RU2527169C2 - GENETIC CONSTRUCT CONTAINING hGDNF CONTROLLED BY TEMPERATURE-SENSITIVE PROMOTOR FOR REGULATED NEUROTROPHIC FACTOR EXPRESSION IN MAMMALIAN CELLS AND BODIES DIRECTLY - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to molecular biology and medicine. What is presented is a genetic construct on a basis of the vector plasmid pEGFP-N1 with a neomycin resistance gene, containing human glial cell-derived neurotrophic factor (GDNF) gene with heat shock elements (HSE) 4-8 and green fluorescent protein (GFP) gene controlled by a thermally regulated promoter of heat shock protein hsp70 gene of Drosophila melanogaster. The invention can be used in therapy of neurodegenerative disorders, traumatic adromia, as well as in cerebral ischemic stroke in mammals (including in humans), as a decrease of temperature activation threshold (39 to 42 degrees Celsius) of GDNF therapeutic gene expression enables reducing a negative effect of high temperatures on human body when using the construct containing the GDNF therapeutic gene for treating the neurodegenerative disorders that is ensured by using hsp 70 of Drosophila melanogaster.

EFFECT: temperature regulated temporary activation of GDNF is ensured by stimulating neural differentiation of neural precursors, as well as prevents the negative consequences of transgenic factor hyperexpression.

26 dwg, 7 ex

Description

The present invention relates to the fields of biology and medicine, in particular to molecular genetics and cell biology. This invention is necessary for the regulated production of human neurotrophic factor GDNF in mammalian cells and tissues. A construct containing the human GDNF gene was obtained under the control of the temperature-regulated promoter of the heat shock protein gene Hsp70 Drosophila melanogaster with a regulatory zone. To obtain a transgenic cell culture, a neomycin resistance gene is introduced into the vector.

It is known that neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, etc.), as well as ischemic stroke and neural injuries are the most important socially significant diseases, one of the significant causes of disability and mortality in people of working and old age. The methods of cell and gene therapy using cells with modified genes that provide a therapeutic effect are widely used in the treatment of these diseases. Particularly promising is the possibility of using regional stem cells of the patient himself, induced to develop in the right direction. The best transplant material is own stem cells from red bone marrow and other tissues containing mesenchymal stem / progenitor cells. However, since intact regional stem / progenitor cells have differentiation potential limitations, the development of ways to control the specialization of transplanted cells and increase their viability, as well as ways to prevent the formation of scar tissue at the sites of transplantation, is essential.

The main factors determining the nature and growth of afferent and efferent fibers of transplants are the environment in which the brain transplants are located, the growth ability of the transplant, the presence or absence of a scar at the border between the transplant and the host brain. However, already in the first week after the operation, the recipient's astrocytes begin to migrate to the graft and replace the wound canal with a glial scar. Moreover, in glial septa and in areas of the glial scar, the density of capillaries growing into the implant is significantly lower than that in the surrounding brain tissue of the recipient. This can lead to a very short experience of the transplant and its complete death as a result of a lack of nutrients within a few months after the operation, which ultimately reduces the effect to zero. To achieve better neurotransplantation efficiency, maximum integration of the transplant with the recipient's brain is required, which is possible only in the absence of a gliomesodermal capsule (Korochkin, 2000).

To achieve the maximum cellular and gene-therapeutic effect and ensure safety for the patient, it is necessary to search for factors capable of transforming stem cells in the direction necessary for treatment. In the case of a successful selection of such elements, it becomes possible to increase the effectiveness of gene and cell therapy in the treatment of many diseases that require the use of tissue transplantation.

The development of gene constructs that shift the differentiation of stem and progenitor cells in the neural direction and favorably affect graft survival, as well as the study of the functioning of the resulting constructs in transfected cells in culture before and after transplantation, are crucial for the development of cell and gene therapy. Of great importance in this case is the choice of factors with neuroprotective properties.

Of particular interest among the factors studied by scientists is the glia derivative neurotrophic factor (GDNF). At the moment, it is known that GDNF plays an important role in the development and survival of neural crest cells, as well as their migration to the kidneys and autonomic nervous system. GDNF is characterized as a growth factor contributing to the survival of dopaminergic midbrain neurons. GDNF is one of the main survival factors for dopaminergic midbrain neurons. Only GDNF can provide axon growth and hypertrophy of neurons of this type (Åkerud, et al., 1999). This factor is involved in the signaling pathway through the GFRα1-RET receptor complex, which is assembled on the membranes of both developing and mature dopaminergic neurons. However, a study on knockout mice showed that signaling involving GFRα1-RET is not necessary at the embryonic stage of development of dopaminergic neurons in vivo.

But GDNF still remains necessary in the process of postnatal development to maintain the vital activity of cells, their plasticity and the directed innervation of target sites. This is indirectly confirmed by the fact that GDNF is expressed in significant quantities in the striatum during the first few postnatal weeks, when directed innervation by dopaminergic neurons of various brain structures forms. In addition, unlike wild-type mouse neurons, transplanted embryonic dopaminergic neurons of GDNF-deficient mice are not capable of surviving and innervating a striatum denervated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine, (MPTP)) (Granholm et al., 2000). In addition, antisense inhibition of GDNF expression dramatically reduces axonal proliferation of dopaminergic neurons in the damaged striatum (Batchelor et al., 2000). Subsequent studies on knockout mice are needed to determine whether GDNF signaling is necessary for postnatal survival of neurons or for their targeted innervation of brain regions.

Thus, scientists and doctors have long been trying to use GDNF in practice. For example, damage to peripheral nervous system neurons usually leads to chronic neuropathic pain. As a rule, with such a disease, disorders occur in the sensory pathways. In laboratory conditions, on the model of neuropathic pain, the involvement of neurotrophic factors was proved, because their blocking by antibodies led to a decrease in hyperalgesia (an abnormally high sensitivity of the body to pain stimuli) (Zhou et al., 2000). A group of scientists led by McMahon showed that exogenous administration of GDNF can prevent some axotomic changes in sensory neurons. In response to peripheral nerve injuries, RET-expressing sensory neurons grow by processes around other sensory neurons, possibly due to local production of GFLs in damaged areas (Li et al., 2001). It is important to note that in models of neuropathic pain during intrathecal administration (under the shell of the spinal cord) of GDNF, an analgesic effect is observed, in particular, the functioning of Na + channels damaged by trauma is restored (Boucher et al., 2000). Damaged axons of sensory neurons in the peripheral system are able to recover normally, but cannot reach their main target - the spinal cord. Continuous intrathecal administration of GDNF can overcome this: in case of injuries of the dorsal root of the spinal cord, the introduction of these neurotrophs leads to the restoration of the structure and functions of sensory neurons, they reach the spinal cord and form correct functioning contacts with the target cells (Ramer et al., 2000). In experimental models of local ischemia, exogenous administration of GDNF before or immediately after anoxia contributed to a significant reduction in ischemic brain damage. In the adult, a strong response to the administration of GDNF is observed, as in neurons of the forebrain, after an ischemic stroke, the number of RET and GFRα1 sharply increases. An interesting fact is that in cerebral cortical neuron cultures, the administration of GDNF is not able to prevent cell death by apoptosis, but is able to prevent necrosis by reducing the influx of Ca ++ ions through Ca-mediated NMDA receptors (N-methyl-D-aspartate) (Nicole et al ., 2001). Therefore, GDNF should be administered immediately after an ischemic stroke.

Currently available methods of treating Parkinson's disease are aimed at eliminating symptoms and do not stop the destruction of dopaminergic neurons of the midbrain. In various models of Parkinson's disease, it has been shown that GDNF can prevent the neurotoxic provoked death of dopaminergic neurons and helps restore their functional activity (Grondin et al., 1998). However, the administration of GDNF into the lateral ventricles of the brain of a patient with Parkinson's disease had no positive effect and led to side effects, for example, weight loss (Kordower et al., 1999). Recent improvements in GDNF delivery tested on mouse models of Parkinson's disease include: 1. co-administration of GDNF and heparin, which increases the spread of GFLs; 2. the introduction of new viral vectors; 3. The introduction of GDNF-producing stem neuronal cells that maintain a constant high level of GDNF expression (Kordower et al., 2000; Åkerud et al., 2001). The issue of proper delivery of GFLs is crucial because continuous delivery of GDNF to the striatum does not result in weight loss and promotes the functionally active innervation of the striatum (Björklund et al., 2000). In combination with safety measures, including methods for controlling gene expression, new GDNF delivery vehicles look promising for treating Parkinson's disease (Zurn et al., 2001). It is extremely important to remember that overexpression of a neurotrophic factor can be just as dangerous as its lack. For example, prolonged subcutaneous administration of GDNF in the postnatal period or transgenic overexpression of GDNF in skeletal muscle leads to hyperinnervation of neuromuscular connections, this effect also persists in the adult state (Keller-Peck et al., 2001; Zwick et al., 2001) . A number of researchers have reported side effects, such as weight loss and the possibility of tumor neoplasms, observed with GDNF overexpression (Meng et al. 2000). These disorders can be eliminated by controlled expression of the neurotrophic factor.

The originality of the proposed invention is to obtain a design where the activation of GDNF in vivo is extremely active for up to 7 days with a gradual attenuation. Thus, the activity of GDNF is limited in time and is involved only in the restoration of innervation, not possessing constant increased expression, leading to negative consequences in the body. This design can be used as a gene therapy drug, or in the form of transgenic cells that produce this neurotrophic factor for a limited time.

Thus, an object of the invention is to obtain a genetic construct for introducing a gene for the neurotrophic factor GDNF into mammalian cells (including humans), where the activation of the gene will be regulated by increasing temperature (in the range from 39 to 42 ° C). GDNF is able to induce neural differentiation and stimulate restoration of innervation. This plasmid can be used both in mammalian cells (in vitro) and in mammals (in vivo) for fundamental and applied purposes. The plasmid can be introduced into the body of mammals both as transgenic cells, and directly as gene therapy material (naked plasmid). The plasmid is applicable in the treatment of neurodegenerative diseases, traumatic disorders of innervation, as well as in ischemic stroke of the brain of mammals (including humans).

The stated technical problem and the technical result are achieved by obtaining the genetic construct expressed in mammalian cells (including humans), constructed on the basis of the vector plasmid pEGFP-N1 with the neomycin resistance gene containing a DNA fragment encoding the regulatory sequence of the promoter with HSE heat shock elements more than 4, promoter of the heat shock shock protein gene hsp70 Drosophila melanogaster, human neurotrophic factor GDNF gene, green fluorescent protein (GFP) gene under control annogo promoter, wherein the promoter is able to be activated under the effect of heat shock temperature mammalian or toxic effects.

Example 1

We have created a genetic engineering construct containing the gene for the neurotrophic factor GDNF under the control of the promoter of the gene for heat shock protein of Drosophila hsp70 and the regulatory zone containing 4HSE.

PCR was used to synthesize the neurotrophic factor gene; primers were used for cloning the insert: T3 (5'-attaaccctcactaaaggga-3 ') and gdnf-BamHI (5'-tggatcccagatacatccacaccttttagcgg-3'). Using these primers from the plasmid phGDNF, the GDNF fragment was cloned by the corresponding restriction sites (Fig. 1). Synthesis program: 94 ° С - 1.5 min, 94 ° С - 15 sec, 57 ° С - 20 sec, 72 ° С - 15 sec, 72 ° С - 10 min, number of cycles: 30. The obtained DNA fragment was isolated from gel using Qiagen reagent.

This fragment was cloned into the proprietary vector pGEM-T Easy (Promega), according to the protocol. The resulting construct was verified by restriction analysis followed by sequencing with primers M13R and M13F. (Fig. 1)

The neurotrophic factor GDNF gene was cut from this construct at the EcoRV-BamHI restriction sites (600bp) and inserted into a plasmid containing the Drosophila hsp70 promoter and a 4 HSE regulatory zone at the SmaI / BamHI restriction sites. The presence of an insert in these constructs was verified by restriction analysis at various sites, including PCR cloning sites with primers gdnf-F (5'-ggaatcggcaggctgcagctg-3 ') and gfp-R (5'-aataaagcttgcatggcggtaatacg-3') .

Sequencing was also performed using gdnf-F primers (5'-ggaatcggcaggctgcagctg-3 ') and gdnf-R (5'-aatgctttcttagaatatggt-3'). (Fig. 2)

A diagram of the genetic engineering design is shown in Fig. 3.

After isolation and purification, this construct was introduced into the HEK293 cell culture by transfection using ExGene 500 reagent (Fermentas, R0511).

Transfection was performed according to the protocol. A HEK-293 cell culture was incubated at 37 ° C in a CO ₂ incubator. After selection on gentamicin for 10 days, transfected cells were scattered in 25 cm ² culture bottles. Next, it was necessary to select, under a fluorescence microscope, colonies of cells that expressed GDNF labeled with a green fluorescent GFP protein. Active luminescence of cells was observed. However, we checked the presence of our construct in the cells. For this, RNA was isolated from this cell culture for subsequent analysis using RT-PCR and Western blot.

For RT-PCR, cells were lysed with trisol according to the protocol, cDNA was synthesized using reverse revertase. The cDNA synthesis program (RT): 70 ° С - 5 min, 37 ° С - 5 min, 37 ° С - 60 min, 70 ° С - 5 min, 20 ° С - 30 sec. The synthesized cDNA was used for PCR reactions with various primers: GAPDH R (5'-cccctggccaaggtcatccatgacaactt-3 ') GAPDH F (5'-ggccatgaggtccaccaccctgttgctgta-3'); gdnf-F (5'-ggaatcggcaggctgcagctg-3 ') and GDNF-BamHI (5'-tggatcccagatacatccacaccttttagcgg-3'). A Western blot hybridization with a transfected cell culture was also performed.

To obtain a transgenic cell culture, a neomycin resistance gene was introduced into the construct, which allows selection of transfected cells on G418.

Transfected HEK293 cells were put on line for neomycin resistance. The presence of the constructed gdnf and gfp genes and protein expression in the transgenic culture derived were confirmed by sequence and Western blot hybridization. Protein was isolated from cell culture in SDS buffer according to the protocol (Amersham Bioscience). Further, these proteins were dispersed in a 10% acrylamide gel and analyzed using Western blot hybridization. A nitrocellulose membrane (Amersham Bioscience, # RPN68D) was used for transfer. This membrane was treated with antibodies: Rabbit polyclonal anti-GFP (Abeam #ab 290, 1: 2000) and secondary anti-Rabbit conjugated to horseradish peroxidase (HRP, 1: 2000). Proteins were detected using ECL reagent (Amersham Biocsience, # RPN2109) for peroxidase. The presence of the construct in the cells is proved.

After 24 hours of cultivation, the effect of temperature on cell cultures was analyzed. Cells were heated in an incubator at temperatures ranging from 37 ° C to 42 ° C for 60 minutes. GFP marker protein expression was observed using an Olympus fluorescence microscope. It was found that a green glow of the cell culture was observed after exposure to temperature. It was found that the maximum expression of GFP protein was observed at 40 ° C 48 hours after heating in the case of using 4 HSE zones (Fig. 2). Another 1/3 of the studied cells after exposure to temperature was recorded in a 4% paraform solution and was used to measure the intensity of luminescence using flow cytometry using a FACScan instrument (Becton Dickinson, USA). The data are for two temperatures of 40 and 42 ° C with different intervals after temperature exposure. It should be noted that, according to the cytofluorimeter, expression of the marker green fluorescent gene was already observed at 39 ° C, which is the result of using 4 heat shock elements (HSE) in the regulation of the promoter. One more fact should be noted: upon activation of 40 ° C, a decrease in luminescence (i.e., promoter activity) is observed after 48 hours, which can also be associated with the use of 4 heat shock elements (HSE) in the regulation of the promoter.

After 72 hours, a decrease in GFP protein synthesis is observed. However, upon repeated heating, secondary activation of protein synthesis is observed. Also, 1/3 of the cells were used to isolate total DNA according to the protocol. The resulting total DNA was examined by PCR analysis for the presence of the GFP gene in the HEK293 cell line. For the PCR method, a BioRad amplifier was used, primers: gfpF (5'-cgtcagatccgctagegctaccgg-3 ') and gfpR (5'-aataaagcttgcatggcggtaatacg-3'). Program: 95 ° C - 3 min, 94 ° C - 20 sec, 55 ° C - 15 sec, 72 ° C - 30 sec, 72 ° C - 5 min, number of cycles: 30.

Thus, it was shown that this system of activated GDNF expression works in HEK293 cell culture. It was found that the optimal time of temperature activation is 60 minutes, and the maximum glow of green fluorescent GFP protein is observed 48 hours after exposure to temperature. The effect of the re-inclusion of the promoter and the appearance of luminescence of the green fluorescent protein after repeated exposure to temperature was revealed (Fig. 4)

To assess the ability of transgenic HEK293 cells expressing gdnf / gfp to survive inside the nervous tissue, they were injected into the brain in black6 mice. Transgenic HEK293 cells (passage 2) were stimulated by heat shock at a temperature of 42 ° C for 60 minutes in DMEM, 10% EBS. Non-transgenic HEK293 cells were used as a control. 1 × 10 ⁶ GFP-labeled cells in 2 μl of Hanks solution were locally injected into the striatum of mice. At the end of the experiment (after 24 hours, 3 days and 5 days), the mice were perfused with 4% paraformaldehyde solution, the brain was removed, and after additional fixation and impregnation in a 30% sucrose solution, they were frozen at -20 ° C. Then sections were prepared with a thickness of 40 μm. The survival of transplanted HEK293 was evaluated under a microscope by the green fluorescence of the OPP protein expressed by them. (Fig. 5)

In the course of studies, it was found that transplanted transgenic cells behave differently from cell culture. Active expression was observed after 24 hours and gradually decreased over three days. Apparently, a longer activity of the promoter is associated with inflammatory processes during transplantation. This result is extremely attractive, since the activity of the gdnf gene is needed precisely for engraftment of the graft and, as a result, reduction of the glial scar during engraftment. (Fig. 6, 7)

Example 2

A genetic engineering construct was created containing the gene for the neurotrophic factor GDNF under the control of the promoter of the heat shock protein gene of Drosophila hsp70 and the regulatory zone containing 8HSE.

PCR was used to synthesize the neurotrophic factor gene; primers were used for cloning the insert: T3 (5'-attaaccctcactaaaggga-3 ') and gdnf-BamHI (5'-tggatcccagatacatccacaccttttagcgg-3'). Using the indicated primers from the plasmid phGDNF, the GDNF fragment was cloned by the corresponding restriction sites. Synthesis program: 94 ° С - 1.5 min, 94 ° С - 15 sec, 57 ° С - 20 sec, 72 ° С - 15 sec, 72 ° С - 10 min, number of cycles: 30. The obtained DNA fragment was isolated from gel using Qiagen reagent.

This fragment was cloned into the pGEM-T Easy (Promega) proprietary vector according to the protocol. The resulting construct was verified by restriction analysis (Fig. 8) followed by sequencing with primers M13R and M13F.

The neurotrophic factor GDNF gene was excised from this construct at the EcoRV-BamHI restriction sites (600bp) and inserted into a plasmid containing the hsp70 Drosophila promoter and, in one case, a regulatory zone with 4 HSE, in the other, 8 HSE, at SmaI / BamHI restriction sites. The presence of an insert in these constructs was verified by restriction analysis at various sites, including PCR cloning sites with primers gdnf-F (5'-ggaatcggcaggctgcagctg-3 ') and gfp-R (5'-aataaagcttgcatggcggtaatacg-3') .

Sequencing was also performed using gdnf-F primers (5'-ggaatcggcaggctgcagctg-3 ') and gdnf-R (5'-aatgctttcttagaatatggt-3') (Fig. 9)

The genetic engineering design is shown in Fig. 10.

After isolation and purification, these constructs were introduced into the HEK293 cell culture by transfection using ExGene 500 reagent (Fermentas, R0511).

Transfection was performed according to the protocol. The HEK293 cell culture was incubated at 37 ° C in a CO ₂ incubator. After selection on gentamicin for 10 days, transfected cells were scattered in 25 cm ² culture bottles. Next, it was necessary to select, under a fluorescence microscope, colonies of cells that expressed GDNF labeled with green fluorescent GFP protein. Active luminescence of cells was observed. However, we checked the presence of our construct in the cells. For this, RNA was isolated from this cell culture, for subsequent analysis using RT-PCR and Western blot.

For RT-PCR, cells were lysed with trisol according to the protocol, cDNA was synthesized using reverse revertase. The synthesized cDNA was used for PCR reactions with various primers: GAPDH R (5'-cccctggccaaggtcatccatgacaactt-3 ') GAPDH F (5'-ggccatgaggtccaccaccctgttgctgta-3'); gdnf-F (5'-ggaatcggcaggctgcagctg-3 ') and GDNF-BamHI (5'-tggatcccagatacatccacaccttttagcgg-3'). A Western blot hybridization with a transfected cell culture was also performed.

To obtain a transgenic cell culture, a neomycin resistance gene was introduced into the construct, which allows selection of transfected cells on G418.

Transfected HEK293 cells were put on line for neomycin resistance. The presence of the inserted gdnf and gfp genes and the expression of the protein in the transgenic culture derived was confirmed by sequence and Western blot hybridization. Protein was isolated from cell culture in SDS buffer according to the protocol (Amersham Bioscience). Further, these proteins were dispersed in a 10% acrylamide gel and analyzed using Western blot hybridization. A nitrocellulose membrane (Amersham Bioscience, # RPN68D) was used for transfer. This membrane was treated with antibodies: Rabbit polyclonal anti-GFP (Abeam #ab 290, 1: 2000) and secondary anti-Rabbit conjugated to horseradish peroxidase (HRP, 1: 2000). Proteins were detected using ECL reagent (Amersham Biocsience, # RPN2109) for peroxidase. The presence of the construct in the cells is proved.

It was found that a green glow of the cell culture was observed after exposure to temperature. After exposure to temperature, the studied cells were fixed in a 4% paraform solution and used to measure the luminescence intensity using flow cytofluorimetry on a FACScan instrument (Becton Dickinson, USA). The data were analyzed for two temperatures of 40 and 42 ° C with different intervals after temperature exposure. The presence of eight heat shock elements (HSE) in the regulated region of the hsp70 heat shock protein gene promoter affects the changes in the activation of this promoter.

The presence of eight heat shock elements (HSE) in the regulated region of the hsp70 heat shock protein gene promoter affects changes in the activation of GDNF expression. In the resulting construct, there is a more intense accumulation of luminous cells in a transgenic culture. So, for example, when a temperature of 40 ° C was applied to the cell culture after 24 hours, 43% of luminous cells were detected, and this percentage increased after 48 hours to 51%, in contrast to the stimulation of the promoter in B7 / gdnf-gfp. When exposed to a temperature of 42 ° C, there is a decrease in the percentage of luminous cells (32%) compared with the percentage of luminous cells when exposed to 40 ° C, but their number increases sharply after 48 hours (53%). A study using cytofluorimetry of the expression of this promoter with eight regulatory sequences (HSE) at lower temperatures showed that the expression of GDNF and the marker gene is already observed at 38 ° C (12%) and increased with increasing temperature. Thus, it can be argued that an increase in heat shock elements (up to eight) in the regulatory zone of the Drosophila hsp70 heat shock protein gene promoter promotes the sensitivity of this promoter to a temperature increase of even 2 ° C (up to 38 ° C) and enhances expression when exposed to an increased threshold temperature in the range of 38 ° C-42 ° C.

After 72 hours, a decrease in GFP protein synthesis is observed. However, upon repeated heating, secondary activation of protein synthesis is observed. Also, 1/3 of the cells were used to isolate total DNA according to the protocol. The resulting total DNA was examined by PCR analysis for the presence of the GFP gene in the HEK293 cell line. For the PCR method, a BioRad amplifier was used, primers: gfpF (5'-cgtcagatccgctagcgctaccgg-3 ') and gfpR (5'-aataaagcttgcatggcggtaatacg-3').

It was found that the optimal duration of temperature activation is 60 minutes, and the maximum luminescence of the green fluorescent GFP protein is observed 48 hours after exposure to temperature. The effect of the re-inclusion of the promoter and the occurrence of luminescence of green fluorescent protein after repeated exposure to temperature was revealed.

To assess the ability of HEK293 transgenic cells expressing gdnf / gfp to survive inside the nerve tissue, they were injected into the brain in black6 mice. Transgenic HEK293 cells (passage 2) were stimulated by heat shock at a temperature of 42 ° C for 60 minutes in DMEM, 10% EBS. Non-transgenic HEK293 cells were used as a control. 1 × 10 ⁶ GFP-labeled cells in 2 μl of Hanks solution were locally injected into the striatum of mice. At the end of the experiment (after 24 hours, 3 days and 5 days), the mice were perfused with 4% paraformaldehyde solution, the brain was removed, and after additional fixation and impregnation in a 30% sucrose solution, they were frozen at -20 ° C. Then sections were prepared with a thickness of 40 μm. The survival of transplanted HEK293 was evaluated under a microscope by the green fluorescence of the GFP protein they expressed. (Fig. 11 and 12)

In the course of studies, it was found that transplanted transgenic cells behave differently from cell culture. Active expression was observed after 24 hours and gradually decreased over three days. Apparently, a longer activity of the promoter is associated with inflammatory processes during transplantation. This result is extremely attractive, since the activity of the gdnf gene is needed precisely for engraftment of the graft and, as a result, reduction of the glial scar during engraftment.

It should be noted that an increase in the regulatory zone of the promoter affected the activation of the promoter and the duration of this activity during transplantation. Moreover, these properties during transplantation differed from the potential of the promoter in the culture. It was found that when using transgenic cells containing F7 / gdnf-gfp, the luminescence efficiency after 24 hours is reduced compared to transgenic cells containing B7 / gdnf-gfp. However, this significantly increases the time of activity of the promoter. So, we observed single luminous cells after five days. It is assumed that this is associated with an increase in HSE zones (from 4 to 8) in the regulatory region of the promoter. For further studies, F7 / gdnf-gfp was used. (Fig. 13-15)

Example 3. The effect of temperature-sensitive expression of GDNF on the morphology of transgenic cells

The Western blot analysis method confirmed the synthesis of the transgenic factor in transfected HEK293 cells after exposure to hitshock temperature. Thus, it can be argued that we obtained a transgenic cell culture that regulated GDNF expression. Further, the method of Western blot analysis was used to study the secretion of the factor from cells into the medium. For this, transgenic cells were exposed to elevated temperature (42 ° C), and after culturing the transgenic cells for two days, the conditioned medium was taken from the plates. Then a protein was isolated from the conditioned medium and analyzed for the presence of GDNF. The results confirmed the secretion of GDNF from cells to the medium (Fig. 16).

During immunohistochemical analysis, it was found that transgenic HEK293 cells did not acquire neural characteristics without exposure to temperature shock (42 ° C), which is attractive enough for transgenic culture.

A study of the transgenic HEK293 culture revealed a significant change in the phenotypic profile of cells after exposure to elevated temperature, stimulating the expression of GDNF. Cells form multiple processes (Fig. 17) and, upon subsequent growth, assemble into spherical structures (Fig. 18). It should be noted that such an effect was not observed under the influence of temperature on the control HEK293 (transgenic for the gfp gene).

It cannot be argued that these acquired properties are associated with the neural differentiation of transgenic cells, since no change in the expression intensity of markers characteristic of neural cells was detected. However, it can be argued that confirmed GDNF expression phenotypically changes the transgenic cell itself. To confirm the effectiveness of the transgenic factor, preliminary experiments were carried out to enhance the expression of GDNF during transplantation. The obtained transgenic HEK293 cultures expressing the chimeric GDNF-GFP protein were transplanted into the mouse striatum. After 7 days, a synthesis of green fluorescent protein in the transplantation zone was detected on sections of the brain. In the study of another group of mice, it was shown that after 10 days the green glow of the transplanted cells attenuated, which is possibly associated with a decrease in the inflammatory process in the transplantation zone.

Example 4. The influence of the conditioned medium from transgenic hsp70 / gdnf-gfp cells on the formation of nerve processes and cell motility in the rat retina

Isolated rat eyes were washed in 70% ethanol and in two shifts of sterile phosphate buffer (DPBS; Sigma). The retina was isolated in chilled DPBS containing 0.8% glucose (G1 - DPBS). The isolated retina was crushed into fragments of 1-2 mm ² in size, the explants were washed with a solution of antibiotics and placed in a culture dish with a complete nutrient medium (DMEM / F12 supplemented with cytokines, antibiotics and 5% serum).

The explants were cultured in attached form in a Petri dish with a diameter of 30 mm.

Cultivation was carried out in an incubator in a humid chamber at a temperature of + 37 ° C and a carbon dioxide content of 5% in a culture medium - DMEM / F12 (1: 1) with the addition of cytokines, antibiotics and 5% fetal calf serum. The change of environment was performed every 2-3 days.

Then the culture medium was changed to a conditioned medium taken from cultured HEK293 / hsp70 / gdnf-gfp transgenic cells. (It should be noted that there was no difference in the use of conditioned media from cultures of transgenic HEK293 / F7 / gdnf-gfp and HEK293 / B7 / gdnf-gfp).

Cultivation was carried out for 21 days, then the samples were fixed and stored in a buffered antifreeze solution until stained with antibodies.

During the experiment, the state of the explant and the behavior of the cells were monitored daily using an inverted phase contrast microscope. For a quantitative assessment of the growth of cultured explants, an area index was used - the ratio of the explant area together with the zone of evicted cells to the original explant area.

At the end of the experiment, the explants were washed with phosphate buffer and fixed with a 4% solution of paraformaldehyde in physiological saline with the addition of 0.01 molar phosphate buffer at pH 7.4 for 20 minutes at 4 ° C. After washing the fixed attached explants in phosphate buffer, the bottom of the culture plate was dried and the explants were surrounded by hydrophobic strips that evicted the cells, and then immunohistochemical staining was performed. For this, we used the method of indirect immunohistochemical staining using a cocktail of monoclonal mouse antibodies to the marker of committed neuroblasts - β-III tubulin at a 1: 100 dilution (Chemicon, MAB1637) and polyclonal rabbit antibodies to the glial cell marker - acid glial fibrillar protein - GFAP in dilution 1 : 80 (Sigma, G9269). The reaction with primary antibodies was carried out for 12 hours at 4 ° C. After washing in phosphate buffer, the explants were coated with a mixture of secondary antibodies to rabbit immunoglobulin labeled with Texas red (red glow in green light) and antibodies to mouse immunoglobulin labeled with Su 2 (green glow in blue light) both at a 1: 100 dilution.

During the first 5-7 days of cultivation, subsidence and spreading of human retinal explants at the bottom of the culture plate occurred. A small number of cells evicted from the explant to the bottom were also observed. When examined in phase contrast at this stage, retinal explants during cultivation with a conditioned medium from HEK293 / hsp70 / gdnf-gfp and without it did not differ in appearance and in the number of cells evicted to the bottom. On the 14th day of cultivation, the cells are further evicted from the explant to the bottom of the culture plate and settled over a significant area. With an explant diameter of 2 mm, the cells settled at a distance of more than 2 mm from the initial external borders of the explant. In explants cultivated with HEK293 / hsp70 / gdnf-gfp conditioned medium, numerous long branched processes begin to form, extending beyond the explants and sometimes reaching the explants attached in the neighborhood. In the control of the processes is not observed. On the 17th and especially on the 21st day of cultivation, the eviction of cells from the explant to the bottom of the cup continues. In explants cultivated with HEK293 / hsp70 / gdnf-gfp conditioned medium, there is a further increase in the number of processes, their length and branching. In explants cultivated with HEK293 / hsp70 / gdnf-gfp conditioned medium, a small number of short processes appear. (Fig. 19 and 20)

Example 5. The introduction of transgenic HEK293 / hsp70 / gdnf-gfp cells into the brain of mice

In order to analyze the ability of HEK293 transfected with the gdnf gene (also expressing the GFP marker protein) to affect the brain cells of the recipient (mouse), these cells were injected into the cortex and striatum of Black6 mice that do not express the GFP protein. To compare the properties of transfected cells with non-transfected ones, experiments were also carried out with injections into the brain of Black6 mice not expressing the GFP protein of control HEK293. The operation to introduce ET-1. The animal was anesthetized with chloral hydrate and placed in stereotaxis, and after a longitudinal section of the scalp opened access to the skull. A hole in the skull with a diameter of 1 mm is made according to the coordinates AP0 L2.5 on the right side. An anesthetized animal is placed in stereotaxis. Using a microsyringe placed in a microfeed device and connected by a capillary tube to a needle fixed in the ET-1 stereotaxis in the amount of 1 microgram in a volume of 1 microliter, it is introduced through the aforementioned hole to a depth of 3 mm. The injection was carried out for 10 minutes while gradually raising the tip of the needle from a depth of 3 mm to a depth of 1 mm. Thus, endothelin was introduced into the caudate nucleus-shell complex and into the sensorimotor region of the mouse cortex.

Introduction of control and transfected HEK293 / hsp70 / gdnf-gfp cells into the ischemic brain. At the end of the ET-1 injection, the injection needle was removed and a cell suspension needle was immersed in its place. About 200-300 thousand cells were injected in 2 μl of Hanks solution. The injection was carried out for 4 minutes while gradually raising the tip of the needle from a depth of 2.5 mm to a depth of 0.7 mm.

3 series of experiments were carried out with the introduction of transfected HEK293 / hsp70 / gdnf-gfp and control HEK293.

Histological examination

10 days after the injection, the mice were euthanized with a lethal dose of urethane. The circulatory system was washed first with a solution of phosphate buffer (pH 7.4) in physiological saline (PBS), and then with a fixing solution: 4% solution of paraformaldehyde in PBS. The brain so fixed was removed from the cranium and immersed in the fixative solution for 5-6 hours, after which the brain was soaked in 30% sucrose in PBS for 12 hours at 4 ° C. Subsequently, the brain is frozen and cut on a freezing microtome. Slices with a thickness of 40 micrometers, containing brain regions subject to ischemia, are mounted on a glass slide and, after drying, are stained according to Giemsa. For immunohistochemical staining, a technique for staining free-floating sections was used. The glial acidic fibrillar protein was stained (anti-GFAP from Sigma, USA, diluted 1: 100 in pH 7.4 phosphate buffer at 4 ° C for 15 hours) to evaluate the accumulation of glial cells surrounding the graft. To assess possible changes in the phenotype of the transplanted cells, antibodies to the neural marker, bIII tubulin isoform, were also used.

Primary antibody-treated brain sections were washed three times in phosphate buffer (pH 7.4) and immersed in a solution of secondary donkey antibodies against rabbit or goat immunoglobulin (respectively, primary antibodies) conjugated with Texas Red fluorescent dye (Texas Red Jackson ImmunoResearch Laboratories, Inc. United Kingdom) at a dilution of 1: 100.

Sections were examined and photographed using an Olympus CX 41 fluorescence microscope (Japan) equipped with a fluorescent nozzle and a digital camera. To quantify reactive gliosis around the cell injection site, sections immunohistochemically stained with GFAP were examined using a Leica confocal microscope (Germany).

HEK293 transfected with the gdnf gene, as well as HEK293 non-transfected survive in the brain of mice after transplantation for 5 days. At least some of the HEK293 transfected with the gdnf gene does not remain at the injection site, but migrates into the surrounding brain tissue ischemic by ET-1 administration, while the control HEK293 remains at the injection site.

A study of drugs stained with an activated glia marker GFAP showed that the introduction of geknf transfected with gdnf gene into the ischemic lesion in the brain causes much less intense gliosis compared to the control HEK293.

In the GFAP control, immunopositive structural elements — reactive astrocytes and their processes adhere closely to the localization site of control injected cells, forming a dense glial scar around it. Around the injection site of HEK293 transfected with gdnf, the density of positive elements was much lower. At the same time, a small number of GFAP profiles are present in the immediate vicinity of the localization site. Differences in means between the two groups of animals are reliable by the criterion and the nonparametric Mann-Whitney criterion. (Fig. 21)

Example 6. The effect of the conditioned medium from transgenic cells on the appearance of neural markers in human gastrointestinal tract.

Conditioned medium was collected from the obtained transgenic cell cultures, in which the secretion of the factor was confirmed by the methods described above. Then, the effect of secreted factor on the stimulation of neural differentiation on stromal cells of mammalian adipose tissue (humans) was investigated. It was found that when the conditioned medium with GDNF was added to the SCLC culture, the expression of the glial marker GFAP sharply increased (Fig. 22). An increase in the expression of neuronal markers such as: nestin (Fig. 23), NeuN (Fig. 24) and βIII-tubulin (Fig. 25) was also found. These results do not indicate the stimulation of neural differentiation in gastrointestinal tract, but it can be argued that the transgenic factor affects the change in cell status and stimulates the appearance of neural markers. (Fig. 22-25)

Thus, it was found that temperature-regulated GDNF shifts the differentiation of transgenic cells in the neural direction. This result looks quite attractive if you use an adjustable promoter with the introduction of a neurotrophic factor. During transplantations, it will be possible to activate the neurotrophic factor directly in the brain and stimulate the differentiation of the transplant in the neural direction.

Example 7. The effect of transgenic cells on immature (embryonic) cells of dopaminergic neurons.

To obtain a culture of dopaminergic neurons, 13-day-old rat embryos were used. Rats were decapitated, sterilized in a 70% alcohol solution. Cells were obtained from the substantia nigra black substantia nigra.

Conducted dissection. Under a binocular microscope, the skull was opened under sterile conditions and the brain was isolated. The black substance of the midbrain (substantia nigra) was separated from other brain structures, the membranes and blood vessels were removed, cut into small pieces.

Next, trypsinization was performed. The tissue was transferred to a 15 ml tube and incubated for 5-1 min in a 0.05% trypsin-EDTA solution (37 ° C, 5% CO ₂ ). Trypsinization was stopped with a similar amount of DMEM / F12 medium with 3% fetal bovine serum.

After that, the tissue was washed with HBSS and dissociated by repeated pipetting in a nutrient medium. The cell suspension was centrifuged for 3-8 minutes depending on the volume of suspension obtained at 1000 rpm, the pellet was resuspended in 1 ml of culture medium. Then repeated centrifugation was performed. The number and viability of dissociated cells were determined in the Goryaev chamber in 100 μl of cell suspension after staining with 0.1% trypan blue.

Then, a suspension of cells with a final concentration of 200,000 cells / well was cultured in 4-well plates on a polyethyleneimine substrate (1 mg / ml, incubated for 30 minutes, washing with distilled water 3-4 times for 10 minutes) for 10 days (5% CO ₂ , 37 ° C) The environment changed every 2-3 days.

To identify the culture, a 4% solution of paraformaldehyde was fixed in phosphate buffer for 30 min at 4 ° C.

Next, immunohistochemical analysis was performed. (Fig. 26)

It was found that temperature-regulated GDNF stimulates significantly more intensive production of tyrosine hydroxylase in brain cells of rat embryo than recombinant GDNF. This can be explained by the more powerful inclusion of the hsp70 promoter in a short time.

Information sources

1. Åkerud, P., Alberch, L, Eketjäll, S., Wagner, J. & Arenas, E. Differential effects of glial cell line-derived neurotrophic factor and neurturin on developing and adult substantia nigra dopaminergic neurons. J. Neurochem. 73 (1999). pp. 70-78.

2. Åkerud, P., Canals, J.M., Snyder, E.Y. & Arenas, E. Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease. J. Neurosci. 21 (2001). pp. 8108-8118.

3. Batchelor, P.E., Liberatore, G.T., Porritt, M.J., Donnan, G.A. & Howells, D.W. Inhibition of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression reduces dopaminergic sprouting in the injured striatum. Eur. J. Neurosci. 12 (2000). pp. 3462-3468.

4. Björklund, A. et al. Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model. Brain Res. 886 (2000). pp. 82-98.

5. Boucher, T.J. et al. Potent analgesic effects of GDNF in neuropathic pain states. Science 290 (2000). pp. 124-127.

6. Granholm, AC, Reyland, M., Albeck, D., Sanders, L., Gerhardt, G., Hoernig, G., Shen, L., Westphal, H., Hoffer, B. Glial cell line-derived neurotrophic factor is essential for postnatal survival of midbrain dopamine neurons. J, Neurosci. 20 (2000). pp. 3182-3190.

7. Grondin, R. & Gash, D.M. Glial cell line-derived neurotrophic factor (Gdnf) - a drug candidate for the treatment of Parkinson's disease. J. Neurol. 245 (1998). pp. 35-42.

8. Keller-Peck, C.R., Feng, G., Sanes, J.R., Yan, Q., Lichtman, J.W., Snider, W.D. Glial cell line-derived neurotrophic factor administration in postnatal life results in motor unit enlargement and continuous synaptic remodeling at the neuromuscular junction. J. Neurosci. 21 (2001). pp. 6136-6146.

9. Kordower, J.H. et al. Clinicopathological findings of the following intraventricular glial-derived neurotrophic factor treatment in a patient with Parkinson's disease. Ann. Neurol. 46 (1999). pp. 419-424.

10. Kordower, J.H. et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 290 (2000). pp. 767-773.

11. Li. L. & Zhou, X.F. Pericellular Griffonia simplicifolia I isolectin B4-binding ring structures in the dorsal root ganglia following peripheral nerve injury in rats. J. Comp. Neurol. 439 (2001). pp. 259-274.

12. Meng, X. et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287 (2000). pp. 1489-1493.

13. Nicole, O. et al. Neuroprotection mediated by glial cell linederived neurotrophic factor: involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway. J. Neurosci. 21 (2001). pp.3024-3033.

14. Ramer, M.S., Priestley, J.V. & McMahon, S.B. Functional regeneration of sensory axons into the adult spinal cord. Nature 403 (2000). pp. 312-316.

15. Zhou, X.F., Deng, Y.S., Xian, C.J. & Zhong, J.H. Neurotrophins from dorsal root ganglia trigger allodynia after spinal nerve injury in rats. Eur. J. Neurosci. 12 (2000). pp. 100-105.

16. Zwick, M., Teng, L., Mu, X., Springer, J.E. & Davis, B.M. Overexpression of GDNF induces and maintains hyperinnervation of muscle fibers and multiple end-plate formation. Exp. Neurol. 171 (2001). pp. 342-350.

17. Zurn, A.D., Widmer, H.R. & Aebischer, P. Sustained delivery of GDNF: towards a treatment for Parkinson's disease. Brain Res. Brain Res. Rev. 36 (2001). pp. 222-229.

Claims (1)

 A genetic construct for the expression of GDNF in cells and tissues of mammals (including humans) at an increase in temperature, constructed on the basis of the vector plasmid pEGFP-N1 with the neomycin resistance gene containing a DNA fragment encoding the regulatory sequence of the promoter of the heat shock protein gene hsp70 Drosophila melanogaster with heat shock elements HSE 4-8, the human neurotrophic factor GDNF gene, the green fluorescent protein (GFP) gene, which are under the control of this promoter, and the promoter can be activated by Under the influence of mammalian heat shock temperature or toxic effects, and regulate the intensity of GDNF gene expression due to the presence of heat shock elements HSE 4-8.

Images