Methods of treating hypertension

Details Methods of treating hypertension Methods of treating hypertension

1999-05-05

2003-02-04

Yucel, Remy (Department: 1636)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

Reexamination Certificate

active

06514935

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to hypertension.
In their normal state, vascular smooth muscle cells regulate vessel tone and blood pressure. Unlike skeletal muscle and cardiac muscle cells, these cells are not terminally differentiated. In response to mechanical, chemical, or immunologic injury (Libby et al., 1991, Lab Invest. 64:5-15; Munro et al., 1988, Lab Invest. 58:249-261; Ross, R., 1993, Nature 362:801-809; Tsai et al., 1994, Proc. Natl. Acad. Sci. USA 91:6369-6373; and Tsai et al., 1996, Clin. Invest. 97:146-153), the phenotype of these cells changes rapidly from that of a differentiated, quiescent cell to that of a dedifferentiated, proliferating cell.
SUMMARY OF THE INVENTION
The invention is based on the identification and characterization of a smooth muscle cell LIM (SmLIM/CRP2) polypeptide which is expressed preferentially in arterial smooth muscle cells. Mammals which are SmLIM-deficient are resistant to developing hypertension. Accordingly, the invention features a method of inhibiting hypertension in a mammal by identifying a mammal suffering from or at risk of developing hypertension and administering to the mammal, e.g., a human patient, a compound that reduces expression of SmLIM. An inhibitory compound inhibits transcription of SmLIM-encoding DNA or translation of an endogenous SmLIM transcript into a SmLIM gene product. For example, to inhibit SmLIM transcription, a compound which binds to a cis-acting regulatory sequence of a SmLIM gene is administered. The cis-acting regulatory sequence is located 5′ to the transcription start site of SmLIM and comprises CANNTG (SEQ ID NO:44), GGGRNTYYC (SEQ ID NO:45), or CACCC (SEQ ID NO:46). The cis-acting regulatory sequence has SmLIM promoter activity and is at least at least 50% identical to SEQ ID NO:3 or 16. Preferably, the regulatory sequence comprises SEQ ID NO:3 or 16.
A compound which inhibits SmLIM transcription is preferably an antisense nucleic acid. For example, the antisense nucleic acid molecule contains at least 10 nucleotides the sequence of which is complementary to an mRNA encoding a SmLIM polypeptide. The antisense nucleic acid is a DNA operatively linked to a smooth muscle cell-specific promoter, and transcription of the DNA yields nucleic acid product which is complementary to an mRNA encoding a SmLIM polypeptide. The cell-specific promoter is preferably at least 50% identical to SEQ ID NO:3 or 16. Most preferably, the promoter contains the nucleic acid sequence of SEQ ID NO:3 or 16.
A DNA construct for production of antisense nucleic acids in a target cell is also within the invention. For example, the invention includes a substantially pure DNA containing a first DNA sequence at least 50% identical to SEQ ID NO:3 or 16, operably linked to a second DNA sequence which is an antisense template, the transcript of which is complementary to a portion of an mRNA encoding a vascular smooth muscle cell polypeptide. The first DNA sequence directs transcription of the second DNA sequence preferentially in a vascular smooth muscle cell compared to in a non-vascular smooth muscle cell. For inhibition of hypertension, the vascular smooth muscle cell polypeptide is SmLIM.
In addition to inhibiting SmLIM transcription, hypertension is reduced by inhibiting SmLIM activity. For example, a compound which inhibits SmLIM activity is a polypeptide that binds to a LIM domain. Preferably, the compound inhibits contraction of smooth muscle cells. The compound inhibits dimerization of SmLIM. Inhibitory compounds include SmLIM-specific antibodies such as an antibody which binds to an epitope comprising the amino acid sequence of residues 91-98 of SEQ ID NO:13. Intrabodies with the same specificity as SmLIM-binding antibodies are expressed intracellularly to inhibit SmLIM dimerization or to inhibit SmLIM binding to an intracellular ligand. Preferably the compound is introduced into an artery of the mammal such as a human patient.
The invention also includes a transgenic non-human mammal the germ cells and somatic cells of which comprise a null mutation in a gene encoding SmLIM. The null mutation is a deletion of part or all of an exon, e.g., exon 3. Preferably, the mammal is a rodent such as a mouse.
Methods of screening for compounds that inhibit SmLIM expression or function are also encompassed by the invention. For example, a method of screening candidate compounds to identify a compound capable of decreasing expression of SmLIM/CRP2 in vascular smooth muscle cells is carried out by (a) providing a vascular smooth muscle cell; (b) contacting the vascular smooth muscle cell with a candidate compound; and (c) determining the amount of SmLIM/CRP2 expression in the vascular smooth muscle cell. A decrease in the amount of expression, e.g, as measured by detecting SmLIM transcripts or gene products in the cell, in the presence of the candidate compound compared to the amount in the absence of the candidate compound indicates that the candidate compound decreases expression of SmLIM/CRP2 in vascular smooth muscle cells, and thus, inhibits hypertension.
The invention features a substantially pure DNA containing a sequence which encodes a SmLIM/CRP2 polypeptide. By the term “SmLIM/CRP2” is meant a polypeptide that contains at least two LIM domains, lacks a homeobox domain and a protein kinase domain, and inhibits proliferation of vascular smooth muscle cells. By “substantially pure DNA” is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the SmLIM/CRP2 gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a procaryote or eucaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. A “LIM domain” is defined by the amino acid consensus sequence CX
2
CX
17±1
HX
2
CX
2
CX
2
CX
17±1
CX
2
C/D/H (SEQ ID NO:18).
The SmLIM/CRP2 polypeptide of the invention preferably has at least 85% sequence identity with SEQ ID NO:1, and more preferably at least 90% (e.g., at least 95%). The DNA may encode a naturally occurring mammalian SmLIM/CRP2 polypeptide such as a human, rat, mouse, guinea pig, hamster, dog, cat, pig, cow, goat, sheep, horse, monkey, or ape SmLIM/CRP2. For example, the SmLIM/CRP2 polypeptide may have the amino acid sequence of the naturally-occurring human polypeptide, e.g., a polypeptide which includes the amino acid sequence of SEQ ID NO:1. Preferably, the DNA includes the nucleotide sequence of SEQ ID NO:2. The DNA may contain a strand which hybridizes at high stringency to a DNA probe having a portion or all of the nucleotide sequence of SEQ ID NO:2, or the complement thereof. The probe to which the DNA of the invention hybridizes preferably consists of at least 20 nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 100 nucleotides or more (up to 100%) of the nucleotide sequence of SEQ ID NO:2, or the complement thereof. Such a probe is useful for detecting expression of a SmLIM/CRP2 transcript in a cell by a method which includes the steps of (a) contacting mRNA obtained from the cell with the labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA transcript. The invention also includes a substantially pure strand of DNA containing at least 15 nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of SEQ ID NO:2.
Hybridization is carried out using standard techniques such as those described in Ausubel et al.,
Current Protocols in Molecular Biology,
John Wiley & Sons, (1989). “High stringency” refers to DNA hybridization and wash conditions characterized by high temperature and l

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