Title:  Methods of treating hypertension

United States Patent:  6,514,935

Issued:  February 4, 2003

Inventors:  Lee; Mu-En (Newton, MA); Yet; Shaw-Fang (Andover, MA)

Assignee:  President and Fellows of Harvard (Cambridge, MA)

Appl. No.:  305839

Filed:  May 5, 1999

The invention features a method of inhibiting hypertension in a mammal by administering to the mammal a compound that reduces expression or activity of SmLIM.

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 CX₂ CX₁₇ + 1 HX₂ CX₂ CX₂ CX₁₇+1 CX₂ 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 low salt concentration, e.g., wash conditions of 650C at a salt concentration of approximately 0.1xSSC. "Low" to "moderate" stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60^oC. at a salt concentration of at least 1.0xSSC. For example, high stringency conditions may include hybridization at about 42^oC., and about 50% formamide; a first wash at about 65^oC., about 2 xSSC, and 1% SDS; followed by a second wash at about 65^oC. and about 0.1%xSSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to a SmLIM/CRP2 gene are detected by, for example, hybridization at about 42^oC. in the absence of formamide; a first wash at about 42^oC., about 6xSSC, and about 1% SDS; and a second wash at about 50^oC., about 6xSSC, and about 1% SDS.

The invention also includes a substantially pure DNA encoding a SmLIM/CRP2 polypeptide, which DNA includes a nucleotide sequence having at least 50% sequence identity to SEQ ID NO:2. Preferably the DNA has at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 99% identity to SEQ ID NO:2. Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity or conservation to a reference polypeptide or nucleic acid molecule, the percent identity or conservation is determined by the algorithm of Myers and Miller, CABIOS (1989), which is embodied in the ALIGN program (version 2.0), or its equivalent, using a gap length penalty of 12 and a gap penalty of 4 where such parameters are required. All other parameters are set to their default positions. Access to ALIGN is readily available. See, e.g., http://www2.igh.cnrs.fr/bin/align-guess.cgi on the Internet.

The DNA may be operably linked to regulatory sequences, e.g., a promoter, for expression of the polypeptide. Preferably, the promoter is vascular cell-specific, more preferably, it is vascular smooth muscle cell-specific, and most preferably, it is arterial smooth muscle cell-specific. By "operably linked" is meant that a coding sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). By "promoter" is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible.

The invention includes a substantially pure DNA containing a sequence at least 50% identical to SEQ ID NO:3 or SEQ ID NO:16, which regulates arterial smooth muscle cell-specific transcription of a polypeptide-encoding sequence to which it is operably linked. Preferably, the DNA is at least 75% identical, more preferably at least 90% identical, more preferably at least 95%, and most preferably 100% identical to SEQ ID NO:3 or SEQ ID NO:16. The DNA may be operably linked to a heterologous polypeptide-encoding sequence and may be used in a method of directing arterial smooth muscle cell-specific expression of the polypeptide, e.g., by introducing the DNA linked to the coding sequence into an arterial cell. By the term "heterologous polypeptide" is meant a polypeptide other than a SmLIM/CRP2 polypeptide.

The invention also includes a substantially pure DNA comprising a first DNA sequence containing a SmLIM/CRP2-derived promoter sequence, e.g., one which is at least 50% identical to SEQ ID NO:3 or 16, operably linked to a second DNA sequence encoding a polypeptide other than SmLIM/CRP2, i.e., a heterlogous peptide, wherein the first DNA sequence directs transcription of the second DNA sequence preferentially in an a vascular smooth muscle cell, e.g., an arterial smooth muscle cell, compared to in a non-vascular smooth muscle cell. Preferably, the second DNA sequence does not encode SmLIM/CRP2. Vascular smooth muscle cell-specific expression of a polypeptide is accomplished by introducing into an vascular smooth muscle cell a vector containing SmLIM promoter sequences operably linked to polypeptide-encoding DNA and maintaining the cell under conditions suitable for expression of the second DNA, e.g., in vitro culture under standard tissue culture conditions or in vivo, i.e., in an animal. For example, the invention provides a method of inhibiting arteriosclerosis in an animal by contacting an artery of an animal with the vector containing DNA encoding a polypeptide which reduces or prevents the development of arteriosclerosis. e.g., a polypeptide which reduces proliferation of smooth muscle cells. Alternatively, the second DNA sequence may be a an antisense template the transcript of which is complementary to a portion of an mRNA encoding a vascular smooth muscle cell polypeptide. As described above, the invention includes a substantially pure DNA comprising a first DNA sequence containing a SmLIM/CRP2-derived promoter sequence, e.g, one which is 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 an 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. By the term "antisense template" is meant a DNA which is transcribed into an RNA which hybridizes to mRNA encoding a polypeptide expressed in vascular smooth muscle cells.

Preferably the level of transcription of a polypeptide-encoding or antisense template in vascular smooth muscle cells under the control of a SmLIM/CRP2-derived promoter sequence is at least 2-fold greater, more preferably 3-fold, more preferably 4-fold, and more preferably 10-fold greater than that in non-vascular smooth muscle cells. Most preferably, the SmLIM/CRP2-derived promoter sequence of the invention direct vascular smooth muscle cell-specific transcription of the DNA to which it is linked.

The invention also includes a vector containing the promoter sequences of the invention, a method of directing vascular smooth muscle cell-specific expression of a polypeptide by introducing the vector into an vascular smooth muscle cell, and a vascular smooth muscle cell containing the vector.

The vector of the invention can be used for gene therapy, such as a method of inhibiting arteriosclerosis in an animal by contacting an artery of the animal with the vector of the invention which directs the production of a polypeptide capable of reducing or preventing the development of arteriosclerosis.

A cell which contains a recombinant SmLIM/CRP2 polypeptide-encoding DNA is also within the invention. The cell may be eucaryotic or procaryotic. A method of making a SmLIM/CRP2 polypeptide includes the steps of (a) providing the cell which contains SmLIM/CRP2 polypeptide-encoding DNA, and (b) culturing it under conditions permitting expression of the DNA. If the polypeptide is secreted by the cell, the SmLIM/CRP2 polypeptide produced can be recovered from the culture supernatant of the cell culture. If the polypeptide is not secreted, the polypeptide can be recovered by lysing the cultured cells.

The invention also includes a substantially pure human SmLIM/CRP2 polypeptide. Preferably, the amino acid sequence of the polypeptide is at least 90% identical, more preferably at least 95% identical, more preferably at least 99% identical to the amino acid sequence of SEQ ID NO:1. Most preferably, the amino acid sequence of the polypeptide includes SEQ ID NO:1. By a "substantially pure polypeptide" is meant a polypeptide which is separated from those components (proteins and other naturally-occurring organic molecules) which naturally accompany it. Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation consists of at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a SmLIM/CRP2 polypeptide. A substantially pure SmLIM/CRP2 polypeptide may be obtained, for example, by extraction from a natural source (e.g., an arterial smooth muscle cell); by expression of a recombinant nucleic acid encoding a SmLIM/CRP2 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components even without further purification steps. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eucaryote but produced in E. coli or another procaryote, or in a eucaryote other than that from which the polypeptide was originally derived.

The invention also includes diagnostic methods. SmLIM/CRP2 expression was found to decrease as vascular smooth muscle cells changed from a quiescent, differentiated phenotype to a proliferative phenotype in response to vascular injury. One can detect injury in a sample of vascular tissue by determining the level of SmLIM/CRP2 gene expression in the tissue sample, and comparing it to the level of expression in a control sample of vascular tissue. This determination may be made using SmLIM/CRP2-specific DNA probes to detect the level of gene transcription or using SmLIM/CRP2-specific antibodies to detect the level of gene product in the cells. A decrease in the level of expression of SmLIM/CRP2 compared to the level in uninjured control vascular tissue indicates the presence of a vascular injury.

Methods of therapy are also within the invention. A method of inhibiting arterial smooth muscle cell proliferation in a mammal may include the steps of identifying a mammal in need of such inhibition, and introducing either SmLIM/CRP2 or a SmLIM/CRP2-encoding DNA into an artery of the mammal. One can inhibit neointima formation after balloon angioplasty in a mammal by contacting an artery of the mammal with a SmLIM/CRP2 or SmLIM/CRP2-encoding DNA prior to, during, or immediately after angioplasty to reduce proliferation of arterial smooth muscle cells in the mammal, particularly at the site of vascular injury treated by the angioplasty procedure. Preferably, the mammal is a human, and the SmLIM/CRP2 polypeptide is a human SmLIM/CRP2 polypeptide.

A method of screening candidate compounds to identify a compound capable of increasing expression of a SmLIM/CRP2 polypeptide in vascular smooth muscle cells is also within the invention. For example, an in vitro method may include the steps of (a) providing a vascular smooth muscle cell, e.g., a human arterial smooth muscle cell; (b) contacting the smooth muscle cell with a candidate compound; and (c) determining the amount of SmLIM/CRP2 expression by the vascular smooth muscle cell. The screening method can also be carried out in vivo, e.g., in an animal subjected to a vascular injury, and then treated with the candidate compound or a placebo. An increase in the amount of expression in the presence of the candidate compound compared to that in the absence of the candidate compound indicates that the candidate compound increases expression of a SmLIM/CRP2 polypeptide in vascular smooth muscle cells. An increase of SmLIM/CRP2 expression correlates with an inhibition in vascular smooth muscle cell proliferation. Expression may be determined by measuring gene transcription, e.g., in a Northern blot assay, or by measuring the amount of SmLIM/CRP2 polypeptide in the cell, e.g., by immunoblotting.

Claim 1 of 3 Claims

What is claimed is:

1. A method of decreasing hypertension in a mammal comprising administering to said mammal a compound that reduces expression of SmLIM by binding to a cis-acting regulatory sequence of a SmLIM gene, wherein said cis-acting regulatory sequence is SEQ ID NO: 3 or SEQ ID NO:16.
 

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