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Title:  Method for generating dopaminergic cells derived from neural precursors

United States Patent:  6,284,539

Inventors:  Bowen; David C. (Washington, DC); Johe; Karl K. (Potomac, MD)

Assignee:  NeuralStem Biopharmaceuticals, Ltd. (College Park, MD)

Appl. No.:  169309

Filed:  October 9, 1998

Abstract

The present invention describes a novel method to direct a particular set of fate choice decisions by multipotential precursor cells from the central nervous system. Specifically we show that introducing the gene coding for the nuclear receptor, Nurr1, into central nervous system (CNS) stem cells causes cells to adopt a dopaminergic cell fate. One use of this technology would be to prepare in vitro neural populations enriched in dopaminergic cells for transplantation in Parkinson's Disease or other neurological disorders. Furthermore, the finding that Nurr1 expression induces a dopaminergic phenotype suggests that introducing this gene into the brains of patients in which dopaminergic cells are degenerating or have been injured may promote the functional recovery of these neurons and thus the clinical recovery of the patient. Finally, the technology described in this application could be incorporated into a program of drug screening or gene discovery.

SUMMARY OF THE INVENTION

As stated above, the present invention discloses a method for generating dopaminergic cells from a culture of mammalian CNS stem cells, methods for treating a patient with a neurological disorder, such as Parkinson's Disease, methods of screening for compounds which generate or increase the number of dopaminergic cells in a culture of mammalian CNS stem cells and methods for discovering genes involved in generating dopaminergic cells.

More specifically, the method for generating dopaminergic cells in a culture of mammalian CNS stem cells comprises culturing mammalian CNS stem cells in vitro; introducing an exogenous gene encoding a transcriptional regulator into the mammalian CNS stem cells in the culture; incubating the mammalian CNS stem cells; and identifying dopaminergic cells in the culture.

In a preferred embodiment of the method, the exogenous gene encoding a transcriptional regulator is selected from the group consisting of Nurr1, RNR-1, HZF-3, TINUR, NOT, Nur77, NGF1-B, NAK-1, N10, NOR-1 and NOR-2. In a more preferred embodiment, the exogenous gene encoding a transcriptional regulator is Nurr1.

In another preferred embodiment, the method further comprises incubating the mammalian CNS stem cells with an agonist of protein kinase A activity. In a more preferred embodiment of the method, the agonist of protein kinase A activity is selected from the group consisting of forskolin; 1,9-dideoxy-forskolin; 6-[[(2-carbethoxyethyl)amino]carbonyl]-forskolin; 6-acetyl-7-deacetyl-forskolin; 6-O-[3'-(piperidino)propionyl]-forskolin hydrochloride; 7-deacetyl-forskolin; 7-deacetyl-6-(N-acetylglycyl)-forskolin; 7-deacetyl-7-[O-(N-methylpiperazino)-g-butyryl]-forskolin, dihydrochloride; 7-deacetyl-7-O-hemisuccinyl-forskolin; 3',5'cyclic monophosphate (cAMP); 8-chloro-cAMP; 8-bromo cAMP; 8-(4-chloropheylthio)-cAMP; dibutyryl-cAMP; dioctanoyl-cAMP; N6-monobutyryl-cAMP; adenosine 3', 5'cyclic monophosphorthioate, Sp-isomer; and 8-bromo-adenosine 3',5'cyclic monophosphorthioate, Sp-isomer. In a most preferred embodiment of the method, the agonist of protein kinase A activity is forskolin.

In another preferred embodiment of the method, the mammalian CNS stem cells are selected from ventral midbrain, dorsal midbrain, lateral ganglionic eminence, hippocampus, cerebral cortex, striatum, septum, diencephalon, mesencephalon, hindbrain and spinal cord. In a more preferred embodiment of the method, the mammalian CNS stem cells are human CNS stem cells.

A method for treating a patient with a neurological disorder comprises culturing mammalian CNS stem cells in vitro; introducing an exogenous gene encoding a transcriptional regulator into the mammalian CNS stem cells in the culture; identifying dopaminergic cells in the culture; and transplanting dopaminergic cells into the brain of a patient in need thereof.

In a preferred embodiment of the method, the exogenous gene encoding a transcriptional regulator is selected from the group consisting of Nurr1, RNR-1, HZF-3, TINUR, NOT, Nur77, NGF1-B, NAK-1, N10, NOR-1 and NOR-2. In a more preferred embodiment of the method, the exogenous gene encoding a transcriptional regulator is Nurr1.

In another preferred embodiment, the method further comprises incubating the mammalian CNS stem cells with an agonist of protein kinase A activity. In a more preferred embodiment of the method, the agonist of protein kinase A activity is selected from the group consisting of forskolin; 1,9-dideoxy-forskolin; 6-[[(2-carbethoxyethyl)amino]carbonyl]-forskolin; 6-acetyl-7-deacetyl-forskolin; 6-O-[3'-(piperidino)propionyl]-forskolin hydrochloride; 7-deacetyl-forskolin; 7-deacetyl-6-(N-acetylglycyl)-forskolin; 7-deacetyl-7-[O-(N-methylpiperazino)-g-butyryl]-forskolin, dihydrochloride; 7-deacetyl-7-O-hemisuccinyl-forskolin; 3',5'cyclic monophosphate (cAMP); 8-chloro-cAMP; 8-bromo cAMP; 8-(4-chloropheylthio)-cAMP; dibutyryl-cAMP; dioctanoyl-cAMP; N6-monobutyryl-cAMP; adenosine 3', 5'cyclic monophosphorthioate, Sp-isomer; and 8-bromo-adenosine 3',5'cyclic monophosphorthioate, Sp-isomer. In a most preferred embodiment of the method, the agonist of protein kinase A activity is forskolin.

In another preferred embodiment of the method, the mammalian CNS stem cells are selected from ventral midbrain, dorsal midbrain, lateral ganglionic eminence, hippocampus, cerebral cortex, striatum, septum, diencephalon, mesencephalon, hindbrain and spinal cord.

In more preferred embodiments of the method, the mammalian CNS stem cells are human CNS stem cells and the neurological disorder is Parkinson's Disease.

A method for treating a patient experiencing compromised dopaminergic function comprises introducing a Nurr1 gene into the brain of a patient experiencing compromised dopaminergic function. In a preferred embodiment of the method, the compromised dopaminergic function results from Parkinson's Disease.

Another method for treating a patient experiencing compromised dopaminergic function comprises administering a substance capable of upregulating Nurr1 expression or activity to a patient experiencing compromised dopaminergic function. In a preferred embodiment of the method, the compromised dopaminergic function results from Parkinson's Disease.

A method of screening for substances that increase Nurr1 expression comprises culturing cells endogenously expressing Nurr1 ; exposing the cultured cells to a substance of interest; and determining whether the substance increased the expression of Nurr1.

Another method of screening for substances that increase Nurr1 expression comprises introducing a construct comprising a Nurr1 promoter linked to a reporter gene into a culture of cells; exposing the cultured cells to a substance of interest; and determining whether the substance increased the expression of the reporter gene.

Another method of screening for substances that increase Nurr1 activity comprises introducing a construct comprising a Nurr1 response element (AAAGGTCA) linked to a reporter gene into a culture of cells, where the cells express Nurr1 either endogenously or due to the introduction of an exogenous Nurr1 gene; exposing the cultured cells to a substance of interest; and determining whether the substance increased expression of the reporter gene.

A method for identifying genes in a signalling pathway by which a cell acquires a dopaminergic phenotype comprises introducing a Nurr1 gene into a culture of cells; examining genes expressed in the cultures into which the Nurr1 gene has been introduced relative to genes expressed in cultures in which the Nurr1 gene has not been introduced; and identifying genes in the signalling pathway by which a cell acquires a dopaminergic phenotype.

Another methods for identifying genes in a signalling pathway by which a cell acquires a dopaminergic phenotype comprises introducing a construct comprising a Nurr1 coding sequence under control of an inducible promoter into a culture of cells; exposing the cells to an inducing agent capable of regulating expression of genes under the control of the inducible promoter or to an inactive control substance; comparing a complement of genes expressed in the cells that have been exposed to the inducing agent with a complement of genes expressed by the cells treated with the inactive control substance; and identifying genes in the signalling pathway by which a cell acquires a dopaminergic phenotype.

Another method for identifying genes in a signalling pathway by which a cell acquires a dopaminergic phenotype comprises introducing a construct coding for a chimeric protein in which a DNA binding domain of Nurr1 is fused to a ligand-binding domain of a nuclear receptor having a known activating ligand; exposing the cells to the activating ligand or to an inactive control substance; comparing a complement of genes expressed in the cells that have been exposed to the activating ligand with a complement of genes expressed by the cells treated with the inactive control substance; and identifying genes in the signalling pathway by which a cell acquires a dopaminergic phenotype.

Another method for identifying genes in a signalling pathway by which a cell acquires a dopaminergic phenotype comprises culturing cells that express Nurr1 ; exposing the cells to a substance capable of upregulating Nurr1 expression or activity or to an inactive control substance; comparing a complement of genes expressed in the cells that have been exposed to the substance capable of upregulating Nurr1 expression or activity with a complement of genes expressed by the cells treated with the inactive control substance; and identifying genes in the signalling pathway by which a cell acquires a dopaminergic phenotype.

Claim 1 of 7 Claims

What is claimed is:

1. A method for generating tyrosine hydroxylase expressing cells in a culture of mammalian CNS stem cells, comprising the steps of:

a) culturing mammalian CNS stem cells in vitro;

b) introducing a polynucleotide that encodes a transcriptional regulator into the mammalian CNS stem cells in the culture,

wherein the polynucleotide encodes a transcriptional regulator selected from the group consisting of Nurr1, RNR-1, HZF-3, TINUR, and NOT, and wherein the polynucleotide is expressed;

c) incubating the mammalian CNS stem cells; and

d) identifying tyrosine hydroxylase expressing cells in the culture.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

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