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    Topic review

    Preventive Triple Gene Therapy

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    Submitted by: Vage Markosyan

    Definition

    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. 

    1. Introduction

    The current options for ischemic stroke treatment are extremely limited and are aimed at restoring blood flow in the ischemic area by intravenous infusion of recombinant tissue plasminogen activator and/or physical removal of the clots[1]. To date, the main fundamental and clinical interest is focused on developing a neuroprotective treatment of the penumbra region within the therapeutic window. The strategy of a cell-, gene-, and gene-cell therapy for neuroprotection in stroke treatment has been proven by numerous experiments in animal models [2][3][4]. Besides brain-specific cell types, umbilical cord blood (UCB) is widely used for neuroprotection in the central nervous system (CNS) for different pathological conditions[5]. UCB cells are considered a valuable source of stem cells, growth, and neurotrophic factors for cell therapy. The mononuclear fraction of UCB contains populations of different immature cells that are capable of differentiating into many cell types[6] and, thus, represent an alternative to embryonic stem cells for transplantation to patients with post-ischemic, post-traumatic and degenerative diseases[7][8]. To date, the following have been discovered in UCB: Hematopoietic stem cells (HSCs), endothelial progenitor cells, mesenchymal stem cells (MSCs), unrestricted somatic stem cells (USSCs), and side population cells (SP)[9][10][11][12].

    Due to the immaturity of the immune system of a new-born, the use of UCB cells for cell therapy does not require matching of genes relating to HLA (Human Leucocyte Antigens) human tissue compatibility, as evidenced by the absence of an acute or chronic form of the disease “graft-versus-host” (graft versus host disease)[13][14]. Besides, with UCB cell transplantation, tumor transformation of cells in the recipient’s body is practically prevented[15].

    Another attractive reason for using UCB cells for cell therapy is their ability to produce various biologically active molecules, such as proteins which are antioxidants, angiogenic, neurotrophic, and growth factors[16][17][18][19][20]. Thus, transplantation of UCB cells can be aimed at replacing dead cells and at preventing the further death of surviving cells due to secreted biologically active molecules. Enhancement of the positive effects of UCB cells on tissue regeneration after their genetic modification is a relatively new and promising gene-cell approach in cell therapy to stimulate post-traumatic or post-ischemic brain injury[21][22]. Gene-modified UCB cells may provide addressed delivery of therapeutic genes and supply the expression of the recombinant molecules at the site of regeneration.

    In our previous studies, we showed the positive effect of gene-modified umbilical cord blood mononuclear cells (UCB-MC), simultaneously producing three recombinant molecules—vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM)—in animal models of amyotrophic lateral sclerosis[23], spinal cord injury[24], and stroke[25]. The rationale of using a combination of two neurotrophic factors with cell adhesion molecules is based on the well-known neuroprotective effects of VEGF and GDNF[26][27], with the expression of NCAM increasing the homing and survivability of UCB-MC at the brain injury site[28] supporting local production of the therapeutic molecules. In the model of middle cerebral artery occlusion (MCAO) in rats, we demonstrated that intrathecal injection of genetically-engineered UCB-MC over-expressing VEGF, GDNF, and NCAM, four hours after MCAO results in a reduction of infarct volume, the positive reaction of neuroglial cells, and an increase in synaptic protein expression. Thus, ex vivo gene modification may enhance the naïve neuroprotective properties of UCB-MC.

    The development of treatment under the threat of stroke is of particular interest. Preventive therapy may highly reduce the consequences of a stroke-induced brain injury. In the present study, we suggest the approach of preventive cell-mediated gene therapy for stroke. The efficacy of gene-engineered UCB-MC overexpressing recombinant molecules-stimulants of neuroregeneration VEGF, GDNF, and NCAM, administered intrathecally 3 days before MCAO in rats, was investigated using morphometric and immunofluorescent methods.

    2.Triple Gene Therapy Reduces the Negative Consequences of Ischemia-Induced Brain Injury

    At present, the most promising of the actively developed strategies to prevent brain cell death in penumbra is the development of gene and cellular technologies. Gene therapy is mainly aimed at the delivery of therapeutic genes encoding neurotrophic factors. Among them, the most promising are genes encoding neurotrophic factors (BDNF, CNTF, GDNF, VEGF), anti-apoptotic proteins (Bcl-2, Bcl-XL), heat shock proteins (Hsp25, Hsp70), and anti-inflammatory molecules (IL-1RA). The neuroprotective effect of these factors has been experimentally proven, but there is no unequivocal, let alone exhaustive, answer to the question of which of these factors may be recommended as neuroprotective factors in practical medicine. It has been established that combinations of several neurotrophic factors may have a more pronounced effect on nerve cell survival[25][28].

    Other equally important issues in the strategy for the gene therapy of stroke include the development of technology to deliver transgenes to the brain. Difficulties in the delivery of therapeutic genes to the cerebral infarction area are one of the main reasons for the unavailability of effective gene therapy in treating post-ischemic negative consequences in the brain. Direct (in vivo) gene therapy provides for the delivery of transgenes into the recipient brain plasmid or viral genetic vectors [33]. Cell-mediated (ex vivo) gene therapy is based on the delivery of therapeutic genes using cells that serve as carriers of transgenes, as well as producers of recombinant protein molecules.

    For the treatment of stroke, various methods of gene therapy are known, the effectiveness of which has been proven in numerous experiments on animals[2][4][34]. Injections of viral vectors carrying transgenes into the ventricles or the infarction area are mainly used to deliver the therapeutic genes to the brain. Genetic vectors based on Sendai virus vectors containing gdnf or ngf genes were injected 30 min after stroke simulation[35]. The adeno-associated viral vector carrying gdnf was injected 2 days after the stroke modeling[36]. In other studies, viral vectors carrying therapeutic genes were delivered before modeling the stroke. The positive effect was shown after local delivery to the brain of gdnf or cntf genes—7 days[37], bdnf—2 weeks[38], gdnf—4 weeks[39], or ngf and bdnf—4 to 5 weeks[40] before stroke modeling.

    The list of genes employed in gene therapy for stroke is quite long. Of these, we find vegf, gdnf, and ncam to be the most promising.

    In addition to angiogenic action, VEGF exhibits the properties of a typical neurotrophic factor. It supports the survival of sensitive[41] and motor neurons[42] and stimulates the proliferation of astrocytes[43], neural stem cells [44], and Schwann’s cells[41]. GDNF has a pronounced neuroprotective effect on dopaminergic brain neurons and cholinergic spinal cord motoneurons[45] and stimulates the growth of nerve processes[46]. NCAM (CD56) is expressed on the surface of neurons and glial cells. Intercellular interactions mediated by NCAM in neuro-ontogenesis and posttraumatic regeneration provide survival and migration of neurons, directed neurite growth and synaptogenesis.

    The efficiency of cell therapy for stroke treatment in experiments using neural precursors derived from embryonic stem cells[47], induced pluripotent cells[48], MSCs isolated from red bone marrow[49], or UCB[50] suggests the use of these cells as carriers of therapeutic genes for delivery to the brain. Thus, MSCs were used for delivery of bdnf[51], pigf[52], and vegf165[53] to the brain. Of particular interest is the transduction of cell carriers by two or more expression vectors[28]. This approach allows simultaneous overexpression of several molecules/stimulants of neuroregeneration to be obtained. In our studies, in addition to the gene encoding neurotrophic factors, a gene encoding NCAM was delivered into UCB-MC, which, according to the obtained data, promoted the addressed migration of transplanted cells into the CNS after intravenous injection, increased their survival in the recipient’s tissues and supported prolonged production of recombinant therapeutic molecules[28]. In clinical investigations, autologous cells isolated from the red bone marrow (mononuclear and MSCs) or peripheral blood (CD34+) are predominantly used in the cell therapy of patients after stroke[54][55].

    The most promising cell carriers of therapeutic genes are UCB-MC[56][57][58]. The basis for their application is the suitability for both allografting and autotransplantation in humans, availability, and the ease of obtaining and storage. An important factor is the absence of legal, ethical, and religious prohibitions related to blood cell transplantation. In our previous study, we showed that intrathecal injection of adenoviral vectors carrying vegf, gdnf, and ncam, or genetically-modified UCB-MC+Ad5-VEGF-GDNF-NCAM, 4 h after stroke modeling in rats, had a positive effect on the morpho-functional recovery of the post-ischemic brain[25]. Adenoviral vectors and genetically-modified UCB-MC with cerebrospinal fluid reached the ischemic area and delivered the production of recombinant VEGF, GDNF, and NCAM, lasting up to 21 days in the experiment.

    Other important issues in the strategy for the treatment of ischemic stroke include the development of approaches to enhance the viability of neurons with the threat of a stroke. Patients with transient ischemic attacks, arterial hypertension, atrial fibrillation, disorders of lipid metabolism with high cholesterol, and diabetes are at high risk of ischemic stroke. Preventive therapy aimed at increasing the survivability of neurons in at-risk patients may prevent severe post-ischemic consequences in the brain, or improve the outcome of the disease. Currently, in medical practice, measures to prevent stroke are based on the use of anticoagulants and prosthetics of blood vessels. At the same time, the preventive methods that can considerably decrease the death of neurons in the “ischemic penumbra” during the 3–6-h “therapeutic window” are unknown. Enhancement of the viability of nerve cells at risk of stroke is also associated with the delivery of therapeutic genes that encode molecules to the brain, which inhibit neuronal death and stimulate neuroregeneration. In this study, for the first time, we propose the approach of preventive gene therapy to improve the viability of brain neurons under threat of ischemic stroke to contain neuronal death in the first hours of a stroke. The use of leucocytes for delivery of therapeutic genes (vegf165, gdnf, and ncam1) in the brain was based on their biological properties. Leucocytes are cells with high secretory and migration potentials, which suggest their exclusive role as cell carriers for addressed delivery and effective expression of transgenes. The results obtained in the study demonstrate that preventive intrathecal adenoviral- or UCB-MC-mediated delivery of vegf165, gdnf, and ncam1 results in a reduction of apoptosis and, consequently, the infraction volume. In addition to the decrease in expression of proteins of cellular stress and restraining neuronal death in the area of ischemic damage, we found evidence of the restoration of functional activity of neurons (increase in expression of synaptic proteins), maintenance of myelinization (increase in the number of oligodendrocytes) and an obstacle to astrogliosis development (decrease in the immunopositive areas for astrocytes and microglial cells markers). Importantly, the transplantation of gene-modified UCB-MC is safer and more efficacious compared with direct gene therapy. These data are in line with our results using the same gene and gene-cell constructs for ischemic stroke treatment in rats and allow us to conclude that preventive gene therapy may be effective in overcoming the negative consequences of ischemic stroke in the rehabilitation period.

    Recently, for personalized ex vivo gene therapy, we suggested the use of gene-modified leukoconcentrate (GML) prepared from the patient’s peripheral blood and chimeric adenoviral vectors (Ad5/35F) carrying one or a combination of therapeutic genes[59]. Taken together with the concept of GML-therapy and the data of this study, we propose the use of GML carrying vegf165, gdnf, and ncam1 for personalized preventive gene therapy in the threat of stroke.

    This entry is adapted from 10.3390/ijms21186858

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