Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model.

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 ßIII-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.

New Therapy for Spinal Cord Injury: Autologous Genetically-Enriched Leucoconcentrate Integrated with Epidural Electrical Stimulation.

The contemporary strategy for spinal cord injury (SCI) therapy aims to combine multiple approaches to control pathogenic mechanisms of neurodegeneration and stimulate neuroregeneration. In this study, a novel regenerative approach using an autologous leucoconcentrate enriched with transgenes encoding vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) combined with supra- and sub-lesional epidural electrical stimulation (EES) was tested on mini-pigs similar in morpho-physiological scale to humans. The complex analysis of the spinal cord recovery after a moderate contusion injury in treated mini-pigs compared to control animals revealed: better performance in behavioural and joint kinematics, restoration of electromyography characteristics, and improvement in selected immunohistology features related to cell survivability, synaptic protein expression, and glial reorganization above and below the injury. These results for the first time demonstrate the positive effect of intravenous infusion of autologous genetically-enriched leucoconcentrate producing recombinant molecules stimulating neuroregeneration combined with neuromodulation by translesional multisite EES on the restoration of the post-traumatic spinal cord in mini-pigs and suggest the high translational potential of this novel regenerative therapy for SCI patients.

Combination of epidural electrical stimulation with ex vivo triple gene therapy for spinal cord injury: a proof of principle study.

Despite emerging contemporary biotechnological methods such as gene- and stem cell-based therapy, there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury. Our previous studies have demonstrated that transplantation of genetically engineered human umbilical cord blood mononuclear cells producing three recombinant therapeutic molecules, including vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) can improve morpho-functional recovery of injured spinal cord in rats and mini-pigs. To investigate the efficacy of human umbilical cord blood mononuclear cells-mediated triple-gene therapy combined with epidural electrical stimulation in the treatment of spinal cord injury, in this study, rats with moderate spinal cord contusion injury were intrathecally infused with human umbilical cord blood mononuclear cells expressing recombinant genes VEGF165, GDNF, NCAM1 at 4 hours after spinal cord injury. Three days after injury, epidural stimulations were given simultaneously above the lesion site at C5 (to stimulate the cervical network related to forelimb functions) and below the lesion site at L2 (to activate the central pattern generators) every other day for 4 weeks. Rats subjected to the combined treatment showed a limited functional improvement of the knee joint, high preservation of muscle fiber area in tibialis anterior muscle and increased H/M ratio in gastrocnemius muscle 30 days after spinal cord injury. However, beneficial cellular outcomes such as reduced apoptosis and increased sparing of the gray and white matters, and enhanced expression of heat shock and synaptic proteins were found in rats with spinal cord injury subjected to the combined epidural electrical stimulation with gene therapy. This study presents the first proof of principle study of combination of the multisite epidural electrical stimulation with ex vivo triple gene therapy (VEGF, GDNF and NCAM) for treatment of spinal cord injury in rat models. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 2.20.02.18) on February 20, 2018.

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury.

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.

Epidural Stimulation Combined with Triple Gene Therapy for Spinal Cord Injury Treatment.

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.

Combined Supra- and Sub-Lesional Epidural Electrical Stimulation for Restoration of the Motor Functions after Spinal Cord Injury in Mini Pigs.

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.

Preventive Triple Gene Therapy Reduces the Negative Consequences of Ischemia-Induced Brain Injury after Modelling Stroke in a Rat.

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.

Triple-Gene Therapy for Stroke: A Proof-of-Concept in Vivo Study in Rats.

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.

Spinal Cord Molecular and Cellular Changes Induced by Adenoviral Vector- and Cell-Mediated Triple Gene Therapy after Severe Contusion.

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).

Evaluation of direct and cell-mediated triple-gene therapy in spinal cord injury in rats.

Current treatment options for spinal cord injury (SCI) are scarce. One of the most promising innovative approaches include gene-therapy, however no single gene has so far been shown to be of clinical relevance. This study investigates the efficacy of various combinations of vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG) and neuronal cell adhesion molecule (NCAM) in rats. Multiple therapeutic genes were administered intrathecally either via adenoviral vectors or by using genetically modified human umbilical cord blood mononuclear cells (hUCBMCs). Following the induction of SCI, serial assessment of cord regeneration was performed, including morphometric analysis of gray and white matters, electrophysiology and behavioral test. The therapeutic gene combinations VEGF+GDNF+NCAM and VEGF+ANG+NCAM had positive outcomes on spinal cord regeneration, with enhanced recovery seen by the cell-based approach when compared to direct gene therapy. The efficacy of the genes and the delivery methods are discussed in this paper, recommending their potential use in SCI.

A pilot study of cell-mediated gene therapy for spinal cord injury in mini pigs.

Currently, in clinical practice there is no efficient way to overcome the sequences of neurodegeneration after spinal cord traumatic injury. Using a new experimental model of spinal cord contusion injury on miniature pigs, we proposed to deliver therapeutic genes encoding vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) to the damaged area, using umbilical cord blood mononuclear cells (UCBC). In this study, genetically engineered UCBC (2×106 cells in 200 ml of saline) were injected intrathecally to mini-pigs 10days after SCI. Control and experimental mini pigs were observed for 60days after surgery. Histological, electrophysiological, and clinical evaluation demonstrated significant improvement in animal treated with genetically engineered UCBCs. Difference in recovery of the somatosensory evoked potentials and in histological findings in control and treated animals support the positive effect of the gene-cell constriction for recovery after spinal cord injury. Results of this study suggest that transplantation of UCBCs simultaneously transduced with three recombinant adenoviruses Ad5-VEGF, Ad5-GDNF and Ad5-NCAM represent a novel potentially successful approach for treatment of spinal cord injury.

Genetically modified human umbilical cord blood cells expressing vascular endothelial growth factor and fibroblast growth factor 2 differentiate into glial cells after transplantation into amyotrophic lateral sclerosis transgenic mice.

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF(165)) and human fibroblast growth factor 2 (FGF(2)) (pBud-VEGF-FGF(2)). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models ((G)93(A) mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal ß(3)-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF(2). Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific ß(3)-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF(2), hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF(2) on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.