Biosafety Evaluation of a Chimeric Adenoviral Vector in Mini-Pigs: Insights into Immune Tolerance and Gene Therapy Potential.

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.

A Biosafety Study of Human Umbilical Cord Blood Mononuclear Cells Transduced with Adenoviral Vector Carrying Human Vascular Endothelial Growth Factor cDNA In Vitro.

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).

Evaluation of the Autologous Genetically Enriched Leucoconcentrate on the Lumbar Spinal Cord Morpho-Functional Recovery in a Mini Pig with Thoracic Spine Contusion Injury.

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.

Induction of Angiogenesis by Genetically Modified Human Umbilical Cord Blood Mononuclear Cells.

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.

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.

Autologous Genetically Enriched Leucoconcentrate in the Preventive and Acute Phases of Stroke Treatment in a Mini-Pig Model.

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.

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.

Influence of Recombinant Codon-Optimized Plasmid DNA Encoding VEGF and FGF2 on Co-Induction of Angiogenesis.

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-165-2 plasmid (gene-cell therapy) and direct gene therapy using pBud-165-2 plasmid to enhance healing of full thickness skin wounds in rats.

Evaluation of Direct and Cell-Mediated Lactoferrin Gene Therapy for the Maxillofacial Area Abscesses in Rats.

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.

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.

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.

Combination of epidural electrical stimulation with 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.

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.

Gene-modified leucoconcentrate for personalized 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.

Bioinformatic Study of Transcriptome Changes in the Mice Lumbar Spinal Cord After the 30-Day Spaceflight and Subsequent 7-Day Readaptation on Earth: New Insights Into Molecular Mechanisms of the Hypogravity Motor Syndrome.

The hypogravity motor syndrome (HMS) is one of the deleterious impacts of weightlessness on the human body in orbital space missions. There is a hypothesis that disorders of musculoskeletal system as part of HMS arise in consequence of changes in spinal motor neurons. The study was aimed at bioinformatic analysis of transcriptome changes in lumbar spinal cords of mice after a 30-day spaceflight aboard biosatellite Bion-M1 (space group, S) and subsequent 7-day readaptation to the Earth's gravity (recovery group, R) when compared with control mice (C group) housed in simulated biosatellite conditions on the Earth.

Triple-Gene Therapy for Stroke: A Proof-of-Concept 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.

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.

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.

Post-spinal cord injury astrocyte-mediated functional recovery in rats after intraspinal injection of the recombinant adenoviral vectors Ad5-VEGF and Ad5-ANG.

OBJECTIVE The most actively explored therapeutic strategy for overcoming spinal cord injury (SCI) is the delivery of genes encoding molecules that stimulate regeneration. In a mouse model of amyotrophic lateral sclerosis and in preliminary clinical trials in patients with amyotrophic lateral sclerosis, the combined administration of recombinant adenoviral vectors (Ad5-VEGF+Ad5-ANG) encoding the neurotrophic/angiogenic factors vascular endothelial growth factor ( VEGF) and angiogenin ( ANG) was found to slow the development of neurological deficits. These results suggest that there may be positive effects of this combination of genes in posttraumatic spinal cord regeneration.

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 (2×10 cells in 200 ml of saline) were injected intrathecally to mini-pigs 10days after SCI.

Tandem Delivery of Multiple Therapeutic Genes Using Umbilical Cord Blood Cells Improves Symptomatic Outcomes in ALS.

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.

Symptomatic improvement, increased life-span and sustained cell homing in amyotrophic lateral sclerosis after transplantation of human umbilical cord blood cells genetically modified with adeno-viral vectors expressing a neuro-protective factor and a neural cell adhesion molecule.

Amyotrophic lateral sclerosis (ALS) is an incurable, chronic, fatal neuro-degenerative disease characterized by progressive loss of moto-neurons and paralysis of skeletal muscles. Reactivating dysfunctional areas is under earnest investigation utilizing various approaches. Here we present an innovative gene-cell construct aimed at reviving inert structure and function.

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo.

Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS).

RNA interference and amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neuro-degenerative disorder characterized by progressive loss of motor neurons. The etiology and molecular pathogenesis of cell death in most sub-types of the disease are largely unknown. The best documented cause of moto-neuron degeneration is the mutation in the superoxide dismutase-1 (SOD1) gene, which occurs in 10% of the familial forms of ALS.