ABSTRACT
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.
ABSTRACT
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.
Subject(s)
CD56 Antigen/genetics , Genetic Therapy/methods , Glial Cell Line-Derived Neurotrophic Factor/genetics , Ribonuclease, Pancreatic/genetics , Spinal Cord Injuries/therapy , Vascular Endothelial Growth Factor A/genetics , Adenoviridae/genetics , Animals , CD56 Antigen/metabolism , Cord Blood Stem Cell Transplantation , Disease Models, Animal , Escherichia coli , Female , Fetal Blood/cytology , Genetic Vectors , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Injections, Spinal , Rats, Wistar , Ribonuclease, Pancreatic/metabolism , Spinal Cord Injuries/pathology , Spinal Cord Injuries/physiopathology , Spinal Cord Regeneration/physiology , Transduction, Genetic , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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.
Subject(s)
Cord Blood Stem Cell Transplantation/methods , Disease Models, Animal , Genetic Therapy/methods , Leukocytes, Mononuclear/transplantation , Spinal Cord Injuries , Adenoviridae/genetics , Animals , Female , Genetic Vectors , Glial Cell Line-Derived Neurotrophic Factor/genetics , Humans , Neural Cell Adhesion Molecules/genetics , Pilot Projects , Recovery of Function , Swine , Swine, Miniature , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Current treatment options of chronic, progressive degenerative neuropsychiatric conditions offer only marginal efficacy, and there is no therapy which arrests or even reverses these diseases. Interest in genetic engineering and cell-based approaches have constantly been increasing, although most of them so far proved to be fruitless or at best provided very slight clinical benefit. In the light of the highly complex patho-mechanisms of these maladies, the failure of drugs aimed at targeting single molecules is not surprising. In order to improve their effectiveness, the role of a unique triple-combination gene therapy was investigated in this study. Intravenous injection of human umbilical cord blood mononuclear cell (hUCBMC) cotransduced with adenoviral vectors expressing vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) resulted in prominent increase of life span and performance in behavioral tests in amyotrophic lateral sclerosis (ALS). Expression of the recombinant genes in hUCBMCs was confirmed as soon as 5 days after transduction by RT-PCR, and cells were detectable for as long as 1 month after grafting in lumbar spinal cord by immunofluorescent staining. Xenotransplantation of cells into mice blood without any immunosuppression demonstrated a high level of hUCBMCs homing and survivability in the central nervous system (CNS), most conspicuously in the spinal cord, but not in the spleen or liver. This study confirms an increased addressed homing and notable survivability of triple-transfected cells in lumbar spinal cord, yielding a remarkably enhanced therapeutic potential of hUCBMCs overexpressing neurotrophic factors.
