Gene- and cell-based therapy of muscle system hereditary disorders: State-of-art

Genetic disorders primarily affecting skeletal muscles can be caused by dysfunction of more than 30 genes. To date there is no effective etiotropic and pathogenetic treatment of such disorders. Investigators focus on search for new therapeutic agents based on gene and cell technologies, small molecules as well. There are numerous preclinical and several dozens of clinical studies in the world. Unfortunately tested technologies did not lead to significant advance in treatment of patients with such disorders. At the same time resulting data allow to determine the most feasible directions of future development combining of genome correction methods with cell delivery of corrected genome to skeletal muscles. This review is intended to give general information about etiology of skeletal muscles genetic disorders, the main directions of biotechnological development and results of the clinical studies

Combined use of plasmid drug pCMV-VEGFA and autodermoplasty for stimulation of skin defects healing in the experiment

© 2018 Human Stem Cell Institute. All rights reserved. To find effective ways to stimulate chronic skin wounds healing (including deep burns, diabetic and trophic ulcers) is an actual multidisciplinary task. The aim of our study was to assess the potential of using autodermoplasty in combination with plasmid drug pCMV-VEGFA to optimize skin defects repair in the experiment. Autodermoplasty was performed on Wistar rats. The size of the skin flap was 22 cm. Immediately after surgery the animals of the test group (n=8) underwent intradermal injection in the periphery of autotransplant with 1 ml solution containing 0.3 mg of supercoiled plasmid DNA pCMV-VEGFA, rats of the control group (n=8) received 1 ml of 0.9 % NaCl. The results were analyzed in 3, 6, 9 12, 18 days using macroscopic evaluation, laser Doppler flowmetry, histological methods. Macroscopically in the test group necrosis of the transplanted skin flap was found at later periods of observation, in one case complete survival of autotransplant was observed. The results of laser Doppler flowmetry in the group with plasmid DNA did not have statistically significant differences with control. The wound defect diameter in the test group at 12 days was 5,52± 4.80 mm, in the control 12,45±0,82 mm (p=0,03); 2,53±of 2,94 mm and 4,23±3,5 mm (p=0,067) at 18 days, respectively. At 18 days, the average number of vessels under the flap in the central zone were: of 26±2,9 in the test group and 20±8 in control; it the peripheral zone 27±3,4 and of 12,1±3,9 (p=0,035), respectively; in the skin muscle 21,2±of 3,9 and 12,4±3,6 (p=0,04), respectively. Thus, the use of plasmid drug pCMV-VEGFA improved the skin healing after autodermoplasty

Serum Cytokine Profile in a Patient Diagnosed with Dysferlinopathy

© 2017 Svetlana F. Khaiboullina et al. Limb-girdle muscular dystrophy type 2 (LGMD2B) is a mild form of dysferlinopathy, characterized by limb weakness and wasting. It is an autosomal recessive disease, with currently 140 mutations in the LGMD2B gene identified. Lack of functional dysferlin inhibits muscle fiber regeneration in voluntary muscles, the main pathological finding in LGMD2B patients. However, the immune system has been suggested to contribute to muscle cell death and tissue regeneration. Serum levels of 27 cytokines wer e evaluated in a dysferlinopathy patient. Levels of 8 cytokines differed in patient serum compared to controls. Five cytokines (IL-10, IL-17, CCL2, CXCL10, and G-CSF) were higher while 3 were lower in the patient than in controls (IL-2, IL-8, and CCL11). Together, these data on serum cytokine profile of this dysferlinopathy patient suggest immune response activation, which could explain leukocyte infiltration in the muscle tissue

Long-term results of autologous peripheral blood hematopoietic stem cell transplantation in patients with peripheral arterial diseases

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, anklebrachial 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). Anklebrachial 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. © Human stem cells institute, 2013

Histopathological Analysis of Skeletal Muscle Biopsy of Patient with Peripheral Arterial Disease before and after Peripheral Blood Stem Cells Intramuscular Injection

© 2016, Springer Science+Business Media New York.Peripheral arterial diseases are characterized by a progressing tissue ischemia which results in the invalidization of patients. The aim of our research was to study the morphological effects of autologous peripheral blood stem cells intramuscular injection into patients with peripheral arterial disease. Peripheral blood stem cells were transplanted intramuscularly into a 48-year-old male patient with peripheral arterial disease stage IIb by Fontaine. The biopsies of his gastrocnemius muscle were taken before the stem cells were transplanted and 3 months after transplantation. These biopsies were stained with H&E and also with antibodies against CD34, myogenin, caspase 3, and bcl-2. Immunohistochemical study results showed an increase of capillary density of 32.7 % (P = 0.005). In muscular biopsies obtained before therapy, we identified single myogenin+ myosatellite cells, while 3 months after transplantation we detected the presence of cells with myogenin-positive nuclei and multinucleated myotubes. We also observed the formation of young myogenin+ muscle fibers with central nuclei. There was no significant difference in the expression of caspase-3 before transplantation and 3 months after transplantation. An increased number of bcl-2+ myosatellite cells, myotubes, and muscle fibers were detected after transplantation. The patient’s ankle-brachial index increased by 13.56 % (0.59 before and 0.67 3 months post transplantation). The patient’s pain-free walking distance by 89.97 % (from 59.56 to 113.77 m). Control arteriograms showed the formation of new collaterals. Transplanted autologous peripheral blood stem cells stimulated the formation of new capillaries, the activation of myosatellite cells and bcl-2 expression in muscle fibers

The first clinical experience of direct gene therapy using VEGF and bFGF in treatment patients with critical lower limb ischemia

In this paper we present the clinical observation of successful treatment of distal form of peripheral artery disease of the lower extremity with symptoms of critical ischemia in a 60 years old patient. Intramuscular injection of dual expression plasmid, encoding vascular endothelial growth factor VEGF and basic fibroblast growth factor FGF2, was performed to the affected lower extremity. The effect of treatment was evaluated by functional tests: measurement of ankle-brachial index, treadmill test, the recovery time, shoulder-ankle index after strain and temporary occlusion. Performed immunohistochemical examination of biopsy samples of the affected lower extremity muscles

Effect of Adenoviral Transduction of Hepatic Stellate Cells with Adv5-optHGF-RFP on their Phenotype

© 2016, Springer Science+Business Media New York.Adenovirus is a promising gene delivery vector that has a high efficiency and relative simplicity of construction. These advantages make this system attractive for diverse research applications. In this project we transduced hepatics stellatecells (HSC) with adenoviral vector containing genes of hepatocytes growth factor (HGF) and red fluorescent protein (RFP) as a reporter (Adv5-optHGF-RFP), which let us to visualize the transduced cells. Further changes of phenotype were studied by real-time PCR and immunocytochemical staining. According to our results, most of the cells were transduced and demonstrated stable expression of RFP. Effectiveness of the transduction was also confirmed by a high expression of HGF (1200 times higher than in control HSC culture). Adenoviral transduction of HSC with HGF gene did not change their morphology, but stimulated expression of HSC marker desmin, leading to HSC activation (α-smooth muscle actin) and appearance of α-fetoprotein, one of the hepatoblasts markers. During the entire experiment, there were Ki-67+ cells, meaning that proliferation of transduced HSC was not affected. Thus, adenoviral transduction of HSC with Adv5-optHGF-RFP is a good gene delivery system with a stimulating effect on HSC

Native and Activated Hepatic Stellate Cells Stimulate Liver Regeneration in Rats After Partial Hepatectomy and 2-Acetylaminofluorene Injection

© 2016, Springer Science+Business Media New York.One the current challenges of modern hepatology is to find new approaches to stimulate liver regeneration and to find new methods for liver disease treatment. Cell therapies, which are based on using regional stem cells for disease treatment, are under active development. However, studies, devoted to their transplantation, are currently scarce. In recent years, hepatic stellate cells are considered to be hepatic stem cells. It is known that activated hepatic stellate cells can transdifferentiate into myofibroblasts and lead to liver fibrosis. The aim of our work was to study the influence of native and activated hepatic stellate cells in vivo by lead nitrate injection after transplantation into partial hepatectomized rats, which is considered to be a classical model to study liver regeneration. Injection of 2-acetylaminofluorene (AAF), which selectively eliminates hepatocyte proliferation, was used to understand the hepatic stellate cells role in liver regeneration process better. Our results suggest that transplanted native and activated hepatic stellate cells can differentiate into hepatocyte-like cells and positively influence liver regeneration without inducing liver fibrosis

Twenty-year clinical progression of dysferlinopathy in patients from Dagestan

© 2017 Umakhanova, Bardakov, Mavlikeev, Chernova, Magomedova, Akhmedova, Yakovlev, Dalgatov, Fedotov, Isaev and Deev.To date, over 30 genes with mutations causing limb-girdle muscle dystrophy have been described. Dysferlinopathies are a form of limb-girdle muscle dystrophy type 2B with an incidence ranging from 1:1,300 to 1:200,000 in different populations. In 1996, Dr. S. N. Illarioshkin described a family from the Botlikhsky district of Dagestan, where limb-girdle muscle dystrophy type 2B and Miyoshi myopathy were diagnosed in 12 members from three generations of a large Avar family. In 2000, a previously undescribed mutation in the DYSF gene (c.TG573/574AT; p. Val67Asp) was detected in the affected members of this family. Twenty years later, in this work, we re-examine five known and seven newly affected family members previously diagnosed with dysferlinopathy. We observed disease progression in family members who were previously diagnosed and noted obvious clinical polymorphism of the disease. A typical clinical case is provided