ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Electric Stimulation/methods , Epidural Space/physiology , Genetic Therapy/methods , Leukocyte Count/methods , Spinal Cord Injuries/therapy , Transgenes/genetics , Animals , Disease Models, Animal , Female , SwineABSTRACT
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.
ABSTRACT
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.
Subject(s)
Electric Stimulation Therapy , Glial Cell Line-Derived Neurotrophic Factor/genetics , Neural Cell Adhesion Molecules/genetics , Spinal Cord Injuries/therapy , Vascular Endothelial Growth Factor A/genetics , Adenoviridae/genetics , Animals , Cell- and Tissue-Based Therapy/methods , Disease Models, Animal , Epidural Space , Genetic Therapy/methods , Genetic Vectors/therapeutic use , Glial Cell Line-Derived Neurotrophic Factor/therapeutic use , Humans , Motor Activity/genetics , Motor Activity/physiology , Neural Cell Adhesion Molecules/therapeutic use , Neuroglia/transplantation , Recovery of Function/genetics , Recovery of Function/radiation effects , Spinal Cord/physiopathology , Spinal Cord/radiation effects , Spinal Cord Injuries/genetics , Spinal Cord Injuries/physiopathology , Swine/genetics , Vascular Endothelial Growth Factor A/therapeutic useABSTRACT
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.
ABSTRACT
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.
Subject(s)
Brain Injuries/genetics , Brain Injuries/prevention & control , Genetic Therapy/methods , Glial Cell Line-Derived Neurotrophic Factor/genetics , Neural Cell Adhesion Molecules/genetics , Stroke/genetics , Vascular Endothelial Growth Factor A/genetics , Adenoviridae , Animals , Astrocytes/metabolism , Brain Injuries/complications , Brain Injuries/metabolism , Caspases/metabolism , Chemokines/blood , Chemokines/cerebrospinal fluid , Cytokines/blood , Cytokines/cerebrospinal fluid , Disease Models, Animal , Female , Genetic Vectors , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Intercellular Signaling Peptides and Proteins/blood , Intercellular Signaling Peptides and Proteins/cerebrospinal fluid , Monocytes/metabolism , Neural Cell Adhesion Molecules/metabolism , Neuroglia/metabolism , Neuroprotection/genetics , Rats , Rats, Wistar , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recovery of Function/genetics , Recovery of Function/physiology , Stroke/complications , Stroke/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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).
