JP2006506086A - Methods, nucleic acid constructs and cells for treating neurodegenerative disorders - Google Patents

Abstract

A method of curing a neurodegenerative disorder is provided. The method is performed by administering to an individual in need thereof a cell capable of exogenously regulatable synthesis of a neurotransmitter, thereby treating a neurodegenerative disorder.

Description

  The present invention relates to neuron-like cells capable of synthesis capable of controlling neurotransmitters, and cell replacement therapy using cells to treat neurodegenerative disorders such as Parkinson's disease.

  Parkinson's disease is an aging characterized by progressive loss of dopaminergic neurons in the substantia nigra, which eventually results in a progressive loss of motor function that manifests through symptoms such as tremor, rigidity and ataxia. Related obstacles. Parkinson's disease can be treated by administering a pharmacological dose of the dopamine precursor L-DOPA (Marsden, Trends Neurosci., 9: 512, 1986; Vinken et al., Handbook of Clinical Neurology, page 185, Elsevier Amrsterdam, 1986). Although such treatment is effective in early stage Parkinson's disease patients, the progressive disappearance of substantia nigra cells may ultimately cause the remaining cells to synthesize sufficient dopamine from the administered precursor. It can not be done and the medicinal effect is reduced.

  In studies of neurodegenerative diseases, symptoms that occur in people suffering from illness are secondary to failures in local neural circuits and cannot be effectively treated using systemic drug delivery. It is suggested. As a result, alternative methods for treating neurodegenerative diseases have emerged, such as transplantation of cells that can replace or supplement the function of damaged neurons. In order for such a cell replacement therapy to work, the transplanted cells must survive in the damaged tissue and be integrated both functionally and structurally.

  Parkinson's disease is the first brain disease for which intracerebral cell replacement therapy has been used in humans. Several attempts have been made to provide the neurotransmitter dopamine to the affected brainstem ganglia in Parkinson's disease patients by allografting adrenal medullary cells into the patient's brain (Backlund et al., J. Biol. Neurosurg., 62: 169-173, 1985; Madrazo et al., New Eng. J. Med., 316: 831-836, 1987). Transplantation of other donor cells, such as fetal brain cells from the substantia nigra, which is a region of the brain rich in cell bodies containing dopamine and also the most affected brain region in Parkinson's disease, It has been shown to be partially effective in reversing behavioral defects induced by selective dopaminergic neurotoxins (Bjorkland et al., Ann. NY Acad. Sci., 457: 53-81, 1986; Dunnett et al., Trends Neurosci., 6: 266-270, 1983).

  Several cell replacement studies utilizing various non-neuronal cell types from different sources have also been conducted over the last few years. In animal models of Parkinson's disease, researchers have transplanted cells such as monocytes, bone marrow stem cells, myoblasts, fibroblasts, astrocytes and Sertoli cells (Costantin et al., 2000; Hwan-Wun et al. 1999; Linder et al., 1995; Patredge & Davies, 1995; Perry & Gordon, 1998; Yadid et al., 1999). In other studies, cells are transplanted after being genetically engineered with growth factor genes (eg, glial and brain-derived growth factors) to increase viability, or tyrosine hydroxylase, aromatic amino acid residues. Transplanted after being genetically engineered with a gene such as carboxylase or GTP cyclohydrolase I, which can increase dopamine synthesis in transformed cells (Choi-Lundberg et al., 1998; Yoshimoto et al., 1995) Schwarz et al., 1999). However, these cells were not able to fully acquire the structural and functional characteristics of damaged neuronal cells and were therefore found to be therapeutically ineffective (Brundin et al., 2000).

  Clinical cell replacement tests for patients with Parkinson's disease have been performed using fetal cells containing only 1-2% dopaminergic neurons (Freeed et al., 1992; Freeed et al., 2001; Freeman et al., 1995; Kordwer et al., 1995; Kordwer et al., 1998; Lindvall O., 1991; Wenning et al., 1997). Freed et al. (2001) found that transplantation of fetal cells to Parkinson's disease patients was beneficial only to young patients (under 60 years). In addition, some patients suffered from severe dyskinesia (“runaway dyskinesia” in the absence of levodopa treatment) due to excessive and uncontrolled production and release of dopamine by transplanted cells. (Freed et al., 2001; Olanow et al., 2003). In addition, the low availability of human fetal tissue substantially limits the number of patients that can benefit from fetal cell transplantation.

  Adult bone marrow stromal cells (BMSc) are non-hematopoietic cells and various neuronal markers (Azizi et al., 1998; Deng et al., 2001; Kopen et al., 1999; Sanchez-Ramos et al., 2000; Schwartz et al., 1999; Woodbury Et al., 2000), can differentiate into neuron-like cells that exhibit electrophysiological functions (Kohyama et al., 2001) and in vivo expression of tyrosine hydroxylase (Schwarz et al., 1999). Moreover, transplantation of BMSc in Parkinson's disease mouse and rat models has had beneficial effects (Li et al., 2001; Schwartz et al., 1999).

  Adult BMSc can differentiate into neuron-like cells that are structurally compatible with transplantation, but engrafted BMSc may uncontrollably release neurotransmitters such as dopamine, which is Severe side effects such as “dyskinesia” may occur over time, thus making the use of BMSc unsuitable for the treatment of neurodegenerative disorders.

  Therefore, it can be integrated into damaged neuronal tissue, and it is further possible to controllably synthesize neurotransmitters such as dopamine, and thus to effectively and safely treat neurodegenerative disorders. It is widely recognized that there is a need for neuron-like cells that can be utilized for, and it would be very advantageous to have such neuron-like cells.

  According to one aspect of the invention, administering to an individual in need thereof a cell capable of exogenously regulatable synthesis of a neurotransmitter, thereby treating a neurodegenerative disorder. A method of treating a neurodegenerative disorder is provided.

  According to another aspect of the invention, (a) administering a cell capable of exogenously regulatable synthesis of a neurotransmitter to an individual in need thereof, and (b) neurotransmission in the cell. Provided is a method of treating a neurodegenerative disorder comprising the step of periodically exposing an individual to an agent or condition capable of modulating the synthesis of the substance, thereby treating the neurodegenerative disorder.

  According to yet another aspect of the invention, a polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis is placed under the control of a regulatory sequence capable of regulating the expression of that enzyme in mammalian cells. Nucleic acid constructs comprising are provided.

  According to yet another aspect of the invention, a first expression construct comprising a first polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis disposed under transcriptional control of a first regulatory sequence; And a second expression construct comprising a second polynucleotide sequence encoding a transactivator disposed under transcriptional control of a second regulatory sequence, wherein a transactivator is provided. Can activate the first regulatory sequence to effect transcription of the first polynucleotide sequence.

  According to a further aspect of the invention, a nucleic acid construct comprising a polynucleotide sequence encoding an enzyme involved in the synthesis of a neurotransmitter, disposed under the control of a regulatory sequence capable of regulating the expression of that enzyme in a cell. Are provided.

  According to yet a further aspect of the invention, a first expression construct comprising a first polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis disposed under transcriptional control of a first regulatory sequence; There is provided a cell comprising a construct system comprising a second expression construct comprising a second polynucleotide sequence encoding a transactivator disposed under transcriptional control of a second regulatory sequence. In this case, the transactivator can activate the first regulatory sequence to cause transcription of the first polynucleotide sequence.

  In accordance with yet a further aspect of the invention, there are provided methods of making cells for use in treating a neurodegenerative disorder. The method comprises: (i) isolating bone marrow cells; (ii) incubating bone marrow cells in a growth medium capable of maintaining and / or expanding bone marrow cells; (iii) obtained from step (ii) Selecting bone marrow stromal cells from the resulting cells; and (iv) incubating the bone marrow stromal cells in a differentiation medium comprising at least one polyunsaturated fatty acid and at least one differentiation agent, whereby neurodegeneration Producing a cell for use in treating the disorder.

  According to yet a further aspect of the invention, there is provided a cell population comprising bone marrow derived stromal cells capable of synthesizing neurotransmitters.

  According to yet a further aspect of the invention, there is provided a mixed cell population comprising bone marrow derived stromal cells capable of synthesizing at least two types of neurotransmitters.

  According to further features in preferred embodiments of the invention described below, the method of treating a neurodegenerative disorder comprises subjecting an individual to an agent or condition capable of modulating neurotransmitter synthesis in a cell. In addition.

  According to still further features in the described preferred embodiments, the cells are genetically modified to allow exogenously regulatable synthesis of neurotransmitters.

  According to still further features in the described preferred embodiments, the cell is transformed with an expression construct comprising a polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis. In this case, the expression construct is designed such that the expression of the polynucleotide can be controlled by the agent.

  According to still further features in the described preferred embodiments, the agent is capable of down-regulating the expression of enzymes involved in neurotransmitter synthesis.

  According to still further features in the described preferred embodiments the agent is capable of upregulating the expression of enzymes involved in neurotransmitter synthesis.

  According to still further features in the described preferred embodiments the cell comprises a first polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis, placed under transcriptional control of the first regulatory sequence; , And at least one expression construct comprising a second polynucleotide sequence encoding a transactivator, placed under transcriptional control of a second regulatory sequence. In this case, the transactivator can activate the first regulatory sequence to cause transcription of the first polynucleotide sequence in the absence of the agent.

  According to still further features in the described preferred embodiments the agent is doxycycline.

  According to still further features in the described preferred embodiments the transactivator is a tetracycline-controlled transactivator.

  According to still further features in the described preferred embodiments the first regulatory sequence comprises a tetracycline response element.

  According to still further features in the described preferred embodiments the enzyme is from tyrosine hydroxylase, DOPA decarboxylase, GTP cyclohydrolase I, dopamine β-hydroxylase, glutamate decarboxylase, tryptophan-5 monooxygenase and choline acetyltransferase. Selected from the group consisting of

  According to still further features in the described preferred embodiments the second regulatory sequence comprises a human neuron specific enolase promoter.

  According to still further features in the described preferred embodiments the neurodegenerative disorder comprises Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, autoimmune encephalomyelitis, Alzheimer's disease and Huntington's disease Selected from the group.

  According to still further features in the described preferred embodiments the neurodegenerative disorder is Parkinson's disease.

  According to still further features in the described preferred embodiments the neurotransmitter is selected from the group consisting of dopamine, norepinephrine, epinephrine, γ-aminobutyric acid, serotonin, acetylcholine and glutamic acid.

  According to still further features in the described preferred embodiments the neurotransmitter is dopamine.

  According to still further features in the described preferred embodiments, the cell is a bone marrow cell.

  According to still further features in the described preferred embodiments, the bone marrow cells are bone marrow stromal cells.

  According to still further features in the described preferred embodiments, the cell is a neuron-like cell.

  According to still further features in the described preferred embodiments, the neuron-like cell expresses at least one neuronal marker.

  According to still further features in the described preferred embodiments, the neuronal marker is CD90, neuron specific nuclear protein, neurofilament heavy chain, neuron specific enolase, β-tubulin 3, tyrosine hydroxylase, microtubule associated protein 2 (MAP-2), nestin and calbindin.

  According to still further features in the described preferred embodiments, administering the cells is accomplished by transplanting the cells into the brain tissue of the individual.

  According to still further features in the described preferred embodiments, administering the cells is accomplished by transplanting the cells into the spinal cord of the individual.

  According to still further features in the described preferred embodiments, exposing the individual is accomplished by orally administering the agent to the individual.

  According to still further features in the described preferred embodiments, exposing the individual is accomplished by injecting the agent into the individual.

  According to still further features in the described preferred embodiments, the cells are genetically modified to allow exogenous regulatable synthesis of neurotransmitters.

  According to still further features in the described preferred embodiments, the cell is transformed with an expression construct comprising a polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis. In this case, the expression construct is designed such that the expression of the polynucleotide can be controlled by the regulatory agent.

  According to still further features in the described preferred embodiments, the agent is capable of down-regulating the expression of enzymes involved in neurotransmitter synthesis.

  According to still further features in the described preferred embodiments the agent is capable of upregulating the expression of enzymes involved in neurotransmitter synthesis.

  According to still further features in the described preferred embodiments, the cell is such that, in the absence of the agent, the activator molecule binds to the response element and thereby upregulates expression of tyrosine hydroxylase. Genetically modified to express tyrosine hydroxylase under regulatable control.

  According to still further features in the described preferred embodiments, the cell is a neuron-like cell that does not have the endogenous activity of enzymes involved in neurotransmitter synthesis.

  According to still further features in the described preferred embodiments, the cell further comprises a polynucleotide encoding an apoptosis-inhibiting polypeptide.

  According to still further features in the described preferred embodiments the growth medium comprises DMEM, SPN, L-glutamine, FCS, 2-β-mercaptoethanol, non-essential amino acids and EGF.

  According to still further features in the described preferred embodiments, the method of making a cell for treating a neurodegenerative disorder comprises, prior to step (iv), using the cell obtained from step (iii) for pre-differentiation. Incubating in the medium, thereby facilitating the differentiation of the cells into neuron-like cells.

  According to still further features in the described preferred embodiments the pre-differentiation medium comprises bFGF.

  According to still further features in the described preferred embodiments the pre-differentiation medium further comprises DMEM, SPN, L-glutamine, N2 supplement and FCS.

  According to still further features in the described preferred embodiments the at least one polyunsaturated fatty acid is docosahexaenoic acid.

  According to still further features in the described preferred embodiments the at least one differentiation agent is selected from the group consisting of BHA, dbcAMP and IBMX.

  According to still further features in the described preferred embodiments the differentiation medium further comprises DMEM, SPN, L-glutamine, N2 supplement and retinoic acid.

  According to still further features in the described preferred embodiments, the method of making a cell for treating a neurodegenerative disorder further comprises prior to step (iv) the cell obtained from step (iii) according to the present invention. This is accomplished by transformation with the nucleic acid construct of

  According to still further features in the described preferred embodiments, step (i) is accomplished by aspiration.

  According to still further features in the described preferred embodiments, step (iii) is accomplished by collecting surface adherent cells.

  According to still further features in the described preferred embodiments the neurotransmitter is dopamine.

  According to still further features in the described preferred embodiments the at least two types of neurotransmitters include dopamine.

  According to still further features in the described preferred embodiments the at least two types of neurotransmitters include serotonin.

  The present invention relates to neuron-like cells capable of controllable synthesis of neurotransmitters, and neuron-like cells using such cells to treat neurodegenerative disorders such as Parkinson's disease. Has been successful in addressing the shortcomings of currently known methods of treating neurodegenerative diseases.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The present invention is described herein by way of example only and with reference to the accompanying drawings. Referring now in detail to the drawings, the items shown are by way of example only and are for illustrative discussion of the preferred embodiments of the invention, and thus the principles of the invention It is emphasized that it has been presented to provide what is considered to be the most useful and readily understood description of conceptual aspects. In this regard, it will be apparent to those skilled in the art, upon understanding the description in conjunction with the drawings, that some forms of the invention may actually be embodied, so that the structural details of the invention can be No attempt has been made to show in more detail than is necessary for a basic understanding of.
BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-C illustrate light micrographs of undifferentiated human bone marrow stromal cells (hBMSc) (FIGS. 1A-1C). hBMSc was cultured in “growth medium” (described in Example 1 in the Examples section below) and grown to a confluency of 80% to 90% over a period of about 15 days. The plastic adhesive hBMSc had a round or spindle body shape (FIG. 1A) or a flat body shape (FIGS. 1B-1C).
FIG. 2 illustrates flow cytometer analysis of undifferentiated hBMSc. Day 15 hBMS cells were stained for the presence of surface markers CD45, CD5, CD20, CD11b and CD34 (which are characteristic of lymphohematopoietic cells) and CD90 (Thy-1; during synapse formation) Protein). Cells were analyzed using a FACSCalibur ™ flow cytometer (Becton Dickinson) equipped with an argon ion laser (which was tuned to an excitation wavelength of 488 nm) and a CELLQuest ™ software program (Becton Dickinson). Analysis shows that hBMS cells do not express any of the lymphocyte-related markers, but clearly express the Thy-1 protein.
FIG. 3 illustrates an optical microscope image of adult hBMSc differentiated into neurons. Twenty-four hours prior to differentiation, the cultured hBMSc was transferred from "Proliferation medium" to "Pre-differentiation medium" (see Examples 1 to 2 in the Examples section below). After 24 hours incubation in “pre-differentiation medium”, hBMSc was transferred to “differentiation medium” (see Example 2 in the Examples below). Plastic-adherent cells were transformed into neuron-like cells with spindle-shaped cell bodies and long branching processes that appeared as early as 3 hours after differentiation induction and continued to appear 72 hours after differentiation induction.
FIG. 4 shows uptake of ³ H-thymidine in hBMSc cultured in “differentiation medium” and hBMSc cultured in “predifferentiation medium” (see Example 2 below). Illustrates proliferation). ³ H-thymidine incorporation into differentiating cells was reduced by approximately 45% and 90%, respectively, after 16 and 39 hour incubation periods compared to non-differentiated cells.
FIGS. 5A-D illustrate reverse transcriptase RT-PCR analysis, real-time PCR analysis and Northern blot analysis of RNA extracted from neuronal markers in undifferentiated and differentiating hBMSc (FIGS. 5A-5D). FIG. 5A illustrates RT-PCR analysis designed to identify neuronal transcripts. FIG. 5B illustrates Northern blot analysis and real-time PCR analysis utilizing a ³² P-labeled PCR product of NEGF2 as a probe. FIG. 5C illustrates Northern blot analysis utilizing a ³² P-labeled PCR product of neurofilament 200 (NF-200) as a probe. FIG. 5D illustrates Northern blot analysis utilizing a ³² P-labeled PCR product of neuron-specific enolase (NSE) as a probe.
6A-B illustrate fluorescence microscopic images of neuronal markers in hBMSc cultured for 12 hours to 5 days in “differentiation medium” (see Example 2 below) (FIGS. 6A-6B). . FIG. 6A shows antibody labeled neuronal nucleus specific markers (NeuN; expressed after 12 hours), neurofilament heavy chain (NF-200; expressed after 24 hours), neuron specific enolase (NSE; 48 hours later). Illustrated) and nestin (expressed after 48 hours). FIG. 6B illustrates antibody labeled glial fibrillary acidic protein (GFAP; expressed after 48 hours) and β-tubulin III (expressed after 5 days).
FIGS. 7A-C illustrate Western blot analysis of differentiated hBMSc showing elevated expression of neuron specific enolase (NSE; FIG. 7A), neuronal nucleus specific marker (NeuN; FIG. 7B) and nestin (FIG. 7C). .
FIG. 8 illustrates a light micrograph of neuron-like differentiated hBMSc after 28 days incubation in “long-term differentiation medium” (see Example 5 below).
FIGS. 9A-C show the optics of hBMSc incubated in “growth medium” (see example 1 below) or “long-term differentiation medium” (see example 5 below). A microscopic image and a fluorescence microscopic image are illustrated (FIGS. 9A to 9C). FIG. 9A illustrates antibody-labeled expression of the neuronal marker β-tubulin III in differentiated cells. FIG. 9B illustrates antibody-labeled expression of the neuronal marker MAP-2 in differentiated cells. FIG. 9C illustrates antibody-labeled expression of the neuronal marker nestin in differentiated cells.
FIG. 10 illustrates an RT-PCR analysis designed to identify dopaminergic markers in hBMSc cultured for 12-72 hours in “differentiation medium” (see Example 2 below). To do.
FIGS. 11A-C illustrate the expression of tyrosine hydroxylase (TH) in differentiated hBMSc. FIG. 11A illustrates real-time PCR analysis showing elevated TH transcription in differentiated cells (FIGS. 11A-11C). FIG. 11B illustrates a Western blot analysis showing elevated levels of TH in differentiated cells. FIG. 11C illustrates a fluorescence microscopic image highlighting antibody-labeled TH expression in differentiated cells.
FIG. 12 was incubated for 5 days in “differentiation medium” (see Example 2 below), highlighting antibody labeled vesicular monoamine transporter 2 (VMAT-2) in differentiated cells. The confocal fluorescence microscope image of hBMSc is illustrated.
FIG. 13 illustrates flow cytometer analysis of hBMSc incubated for 48 hours in “differentiation medium” (see Example 2 in the Examples below). Cells were stained for the presence of D2 dopamine receptors and analyzed using a FACSCalibur ™ flow cytometer (Becton Dickinson). Analysis shows that D2 dopamine receptors were present in differentiated cells.
Figures 14A-D illustrate HPLC analysis of differentiating hBMSc (Figures 14A-14D). FIG. 14A illustrates measured dopamine levels in hBMSc supernatants incubated for 0-96 hours in “differentiation medium” (see Example 2 below). FIG. 14B illustrates the measured dopamine levels in the supernatant of hBMSc incubated in “differentiation medium” for 0-96 hours and then further incubated in 56 mM KCl solution for 10 minutes. FIG. 14C illustrates the measured level of dopamine precursor DOPA in the supernatant of hBMSc incubated for 0-72 hours in “differentiation medium”. FIG. 14D illustrates the measured level of dopamine metabolite DOPAC in the supernatant of hBMSc cultured for 0-50 hours in “differentiation medium”.
Figures 15A-D illustrate the effect of mouse bone marrow stromal cell (mBMSC) transplantation on restoration of amphetamine-induced motor rotation in a rat model for Parkinson's disease (Figures 15A-15D). mBMSc was isolated from a transgenic mouse (GFT-Tg) using green fluorescent protein as a landmark. Isolated mBMSc was induced for neural differentiation and transplanted into the substantia nigra of 6-OHDA-damaged rats. Rats were then treated with amphetamine and examined for a rotational response over a period of 45 days after transplantation. FIG. 15A illustrates the change in rotational speed over time after transplantation, showing a decreasing rotation (which indicates improved motor function) at day 45 post-transplant. FIG. 15B illustrates the change in relative rotation over time (as compared to untreated mice) after transplantation, showing a 97.9% decrease in relative rotation 45 days after transplantation. FIG. 15C illustrates a fluorescence microscopic image highlighting transplanted mBMSc present in the substantia nigra of treated mice 45 days after transplantation. FIG. 15D illustrates a fluorescence microscopic image highlighting transplanted mBMSc present in the striatum of treated mice 45 days after substantia nigra transplantation.
FIGS. 16A-C illustrate dopaminergic and serotonergic activity in differentiated human bone marrow stromal cells (hBMSc) (FIGS. 16A-16C). FIG. 16A illustrates Western blot analysis showing expression of tryptophan hydroxylase. FIG. 16B illustrates RT-PCR analysis showing transcription of tryptophan hydroxylase. FIG. 16C illustrates an HPLC analysis showing the synthesis of DOPAC (dopamine metabolite) and 5HIAA (serotonin metabolite).
FIG. 17 illustrates the construction of an expression vector containing the Nurr-1 coding sequence inserted into pcDNA-3.1A (Invitrogene).
FIG. 18 illustrates a construct designed for negative selection of dopaminergic cells. This construct contains the human TH (tyrosine hydroxylase) promoter inserted into pMOD (InvivoGene) upstream of the “suicide gene” HSV1-tk (a type 1 herpes simplex virus thymidine kinase encoding toxic ganciclovir). .
19A-B illustrate the Tet-off / Tet-on system for doxycycline-controlled expression of tyrosine hydroxylase (TH) (FIGS. 19A-19B). FIG. 19A illustrates the regulator and response constructs of this system. FIG. 19B illustrates a schematic diagram describing the mode of operation of this system.
20A-B illustrate fluorescence microscopic images of differentiated BMSc (mBMSc) of GFP-Tg mice (FIGS. 20A-20B). FIG. 20A illustrates a fluorescence microscopic image of morphological changes induced by “differentiation medium” (see Example 1). FIG. 20B illustrates a fluorescence microscopic image highlighting antibody-labeled A2B5 (marker of oligodendrocyte precursor) expressed in NT-3 induced mBMSc.
FIG. 21 illustrates the change over time in the rotation results of mice expressing SOD1 (an animal model of amyotrophic lateral sclerosis (ALS)). SOD1 mice experienced a substantial decrease in rotation performance compared to wild type mice and became completely paralyzed at 4-5 months of age.
FIG. 22 illustrates PCR analysis of various tissues of female mice sampled one week after the male-derived mBMSc was transplanted into the cisterna magna. In the analysis, the Y chromosome (indicating that it is a transplanted cell) is detected in the spinal cord of female mice and not in other tissues.
FIG. 23 illustrates the rotarod performance (indicating rotational behavior) of wild-type mice that received mBMSc transplantation into their spinal cord at the age of 7 weeks. Mice treated with saline injection were used as controls. Transplantation of mBMSc did not affect the rotational behavior of wild type mice.
FIG. 24 illustrates the rotarod performance (showing rotational behavior) of SOD1 mice (a model of amyotrophic lateral sclerosis) that received mBMSc transplantation into the spinal cord at the age of 7 weeks. Mice treated with saline injection were used as controls. Transplantation of mBMSc significantly improved the rotational behavior of SOD1 mice compared to saline treated controls.

  The present invention relates to genetically modified cells capable of controllable synthesis of neurotransmitters, methods of making such cells, and methods of using such cells in cell replacement therapy for neurodegenerative disorders About.

  The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

  Before describing at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited in its application to the details set forth in the following description or the details illustrated by the examples. The invention is capable of other embodiments or of being practiced in various ways or being carried out in various ways. It should also be understood that the wording and terminology used herein is for description only and should not be considered limiting.

  Neurodegenerative disorders characterized by loss of neuronal function (such as Parkinson's disease) cannot be treated efficiently using conventional drug treatments. This is because such drugs have no effect on the underlying disease process typically caused by neuronal degeneration. As a result, drug treatment cannot completely compensate for the increasing loss of neuronal cells.

  Various cell replacement methods of the prior art have been successful when tested in animal models (US Pat. No. 6,528,245; Schwartz et al., 1999; Li et al., 2001; Costantini et al., 2000; Hwan-Wun et al., 1999; Linder et al., 1995; Patredge & Davies, 1995; Perry & Gordon, 1998; Yadid et al., 1999; Choi-Lundberg et al., 1998; Yoshimoto et al., 1995), although these methods may preclude their use in human patients. It is damaged by general restrictions.

  When considering the present invention, the inventors have found that cells that can be easily harvested and manipulated to provide a safe and effective cell replacement therapy for neurodegenerative diseases such as Parkinson's disease, especially external I realized the need for cells capable of synthesizing neurotransmitters such as dopamine in response to stimuli.

  Neuron-like bone marrow stromal cells (BMSc) capable of synthesizing neurotransmitters have been studied in various prior art studies (eg, US Pat. No. 6,528,245; Sanchez-Ramos et al. (2000); Woodbury et al. (2000); (See J. Neurosci. Res., 96: 908-917, 2002); Deng et al. (Biophys. Res. Commun., 282: 148-152, 2001)), but these studies Did not reveal neurotransmitter production by such differentiated BMSc.

  Nonetheless, even though such prior art cells produced neurotransmitters, their use in cell replacement therapy is severe in the synthesis of constitutive neurotransmitters from transplanted cells. Not aware as it can lead to the formation of side effects (eg, skeletal dyskinesia).

  Thus, according to one aspect of the invention, a method for treating a neurodegenerative disorder is provided. As used herein, the expression “neurodegenerative disorder” refers to any disorder or disease or condition of the nervous system (preferably the CNS) that is characterized by a gradual loss of neural tissue, neurotransmitter or nerve function. Indicates. Examples of neurodegenerative disorders include Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, autoimmune encephalomyelitis, Alzheimer's disease and Huntington's disease.

  As used herein, the term “treating” refers to reversing, reducing, or inhibiting the progression of a disorder or disease or condition to which such term applies, or such term It indicates preventing an applied disorder or disease or condition or preventing one or more symptoms of such disorder or condition. As used herein, the term “treatment” or the term “therapy” refers to the effect of treating.

  The method of the present invention is achieved by administering to an individual in need thereof a cell capable of exogenously controllable synthesis of a neurotransmitter, thereby treating a neurodegenerative disorder. .

  Cells capable of exogenously controllable synthesis of neurotransmitters (ie, cells that produce neurotransmitters on demand), such as the cells described in more detail herein below, It is better suited for cell replacement therapy because undesirable side effects associated with cells utilized by the prior art methods described above can be eliminated.

  Neurotransmitters according to the teachings of the present invention are released upon excitation from the axon endings of presynaptic neurons in the central or peripheral nervous system and migrate across the synaptic gap, either exciting or inhibiting target cells Can be any substance that produces The neurotransmitter can be, for example, dopamine, norepinephrine, epinephrine, γ-aminobutyric acid, serotonin, acetylcholine or glutamic acid. Preferably, the neurotransmitter is dopamine.

  The cellular synthesis of such neurotransmitters is guided by a pathway consisting of various enzymes that cooperate in converting precursor molecules into active neurotransmitters. For example, dopamine is synthesized in dopaminergic neurons from L-dihydroxyphenylalanine (L-DOPA) by the action of DOPA decarboxylase, while L-DOPA is produced from tyrosine by the action of tyrosine hydroxylase. Norepinephrine is produced from dopamine by the action of dopamine β-hydroxylase in norepinephrine neurons. Epinephrine is produced from norepinephrine by the action of phenylethanolamine N-methyltransferase in epinephrine neurons. γ-Aminobutyric acid (GABA) is produced from glutamate by the action of glutamate decarboxylase in GABAergic neurons. Serotonin is produced in serotonergic neurons from tryptophan by a two-step process with tryptophan-5-monooxygenase (hydroxylation) and aromatic L-amino acid decarboxylase (decarboxylation). Acetylcholine is produced in cholinergic neurons from choline and acetyl-CoA by the action of choline acetyltransferase (http://www.indstate.edu/theme/mwking/nerves.html).

  The cells utilized by the present invention can be any actively growing cells, but are preferably bone marrow derived cells, more preferably bone marrow stromal cells (BMSc). BMSc can be isolated from the iliac crest of an individual by aspiration and then culturing in a growth medium that can maintain and / or expand the isolated cells ex vivo. Preferably, the growth medium comprises DMEM, SPN, L-glutamine, FCS, 2-β-mercaptoethanol, non-essential amino acids and EGF as described in Example 1 of the Examples section below.

  Proliferating cells can be differentiated directly into neuron-like cells or transformed using the constructs and transformation methods described herein below prior to their differentiation into neuron-like cells. can do.

  Differentiation into neuron-like cells is described in US Pat. No. 6,528,245, and Sanchez-Ramos et al. (2000); Woodbury et al. (2000); Woodbury et al. (J. Neurosci. Res., 96: 908-917, 2001); Black and Woodburry (Blood Cells Mol. Dis., 27: 632-635, 2001); Deng et al. (2001); Kohyama et al. (2001); Reies and Verfatile (Ann. N. Y. Acad. Sci., 938: 231). 235, 2001); Jiang et al. (Nature, 418: 47-49, 2002) can be achieved by incubating the cells in a differentiation medium such as the medium described.

  Preferably, differentiation is achieved in the presence of at least one type of long chain polyunsaturated fatty acid. Long chain polyunsaturated fatty acids such as docosahexaenoic acid (DHA) are known to be essential for proper neuronal development and function. Such polyunsaturated fatty acids have been implicated in regulating the biosynthesis and accumulation of major anionic phospholipids in neuronal membranes [Connor and Neuringer, “Biological Membranes and Aberrations in Membrane Structure and Function”, Karnovs , M.M. Leaf, A .; And Bollis, L .; Ed., Pages 267-292, Alan R. Liss, New York, 1988; L. "The Effect of n-3 Fatty Acid Defectivity and Replication up the Fatty Acid Composition and Function of the Brain and Retina", New York. Neurochem. 65: 2555-2560, 1995]. In neuronal cell culture studies, DHA has been shown to form part of the phosphatidylethanolamine and phosphatidylserine phospholipids around the synapse (Kim et al., J. Biol. Chem., 275: 35215). Kim et al., J. Mol. Neurosci., 16: 223-227, 2002; Akbar and Kim, J. Neurochem., 82: 655-665, 2002).

  When putting the invention into practice, we added 20 μM to 100 μM DHA (Sigma) or ethyl-DHA (Nu-Chek-Preo, Elysian, MN) to the cultured BMSc, It was found that the formation of neurotransmitter secretory vesicles at the synapse of neuron-like BMSc was substantially promoted (data not shown).

  Thus, according to a preferred embodiment of the present invention, differentiation of BMSc into neuron-like cells is achieved by incubating the cells in a differentiation medium containing at least one polyunsaturated fatty acid (eg, DHA). . Preferably, the differentiation medium also contains at least one neuronal differentiation agent, such as BHA, dbcAMP or IBMX. More preferably, the differentiation medium further comprises DMEM, SPN, L-glutamine, N2 supplement and retinoic acid (see Example 2 in the Examples section below).

  In order to further increase the differentiation efficiency, BMSc is preferably incubated in a “pre-differentiation medium” prior to its incubation in the differentiation medium described above. A suitable pre-differentiation medium may be any growth medium that allows cells to be susceptible to neuron-like differentiation, such as a growth medium supplemented with mitogenic basic fibroblast growth factor (bFGF). Preferably, the pre-differentiation medium further comprises DMEM, SPN, L-glutamine, N2 supplement and FCS, as described in Example 2 etc. of the Examples section below.

  Neuron-like cells obtained from the procedures described hereinabove typically exhibit neuronal cell morphology (this is illustrated in FIG. 3) and include at least one neuronal marker (eg, CD90, Neuron-specific nuclear proteins, neurofilament heavy chains, neuron-specific enolase, β-tubulin 3, MAP-2, tyrosine hydroxylase, microtubule-binding protein, nestin and calbindin). Expression of neuronal markers can be determined by various methods such as immunoassays, flow cytometry, RT-PCR, real-time PCR and HPLC methods as described in Examples 3 to 9 and 12 in the Examples section below. Be confirmed using.

  The differentiated cells of the present invention are preferably capable of synthesizing neurotransmitters. Cells that synthesize neurotransmitters are prepared by incubating in a neuron-like differentiation medium as described above, or “dopaminergic differentiation medium” as described in Example 9 of the Examples section below. can do.

  Replacement therapy for Parkinson's disease using only dopaminergic cells leads to imbalances in non-dopaminergic transmitter systems, followed by side effects such as reduced efficacy and dyskinesia, in long-term treatment. (Nicholson and Brotchie, Eur J Neurol. 3: 1-6, 2002). Therefore, agents that can target non-dopaminergic systems and prevent or limit the development of involuntary movements in Parkinson's disease have been proposed for use in treating Parkinson's disease patients (Djaldetti and Melamed, J Neurol., 2: 30-5, 2002; Muller, T., Expert Opin. Pharmacother., 2: 557-72, 2001; Jenner, P., J. Neurol., 2: 43-50, 2000. ). In addition, various serotonin receptors have been identified as potential therapeutic targets in Parkinson's disease (Nicholson and Brotchie, Eur J Neurol., 3: 1-6, 2002).

  Thus, according to another embodiment of the present invention, the population of cells utilized by the present invention produces two or more different neurotransmitter production directed to provide balanced neurotransmitter production. A mixed cell population containing cells. Preferably, the mixed cell population comprises dopaminergic cells such as those illustrated in FIGS. 16A-16C as well as serotonergic cells.

  As stated hereinabove, the cells utilized by this aspect of the invention are preferably capable of controllable synthesis of neurotransmitters.

  Several methods can be utilized to create cells that are capable of such controlled synthesis.

  Preferably, cells suitable for neuronal transplantation are harvested or generated as described herein above and genetically modified to allow controllable expression of neurotransmitters.

  Genetic modification is preferably accomplished by transforming such cells with an expression construct designed for controllable expression of enzymes involved in neurotransmitter synthesis.

  The expression constructs of the present invention preferably comprise a polynucleotide sequence encoding an enzyme involved in the synthesis of neurotransmitters, but the expression construct is obtained by subjecting the expression of the polynucleotide to a particular agent or condition. Designed to be adjusted. Controllable expression of enzymes involved in neurotransmitter synthesis can switch the polynucleotide sequence encoding the enzyme involved in neurotransmitter synthesis "on" (induction) or "off" (inhibition). This can be accomplished by utilizing an expression construct that is placed under the transcriptional control of a promoter or regulatory element.

  The expression construct can be designed as a gene knock-in construct that results in genomic integration of the construct sequence, or it can be designed as an episomal expression vector.

  In any case, expression constructs can be made using standard ligation and restriction techniques well known in the art (Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory). , New York, 1982)). Isolated plasmids, DNA sequences or synthetic oligonucleotides are cleaved, adjusted and religated in the desired form.

  Polynucleotide sequences that can be utilized in the expression constructs of the present invention are described in various databases such as databases managed by the National Molecular Biology Information Resource (http://www.ncbi.nlm.nih.gov/). Yes. Examples include GenBank accession numbers NM_000360 (which encodes tyrosine hydroxylase), NM_000790 (which encodes DOPA decarboxylase), NM_000161 (which encodes GTP cyclohydrolase I), NM_000787 ( This encodes dopamine β-hydroxylase), NM_002686 (which encodes glutamate decarboxylase), NM_003450 (which encodes tryptophan-5 monooxygenase) and NM_020549 (which encodes choline acetyltransferase) Sequence) is included.

  Suitable promoters for use with the present invention are preferably response elements that can cause transcription of polynucleotide sequences to effect regulatable expression of neurotransmitters. Suitable response elements include, for example, tetracycline response elements such as those described by Gossen and Bujard (Proc. Natl. Acad. Sci. USA, 89: 5547-551, 1992); Element (No D et al. (Proc. Natl. Acad. Sci. USA, 93: 3346-3351, 1996)); metal ion responsive elements such as Mayo et al. (Cell, 29: 99-108, 1982), Brinster et al. ( Nature, 296: 39-42, 1982) and Seale et al. (Mol. Cell. Biol., 5: 1480-1489, 1985) and the like; heat shock response elements such as Response elements as described by Nouer et al. (Heat Shock Response, Nouer, L. ed., CRC, Boca Raton, Fla, pages 167-220, 1991); or hormone response elements such as Lee et al. (Nature, 294 : 228-232, 1981), Hynes et al. (Proc. Natl. Acad. Sci. USA, 78: 2038-2042, 1981), Klock et al. (Nature, 329: 734-736, 1987), Israel and Kaufman (Nucl. Acids Res., 17: 2589-2604, 1989) and the like. Preferably, the response element is an ectizone-inducible response element, more preferably the response element is a tetracycline response element.

  The expression constructs of the present invention can also include one or more elements. Enhancer elements can stimulate transcription up to 1000-fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream of the transcription start site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or mouse cytomegalovirus (CMV), various retroviruses (eg, murine leukemia virus, mouse sarcoma). Long terminal repeats derived from viruses or Rous sarcoma virus and HIV). See Enhancers and Eukaryotic Expression (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1983), which is incorporated herein by reference.

  Polyadenylation sequences can also be added to the expression construct to increase the translation efficiency of the enzyme expressed from the expression construct of the present invention. Two different sequence elements are required for accurate and efficient polyadenylation: a GU-rich or U-rich sequence that is downstream of the polyadenylation site and 11 to 30 nucleotides upstream 6 Nucleotide highly conserved sequence AAUAAAA. Termination and polyadenylation signals suitable for the present invention include signals derived from SV40.

  In addition to the elements already described, the expression constructs of the present invention typically facilitate the identification of cells carrying recombinant DNA or to increase the expression level of cloned polynucleotides. Other specialized elements intended for the purpose can be included. For example, many animal viruses contain DNA sequences that promote extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying these viral replicons will replicate episomally as long as the appropriate factor is provided either by the gene carried by the plasmid or by the gene carried with the host cell genome.

  The expression construct may include a eukaryotic replicon or may not include a eukaryotic replicon. If a eukaryotic replicon is present, the vector can be amplified in eukaryotic cells using an appropriate selectable marker. If the construct does not contain a eukaryotic replicon, episomal amplification cannot be performed. Instead, the recombinant DNA is integrated into the engineered cell's genome, where the desired polynucleotide is expressed by the promoter.

  The expression constructs of the present invention further include, for example, additional polynucleotide sequences that allow translation of several proteins from a single mRNA (eg, internal ribosome entry site (IRES), and genomic integration of a polypeptide whose promoter is chimeric) For example). For example, one expression construct can be designed to co-express two different enzymes involved in neurotransmitter synthesis, such as the tyrosine hydroxylase and DOPA decarboxylase enzymes involved in dopamine synthesis.

  Examples for mammalian expression constructs include pcDNA3, pcDNA3.1 (+/−), pGL3, pZeoSV2 (+/−), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1. , PSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (which are available from Invitrogen), pCI (which is available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (these are Strate ), PTRES (which is available from Clontech), and derivatives thereof, including but not limited to.

Expression constructs containing regulatory elements derived from eukaryotic viruses such as retroviruses can also be used by the present invention. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA. Vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A ⁺ , pMTO10 / A ⁺ , pMANne-5, baculovirus pDSVE, and SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus Any other that allows expression of the protein under the direction of a promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoter that has been shown to be effective for expression in eukaryotic cells Of vectors.

  Viruses are specialized infectious agents that have often evolved to circumvent host defense mechanisms. Typically, viruses infect and propagate in specific cell types. With the targeting specificity of a viral vector, its natural specificity is exploited to specifically target a given cell type and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by the present invention depends on the host cell to be transformed. The ability to select a suitable vector according to the cell type to be transformed is well within the ability of those skilled in the art, so a general description of selection considerations is presented herein. Not. For example, bone marrow cells can be targeted using human T cell leukemia virus type I (HTLV-I).

  Recombinant viral vectors are useful for in vivo expression of genetically modified polynucleotides because they provide advantages such as lateral infection and targeting specificity. Lateral infection is, for example, a process that is unique in the life cycle of a retrovirus and in which only one infected cell buds and produces many progeny virions that infect neighboring cells. The result is that large areas, most of which were not initially infected by the very first virus particles, are rapidly infected. This is in contrast to a vertical infection where the infectious agent spreads only through daughter offspring. Viral vectors that cannot spread laterally can also be made. This feature may be useful if the desired purpose is to introduce the designated gene only into a number of localized targeted cells.

  As described in the Examples section below, the cells of the invention also include a first polynucleotide sequence that is regulated by a transactivator disposed under transcriptional control of a second regulatory sequence. It can be transformed with a construct or construct system. In such an expression scheme, the transactivator can activate the first regulatory sequence to effect transcription of the first polynucleotide sequence in the absence of the agent.

  Preferably, the first polynucleotide sequence of the expression construct or construct system functions in an ecdysone responsive promoter as described by No et al. (Proc. Natl. Acad. Sci. USA, 93: 3346-3351, 1996) and the like. A sequence encoding an enzyme involved in the synthesis of neurotransmitters, which are linked together.

  More preferably, the first polynucleotide sequence of the expression construct or construct system functions in a tetracycline regulatory element as described by Gossen and Bujard (Proc. Natl. Acad. Sci. USA, 89: 5547-551, 1992), etc. A sequence encoding an enzyme involved in the synthesis of neurotransmitters, which are linked together. See Example 15 in the Examples section below for further details.

  The transactivator is preferably tetracycline-regulated, such as described by Gossen and Bujard (Proc. Natl. Acad. Sci. USA, 89: 5547-551, 1992), operably linked to a human enolase promoter. It is a transactivator. See Example 15 in the Examples section below for further details.

  A variety of methods can be used to introduce the expression constructs of the invention into mammalian cells. Various such methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York (1989, 1992)); Ausubel et al., Current Protocol in Mole in Sole. (1989)); Chang et al., Somatic Gene Therapy (CRC Press, Ann Arbor, Mich. (1995)); Vega et al., Gene Targeting (CRC Press, Ann Arbor, Mich. (1995)); Cloning Vectors and Ther Uses (Butterworths, Boston, Mass. (1988)); Gilboa et al. [Biotechniques, 4 (6): 504-512, 1986], such methods include, for example, Stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. For the positive / negative selection method, see US Pat. Nos. 5,464,764 and 5,487,992.

  Once transformed cells are made, the transformed cells are cultured for their ability to synthesize functional neurotransmitters in response to external signals (eg, the presence or absence of an agent). Be examined). Preferably, the concentration of neurotransmitter is comparatively analyzed (in its presence relative to the absence of the agent) using standard chemical analysis methods such as, for example, HPLC or GC-MS. Alternatively, the culture broth can be used using biochemical analysis methods such as immunoassays, Western blots and real-time PCR, using the procedures described in Example 7 etc. in the Examples section below, or enzyme activity. Comparative analysis for the expression of recombinant enzymes (eg tyrosine hydroxylase).

  In the case where cells capable of endogenously producing neurotransmitters are selected for use with the present invention (eg, neuron-derived BMSc), such cells are preferably neurotransmitters. It is understood that the engineered to delete or mutate the endogenous coding sequence of an enzyme involved in the synthesis of (eg, an endogenous tyrosine hydroxylase). Such genetic manipulations include, for example, the techniques and vectors described by Galli-Taliadoros et al. (J. Immunol. Methods, 181: 1-15, 1995) and Harris & Ford (Pharmacogenomics, 1: 433-43, 2000). Can be achieved by using techniques and vectors for gene knockout or site-directed mutagenesis. Alternatively, cells capable of endogenously producing neurotransmitters can be removed by exposure to a neurotoxin (eg, MPTP) or by transformation with a suicide vector exemplified in FIG. .

  Endogenous sequence deletions are the genome of an exogenous sequence encoding an enzyme that simultaneously loses the ability of the cell to synthesize neurotransmitter endogenously and is placed under the transcriptional control of a controllable regulatory sequence. It can be combined with the knock-in of the coding sequence of the exogenous enzyme (eg, the coding sequence described above) so as to acquire such ability (regulatable) by integration.

  Alternatively, such cells can also be engineered such that the coding sequence for the endogenous enzyme is brought under the control of a regulatable promoter sequence. Such manipulation can be accomplished by replacing the enzyme's endogenous promoter sequence (eg, TH promoter sequence) with a gene knock-in of a regulatable promoter sequence.

  Optionally, the cells of the invention are transformed to acquire resistance to cell death that occurs during brain transplantation. Cells transplanted into brain tissue can undergo apoptosis initiated by hypoxia, hypoglycemia, mechanical trauma, free radicals, growth factor depletion, and excessive extracellular concentrations of excitatory amino acids in the host brain Have been found (Brundin et al., Cell Transolant. 9: 179-195, 2000). Under circumstances where there is a high risk of cell death due to apoptosis induction, the cells of the present invention may contain an apoptosis-inhibiting polypeptide (eg, human bcl-2 gene (Adams and Cory, Science, 281: 1322-1326, 1998), etc. ). This polypeptide is under the control of a constitutive promoter as described herein above or preferably as described, for example, by Levy et al. (Journal of Molecular Neuroscience, 21: 121-132, 2003). It can be expressed under the control of a neuron tissue specific promoter such as the human neuron specific enolase (NSE) promoter.

  The cells of the invention can be administered to a therapist using various transplantation methods, the type of which depends on the site of transplantation.

  The terms or expressions “transplant”, “cell replacement” or “transplant” are used interchangeably herein to indicate introduction of a cell of the present invention into a target tissue. The cells can be derived from the transplanter or can be derived from the same or different donor.

  The cells can be transplanted into the central nervous system, or into the ventricular cavity of the host brain, or subdurally on the surface of the host brain. Conditions for successful transplantation include (i) survival of the transplanted tissue; (ii) retention of the graft at the transplant site; and (iii) minimal amount of pathological reaction at the transplant site. Various methods for transplanting various neural tissues (eg, embryonic brain tissue) into the host brain are described in “Neural grafting in the mammarian CNS” (Bjorklund and Steinvi, 1985). These techniques include intra-parenchymal, ie, injection or placement of tissue in the host brain as opposed to brain parenchymal tissue at the time of transplantation (transplant outside or outside the brain. In parenchymal transplantation in the host brain).

  Parenchymal transplantation can be accomplished using two methods: (i) injecting cells into the host brain parenchyma, or (ii) to expose the host brain parenchyma. Prepare the cavity by surgical means, and then place the implant in the cavity. In both methods, parenchymal tissue placement is provided between the graft and the host brain tissue at the time of transplantation, and in both methods the anatomical integration between the graft and the host brain tissue. Is promoted. This is important if the graft becomes a component of the host brain and is required to survive the life of the host.

  Alternatively, the graft can be placed in the ventricle (eg, cerebral ventricle) or subdurally, ie, on the surface of the host's brain, where the graft is an interstitial buffy coat or arachnoid membrane and It is separated from the parenchyma of the host brain by the buffy coat. Transplantation into the ventricles can be accomplished by injection of donor cells or transplanted into the ventricles to grow the cells in a substrate such as 3% collagen to prevent changes in the location of the graft. This can be achieved by forming a plug of the resulting solid tissue. In the case of a subdural transplant, cells can be injected around the surface of the brain after creating a gap in the dura mater. Injection into selected areas of the host brain can be made by drilling and piercing the dura mater to allow the needle of a microsyringe to be inserted. The microsyringe is preferably attached to a stereotaxic frame and three-dimensional stereotaxic coordinates are selected to place the needle at the desired location in the brain or spinal cord. Cells can also be introduced into the brain putamen, basal ganglia, hippocampal cortex, striatum, substantia nigra or caudate region, and the spinal cord.

  For implantation, the cell suspension is drawn into a syringe and administered to an anesthetized transplanter. Multiple injections can be made using this approach.

Thus, such cell suspension techniques allow transplantation of cells to any given site in the brain or spinal cord, are relatively atraumatic, and use several different sites or the same cell suspension. Allows multiple transplants at the same site at the same time and allows a mixture of cells from different anatomical regions. Many grafts can consist of a mixture of cell types and / or a mixture of transgenes inserted into the cells. Preferably, about 10 ⁴ to about 10 ⁸ cells are introduced per graft.

  In the case of transplantation into the cavity, this may be preferred for spinal cord transplantation, but the tissue may be the brain as described, for example, by Stenevi et al. (Brain Res., 114: 1-20, 1976). Is removed from the area close to the outer surface of the central nervous system (CNS) to form a graft cavity by stopping bleeding with a material such as gel foam. Suction can be used to create the cavity. Thereafter, the implant is placed in the cavity. Two or more grafts can be placed in the same cavity using injection of cells or solid tissue transplant. Preferably, the transplant site is determined by the type of neurotransmitter synthesized by the cells of the invention. For example, dopaminergic cells are preferably transplanted into the substantia nigra of Parkinson's disease patients.

  The cells of the present invention can be co-administered with therapeutic agents useful in treating neurodegenerative disorders, such as growth factors (eg, nerve growth factor), gangliosides, antibiotics, neurotransmitters, neurohormones, It can be co-administered with toxins, neurite promoting molecules, and antimetabolites, and precursors of these molecules, such as L-DOPA.

  After transplantation, the treated individual is carefully and continuously monitored for the level of neurotransmitter released by the transplanted cells. Neurotransmitter levels are preferably estimated indirectly by using suitable clinical trials to diagnose neurodegenerative disorders. For example, release of dopamine by transplanted cells in Parkinson's disease patients is described, for example, in Adker, C. et al. H. And Ahlskog, J. et al. E. Ed., “Parkinson's Disease and Movement Disorders, Diagnosis and Treatment Guidelines for the Practicing Physician” (Human Press, New Jersey, 2000) . Based on the monitored display value, the release rate of the neurotransmitter can be administered to the individual by administering an agent that can modulate neurotransmitter synthesis in the transplanted cells, or such an agent It is adjusted by removing it from the individual. An agent can be any molecule that can up- or down-regulate the expression of enzymes involved in neurotransmitter synthesis, such as described hereinabove.

  The agent can be administered directly to the individual or can be administered as part of the pharmaceutical composition (active ingredient).

  As used herein, a “pharmaceutical composition” refers to one or more of the active ingredients or agents described herein and other chemistries such as physiologically suitable carriers and excipients. The preparation with the active ingredient is shown. The purpose of a pharmaceutical composition is to facilitate administration of a formulation to an organism.

  Hereinafter, the expressions “physiologically acceptable carrier” and the expression “pharmaceutically acceptable carrier” may be used interchangeably, but do not cause significant irritation to an organism and are administered. A carrier or diluent that does not interfere with the biological activity and properties of the formulated formulation. An adjuvant is included in these expressions.

  As used herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

  Various techniques for drug formulation and administration can be found in “Remington's Pharmaceutical Sciences” (Mack Publishing Co., Easton, PA, latest edition), which is incorporated herein by reference. ).

  For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. Preferably, doses are defined in animal models to achieve the desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

  Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. Data obtained from these in vitro and cell culture assays and animal studies can be used in defining dosage ranges for use in humans. The dosage can vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, eg, Fingl et al., 1975, “The Pharmaceutical Basis of Therapeutics”, Chapter 1, page 1. ) For example, Parkinson's disease patients can be monitored symptomatically for improved motor function that shows a positive response to treatment and for staggered dyskinesia symptoms that show excessive dopamine expression.

  The agent can be administered to the patient in a variety of ways including, but not limited to, oral administration, parenteral administration, subarachnoid administration, intracerebroventricular administration, and intranigal application. included. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, eg, Fingl et al., 1975, “The Pharmaceutical Basis of Therapeutics”, Chapter 1, page 1. )

  Dosage amount and interval may be adjusted individually to the levels of the active ingredient that are sufficient to effectively modulate the synthesis of neurotransmitters by the transplanted cells. The dosage required to achieve the desired effect will depend on the individual's characteristics and route of administration. A variety of detection assays can be used to measure plasma concentrations.

  Depending on the severity and responsiveness of the condition being treated, the medication can be administered in single or multiple doses, and the course of treatment can range from days to weeks or a reduction in disease state. Continue until

  The amount of composition administered will, of course, depend on the individual being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like. Dosage and time of administration respond to careful and continuous monitoring of the individual's changing conditions. For example, a Parkinson's disease patient being treated is administered an amount of an agent that is sufficient to promote or inhibit dopamine synthesis to a desired level based on the displayed value during monitoring.

  Accordingly, the present invention provides novel nucleic acid constructs, construct systems, cells and methods for cell therapy of neurodegenerative diseases that are effective, safe and clinically feasible.

  Further objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon review of the following examples, which are not intended to be limiting. Also, each of the various embodiments and aspects of the invention as described hereinabove and as claimed in the claims section below finds experimental support in the examples below. .

  Together with the above description, the invention is illustrated with reference to the following examples. In addition, this invention is not limited by these Examples.

  The terms used in the present application and the experimental methods utilized in the present invention broadly include molecular biochemistry, microbiology and recombinant DNA techniques. These techniques are explained in detail in the literature [see for example the following references. “Molecular Cloning: A laboratory Manual” Sambrook et al. 1989; Ausubel, R .; M.M. 1994 "Current Protocols in Molecular Biology" I-III; Ausubel et al., 1989 "Current Protocols in Molecular Biology" John Wiley and Sons, Baltimore, Maryland, USA & Sons, New York, USA 1988; Watson et al., “Recombinant DNA” Scientific American Books, New York, USA; edited by Birren et al., “Genome Analysis: A Laboratory Manual Series,” Volumes 1-4. Spring Harbor Laboratory Press, New York 1998, USA; methods described in US Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5192659, and 5,272,057; E. Ed. "Cell Biology: A Laboratory Handbook" I-III 1994; Coligan, J. et al. E. Ed. “Current Protocols in Immunology”, I-III, 1994; Stites et al., “Basic and Clinical Immunology” (8th edition), Appleton & Lange, United States, United States, Norwalk, Connecticut, 1994; Immunology ", W.W. H. Freeman and Co. New York, 1980; available immunoassays are extensively described, for example, in the following patent and scientific literature. U.S. Pat. Gait, M .; J. et al. Ed. "Oligonucleotide Synthesis" 1984; Hames, B .; D. And Higgins S .; J. et al. Ed. “Nucleic Acid Hybridization” 1985; Hames, B .; D. And Higgins S .; J. et al. Ed. “Transscription and Translation” 1984; Freshney, R .; I. Ed. “Animal Cell Culture” 1986; “Immobilized Cells and Enzymes” IRL Press 1986; Perbal, B. et al. “A Practical Guide to Molecular Cloning”, 1984 and “Methods in Enzymology”, 1-31; Academic Press; “PCR Protocols: A Guide To Mes. Ap. "Stratesies for Protein Purification and Characterization-A Laboratory Course Manual", CSHL Press, 1996 and Parfitt et al. (1987). Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2 (6), 595-610; these references are incorporated as if fully set forth in this application]. Other general literature is provided throughout this specification. The methods described herein are considered well known in the art and are provided for the convenience of the reader. All information contained herein is incorporated herein by reference.

Example 1
Isolation and culture of human bone marrow stromal cells (hBMSC)
Proliferation culture: Bone marrow aspirate (10 ml) was obtained from the iliac crest of a healthy human donor with informed consent. Mononuclear cells were isolated by centrifugation through a density gradient (Histopaque®-1077), “Growth Medium” [Dulbecco's Modified Eagle Medium (DMEM; Biological Industries); 100 μg / ml streptomycin, 100 U / ml penicillin. 12.5 units / ml nystatin (SPN; Biological Industries); 2 mM L-glutamine; 5% horse serum; 15% fetal calf serum (FCS; Biological Industries); 0.001% 2-β- Mercaptoethanol (Sigma); 1 × nonessential amino acid; 10 ng / ml human epidermal growth factor (EGF)]. Cells were incubated for 2 days at 37 ° C. in a humidified 5% CO ₂ incubator, after which non-adherent cells were discarded. The remaining plastic adherent cells were washed twice with Dulbecco's phosphate buffered saline (PBS; Biological Industries) and fresh growth medium was added. The medium was changed every 3 or 4 days. Cells grew to 80% -90% confluence within 15 days and appeared as circular or spindle shapes (FIG. 1A) or flat shapes (FIGS. 1B-1C).

Flow cytometry: After 15 days under growth culture conditions, cells were collected and suspended in phosphate buffered saline (PBS) containing 0.05% trypsin and 25 mM EDTA. Cells in a solution at a concentration of 0.5 × 10 ⁶ cells / ml, antibodies specific to the cell surface markers CD45, CD5, CD20, CD11b and CD34 (which are associated with lympho-hematopoietic cells) (Beckton) Dickinson) was stained for 20 minutes with an empirically determined amount of each antibody (generally 10 μl to 20 μl). Antibody labeled cells were washed thoroughly with 2 volumes of PBS and fixed in flow buffer (1% paraformaldehyde, 0.1% sodium azide and 0.5% bovine serum albumin in PBS). Washed cells were 10000 events using a FACSCalibur ™ flow cytometer (Beckton Dickinson) equipped with an argon ion laser tuned to an excitation wavelength of 488 nm and using the CELLQuest ™ software program (Beckton Dickinson). Was analyzed by collecting.

result:
Cultured hBMSc does not express any of the surface markers associated with lymphohematopoietic cells (ie, CD45, CD5, CD20, CD11b and CD34), but rather a CD90 surface marker (Thy-1) indicating synaptogenesis. ) Was expressed (FIG. 2).

Example 2
In vitro differentiation of human bone marrow stromal cells (hBMSCs) Methods:
Differentiation culture: hBMSc were cultured in “growth medium” (described in Example 1 hereinabove) for up to 3 months prior to induction of differentiation. The plastic adherent cells were then transformed into “pre-differentiation medium” [SPN, 2 mM glutamine, N2 supplement (insulin 25 μg / ml; progesterone 20 nM; putrescine 100 μM; selenium 30 nM; transferrin 100 μg / ml), 10% FCS, 10 ng In DMEM supplemented with 10 ml / ml basic fibroblast growth factor (bFGF; R & D Systems, MN) and 10 ng / ml human epidermal growth factor (EGF; R & D Systems). After 24 hours incubation at 37 ° C., the cells were brought to a density of “differentiation medium” [SPN, 2 mM glutamine, N2 supplement (insulin 25 μg / ml; progesterone 20 nM; putrescine 100 μM; selenium 30 nM; 1 × 10 ⁵ cells / ml). Transfer medium 100 μg / ml), 200 μM butylated hydroxyanisole (BHA; Sigma), 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma), 0.5 mM isobutylmethylxanthine (IBMX; Sigma), 10 μM docosahexaenoic acid ( DHA; Sigma), and DMEM supplemented with 10 μM retinoic acid (RA; Sigma)] and incubated at 37 ° C. for 12-72 hours.

Proliferation evaluation: hMBSc was suspended in “pre-differentiation medium” and “differentiation medium” dispensed in 96-well microtiter plates (100 μl / well) and incubated at 37 ° C. for 16 hours and 39 hours. The culture was then supplemented with 10 μCi / ml ³ H-thymidine and the culture was further incubated for 4 hours. The cells were then collected and suspended in phosphate buffered saline (PBS) containing 0.05% trypsin and 25 mM EDTA and analyzed using a liquid scintillation counter to determine ³ H-thymidine in the cells. Uptake level (which indicates proliferative activity) was measured.

result:
The plastic adherent cells appeared as early as 3 hours after differentiation induction and showed a neuron-like spindle shape with a long branching process that continued to appear at 72 hours after differentiation induction (FIGS. 3A-3F).

Uptake of ³ H-thymidine was substantially reduced in differentiated cells (FIG. 4). This therefore indicates that proliferation was attenuated with neuron-like differentiated bBMSC.

Example 3
Identification of Neuronal Transcripts in Differentiating hBMSc Method RT-PCR: hBMSc in “growth medium” or “differentiation medium” (see Example 1 to Example 2 hereinabove) at 37 ° C. 3 Incubated for time-72 hours. Total RNA was extracted from hBMSc by using the guanidine isothiocyanate method as described by Chomczynski & Sacchi (1987). Total RNA was also extracted from fresh human lymphocytes (from donors) using an RNA isolation kit (Puregene Gentra, Manneapolis, USA). These RNA samples were separated by 1% agarose formaldehyde denaturing gel electrophoresis to confirm their integrity. To make cDNA, an RNA sample (0.5 μg) was added to the reaction mixture [1.3 μM random primer, 1 × buffer (supplied by InvitroGene), 10 mM DTT, 20 μM dNTPs and RNase inhibitors. The RT-superscript II enzyme (10 units) contained in the mixture was incubated at 25 ° C. for 10 minutes, 42 ° C. for 2 hours, 70 ° C. for 15 minutes, and 95 ° C. for 5 minutes. Primers indicated by SEQ ID NO: 1-2, SEQ ID NO: 11-12, SEQ ID NO: 21-22, SEQ ID NO: 26-26 and SEQ ID NO: 29-30 (see Table 1 below) And amplified under 35 cycles of 94 ° C. for 1 minute, 55 ° C. to 58 ° C. for 1 minute and 72 ° C. for 1 minute.
Table 1
Upstream sense and downstream antisense primers derived from different exons for detecting neuronal and dopaminergic transcripts in differentiated hBMSc

Northern blot analysis: RNA samples extracted from hBMSc were size fractionated on a 1% agarose gel supplemented with 3% formaldehyde and MOPS and transferred to a Duralon-UVTM membrane (Stratagene). The membrane was then hybridized overnight with purified ³² P-labeled probes for the neuronal markers NEGF2 (neurite growth promoting factor 2), NF-200 (neurofilament heavy chain) and NSE (neuron specific enolase). I let you. The hybridized membrane is washed several times, exposed to a storage phosphor screen, autoradiographed with a phosphor imager (Cyclone, Packard), and the probe is used to confirm equal loading and transcription of RNA. Except for rehybridization with a ³² P-labeled probe for GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

  Real-time PCR: Real-time quantitative PCR analysis of the neuronal marker NEGF2 with “Rotor-Gene DNA sample analysis system” version 4.6 (Corbett Research) using the Sybergreen “PCR master mix” and primers SEQ ID NOs: 17-18. went. A real-time PCR analysis of GAPDH was also performed to provide accelerated conditions for sample normalization using primers of SEQ ID NOs: 9-10. The amplification protocol was 40 cycles of 95 ° C. for 15 seconds, 55 ° C. for 40 seconds, 72 ° C. for 40 seconds and 77 ° C. for 20 seconds. Accelerated conditions for sample normalization were applied by amplification of 18S rRNA. The amplification protocol was 80 cycles of 95 ° C. for 20 seconds and 61 ° C. for 1 minute. Quantification of gene expression for 18S rRNA was calculated by the protocol ΔΔCT method and from the standard curve method.

result:
RT-PCR analysis shows the transcriptional expression of the neuronal markers nestin, NSE, NF-M, CD90, RA-R and GPC4 in both differentiated and non-differentiated hBMSc (FIG. 5A). However, transcriptional expression of the neuronal markers NF-H and necdin occurred only in differentiated hBMSc (FIG. 5A).

  Real-time PCR analysis shows a 7-fold increase in NEGF2 mRNA in differentiated cells when compared to non-differentiated cells after 50 hours of incubation (FIG. 5B).

  Northern blot analysis shows that the transcriptional expression of neuronal markers NEGF2, NF-200, and NSE was significantly increased in differentiating hBMSc (FIGS. 5B-5D, respectively).

Example 4
Identification of neuronal proteins in differentiated hBMSc Methods:
Western Blot: 50 micrograms of protein extract obtained from hBMSC was sample buffer (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 5% 2-β-mercaptoethanol. Denatured in 0.0025% bromophenol blue (SIGMA) (diluted 1: 5 with sample and boiled for 5 minutes) Each sample was 12.5% SDS-polyacrylamide gel (Bio -Rad Laboratories) After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories) followed by Tris-buffered saline (TSB containing 0.1% Tween-20). 10 mM Tris (7.5), Blocked with 5% non-fat milk (blocking solution) in 150 mM NaCl) Membranes were probed overnight at 4 ° C. with neuron marker specific antibodies as described herein below in Table 2. After incubation, the membrane is washed twice with blocking solution (15 minutes each) and then once with TBS-T for 15 minutes, followed by horseradish as described in Table 3 herein below. Exposed to peroxidase conjugated secondary antibody, then the membrane was washed twice with blocking solution (15 minutes each) and then once with TBS-T for 15 minutes, followed by enhanced SuperSignal® chemiluminescence Staining using a detection kit (Pierce) and medical X-ray film (Fuji Photo Fi) a) was used to assess and quantify changes upon induction, and density measurements of specific protein bands were performed using the VersaDoc® imaging system (Bio-Rad Laboratories) and Quantity. This was done by using One® software (Bio-Rad).
Table 2
Neuron marker specific antibody and dopaminergic marker specific antibody
Table 3
Anti-mouse secondary antibody and anti-rabbit secondary antibody

result:
Immunostaining revealed the presence of neuronal markers (NeuN, NF-200, NSE) in hBMSc during differentiation at 24 hours, 24 hours, 48 hours and 48 hours after differentiation induction, respectively (FIG. 6A). Antibody labeled GFAP and β-tubulin III were observed in hBMSc at 48 hours and 5 days after differentiation induction, respectively (FIG. 6B).

  Expression of Neu-N protein, NSE protein and nestin protein (relative to actin) was substantially increased after differentiation induction, as shown by Western blot analysis (FIGS. 7A-7C).

Example 5
Long-term survival of differentiating hBMSc Methods:
Long-term differentiation culture: hBMSCs were incubated in “pre-differentiation medium” (see Example 2 above) at 37 ° C. for 24 hours, followed by “long-term differentiation medium” [SPN, 2 mM glutamine, N2 supplement (insulin 25 μg / ml; progesterone 20 nM; putrescine 100 μM; selenium 30 nM; transferrin 100 μg / ml), 1 mM dbcAMP, 0.5 mM IBMX, 10 μM docosahexaenoic acid (DHA; Sigma), 10 ng / ml bFGF, 10 ng / ml human glia Cell line-derived neurotrophic factor (GDNF) and DMEM supplemented with 10 ng / ml human β-nerve growth factor (β-NGF). hBMSc was incubated in this “long-term differentiation medium” at 37 ° C. for 28 days.

Immunoassay: Cells were placed in a slide chamber (Nalge Nunc International) aseptically pretreated with poly-L-lysine (Sigma) and processed. The cells were fixed with 4% paraformaldehyde in PBS (pH 7.3) for 30 minutes at 4 ° C. and then for 30 minutes at room temperature. The slides were then washed 3 times with PBS (5 min each), 10 min at 4 ° C. with PBS containing 0.1% Triton X-100 (Sigma) and 10% goat serum (Biological Industries), then at room temperature For 10 minutes. The slides were then washed 3 times with PBS (5 minutes each). Endogenous peroxide was blocked for 20 minutes at room temperature by adding 3% H ₂ O ₂ (Merck) in anhydrous methanol (Bio-Lab, Israel). After 3 washes in PBS (5 minutes each), the slide chamber was diluted with anti-MAP2, anti-β-tubulin diluted as described in Tables 2 to 3 in Example 4 hereinabove. Incubated overnight at 4 ° C. with III or anti-nestin. The next day, the slides are thoroughly washed 3 times in PBS (10 minutes each) and then Cy ™ 3-conjugated goat anti-rabbit IgG in PBS containing 10% goat serum and 0.2% Tween-20 or Incubated with Cy ™ 2-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 30 minutes at room temperature. After incubation, the slides were washed 3 times with PBS (5 minutes each), fixed with glycerol vinyl alcohol fixative (Zymed Laboratories), covered with a cover glass, and examined with a fluorescence microscope.

result:
hBMSc showed typical neuron-like cell morphology after 28 days incubation in “long-term differentiation medium” (FIG. 8).

  The antibody-labeled neuronal markers MAP2 (microtubule binding protein 2), β-tubulin III and nestin were observed in hBMSc after 28 days incubation (FIGS. 9A-9C, respectively).

Example 6
Expression of dopaminergic mRNA in differentiated hBMSc Methods:
RT-PCR: hBMSc was incubated for 12-72 hours in “Growth Medium” and “Differentiation Medium” (see Examples 1 to 2 above). Total RNA was extracted from hBMSc by using the guanidine isothiocyanate method as described by Chomczynski & Sacchi (1987). The RT-PCR method designed to identify dopaminergic markers is essentially the same except that it uses primers of SEQ ID NOs: 3-8, SEQ ID NOs: 13-14 and SEQ ID NOs: 27-28. Performed as described in Example 3 above in the specification.

result:
As can be seen in FIG. 10, several dopaminergic marker transcripts were expressed in differentiated and / or undifferentiated hBMSc. These include Nurr1 [nuclear receptor-related 1; a transcription factor having a role in the differentiation of midbrain progenitors into dopamine neurons] and AADC [aromatic L-amino acid decarboxylase; L-3,4-dihydroxyphenylalanine (L -DOPA) decarboxylation to dopamine, L-5-hydroxytryptophan decarboxylation to serotonin, and L-tryptophan decarboxylation to tryptamine decarboxylation]. Transcriptional expression of GTP [cyclohydrolase 1; an enzyme required to produce a tetrahydrobiopterin (BH4) cofactor for TH] is significantly higher in differentiated hBMSc when compared to undifferentiated hBMSc, whereas , D2 dopamine receptor and DAT dopamine transporter transcripts were expressed only in differentiated hBMSc.

Example 7
Induction of tyrosine hydroxylase in differentiated hBMSC Methods:
Real-time PCR: Total RNA was extracted from hBMSc using the guanidine isothiocyanate method as described by Chomczynski & Sacchi (1987). cDNA was made as described in Example 3 hereinabove by performing an RT reaction using random primers. Amplification of cDNA was performed in an ABI Prism 7700 sequence detection system (Applied Biosystems) using TaqMan® universal PCR master mix (Applied Biosystems) using specific primers for human TH and 18S rRNA. Accelerated conditions for sample normalization were applied by amplification of 18S rRNA. The amplification protocol was 80 cycles of 95 ° C. for 20 seconds and 61 ° C. for 1 minute. Quantification of gene expression for 18S rRNA was calculated by the protocol ΔΔCT method and from the standard curve method.

  Western blot analysis: The assay was performed as described in Example 4 hereinabove except that immunoblotting was performed using anti-TH and anti-actin antibodies.

  Immunoassay: The assay was performed as described in Example 5 hereinabove using anti-TH antibodies as described in Tables 2 to 3 in Example 4 above. went.

Results Tyrosine hydroxylase mRNA and protein levels were substantially elevated during neuronal differentiation of hBMSc (FIGS. 11A-11B, respectively). In addition, the presence of antibody-labeled tyrosine hydroxylase was observed in hBMSc during differentiation 6 to 48 hours after differentiation induction (FIG. 11C).

Example 8
Identification of dopamine-related proteins in hBMSc Methods:
Immunoassay: hBMSCs are incubated in “differentiation medium” (see Example 2 hereinabove) for 5 days, then collected and vesicles using the procedure described in Example 5 above. Staining was performed with an antibody specific for monoamine transporter 2 (VMAT-2). The antibody binding to VMAT-2 in the cells was visualized by using a secondary Cy ™ 3-conjugated antibody. Stained cells were observed with a laser confocal microscope LSM510 (ZEISS, Germany).

Flow cytometry: After 48 hours incubation in “differentiation medium” (see Example 2 above), cells were collected and phosphate buffered with 0.05% trypsin and 25 mM EDTA. Suspended in physiological saline (PBS). The cell suspension (0.5 × 10 ⁶ cells / ml) was incubated for 30 minutes with antibodies specific for the D2 dopamine receptor as described in Tables 2 to 3 in Example 4 above. Antibody labeled cells were washed thoroughly with 2 volumes of PBS and fixed in flow buffer (1% paraformaldehyde, 0.1% sodium azide and 0.5% bovine serum albumin in PBS). Washed cells were analyzed by FACSCalibur ™ flow cytometer (Beckton Dickinson).

result:
Fluorescence microscopic images of hBMSc revealed that antibody-labeled VMAT2 dopaminergic markers were present in differentiated hBMSc and not in non-differentiated cells (FIG. 12).

  Similarly, flow cytometer analysis shows that dopaminogenin marker D2 is expressed in differentiated hBMSc and not in non-differentiated cells (FIG. 13).

Example 9
Secretion of dopamine by hBMSc during differentiation is induced by neurotrophic factors Method:
Cell culture: hBMSc were cultured as described in Table 4 below.
Table 4
Culture medium used to induce dopamine secretion by hBMSc

  HPLC analysis: The sample was stabilized by adding 88 μl of 85% orthophosphoric acid and 4.4 mg metabisulfite to the ml sample. Dopamine was extracted by aluminum adsorption (alumina, Bioanalytical Systems Inc.). Separation of the injected sample (50 μl) was performed using an isocratic HPLC-electrochemical detection (HPLC-ECD) system using a reverse phase C18 column (125 × 4.6 mm diameter, Hichrom, Inc.) in a monochloroacetate buffer mobile phase. Achieved by tick elution. The flow rate was set at 1.2 ml / min and the oxidation potential of the analysis cell was set at +650 mV. Results were confirmed by co-elution with dopamine standards under various buffer conditions and detector settings.

result:
The measured amount of dopamine in the supernatant of differentiating hBMSc increased from an undetectable level to about 23 ng / ml (10 ⁵ cells) during a 72 hour incubation period in “Dopaminergic differentiation medium” ( FIG. 14A). Induction of cell polarization by KCl (supplementing the medium with 56 mM KCl followed by a 10 minute incubation) further enhanced dopamine secretion (FIG. 14B). The amount of DOPA synthesized by hBMSc differentiating (dopamine precursor) during the incubation period of 72 hours in the "dopaminergic differentiating medium", increased from about 10 pg / ml to 300 pg / ml (10 ⁵ cells) However, the amount of DOPAC (dopamine metabolite) increased from an undetectable level to about 105 ng / ml (10 ⁵ cells) during the 50 hour incubation period in “dopaminergic differentiation medium”. (FIG. 14D).

Example 10
Transplantation of mouse BMSc in the striatum of a rat model for Parkinson's disease improves rotational behavior Method:
Generation of mouse bone marrow stromal cells (mouse BMSc): Mouse BMS cells were obtained from transgenic male mice (Tg-EGFP; Hadjantatonakis et al., 1998) carrying enhanced green fluorescent protein. Mice were sacrificed by cervical dislocation and the tibia and femur were removed and placed in Hanks Balanced Salt Solution (HBSS). Mouse bone marrow cells were harvested by flushing the bone marrow using a syringe with a 25G needle (1 ml) packed with 0.5 ml sterile HBSS. Harvested cells were dissociated by gently pipetting until a homogenous single cell suspension such as milk was achieved. Single cell suspensions were washed in 5 ml HBSS and centrifuged at 1000 g for 20 minutes at room temperature. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in 10 ml growth medium.

  Cell culture: Isolated mouse bone marrow cells were cultured in “growth medium” (see Example 1 above) and incubated at 37 ° C. for 48 hours. Thereafter, the non-adhesive layer was discarded, and the strongly adhered cells were washed twice with PBS and cultured in fresh “growth medium”. The growth medium was changed every 3-4 days until the cells reached 70% -90% confluency. The cells were then collected and mixed in a trypsin-EDTA solution (0.05% trypsin in PBS and 25 mM EDTA) and incubated at 37 ° C. for 5 minutes before “pre-differentiation medium” (as described herein above). (See Example 2) and further incubated at 37 ° C. for 72 hours. Predifferentiated cells are washed in PBS and induced for neuronal differentiation by incubation at 37 ° C. for 12-72 hours in “differentiation medium” (see Example 1 above) did.

  Transplantation of mouse BMSc: Neurodifferentiated bone marrow stromal cells (mBMSc) were injected into the substantia nigra of female 6-OHDA-damaged rats using a stereotaxic frame (as described by Bjorkrund et al. (2002)). Saline injection was used as a control.

  Analysis of rotational behavior: 6-OHDA-damaged rats were treated with amphetamine (5 mg / kg) to induce rotational behavior. The rotational response to amphetamine was examined on days 3, 15, 30, and 45 after transplantation using a computerized rotometer (San Diego Instrument).

result:
Transplantation of neuron-induced mBMSCs into amphetamine-induced 6-OHDA rats significantly reduced the rat's rotational behavior from about 340 to only 25 revolutions per 2 hours 45 days after implantation (FIG. 15A). The relative rotational speed of the treated rats was reduced by 97.9% compared to rats treated with saline at day 45 after transplantation (FIG. 15B).

Example 11
Survival and migration of transplanted mouse BMSc in rat brain Method:
Neuronal differentiated Tg-EGEF mouse BMSc was prepared and implanted into the substantia nigra (both brain hemispheres) of 6-OHDA rats as described in Example 10 hereinabove. Treated and untreated rats (saline alone) were sacrificed 45 days after transplantation. Tissue samples obtained from brain hemispheres of damaged and non-damaged rats were sectioned and observed with a fluorescent microscope for the presence of green fluorescent protein (GFP) that was marked by the implanted mBMSc.

result:
mBMSc survived in the substantia nigra of treated rats 45 days after substantia nigra transplant (FIG. 15C) and migrated to the striatum of treated rats (FIG. 15D). Also. The transplanted cells were successfully transferred to the cortex and striatum (FIGS. 15C-15D). In rats injected with saline, the rotational behavior was unchanged.

Example 12
Mouse BMSc differentiated into oligodendrocyte precursors
Method:
Cell culture: Mouse B5 / EGFP (male) was sacrificed by cervical dislocation and prepared with 70% alcohol solution. The tibia and femur are removed and placed in Hank's Balanced Salt Solution (HBSS; Biological Industries, Bet-Haemek, Israel) and the mouse bone marrow cells are filled with a syringe (1 ml) with a 25G needle packed with 0.5 ml of sterile HBSS. Used to recover by flushing out bone marrow. Cells were dissociated by gentle pipetting several times until a homogeneous single cell suspension such as milk was achieved. The bone marrow aspirate was diluted and washed by adding 5 ml HBSS, centrifuging at 1000 g for 20 minutes at room temperature (RT), and removing the supernatant. The cell pellet was resuspended in 1 ml growth medium and diluted to 10 ml. Cells were placed in “growth medium” (see Example 1 herein above) in a 75 cm ² flask (Corning Incorporated, Corning, NY) for plastic tissue culture of polystyrene for 1 week. The cells were then transferred to a polylysine-coated slide chamber (3200 cells / well) supplemented with “growth medium” and incubated at 37 ° C. for 24 hours. The growth medium was then replaced with “oligodendrocyte differentiation medium” composed of DMEM supplemented with 2 mM glutamine, SPN, one or more of the following: bFGF (10 ng / ml), EGF ( 10 ng / ml to 20 ng / ml), interleukin-1b (20 ng / ml to 40 ng / ml), dbcAMP (1 mM to 2 mM), retinoic acid (0.5 μM or 1 μM), neurotrophin-3 (50 ng / ml or 100 ng / ml), human platelet derived growth factor (PDGF-AA; 5 ng / ml to 20 ng / ml), N2 supplement, triiodothyronien (T3; 40 ng / ml) and ciliary neurotrophic factor (20 ng / ml) CNTF).

  Immunoassay: Cultures were incubated at 37 ° C. for 1 day, 2 days or 6 days (with growth medium replaced every 2 days with fresh medium) and fixed in 4% PFA. Cells were blocked in 10% FCS solution and then incubated overnight at 4 ° C. with 5 ug / ml anti-A2B5 monoclonal antibody (1: 200; R & D systems; 1: 200). Cells were then washed twice in PBS for 10 minutes and incubated with goat anti-mouse Cy-3 secondary antibody (Jackson laboratories; 1: 500) for 20 minutes at room temperature. The appearance of cells that stained positive for A2B5 (an early marker of oligodendrocyte precursors) was measured using a fluorescence microscope equipped with the Image ProPlus cell counting program (Cybernetics).

result:
Neuronal cell morphology was observed in cells after 24 hours incubation cultured with any one of the inducers (IL-1b, dbcAMP, retinoic acid or NT-3) alone. The most prominent effect on cell morphology was induced by dbcAMP and NT-3 (FIG. 20A) as well as by IL-6 and thyroid factor 3 (data not shown).

  Cell survival was normal after 6 days of incubation with any one of the inducers alone (at any concentration). On the other hand, cell survival was reduced when inducers were combined.

  The appearance of cells that stained positive for the oligodendrocyte progenitor cell marker A2B5 was 8% overall. The highest appearance of antibody labeled A2B5 was found in cells treated with NT-3 (FIG. 20B).

  Thus, BMSc can be induced to differentiate into precursors of oligodendrocytes (myelin producing cells) that can be utilized to treat multiple sclerosis.

Example 13
Cell replacement in amyotrophic notochord sclerosis (ALS) Method:
Animals: TgN (SOD1-G93A) 1Gur transgenic mice expressing a mutated human superoxide dismutase-1 gene (SOD1; Gurney et al., 1994) were mated in CSJF1. This transgenic mouse was healthy until 3 months of age, but then ALS worsened, and was completely paralyzed at 4 to 5 months of age.

Transplantation: Neuron differentiated male mice BMSc were obtained as described in Example 10 hereinabove. Cells were injected into the spinal cord (cisterna) of female mutant SOD1 transgenic mice and wild type mice (10 ⁵ cells / injection; repeated with 5 animals per treatment group). Saline injection was used as a control.

  Assessment of motor function: Rotational behavior of treated and untreated mice (physiological saline only) was assessed weekly by using a rotometer (San Diego Instrument).

result:
Mice expressing SOD1 suffered from amyotrophic chordal sclerosis (ALS) as indicated by a substantial decline in convolution performance from week 7 onwards and complete paralysis after 4 to 5 months (FIG. 21).

  PCR analysis detected that the Y chromosome (representing male transplanted cells) was present in the spinal cord of treated female mice. The Y chromosome was not detected in any other tissue of treated female mice (FIG. 22).

  Seven-week-old treated wild-type rotational behavior was not significantly different from untreated (saline only) wild-type mice (FIG. 23). On the other hand, treated SOD1 mice showed a substantial reduction in rotational behavior indicating improved motor function (FIG. 24).

Example 14
Construction of Nurr1 expression vector Nuclear receptor associated 1 (Nurr-1) is a transcription factor involved in the differentiation of midbrain progenitors into dopamine neurons. Using human Nurr1 full-length cDNA (GeneBank Accession No. NM — 173171) using high fidelity Taq polymerase (TaKaRa, Japan) using 5′BamHI primer and 3′XbaI primer (primers shown by SEQ ID NOs: 31 to 32) And amplified. PCR conditions for amplification were as follows: 10 cycles of 95 ° C for 1 minute, 56 ° C for 1 minute, 72 ° C for 1 minute; 95 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute 10 cycles of 95 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute. The PCR product was digested with BamHI and XbaI restriction enzymes and the resulting fragment was expressed into the expression vector pcDNA-3.1A (Invitrogene) as illustrated in FIG. 17 using T4 DNA ligase (New England BioLabs). Inserted and cloned.

  Human bone marrow stromal cells (hBMSc; 60% -80% confluence) were transfected with pcDNANurr1 using FuGENE-6 transfection reagent according to manufacturer's recommendations (Roche Applied Science). Stably transfected cells were isolated in growth medium containing 500 μg / mL neomycin (G418 sulfate, Clontech, Palo Alto, Calif.). Total RNA was extracted from isolated neomycin resistant hBMSc as described by Chomczynski & Sacchi (1987) and the presence of Nurr1 transcript was determined by RT as described in Example 3 hereinabove. -Confirmed using PCR technique.

Example 15
Transformation of hBMSc for doxycycline-regulated expression of tyrosine hydroxylase Inducible tyrosine hydroxylase (TH) expression is achieved by transforming hBMSc with a responsive and regulatory vector that can be constructed as follows. be able to.

  TH responsive vector: 1.5 kb human tyrosine hydroxylase gene (TH, GenBank accession number NM_000360), human by PCR using primers shown by high fidelity Taq polymerase (TaKaRa, Japan), SEQ ID NOs: 39-40 It can be isolated from cDNA. The TH cDNA is inserted into pBI-EGFP (Clontech Tet-Off ™ gene expression system and Tet-On ™ gene expression system) as illustrated in FIG. 19A.

TH regulatory vector: The promoter of the 1.3 kb human NSE gene (HSENO2, GeneBank Accession No. X51956) is isolated from human cDNA by PCR using the promoters of SEQ ID NOs: 37-38. The NSE promoter cDNA was then transferred to the transcriptional activator gene (tTA; Gossen, M. and Bujard) in pRevTet-Off-IN (Clontech) instead of 5-LTR-Ψ ⁺ , as illustrated in FIG. , H., Proc. Natl. Acad. Sci. USA, 89: 5547-551, 1992). Positive clones carrying the neo ^r gene are selected using the antibiotic neomycin.

hBMSc can be transformed with both responsive and regulatory vectors (Tet-off / Tet-on system) using any of the transformation methods described hereinabove. When introduced into a cell, a regulatory vector containing an internal ribosome entry site (IRES) that exists between a transactivator (tTA) regulated by tetracycline and a gene encoding neomycin resistance (Neo ^r ). These two elements are expressed simultaneously. The expressed tTA binds to the tetracycline response element (TRE) present in the response vector, thereby activating TH transcription. However, when doxycycline (an antibiotic that crosses the blood-brain barrier) is present, iTA binding to TRE is blocked, thereby stopping TH transcription. This drug-controlled expression of TH is schematically illustrated in FIG. 19B.

  Thus, hBMSc can be genetically modified to express TH under the control of negative regulators such as doxycycline that can be administered orally.

  Since TH expression results in dopamine synthesis, genetically modified hBMSc can be used in cell replacement therapy to provide a safe and effective treatment for neurodegenerative diseases such as Parkinson's disease.

  Positive regulation using an agent that induces the expression of TH also includes, for example, an ecdysone-inducible mammalian expression system (Invitrogen) that utilizes a responsive vector pDHSP containing the TH gene and a regulator vector pVgRXR. It is understood that it can be achieved using. In the presence of an inducer (eg, ponasterone A or muristerone A), a functional ecdysone receptor binds upstream of the ecdysone responsive promoter and activates expression of TH.

  It will be appreciated that several features of the invention described in separate embodiments for the sake of clarity can also be combined and provided in a single embodiment. Conversely, the various features of the invention described in a single embodiment for simplicity can also be provided separately or in appropriate subcombinations.

While the invention has been described with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications and GenBanks mentioned herein are presented as if each individual publication, patent or patent application and GenBank were specifically and individually referenced in the present application. As such, it is incorporated into this application. Furthermore, citation or confirmation of any document in this application should not be regarded as a confession that such document can be used as prior art to the present invention.

The optical microscope image of a non-differentiation human bone marrow stromal cell (hBMSc) is illustrated (FIG. 1A-FIG. 1C). 2 illustrates a flow cytometer analysis of undifferentiated hBMSc. The optical microscope image of adult hBMSc differentiated into the neuron is illustrated. ³ H-thymidine uptake in hBMSc cultured in “differentiation medium” and hBMSc cultured in “pre-differentiation medium” (see Example 2 below), which indicates cell proliferation ). 2 illustrates RT-PCR analysis designed to identify neuronal transcripts. Illustrated are Northern blot analysis and real-time PCR analysis using a ³² P-labeled PCR product of NEGF2 as a probe. FIG. 5C illustrates Northern blot analysis utilizing a ³² P-labeled PCR product of neurofilament 200 (NF-200) as a probe. FIG. 5D illustrates Northern blot analysis utilizing a ³² P-labeled PCR product of neuron-specific enolase (NSE) as a probe. Illustrated are fluorescence micrographs of neuronal markers in hBMSc cultured for 12 hours to 5 days in “differentiation medium” (see Example 2 below) (FIGS. 6A-6B). FIG. 6A shows antibody labeled neuronal nucleus specific markers (NeuN; expressed after 12 hours), neurofilament heavy chain (NF-200; expressed after 24 hours), neuron specific enolase (NSE; 48 hours later). Illustrated) and nestin (expressed after 48 hours). FIG. 6B illustrates antibody labeled glial fibrillary acidic protein (GFAP; expressed after 48 hours) and β-tubulin III (expressed after 5 days). 7 illustrates Western blot analysis of differentiated hBMSc showing elevated expression of neuron specific enolase (NSE; FIG. 7A), neuron nucleus specific marker (NeuN; FIG. 7B) and nestin (FIG. 7C). 7 illustrates Western blot analysis of differentiated hBMSc showing elevated expression of neuron specific enolase (NSE; FIG. 7A), neuron nucleus specific marker (NeuN; FIG. 7B) and nestin (FIG. 7C). 2 illustrates a light micrograph of neuron-like differentiated hBMSc after incubation for 28 days in “long-term differentiation medium” (see Example 5 below). FIG. 6 illustrates antibody-labeled expression of the neuronal marker β-tubulin III in differentiated cells. FIG. 6 illustrates antibody-labeled expression of the neuronal marker MAP-2 in differentiated cells. FIG. 6 illustrates antibody-labeled expression of the neuronal marker nestin in differentiated cells. 2 illustrates RT-PCR analysis designed to identify dopaminergic markers in hBMSc cultured for 12-72 hours in “differentiation medium” (see Example 2 below). FIG. 11A illustrates real-time PCR analysis showing elevated TH transcription in differentiated cells (FIGS. 11A-11C). FIG. 11B illustrates a Western blot analysis showing elevated levels of TH in differentiated cells. FIG. 6 illustrates a fluorescence microscopic image highlighting antibody-labeled TH expression in differentiated cells. Co-expression of hBMSc incubated for 5 days in “differentiation medium” (see example 2 below) highlighting antibody-labeled vesicular monoamine transporter 2 (VMAT-2) in differentiated cells. The focused fluorescence microscope image is illustrated. FIG. 4 illustrates flow cytometer analysis of hBMSc incubated for 48 hours in “differentiation medium” (see Example 2 in the Examples below). FIG. 14A illustrates measured dopamine levels in hBMSc supernatants incubated for 0-96 hours in “differentiation medium” (see Example 2 below). FIG. 14B illustrates the measured dopamine levels in the supernatant of hBMSc incubated in “differentiation medium” for 0-96 hours and then further incubated in 56 mM KCl solution for 10 minutes. FIG. 14C illustrates the measured level of dopamine precursor DOPA in the supernatant of hBMSc incubated for 0-72 hours in “differentiation medium”. FIG. 14D illustrates the measured level of dopamine metabolite DOPAC in the supernatant of hBMSc cultured for 0-50 hours in “differentiation medium”. FIG. 15A illustrates the change in rotational speed over time after transplantation, showing a decreasing rotation (which indicates improved motor function) at day 45 post-transplant. FIG. 15B illustrates the change in relative rotation over time (as compared to untreated mice) after transplantation, showing a 97.9% decrease in relative rotation 45 days after transplantation. FIG. 15C illustrates a fluorescence microscopic image highlighting transplanted mBMSc present in the substantia nigra of treated mice 45 days after transplantation. FIG. 15D illustrates a fluorescence microscopic image highlighting transplanted mBMSc present in the striatum of treated mice 45 days after substantia nigra transplantation. FIG. 16A illustrates Western blot analysis showing expression of tryptophan hydroxylase. FIG. 16B illustrates RT-PCR analysis showing tryptophan hydroxylase transcription. 2 illustrates HPLC analysis showing the synthesis of DOPAC (dopamine metabolite) and 5HIAA (serotonin metabolite). Illustrates the construction of an expression vector containing the Nurr-1 coding sequence inserted into pcDNA-3.1A (Invitrogene). Illustrates a construct designed for negative selection of dopaminergic cells. The regulator constructs and response constructs of this system are illustrated. Figure 2 illustrates a schematic diagram describing the mode of action of this system. The fluorescence-microscope image of BMSc (mBMSc) which the GFP-Tg mouse differentiated is illustrated (FIG. 20A-FIG. 20B). FIG. 20A illustrates a fluorescence microscopic image of morphological changes induced by “differentiation medium” (see Example 1). FIG. 20B illustrates a fluorescence microscopic image highlighting antibody-labeled A2B5 (marker of oligodendrocyte precursor) expressed in NT-3 induced mBMSc. The time-dependent change in the rotation result of the mouse | mouth (Animal model of amyotrophic lateral sclerosis (ALS)) which expresses SOD1 is illustrated. 2 illustrates PCR analysis of various tissues of female mice sampled one week after male-derived mBMSc was transplanted into the cisterna magna. 7 illustrates the rotarod performance (indicating rotational behavior) of wild type mice that received mBMSc transplantation into their spinal cord at 7 weeks of age. 7 illustrates the rotarod performance (showing rotational behavior) of SOD1 mice (a model of amyotrophic lateral sclerosis) that received mBMSc transplantation into the spinal cord at the age of 7 weeks.

  SEQ ID NOs: 1-40 are single-stranded DNA oligonucleotide sequences.

[Sequence Listing]

Claims (75)

  A method of treating a neurodegenerative disorder comprising administering to an individual in need thereof a cell capable of exogenously regulatable synthesis of a neurotransmitter and thereby capable of treating the neurodegenerative disorder .   2. The method of claim 1, further comprising subjecting the individual to an agent or condition capable of modulating the synthesis of the neurotransmitter in the cell.   3. The method of claim 2, wherein the cells are genetically modified to allow exogenously regulatable synthesis of the neurotransmitter.   The cell is transformed with an expression construct comprising a polynucleotide sequence encoding an enzyme involved in the synthesis of the neurotransmitter, wherein the expression construct is capable of controlling expression of the polynucleotide by the agent. The method of claim 2, wherein the method is designed to be   5. The method of claim 4, wherein the agent is capable of down-regulating the expression of the enzyme involved in the synthesis of the neurotransmitter.   5. The method of claim 4, wherein the agent is capable of upregulating expression of the enzyme involved in the synthesis of the neurotransmitter.   The cell is placed under transcriptional control of a first polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis, placed under transcriptional control of a first regulatory sequence, and a second regulatory sequence. And at least one expression construct comprising a second polynucleotide sequence encoding a transactivator, wherein the transactivator is in the absence of the agent, the first activator. 4. The method of claim 3, wherein the regulatory sequence of can be activated to cause transcription of the first polynucleotide sequence.   8. The method of claim 7, wherein the agent is doxycycline.   8. The method of claim 7, wherein the transactivator is a tetracycline-controlled transactivator.   8. The method of claim 7, wherein the first regulatory sequence comprises a tetracycline response element.   8. The method of claim 7, wherein the enzyme is selected from the group consisting of tyrosine hydroxylase, DOPA decarboxylase, GTP cyclohydrolase I, dopamine β-hydroxylase, glutamate decarboxylase, tryptophan-5 monooxygenase, and choline acetyltransferase. .   8. The method of claim 7, wherein the second regulatory sequence comprises a human specific enolase promoter.   The method of claim 1, wherein the neurodegenerative disorder is selected from the group consisting of Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, autoimmune encephalomyelitis, Alzheimer's disease and Huntington's disease.   14. The method of claim 13, wherein the neurodegenerative disorder is Parkinson's disease.   2. The method of claim 1, wherein the neurotransmitter is selected from the group consisting of dopamine, norepinephrine, epinephrine, [gamma] -aminobutyric acid, serotonin, acetylcholine and glutamic acid.   The method of claim 15, wherein the neurotransmitter is dopamine.   The method of claim 1, wherein the cell is a bone marrow cell.   The method according to claim 17, wherein the bone marrow cells are bone marrow stromal cells.   The method of claim 1, wherein the cell is a neuron-like cell.   20. The method of claim 19, wherein the neuron-like cell does not have an endogenous activity of the enzyme involved in the synthesis of the neurotransmitter.   20. The method of claim 19, wherein the neuron-like cell expresses at least one neuronal marker.   The neuronal marker is selected from the group consisting of CD90, neuron specific nucleoprotein, neurofilament heavy chain, neuron specific enolase, β-tubulin 3, MAP-2, tyrosine hydroxylase, microtubule binding protein, nestin and calbindin 24. The method of claim 21, wherein:   2. The method of claim 1, wherein administering the cell is accomplished by transplanting the cell into the brain tissue of the individual.   2. The method of claim 1, wherein administering the cell is accomplished by transplanting the cell into the spinal cord of the individual.   3. The method of claim 2, wherein exposing the individual is accomplished by orally administering the agent to the individual.   The method of claim 2, wherein exposing the individual is accomplished by injecting the agent into the individual.   (A) administering a cell capable of exogenously regulatable synthesis of a neurotransmitter to an individual in need thereof; and (b) modulating the synthesis of the neurotransmitter in the cell. A method of treating a neurodegenerative disorder comprising the step of periodically exposing the individual to an agent or condition capable of treating the neurodegenerative disorder.   28. The method of claim 27, wherein the cells are genetically modified to allow exogenously regulatable synthesis of the neurotransmitter.   The cell is transformed with an expression construct comprising a polynucleotide sequence encoding an enzyme involved in the synthesis of the neurotransmitter, wherein the expression construct is capable of controlling expression of the polynucleotide by the agent. 30. The method of claim 28, designed to be   30. The method of claim 29, wherein the agent is capable of down-regulating the expression of the enzyme involved in the synthesis of the neurotransmitter.   30. The method of claim 29, wherein the agent is capable of upregulating expression of the enzyme involved in the synthesis of the neurotransmitter.   28. The method of claim 27, wherein the neurodegenerative disorder is selected from the group consisting of Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, autoimmune encephalomyelitis, Alzheimer's disease, and Huntington's disease.   35. The method of claim 32, wherein the neurodegenerative disorder is Parkinson's disease.   28. The method of claim 27, wherein the neurotransmitter is selected from the group consisting of dopamine, norepinephrine, epinephrine, [gamma] -aminobutyric acid, serotonin, acetylcholine and glutamic acid.   35. The method of claim 34, wherein the neurotransmitter is dopamine.   30. The method of claim 29, wherein the enzyme is selected from the group consisting of tyrosine hydroxylase, DOPA decarboxylase, GTP cyclohydrolase I, dopamine β-hydroxylase, glutamate decarboxylase, tryptophan-5 monooxygenase, and choline acetyltransferase. .   28. The method of claim 27, wherein the cell is a bone marrow cell.   38. The method of claim 37, wherein the bone marrow cells are bone marrow stromal cells.   28. The method of claim 27, wherein the cell is a neuron-like cell.   40. The method of claim 39, wherein the neuron-like cell expresses at least one neuronal marker.   The neuronal marker is selected from the group consisting of CD90, neuron specific nucleoprotein, neurofilament heavy chain, neuron specific enolase, β-tubulin 3, MAP-2, tyrosine hydroxylase, microtubule binding protein, nestin and calbindin 42. The method of claim 40, wherein:   The cell has a tyrosine hydroxylase under regulatable control of the agent such that in the absence of the agent, an activator molecule binds to a response element, thereby up-regulating the expression of the tyrosine hydroxylase. 28. The method of claim 27, wherein the method is genetically modified to cause expression.   43. The method of claim 42, wherein the agent is doxycycline.   43. The method of claim 42, wherein the activator molecule is a tetracycline-controlled transactivator.   43. The method of claim 42, wherein the response element is a tetracycline response element.   A nucleic acid construct comprising a polynucleotide sequence encoding an enzyme involved in the synthesis of a neurotransmitter, disposed under the control of a regulatory sequence capable of regulating the expression of said enzyme in a mammalian cell.   47. The nucleic acid construct of claim 46, wherein the regulatory sequence comprises a tetracycline response element.   47. The nucleic acid of claim 46, wherein the enzyme is selected from the group consisting of tyrosine hydroxylase, DOPA decarboxylase, GTP cyclohydrolase I, dopamine β-hydroxylase, glutamate decarboxylase, tryptophan-5 monooxygenase, and choline acetyltransferase. Constructs.   A first expression construct comprising a first polynucleotide sequence encoding an enzyme involved in neurotransmitter synthesis disposed under transcriptional control of a first regulatory sequence; and a second encoding a transactivator. And a second expression construct comprising a polynucleotide sequence disposed under transcriptional control of a second regulatory sequence, wherein the transactivator activates the first regulatory sequence. A construct system capable of transcribing the first polynucleotide sequence.   50. The construct system of claim 49, wherein the neurotransmitter is dopamine.   50. The construct system of claim 49, wherein the enzyme is a tyrosine hydroxylase.   50. The construct system of claim 49, wherein the first regulatory sequence comprises a tetracycline response element.   50. The construct system of claim 49, wherein the transactivator is a tetracycline-controlled transactivator.   49. A cell comprising the nucleic acid construct of claim 46.   55. The cell of claim 54, wherein the cell is a neuron-like cell that does not have an endogenous activity of the enzyme involved in the synthesis of the neurotransmitter.   55. The cell of claim 54, further comprising a polynucleotide encoding an apoptosis inhibiting polypeptide.   50. A cell comprising the construct system of claim 49.   57. The cell of claim 56, wherein the cell is a neuron-like cell that does not have an endogenous activity of the enzyme involved in the synthesis of the neurotransmitter.   58. The cell of claim 57 further comprising a polynucleotide encoding an apoptosis inhibiting polypeptide.   A method for producing cells used in treating a neurodegenerative disorder, comprising: (a) a step of isolating bone marrow cells; (b) a growth medium capable of maintaining and / or expanding said bone marrow cells (C) selecting bone marrow stromal cells from cells obtained from step (b), and (d) at least one polyunsaturated fatty acid and at least one differentiation agent. Incubating said bone marrow stromal cells in a differentiation medium comprising, thereby producing cells used in treating neurodegenerative disorders.   61. The method of claim 60, wherein the growth medium comprises DMEM, SPN, L-glutamine, FCS, 2- [beta] -mercaptoethanol, non-essential amino acids and EGF.   61. The method of claim 60 further comprising incubating the cells obtained from step (c) in a pre-differentiation medium prior to step (d), thereby facilitating the differentiation of the cells into neuron-like cells. .   63. The method of claim 62, wherein the pre-differentiation medium contains bFGF.   64. The method of claim 63, wherein the pre-differentiation medium further comprises DMEM, SPN, L-glutamine, N2 supplement and FCS.   61. The method of claim 60, wherein the at least one polyunsaturated fatty acid is docosahexaenoic acid.   61. The method of claim 60, wherein the at least one differentiation agent is selected from the group consisting of BHA, dbcAMP, and IBMX.   61. The method of claim 60, wherein the differentiation medium further comprises DMEM, SPN, L-glutamine, N2 supplement and retinoic acid.   61. The method of claim 60, wherein step (a) is accomplished by aspiration.   61. The method of claim 60, wherein step (c) is accomplished by collecting surface adherent cells.   A cell population comprising bone marrow-derived stromal cells capable of synthesizing neurotransmitters.   71. The cell population of claim 70, wherein the neurotransmitter is dopamine.   71. The cell population of claim 70, wherein the neurotransmitter is serotonin.   A mixed cell population comprising bone marrow derived neuron-like cells capable of synthesizing at least two types of neurotransmitters.   74. The mixed cell population of claim 73, wherein the at least two types of neurotransmitters include dopamine.   74. The mixed cell population of claim 73, wherein the at least two types of neurotransmitters comprise serotonin.