoxylin and eosin, azur by Romanovsky and by Van Gieson. Bone marrow smears were stained by Romanovsky-Giemsa. The presence of the mutation c.807 + 5G>A in the gene TCIRG1 in patients with autosomal recessive osteopetrosis causes a complete loss of osteoclastic resorption, which is accompanied by pronounced early structural changes in the hematopoietic microenvironment already at the time of diagnosis. This is manifested by an excessive amount of lamellar bone at the same time as the almost complete obliteration of the medullar lacunae, as well as fibrosis of the bone marrow stroma. A common morphological phenomenon among this group of patients is the presence in the bone marrow of an excessive number of osteoclast differentiation cells without the formation of resorption lacunae. This causes a complete suppression of hemopoiesis, which clinically manifests cytopenia and the formation of foci of extramedullary hematopoiesis. Such pronounced structural changes in the hematopoietic microenvironment result in a high risk of primary graft failure during after transplantation of bone marrow hematopoietic stem cells, the effectiveness of which decreases as the child's age increases. Early development of pronounced structural changes in the hematopoietic microenvironment in patients with TCIRG1-mediated osteopetrosis determines the need for diagnosis and transplantation as soon as possible after birth.

Scientific progress and advances in medicine and biology have contributed to emergence of new treatments based on delivery of living cells, genetic material and tissue engineering. The Federal Law N180-FZ “On Biomedical Cell Products” has taken effect in Russia opening a new stage in development of biomedical technologies, the main objective of which is to create a unified harmonious environment for the development, production and sale of safe and effective cell therapy products. At the same time, the system of legal regulation of the market of cellular products abroad has been functioning for more than 10 years and continues to develop actively. This review analyses international experience in regulation of medical biotechnologies market and identifies main features and current problems of existing scheme of biomedical products marketing as well as highlights main issues to be addressed while forming regulatory and legal filed for biomedical cellular products (BMCP) in Russia.