WO2001012126A2 - Cardiomyocyte regeneration - Google Patents

Abstract

Methods and compositions for cardiomyocyte proliferation and regeneration by contacting cardiomyocytes with Cardiac Helix-loop-helix Factor polypeptide or DNA encoding the polypeptide.

Description

CARDIOMYOCYTE REGENERATION

RELATED INVENTIONS

This application claims priority to U.S. Provisional Patent Application Serial Number 60/148,974, filed August 13, 1999, which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under National Institutes of Health grants HL57666 and HL03745. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to cardiovascular injury.

BACKGROUND OF THE INVENTION

Cardiomyocytes in an adult mammal retain little or none of their developmental capacity for hyperplastic growth. As a consequence of this differentiated, nonproliferative phenotype, cardiomyocyte loss due to injury, such as myocardial infarction, or disease, such as cardiomyopathy, is irreversible. Cardiomyopathy is a progressive disease of the heart muscle, which affects not only middle age and older individuals, but children as well. This disease may result in a decrease in the capability of the heart to pump blood and arrhythmias.

SUMMARY OF THE INVENTION

The invention provides novel methods of therapeutically regenerating heart tissue and promoting smooth muscle differentiation to treat heart disease or injury to heart tissue by administering to a mammal a Cardiac Helix-loop-helix Factor (CHF) polypeptide or DNA encoding a CHF polypeptide. Expansion of an endogenous population of proliferative cardiomyocytes is accomplished by contacting cardiac tissue with a CHF polypeptide or CHF DNA. Increasing the number of cardiomyocytes in a diseased or injured heart improves overall function of the heart, and thus, improves the patient's condition and survival.

The invention features a substantially pure DNA comprising a sequence encoding a

CHF polypeptide, e.g., CHF-1 or CHF-2. A CHF polypeptide is a transcription factor that comprises a basic helix-loop-helix domain and is expressed in cardiovascular tissue. Preferably, the CHF polypeptide contains an amino acid sequence that is at least 50% identical to SEQ ID NO: 5. More preferably a CHF polypeptide contains an amino acid sequence that is at least 75%, more preferably at least 85%, more preferably at least 95% identical to SEQ ID NO:5. For example, the sequence includes the amino acid sequence RKKRRGIIEKRRRDPJNNSLSELRRLVPTAFEKQGSAKLEKAEILQMTVDLHKMLQA T (SEQ ID NO:5).

The DNA may encode a naturally-occurring mammalian CHF-1 polypeptide such as a human, rat, mouse, guinea pig, hamster, dog, cat, pig, cow, goat, sheep, horse, monkey, or ape CHF-1. Preferably, the DNA encodes a human CHF-1 polypeptide, e.g., a polypeptide which contains the amino acid sequence of SEQ ID NO: 1. For example, the invention includes degenerate variants of the human cDNA (SEQ ID NO:3) or the mouse cDNA (SEQ ID NO:4). The DNA contains a nucleotide sequence having at least 50% sequence identity to SEQ ID NO: 3. For example, the DNA contains a sequence which encodes a human CHF- 1 polypeptide, such as the coding sequence of SEQ ID NO:3 (nucleotides 298 to 1170 of SEQ ID NO:3). Alternatively, the DNA contains a sequence which encodes a mouse CHF-1 polypeptide, such as the coding sequence of SEQ ID NO:4 (nucleotides 266 to 1144 of SEQ ID NO:4). The DNA contains a strand which hybridizes at high stringency to a strand of DNA having the sequence of SEQ ID NO:3 or 4, or the complement thereof. The DNA has at least 50% sequence identity to SEQ ID NO:3 or 4, and encodes a polypeptide having the biological activity of a CHF-1 polypeptide, e.g., the ability to inhibit ARNT-dependent activation of the VEGF promoter. Preferably, the DNA has at least 75% identity, more preferably 85% identity, more preferably 90% identity, more preferably 95% identity, more preferably 99% identity, and most preferably 100% identity to the coding sequence of SEQ ID NO:3 or 4.

CHF polypeptides preferably contain at least one of the following domains: a bHLH domain, an Orange domain, and carboxy-terminal YRPW domain. The bHLH domain contains a basis helix-loop-helix structure. A bHLH domain may bind to a class B E-box or N-box. For example, the polypeptide contains amino acids 49-106 of SEQ ID NO: 2, amino acids 52-104 of SEQ ID NO: 1, or amino acids 52-104 of SEQ ID NO: 12.

The polypeptide contains an Orange domain; the Orange domain mediates interaction with one or more transcription factors. For example, the polypeptide contains amino acids 121-162 of SEQ ID NO: 1 or amino acids 121-162 of SEQ ID NO: 12. Other examples, of CHF polypeptides include those which contain amino acids 38-337 of SEQ ID NO. 1, amino acids 38-337 of SEQ ID NO. 12, amino acids 147-337 of SEQ ID NO. 1, or amino acids 147- 337 of SEQ ID NO. 12. Such peptides promote cardiomyocyte regeneration inhibit VEGF- mediated angiogenesis or promote the growth and differentiation of vascular smooth muscle cells.

Nucleotide and amino acid comparisons were carried out using the Lasergene software package (DNASTAR, Inc., Madison, WI). The MegAlign module used was the Clustal V method (Higgins et al., 1989, CABIOS 5(2):151-153). The parameter used were gap penalty 10, gap length penalty 10.

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 nucleic acid hybridization and wash conditions characterized by high temperature and low salt concentration, i.e., wash conditions of 65°C at a salt concentration of approximately 0.1 X SSC. "Low" to "moderate" stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, i.e., wash conditions of less than 60°C at a salt concentration of at least 1.0 X SSC. For example, high stringency conditions include hybridization at 42°C, and 50% formamide; a first wash at 65°C in 2 X SSC, and 1% SDS; followed by a second wash at 65°C and 0.1% x SSC. Lower stringency conditions suitable for detecting DNA sequences 50% sequence identity (or lower) to an CHF-1 gene are detected by, for example, hybridization at 42°C in the absence of formamide; a first wash at 42°C, about 6X SSC, and 1% SDS; and a second wash at 50°C in 6 X SSC, and 1% SDS.

A vector containing a CHF-encoding DNA is also within the invention. Preferably the DNA which includes a CHF-encoding DNA is less than 5 kilobases in length; more preferably, the DNA is less than 4 kilobases in length, more preferably the DNA is less than 3 kilobases in length, and most preferably the DNA is approximately 2 kilobases or less in length. The invention also provides a method of directing cardiac-specific or smooth muscle cell-specific expression of a protein by introducing into a cell an isolated DNA containing a sequence encoding the protein operably linked to the tissue-specific promoter. A cell containing the DNA or vector of the invention is also within the invention.

Also within the invention is polypeptide which includes the amino acid sequence of SEQ ID NO: 5, e.g., a substantially pure human CHF polypeptide such as a human or mouse CHF-1 or CHF-2 polypeptide. Preferably, the polypeptide is at least 50 amino acids in length, more preferably, the polypeptide is at least 100 amino acids in length, more preferably, the polypeptide is at least 150 or 200 amino acids in length. For example, the polypeptide is at least 58 amino acids in length but less than about 340 amino acids in length.

The polypeptide contains the amino acid sequence of SEQ ID NO: 5 (bHLH region of human CHF-1) or the amino acid sequence of SEQ ID NO: 6 or both. Preferably the polypeptide contains an amino acid sequence which is at least 50% identical to SEQ ID

NO: 1. Preferably, the amino acid sequence has at least 75% identity, more preferably 85% identity, more preferably 90% identity, more preferably 95% identity, more preferably 99% identity, and most preferably 100% identity to the amino acid sequence of SEQ ID NO:l. For example, the CHF-1 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.

For therapeutic application, vascular cells are contacted ex vio or in vivo with CHF polypeptides or DNA encoding CHF polypeptides. For example, augmentation of a cardiomyocyte population in a mammal is achieved by increasing the amount of a CHF-1 polypeptide in mature heart tissue, e.g., by administering a CHF-1 polypeptide either locally to the myocardium or systemically. A method of promoting proliferation of a cardiomyocyte in a mammal is carried out by administering to the mammal a CHF-1 polypeptide, a DNA encoding a CHF-1 polypeptide, or cells, e.g., genetically-altered heterologous or autologous cardiovascular cells, which express a CHF-1 polypeptide. A CHF-1 polypeptide, DNA encoding such a polypeptide, of CHF-1 expressing cells are administered systemically or locally to the myocardium of the mammal in need of such therapy, e.g., one which has suffered a cardiovascular injury such as a myocardial infarction or one which has been diagnosed as suffering from myocarditis. CHF-1 is useful to regenerate cardiomyocytes by contacting cardiovascular tissue with a CHF-1 polypeptide, or DNA encoding such a polypeptide, in vivo or in vitro. For example, cardiovascular cells such as cardiomyocytes or vascular smooth muscle cells are harvested from a patient, treated ex vivo, and then returned to the patient. Promoting proliferation of cardiomyocytes in a mature animal is therapeutically beneficial to individuals have suffered a injury to cardiovascular tissue such as a myocardial infarction or who are affected by a heart disease such as cardiomyopathy. In the latter case, the invention offers the advantage of treating the disease in a less invasive manner compared to cardiac transplantation, an approach often used with patients suffering from advanced cardiomyopathy. Other advantages include greater effectiveness than conventional drug therapies, longer lasting beneficial clinical effects (and fewer complications) than transplantation. Expansion of an endogenous population of proliferative cardiomyocytes is the only methods of regenerating damaged or dead heart tissue after myocardial infarction.

The invention also includes a method for inhibiting angiogenesis in a tissue, e.g., a solid tumor, by contacting the tissue with a DNA containing a sequence encoding a CHF polypeptide.

A method of inducing differentiation of smooth muscle cells in a tissue is also within the invention. Growth and differentiation of smooth muscle cells in a given tissue is induced by contacting the tissue with DNA encoding a CHF polypeptide. The tissue is veinous tissue or arterial tissue. In the case of veinous tissue, inducing growth and differentiation of smooth muscle cells in the tissue results in "arterialization" of the veinous tissue, i.e., the veinous tissue takes on the characteristics of arterial tissue. For example a CHF -treated vein graft includes a layer of smooth muscle cells. Accordingly, the invention provides a method of inducing growth of smooth muscle cells in a antologous ein explant by contacting the explant with a DNA encoding a CHF polypeptide. The same procedure is used to induce smooth muscle cell growth in heterologous tissue.

Arterialization of veinous tissue prior to or after surgical implantation into an artery allows greater compatibility of the vein tissue with the arterial tissue. Thus, the invention includes a method of reducing vein graft stenosis in a mammal by contacting ex vivo a veinous tissue with a DNA encoding a CHF polypeptide, e.g., human CHF-1 or 2, prior to implantation of the vein explant into an artery of the mammal. For example, prior to bypass surgery, a vein explant is obtained from an individual and bathed in a solution containing DNA encoding a CHF polypeptide. The explant is engrafted into an artery in a standard bypass surgical procedure, and optionally, the vein graft is contacted with CHF DNA subsequent to the surgery. The invention also includes a method of promoting smooth muscle cell regeneration in an injured or diseased vascular tissue by contacting the tissue with a DNA encoding a CHF polypeptide. The vascular tissue is veinous tissue or arterial tissue. For example, the tissue is a vein graft or arterial tissue injured in the course of angioplasty. The DNA is provided to the tissue intravascularly, e.g., using a catheter. For longer term delivery of CHF DNA, a stent is coated with the DNA and implanted in the tissue.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram of a comparison of the amino acid sequences of hCHF-1 (SEQ ID NO:l), hairy (SEQ ID NO:8), and human hairy and enhancer of split homologue (hHES-1; SEQ ID NO:9).

FIG. 2 is a diagram of a map of the mouse CHF-1 cDNA and deduced amino acid sequence of the CHF-1 polypeptide. The bHLH domain and YQPW (SEQ ID NO:6) domain of the translated amino acid sequence is underlined. Nucleotides in upper case letters denote coding sequence, with the termination codon denoted by an asterisk.

FIG. 3 is a diagram of a map of the human CHF-1 cDNA and the deduced amino acid sequence for the CHF-1 polypeptide. The bHLH domain and YRPW (SEQ ID NO:7) domain of the translated amino acid sequence is underlined. Nucleotides in upper case letters denote coding sequence, with the termination codon denoted by an asterisk.

FIG. 4 is a diagram of the phylogenetic tree of Hairy family members. FIG. 5 is an autoradiograph of a Northern blot analysis showing that CHF-1 is highly expressed in the adult cardiovascular system.

FIG. 6 is an autoradiograph of a Northern blot analysis showing that CHF-1 is highly expressed in the developing mouse embryo.

FIG. 7 is an autoradiograph of a Northern blot analysis showing that CHF-1 is downregulated in cardiac myocytes during maturation.

FIG. 8 is an autoradiograph of a Northern blot analysis showing that CHF-1 is induced during Monc-1 cell differentiation into a smooth muscle cell phenotype.

FIG. 9 is a bar graph showing that CHF-1 inhibits ARNT-dependent transactivation of the VEGF promoter.

FIG. 10 is a diagram of the gene targeting construct used to make CHF-1 -deficient (knockout) mice.

FIG. 11 is a diagram of a comparison of the amino acid sequences of hCHF-1 (SEQ ID NO:l), hHES-1 (SEQ ID NO:9), and hCHF-2 (SEQ ID NO:12). The amino acid sequences for hCHF-1 and hCHF-2 were deduced from the human cDNA sequences, while the hHES-1 amino acid sequence was obtained from GenBank. Bar denotes bHLH region and dashed bar denotes Orange domain. **** denotes YRPW motif. Alignment of amino acid sequences was performed using the Clustal V method included in the MegAlign module of the Lasergene software package (DNASTAR, Inc., Madison, WI). Phylogenetic analysis was performed using the MegAlign module of the Lasergene software package (DNASTAR, Inc.).

FIG. 12 is a diagram of the phylogenetic tree which shows the relationship of hCHF- 1 and hCHF-2 relative to other members of the protein family.

FIG. 13 is an autoradiograph of a Northern blot analysis showing that CHF-2 is highly expressed in adult brain and lung tissue.

FIG. 14 is an autoradiograph of a Northern blot analysis showing that CHF-2 is highly expressed in the developing mouse embryo. DETAILED DESCRIPTION

The present invention includes a novel bHLH protein, CHF-1, that has been cloned using a yeast 2-hybrid screen with the bait protein ARNT. CHF-1 has been discovered to be highly expressed in the developing aorta and vasculature. This expression precedes the appearance of most of the known smooth muscle-specific markers and persists into adulthood.

Structural homology analysis indicates that CHF1 is a distant relative of hairy, a drosophila bHLH protein essential for proper development of the peripheral nervous system. Members of the hairy family of transcriptional repressors have three distinct characteristics. They contain a proline in the basic region that affects the DNA-binding specificity, an Orange domain and a WRPW motif at or adjacent to the carboxyl-terminus. The bHLH region of the hairy proteins binds to class B E-boxes or N-boxes. The Orange domain is required for interaction with specific transcriptional activators. Of note, CHF-1 possesses neither a proline in the basic region nor a canonical WRPW motif at the carboxyl-terminus. Instead, a glycine residue is present in place of proline and the WRPW motif is replaced by the sequence YRPWGTEVGAF (SEQ ID NO: 16).

Included in the invention is a substantially pure DNA that includes a sequence encoding a Cardiovascular Helix-loop-helix Factor (CHF). The term "substantially pure" refers to molecules separated from other DNA or RNA molecules that are present in the natural source of the sequence. Specifically, 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 a CHF-1 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 prokaryote or eukaryote 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.

The term also refers to a DNA or polypeptide that is substantially free of, or isolated from, cellular material, viral material, or culture medium when produced by recombinant

DNA techniques, or chemical precursors or other chemicals when chemically synthesized. A substantially pure DNA includes nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" and "substantially pure" is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.

A substantially pure CHF polypeptide is obtained by extraction from a natural source

(e.g., cardiac tissue); by expression of a recombinant nucleic acid encoding a CHF polypeptide; or by chemically synthesizing the protein. A polypeptide or protein is substantially pure when it is separated from those contaminants which accompany it in its natural state (proteins and other naturally-occurring organic molecules). 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 is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, CHF. Purity is measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eukaryote but produced in E. coli or another prokaryote, or in a eukaryote other than that from which the polypeptide was originally derived.

"Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. Such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent eel.

"Complementary" sequences or "complements" as used herein refer to sequences which have sufficient complementarity to be able to hybridize under appropriate conditions to a specified nucleic acid, thereby forming a stable duplex.

As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence. The term "intron" refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.

A nucleic acid is transcribed from a promoter if it is operably linked to the promoter. The term "operably linked" is intended to mean that the promoter is associated with the nucleic acid in such a manner as to facilitate transcription of the nucleic acid from the promoter.

"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").

The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.

The term "treating" as used herein is intended to encompass curing as well as ameliorating at least one symptom of a condition or disease.

CHF-1 Polypeptides

CHF-1 is a novel helix-loop-helix protein that is highly expressed in the developing cardiovascular system. Its expression pattern is unique among cardiovascular specific transcription factors in that it is highly expressed both in the developing ventricle and in the developing aorta. Homology searching revealed that CHF-1 is most closely related to the Hairy family of transcriptional repressors, which have been shown to play an important role in neuronal cell fate decisions. Homology searching was carried out using the DNA* package from Lasergene. The MegAlign module uses the Clustal V method as described above. The amino acid sequences of human CHF-1 and hairy are 19.0 % similar, while those of hCHF-1 and hHES-1 are 22.9% similar (FIG. 1). Percent identity is even lower. Homology searching also indicated that CHF-1 is phylogenetically distinct from other hairy family members (FIG. 4). A phylogenetic tree showing the relationship of hCHF-1 and hCHF-2 among other family members is shown in FIG. 12.

As used herein, the term "transcription factor" refers to a polypeptide that directly or indirectly activates and/or regulates expression of a DNA sequence. For example, a transcription factor may bind alone, (or complexed with another transcription factor), to a 5' flanking sequence of the gene which expression it regulates.

Transcription factors include proteins, fragments or modified forms thereof, which interact preferentially with specific nucleic acid sequences, i.e., regulatory elements, and which in appropriate conditions stimulate or repress transcription. For example, factors ARNT and EPAS1 form a heterodimer and activate the VEGF promoter. CHF polypeptides bind to ARNT, thereby inhibiting formation of a heterodimer which activates VEGF transcription. Angiogenesis is inhibited by increasing the level of CHF in cells of a tissue undergoing angiogenesis.

Hairy Family of Transcriptional Regulators

Hairy is a drosophila protein, which is involved in embryo patterning and cell fate decisions in the developing nervous system. Hairy proteins typically contain a proline bHLH domain and a carboxyterminal WRPW (SEQ ID NO: 10) motif, which is involved in interacting with and recruiting members of the groucho family of transcriptional co- repressors. Hairy proteins typically bind to the N box sequence, CACNAG (SEQ ID NO:l 1) and function as long range repressors of transcription, and can also inhibit heterodimer formation of transcriptional activators. The mammalian homologue of the drosophila Hairy protein is HES-1. Targeted disruption of HES-1 leads to upregulation of neurogenic bHLH factors and neurogenesis. HES-1 has been implicated in control of timing of nervous system development, and retroviral overexpression of HES-1 in mouse embryos leads to delayed neuronal differentiation.

CHF-1 differs in structure and function from Hairy transcriptional regulators

CHF-1 differs from other Hairy family members structurally in that it lacks the canonical proline in its helix-loop-helix region and also lacks the canonical WRPW motif at the C-terminus. As a result, the function of CHF-1 differs from other Hairy family members. For example, its binding site specificity and ability to recruit the groucho family of transcriptional corepressors is altered, e.g., CHF-1 does not bind to E-box sequences. CHF-

1 expression in the developing cardiovascular system is unique. Based on its temporal expression in the cardiovascular system, CHF-1 functions as a regulator of cardiovascular differentiation.

Molecular cloning and characterization of CHF-1

A standard yeast 2-hybrid system was used to screen a human heart library for proteins which interact with the bHLH-PAS domain protein, Aryl-hydrocarbon Receptor Nuclear Translocator (ARNT). The screen identified a novel bHLH protein, CHF-1. The human CHF-1 nucleotide and amino acid sequence is shown in FIG. 3, and the mouse CHF- 1 nucleotide and amino acid sequence is shown in FIG. 2. Human CHF-1 is a 337 amino acid protein that contains a bHLH region near its amino terminus. By comparison of the CHF-1 amino acid sequence with other known sequences, CHF-1 was found to be a member of the Hairylike protein family. In contrast to other Hairylike proteins, CHF-1 is a nodal regulator of development and differentiation of cardiomyocytes and vascular smooth muscle cells.

Another bHLH cDNA which is expressed in the cardiac atria and in the developing aorta was also cloned. The nucleotide and amino acid sequences of human and mouse CHF-

2 are shown below in Tables 1-4. Table 1 : Human CHF-2 Amino Acid Sequence

MKRAHPEYSSSDSELDETIEVEKESADENGNLSSALGSMS PTTSSQILARKRRRGIIEKRRRDRI NSLSELRRLVPSAF EKQGSAKLEKAEILQMTVDHLK LHTAGGKGYFDAHALAM DYRSLGFRECLAEVARYLSIIEGLDASDPLRVRLVSHLNN YASQREAASGAHAGLGHIPWGTVFGHHPHIAHPLLLPQNG HGNAGTTASPTEPHHQGRLGSAHPEAPALRAPPSGSLGPV PWTSASKLSPPLLSSVASLSAFPFSFGSFHLLSPNALS PSAPTQAANLGKPYRP GTEIGAF (SEQ ID NO: 12)

Table 2: Human CHF-2 cDNA sequence gaattcgcggccgcgtcgaccgagagcggagaggcgccgctgtagttaactcctccctgccc gccgcgccgaccctccccaggaacccccagggagccagcATGAAGCGAGCTCACCCCGAGTA CAGCTCCTCGGACAGCGAGCTGGACGAGACCATCGAGGTGGAGAAGGAGAGTGCGGACGAGA ATGGAAACTTGAGTTCGGCTCTAGGTTCCATGTCCCCAACTACATCTTCCCAGATTTTGGCC AGAAAAAGACGGAGAGGAATAATTGAGAAGCGCCGACGAGACCGGATCAATAACAGTTTGTC TGAGCTGAGAAGGCTGGTACCCAGTGCTTTTGAGAAGCAGGGATCTGCTAAGCTAGAAAAAG CCGAGATCCTGCAGATGACCGTGGATCACCTGAAAATGCTGCATACGGCAGGAGGGAAAGGT TACTTTGACGCGCACGCCCTTGCTATGGACTATCGGAGTTTGGGATTTCGGGAATGCCTGGC AGAAGTTGCGCGTTATCTGAGCATCATTGAAGGACTAGATGCCTCTGACCCGCTTCGAGTTC

GACTGGTTTCGCATCTCAACAACTACGCTTCCCAGCGGGAAGCCGCGAGCGGCGCCCACGCG GGCCTCGGACACATTCCCTGGGGGACCGTCTTCGGACATCACCCGCACATCGCGCACCCGCT GTTGCTGCCCCAGAACGGCCACGGGAACGCGGGCACCACGGCCTCACCCACGGAACCGCACC ACCAGGGCAGGCTGGGCTCGGCACATCCGGAGGCGCCTGCTTTGCGAGCGCCCCCTAGCGGC AGCCTCGGACCGGTGCTCCCTGTGGTCACCTCCGCCTCCAAACTGTCGCCGCCTCTGCTCTC CTCAGTGGCCTCCCTGTCGGCCTTCCCCTTCTCTTTCGGCTCCTTCCACTTACTGTCTCCCA ATGCACTGAGCCCTTCAGCACCCACGCAGGCTGCAAACCTTGGCAAGCCCTATAGACCTTGG GGGACGGAGATCGGAGCTTTTTAAagaactgatgtagaatgagggaggggaaagtttaaaat ccagctgggctggactgttgccaacatcaccttaaagtcgtcagtaaaagtaaaaaggaaaa aggtacactttcagataattttttttttaaagactaaaggtttgttggtttacttttttctt ttttaatgtttttttcatcatgtcatgtattagcagtttttaaaaaactagttgttaaattt tgttcaagacattaaattgaaatagtgagtataagccaacactttgtgataggtttgtactg tgcctaatttactttgtaaaccagaatgattccgtttttgcctcaaaatttggggaatctta acatttagtatttttggtctgtttttctccttgtatagttatggtctgtttttagaattaat tttccaaaccactatgcttaatgttaacatgattctgtttgttaatattttgacagattaag gtgttgtataaataatattcttttggggggaggggaactatattgaattttatatttctgag caaagcgttgacaaatcagatgatcagctttatccaagaaagaagactagtaaattgtctgc ctccta agcagaaaggtgaatgtacaaactgttggtggccctgaatccatctgaccagctg ctgg atctgccaggactggcagttctgatttagttaggagagagccgctgataggttaggt ctca tggagtgttggtggaaaggaaactgaaggtaattgaatagaatacgcctgcattta ccagccccagcaacacaaagaatttttaatcacacggatctcaaattcacaaatgttaacat ggataagtgatcatggtgtgcgagtggtcaattgagtagtacagtggaacctgttaaatgca taacctaattttcctgggactgccatattttcttttaactggaaatttttatgtgagttttc cttttggtgcatggaactgtggttgccaagg a taaaagggctttcctgcctccttctct ttgatttatttaatttgatttgggctataaaatatcatttttcaggtttattcttttagcag gtgtagttaaacgacctccactgaactgggtttgacctctgttgtactgatgtgttgtgact aaataaaaaagaaagaacaaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aa (SEQ ID NO: 13; coding sequence indicated by upper case letters; termination codon underlined) Table 3: Mouse CHF-2 Amino Acid Sequence

MKRAHPEYSSSDSELDETIEVEKESADENGNLSSALGSMS PTTSSQILARKRRRGIIEKRRRDRINNS SELRRLVPSAF EKQGSAKLEKAEILQMTVDHLK LHTAGGKGYFDAHALAM DYRSLGFRECLAEVARYLSIIEGLDASDPLRVRLVSHLNN YASQREAASGAHAGLGHIP GTVFGHHPHIAHPLLLPQNG HGNAGTTASPTEPHHQGRLGSAHPEAPALRAPPSGSLGPV LPWTSASKLSPPLLSSVASLSAFPFSFGSFHLLSPNALS PSAPTQAANLGKPYRPWGTEIGAF (SEQ ID NO: 14)

Table 4: Mouse CHF-2 cDNA sequence gaattcgcggccgcgtcgaccgagagcggagaggcgccgctgtagttaactcctccctgccc gccgcgccgaccctccccaggaacccccagggagccagcATGAAGCGAGCTCACCCCGAGTA CAGCTCCTCGGACAGCGAGCTGGACGAGACCATCGAGGTGGAGAAGGAGAGTGCGGACGAGA ATGGAAACTTGAGTTCGGCTCTAGGTTCCATGTCCCCAACTACATCTTCCCAGATTTTGGCC AGAAAAAGACGGAGAGGAATAATTGAGAAGCGCCGACGAGACCGGATCAATAACAGTTTGTC TGAGCTGAGAAGGCTGGTACCCAGTGCTTTTGAGAAGCAGGGATCTGCTAAGCTAGAAAAAG CCGAGATCCTGCAGATGACCGTGGATCACCTGAAAATGCTGCATACGGCAGGAGGGAAAGGT TACTTTGACGCGCACGCCCTTGCTATGGACTATCGGAGTTTGGGATTTCGGGAATGCCTGGC AGAAGTTGCGCGTTATCTGAGCATCATTGAAGGACTAGATGCCTCTGACCCGCTTCGAGTTC

GACTGGTTTCGCATCTCAACAACTACGCTTCCCAGCGGGAAGCCGCGAGCGGCGCCCACGCG GGCCTCGGACACATTCCCTGGGGGACCGTCTTCGGACATCACCCGCACATCGCGCACCCGCT GTTGCTGCCCCAGAACGGCCACGGGAACGCGGGCACCACGGCCTCACCCACGGAACCGCACC ACCAGGGCAGGCTGGGCTCGGCACATCCGGAGGCGCCTGCTTTGCGAGCGCCCCCTAGCGGC AGCCTCGGACCGGTGCTCCCTGTGGTCACCTCCGCCTCCAAACTGTCGCCGCCTCTGCTCTC CTCAGTGGCCTCCCTGTCGGCCTTCCCCTTCTCTTTCGGCTCCTTCCACTTACTGTCTCCCA ATGCACTGAGCCCTTCAGCACCCACGCAGGCTGCAAACCTTGGCAAGCCCTATAGACCTTGG GGGACGGAGATCGGAGCTTTTTAAagaactgatgtagaatgagggaggggaaagtttaaaat ccagctgggctggactgttgccaacatcaccttaaagtcgtcagtaaaagtaaaaaggaaaa aggtacactttcagataattttttttttaaagactaaaggtttgttggtttacttttttctt ttttaatgtttttttcatcatgtcatgtattagcagtttttaaaaaactagttgttaaattt tgttcaagacattaaattgaaatagtgagtataagccaacactttgtgataggtttgtactg tgcctaatttactttgtaaaccagaatgattccgtttttgcctcaaaatttggggaatctta acatttagtatttttggtctgtttttctccttgtatagttatggtctgtttttagaattaat tttccaaaccactatgcttaatgttaacatgattctgtttgttaatattttgacagattaag gtgttgtataaataatattcttttggggggaggggaactatattgaattttata ttctgag caaagcgttgacaaatcagatgatcagctttatccaagaaagaagactagtaaattgtctgc ctcctatagcagaaaggtgaatgtacaaactgttggtggccctgaatccatctgaccagctg ctggtatctgccaggactggcagttctgatttagttaggagagagccgctgataggttaggt ctcatttggagtgttggtggaaaggaaactgaaggtaattgaatagaatacgcctgcattta ccagccccagcaacacaaagaatttttaatcacacggatctcaaattcacaaatgttaacat ggataagtgatcatggtgtgcgagtggtcaattgagtagtacagtggaacctgttaaatgca taacctaattttcctgggactgccatattttcttttaactggaaatttttatgtgagttttc cttttggtgcatggaactgtggttgccaaggta ttaaaagggctttcctgcctccttctct ttgatttatttaatttgatttgggctataaaatatcatttttcaggtttattcttttagcag gtgtagttaaacgacctccactgaactgggtttgacctctgttgtactgatgtgttgtgact aaataaaaaagaaagaacaaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aa (SEQ ID NO: 15; coding sequence indicated by upper case letters; termination codon underlined) CHF-1 is highly expressed in the developing cardiovascular system

Northern blot analysis revealed that CHF-1 is expressed as a 2.6 kb mRNA. Expression was found to highest in the adult aorta, followed by heart, lung and brain (FIG. 5). CHF-1 is also highly expressed in the developing mouse embryo. In situ hybridization revealed that CHF-1 is expressed in the ventricular segment of the primitive heart tube at day 8.5 in mouse embryos and appears in the dorsal aorta at day 9.5 (FIG. 6). This pattern of ventricular and arterial vascular expression continued throughout embryogenesis and persisted into adulthood, indicating that CHF-1 plays an important role in regulating differentiation of cardiac and vascular smooth muscle cells. CHF-1 expression is downregulated in cardiac myocytes during maturation (FIG. 7). Thus, although CHF-1 expression persists into adulthood, it remains at a low level in the adult cardiovascular system.

CHF-2 was found to be highly expressed in embryonic tissue (FIG. 14) as well as in brain and lung (FIG. 13).

CHF-1 is induced during smooth muscle cell differentiation

To investigate whether CHF-1 plays a role in smooth muscle ontogeny, CHF-1 expression in Monc-1 cells (Jain et al., 1998, J. Biol. Chem. 273:5993-5996) was evaluated. Monc-1 cells are mouse neural crest cells which can be induced to differentiate into smooth muscle cells in vitro. Monc-1 cells differentiate into smooth muscle cells when tissue culture medium supplemented with chick embryo extract is replaced with differentiation medium. CHF-1 was found to be undetectable in undifferentiated Monc-1 cells, but was induced within 24 hours after culture in smooth muscle differentiation medium (FIG. 8). These data indicate that CHF-1 is highly expressed during smooth muscle differentiation. CHF is the only tissue-specific bHLH protein identified in vascular smooth muscle cells. Based on this pattern of expression, the data indicate that CHF-1 is involved in the development of the heart and vasculature. The data described above indicate that CHF-1 regulates the timing of cardiac and vascular muscle differentiation both in vivo and in vitro. CHF-1 inhibits ARNT-dependent transactivation of the VEGF promoter

To investigate the effect of CHF- 1 on transcription, co-transfection experiments were carried out with ARNT, EPAS 1 , and the VEGF promoter. ARNT and EPAS 1 form a heterodimer and activate the VEGF promoter. As described above, CHF-1 interacts with ARNT in a yeast 2-hybrid assay. The functional significance of the interaction was investigated as follows. Plasmids encoding CHF-1, ARNT, and EPAS1 were transfected into bovine aortic endothelial cells (BAEC) with another plasmid containing the promoter region of the human VEGF gene upstream of a luciferase reporter gene. CHF-1 was found to inhibit activation of VEGF promoter activity (as measured by a standard luciferase assay) to 25% of its activity in the absence of CHF- 1 (FIG. 9).

Mapping CHF functional domains

Domains of CHF 1 required for transcriptional repression were identified by generating a series of deletion mutants. In transient transfection analysis, full-length CHF1 abolished ARNT/EPAS1 -dependent transcription. Deletion of amino acids 1-37 had no effect. In contrast, deletion of amino acids 1-146, removing the bHLH domain and part of the Orange domain, abolished repression. Deletion of amino acids 1-37 and 147-337, leaving primarily the bHLH domain, also abolished repression. Taken together, these results indicate that the bHLH domain is necessary for transcriptional repression, e.g., ARNT- dependent transcription. An intact Orange domain or other domains in the carboxy-terminus of CHF also function to mediate CHF repression of ARNT-dependent transcription.

CHF-deficient mice

To further investigate the role of CHF-1 in vivo, CHF-1 knockout mice (CHF-1- deficient mice) were generated by homologous recombination. A gene-targeting construct for generating CHF-1 -deficient mice was made using a targeted gene deletion strategy using standard methods (FIG. 10). The deletion in the CHF-1 gene renders the CHF-1 protein non-functional. The linearized targeting construct is transfected into murine D3 embryonic stem cells (murine W9.5 ES cells), and a clone with the correct homologous recombination (yielding the appropriately disrupted CHF-1 gene) is injected into blastocysts and used to generate CHF-1 deficient mice. Myocardial and smooth muscle cell regeneration

Although CHF-1 is highly expressed in fetal heart, its expression is markedly decreased in neonatal heart, a period when cardiomyocytes undergo terminal differentiation. Overexpression of CHF-1 inhibits terminal differentiation of cardiomyocytes (thereby promoting proliferation of cardiomyocytes), and thus administering a CHF-1 polypeptide (or a nucleic acid encoding a CHF-1 polypeptide) is useful as a therapeutic approach to regenerating cardiomyocytes in injured or diseased heart tissue. In addition, expression of CHF-1 in the dorsal aorta prior to the expression of smooth muscle markers indicates that CHF-1 plays an important role in cell fate determination in the vessel wall. CHF-1 is used to promote myocardial and smooth muscle regeneration after injury. Downregulation of CHF- 1 (e.g., by antisense therapy) to prevent cardiac hypertrophy and in amelioration of vascular occlusive disorders such as arteriosclerosis and Buerger's disease).

Production of CHF-specific antibodies

Anti-CHF antibodies are obtained by techniques well known in the art. Such antibodies can be polyclonal or monoclonal. Thus, the invention encompasses antibodies and antibody fragments, such as F_ab or (F_ab)₂ that bind immunospecifically to any of the CHF polypeptides of said invention.

An isolated CHF protein, or a portion or fragment thereof, e.g., a fragment containing a functional domain such as bHLH, can be used as an immunogen to generate antibodies that bind to CHF polypeptides using standard techniques for polyclonal and monoclonal antibody preparation. The full-length CHF proteins can be used or, alternatively, the invention provides antigenic peptide fragments of CHF proteins for use as immunogens. An antigenic peptide contains at least 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to someone skilled in the art. More preferably, the antibody will have an affinity of at least about 10⁸ liters/mole and more preferably, an affinity of at least about 10⁹ liters/mole. Antigenic polypeptides useful as immunogens include polypeptides which contain a bHLH domain and/or a YQPW (SEQ ID NO:7) or YRPW (SEQ ID NO:6) motif of a CHF polypeptide.

In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of CHF that is located on the surface of the protein (e.g., a hydrophilic region). As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte-Doolittle or the Hopp- Woods methods, either with or without Fourier transformation (see, e.g., Hopp and Woods, 1981. Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle, 1982. J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety).

As disclosed herein, CHF protein sequences of SEQ ID NO: 1 and 12, or derivatives, fragments, analogs, or homologs thereof, may be utilized as immunogens in the generation of antibodies that immunospecifically-bind these protein components. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically-binds (immunoreacts with) an antigen, such as CHF. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_ab and F_(ab)2 fragments, and an F_ab expression library. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to a CHF protein sequence of SEQ ID NO: 1 and 12, or a derivative, fragment, analog, or homolog thereof.

For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed CHF protein or a chemically- synthesized CHF polypeptide. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. If desired, the antibody molecules directed against CHF can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.

The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of CHF. A monoclonal antibody composition thus typically displays a single binding affinity for a particular CHF protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular CHF protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see, e.g., Kozbor, et al, 1983. Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see, e.g., Cole, et al, 1985. In: MONOCLONAL ANTIBODIES AND CANCER

THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the invention and may be produced by using human hybridomas (see, e.g., Cote, et al, 1983. Proc. Natl Acad. Sci. USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see, e.g., Cole, et al, 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Each of the above citations is incorporated herein by reference in their entirety.

According to the invention, techniques can be adapted for the production of single-chain antibodies specific to a CHF protein (see, e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F_ab expression libraries (see, e.g., Huse, et al, 1989. Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F_ab fragments with the desired specificity for a CHF protein or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be "humanized" by techniques well known in the art. See, e.g., U.S. Patent No. 5,225,539. Antibody fragments that contain the idiotypes to a CHF protein may be produced by techniques known in the art including, but not limited to: (/) an F_(ab.₎₂ fragment produced by pepsin digestion of an antibody molecule; (ii) an F_ab fragment generated by reducing the disulfide bridges of an F_(ab)2 fragment; (Hi) an F_ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F_v fragments.

Additionally, recombinant anti-CHF antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No. 125,023; Better, et al, 1988. Science 240:

1041-1043; Liu, et al, 1987. Proc. Natl Acad. Sci. USA 84: 3439-3443; Liu, et al, 1987. J Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl Acad. Sci. USA 84: 214-218; Nishimura, et al, 1987. Cancer Res. 47: 999-1005; Wood, et al, 1985. Nature 314 :446-449; Shaw, et al, 1988. J. Natl. Cancer Inst. 80: 1553-1559); Morrison(1985) Science 229:1202-1207; Oi, et al. (1986) BioTechniques 4:214; Jones, et al, 1986. Nature 321:

552-525; Verhoeyan, et al, 1988. Science 239: 1534; and Beidler, et al, 1988. J. Immunol 141 : 4053-4060. Each of the above citations are incorporated herein by reference in their entirety.

Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of a CHF protein is facilitated by generation of hybridomas that bind to the fragment of a CHF protein possessing such a domain. Thus, antibodies that are specific for a desired domain within a CHF protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

Anti-CHF antibodies may be used in methods known within the art relating to the localization and/or quantitation of a CHF protein (e.g., for use in measuring levels of the CHF protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for CHF proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").

An anti-CHF antibody (e.g., monoclonal antibody) is used to isolate a CHF polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-CHF antibody facilitates the purification of natural CHF polypeptide from cells and of recombinantly-produced CHF polypeptide expressed in host cells. Moreover, an anti-CHF antibody can be used to detect CHF protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the CHF protein.

CHF Recombinant Expression Vectors and Host Cells

Recombinant expression vectors contain a CHF nucleic acid cloned in-frame with one or more regulatory sequences for expression in a desired cell, e.g., vascular cells. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). CHF DNA is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBOJ. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Preferably, CHF DNA is operably linked to a promoter or other regulatory sequences for preferential expression in vascular smooth muscle cells. Suitable promoter sequences include an alpha-mysosin heavy chain (MHC) promoter and an SmLIM promoter.

For example, a SmLIM is described in United States Patent No. 5,767,262, which is incorporated herewith by reference. Vascular tissue-specific promoters are used to target expression of CHF gene products to heart tissue and and blood vessel walls.

Table 5 : Human SmLIM Promoter Sequence

TGAGGAATGCAGCTCTTTCGCGACAGGAAAGCTGCGGATTCCAGAAGCCGGGATTCTGAC60 CAGAGACTATCTGCACCGGGGAGTCCTGCACCCCGAGCTAACATATGGCGTTTGTGCAGT120 AAAAGGGTGGCGGGAATCCCACGGGGCGACACCGGATCTCGCTGGCTCCGGGCCGATCCT180 GAGTGCTCCGGACGTCCCGGGACCGCGGGTAGGAGCAGCCGAGACGTGGGAGACTCGGAC240 GCGGGAAGCCGCAGGAAGAGGCGGATTCCGGTCTTTTTGTCTCGGGGCCAGAGCACGAAA300 CCCGCATCGGATCCCCGAGCTCACGCCGGGCGGAGACCATCGCACACCCGAGGGGCATGA360 CCGATGGCTGAGTCGGAACAAGCCACGCCCAACATAAGTCTTTAAAAGCGGGCACACGCG420 TCCCGCC427

(SEQ ID NO: 17) The nucleic acid sequence of a human alpha-MHC gene promoter (GenBank Accession No. X05632 Y00362) is shown in Table 6 below.

Table 6: Human alpha-MHC sequence

1 cggtgtgaga aggtccagtc ttcccagcta tctgctcatc agccctttga aggggaggaa 61 tgtgcccaag gactaaaaaa aggccgtgga gccagagagg ctggggcagc agacctttca

121 agggcaaatc aggggccctg ctgtcctcct gtcacctcca gagccaaagg atcaaaggag

181 gaggagccag gagggagaga ggtgggaggg agggtcctcc ggaaggactc caaatttaga

241 cagagggtgg gggaaacggg atataaagga actggagctt tgaggagaga tagagagact

₃01 ctgcggccca ggtaagagga ggtttggggt gggatgccct gcagcccgtc cacagagccc 361 ccaccgtgag ggacctcctt caccaggagt ggggtgcagg tcagttggag gcctaagggc

421 tctattaaaa ctgcctatct ccaggcccag ggaagttccc cctgacacag gaggttccac

481 aggaaaccca gaaacctctt ttctccttct ctgactctcc atttctttct ctgcatcatt

541 ctgagtctcc tgcatgttgt ctccatcttt ccatcttcac ttcctccttt ggatggcttc

601 cttcccttga tcctggcttt tatcttgcct cttggtcttc atcgacactt gtcacaatca 661 tgcttctttg tctctctccc ttgtccttcc ttcttggcac ttgttctcac ctccctgcct

721 ctctgcttct aaccctgttt ccacaccccg tcccacctgg ggctgcctcc atccccgggt

781 ggcctgcctn tggtgttctt cactctcctc atttgttctt ctctctgccc ggctctacct

841 ntggtgttcc ttgctccacc cacggtccag attcttcagg attctccgtg aagggataac

901 caggtgagaa ctgcccccat tttctctgca gagactgggg catgcttctc ctgggagccg 961 gattgctgga ccaggggtct gctgtcccaa gcactcagcg ccaaccctta gcatactcca

1021 gccaatgcca ccccagggaa accccttaca gagattgtcc ttcagcatca cctcagaggg

1081 caggagaagc agagccctga gtaggggagg tgcaacagca gtgcctctcc cagggtggag

1141 gagaggagcg ggggtaggga gggggtctgc agaggacaaa gccactcgct ggagcctggg

1201 ctccctcagg agtaacatag ccctcctgtc tctgacccag ggaagcacca agatgaccga 1261 tagccagatg gctgactttg gcacggccca gtacctccgc aagtcagaga aggagcgtct

1321 agaggcccag acccggccct ttgacattcg cactgagtgc ttcgtgcccg atgacaagga

1381 agagtttgtc aaagccaaga ttttgtcccg ggagggaggc aaggtcattg ctgaaaccga

1441 gaatgggaag gtgagtaggg catggcgccg gggcagaagg gaaggaggtc tgggaaagaa

1501 gatgcaggag gaggtgccac ttgcaggggg agctgagagg gctggagaaa agccaaggcc 1561 agtggggatg ccaggacatg ctcctttgag gagcccagaa tctgatccct ctcaaattag

1621 cctgagctgg tgcaacaggt gccacccagg gccatgttcc ccctgccaga gaggatgctg

1681 aggaagaaga acctcagtgt tcgcctaaga ggggtcttgt agataaagag ggcacagaca 1741 cagcattaaa tgatgccccc ttcttgcact tgtatccctc caccctgtgc ctcagttcct 1801 ccatgagtcc accctctcaa attccgttca cccaaatcaa gagcaattct tagacccaga 1861 tgaacacaaa gatcagaaac ttttgagctg atgcactctc cttgactggc gactcagaag 1921 ctctggtccc tggtttgctc acaccagcca atagaatcac ccctggttac cagctgcggc 1981 tcaggctgtg tgcctcatga actcgttgac tgaatgttac aacccattga agtgtagaat 2041 aacaggccac aatcccctgg ggcttttgac tctgatccca gctcagccac ccgctagtca 2101 ctgtgcaggc aaatcattta gtcatttaga gccacggatt tctccactat aaaaaacact 2161 ggaataccta ctggcaggat ctaatgacat cagggcatgg caaactgact gctgccaatc 2221 aaaccacacc aacagtgatg gatggggagt gtggagtaga tgggtgaact acttttccag 2281 caggggtgaa ggtttgccct gagcaacaga taccctaaag gcctgcccgc gggagacagc 2341 ctcggggtca gcataaggtg tgcaca (SEQ ID NO: 18)

Therapeutic Administration

CHF-1 is administered locally to injured or diseased heart tissue. One means for accomplishing local delivery is providing a CHF-1 polypeptide or DNA encoding a CHF-1 polypeptide on a surface of a vascular catheter, e.g., a balloon catheter or perfusion cathetor, which contacts the wall of a blood vessel to deliver therapeutic compositions at the site of contact. Drug delivery catheters or vascular stents are also be used to administer solutions of therapeutic compositions.

CHF-1 is therapeutically overexpressed (e.g., by administering an inducing agent) to increase expression from the endogenous gene or by administering DNA (alone or in a plasmid) encoding an CHF-1 under the control of a strong inducible or constitutive promoter.

For local administration of DNA to cardiovascular tissue, standard gene therapy vectors used. Suitable vectors include adenovirus vectors known in the art, e.g., those described by He et al., 1998, Proc Natl Acad Sci U S A 95:2509-14. Other suitable vectors include those derived from replication-defective hepatitis viruses (e.g., HBV and HCV), retroviruses (see, e.g., WO 89/07136; Rosenberg et al., 1990, N. Eng. J. Med. 323(9):570- 578), adenovirus (see, e.g., Morsey et al., 1993, J. Cell. Biochem., Supp. 17E,), adeno- associated virus (Kotin et al., 1990, Proc. Natl. Acad. Sci. USA 87:2211-2215,), replication defective herpes simplex viruses (HSV; Lu et al., 1992, Abstract, page 66, Abstracts of the Meeting on Gene Therapy, Sept. 22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), and any modified versions of these vectors. The invention may utilize any other delivery system which accomplishes in vivo transfer of nucleic acids into eucaryotic cells. For example, the nucleic acids may be packaged into liposomes, e.g., cationic liposomes (Lipofectin), receptor-mediated delivery systems, non-viral nucleic acid-based vectors, erythrocyte ghosts, or microspheres (e.g., microparticles; see, e.g., U.S. Patent No. 4,789,734; U.S. Patent No. 4,925,673; U.S. Patent No. 3,625,214; Gregoriadis, 1979, Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press,). Naked DNA may also be administered. Alternatively, a plasmid which directs cardiospecific expression (e.g., a plasmid containing a myosin heavy chain ( MHC) promoter) of a CHF-1 -encoding sequence can be used for gene therapy. Overexpression of a CHF-1 polypeptide, e.g, from such a constitutive promoter, is useful to inhibit terminal differentiation, and thus, promote proliferation of cardiomyocytes in vivo. For gene therapy of cardiovascular tissue, fusigenic viral liposome delivery systems known in the art (e.g., hemagglutinating virus of Japan (HVJ) liposomes or Sendai virus-liposomes) are useful for efficiency of plasmid DNA transfer (Dzau et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:11421-11425). Using HVJ-liposomes, genes are expressed from plasmid DNA delivered to target tissues in vivo for extended periods of time (e.g., greater than two weeks for heart and arterial tissue and up to several months in other tissues).

CHF-1 promoter DNA is used to target expression of heterolous gene products (polypeptides other than CHF-1) to heart tissue and and the blood vessel walls. Promoter regulatory sequences of the CHF-1 gene, e.g, sequences located approximately 500 nucleotides upstream of exon 1, are used in targeting vectors for enhanced expression of therapeutic polypeptides in the heart and vasculature.

DNA for gene therapy is administered to patients parenterally, e.g., intravenously, subcutaneously, intramuscularly, and intraperitoneally. Sustained release administration such as depot injections or erodible or indwelling implants, e.g., vascular stents coated with DNA encoding a CHF-1 polypeptide, are also be used. The compounds may also be directly applied during surgery, e.g, bypass surgery, or during angioplasty, e.g, an angioplasty catheter may be coated with DNA encoding a CHF polypeptide. The DNA is then deposited at the site of angioplasty. DNA or an inducing agent is administered in a pharmaceutically acceptable carrier, i.e., a biologically compatible vehicle which is suitable for administration to an animal e.g., physiological saline. A therapeutically effective amount is an amount

Claims

which is capable of producing a medically desirable result, e.g., expression or overexpression of CHF-1, in a treated animal. Such an amount can be determined by one of ordinary skill in the art. As is well known in the medical arts, dosage for any given patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, severity of arteriosclerosis or vascular injury, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for intravenous administration of DNA is approximately 106 to 1022 copies of the DNA molecule.Ex vivo therapyExplanted veinous tissue is bathed in a solution of a DNA encoding a human CHF polypeptide, e.g., cloned into a standard adenovirus gene therapy vector, to arterialize a veinous tissue prior to engraftment into an autologous or heterologous host. A segment of vein is surgically removed from an individual and placed in a tissue culture vessel in a solution of a physiologically-acceptable buffer, e.g., phosphate-buffered saline, and DNA encoding the CHF polypeptide. The vein graft is allowed to soak in the solution at room temperature or at 37 degrees C for at least 5 minutes and up to several days to allow uptake of the DNA by vascular cells and expression of the encoded CHF polypeptide. After soaking the veinous tissue in the CHF DNA bath, the vein graft is rinsed and processed for surgical engraftment using standard methods. For example, the vein graft is used in a standard bypass surgical procedure.In addition to presoaking the vein graft, the graft is optionally contacted with CHF DNA after engraftment. CHF DNA is administered in vivo using a perfusion catheter or vascular stent.Other embodiments are within the following claims. What is claimed is:

1. A substantially pure DNA comprising a sequence encoding a Cardiovascular Helix-loop-helix Factor (CHF) polypeptide.

2. The DNA of claim 1, wherein said CHF polypeptide is CHF-1.

3. The DNA of claim 1, wherein said polypeptide is human CHF-2.

4. The DNA of claim 1, wherein said DNA is operably linked to regulatory sequences for expression of said polypeptide, said regulatory sequences comprising a promoter.

5. The DNA of claim 1 , wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

6. The DNA of claim 1, wherein said polypeptide comprises amino acids 49-106 of SEQ ID NO: 2.

7. The DNA of claim 1, wherein said polypeptide comprises amino acids 52-104 of SEQ ID NO: 1.

8. The DNA of claim 1, wherein said polypeptide comprises amino acids 52-104 of SEQ ID NO: 12.

9. The DNA of claim 1, wherein said polypeptide comprises amino acids 121- 162 of SEQ ID NO: 1.

10. The DNA of claim 1, wherein said polypeptide comprises amino acids 121- 162 of SEQ ID NO: 12.

11. The DNA of claim 1, wherein said polypeptide comprises amino acids 38-337 of SEQ ID NO. 1.

12. The DNA of claim 1, wherein said polypeptide comprises amino acids 38-337 of SEQ ID NO. 12.

13. The DNA of claim 1, wherein said polypeptide comprises amino acids 147- 337 ofSEQ ID NO. 1.

14. The DNA of claim 1, wherein said polypeptide comprises amino acids 147- 337 ofSEQ ID NO. 12.

15. A substantially pure DNA comprising a nucleotide sequence having at least

50% sequence identity to SEQ ID NO: 3.

16. The DNA of claim 1, wherein said DNA comprises the coding sequences of SEQ ID NO: 3.

17. The DNA of claim 1, wherein said DNA comprises the coding sequences of SEQ ID NO: 4.

18. The DNA of claim 1 , wherein said polypeptide is mouse CHF-1.

19. The DNA of claim 18, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:2.

20. The DNA of claim 18, wherein said polypeptide comprises amino acids 49- 106 of SEQ ID NO:2.

21. A substantially pure DNA comprising a strand which hybridizes at high stringency to a strand of DNA having the sequence of SEQ ID NO:3, or the complement thereof.

22. A substantially pure DNA having at least 50% sequence identity to SEQ ID NO:3, and encoding a polypeptide having the biological activity of a CHF-1 polypeptide.

23. A substantially pure human CHF polypeptide.

24. The polypeptide of claim 23, wherein said polypeptide is a CHF-1 polypeptide.

25. The polypeptide of claim 23, wherein said polypeptide is human CHF-1.

26. The polypeptide of claim 23, wherein said polypeptide comprises amino acids

52-104 of SEQ ID NO:l.

27. The polypeptide of claim 23, wherein said polypeptide comprises amino acids 52-104 of SEQ ID NO:12.

28. The polypeptide of claim 23 wherein said polypeptide comprises amino acids 52-104 of SEQ ID NO: 1.

29. The polypeptide of claim 23 wherein said polypeptide comprises amino acids

52-104 of SEQ ID NO: 12.

30. The polypeptide of claim 23 wherein said polypeptide comprises amino acids 121-162 of SEQ ID NO: 1.

31. The polypeptide of claim 23 wherein said polypeptide comprises amino acids 121-162 of SEQ ID NO: 12.

32. The polypeptide of claim 24 wherein said polypeptide comprises amino acids 38-337 of SEQ ID NO. 1.

33. The polypeptide of claim 23 wherein said polypeptide comprises amino acids 38-337 of SEQ ID NO. 12.

34. The polypeptide of claim 23 wherein said polypeptide comprises amino acids

147-337 of SEQ ID NO. 1.

35. The polypeptide of claim 23 wherein said polypeptide comprises amino acids 147-337 of SEQ ID NO. 12.

36. The polypeptide of claim 23, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:5.

37. The polypeptide of claim 23, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:6.

38. The polypeptide of claim 23, wherein said polypeptide comprises an amino acid sequence at least 50% identical to SEQ ID NO:l.

39. The polypeptide of claim 23, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

40. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

41. A substantially pure mouse CHF polypeptide.

42. The polypeptide of claim 41 , wherein said polypeptide comprises amino acids 49-106 of SEQ ID NO:2.

43. A vector comprising the DNA of claim 1.

44. A method of expanding an endogenous population of proliferative cardiomyocytes in a heart tissue of a mammal, comprising contacting said tissue with a CHF polypeptide.

45. A method of promoting proliferation of a cardiomyocyte in a mammal comprising administering to said mammal a CHF-1 polypeptide.

46. The method of claim 45, wherein said CHF-1 polypeptide comprises the amino acid sequence of SEQ ID NO:5.

47. The method of claim 45, wherein said CHF-1 polypeptide further comprises the amino acid sequence of SEQ ID NO:6.

48. The method of claim 45, wherein said CHF-1 polypeptide is administered locally to the myocardium of said mammal.

49. The method of claim 45, wherein said mammal has suffered a myocardial infarction.

50. The method of claim 45, wherein said mammal is characterized as having myocarditis.

51. A method of promoting proliferation of a cardiomyocyte in a mammal comprising locally administering to said mammal a DNA encoding a CHF-1 polypeptide.

52. A method of regenerating cardiomyocytes in vivo, comprising contacting cardiomyocytes with a CHF-1 polypeptide.

53. A method of regenerating cardiomyocytes in vitro, comprising contacting cardiomyocytes with DNA encoding a CHF-1 polypeptide.

54. A method inhibiting angiogenesis in a tissue, comprising contacting said tissue with a DNA comprising a sequence encoding a Cardiovascular Helix-loop-helix Factor (CHF) polypeptide.

55. A method of inducing differentiation of smooth muscle cells in a mammalian tissue, comprising contacting said tissue with DNA comprising a sequence encoding a

Cardiovascular Helix-loop-helix Factor (CHF) polypeptide.

56. The method of claim 55, wherein said tissue is veinous tissue.

57. A method of inducing growth of smooth muscle cells in a vein explant, comprising contacting said explant with a DNA comprising a sequence encoding a Cardiovascular Helix-loop-helix Factor (CHF) polypeptide.

58. A method of reducing vein graft stenosis in a mammal, comprising contacting ex vivo a veinous tissue with a DNA encoding a Cardiovascular Helix-loop-helix Factor (CHF) polypeptide prior to implantation of said veinous tissue into an artery of said mammal.

59. A method of promoting smooth muscle cell regeneration in an injured or diseased vascular tissue, comprising contacting the tissue with a DNA encoding a

Cardiovascular Helix-loop-helix Factor (CHF) polypeptide.

60. The method of claims 59, wherein said tissue is veinous or arterial tissue.