Method for stimulating spinal cord regeneration with genetically modified human umbilical cord blood cells

FIELD: medicine.

SUBSTANCE: what is presented is a method for stimulating the post-traumatic spinal cord regeneration consisting in a single-stage transplantation of human umbilical cord blood mononuclear cells pre-transduced with recombinant adenovirus with a cloned gene of glial derived neurotrophic factor (gdnf), in the damage area.

EFFECT: using the invention enables providing a better outcome of the post-traumatic spinal cord regeneration, reduced length of staying in hospital of patients suffering from a spinal cord injury and improving the patients' quality of life.

1 ex

The invention relates to medicine, in particular to methods of treatment of spinal cord injuries using the introduction in the area of corruption genetically modified mononuclear blood cells of the human umbilical cord, and can be used in neurosurgery and traumatology.

Methods of treatment of the consequences of multiple trauma remain ineffective due to low regeneration potential in the brain. Enhancing the effectiveness of treatment for this disease is associated with gene-cell therapy. Cell transplantation in experimental spinal cord injury slightly stimulates regeneration and provides partial recovery of lost functions. Enhancement of therapeutic effect of transplantation of cells reached by genetic modification using non-viral and viral vectors. Transplantation of these cells provides long-lasting overexpression of key molecules that support the survival and differentiation of neural cells. In experimental spinal cord injury shown that transplantation of genetically modified cells using viral and plasmid vectors with cloned genes neurotrophic factors, protivopolozhnyh molecules, adhesion molecules and other stimulant neuroregenerative, is more effective than transplantat the I same native cells.

For delivery in the area of traumatic spinal cord injury therapeutic genes used differentiated, stem, induced pluripotent stem and progenitor cells. Quite promising are the blood cells of the umbilical cord, because of their low immunogenicity, availability, simplicity and security to obtain, the ability to withstand long-term storage. These cells have been intensively investigating for transplantation with brain injury (Cao, F. J., S. Q. Feng. "Human umbilical cord mesenchymal stem cells and the treatment of spinal cord injury." ChinMed J (Engl) 122(2). - 2009. - P.225-231; Schira, J., M. Gasis, et al. "Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood." Brain 135(Pt 2). - 2012. - P.431-446). The gene delivery using viral vectors in different experimental models of neurodegenerative diseases have shown improvement in function, that allows to consider this method as the most promising for targeted therapies in the pathology of the Central nervous system.

Member of the family of transforming growth factor beta (TGFβ) glial-derived neurotrophic factor (eng. glial cell line-derived neurotrophic factor, GDNF) is a potent factor in maintaining the viability of many populations of neurons, including motoneurons. That is why this neurotrophic factor associated with the future treatment of neurological R is straist.

In experimental spinal cord injury introduction in the area of corruption GDNF exerts a neuroprotective effect, supports the expression of proteins neurofilament, peptide associated with calcitonin genome (CGRP) and is associated with axonal protein 43 (GAP-43) (Cheng, N., J. P. Wu, et al. "Neuroprotection of glial cell line-derived neurotrophic factor in damaged spinal cords following contusive injury." J Neurosci Res 69(3). - 2002. - P.397-405). In combination with transplantation of Schwann cells GDNF inhibits the production of astrocytes molecules inhibitors of axon growth, which reduces the inhibitory effects of glial scar regeneration of nerve fibers (Deng, L.-X., J. Ni, et al. "GDNF modifies reactive astrogliosis allowing robust axonal regeneration through Schwann cell-seeded guidance channels after spinal cord injury." Experimental neurology 229(2). - 2011. - P.238-250). Many molecules produced GFAP-immunopositive astrocytes exert a neurotrophic effect, and the astrocytes form a potential space for the growth of axons. The results formed the basis for the concept of supporting axonal phenotype GFAP-immunopositive of astrocytes (growth-supportive phenotype) (White, R. E., M. Rao, et al. (2011). "Transforming growth factor alpha transforms astrocytes to a growth-supportive phenotype after spinal cord injury." JNeurosci 31(42): 15173-15187). GDNF has a direct stimulating effect on the growth of axons in the injured spinal cord stimulates their myelination, reduces apoptosis and degeneration of tissue (Iannotti, S., N. Li, et a. "Glial cell line-derived neurotrophic factor-enriched bridging transplants promote propriospinal axonal regeneration and enhance myelination after spinal cord injury." Exp Neurol 183(2). 2003. - P.379-393). In experimental spinal cord injury not only local but also systemic administration of GDNF inhibits the development of pathological manifestations (Kao, C.-N., S.-H. Chen, et al. "Exogenous administration of glial cell line-derived neurotrophic factor improves recovery after spinal cord injury." Resuscitation 77(3). - 2008. - P.395-400). While GDNF increases the number and caliber of regenerating axons, and stimulates the growth of axons in culture neurons in spinal ganglia (Zhang, L., Z. Ma, et al. "GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons." Glia 57(11). - 2009. - P.1178-1191).

There is a method of stimulating post-traumatic regeneration of the spinal cord by delivery to the site of injury is not neurotropism factor, and its gene. In the experiment with spinal cord injury have been shown to improve functional and structural indicators for local delivery of a gdnf gene in the composition of the plasmid vector using cationic liposomes (Lu, K. W., Z. Y. Chen, et al. "Protective effect of liposome-mediated glial cell line-derived neurotrophic factor gene transfer in vivo on motoneurons following spinal cord injury in rats." Chin J Traumatol 7(5). - 2004. - P.275-279), nanoparticles biorastvorimym polymer (Wang, Y.-C., Y.-T. Wu, et al. "Sustained intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles following contusive spinal cord injury." Biomaterials 29(34). - 2008. - P.4546-4553) or in complex with what ntially against receptors for neurotrophic factors (Barati, S., P. R. Hurtado, et al. "GDNF gene delivery via the p75(NTR) receptor rescues injured motor neurons." Exp Neurol 202(1). - 2006. - P.179-188). Compared to non-viral vectors viral vectors are more transfectional activity. Injection in the area of traumatic injury of the spinal cord genetic constructions on the basis of herpes simplex virus gene gdnf improves recovery of function (Natsume, A., D. Wolfe, et al. "Enhanced functional recovery after proximal nerve root injury by vector-mediated gene transfer." Exp Neurol 184(2). - 2003. - P.878-886). Local delivery in the area of spinal cord injury of the same gene using adenoviral vector prevents retrograde atrophy corticospinal of motoneurons and promotes recovery of motor functions (Tang, X.-Q., Y. Wang, et al. "Adenovirus-mediated delivery of GDNF ameliorates corticospinal neuronal atrophy and motor function deficits in rats with spinal cord injury." Neuroreport 15(3). - 2004. - P.425-429). Injection of the transgene allows transliterate cells, the localization of which is limited to a fairly narrow area of introduction. However, when spinal cord injury in the pathological process involves an extensive area, adjacent to the epicenter of injury. So for the most complete manifestation of therapeutic effects of the introduced gene is necessary to ensure his presence not only in the midst of traumatic injury, but also in adjacent areas, as a rule, quite remote from this zone. To solve this problem, n is the most promising approach is the delivery of therapeutic genes at the cellular carriers. Numerous experimental studies have established that transplantation of transfected cells carrying the transgene, has a stimulating effect on post-traumatic spinal cord regeneration. Delivery of gdnf gene using transduced by retrovirus fibroblasts in the area of complete transection or dorsal hemisection spinal cord of rats was carried out in rats (Blesch, A., M. N. Tuszynski. "Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and dosage remyelination." J Comp Neurol 467(3). - 2003. - P.403-417), which failed to reveal signs of regeneration of nerve fibers and their remyelinization. However, fiber is not germinated after the injury area, and was not restored ties with target cells that had not led to an improvement in motor or sensory function. Cell-mediated delivery of gdnf gene carried out in the area of complete transection of the spinal cord of the rat thoracic by transplantation of transfected glial cells of the olfactory structures (Yan, N. C., Z. M. Zhang, et al. "The repair of acute spinal cord injury in rats by humans ensheathing cells graft modified by glia cell line-derived neurotrophic factor gene in combination with the injection of monoclonal antibody IN-1." Zhonghua Wai Ke Za Zhi 47(23). - 2009. - P.1817-1820). When comparing groups of animals with transplantation of transfected and native cells, the authors were able to show a steady germination regenerating fiber is n through the region of the gap only in the first case, which was accompanied by improved recovery of motor function.

Cell transplantation umbilical cord blood of a person with spinal cord injury inhibits inflammatory response, has neurotrophic activity and stimulates neovascularization (Chen, C. T., N. N. Foo, et al. "Infusion of human umbilical cord blood cells ameliorates hind limb dysfunction in experimental spinal cord injury through anti-inflammatory, vasculogenic and neurotrophic mechanisms." Pediatr Neonatol 49(3). - 2008. - P.77-83), reduces the expression of proapoptotic genes and supports the survival of neurons (Dasari, V. R., K. K. Veeravalli, et al. "Neuronal apoptosis is inhibited by cord blood stem cells after spinal cord injury " J. Neurotrauma 26(11). - 2009. - P.2057-2069). Several experimental models have established that the blood cells of the umbilical cord, producing colony-stimulating factor 1, thrombopoietin and interleukin-11, have immunomodulatory effects (Suen, Y., S. M. Lee, et al. "Decreased macrophage colony-stimulating factor mRNA expression from activated cord versus adult mononuclear cells: altered posttranscriptional stability." Blood 84(12). - 1994. - P.4269-4277; Taguchi, A., T. Soma, et al. "Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model." J Clin Invest 114(3). - 2004. - P.330-338; Vendrame, M., J. Cassady, et al. "Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct dementia volume." Stroke 35(10). - 2004. - P.2390-2395). According to our records transplantation of these cells, transfected with a plasmid with the genes of neurotrophic factors in the injury of the spinal cord of the rat has a marked stimulating effect on near the regeneration (Shaymardanova GF, Muhamedshina YAO and others, "Effect of transplantation in the area of traumatic injury of the spinal cord of the rat mononuclear blood cells of the umbilical cord human, recombinant genes VEGF and FGF2." J.Morphology 142(4). - 2012. - R.31-36).

Closest to the claimed invention is a method of stimulating post-traumatic regeneration of the spinal cord, used in the study (Yan, N. C., Z. M. Zhang, et al. "The repair of acute spinal cord injury in rats by humans ensheathing cells graft modified by glia cell line-derived neurotrophic factor gene in combination with the injection of monoclonal antibody IN-1." Zhonghua Wai Ke Za Zhi 47(23). - 2009. - P.1817 - 1820), according to which isolated from the body glial cells of the olfactory structures were transfusional in vitro lentiviruses vector with the gene gdnf. These cells transplanted rats in the injury area immediately after complete transection of the spinal cord in the thoracic region. Using a behavioral test "BBB" shows a more pronounced recovery of motor function than in animals with the same injury, but without the introduction of cells and in animals with the same injury, but with the introduction of nitrostilbene the same cells. Applied research method cell-mediated delivery of gdnf gene has the following disadvantages:

• the method of delivery of gdnf gene using lentiviruses vector, compared to the other most studied viral vectors, such as simple virus is on herpes, adenovirus, adeno-associated virus, is the least safe from the point of view uncontrolled differentiation of the transplanted cells and oncogenic potential;

• applied as carriers of the transgene glial cells of the olfactory structures, as well as Schwann cells, are characterized by low migration potential, which limits the delivery of therapeutic gene in the target tissue located away from the epicenter of traumatic injury;

• glial cells of the olfactory structures, as well as Schwann cells, as compared to stem cells and early cells predecessors are characterized by poor survival, which reduces the duration of therapeutic action deliver genes with them.

The objective of the proposed method is the stimulation of post-traumatic regeneration of the spinal cord by transplantation into the damage in mononuclear blood cells of the human umbilical cord, transduced recombinant replication-defective adenovirus carrying the gene for glial-derived neurotrophic factor (gdnf), which will overcome the aforementioned disadvantages of the nearest analogue of the invention and to achieve new technical result, namely:

• to provide a more complete restoration of structures of the spinal cord responsible for the implementation of the motor and sensory functions;

• reduce length of stay of patients with spinal cord injury in the hospital and improve the quality of life of patients of this cohort.

The problem is solved by a method of stimulating post-traumatic regeneration of the spinal cord by means of a single transplant immediately after damage in mononuclear blood cells of the human umbilical cord, transduced with recombinant adenovirus with the cloned gene glial derived neurotrophic factor (gdnf).

Research on the effectiveness of gene-cell therapy in spinal cord brain transplantation into the damaged transduced with recombinant adenovirus mononuclear blood cells of the umbilical cord as carriers of therapeutic gene cloned glial derived neurotrophic factor (gdnf) is available to the applicant the information sources are not identified.

The essence of the claimed technical solution is to promote post-traumatic regeneration of the spinal cord by means of a single transplant to the area of damage in mononuclear blood cells of the umbilical cord of a person previously transduced with recombinant adenovirus with the cloned gene glial derived neurotrophic factor (gdnf).

The inventive method operates on the known sequence of stages. To create recombinant the adenovirus used expression plasmid vector pAd/CMV/V5-DEST (Invitrogen, Catalog #V493-20, USA), in which the cloned gene gdnf technology Gateway® (Invitrogen, USA) using a reaction LR recombination (reaction-based site-specific recombination system of phage λ). Thus obtained recombinant plasmid encoding adenovirus genome with the insertion of a gene gdnf (pAd-GDNF), allows to obtain a recombinant adenovirus containing the desired transgene. Using restriction enzyme Pad was obtained linear plasmid pAd-GDNF, which was transfusional cells SOME A (Invitrogen, Catalog #R705-07, USA) for the Assembly and replication of adenovirus. Splitting vector contributes to the interaction of the left and right inverted terminal repeats and the removal of bacterial sequences (namely, the plot of the beginning of the pUC replication and gene resistance to ampicillin). The Assembly and replication of recombinant adenovirus occurs in the cell line SOME A - immortalization line primary embryonic cells of human kidney transformed with DNA fragments of adenovirus serotype 5. Cell line contains stably integrated into the genome of the e1 gene, which expresses proteins E1 (EA and E1b), necessary for the preparation of recombinant adenovirus. After receiving the crude viral lysate to increase the virus titer was used to amplify adenovirus Ad5-GDNF cells SOME A. 2 days after which argenia in cell culture was observed cytopathic effect that indicates that cells produce viral particles. Umbilical cord blood were taken after obtaining informed consent of the pregnant woman and prenatal screening for contraindications to donation of blood. Mononuclear blood cells from human umbilical cord was obtained by the method of sedimentation in a density gradient ficoll (Hawley, Hawley et al. 2004). After separation of mononuclear cells from umbilical cord blood were sown in the amount of 2×10⁷cells in 10 cm culture cups for suspension cells (Jet Biofil") in the environment of the Needle, the modified Dulbecco (DMEM) containing 10% serum fruits cows (FBS).

The cells were transducible by adding 100 μl of recombinant adenovirus Ad5-GDNF (8×10⁸PFU/ml) in 10 cm culture Cup. After transduction, the cells were incubated overnight at 37°C in a humid atmosphere containing 5% CO₂. The cells were concentrated by tsentrifugirovaniem and purified from the culture medium by washing in a solution of Dulbecco (DPBS) without ions of CA²⁺and Mg²⁺. The washed cells were resuspendable in DPBS and the camera Goryaeva estimated their number and viability after coloring Trifanova blue. Further genetically modified cells are used for transplantation into the damaged rats with contusion model of spinal cord injuries.

The claimed method of stimulating Regener the tion of the spinal cord by transplantation of transduced with recombinant adenovirus with gdnf gene in mononuclear blood cells of human umbilical cord has been studied in laboratory rats and described in detail in the following examples.

The experiments were carried out on white rats-males weighing 150-200 g in accordance with the requirements of the local ethics Committee of the GOU VPO "Kazan state medical University". Animals were kept in plastic cages at a temperature of 18-20°C with free access to food and water. Surgical procedures rats were performed after narkotizirovannyh by intraperitoneal injection of chloral hydrate (Sigma) (80 mg/ml, 0.4 ml per 100 g).

Model dosed contusion injury of the spinal cord of the rat studied the effect on neuroregeneration transplantation in the area of damage in mononuclear blood cells of the human umbilical cord, transduced with recombinant adenovirus gene glial derived neurotrophic factor (gdnf). Rats under anesthesia at the level of the eighth segment of the spinal cord after laminectomy inflicted dosed contusion injury vertically falling metal rod weighing 10 g from a height of 25 mm Immediately after sewing injury in two points at a distance of 1 mm rostralnie and Caudalie from the epicenter of the injury and 0.5 mm lies lateral to the midline using a Hamiltonian of the syringe were injected with previously transduced with recombinant adenovirus gene gdnf mononuclear blood cells from human umbilical cord 1 million cells in 5 μl of DPBS (phosphate-saline buffer Dulbecco, sterile, the ez ions of CA ²⁺and Mg²⁺Biolot, Russia) in each point. Animals of the control group in the same experimental conditions, was introduced the same cells, transfetsirovannyh adenoviral vector with the gene of green fluorescent protein (egfp).

To evaluate the efficiency of recovery of motor function used behavioral test in the open field BBB (Basso, D. M., M. S. Beattie, J. C. Bresnahan. "A sensitive and reliable locomotor rating scale for open field testing in rats." Journal ofNeurotrauma 12. - 1995. - P.1-21).

Ha 30 days after application contusion injury were isolated spinal cord. On cryostatic transverse sections of the spinal cord at a distance of 1.5 cm from the epicenter of the injury carried out immunofluorescence reaction. To identify the antigen, the sections were incubated with primary antibodies against glial fibrillar acidic protein (GFAP, Santa Cruz, 1:200) overnight at 4°C, washed in phosphate-buffered saline and then incubated with secondary antibodies conjugated with fluorescent dye anti-mouse Alexa 647 (Invitrogen, 1:200) for 2 hours at room temperature. To visualize the nuclei of cells, the sections were additionally stained for 10 minutes at room temperature, 4',6-diamidino-2-phenylindole (DAPI, 10 μg/ml in phosphate buffer, Sigma). Stained sections were made in an environment that supports the fluorescence, and studied using confocal scanning microscope LSM 510Meta (Carl Zeiss).

1. Testing motor function using behavioral test "BBB"

The rate of recovery of motor function (BBB) for transplantation into the damaged spinal cord mononuclear blood cells of the human umbilical cord, transduced with recombinant adenoviruses with therapeutic gene gdnf increases by 58% (P<0.05) in comparison with the corresponding figure in animals of the control group with the introduction of the same cells transfected with the gene for green fluorescent protein (egfp). On term of 9, 11 and 13 days the indicator BBB in the experimental group compared with the control, respectively is increased by 62.7 percent, 59.5 per cent and 53.5 per cent.

2. Immunohistochemistry of spinal cord

To 30 days after contusion injury of the spinal cord of the rat and the introduction into the area of damage in mononuclear blood cells of the human umbilical cord, transduced with recombinant adenovirus genes with gdnf (experimental group) and egfp (control group), in white and gray matter of the detected GFAP⁺-cells. In the experimental group in the white matter (ventro-medial part of the anterior cord and the lateral part of the lateral cord) at a distance of 0.5 cm from the epicenter of the injury in a caudal direction population of GFAP⁺cells in 2 times higher than in the control group.

Thus, the results show is about that the inventive method for local delivery of a therapeutic gene glial derived neurotrophic factor (gdnf) into the injury of the spinal cord allows you to effectively stimulate post-traumatic spinal cord regeneration, which is manifested in the form of improvements to restore function and increase the number of reactive astrocytes, essential to the process of neurodegeneration. Revealed as a result of carrying out gene-cell therapy, the increase in the number of GFAP⁺-astrocytes should be considered as a positive factor encouraging neuroregenerative. This conclusion is based on the well-known view on the role of reactive astrocytes, which have antioxidant and cytoprotective action on neurons and melanoblastoma oligodendrocytes, including by increasing the expression of membrane Transporter of glutamate and reduction of the content of this excitatory neurotransmitter in the extracellular space (Lepore, A., J. O'donnell, et al. "Reduction in expression of the astrocyte glutamate transporter, GLT1, worsens functional and histological outcomes following traumatic spinal cord injury." Glia. -2011).

The applicant for the first time:

- applied method for local delivery in the area of traumatic spinal cord injury therapeutic gene glial derived neurotrophic factor (gdnf) by mononuclear blood cells of the umbilical cord che is oweka, translotsirovannoi recombinant adenovirus with these cloned genome;

- shows the stimulating effect of gene-cell therapy that combines the delivery in the area of damage in mononuclear blood cells of human umbilical cord that has its own therapeutic effect, and the cloned gene gdnf on posttraumatic restoration of the structure and function of the spinal cord.

Using the proposed method of stimulating post-traumatic regeneration of the spinal cord by the method of transplantation of mononuclear blood cells of the human umbilical cord, transduced with recombinant adenovirus with the cloned gene glial derived neurotrophic factor (gdnf), allows you to:

to improve the results of post-traumatic regeneration of the spinal cord in the form of a more complete recovery of the structure and functions of the authority;

- reduce the time of stay of patients with spinal cord injury in the hospital and improve the quality of life of patients of this cohort.

The way to promote post-traumatic regeneration of the spinal cord, which consists in a single transplant to the area of damage in mononuclear blood cells of the umbilical cord of a person previously transduced with recombinant adenovirus with the cloned gene glial derived neurotrophic factor (gdnf).