Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare inherited disorder in which thickening of the walls of small and medium-sized blood vessels blocks blood flow to the brain. Diagnosis of CADASIL is based on clinical presentation, neuroimaging findings, and genetic predisposition. This disease is uncommon in children; typically, symptoms manifest in individuals between the ages of 20 and 40, though some may exhibit symptoms later in life. Currently, the diagnosis of CADASIL is of significant interest as there is no specific treatment targeting its etiopathogenesis. This article describes the case of a 51-year-old patient with CADASIL who was diagnosed with recurrent ischemic stroke. The patient has a history of multiple strokes: in 2019 (at age 47), 2020 (at age 48), and 2021 (at age 49). The consequences of these strokes include mild spastic right-sided hemiparesis, moderate complex motor aphasia, mild sensory aphasia, and progressive cognitive impairment. Suspicion of CADASIL was based on the patient's medical history, clinical presentation, and typical neuroimaging findings.

We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases, neurotrauma, and stroke. In this study, using a mini pig model of spinal cord injury, we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy. Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector (Ad5/35F) that carried an enhanced green fluorescent protein (EGFP) reporter gene in the plastic blood bag. The day after blood donation, the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate. A week after gene-modified leucoconcentrate therapy, fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury. In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed. In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes. Thus, the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions. This paper presents a proof-of-concept of simple, safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 5) on May 27, 2014.

This study evaluates the effect of combined epidural electrical stimulation (EES) applied above (C5) and below (L2) the spinal cord injury (SCI) at T8–9 combined with motor training on the restoration of sensorimotor function in mini pigs. The motor evoked potentials (MEP) induced by EES applied at C5 and L2 levels were recorded in soleus muscles before and two weeks after SCI. EES treatment started two weeks after SCI and continued for 6 weeks led to improvement in multiple metrics, including behavioral, electrophysiological, and joint kinematics outcomes. In control animals after SCI a multiphasic M-response was observed during M/H-response testing, while animals received EES-enable training demonstrated the restoration of the M-response and H-reflex, although at a lower amplitude. The joint kinematic and assessment with Porcine Thoracic Injury Behavior scale (PTIBS) motor recovery scale demonstrated improvement in animals that received EES-enable training compared to animals with no treatment. The positive effect of two-level (cervical and lumbar) epidural electrical stimulation on functional restoration in mini pigs following spinal cord contusion injury in mini pigs could be related with facilitation of spinal circuitry at both levels and activation of multisegmental coordination. This approach can be taken as a basis for the future development of neuromodulation and neurorehabilitation therapy for patients with spinal cord injury.

Neural networks disturbed due to spinal cord injury are capable to restore that is largely determined by post-traumatic remodeling. It is known that information exchange between neurons is carried out by electrical impulse, which ensures the transmission of excitation in synapses, that is realized through neurotrophic factors according to the concept of neurotrophic interactions. Objective: to study the effect of a combination of epidural electrostimulation above and below the site of neurotrauma during training on the treadmill and intrathecal administration of human umbilical cord blood mononuclear cells, which simultaneously delivered three therapeutic genes encoding vascular endothelial growth factor (VEGF165), glial neurotrophic factor (GDNF) and neuronal cell adhesion molecule (NCAM1), to post-traumatic reorganization of neuroglia cells in a model of dosed concussion injury of rat spinal cord at the Th8-Th9 level. 30 days after the simulation of neurotrauma by the immunofluorescence method, a change in the number of macro- and microglia cells in the segment caudal from the damage epicenter was revealed. Electrostimulation did not affect the number of GFAP+-cells in the gray matter, but the combined effect of gene and electrotherapy restrained the increase in their number. Differences were found in the reactions of astrocytes in white and gray matter in response to electrical stimulation. In the zones of gray matter, the supporting effect of the combination of gene and electrotherapy on the number of Olig2+-cells was most clearly manifested. In this group of animals, the inhibition of the increase in the number of Iba1+-microglia cells in the gray matter can also be interpreted as a positive factor contributing to neuroregeneration.

The translation of new therapies for spinal cord injury to clinical trials can be facilitated with large animal models close in morpho-physiological scale to humans. Here, we report functional restoration and morphological reorganization after spinal contusion in pigs, following a combined treatment of locomotor training facilitated with epidural electrical stimulation (EES) and cell-mediated triple gene therapy with umbilical cord blood mononuclear cells overexpressing recombinant vascular endothelial growth factor, glial-derived neurotrophic factor, and neural cell adhesion molecule. Preliminary results obtained on a small sample of pigs 2 months after spinal contusion revealed the difference in post-traumatic spinal cord outcomes in control and treated animals. In treated pigs, motor performance was enabled by EES and the corresponding morpho-functional changes in hind limb skeletal muscles were accompanied by the reorganization of the glial cell, the reaction of stress cell, and synaptic proteins. Our data demonstrate effects of combined EES-facilitated motor training and cell-mediated triple gene therapy after spinal contusion in large animals, informing a background for further animal studies and clinical translation.

Nowadays gene and cell therapy become the basic methods in regenerative medicine. However only few gene and cell products are currently approved for clinical usage. Biosafety problems, complexity of cell and gene technologies and high cost of manufacturing are the main reasons for the slow introduction of such approaches in practical medicine. Treatment of hereditary diseases of the immune system based on the correction of the mutant gene by delivering functional recombinant gene into WBC is the first successfully employed in the clinical practice approach of cell-mediated or ex vivo gene therapy. Earlier we have reported the strategy of the cell-mediated gene therapy based on umbilical cord blood mononuclear cells transduced with adenoviral vectors carrying recombinant genes encoding neurotrophic factors for treatment neurodegenerative diseases, neurotrauma and stroke. Significant disadvantage of this method is the usage of the umbilical cord blood mononuclear cells as a cell carrier for the therapeutic genes. Considering immunodeficiency treatment and our own data we developed a new approach of recombinant gene delivery for personalized ex vivo gene therapy. The method is based on autoinfusion of patient's WBC transduced with recombinant therapeutic genes for correction of certain pathological conditions. In the present study for the first time the human gene-modified leucoconcentrate (GML) producing recombinant reporter gene encoding green fluorescent protein (GFP) was obtained without culturing WBC in vitro. The routine unit of peripheral blood (450 ml) was collected into the plastic blood bag and the leucocyte- and platelet-rich concentrates (50 ml) were obtained by standard method using Macopress Smart (Macopharma, France). Afterwards the equal volume of hydroxyethyl starch 6% was added into the plastic blood bag which was centrifuged (DP-2065 R PLUS, Centrifugal Presvac RV; Presvac, Buenos Aires, Argentina) at 350 rpm for 10 min at 10°C. The obtained supernatant was transferred into the new plastic blood bag using manual plasma extractor FK-01 (Leadcore, Russia) and 200 ml of saline was added into the bag which was centrifuged at 1300 rpm for 10 min at 10°C and the supernatant was expressed out of the bag so that the remaining solution in the bag (30 ml) contained leucoconcentrate (WBC - 45.56 ± 23.93 × 106/ml and RBC - 1.76 ± 3.33 × 109/ml). Transduction of WBC with chimeric adenoviral vector (Ad5/35) carrying GFP gene was performed in the plastic bag with MOI 5 according to the count of WBC in the leucoconcentrate. After transduction for 12 hours, 200 ml of saline was added to the bag with leucoconcentrate, the mixture was centrifuged at 1000 rpm for 10 min at 10°C and the supernatant was squeezed out of the bag. The remained in the bag solution (30 ml) was considered as gene-modified leucoconcentrate carrying GFP gen (WBC - 22.63 ± 8.90 × 106/ml and RBC - 1.77 ± 1.21 × 109/ml). For in vitro study of GFP gene expression the samples of GML-GFP were cultivated for 60 hours after GML-GFP preparation. Fluorescent microscopy in the cytoplasm of the transduced WBC showed specific intensive green fluorescence. Flow cytometry analysis demonstrated that 2.5% of WBC from the GML-GFP efficiently expressed GFP. Thus leucoconcentrate after 72 h of transduction with Ad5/35-GFP with MOI 5 resulted in 2.5% of the GFP-positive cells. Thus the results of this study represent a simple, safe and effective approach for preparation of GML for personalized ex vivo gene therapy aimed at temporary production of the specific recombinant biologically active molecules for pathogenetic therapy of the varied nosological form, such as trauma, ischemic, degenerative, autoimmune, infection and other diseases. This study was supported by the grant of Russian Science Foundation 19-75-10030. Disclosures No relevant conflicts of interest to declare.

Currently, the treatments for spinal cord injury are limited. Gene therapy is one of the most promising approaches aimed at overcoming negative post-traumatic consequences in the spinal cord. Numerous studies performed in rodents indicate a positive effect of the delivery of therapeutic genes to the spinal cord to stimulate neuroregeneration. However, to bring the developed protocols of gene therapy to the stage of clinical trials, it is necessary to verify the results obtained in experiments on large laboratory animals. Objective: Immunofluorescence analysis of the response of markers of cell stress and apoptosis, synaptic proteins and neuroglia in the spinal cord of female vietnamese pot-bellied pigs after intrathecal delivery of genes encoding vascular endothelial growth factor (VEGF165), glial-derived neurotrophic factor and neuronal cell adhesion molecule (NCAM1), using human umbilical cord blood mononuclear cells (UCBMC). In experimental pigs (n = 2), 4 hours after modeling a dosed contusion injury of the spinal cord at the Th8-Th9 level, 2х106 genetically modified UCBMCs overexpressing recombinant VEGF, GDNF, and NCAM molecules in 200 |jl of saline were intrathecally injected. Control animals (n = 2) were injected with 200 jl of saline into the cerebrospinal fluid. Intact pigs (n = 2) were used to obtain baseline values for immunofluorescence analysis of post-traumatic molecular and cellular responses. After 60 days, immunofluorescence analysis in the rostral and caudal parts of the spinal cord relative to the epicenter of injury revealed positive changes in experimental pigs against the background of cell-mediated delivery of the VeGf165, GDNF, and NCAM1 genes. In the anterior horns of the rostral and caudal spinal cord of animals from the therapeutic group, a higher level of fluorescence of the synaptic protein synaptophysin, a lower number of astrocytes and microglial cells were found, which may indicate functional recovery of neurons and suppression of the development of astrogliosis. In the rostral section, in the area of the corticospinal tract, gene therapy maintained the number of oligodendrocytes, which ensure myelination of regenerating axons. The results obtained suggest that genetically modified UCBMCs, overexpressing recombinant molecules VEGF and GDNF (as therapeutic molecules) and NCAM (as a molecule providing survival and targeted targeting of cell carriers), contribute to post-traumatic regeneration of the spinal cord.

Currently, the main fundamental and clinical interest for stroke therapy is focused on developing a neuroprotective treatment of a penumbra region within the therapeutic window. The development of treatments for ischemic stroke in at-risk patients is of particular interest. Preventive gene therapy may significantly reduce the negative consequences of ischemia-induced brain injury. In the present study, we suggest the approach of preventive gene therapy for stroke. Adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) or gene engineered umbilical cord blood mononuclear cells (UCB-MC) overexpressing recombinant VEGF, GDNF, and NCAM were intrathecally injected before distal occlusion of the middle cerebral artery in rats. Post-ischemic brain recovery was investigated 21 days after stroke modelling. Morphometric and immunofluorescent analysis revealed a reduction of infarction volume accompanied with a lower number of apoptotic cells and decreased expression of Hsp70 in the peri-infarct region in gene-treated animals. The lower immunopositive areas for astrocytes and microglial cells markers, higher number of oligodendrocytes and increased expression of synaptic proteins suggest the inhibition of astrogliosis, supporting the corresponding myelination and functional recovery of neurons in animals receiving preventive gene therapy. In this study, for the first time, we provide evidence of the beneficial effects of preventive triple gene therapy by an adenoviral- or UCB-MC-mediated intrathecal simultaneous delivery combination of vegf165, gdnf, and ncam1 on the preservation and recovery of the brain in rats with subsequent modelling of stroke.

Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC) transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury. This proof-of-principle project was aimed at evaluating the beneficial effects of the same triple-gene approach in stroke. Rats subjected to distal occlusion of the middle cerebral artery were treated intrathecally with a combination of these genes either directly or using our cell-based (UCB-MC) approach. Various techniques and markers were employed to evaluate brain injury and subsequent recovery after treatment. Brain repair was most prominent when therapeutic genes were delivered via adenoviral vector- or UCB-MC-mediated approach. Remodeling of brain cortex in the stroke area was confirmed by reduction of infarct volume and attenuated neural cell death, depletion of astrocytes and microglial cells, and increase in the number of oligodendroglial cells and synaptic proteins expression. These results imply that intrathecal injection of genetically engineered UCB-MC over-expressing therapeutic molecules (VEGF, GDNF, and NCAM) following cerebral blood vessel occlusion might represent a novel avenue for future research into treating stroke.

Cell-mediated (ex-vivo) gene therapy for the treatment of adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) had started in 1990 and nowadays it is the first marketing approval of an ex vivo gene therapy in Europe. The method based on ex-vivo transduction of peripheral blood lymphocytes with retroviral vector carrying the functional ADA gene in 2002 have been improved to use hematopoietic stem cell (HSC) for ex-vivo transduction with 100% survival and the evidence of safety and efficacy. Remarkably, umbilical cord blood mononuclear cells (UCB-MC) were successfully used for treatment of ADA deficiency in neonates as well. Meanwhile SCID is a very rare congenital disorder of the immune system although the option to use peripheral blood lymphocytes as cell carriers of the therapeutic genes for regenerative medicine is highly attractive. In our studies to overcome the neural cells death and stimulate neuroregeneration at neurodegenerative diseases (ALS), spinal cord injury (SCI), and stroke in animal models we employed ex-vivo triple gene therapy based on human UCB-MC transduced with adenoviral vectors carrying vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM). The reason for clinical application of UCB-MC is based on their availability, ease of preparation and potential for long term storage, as well as legislative, ethical and religious benefits for the transplantation. In our gene-cell construct NCAM was used for homing and survival of UCB-MC at the site of neurodegeneration. VEGF and GDNF are the molecules with well-known neuroprotective function. Moreover VEGF is useful in restoring of the microcirculation as well.

The gene therapy has been successful in treatment of spinal cord injury (SCI) in several animal models, although it still remains unavailable for clinical practice. Surprisingly, regardless the fact that multiple reports showed motor recovery with gene therapy, little is known about molecular and cellular changes in the post-traumatic spinal cord following viral vector- or cell-mediated gene therapy. In this study we evaluated the therapeutic efficacy and changes in spinal cord after treatment with the genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and neuronal cell adhesion molecule (NCAM) applied using both approaches. Therapeutic genes were used for viral vector- and cell-mediated gene therapy in two combinations: (1) VEGF+GDNF+NCAM and (2) VEGF+ANG+NCAM. For direct gene therapy adenoviral vectors based on serotype 5 (Ad5) were injected intrathecally and for cell-mediated gene delivery human umbilical cord blood mononuclear cells (UCB-MC) were simultaneously transduced with three Ad5 vectors and injected intrathecally 4 h after the SCI. The efficacy of both treatments was confirmed by improvement in behavioral (BBB) test. Molecular and cellular changes following post-traumatic recovery were evaluated with immunofluorescent staining using antibodies against the functional markers of motorneurons (Hsp27, synaptophysin, PSD95), astrocytes (GFAP, vimentin), oligodendrocytes (Olig2, NG2, Cx47) and microglial cells (Iba1). Our results suggest that both approaches with intrathecal delivery of therapeutic genes may support functional recovery of post-traumatic spinal cord via lowering the stress (down regulation of Hsp25) and enhancing the synaptic plasticity (up regulation of PSD95 and synaptophysin), supporting oligodendrocyte proliferation (up regulation of NG2) and myelination (up regulation of Olig2 and Cx47), modulating astrogliosis by reducing number of astrocytes (down regulation of GFAP and vimetin) and microglial cells (down regulation of Iba1).

Aim. To develop a protocol of direct and cell-mediated gene therapy for ischemic stroke. Methods. Viral vector carrying green fluorescent protein (GFP) reporter gene was created on the basis of human adenovirus serotype 5 (Ad5). The umbilical blood supply was preserved according to instructions of Kazan State Medical Uuniversity Stem cell bank. Umbilical cord blood mononuclear cells were isolated in a ficoll density gradient by standard procedure and transduced with Ad5-GFP. Ischemic cerebral stroke in rats was caused by distal occlusion of the middle cerebral artery through trephination hole in a temporal bone under surgical microscope. Within four hours after modeling stroke in the anesthetized animals laminectomy was performed at the L4-L5 level, and (1) 0.9% sodium chloride solution, (2) Ad5-GFP and (3) umbilical cord blood mononuclear cells + Ad5-GFP were inserted intrathecally. Survival, targeted migration to the focus of neurodegeneration, the ability to synthesize recombinant protein and the effect of umbilical cord blood mononuclear cells on the infarction area were assessed using luminescent microscopy and morphometric analysis. Results. GFP expression in the area of the stroke was established 3 weeks after stroke modeling, both after intrathecal insertion of Ad5-GFP and after xenotransplantation of umbilical cord blood mononuclear cells Ad5-GFP transduced ex vivo. When comparing the areas of cerebral infarction 3 weeks after modeling the stroke, in animals from umbilical cord blood mononuclear cells + Ad5-GFP group the median of the infarction area was 47.4% less than in animals receiving isotonic saline solution. Conclusion. Umbilical cord blood mononuclear cells + Ad5-GFP after intrathecal insertion to animals with ischemic stroke, are capable of targeted migration to the neurodegeneration site as well as of recombinant protein synthesis; the results suggest the expediency of delivering therapeutic genes to ischemic zone via umbilical cord blood mononuclear cells overexpressing neurotrophic factors.

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.