The blood–brain barrier is a highly selective morphofunctional structure that maintains central nervous system homeostasis and protects it from toxic and foreign substances in the systemic circulation. At the same time, the blood–brain barrier remains a major obstacle to effective pharmacotherapy of central nervous system diseases, as most drugs are unable to cross it. This review discusses current approaches to overcoming the blood–brain barrier, which rely on endogenous transport systems such as adsorptive transcytosis and receptor- or carrier-mediated transcytosis, as well as advanced nano- and biotechnologies. Of particular interest are promising strategies such as modification of therapeutic molecules with cell-penetrating peptides, the use of exosomes and biomimetic nanoplatforms, and cell-mediated delivery of biologically active molecules using autologous leukocytes as “Trojan horses.” Methods for transient disruption of blood–brain barrier integrity (e.g., osmotic modulation and focused ultrasound) and alternative delivery routes, such as intranasal administration providing direct access to the brain via the olfactory and trigeminal nerves, are discussed separately. It is emphasized that successful therapy of central nervous system diseases is not possible without safe, effective, and targeted delivery systems capable of crossing the blood–brain barrier without compromising its protective function. Recent advances in molecular and cellular biology, nanomedicine, and genetic engineering open new horizons for personalized therapeutic approaches aimed at effective traversal of the blood–brain barrier.

Morphological confirmation of the recovery of the spinal cord (SC) and the skeletal muscle (m. soleus) in both hind limbs was achieved in pigs with contusion injury in the lower thoracic region (Th8-Th9), following transtraumatic epidural electrical stimulation (TEES). Sixty days after the neurotrauma model, the anterior and posterior horns of the rostral and caudal spinal cord segments were examined using histological and immunofluorescent techniques relative to the injury epicenter. In animals with a 6-week TEES regimen at the Th5 and L2 levels, a larger area of intact gray matter, a smaller number of caspase-3+ cells undergoing apoptosis, decreased expression of the heat shock protein 27 (HSP27), inhibition of astrogliosis development, and an increase in the number of oligodendroglial cells were observed. This agrees with data on the suppression of m. soleus atrophy and the maintenance of its original phenotype. The information we previously received about the functional recovery of the spinal cord and the results of this study allow us to make conclusion about the morphofunctional post-traumatic recovery of the spinal cord under TEES conditions.

Aim. To study the effectiveness of preventive gene therapy (within 2 days) and gene therapy in the acute phase (after 4 hours) of ischemic stroke in mini-pigs using an autologous leucoconcentrate (AutoLeuc) enriched with recombinant genes of vascular endothelial growth factor (VEGF165), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule 1 (NCAM1), as well as the migration of leukocytes transduced with a chimeric adenoviral vector serotype 5 with fiber 35 serotype (Ad5/F35) and the green fluorescent protein (GFP) genome into immune defense organs.Materials and methods. The experiment was conducted on 8-month-old Vietnamese lop-bellied mini-pigs (n=16). An ischemic stroke was created by occlusion of the distal branches of the left middle cerebral artery and the right common carotid artery. Genetically modified AutoLeuc was administered preventively intravenously 2 days before or in the acute phase 4 hours after stroke modelling; the control group was injected with 30 ml of saline solution. The morphology of the cerebral cortex was assessed using histological methods in the areas bordering the infarction and peri-infarction after 21 days. The migration of genetically modified Ad5/F35-GFP leukocytes into the brain, spleen, and submandibular lymph nodes was studied a week after stroke modelling.Results. In the peri-infarction zone, the content of pyknotic neurons in control animals was higher, while the number of capillaries was lower than in the gene therapy groups. In the latter, neurons had a typical morphology with preserved outgrowths; in the control group, the outgrowths were tortuous and fragmented. Fluorescence microscopy after injection of AutoLeuc with Ad5/F35-GFP revealed GFP-positive cells in the spleen and submandibular lymph nodes.Conclusion. 21 days after modeling a stroke in mini-pigs against the background of preventive gene therapy or gene therapy in the acute phase using VEGF165/GDNF/NCAM1 AutoLeuc, greater preservation of neurons and a higher density of capillaries in the peri-infarction zone of ischemic brain damage were established. Leukocytes with Ad5/ F35-GFP were found in the spleen and submandibular lymph nodes.

Background: The biosafety of gene therapy products remains a major challenge to their introduction into the clinic. In particular, the problem of immunogenicity of viral vectors is the focus of attention. Large animals such as pigs, whose anatomical and physiological characteristics are similar to those of humans, have an advantage in testing vector systems. Methods: We performed a comprehensive in vitro and in vivo study to evaluate the biosafety of a chimeric adenoviral vector carrying a green fluorescent protein gene (Ad5/35F-GFP) in a mini-pig model. Results: Transcriptome and secretome analyses of mini-pig leucocytes transduced with Ad5/35F-GFP revealed changes restraining pro-inflammatory processes and cytokine production. No adverse effects were revealed through the clinical, instrumental, laboratory, and histological examinations conducted within a week after the direct or autologous leucocyte-mediated administration of Ad5/35F-GFP to mini-pigs. The decrease in cytokine levels in the blood of experimental animals is also consistent with the in vitro data and confirms the immune tolerance of mini-pigs to Ad5/35F-GFP. Conclusions: Here, we show the safety of Ad5/35F in a mini-pig model and provide evidence that Ad5/35F is a promising vector for gene therapy. These results advance our understanding of vector–host interactions and offer a solid foundation for the clinical application of this vector.

BACKGROUND:Pathological changes associated with spinal cord injury (SCI) can be observed distant, rostral, or caudal to the epicenter of injury. These remote areas represent important therapeutic targets for post-traumatic spinal cord repair. The present study aimed to investigate the following in relation to SCI: distant changes in the spinal cord, peripheral nerve, and muscles.METHODS:The changes in the spinal cord, the tibial nerve, and the hind limb muscles were evaluated in control SCI animals and after intravenous infusion of autologous leucoconcentrate enriched with genes encoding neuroprotective factors (VEGF, GDNF, and NCAM), which previously demonstrated a positive effect on post-traumatic restoration.RESULTS:Two months after thoracic contusion in the treated mini pigs, a positive remodeling of the macro- and microglial cells, expression of PSD95 and Chat in the lumbar spinal cord, and preservation of the number and morphological characteristics of the myelinated fibers in the tibial nerve were observed and were aligned with hind limb motor recovery and reduced soleus muscle atrophy.CONCLUSION:Here, we show the positive effect of autologous genetically enriched leucoconcentrate-producing recombinant neuroprotective factors on targets distant to the primary lesion site in mini pigs with SCI. These findings open new perspectives for the therapy of SCI.

The biosafety of gene therapy remains a crucial issue for both the direct and cell-mediated delivery of recombinant cDNA encoding biologically active molecules for the pathogenetic correction of congenital or acquired disorders. The diversity of vector systems and cell carriers for the delivery of therapeutic genes revealed the difficulty of developing and implementing a safe and effective drug containing artificial genetic material for the treatment of human diseases in practical medicine. Therefore, in this study we assessed changes in the transcriptome and secretome of umbilical cord blood mononuclear cells (UCB-MCs) genetically modified using adenoviral vector (Ad5) carrying cDNA encoding human vascular endothelial growth factor (VEGF165) or reporter green fluorescent protein (GFP). A preliminary analysis of UCB-MCs transduced with Ad5-VEGF165 and Ad5-GFP with MOI of 10 showed efficient transgene expression in gene-modified UCB-MCs at mRNA and protein levels. The whole transcriptome sequencing of native UCB-MCs, UCB-MC+Ad5-VEGF165, and UCB-MC+Ad5-GFP demonstrated individual sample variability rather than the effect of Ad5 or the expression of recombinant vegf165 on UCB-MC transcriptomes. A multiplex secretome analysis indicated that neither the transduction of UCB-MCs with Ad5-GFP nor with Ad5-VEGF165 affects the secretion of the studied cytokines, chemokines, and growth factors by gene-modified cells. Here, we show that UCB-MCs transduced with Ad5 carrying cDNA encoding human VEGF165 efficiently express transgenes and preserve transcriptome and secretome patterns. This data demonstrates the biosafety of using UCB-MCs as cell carriers of therapeutic genes.

The effect of transtraumatic epidural electrostimulation (TEES) above (T5) and below (L2) spinal cord injury in the lower thoracic region (T8-T9) in combination with treadmill exercise in pigs was evaluated using electrophysiological examination methods and behavioral tests. Two weeks after spinal cord injury, motor evoked potentials of m. soleus were recorded during electrostimulation at the level of T5 and L2 segments, which indicated activation of spinal cord structures above and below the focus of injury. After 6 weeks of TEES in combination with physical training, restoration of the characteristics of M-response and H-reflex of the soleus muscle in response to stimulation of the sciatic nerve, improvement of joint mobility, and appearance of voluntary motor activity in the hindlimbs were observed. Neuromodulation with TEES had been proven to be an effective way to stimulate posttraumatic spinal cord regeneration and can be used in the development of a neurorehabilitation protocol for patients with spinal cord injury.

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes, which affect the potency of the functional recovery after spinal cord injury (SCI). Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue. In our previous studies for delivering the therapeutic genes at the site of spinal cord injury, we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses (Ad5/35) carrying recombinant cDNA. In the present study, the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury. Experimental animals were randomly divided into two groups of 4 pigs each: the therapeutic (infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35-VEGF165, Ad5/35-GDNF, and Ad5/35-NCAM1) and control groups (infused with intact leucoconcentrate). The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed: (1) higher sparing of the grey matter and increased survivability of the spinal cord cells (lower number of Caspase-3-positive cells and decreased expression of Hsp27); (2) recovery of synaptophysin expression; (3) prevention of astrogliosis (lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells); (4) higher growth rates of regenerating βIII-tubulin- positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region. These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF, GDNF, and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury. Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

Stimulating the process of angiogenesis in treating ischemia-related diseases is an urgent task for modern medicine, which can be achieved through the use of different cell types. Umbilical cord blood (UCB) continues to be one of the attractive cell sources for transplantation. The goal of this study was to investigate the role and therapeutic potential of gene-engineered umbilical cord blood mononuclear cells (UCB-MC) as a forward-looking strategy for the activation of angiogenesis. Adenovirus constructs Ad-VEGF, Ad-FGF2, Ad-SDF1α, and Ad-EGFP were synthesized and used for cell modification. UCB-MCs were isolated from UCB and transduced with adenoviral vectors. As part of our in vitro experiments, we evaluated the efficiency of transfection, the expression of recombinant genes, and the secretome profile. Later, we applied an in vivo Matrigel plug assay to assess engineered UCB-MC’s angiogenic potential. We conclude that hUCB-MCs can be efficiently modified simultaneously with several adenoviral vectors. Modified UCB-MCs overexpress recombinant genes and proteins. Genetic modification of cells with recombinant adenoviruses does not affect the profile of secreted pro- and anti-inflammatory cytokines, chemokines, and growth factors, except for an increase in the synthesis of recombinant proteins. hUCB-MCs genetically modified with therapeutic genes induced the formation of new vessels. An increase in the expression of endothelial cells marker (CD31) was revealed, which correlated with the data of visual examination and histological analysis. The present study demonstrates that gene-engineered UCB-MC can be used to stimulate angiogenesis and possibly treat cardiovascular disease and diabetic cardiomyopathy.

Aim . To determine the delayed (after 2 months) effect of spinal cord injury (SCI) in the lower thoracic region in the mini-pigs on the morphologic state of macro- and microglia in nearby and remote caudal areas. Materials and methods . Sexually mature female Vietnamese pot-bellied pigs were randomly divided into two groups: SCI (n = 3) and intact (n = 3). Dosed contusion SCI was modelled at the level of the Th8–Th9 vertebrae, and transverse cryostat sections of the caudal segment adjacent to the epicenter of injury and the lumbar thickening (L4–S2) were examined 2 months later. The expression of astrocyte markers (glial fibrillary acidic protein, GFAP) and microglial markers (ionized calcium-binding adapter molecule 1, Iba1) was assessed as the relative immunopositive area occupied by cells. When counting the number of oligodendroglial cells (oligodendrocyte transcription factor 2, Olig2), the presence of nuclei detectable with 4’,6-diamidino-2-phenylindole (DAPI) was taken into account. Results . After SCI, an increase in the relative areas occupied by GFAP-positive astrocytes and Iba1-positive microglia and a decrease in Olig2-positive oligodendrocytes were detected in both the lesion area and lumbar thickening. In both regions, 2 months after SCI, the proportion of astrocytes was not significantly different in the anterior horns and doubled in the posterior horns. Microglia cells with SCI were 2.5 times more in the anterior horns of both regions and in the posterior horns of the lumbar thickening, while the presence of microglia increased slightly (1.2 times) in the posterior horns in the SCI region. The number of oligodendrocytes decreased in the area of the epicenter of SCI in the anterior and posterior horns by 1.5–1.75 times, and in the lumbar thickening more significantly: the number decreased by 2.5 times in the anterior horn and 5.5 times in the posterior horn. Conclusion. The results of the study revealed a similar pattern of macro- and microglial cell distribution both in the SCI region and in remote areas. The obtained data testify to the necessity to take into account the state of the areas of nervous tissue remote from the epicenter of SCI when stimulating neuroregeneration in such patients

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The natural limitations of regeneration in the CNS are major problems for the treatment of neurological disorders, including ischaemic brain strokes. Among the approaches being actively developed to inhibit post-ischaemic negative consequences is the delivery of therapeutic genes encoding neuroprotective molecules to the brain. Unfortunately, there are currently no proven and available medicines that contain recombinant human genes for the treatment of ischaemic cerebral stroke. Of particular interest is the development of treatments for patients at risk of ischaemic stroke. In the present study, we propose a proof of concept for the use of an autologous, genetically enriched leucoconcentrate temporally secreting recombinant vascular endothelial growth factor (VEGF), glial-cell-line-derived neurotrophic factor (GDNF) and the neural cell adhesion molecule (NCAM) for the treatment of stroke. In a mini-pig ischaemic stroke model, genetically enriched leucoconcentrate was infused 4 h after surgery (gene therapy in acute phase) or 2 days before stroke modelling (preventive gene therapy). On day 21, after the stroke modelling, the post-ischaemic brain recovery was examined by morphologic and immunofluorescence analysis. The benefits of treating a stroke with genetically enriched leucoconcentrate both for preventive purposes and in the acute phase were confirmed by an improved performance in behavioural tests, higher preservation of brain tissue and positive post-ischaemic brain remodelling in the peri-infarct area. These results suggest that the employment of autologous leucocytes enabling the temporary production of the recombinant therapeutic molecules to correct the pathological process in the CNS may be one of the breakthrough approaches in gene therapy.

The development of hypogravitational motor syndrome is an essential negative consequence of weightlessness for humans; an important role in the pathogenesis of this syndrome is played by changes in axons of the spinal tracts. Myelinated fibers and transcriptome of the spinal cord were studied in mice exposed to hypogravity during a 30-day flight on a biosatellite. Morphometric analysis of myelinated fibers of the spinal tracts showed a decrease in the thickness of the myelin sheath. Analysis of spinal cord transcriptome revealed a decrease in the expression of genes involved in the myelination of nerve fibers. These results suggest that the processes of nerve fiber myelination are involved in the development of the hypogravitational motor syndrome under weightless conditions; the 7-day readaptation period was found to be insufficient for reversion of the negative changes in the myelinated fibers of the spinal cord.

Several methods for the stimulation of skin wound repair have been proposed over the last few decades. The most promising among them are gene and stem cell therapy. Our present experiments combined several approaches via the application of human umbilical cord blood mononuclear cells (hUCB-MC) that were transfected with pBud-VEGF165-FGF2 plasmid (gene-cell therapy) and direct gene therapy using pBud-VEGF165-FGF2 plasmid to enhance healing of full thickness skin wounds in rats. The dual expression cassette plasmid pBud-VEGF165-FGF2 encodes both VEGF and FGF2 therapeutic genes, expressing pro-angiogenic growth factors. Our results showed that, with two weeks post-transplantation, some transplanted cells still retained expression of the stem cell and hematopoietic markers C-kit and CD34. Other transplanted cells were found among keratinocytes, hair follicle cells, endothelial cells, and in the derma. PCNA expression studies revealed that transplantation of transfected cells terminated proliferative processes in regenerating wounds earlier than transplantation of untransfected cells. In the direct gene therapy group, four days post-operatively, the processes of flap revascularization, while using Easy LDI Microcirculation Camera, was higher than in control wounded skin. We concluded that hUCB-MC can be used for the treatment of skin wounds and transfection these cells with VEGF and FGF2 genes enhances their regenerative abilities. We also concluded that the application of pBud-VEGF165-FGF2 plasmids is efficient for the direct gene therapy of skin wounds by stimulation of wound revascularization.

Resistance to antibacterial therapy requires the discovery of new methods for the treatment of infectious diseases. Lactoferrin (LTF) is a well-known naïve first-line defense protein. In the present study, we suggested the use of an adenoviral vector (Ad5) carrying the human gene encoding LTF for direct and cell-mediated gene therapy of maxillofacial area phlegmon in rats. Abscesses were developed by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. At 3–4 days after phlegmon maturation, all rats received ceftriaxone and afterward were subcutaneously injected around the phlegmon with: (1) Ad5 carrying reporter gfp gene encoding green fluorescent protein (Ad5-GFP control group), (2) Ad5 carrying LTF gene (Ad5-LTF group), (3) human umbilical cord blood mononuclear cells (UCBC) transduced with Ad5-GFP (UCBC + Ad5-GFP group), and (4) UCBC transduced with Ad5-LTF (UCBC + Ad5-LTF group). Control rats developed symptoms considered to be related to systemic inflammation and were euthanized at 4–5 days from the beginning of the treatment. Rats from therapeutic groups demonstrated wound healing and recovery from the fifth to seventh day based on the type of therapy. Histological investigation of cervical lymph nodes revealed purulent lymphadenitis in control rats and activated lymphatic tissue in rats from the UCBC + Ad5-LTF group. Our results propose that both approaches of LTF gene delivery are efficient for maxillofacial area phlegmon recovery in rats. However, earlier wound healing and better outcomes in cervical lymph node remodeling in the UCBC + Ad5-LTF group, as well as the lack of direct exposure of the viral vector to the organism, which may cause toxic and immunogenic effects, suggest the benefit of cell-mediated gene therapy.

Earlier, in mice after a 30-day space flight on the Bion-M1 biosatellite, we found signs of a negative effect of weightlessness on the structure of myelinated fibers of the spinal cord tracts; these findings indicate their involvement in the pathogenesis of hypogravitational motor syndrome (HMS). In the present study, under conditions of hypogravity modeling by the hindlimb unloading, we obtained data on destructive changes in the myelinated fibers of the motor posterior corticospinal tract (tractus corticospinalis posterior), sensitive anterior spinocerebellar tract (tractus spino-cerebellaris anterior), and the gracile fascicle (fasciculus gracilis), as well as in the tibial fascicle (fasciculus tibialis) of the sciatic nerve of mice 30 days after unloading. The obtained data confirm our hypothesis on the role of disturbance in the processes of myelination of nerve fibers during the development of HMS, both during space flight and under conditions of simulating hypogravity on Earth. Morphometric analysis after a 7-day period of readaptation did not reveal signs of restoration of pathological changes in myelinated fibers that arose after 30 days of hanging. However, preventive gene therapy (administration of a gene construct providing the synthesis of recombinant vascular endothelial growth factor, glial cell line-derived neurotrophic factor, and neural cell adhesion molecule, prior to hindlimb unloading) has been shown to be effective in the preservation of myelinated fibers in projection anterior spininocerebellar tract, compared with control animals that did not receive gene therapy. The research carried out at this stage gives ground to make a preliminary conclusion about the advisability of developing methods of preventive gene therapy to prevent the development of GDS during long-term space flights.

Nowadays, the use of a digital prototype in numerical modeling is one of the main approaches to calculating the elements of an inhomogeneous structure under the influence of external forces. The article considers a finite element analysis method based on computed tomography data. The calculations used a three-dimensional isoparametric finite element of a continuous medium developed by the authors with a linear approximation, based on weighted integration of the local stiffness matrix. The purpose of this study is to describe a general algorithm for constructing a numerical model that allows static calculation of objects with a porous structure according to its computed tomography data. Numerical modeling was carried out using kinematic boundary conditions. To evaluate the results obtained, computational and postprocessor grids were introduced. The qualitative assessment of the modeling data was based on the normalized error. Three-point bending of bone specimens of the pig forelimbs was considered as a model problem. The numerical simulation results were compared with the data obtained from a physical experiment. The relative error ranged from 3 to 15%, and the crack location, determined by the physical experiment, corresponded to the area where the ultimate strength values were exceeded, determined by numerical modeling. The results obtained reflect not only the effectiveness of the proposed approach, but also the agreement with experimental data. This method turned out to be relatively non-resource-intensive and time-efficient.