Vol 8, No 3 (2013)

Dysferlinopathies is a group of autosomal-recessive inherited neuromuscular diseases, which are characterized by defect in mRNA expression or in functionioning of dysferlin protein, appearing in about 1/200 000 births. Dysferlin is encoded by DYSF gene (Dystrophy-associated fer-1-like). It's disruption can cause various types of primary dysferlinopathies, which include Miyoshi myopathy (MM), Limb-girdle Muscular Dystrophy type 2B (LGMD2B) and distal myopathy with anterior tibial onset. Also, dysferlin deficiency can be associated with other diseases, such as caveolin- and calpainopathies. Here we discuss dysferlin protein structure and function, it's clinical phenotypes, known animal models and developing treatment strategies for dysferlinopathies.

Development of new effective osteoplastic materials is highly requested in practice of traumatology and orthopedics, oral and maxillofacial surgery. The goals of our research were design and construction of gene-activated bone graft (GABG) consisting of collagen/hydroxyapatite scaffold and plasmid DNA encoding vegf-a165 and evaluation of its biological effect in vitro and in vivo. We have shown that GABG co-incubation with multipotent mesenchymal stromal cells increased their VEGF protein expression. After GABG implantation into parietal bones defects the transfection of «recipient bed cells» was observed and accompanied by more pronounced angiogenesis as compared with control. The lager volume of bone regenerate was in case of GABG on 15 and 30 days after application. The source of reparative osteogenesis was not only parietal bones but also the GABG fragments (even from central part of the defect) majority of which were surrounded by newly formed bone tissue. In control group no osteoinductive effect has been observed. Thus, GABG with plasmid DNA encoding VEGF-A165 possesses angiogenic activity providing osteoinductive properties.

It is known that human cord blood hematopoietic stem cells (HSC) are able to differentiate into hepatocytes. This ability can be widely used in treatment of various liver diseases. However, there are some genetic diseases of liver, when the application of autologous stem cells is not possible. So it could be very helpful to develop methods of genetic modification of stem/progenitor cells. However, it should be proved that genetic modification does not change the properties of HSC. We performed partial hepatectomy for the white mongrel male rats and injected human umbilical cord blood mononuclear cells transfected by gene of green fluorescent protein (GFP) into the spleen. Paraffin sections of the liver were stained with antibodies to stem cell factor receptor, human leukocyte antigen, a-smooth muscle actin, enhanced GFP, cytokeratin 19, hepatocyte specific antigen, human a-fetoprotein. Also we used a double-immunohistochemical staining to detect expression of stem cell factor receptor and desmin, enhanced GFP and cytokeratin 19. Our study showed that human cord blood mononuclear cells transfected by gfp transplanted into the spleen of rats after partial hepatectomy migrated to the liver and acquired the phenotype of hepatocytes, cholangiocytes and sinusoidal cells. At the same time the differentiation of such transplanted cells into myofibroblasts, as it was previously shown, does not occur. Hepatoblasts and hepatocytes found in the liver of rats after transplantation of genetically modified and native cells express human hepatocyte specific antigen and a-fetoprotein that means they are functionally active.

Gene-cell therapy is a new step for the treatment of different human disorders including central nervous system degenerative diseases. In this review we focused on the last challenges in the field of human umbilical cord blood mononuclear cells transplantation - an attempt to support neuronal cells survival and to stimulate the neuroregeneration. As a potential therapy for the treatment of neurodegenerative diseases we reviewed the latest advances in gene modification of human umbilical cord blood mononuclear cells as a novel tool for the effective delivery of neuroprotective factors and growth factors in the injured or degenerative areas of the central nervous system under pathological conditions. The main topic of this review is the potential therapy of the amyotrophic lateral sclerosis - the progressive neurodegenerative disorder affecting primarily upper and lower motoneurons - by using genetically modified human umbilical cord blood mononuclear cells. The results from the up-to-date experiments indicated the opportunity to obtain differentiated macrophages, endothelial cells, or astrocytes from the genetically modified human umbilical cord blood mononuclear cells after their transplantation in the mouse model of the amyotrophic lateral sclerosis. Taken together, these data build the high-capacity platform for the supporting of degenerating neurons, structural and functional recovery of the brain and spinal cord after trauma, ischemia and other neurodegenerative disorders.

The article presents a case report of successful treatment of pseudarthrosis of the femur with the use of autologous cells of the stromal vascular fraction (SVF) derived from adipose tissue. Autologous SVF cells mixed with fibrin glue were transplanted into bone defect by injection under the control of electro-optical converter. Two months after injection, the patient reported the disappearance of pain. After 4 months we observed the recovery of function of the knee, X-ray shows signs of the fracture healing through the formation of bone callus.

In this article we describe a clinical case of successful treatment of neurotrophic ulcers of heel region using classical surgical techniques such as debridement and autodermoplastics combined with vacuum therapy and injection of double cassette expression plasmid encoding cDNAs of vascular endothelial growth factor and fibroblast growth factor 2. The outcome of therapy opens new horizons in the treatment of such diseases in short period of time.

The aim of our work was to assess long-term results of autologous peripheral blood hematopoietic stem cell application in patients with peripheral arterial diseases. Peripheral blood hematopoietic stem cells mobilized by granulocyte colony-stimulating factor were transplanted intramuscularly to 30 patients with peripheral arterial diseases (IIb stage by Pokrovsky). Standart tredmill test, ankle-brachial index estimation and ankle-brachial index restoration time estimation after loading were performed on 0, 3, 6, 12 and 60 months after transplantation. Immunohistochemical study of injured gastrocnemius muscle biopsies taken before peripheral blood hematopoietic stem cells transplantation and 3 months after the procedure was performed. Peripheral blood hematopoietic stem cells transplantation increases capillary density (22,4%, p = 0,0005). Ankle- brachial index increased by 18,1% on month 6 after transplantation without a tendency to change on month 12. 60 months after transplantation initial to hematopoietic stem cells transplantation ankle-brachial index rates were marked. Painless walking distance was increasing at all times of observation progressively, on month 60 no walking distance limitation was marked by most patients. Ankle-brachial index restoring time shows positive trend of the functional state of limb during the first year after transplantation, 60 months after transplantation it showed no «walking reserve» limitation in most patients. 5-years survival was 79%, death causes were stroke, cardiac pathology (3 cases), lung cancer. So, peripheral blood hematopoietic stem cells transplantation allows eliminating peripheral arterial diseases symptoms and preserving limb in long-term period. Autologous transplantation of peripheral blood hematopoietic stem cells has no complications and is safe for therapy of patients with peripheral arterial diseases II stage.

To succed in gene therapy it is necessary to observe few basic conditions, such as targeted delivery of the therapeutic gene into the target organ and its effective expression. Nowadays targeted delivery of therapeutic genes is one of the critical problems of gene therapy. Delivery of recombinant genes using cell carriers (vectors), expressing tissue-specific cell adhesion molecules, allows us to move toward solving this problem. Using Gateway cloning technology (Invitrogen) we have created recombinant expression constructs pAd-NCAM1, pAd-NCAM2 and pAd-L1CAM, encoding neural cell adhesion molecules. Expression of recombinant proteins has been confirmed by immunofluorescent analysis. Based on these genetic constructs recombinant adenoviruses (serotype 5) were generated and titered. Obtained viral vectors encoding neural cell adhesion molecules may be subsequently used to modify cells carrying additional therapeutic genes to increase the efficiency of gene-cell therapeutics delivery to target organ.