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Title:  Genetic demonstration of requirement for nkx6.1, nkx2.2 and nkx6.2 in ventral neuron generation
United States Patent: 
7,312,081
Issued: 
December 25, 2007

Inventors: 
Jessell; Thomas M. (Bronx, NY), Briscoe; James (London, GB), Ericson; Johan (Hasselby, SE), Rubenstein; John L. R. (San Francisco, CA), Sander; Maike (Hamburg, DE)
Assignee: 
The Trustees of Columbia University in the City of New York (New York, NY), The Regents of the University of California (Alameda, CA)
Appl. No.: 
10/362,437
Filed: 
August 31, 2001
PCT Filed: 
August 31, 2001
PCT No.: 
PCT/US01/27256
371(c)(1),(2),(4) Date: 
August 01, 2003
PCT Pub. No.: 
WO02/18545
PCT Pub. Date: 
March 07, 2002


 

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Abstract

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.1 or Nkx6.2 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron. Provided are methods of diagnosing a motor neuron degenerative disease in a subject. Also provides is a method of treating neuronal degeneration in a subject which comprises implanting in diseased neural tissue of the subject a neural stem cell which is capable of expressing homeodomain Nkx6.1 or Nkx6.2 protein under conditions such that the stem cell is converted into a motor neuron after implantation, thereby treating neuronal degeneration in the subject.

Description of the Invention

SUMMARY OF THE INVENTION

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.1 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention also provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) sequencing the nucleic acid sample; and c) comparing the nucleic acid sequence of step (b) with a Nkx6.1 nucleic acid sequence from a subject without motor neuron degenerative disease, wherein a difference in the nucleic acid sequence of step (b) from the Nkx6.1 nucleic acid sequence from the subject without motor neuron degenerative disease indicates that the subject has the motor neuron degenerative disease.

This invention provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) performing a restriction digest of the nucleic acid sample with a panel of restriction enzymes; c) separating the resulting nucleic acid fragments by size fractionation; d) hybridizing the resulting separated nucleic acid fragments with a nucleic acid probe(s) of at least 15 nucleotide capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human Nkx6.1 protein, wherein the sequence of the nucleic acid probe is labeled with a detectable marker, and hybridization of the nucleic acid probe(s) with the separated nucleic acid fragments results in labeled probe-fragment bands; e) detecting labeled probe-fragment bands, wherein the labeled probe-fragment bands have a band pattern specific to the nucleic acid of the subject; and f) comparing the band pattern of the detected labeled probe-fragment bands of step (d) with a previously determined control sample, wherein the control sample has a unique band pattern specific to the nucleic acid of a subject having the motor neuron degenerative disease, wherein identity of the band pattern of the detected labeled probe-fragment bands of step (d) to the control sample indicates that the subject has the motor neuron degenerative disease.

This invention provides a method of treating neuronal degeneration in a subject which comprises implanting in diseased neural tissue of the subject a neural stem cell which comprises an isolated nucleic acid molecule which is capable of expressing homeodomain Nkx6.1 protein under conditions such that the stem cell is converted into a motor neuron after implantation, thereby treating neuronal degeneration in the subject.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.2 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.1 in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.2 in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention provides a method of diagnosing a neurodegenerative disease in a subject which comprises: a) obtaining a suitable sample from the subject; b) extracting nucleic acid from the suitable sample; c) contacting the resulting nucleic acid with a nucleic acid probe, which nucleic acid probe (i) is capable of hybridizing with the nucleic acid of Nkx6.1 or Nkx6.2 and (ii) is labeled with a detectable marker; d) removing unbound labeled nucleic acid probe; and e) detecting the presence of labeled nucleic acid, wherein the presence of labeled nucleic acid indicates that the subject is afflicted with a chronic neurodegenerative disease, thereby diagnosing a chronic neurodegenerative disease in the subject.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) sequencing the nucleic acid sample; and c) comparing the nucleic acid sequence of step (b) with a Nkx6.1 nucleic acid sequence from a subject without motor neuron degenerative disease, wherein a difference in the nucleic acid sequence of step (b) from the Nkx6.1 nucleic acid sequence from the subject without motor neuron degenerative disease indicates that the subject has the motor neuron degenerative disease.

In an embodiment of the above-described method of diagnosing a motor neuron degenerative disease in a subject the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.

As used herein, the term "sample" includes but is not limited to tonsil tissue, lymph nodes, spleen, skin lesions, blood, serum, plasma, cerebrospinal fluid, lymphocytes, urine, transudates, exudates, bone marrow cells, or supernatant from a cell culture.

As used herein, "subject" means any animal or artificially modified animal. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. The subjects include but are not limited to mice, rats, dogs, guinea pigs, ferrets, rabbits, chicken and primates. In the preferred embodiment, the subject is a human being.

This invention provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) performing a restriction digest of the nucleic acid sample with a panel of restriction enzymes; c) separating the resulting nucleic acid fragments by size fractionation; d) hybridizing the resulting separated nucleic acid fragments with a nucleic acid probe(s) of at least 15 nucleotide capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human Nkx6.1 protein, wherein the sequence of the nucleic acid probe is labeled with a detectable marker, and hybridization of the nucleic acid probe(s) with the separated nucleic acid fragments results in labeled probe-fragment bands; e) detecting labeled probe-fragment bands, wherein the labeled probe-fragment bands have a band pattern specific to the nucleic acid of the subject; and f) comparing the band pattern of the detected labeled probe-fragment bands of step (d) with a previously determined control sample, wherein the control sample has a unique band pattern specific to the nucleic acid of a subject having the motor neuron degenerative disease, wherein identity of the band pattern of the detected labeled probe-fragment bands of step (d) to the control sample indicates that the subject has the motor neuron degenerative disease.

In an embodiment of the above-described method of diagnosing a motor neuron degenerative disease in a subject the nucleic acid is DNA. In a further embodiment of the above-described method the nucleic acid is RNA. In another embodiment the size fractionation in step (c) is effected by a polyacrylamide or agarose gel. In another embodiment the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label. In yet another embodiment the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.

As used herein, "detectable marker" includes but is not limited to a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker. As used herein, "labels" include radioactive isotopes, fluorescent groups and affinity moieties such as biotin that facilitate detection of the labeled peptide. Other labels and methods for attaching labels to compounds are well-known to those skilled in the art.

The phrase "specifically hybridizing" and the phrase "selectively hybridizing" describe a nucleic acid that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA. By selectively hybridizing it is meant that a nucleic acid binds to a given target in a manner that is detectable in a different manner from non-target sequence under high stringency conditions of hybridization. "Complementary", "antisense" or "target" nucleic acid sequences refer to those nucleic acid sequences which selectively and specifically hybridize to a nucleic acid. Proper annealing conditions depend, for example, upon a nucleic acid's length, base composition, and the number of mismatches and their position on the nucleic acid, and must often be determined empirically. For discussions of nucleic acid design and annealing conditions for hybridization, see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3 or Ausubel, F., et al. (1987) Current Protocols in Molecular Biology, New York. The above hybridizing nucleic acids may vary in length. The hybridizing nucleic acid length includes but is not limited to a nucleic acid of at least 15 nucleotides in length, of at least 25 nucleotides in length, or at least 50 nucleotides in length.

This invention provides a method of treating neuronal degeneration in a subject which comprises implanting in diseased neural tissue of the subject a neural stem cell which comprises an isolated nucleic acid molecule which is capable of expressing homeodomain Nkx6.1 protein under conditions such that the stem cell is converted into a motor neuron after implantation, thereby treating neuronal degeneration in the subject.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.2 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

In one embodiment of the above method, the nucleic acid introduced into the stem cell incorporates into the chromosomal DNA of the stem cell. In another embodiment of the above method, the nucleic acid is introduced by transfection or transduction. In a further embodiment of the above method, the ventral neuron is a motor neuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.1 in the stem cell so as to thereby convert the stem cell into the ventral neuron. In one embodiment of the above method, the ventral neuron is a motor neuron, a V2 interneuron or a V3 interneuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.2 in the stem cell so as to thereby convert the stem cell into the ventral neuron. In one embodiment of the above method, the ventral neuron is a motor neuron.

This invention provides a method of diagnosing a neurodegenerative disease in a subject which comprises: a) obtaining a suitable sample from the subject; b) extracting nucleic acid from the suitable sample; c) contacting the resulting nucleic acid with a nucleic acid probe, which nucleic acid probe (i) is capable of hybridizing with the nucleic acid of Nkx6.1 or Nkx6.2 and (ii) is labeled with a detectable marker; d) removing unbound labeled nucleic acid probe; and e) detecting the presence of labeled nucleic acid, wherein the presence of labeled nucleic acid indicates that the subject is afflicted with a chronic neurodegenerative disease, thereby diagnosing a chronic neurodegenerative disease in the subject.

In one embodiment of the above method, the suitable sample is spinal fluid. In another embodiment of the above method, the nucleic acid is DNA. In a further embodiment of the above method, the nucleic acid is RNA.
 

Claim 1 of 4 Claims

1. A method of converting a neural stem cell into a ventral neuron which comprises introducing into the neural stem cell, in vitro, a nucleic acid encoding homeodomain transcription factor Nkx6.2 protein, wherein the encoded protein is expressed in the neural stem cell so as to thereby convert the neural stem cell into the ventral neuron.
 

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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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