Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Nitrogen containing other than solely as a nitrogen in an...
Reexamination Certificate
2001-08-24
2003-06-03
Spivack, Phyllis G. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Nitrogen containing other than solely as a nitrogen in an...
Reexamination Certificate
active
06573300
ABSTRACT:
BACKGROUND OF THE INVENTION
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by degeneration of spinal cord anterior horn cells, which lead to muscular paralysis with muscular atrophy. SMA patients are afflicted to varying degrees of severity and therefore clinically categorized as type 1 (severe), 2 (intermediate), or 3 (mild), according to age of onset and rate of progression. The disorder is found in approximately 1 in 10,000 live births and has a carrier frequency of 1 in 50 (Zerres (1997)
J. Neurol. Sci.
146:67-72). Type 1 patients have a life expectancy of 18 months or less, whereas type 3 patients can survive into adulthood.
All types of human spinal muscular atrophy are due to mutations in the SMN1 gene of the 5q13 locus on chromosome 5. In most individuals, there exists a second gene, SMN2, adjacent to SMN1. Both SMN1 and SMN2 encode SMN, a 294 amino acid RNA-binding protein (Lefebvre et al. (1995)
Cell
80:155-165; Monani et al. (1999)
Hum. Mol. Genet.
8:1177-1183). At the genomic level, only five nucleotides have been found that differentiate the SMN1 gene from the SMN2 gene. Furthermore, the two genes produce identical mRNAs, except for a silent nucleotide change in exon 7, namely, a C→T change six base pairs inside exon 7 in SMN2 as compared to SMN1. This mutation modulates the activity of an exon splicing enhancer (Lorson and Androphy (2000)
Hum. Mol. Genet.
9:259-265). The result of this and the other nucleotide changes in the intronic and promoter regions is that most SMN2 transcripts lack exons 3, 5, or 7. In contrast, the mRNA transcribed from the SMN1 gene is generally a full-length mRNA with only a small fraction of its transcripts spliced to remove exon 3, 5, or 7 (Gennarelli et al. (1995)
Biochem. Biophys. Res. Commun.
213:342-348; Jong et al. (2000)
J. Neurol. Sci.
173:147-153).
Furthermore, there is substantially less transcription of SMN2 than SMN1 in most individuals. As the severity of deletions of the SMN1 indicates, the low level of full-length SMN protein produced by SMN2 is insufficient to protect against spinal muscular atrophy disease (Lefebvre, supra; Coovert et al. (1997)
Hum. Mol. Genet.
6:1205-1214).
There is no effective treatment to date for spinal muscular atrophy disease.
SUMMARY OF THE INVENTION
The invention is based on the discovery that different classes of compounds have been identified, using new methods, as being useful in the modulation of SMN exon 7 gene expression, and therefore as being useful in the treatment of SMA. It has also been discovered that cells harvested from SMA patients and transgenic animals having particular genotypes and phenotypes are useful in the new screening methods.
Accordingly, the invention features a method for modulating SMN gene expression in a subject. The method includes administering to the subject an amount of hydroxyurea sufficient to increase the expression level of SMN exon 7 in a cell of the subject, relative to a reference expression level of SMN exon 7. For example, the method can increase the ratio of SMN transcripts having exon 7 to those lacking exon 7 by at least 30, 50, 60, 70, 80, 90, 100, or 150%. The cell can be a mammalian cell, e.g., a human cell, e.g., a cell from a human SMA patient. For example, the cell can be homozygous for an SMN1 mutation.
In another aspect, the invention features a method of treating spinal muscular atrophy in a subject. The method includes administering to the subject hydroxyurea in an amount sufficient to ameliorate a symptom of spinal muscular atrophy. The subject can be human, e.g., a human homozygous for mutations in SMN1. The ameliorated symptom can be muscular paralysis, muscular atrophy, breathing, walking gait, or a symptom described herein. The symptom can also be decreased expression of SMN exon 7 in a cell of the subject. The administered amount can be about 0.2 to 100, 0.4 to 80, 5 to 50, or 15 to 25 mg/kg/day. The amount can be administered orally or parentally.
In one embodiment, the subject is a fetus, and the hydroxyurea is administered to the subject in utero.
In still another aspect, the invention features a method of for modulating SMN gene expression in a cell. The method includes contacting an amount of hydroxyurea to the cell. The amount is sufficient to increase the expression level of SMN exon 7 in the cell, relative to a reference expression level of SMN exon 7. The cell can be a human cell, e.g., a cell in a patient. The cell can treated in vivo, in vitro, or ex vivo. Following ex vivo treatment, the cell can be administered (e.g., restored) to a subject, e.g., a human patient.
In yet another aspect, the invention features a method of treating spinal muscular atrophy in a subject. The method includes administering to the subject a DNA replication inhibitor, e.g., a ribonucleotide reductases inhibitor (e.g., hydroxyurea or a compound other than hydroxyurea) in an amount sufficient to ameliorate a symptom of spinal muscular atrophy. The subject can be human, e.g., a human homozygous for mutations in SMN1. The ameliorated symptom can be muscular paralysis, muscular atrophy, breathing, walking gait, or a symptom described herein. The symptom can also be decreased expression of SMN exon 7 in a cell of the subject.
As used herein, the term “transgene” refers to a nucleic acid sequence (e.g., encoding one or more human proteins), which is inserted by artifice into a cell. The transgene is integrated into a chromosomal genome. A transgenic sequence can be partly or entirely species-heterologous, i.e., the transgenic sequence, or a portion thereof, can be from a species which is different from the cell into which it is introduced. A transgenic sequence can be partly or entirely species-homologous, i.e., the transgenic sequence, or a portion thereof, can be from the same species as is the cell into which it is introduced. If a transgenic sequence is homologous (in the sequence sense or in the species-homologous sense) to an endogenous gene of the cell into which it is introduced, then the transgenic sequence has one or more of the following characteristics: it is designed for insertion, or is inserted, into the cell's genome in such a way as to alter the sequence of the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the endogenous gene or its insertion results in a change in the sequence of the endogenous gene); it includes a mutation, e.g., a mutation which results in misexpression of the transgenic sequence; by virtue of its insertion, it can result in misexpression of the gene into which it is inserted, e.g., the insertion can result in a knockout of the gene into which it is inserted. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid sequences, such as introns, that may be necessary for a desired level or pattern of expression of a selected nucleic acid. A transgene can provide an antisense transcript or a sense transcript, e.g., a transcript encoding a protein.
As used herein, the term “transgenic cell” refers to a cell containing a transgene.
As used herein, a “transgenic animal” is a non-human animal in which one or more (e.g., all) of the cells of the animal contain a heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques known in the art. The transgene can be introduced into the cell directly, indirectly by introduction into a precursor of the cell, or by way of deliberate genetic manipulation, such as by microinjection, transformation, electroporation, lipofection, or infection with a recombinant virus. In one example, where the transgene is introduced indirectly, the transgene is introduced into a cultured cell, and the nucleus of the cultured cell or of a descendant of the cultured cell is microinjected into an enucleated oocyte to produce a nucleated oocyte which develops into an animal.
As used herein, a “disruption” in reference to an endogenous gene refers to any type of mutation that inactivates an
Chang Jan-Gowth
Jong Yuh-Jyh
Tsai Fuu-Jen
China Medical College Hospital
Spivack Phyllis G.
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