Annotation of the results obtained in 2020
In accordance with the plan of the project, the following specific tasks were carried out this year: (I) production in preparative amounts of chimeric adenoviral vectors Ad5/F35-VEGF165, Ad5/F35-GDNF, and Ad5/F35-NCAM1 to obtain genetically modified leucoconcentrate (GML); (II) in vitro analysis of the therapeutic genes expression in GMF; (III) a comprehensive analysis of the morpho-functional recovery of the mini-pigs spinal cord after contusion injury and: (1) autoinfusion of GML (leucoconcentrate transduced with Ad5/F35-VEGF165 + Ad5/F35-GDNF + Ad5/F35-NCAM1); (2) combined epidural electrical stimulation above (Th5) and below (L2) the epicenter of the trauma with GML-GFP infusion; (3) combined therapy: autoinfusion of GML (leucoconcentrate transduced with Ad5/F35-VEGF165 + Ad5/F35-GDNF + Ad5/F35-NCAM1, in combination with two-level epidural electrostimulation. I. Obtaining preparative amounts of genetic vectors based on recombinant replicative-defective human adenovirus serotype 5 (Ad5) with modified fibers (Ad5/35F), carrying cDNA encoding VEGF165, GDNF and NCAM1 As a result, recombinant replicative-defective viral vectors based on human adenovirus serotype 5 (Ad5) with adenovirus 35 serotype fiber (Ad5/F35) carrying target transgenes (Ad5/F35-VEGF165, Ad5/F35-GDNF and Ad5/F35-NCAM1) for cell-mediated gene therapy of spinal cord injury were obtained and analyzed. II. In vitro analysis of therapeutic genes expression It was established by RT-PCR that vegf165, gdnf, and ncam1 genes mRNA in GML during simultaneous transduction with three adenoviral vectors (MOI = 10) was 324.2 [55.5-1895.6] for vegf16 (P = 0, 0002), 217.9 [61.1-777.0] for gdnf (P = 0.0001) and 190.5 [23.4-1549.7] for ncam1 (P = 0.0009) times higher compared to mRNA level in non-transduced leukocytes. The production of recombinant molecules (VEGF, GDNF, NCAM) was analyzed in the supernatant after 72 hours of incubation of in vitro genetically modified leukocytes using ELISA. A significant increase of recombinant GDNF (23.8 pg/ml) by 43 times, VEGF (2620.9 pg/ml) by 163 times and NCAM (1523.9 pg/ml) by 2.2 times was found in the supernatant after incubation of genetically modified leukocytes, when compared with the supernatant obtained after incubation of non-transduced leukocytes (0.55; 15.68 and 687.0, respectively). Immunofluorescent staining of genetically modified leukocytes after 72 hours of incubation using antibodies to VEGF, GDNF, NCAM revealed immunopositive leukocytes, which indicates the efficient expression of transgenes in genetically modified cells. III. Оценка двигательной активности у мини-свиней с контузионной травмой спинного мозга III. Evaluation of motor activity in mini-pigs with spinal cord contusion injury A preliminary analysis of the PTIBS test revealed that in experimental animals after implantation of electrodes the test values were 9.5 points (on a 10-point scale), which corresponds to normal motor activity. 4 weeks after neurotrauma in control animals, the PTIBS test value was assessed as 1.8 points, in mini-pigs from the therapeutic groups – from 2 to 2.5 points. After 8 weeks of the experiment, before euthanasia, the motor activity in the control mini-pigs was assessed as 2.0 points, in animals after combined therapy – as 2.5 points. Assessment of the joints kinematic one of the effective methods for analyzing the functional recovery after contusion injury of the spinal cord. A preliminary analysis of the joints kinematics in control animals revealed a significant decrease in the range of locomotion in the ankle and knee on day 60 after neurotrauma modeling. At the same time, the range of the hip locomotion in control mini-pigs approached the value before neurotrauma modeling. In the treatment groups, the range of locomotion in the ankle 8 weeks after neurotrauma were higher than in the control mini-pigs. At the same time, animals with combined therapy did not differ when training on a treadmill, both during electrical stimulation and without electrical stimulation. The results of weighing skeletal muscles (m. tibialis anterior and m. soleus) from both extremities of intact and experimental animals made it possible to establish that the muscle weight of the muscles on both extremities in animals from the control group was less than in intact animals. At the same time, the muscle weight in animals from all therapeutic groups did not differ from intact values, which can serve as a prognostic sign of morpho-functional restoration of the spinal cord in mini-pigs with neurotrauma during treatment. IV. Electrophysiological assesment of the m. soleus of experimental animals During electromyography prior to spinal cord injury, a motor (M) response of a standard form was recorded with stimulation of the sciatic nerve, including negative and positive peaks. With an increase in the intensity of stimulation, the amplitude of the M-response increased, reaching a maximum and did not change further. After spinal cord injury, the parameters of the evoked muscle potentials changed depending on the study period. So, after 2 weeks, the M-response curve in the control animals had several phases and this polyphasicity remained at the subsequent stages of the study: after 4, 6 and 8 weeks. In this case, the amplitude of the M response increased, starting from 2 weeks after the injury, and decreased by 8 weeks of the experiment. The form of the M-response in animals with combined therapy tended to recover, starting from 4 weeks after modeling of neurotrauma. The maximum amplitude of the M-response in these animals increased at all periods of the study, reaching a maximum value at 8 weeks. M-response duration in control animals increased from the first weeks after the contusion injury, reaching a maximum at 8 weeks. M-response duration in animals with combined therapy remained without significant fluctuations throughout the experiment and approached the values of intact animals. The latent period did not differ in intact and experimental animals at all periods of the study. A preliminary analysis of the H-response data revealed its absence on the electromyogram in control animals at all periods of the study. In animals treated with combination therapy, the H-response appeared 2 weeks after neurotrauma. V. Histological examination of the spinal cord of experimental animals In the gray matter of the mini-pig 60 days after modeling the contusion injury of the spinal cord, morphometric analysis of the pathological cavities revealed a decrease in the area of preserved tissue in the rostral and caudal segments. In mini-pigs after combined therapy, the values for the rostral segment did not differ from those in intact animals. In the lateral corticospinal tract of all animals, except for mini-pigs, after combined therapy, a significant decrease in the number of myelinated fibers in the caudal and rostral segments of the spinal cord was revealed, when compared with intact animals. The diameter of the axial cylinder in intact mini-pigs was smaller than in experimental animals, both in the rostral and caudal segments. The thickness of the myelin sheath in intact animals in the rostral and caudal segments was less in comparison with all experimental animals. Immunofluorescent staining of the spinal cord of experimental animals with antibodies against the apoptosis effector protein in the rostral and caudal segments revealed Caspase3-positive cells in the anterior and posterior horns of the experimental animals. Preliminary analysis of the data indicates that the control animals had more cells that entered apoptosis than the animals after combination therapy. The level of Hsp27 and KCC2 immunoexpression in all experimental groups did not reveal significant differences in the corresponding areas of the spinal cord of experimental animals, relative to intact mini-pigs. However, higher values of the fluorescence intensity of Hsp27 and KCC2 were found in the rostral region of control animals. Analysis of the immunoexpression of the postsynaptic density protein 95 in neurons of the anterior and posterior horns of the spinal cord in the rostral and caudal segments relative to the epicenter of injury revealed insignificant differences between intact and experimental animals. However, mini-pigs from the control group showed a lower level of PSD95 immunoexpression in all studied areas when compared with intact animals and animals from the therapeutic groups. The level of Synaptophysin immunoexpression in the anterior and posterior horns of the spinal cord in the caudal and rostral segments also did not differ in experimental and intact animals. However, lower values in all assessed areas were in control animals and higher in mini-pigs after combined therapy. Immunofluorescent staining of the spinal cord with antibodies against glial fibrillary acidic protein (GFAP) revealed an increase in the GFAP-positive area in the posterior horns, both in the rostral and caudal segments in control animals, relative to intact mini-pigs. In animals after combined therapy, the values did not differ from intact animals. Analysis of the microglial cell marker (Iba1) revealed an increase in Iba1-positive cells in the anterior and posterior horns in the rostral and caudal segments of control animals, relative to intact animals. A smaller increase of Iba1-positive cells number was found in mini-pigs after combined therapy. Analysis of the immunoexpression of the myelin-forming cells marker (Olig2) in the anterior and posterior horns and the corticospinal tract revealed a decrease in the number of Olig2-positive cells in all assessed areas, both in the rostral and caudal segments of the spinal cord in control mini-pigs compared with intact animals. In mini-pigs after combined therapy, the number of Olig2-positive cells did not differ from the values in intact animals. VI. Molecular genetic analysis of the spinal cord of experimental animals A preliminary analysis of the expression of genes encoding presynaptic and postsynaptic proteins of neurotransmitter systems allows us to conclude that in the rostral segment of the spinal cord, there are less pronounced changes in the expression of target genes in animals from the therapeutic groups, relative to the control animals. In the caudal segment, there is a decrease in the expression of all target genes in mini-pigs after gene and combined therapy, when compared with control animals. The results suggest a possible effect of therapeutic recombinant molecules (VEGF, GDNF, NCAM) on the plasticity of excitatory (cholinergic, glutamatergic), inhibitory (glycinergic and GABAergic), and serotoninergic neurotransmitter systems of the spinal cord below the epicenter of neurotrauma. The data obtained can serve as a prognostic sign of functional recovery of the spinal cord in mini-pigs with neurotrauma during treatment. Conclusion In the present study for the first time, preclinical trial of autologous genetically modified leukoconcentrate for the treatment of spinal cord injury in combination with two-level epidural electrical stimulation above and below the epicenter of neurotrauma in a mini-pig was carried out. Autologous genetically modified leucoconcentrate was prepared from the peripheral blood of a mini-pig and a mixture of three adenoviral vectors Ad5/F35-VEGF165, Ad5/F35-GDNF and Ad5/F35-NCAM1 in equal proportions. Molecular genetic analysis confirmed the efficiency of in vitro expression of transgenes in GML. In accordance with the plan of the project, at this stage of the work, data were obtained by of behavioral tests, electrophysiological, molecular and histological research methods, indicating more pronounced positive effect of combined therapy on the morpho-functional recovery of the spinal cord of a mini-pig with contusion injury, in comparison with control animals and mini-pigs with gene or electrotherapy. Thus at this stage, the stated result of the study was obtained.

 

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Annotation of the results obtained in 2019
During the implementation of the project in 2016-2018 we obtained results indicating the therapeutic effectiveness of epidural electrical stimulation in combination with cell-mediated gene therapy using genetically modified mononuclear umbilical cord blood cells (UCBC) producing recombinant vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) in a mini-pigs with traumatic spinal cord injury. Taking into account the fact that UCBC cannot be widely used as cell carriers of therapeutic genes, we hypothesized the use of leukoconcentrate obtained from peripheral blood for its genetic modification and subsequent autoinfusion for therapeutic purposes. In 2019, following the main plan of the project, we performed particular works, namely: I. Creation and production of genetic vectors based on recombinant replication-defective human adenovirus serotype 5 (Ad5) with modified fiber (Ad5/35F) carrying green fluorescent protein reporter gene (EGFP), vascular endothelial growth factor gene (VEGF), neural cell adhesion molecule gene (GDNF) and neural cell adhesion molecule gene (NCAM) in preparative quantities. As a result a recombinant replication-defective human adenovirus serotype 5 with modified fiber (Ad5/35F) carrying the green fluorescent protein reporter gene (EGFP) was obtained. For the first time, plasmid constructs pAd5/35-out4-GDNF, pAd5/35-out4-hVEGF165 and pAd5/35-out4-NCAM1 with a self-cutting bacterial part, carrying the adenovirus 5 serotype genome with the adenovirus 35 serotype fiber and target genes in the expression cassette. Recombinant adenoviruses 5 serotype with the adenovirus 35 serotype fiber (Ad5/F35-GDNF, Ad5/F35-hVEGF165 and Ad5/F35-NCAM1) carrying expression cassette containing gdnf, vegf и ncam genes respectively were first obtained by transfection of HEK293 cells with pAd5/35-out4-GDNF, pAd5/35-out4-hVEGF165 and pAd5/35-out4-NCAM1 plasmid constructs. II. Development of a protocol for the genetically modified leukoconcentrate (GML) production from the peripheral blood of a mini-pig. Under aseptic conditions, 100 ml of blood was taken from the subclavian vein into sterile closed 250 ml containers (hemacon) with СРDА-1 anticoagulant (Green Cross, Korea) (35 ml of preservative in each bag) and transferred into the «Komplast-300» blood components container («Synthes», Russia). To precipitate red blood cells, a hydroxyethyl starch 6% solution (Stabisol® GAK 6%, Berlin-Chem AG, Germany) was added to the container in a 1:1 ratio and centrifuged at 350 rpm in a Presvac centrifuge (DP-2065 R PLUS) for 10 minutes at 10°C. After removing the red blood cells, the container was centrifuged again at 350 rpm on a Presvac centrifuge for 10 minutes at 10°C, and the supernatant containing leukocytes, plasma and Stabisol was extracted from the container by extrusion of blood components using FC-01 fractionator («Lidkor», Russia) into the unutilized «Komplast-300» container. In this container with supernatant, naomal saline was added in a 1:9 ratio and centrifuged at 1300 rpm in a Presvac centrifuge for 10 minutes at 10°C. The obtained supernatant from the container was extracted by extruding using FC-01 fractionator so that 30 ml of normal saline containing cell suspension (leukoconcentrate) remains in the blood container. Transduction of the obtained leukoconcentrate was carried out in the container. To do this, the required volume of adenoviral vectors (Ad5/35F) containing the green fluorescent protein reporter gene (egfp) or therapeutic vegf, gdnf, ncam genes in normal saline (MOI = 3) was injected using sterile syringe. After 12 hours of incubation at room temperature under continuous shaking (ELMI shaker), 200 ml of sterile normal saline was added into a container with genetically modified leukoconcentrate and centrifuged at 1000 rpm and 10°C for 10 minutes using a Presvac centrifuge. The supernatant from was extracted by extrusion using a fractionator so that 26 ml of normal saline remain in the container, including the cell suspension (14.8±2.07×109 cells/l) – genetically modified leukoconcentrate (GML). To study the in vitro EGFP gene expression GML-EGFP samples were cultured for 72 hours after transduction. Microscopy of the transduced leukocytes cytoplasm showed intense green fluorescence. Analysis of GDF-EGFP using flow cytometry showed that 0.6% of the white blood cells in GDF-EGFP efficiently expressed green fluorescent protein. Thus, after the transduction of the leukoconcentrate with Ad5/35F-EGFP (MOI = 3), it is possible to obtain GDF containing 0.6% EGFP-positive cells. Based on the protocol for the production of GMF-EGFP, a leukoconcentrate was obtained from mini-pig blood transduced with adenoviral vectors carrying recombinant human VEGF, GDNF and NCAM genes in equal proportions (GMF-1/3 VEGF + 1/3 GDNF + 1/3 NCAM). III. Development of a protocol for cell-mediated gene therapy using genetically modified leukoconcentrate on mini-pigs after modeling a dosed contusion spinal cord injury. 1. Blood sampling and genetically modified leukoconcentrate production according to the original protocol was performed a day before the neurotrauma simulation. In this study, white blood cells was transduced with an adenoviral vector (Ad5/35F) carrying the green fluorescent protein reporter gene. 2. The contusion spinal cord injury (CSCI) in the mini-pig was modeled 2 weeks after implantation of the electrodes and one day after the blood sampling. 3. Autoinfusion of genetically modified leukoconcentrate. Genetically modified leukoconcentrate (30 ml) was injected through v. auricularis 4 hours after neurotrauma modeling. 4. Expression of the EGFP reporter gene was studied in the post-traumatic spinal cord and spleen 7 days after intravenous auto-infection of GML-EGFP to mini-pigs with spinal cord contusion. Using fluorescence microscopy in the spinal cord (at the epicenter, rostral and caudal segments), GFP-positive leukocytes were detected. In the spleen of mini-pigs, EGFP-positive cells were detected in the lymphatic follicles of the white pulp. Thus, genetically modified white blood cells are able circulate in the blood throughout the body, migrate from the bloodstream to the spinal cord, and efficiently produce recombinant molecules. IV. Comprehensive analysis of the morphological and functional recovery of the spinal cord of mini-pigs after contusion spinal cord injury on the background of GML infusion. In the study, females of mini-pigs (Vietnamese Pot-bellied) with a body weight of 20-25 kg were used. The experimental protocol, including anesthesia, surgical procedures, postoperative care and euthanasia at the endpoints of the experiment, was approved by the Local Ethics Committee of Kazan State Medical University (permit number 2.20.02.18 dated February 20, 2018). At this stage of the study, the animals were divided into 2 groups: (1) intact group – healthy animals (n = 4); (2) experimental group – animals were implanted with wires for epidural electrical stimulation (ES), contusion spinal cord injury (CSCI) was modeled and genetically modified leukoconcentrate (GML-EGFP) was injected intravenously (n = 4). A morphological and functional study of the spinal cord was performed 2 months after neurotrauma modeling. 1. Motor activity assessment 1) Behavioral tests. Positive changes of the PTIBS test scores in experimental animals after the introduction of GDF were not detected. In this case, the weight of the tibial and soleus muscles on both limbs in animals from the experimental group was less than in intact animals. 2) Kinematics of the joints. A comparative analysis revealed a significant decrease in the range of motion in the ankle and knee joints on days 30 and 60 after neurotrauma modeling. 3) Electrophysiological study. Transformations of motor evoked potentials parameters found in experimental animals (polyphase curve of the M-response, increased duration of the motor potential and a decreased maximum amplitude of the M-response) indicate post-traumatic dysfunction of the central structures of the neuromotor apparatus, which may be a result of degenerative changes in part of the motor units developing to 8 weeks after neurotrauma. 2. Histological examination A morphometric analysis of the gray and white matter preservation revealed significant pathological changes in experimental animals 2 months after neurotrauma modeling, namely: (1) cavitation in the gray matter of the rostral and caudal segments; (2) reducing the number and increasing the diameter of the axial cylinder of myelinated fibers in the lateral corticospinal tract. An immunofluorescence analysis of the spinal cord of mini-pigs 60 day after the contusion allowed us to establish: (1) increased potassium-chlorine cotransporter immunoexpression; (2) decreased levels of synaptic proteins (synaptophysin and postsynaptic density protein); (3) ongoing apoptosis and increased heat shock protein expression; (4) signs of astrogliosis and decreased number of myelin forming cells. 3. Molecular investigation For the first time, analysis of the spinal cord neurotransmitter systems genes expression in mini-pigs 60 days after modeling of spinal cord injury revealed specific changes in the cholinergic, glutamatergic, glycerinergic and serotonergic neurotransmitter systems. Multiplex bioassay did not reveal significant differences in the endogenous cytokines, chemokines and growth factors content in animals with contusion spinal cord injury when compared with intact mini-pigs. Conclusion In this study, for the first time, a protocol for the production of genetically modified leukoconcentrate from peripheral blood of mini-pigs for autoinfusion was developed. Based on the obtained genetically modified leukoconcentrate, a protocol for gene therapy of contusion spinal cord injury in a mini-pig, based on autoinfusion of genetically modified leukoconcentrate was developed. In accordance with the main plan of the project at this stage, using behavioral tests, electrophysiological, molecular and histological methods, data on the effect of a genetically modified leukoconcentrate expressing a recombinant green fluorescent protein on the morphological and functional recovery of a mini-pig with contusion spinal cord injury in comparison with intact animals were obtained. It was found that pathological changes in the neuromuscular system of mini-pigs with contusion spinal cord injury 60 days after autoinfusion of a genetically modified leukoconcentrate producing a recombinant green fluorescent protein are preserved. In other words, intravenous autoinfusion of a leukoconcentrate transduced with Ad5/ 35F-EGFP has no therapeutic effect, and the obtained results are the basis for evaluating the therapeutic effect of a genetically modified leukoconcentrate expressing vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) recombinant molecules combined with epidural stimulation in the next stage of the project. Thus, the claimed result at this stage of the study is obtained.

 

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