WO2008005253A2 - Methods for diagnosing and treating heart disease - Google Patents

Abstract

The invention provides methods of diagnosing, preventing, and treating heart disease, and screening methods for identifying compounds that can be used to treat or prevent heart disease. The diagnostic methods of the invention involve detection of mutations in genes encoding san or vtn proteins (and/or altered san or vtn levels), while the compound identification methods involve screening for compounds that affect the phenotype of organisms having mutations in genes encoding san or vtn or other appropriate models. The invention also provides for compounds identified in this manner, as well as san or vtn genes, siRNA molecules, proteins, and antibodies.

Description

METHODS FOR DIAGNOSING AND TREATING HEART DISEASE

Field of the Invention This invention relates to methods for diagnosing and treating heart disease.

Background of the Invention

Heart disease is a general term used to describe many different heart conditions, which affect over 50 million Americans. For example, coronary artery disease, which is the most common form of heart disease, is characterized by constriction or narrowing of the arteries supplying the heart with oxygen-rich blood, and can lead to myocardial infarction, which is the death of a portion of the heart muscle. Heart failure is a condition resulting from the inability of the heart to pump an adequate amount of blood through the body. Heart failure is not a sudden, abrupt stop of heart activity but, rather, typically develops slowly over many years, as the heart gradually loses its ability to pump blood efficiently. Risk factors for heart failure include coronary artery disease, atherosclerosis, hypertension, valvular heart disease, cardiomyopathy, disease of the heart muscle, obesity, diabetes, and a family history of heart failure. There is a great need for new methods of diagnosing and treating heart disease, in light of the prevalence and medical impact of the disease.

Summary of the Invention

We have identified two genes, herein referred to as santa (san) and valentine (vtn), which play a role in the proper growth and development of the heart and vascular system.

Accordingly, the invention provides methods of determining whether a test subject (e.g., a mammal, such as a human) has or is at risk of developing a heart disease or condition related to san or vtn (e.g., heart failure). These methods can include analyzing a nucleic acid molecule of a sample from the test subject to determine whether the test subject has a mutation in a gene encoding san or vtn.

Detection of the presence of a mutation indicates that the test subject has or is at risk of developing a heart disease or condition related to san or vtn. The invention also provides methods for identifying compounds that modulate the activity of san or vtn. These methods can include incubating san or vtn, or a gene encoding san or vtn, with a candidate compound. The incubating can be carried out, e.g., in a cell-free mixture, a cell-based mixture, a recombinant cell, or an animal (e.g., a human, mouse, or zebrafish). These screening methods also can involve comparing the activity of san or vtn (e.g., the onset of concentric growth in a developing heart) in the presence of a candidate compound with the activity of san or vtn in the absence of the candidate compound.

Also included in the invention are methods of treating or preventing a disease or condition associated with san or vtn (e.g., heart disease, such as heart failure) in patients. These methods involve administering to the patient an expression vector (e.g., an adeno-associated virus) encoding san or vtn (e.g., human san or human vtn). The invention also includes use of such expression vectors in the preparation of medicaments for preventing or treating these diseases and conditions.

The invention also provides methods of treating or preventing a disease or condition associated with san or vtn in patients, which involve administration of a protein preparation of san or vtn (e.g., human san or human vtn). Optionally, the protein preparation can include a protein transduction domain-san or -vtn fusion. Examples of protein transduction domains that can be used in the invention include Tat, Antp, and VP22. The invention also includes use of such protein preparations in the preparation of medicaments for preventing or treating these diseases and conditions.

Further, the invention provides isolated nucleic acid molecules encoding zebrafish san (SEQ ID NO:6), isolated nucleic acid molecules encoding the amino acid sequence set forth in SEQ ID NO: 3, and isolated zebrafish san proteins (SEQ ID NO:3). The nucleic acid molecules can, optionally, be operatively linked to expression control sequences and/or be present in vectors. The invention also includes host cells containing such vectors. The invention also provides isolated nucleic acid molecules encoding zebrafish vtn (SEQ ID NO: 12), isolated nucleic acid molecules encoding the amino acid sequence set forth in SEQ ID NO: 9, and isolated zebrafish vtn proteins (SEQ ID NO: 9). As with the san nucleic acid molecules noted above, the vtn nucleic acid molecules can, optionally, be operatively linked to expression control sequences and/or be present in vectors. Further, the invention provides host cells containing such vectors.

In addition, the invention provides non-human transgenic animals (e.g., zebrafish or mice) including nucleic acid molecules encoding san or vtn. The invention also includes non-human animals having knockout mutations in one or both alleles encoding a san or vtn polypeptide, as well as cells from such non-human knockout animals.

The invention also provides methods of treating or preventing diseases or conditions associated with san or vtn in patients (e.g., heart disease, such as heart failure), which involve administration of host cells (e.g., an autologous host cell) expressing san or vtn, such as host cells as described herein. The invention also includes use of such host cells in the preparation of medicaments for preventing or treating these diseases and conditions.

Isolated nucleic acid molecules including an siRNA that inhibits expression of san or vtn are also included in the invention. Such molecules can be, optionally, operatively linked to expression control sequences, for example, as part of a vector. Further, the invention includes host cells containing such vectors. The siRNA molecules, vectors, and/or host cells can be administered to a subject in methods of treating or preventing a disease or condition associated with san or vtn, as described herein.

Also included in the invention are antibodies that specifically bind to san or vtn, or fragments thereof. By "polypeptide" or "polypeptide fragment" is meant a chain of two or more

(e.g., 10, 15, 20, 30, 50, 100, 200, or more) amino acids, regardless of any post- translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally or non-naturally occurring polypeptide. By "post-translational modification" is meant any change to a polypeptide or polypeptide fragment during or after synthesis. Post-translational modifications can be produced naturally (such as during synthesis within a cell) or generated artificially (such as by recombinant or chemical means). A "protein" can be made up of one or more polypeptides.

By "santa" or "san" is meant a polypeptide that has at least 45%, 60%, 75%, or 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) amino acid sequence identity to the sequence of a human santa (also named CCMl, SEQ ID NO:1), mouse santa (SEQ ID NO:2), or zebrafish santa (SEQ ID NO:3) polypeptide. Polypeptide products from splice variants of san gene sequences and san genes containing mutations are also included in this definition. A san polypeptide as defined herein can play a role in heart development, modeling, and function. It can be used as a marker of diseases and conditions associated with san, such as heart disease (e.g., heart failure; also see below).

By a "santa nucleic acid molecule" or "san nucleic acid molecule" is meant a nucleic acid molecule, such as a genomic DNA, cDNA, or RNA (e.g., mRNA) molecule, that encodes a san protein (e.g., a human (also named CCMl, encoded by SEQ ID NO:4), a mouse (encoded by SEQ ID NO:5) or a zebrafish (encoded by SEQ ID NO:6) san protein), a san polypeptide, or a portion thereof, as defined above. A mutation in a san nucleic acid molecule can be characterized, for example, by the insertion of a premature stop codon anywhere in the san gene, or by a mutation in a splice donor and/or acceptor site, which leads to aberrant transcript production (e.g., transcripts with premature stop codons). The invention includes any mutation that results in aberrant san message or protein production or function, including, only as examples, null mutations and additional mutations causing truncations.

By "valentine" or "vtn" is meant a polypeptide that has at least 45%, 60%, 75%, or 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) amino acid sequence identity to the sequence of a human valentine (CCM2, SEQ ID NO:7), mouse valentine (SEQ ID NO: 8), or zebrafish valentine (SEQ ID NO:9) polypeptide. Polypeptide products from splice variants of vtn gene sequences and vtn genes containing mutations are also included in this definition. A vtn polypeptide as defined herein can play a role in heart development, modeling, and function. It can be used as a marker of diseases and conditions associated with vtn, such as heart disease (e.g., heart failure; also see below).

By a "valentine nucleic acid molecule" or "vtn nucleic acid molecule" is meant a nucleic acid molecule, such as a genomic DNA, cDNA, or RNA (e.g., mRNA) molecule, that encodes a vtn protein (e.g., a human (also named CCM2, encoded by SEQ ID NO: 10), mouse (encoded by SEQ ID NO:11) or a zebrafish (encoded by SEQ ID NO: 12) vtn protein), a vtn polypeptide, or a portion thereof, as defined above. A mutation in a vtn nucleic acid molecule can be characterized, for example, by the insertion of a premature stop codon anywhere in the vtn gene, or by a mutation in a splice donor and/or acceptor site, which leads to aberrant transcript production (e.g., transcripts with premature stop codons). The invention includes any mutation that results in aberrant vtn message or protein production or function, including, only as examples, null mutations and additional mutations causing truncations.

The term "identity" is used herein to describe the relationship of the s^'equence of a particular nucleic acid molecule or polypeptide to the sequence of a reference molecule of the same type. For example, if a polypeptide or a nucleic acid molecule has the same amino acid or nucleotide residue at a given position, as compared to a reference molecule to which it is aligned, there is said to be "identity" at that position. The level of sequence identity of a nucleic acid molecule or a polypeptide to a reference molecule is typically measured using sequence analysis software with the default parameters specified therein, such as the introduction of gaps to achieve an optimal alignment (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, or PILEUP/PRETTYBOX programs). These software programs match identical or similar sequences by assigning degrees of identity to various substitutions, deletions, or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, and leucine; aspartic acid, glutamic acid, asparagine, and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. The sequence of a nucleic acid molecule or polypeptide is said to be "substantially identical" to that of a reference molecule if it exhibits, over its entire length, at least 51%, 55%, 60%, 65%, 75%, 85%, 90%, or 95% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the sequence of the reference molecule. For polypeptides, the length of comparison is typically at least 16 , 20, 25, or 35 amino acids. For nucleic acid molecules, the length of comparison is typically at least 25, 40, 50, 60, 75, or 110 nucleotides. Of course, the length of comparison can be any length up to and including full length.

A san or vtn nucleic acid molecule or a san or vtn polypeptide is "analyzed" or subject to "analysis" if a test procedure is carried out on it that allows the determination of its biological activity or whether it is wild type or mutated. For example, one can analyze the san or vtn genes of an animal (e.g., a human or a zebrafish) by amplifying genomic DNA of the animal using the polymerase chain reaction, and then determining whether the amplified DNA contains a mutation by, e.g., nucleotide sequence or restriction fragment analysis.

By "probe" or "primer" is meant a single-stranded DNA or RNA molecule of defined sequence that can hybridize to a second DNA or RNA molecule that contains a complementary sequence (a "target"). The stability of the resulting hybrid depends upon the extent of the base pairing that occurs. This stability is affected by parameters such as the degree of complementarity between the probe/primer and target molecule, and the degree of stringency of the hybridization conditions, the latter of which is affected by parameters such as the temperature, salt concentration, and concentration of organic molecules, such as formamide, and is determined by methods that are well known to those skilled in the art. Probes or primers specific for san or vtn nucleic acid molecules can have, e.g., greater than 45%, 55-75%, 75-85%, 85-99%, or 100% sequence identity to the sequences of human san or vtn or zebrafish san or vtn, or fragments thereof.

Probes can be detectably labeled, either radioactively or non-radioactively, by methods that are well known to those skilled in the art. Probes can be used for methods involving nucleic acid hybridization, such as nucleic acid sequencing, nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RPLP) analysis, Southern hybridization, northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA), and other methods that are well known to those skilled in the art.

A molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, a cDNA molecule, a polypeptide, or an antibody, can be said to be "detectably-labeled" if it is marked in such a way that its presence can be directly identified in a sample. Methods for detectably labeling molecules are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope, such as ³²P or ³⁵S) and nonradioactive labeling (e.g., with a fluorescent label, such as fluorescein).

By a "substantially pure" polypeptide is meant a polypeptide (or a fragment thereof) that has been separated from proteins and organic molecules that naturally accompany it. Typically, a polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. For example, a san or vtn polypeptide can be at least 75%, 90%, or 99%, by weight, pure. A substantially pure san or vtn polypeptide can be obtained, for example, by extraction from a natural source, expression of a recombinant nucleic acid molecule encoding a san or vtn polypeptide, or chemical synthesis. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A polypeptide is substantially free of naturally associated components when it is separated from those proteins and organic molecules that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system that is different from the cell in which it is naturally produced is substantially free from its naturally associated components. Accordingly, substantially pure polypeptides not only include those that are derived from eukaryotic organisms, but also those synthesized in E. coli, other prokaryotes, and in other such systems. By an "isolated" nucleic acid molecule is meant a nucleic acid molecule that is removed from the environment in which it naturally occurs. For example, a naturally- occurring nucleic acid molecule present in the genome of cell or as part of a gene bank is not isolated, but the same molecule, separated from the remaining part of the genome, as a result of, e.g., a cloning event (amplification), is "isolated." Typically, an isolated nucleic acid molecule is free from nucleic acid regions (e.g., coding regions) with which it is immediately contiguous, at the 5' or 3' ends, in the naturally occurring genome. Such isolated nucleic acid molecules can be part of a vector or a composition and still be isolated, as such a vector or composition is not part of its natural environment.

An antibody is said to "specifically bind" to a polypeptide if it recognizes and binds to the polypeptide (e.g., a san or vtn polypeptide, or a fragment thereof), but does not substantially recognize and bind to other molecules (e.g., non-san or non-vtn- related polypeptides) in a sample, e.g., a biological sample, which naturally includes the polypeptide.

By "high stringency" conditions is meant conditions that allow hybridization comparable with the hybridization that occurs using a DNA probe of at least 100, e.g., 200, 350, or 500, nucleotides in length, in a buffer containing 0.5 M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V), at a temperature of 65⁰C, or a buffer containing 48% formamide, 4.8 x SSC, 0.2 M Tris-Cl, pH 7.6, Ix Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C. (These are typical conditions for high stringency northern or Southern hybridizations.) High stringency hybridization is also relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization. In contrast to northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually 16 nucleotides or longer for PCR or sequencing, and 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998, which is incorporated herein by reference.

By "sample" is meant a tissue biopsy, amniotic fluid, cell, blood, serum, urine, stool, or other specimen obtained from a patient or a test subject. The sample can be S analyzed to detect a mutation in a san or vtn gene, or expression levels of a san or vtn gene, by methods that are known in the art. For example, methods such as sequencing, single-strand conformational polymorphism (SSCP) analysis, or restriction fragment length polymorphism (RFLP) analysis of PCR products derived from a patient sample can be used to detect a mutation in a san or vtn gene; ELISA0 and other immunoassays can be used to measure levels of a san or vtn polypeptide; and PCR can be used to measure the level of a san or vtn nucleic acid molecule.

By "santa-related disease," "santa-related condition," "san-related disease," "san-related condition," "valentine-related disease," "valentine-related disease," "valentine-related condition," "vtn-related disease," or "vtn-related condition" isS meant a disease or condition that results from inappropriately high or low expression of a san or vtn gene, or a mutation in a san or vtn gene (including control sequences, such as promoters) that alters the biological activity of a san or vtn nucleic acid molecule or polypeptide. San- or vtn-related diseases and conditions can arise in any tissue in which san is expressed during prenatal or post-natal life. San- or vtn-related0 diseases and conditions can include diseases or conditions of the heart (e.g., heart failure, hypertrophy, myopathy, or arteriosclerosis).

.The invention provides several advantages. For example, using the diagnostic methods of the invention it is possible to detect an increased likelihood of diseases or conditions associated with san or vtn, such as diseases of the heart, in a patient, so that5 appropriate intervention can be instituted before any symptoms occur. This may be useful, for example, with patients in high-risk groups for such diseases or conditions. Also, the diagnostic methods of the invention facilitate determination of the etiology of such an existing disease or condition in a patient, so that an appropriate approach to treatment can be selected. In addition, the screening methods of the invention can be0 used to identify compounds that can be used to treat or to prevent these diseases or conditions. The invention can also be used to treat diseases or conditions for which, prior to the invention, the only treatment was organ transplantation, which is limited by the availability of donor organs and the possibility of organ rejection.

Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.

Brief Description of the Drawings

Figure 1. Morphological analysis of cardiac chambers of san and vtn mutant embryos. Hematoxylin and eosin stained sagittal sections through 72 hours postfertilization (hpf) hearts was used to assess cardiac histology (A,C,E). Double- staining using an atrial-specific antibody (S46) and an in situ RNA probe to ventricular myosin heavy chain (vMHC) were used to distinguish the atrial and ventricular chambers (B,D,F). The myocardial wall (indicated by arrows) in wt hearts (A) is several cell layers thick. However, the myocardium in both san (C) and vtn (E') mutant hearts does not thicken and remains a single cell layer. Both mutants are shown at a reduced magnification in C and E to illustrate the dramatic dilation of the cardiac chambers. The hearts of (B) wild-type, (D) san -/-, and (F) vtn -/- embryos at 48 hpf express markers specific for the two cardiac chambers, although the hearts of both mutants are enlarged, wt, wild-type; -/-, homozygous mutant; v, ventricle; a, atrium; m, myocardium; e, endocardium; Bars, 25 μm.

Figure 2. Valve formation is perturbed in santa. The sites of formation of the endocardial cushions in day 2 embryos of the ty219c san allele are indicated by staining with an in situ RNA probe to the neuregulin gene (Milan et al., Development 133:1125, 2006). (A₅A') Wild-type sibs of the san ty219c allele demonstrate distinct staining at the sites of future valve formation, indicated by the arrow. (B, B') No staining is observed in the hearts of stage-matched san mutant embryos. The position of the atrial and ventricular chambers are outlined in A' to better illustrate the location of the neuregulin staining, wt, wild-type; -/-, homozygous mutant.

Figure 3. Sarcomeres are present in the hearts of san and vtn mutant embryos. Transmission EM shows sarcomeres (arrows) in the hearts of wild-type (A-C), san (D,E), and vtn (F,G) embryos, but with some disarray in the mutants (D₁F). Sarcomeres are shown in cross-section (indicated by circles) in wild-type (B), san (E), and vtn (G) mutants. Both san and vtn mutant hearts are still able to contract, although circulation is not generated, suggesting that actin/myosin arrangement is S normal. Intercalated discs (indicated by boxes) are detectable in both mutants (E,G) although their structure in wild-type hearts is better defined (C). Comparison with similar EM analyses of cardiac tissue suggests that the electron-dense material along and between the myofilaments is glycogen.

Figure 4. Positional cloning of the san and vtn genes. (A) Initial bulk 0 segregant analysis established linkage to SSR marker z5183 on zebrafish chromosome 19. The san mutation was fine mapped to the region between SSR markers z9512 and z5183. Another SSR in the interval (z5583) was identified from the zebrafish RH map and was used to initiate a walk to create a physical map over the san gene consisting of YACs and BACs. A BAC, 92il2, was isolated using recombinant5 markers on either side of the mutation and was partially sequenced. Another BAC (184dO7) was sequenced to obtain the complete sequence of the san gene, y, YAC clone; b, BAC clone. (B) Initial bulk segregant analysis established linkage to SSR marker z3824 on zebrafish chromosome 20. Other SSR markers in the region were tested for linkage and the vtn interval was defined to the region between 0 z21067/z29926 and z22659. With the availability of the complete fugu genome sequence, a more effective analysis of conserved synteny became possible. Several of the closest BACs had been partially shotgun sequenced to provide more information for the analysis of synteny and generation of genetic markers. A single non-chimeric BAC contained the zebrafish homologs of at least three genes, SOX7 (SRY (sex5 determining region Y)-box 7), PINXl (PIN2 -interacting protein 1), and TDH (L- threonine dehydrogenase). (BAC 8m8 was nonchimeric based on mapping of subcloned regions on both the RH panel and the vtn map cross.) These three genes also mapped to a single scaffold on the fugu genome map. When these genes were compared back to their location within sequenced zebrafish BACs placed on the0 zebrafish BAC fingerprint (fpc) map, a single fpc contig was identified that contained

I l two of the three genes (PINXl and TDH). Sequence analysis of both the ftigu scaffold and the zebrafish fpc contig revealed another closely linked gene, MGC4607, which was tested as a candidate and confirmed as the vtn gene by both morpholino antisense oligomer phenocopy of the mutation and determination of the genetic lesion in the m201 allele of vtn.

Figure 5. The san and vtn loci encode the zebrafish homologs of CCMl and CCM2. (A) The zebrafish (Dr) San (CCMl) (SEQ ID NO: 15) protein sequence is aligned with the C. elegans (Ce) (SEQ ID NO: 16), mouse (Mm) (SEQ ID NO: 14), and human (Hs) (SEQ ID NO: 13) homologs, demonstrating strong identity among the three vertebrate genes. The N-terminal NPxY motif, indicated by the single line

(NPAY, residues 191-194 of the zebrafish protein and residues 192-195 of the mouse and human protein), is altered in the C. elegans homolog to NPAF. This motif is associated with interaction of san with both ICAPl and vtn (Zawistowski et al., Hum. MoI. Genet. 11:389, 2002; Zawistowski et al., Hum. MoI. Genet. 10:2953, 2005; Zhang et al., Hum. MoI. Genet. 10:2953, 2001). The other two NPxF/Y motifs, indicated by double lines, were not required for interaction of san with vtn (Zawistowski et al., Hum. MoI. Genet. 10:2953, 2005). The three "A" boxes indicate the location of the ankyrin repeats and the single "FERM" box denotes the FERM domain. The asterisk indicates the position of the Y -> stop mutation in the zebrafish san ty219c allele. The protein motifs are shown schematically below the sequence. The asterisk again denotes the san ty219c mutation and the region of the protein shown in to the left of the asterisk corresponds to exon 14, deleted in the san m775 allele. (B) Real-time PCR analysis was used to examine the level of san mRNA message containing exon 14. mRNA isolated from m775 wt siblings, and both wt and mutant ty219c embryos show levels of exon 14-containing message 10-fold greater than in the san m775 mutant embryos (m775 -/- level set to 1 for comparison). (C) The Vtn (CCM2) protein is very well conserved among vertebrates (the zebrafish (Dr) Vtn protein sequence (SEQ ID NO: 19) is aligned with the mouse (Mm) (SEQ ID NO: 18) and human (Hs) (SEQ ID NO: 17) homologs, no C. elegans homolog is detectable). The single box indicates the sequence corresponding to the PTB domain and the asterisk indicates the position of the Y -> stop mutation in the zebrafish vtn m201 allele. The Vtn protein shown schematically below the sequence illustrates the position of the PTB domain and site of the vtn m201 mutation.

Figure 6. In situ analysis of san mRN A expression. The san mRNA is expressed in the ventricular zone (black arrow) and diffusely throughout the brain at 28hpf (A₅B). Expression is also detectable at this stage in the posterior cardinal vein (arrow) as illustrated by wholemount (C) and in section (D) (lower arrow). At 48 hpf, san expression is detectable in the notochord with patchy expression in the vein (E). The staining in the vein is clearly visible from sections through the trunk (F). The position of the dorsal aorta is indicated by the upper arrow. The boxed region in C and E represents the region shown in the sections, nt, neural tube; nc, notochord.

Figure 7. In situ analysis of vtn mRN A expression. The vtn mRNA is robustly expressed in the ventricular zone (black arrow) at 28hpf (A-C), also shown in section in D. Expression is also strong in the intermediate cell mass (A,E,F, white arrow). Lengthened staining also reveals vtn message in the vein (E, gray arrow) confirmed by sectioning (G). At 48 hpf, vtn is expressed in the brain and diffusely in the branchial arches (H). Expression in the vein is detectable by wholemount (I) and from sections (J), but is weaker than expression of san at this stage. The position of the dorsal aorta is indicated by the left arrow. The boxed region in E and I represents the region shown in the sections, nt, neural tube; nc, notochord.

Figure 8. Morpholino co-injections reveal evidence of interactions between san, vtn, and heg. Injection of low doses (10 μM and 15 μM) of each morpholino alone is unable to produce a large number of embryos with the characteristic enlarged heart and thin- walled myocardium. However, by injection of combinations of any two of the san, vtn, and heg morpholinos, a dramatic increase in the level of phenocopy is observed (indicated by the increase in the white bars) with a concomitant decrease in the level of wild-type embryos (light gray bar). A number of embryos (indicated by the dark gray bar) display a phenotype intermediate between wild-type and mutant, characterized by a less dramatic dilation of the heart and some with weak circulation. heg, heg exon 11 donor morpholino injected; san, san exon 1 donor morpholino injected; vtn, vtn exon 2 morpholino injected.

Detailed Description We have identified two zebrafish genes, herein termed santa (san) and valentine (vtn), which are necessary for the thickening of the myocardium during development. Mutations in these genes have been found to be associated with abnormal heart phenotypes. Detection of mutations in these genes can therefore be used in methods to diagnose heart conditions as described herein. In addition, compounds that modify the levels or activities of the proteins encoded by these genes can be used in methods of treating or preventing heart disease and related conditions. The invention thus provides methods of diagnosing, preventing, and treating heart disease, and screening methods for identifying compounds that can be used to treat or prevent heart disease. The diagnostic methods of the invention thus involve detection of mutations in genes encoding san or vtn proteins (and/or altered san or vtn levels), while the compound identification methods involve screening for compounds that affect the phenotype of organisms having mutations in genes encoding san or vtn or other appropriate models. Compounds identified in this manner, as well as san or vtn genes, siRNA molecules, proteins, or antibodies, can be used in methods to diagnose, treat, or prevent diseases and conditions associated with san or vtn.

The invention also provides animal model systems (e.g., zebrafish having mutations in san or vtn genes, or mice (or other animals) having such mutations) that can be used in the screening methods mentioned above, as well as san or vtn proteins, and genes encoding these proteins. Also included in the invention are genes encoding mutant zebrafish san or vtn proteins and proteins encoded by these genes. Antibodies that specifically bind to these proteins (wild type or mutant) are also included in the invention. Additionally, the invention includes RNAi molecules against san or vtn and the use of these molecules in animal models and therapy. The diagnostic, screening, and therapeutic methods of the invention, as well as the animal model systems, proteins, and genes of the invention, are described further, as follows, after a brief description of diseases and conditions associated with san or vtn, which can be diagnosed, prevented, or treated according to the invention.

San or Vtn-Associated Diseases or Conditions

Abnormalities in san or vtn genes or proteins can be associated with any of a wide variety of diseases or conditions, all of which can thus be diagnosed, prevented, or treated using the methods of the invention. For example, as discussed above, mutations in san or vtn are implicated in concentric growth of the myocardium. Thus, detection of abnormalities in san or vtn genes or their expression can be used in methods to diagnose, or to monitor the treatment or development of, diseases or conditions of heart (e.g., heart failure or cardiac hypertrophy). In addition, compounds that are identified in the screening methods described herein, as well as san or vtn nucleic acid molecules, proteins, and antibodies themselves, can be used in methods to prevent or treat such diseases or conditions.

Examples of heart failure that can be diagnosed, prevented, or treated using the methods of the invention include congestive heart failure, which is characterized by fluid in the lungs or body, resulting from failure of the heart in acting as a pump; right-sided heart failure (right ventricular), which is characterized by failure of the pumping action of the right ventricle, resulting in swelling of the body, especially the legs and abdomen; left-sided heart failure (left ventricular), which is caused by failure of the pumping action of the left side of the heart, resulting in congestion of the lungs; forward heart failure, which is characterized by the inability of the heart to pump blood forward at a sufficient rate to meet the oxygen needs of the body at rest or during exercise; backward heart failure, which is characterized by the ability of the heart to meet the needs of the body only if heart filling pressures are abnormally high; low-output, which is characterized by failure to maintain blood output; and high-output, which is characterized by heart failure symptoms, even when cardiac output is high. san or vtn also may play roles in cardiovascular diseases other than heart failure, such as coronary artery disease, hypertension, hypotension, heart fibrillation (e.g., atrial fibrillation), arteriosclerosis, or conditions associated with valve formation defects, and, thus, detection of abnormalities in san or vtn genes or their expression can be used in methods to diagnose and monitor these conditions as well.

Diagnostic Methods

Nucleic acid molecules encoding san or vtn proteins, as well as polypeptides encoded by these nucleic acid molecules and antibodies specific for these polypeptides, can be used in methods to diagnose or to monitor diseases and conditions involving mutations in, or inappropriate expression of, genes encoding these proteins.

The diagnostic methods of the invention can be used, for example, with patients that have a disease or condition associated with san or vtn, in an effort to determine its etiology and, thus, to facilitate selection of an appropriate course of treatment. The diagnostic methods can also be used with patients who have not yet developed, but who are at risk of developing, such a disease or condition, or with patients that are at an early stage of developing such a disease or condition. Also, the diagnostic methods of the invention can be used in prenatal genetic screening, for example, to identify parents who may be carriers of a recessive mutation in a gene encoding san or vtn proteins. In addition, the methods can be used to investigate whether a san or vtn mutation may be contributing to a disease or condition (e.g., heart disease) in a patient, by determining whether a san or vtn gene of a patient includes a mutation. The methods of the invention can be used to diagnose (or to prevent or treat) the disorders described herein in any mammal, for example, humans, domestic pets, or livestock.

Abnormalities in san or vtn that can be detected using the diagnostic methods of the invention include those characterized by, for example, (i) a gene encoding a san or vtn protein containing a mutation that results in the production of an abnormal san or vtn protein, (ii) an abnormal san or vtn polypeptide itself (e.g., a truncated protein), and (iii) a mutation in a san or vtn gene that results in production of an abnormal amount of the protein. Detection of such abnormalities can be used to diagnose human diseases or conditions related to san or vtn, such as those affecting the heart.

A mutation in a san or vtn gene can be detected in any tissue of a subject, even one in which this protein is not expressed. Because of the possibly limited number of tissues in which these proteins maybe expressed, for limited time periods, and because of the possible undesirability of sampling such tissues (e.g., heart tissue) for assays, it may be preferable to detect mutant genes in other, more easily obtained sample types, such as in blood or amniotic fluid samples. Detection of a mutation in a gene encoding a san or vtn protein can be carried out using any standard diagnostic technique. For example, a biological sample obtained from a patient can be analyzed for one or more mutations in nucleic acid molecules encoding a san or vtn protein using a mismatch detection approach. Generally, this approach involves polymerase chain reaction (PCR) amplification of nucleic acid molecules from a patient sample, followed by identification of a mutation (i.e., a mismatch) by detection of altered hybridization, aberrant electrophoretic gel migration, binding, or cleavage mediated by mismatch binding proteins, or by direct nucleic acid molecule sequencing. Any of these techniques can be used to facilitate detection of a mutant gene encoding a san or vtn protein, and each is well known in the art. For instance, examples of these techniques are described by Orita et al. (Proc. Natl. Acad. Sci. U.S.A. 86:2766, 1989) and Sheffield et al. (Proc. Natl. Acad. Sci. U.S.A. 86:232, 1989).

As noted above, in addition to facilitating diagnosis of an existing disease or condition, mutation detection assays also provide an opportunity to diagnose a predisposition to disease related to a mutation in a san or vtn gene before the onset of symptoms. For example, a patient who is heterozygous for a gene encoding an abnormal san or vtn protein (or an abnormal amount thereof) that suppresses normal san or vtn biological activity or expression may show no clinical symptoms of a disease related to such proteins, and yet possess a higher than normal probability of developing such disease. Given such a diagnosis, a patient can take precautions to minimize exposure to adverse environmental factors, and can carefully monitor their medical condition, for example, through frequent physical examinations. As mentioned above, this type of diagnostic approach can also be used to detect a mutation in a gene encoding the san or vtn protein in prenatal screens. While it may be preferable to carry out diagnostic methods for detecting a mutation in a san or vtn gene using genomic DNA from readily accessible tissues, as noted above, mRNA encoding the protein, or the protein itself, can also be assayed from tissue samples in which it is expressed. Expression levels of a gene encoding san or vtn in such a tissue sample from a patient can be determined by using any of a number of standard techniques that are well known in the art, including northern blot analysis and quantitative PCR (see, e.g., Ausubel et al., supra; PCR Technology: Principles and Applications for DNA Amplification, H.A. Ehrlich, Ed., Stockton Press, NY; Yap et al. Nucl. Acids. Res. 19:4294, 1991).

In another diagnostic approach of the invention, an immunoassay is used to detect or to monitor the level of a san or vtn protein in a biological sample.

Polyclonal or monoclonal antibodies specific for the san or vtn protein can be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA; see, e.g., Ausubel et al., supra) to measure the levels of san or vtn. These levels can be compared to levels of san or vtn in a sample from an unaffected individual. Detection of a decrease in production of san or vtn using this method, for example, may be indicative of a condition or a predisposition to a condition involving insufficient biological activity of the san or vtn protein.

Immunohistochemical techniques can also be utilized for detection of san or vtn proteins in patient samples. For example, a tissue sample can be obtained from a patient, sectioned, and stained for the presence of san or vtn using an anti-san or anti- vtn antibody and any standard detection system (e.g., one that includes a secondary antibody conjugated to an enzyme, such as horseradish peroxidase). General guidance regarding such techniques can be found in, e.g., Bancroft et al., Theory and Practice of Histological Techniques, Churchill Livingstone, 1982, and Ausubel et al., supra. Identification of Molecules that can be used to Treat or to Prevent Diseases or Conditions Associated with san or vtn

Identification of a mutation in the gene encoding san or vtn as resulting in a phenotype that results in abnormal concentric growth of the myocardium facilitates the identification of molecules (e.g., small organic or inorganic molecules, antibodies, peptides, or nucleic acid molecules) that can be used to treat or to prevent diseases or conditions associated with san or vtn, as discussed above. The effects of candidate compounds on such diseases or conditions can be investigated using, for example, the zebrafish system. The zebrafish, Danio rerio, is a convenient organism to use in the genetic analysis of development. It has an accessible and transparent embryo, allowing direct observation of organ function from the earliest stages of development, has a short generation time, and is fecund. As discussed further below, zebrafish and other animals having a san or vtn mutation, such as the mil S or m201 mutations, which can be used in these methods, are also included in the invention. In one example of the screening methods of the invention, a zebrafish having a mutation in a gene encoding the san or vtn protein is contacted with a candidate compound, and the effect of the compound on the development of abnormal concentric growth of the myocardium, or on the status of such an existing abnormality, is monitored relative to an untreated, identically mutant control. After a compound has been shown to have a desired effect in the zebrafish system, it can be tested in other models of heart disease, for example, in mice or other animals having a mutation in a gene encoding san or vtn. Alternatively, testing in such animal model systems can be carried out in the absence of zebrafish testing. Cell culture-based assays can also be used in the identification of molecules that increase or decrease san or vtn levels or biological activity. According to one approach, candidate molecules are added at varying concentrations to the culture medium of cells expressing san or vtn mRNA. San or vtn biological activity is then measured using standard techniques. The measurement of biological activity can include the measurement of san or vtn protein and nucleic acid molecule levels. In general, novel drugs for the prevention or treatment of diseases related to mutations in genes encoding san or vtn can be identified from large libraries of natural products, synthetic (or semi-synthetic) extracts, and chemical libraries using methods that are well known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening methods of the invention and that dereplication, or the elimination of replicates or repeats of materials already known for their therapeutic activities for san or vtn, can be employed whenever possible.

Candidate compounds to be tested include purified (or substantially purified) molecules or one or more components of a mixture of compounds (e.g., an extract or supernatant obtained from cells; Ausubel et al., supra), and such compounds further include both naturally occurring or artificially derived chemicals and modifications of existing compounds. For example, candidate compounds can be polypeptides, synthesized organic or inorganic molecules, naturally occurring organic or inorganic molecules, nucleic acid molecules, and components thereof.

Numerous sources of naturally occurring candidate compounds are readily available to those skilled in the art. For example, naturally occurring compounds can be found in cell (including plant, fungal, prokaryotic, and animal) extracts, mammalian serum, growth medium in which mammalian cells have been cultured, protein expression libraries, or fermentation broths. In addition, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, LJK), Harbor Branch Oceanographic Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). Further, libraries of natural compounds can be produced, if desired, according to methods that are known in the art, e.g., by standard extraction and fractionation.

Artificially derived candidate compounds are also readily available to those skilled in the art. Numerous methods are available for random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, for example, saccharide-, lipid-, peptide-, and nucleic acid molecule-based compounds. In addition, synthetic compound libraries are commercially available from Brandon Associates (Meπϊmack, NH) and Aldrich Chemicals (Milwaukee, WI). Libraries of synthetic compounds can also be produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation. Further, if desired, any library or compound can be readily modified using standard chemical, physical, or biochemical methods. The techniques of modern synthetic chemistry, including combinatorial chemistry, can also be used (reviewed in Schreiber, Bioorganic and Medicinal Chemistry 6:1172-1152, 1998; Schreiber, Science 287:1964-1969, 2000). When a crude extract is found to have an effect on the development or persistence of a san or vtn-associated disease, further fractionation of the positive lead extract can be carried out to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having a desired activity. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives of these compounds. Methods of fractionation and purification of such heterogeneous extracts are well known in the art. If desired, compounds shown to be useful agents for treatment can be chemically modified according to methods known in the art.

In general, compounds that are found to activate san or vtn expression or activity may be used in the prevention or treatment of diseases or conditions of heart, such as those that are characterized by abnormal growth or development, weakened blood vessels, or heart failure (also see above). Compounds found to increase san or vtn activity in zebrafish (or to counteract the phenotypes described herein) can be also be used, for example, in the treatment of cerebral cavernous malformations.

A compound affecting san activity can, optionally, be used in combination with one or more compounds affecting san and/or vtn. Similarly, a compound affecting vtn activity can, optionally, be used in combination with another compound affecting vtn and/or one or more compounds affecting san. Further, treatment according to the methods of the present invention can be combined with treatment focused on increasing the activity or levels of a heart of glass (heg) protein, mutations in which result in a zebrafish phenotype similar to those associated with san and vtn mutations (see, e.g., WO 02/062205).

Animal Model Systems

The invention also provides animal model systems for use in carrying out the screening methods described above. Examples of these model systems include zebrafish and other animals, such as mice, that have a mutation (e.g., the m775 or m201 mutations) in a san or vtn gene. For example, a zebrafish model that can be used in the invention can include a mutation that results in a lack of san or vtn protein production or production of a truncated (e.g., by introduction of a stop codon or a splice site mutation) or otherwise altered san or vtn gene product. As a specific example, a zebrafish having the m775 or m201 mutations can be used (see below). Additional animal models include animals treated with RNAi molecules designed to inhibit the expression of san or vtn. Methods for RNA interference are described below.

Treatment or Prevention of San or Vtn- Associated Diseases or Conditions Compounds identified using the screening methods described above can be used to treat patients that have or are at risk of developing diseases or conditions of the heart (e.g., heart failure or cardiac hypertrophy; also see above). Nucleic acid molecules encoding the san or vtn protein, as well as these proteins themselves, alone or in combination, can also be used in such methods. Further, san and/or vtn treatment can, optionally, be combined with heart of glass (heg) treatment (see above). Treatment may be required only for a short period of time or may, in some form, be required throughout a patient's lifetime. Any continued need for treatment, however, can be determined using, for example, the diagnostic methods described above. In considering various therapies, it is to be understood that such therapies are, preferably, targeted to the affected or potentially affected organ (e.g., the heart). Such targeting can be achieved using standard methods.

Treatment or prevention of diseases resulting from a mutated san or vtn gene can be accomplished, for example, by modulating the function of a mutant san or vtn protein. Treatment can also be accomplished by delivering normal san or vtn protein to appropriate cells, altering the levels of normal or mutant san or vtn proteins, replacing a mutant gene encoding a san or vtn protein with a normal gene encoding a san or vtn protein, or administering a normal gene encoding a san or vtn protein. It is also possible to correct the effects of a defect in a gene encoding a san or vtn protein by modifying the physiological pathway (e.g., a signal transduction pathway) in which a san or vtn protein participates.

In a patient diagnosed as being heterozygous for a gene encoding a mutant san or vtn protein, or as susceptible to such mutations or aberrant san or vtn expression (even if those mutations or expression patterns do not yet result in alterations in expression or biological activity of san or vtn), any of the therapies described herein can be administered before the occurrence of the disease phenotype. In particular, compounds shown to have an effect on the phenotype of mutants, or to modulate expression of san or vtn proteins, can be administered to patients diagnosed with potential or actual disease by any standard dosage and route of administration. Any appropriate route of administration can be employed to administer a compound identified as described above, a san or vtn gene, siRNA, prcttein, or antibody, according to the invention. For example, administration can be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, by aerosol, by suppository, or oral.

A therapeutic compound of the invention can be administered within a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Administration can begin before or after the patient is symptomatic. Methods that are well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA. Therapeutic formulations can be in the form of liquid solutions or suspensions. Formulations for parenteral administration can contain, for example, excipients, sterile water, or saline; polyalkylene glycols, such as polyethylene glycol; oils of vegetable origin; or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. For oral administration, formulations can be in the form of tablets or capsules. Formulations for inhalation can contain excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or can be oily solutions for administration in the form of nasal drops or as a gel. Alternatively, intranasal formulations can be in the form of powders or aerosols. To replace a mutant protein with normal protein, or to add protein to cells that do not express a sufficient amount of san or vtn or normal san or vtn, it may be necessary to obtain large amounts of pure san or vtn protein from cell culture systems in which the protein is expressed (see, e.g., below). Delivery of the protein to the affected tissue can then be accomplished using appropriate packaging or administration systems.

Gene therapy is another therapeutic approach for preventing or ameliorating diseases caused by san or vtn gene defects. Nucleic acid molecules encoding wild type san or vtn protein can be delivered to cells that lack sufficient, normal san or vtn protein biological activity (e.g., cells carrying mutations (e.g., the m775 or m201 mutations) in san or vtn genes). The nucleic acid molecules must be delivered to those cells in a form in which they can be taken up by the cells and so that sufficient levels of protein, to provide effective san or vtri protein function, can be produced. Alternatively, for some san or vtn mutations, it may be possible to slow the progression of the resulting disease or to modulate san or vtn protein activity by introducing another copy of a homologous gene bearing a second mutation in that gene, to alter the mutation, or to use another gene to block any negative effect.

Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression. For example, the full length san or vtn gene, or a portion thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275, 1989; Eglitis et al., BioTechniques 6:608, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1 :55, 1990; Sharp, The Lancet 337:1277, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311, 1987; Anderson, Science 226:401, 1984; Moen, Blood Cells 17:407, 1991; Miller et al., Biotechnology 7:980, 1989; Le Gal La Salle et al., Science 259:988, 1993; and Johnson, Chest 107:77S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346). Non-viral approaches can also be employed for the introduction of therapeutic

DNA into cells predicted to be subject to diseases involving the san or vtn protein. For example, a san or vtn nucleic acid molecule or an antisense nucleic acid molecule can be introduced into a cell by lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101 :512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal san or vtn protein into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue.

San or vtn cDNA expression for use in gene therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct san or vtn expression. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a san or vtn genomic clone is used as a therapeutic construct (such clones can be identified by hybridization with san or vtn cDNA, as described herein), regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above. Molecules for effecting RNA interference (herein termed RNAi)-based strategies can be employed to explore san or vtn protein gene function, as a basis for therapeutic drug design, as well as to treat san or vtn-associated diseases. These strategies are based on the principle that sequence-specific suppression of gene expression (via transcription or translation) can be achieved by intracellular hybridization between genomic DNA or mRNA and a complementary antisense species. The formation of a hybrid RNA duplex interferes with transcription of the target san or vtn-encoding genomic DNA molecule, or processing, transport, translation, or stability of the target san or vtn mRNA molecule.

RNAi strategies can be delivered by a variety of approaches. For example, antisense oligonucleotides or antisense RNA can be directly administered (e.g., by intravenous injection) to a subject in a form that allows uptake into cells. Alternatively, viral or plasmid vectors that encode antisense RNA (or antisense RNA fragments) can be introduced into a cell in vivo or ex vivo. RNAi effects can be induced by control (sense) sequences; however, the extent of phenotypic changes is highly variable. Phenotypic effects induced by antisense molecules are based on changes in criteria such as protein levels, protein activity measurement, and target mRNA levels.

San or vtn gene therapy can also be accomplished by direct administration of antisense san or vtn mRNA to a cell that is expected to be adversely affected by the expression of wild type or mutant san or vtn protein. The antisense san or vtn mRNA can be produced and isolated by any standard technique, but is most readily produced by in vitro transcription using an antisense san or vtn cDNA under the control of a high efficiency promoter (e.g., the T7 promoter). Administration of antisense san or vtn mRNA to cells can be carried out by any of the methods for direct nucleic acid molecule administration described above.

The term "RNAi" is used herein to refer collectively to several gene silencing techniques, including the use of siRN A (short interfering RNAs), shRN A (short hairpin RNA: an RNA bearing a fold-back stem-loop structure), dsRNA (double- stranded RNA; see, for example, Williams, Biochem. Soc. Trans. 25:509, 1997; Gil and Esteban, Apoptosis 5:107, 2000; Clarke and Mathews, RNA 1 :7, 1995; Baglioni and Nilsen, Interferon 5:23, 1983), miRNA (micro RNAs), StRNAs (short (or "small") temporal RNAs), and the like, all of which can be used in the methods of the present invention. A number of methods for producing and selecting RNAi molecules, such as shRNAs, siRNAs, and dsRNAs, have been developed and can be used in the present invention (see, e.g., Paddison et al., Methods MoI. Biol. 265:85, 2004; and Kakare et al., Appl. Biochem. Biotechnol. 119:1, 2004). In addition, commercially available kits can be used to make RNAi for use in the methods of the invention (e.g., GeneEraser™ (catalog # 240090) from Stratagene, La Jolla, CA).

San or vtn nucleic acid molecules as described herein can be used as guide sequences in the design of RNAi molecules of the invention, which can include sense and/or antisense sequences or regions that are generally covalently linked by nucleotide or non-nucleotide linker molecules, as is known in the art. Alternatively, the linkages can be non-covalent, involving, for example, ionic, hydrogen bonding, Van der Waals, hydrophobic, and/or stacking interactions. siRNAs of the invention can be, e.g., between 19 and 29 nucleotides in length, while dsRNAs can be at least 30, 50, 100, or 500 nucleotides in length. As is known in the art, shRNAs are generally designed to form double-stranded regions of 19 to 29 nucleotides in length, although these lengths can vary (see Paddison et al., Genes Dev. 16:948, 2002). Exemplary requirements for siRNA length, structure, chemical composition, cleavage site position, and sequences essential to mediate efficient RNAi activity are described, for example, by Elbashir et al., EMBO J. 20:6877, 2001; and Nykanen et al., Cell 107:309, 2001.

RNAi molecules of the present invention include any form of RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material to, e.g., the end(s) of the RNA or internally (at one or more nucleotides of the RNA), or the RNA molecule can contain a 3'hydroxyl group. RNAi molecules of the present invention can also include non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. Examples of modified nucleotides that can be included in RNAi molecules of the invention, such as 2'-O-methyl ribonucleotides, 2'-deoxy-2'- fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, nucleotides with phosphorothioate internucleotide linkages, and inverted deoxyabasic residues, are described, for example, in U.S. Patent Application Publication No.

20040019001. RNAi molecules directed against san or vtn can be used individually, or in combination with other RNAi constructs, for example, constructs against heart of glass (heg).

An alternative strategy for inhibiting san or vtn protein function using gene therapy involves intracellular expression of an anti-san or anti-vtn protein antibody or a portion thereof. For example, the gene (or gene fragment) encoding a monoclonal antibody that specifically binds to a san or vtn protein and inhibits its biological activity can be placed under the transcriptional control of a tissue-specific gene regulatory sequence. Another therapeutic approach included in the invention involves administration of a recombinant san or vtn polypeptide, either directly to the site of a potential or actual disease-affected tissue (for example, by injection) or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the san or vtn protein depends on a number of factors, including the size and health of the individual patient but, generally, between 0.1 mg and 100 mg, inclusive, is administered per day to an adult in any pharmaceutically acceptable formulation.

In addition to the therapeutic methods described herein, involving administration of san or vtn-modulating compounds, san or vtn proteins, or san or vtn nucleic acids to patients, the invention provides methods of culturing organs in the presence of such molecules. In particular, as is noted above, a san or vtn mutation is associated with abnormal concentric growth of the myocardium. Thus, culturing heart tissue in the presence of these molecules can be used to promote its proper concentric growth. This tissue can be that which is being prepared for transplant from, e.g., an allogeneic or xenogeneic donor, as well as synthetic tissue or organs.

Synthesis of San or Vtn Proteins. Polypeptides, and Polypeptide Fragments

Those skilled in the art of molecular biology will understand that a wide variety of expression systems can be used to produce recombinant san or vtn proteins. As discussed further below, the precise host cell used is not critical to the invention. The san or vtn proteins can be produced in a prokaryotic host (e.g., E. colϊ) or in a eukaryotic host (e.g., S. cerevisiae, insect cells, such as Sf9 cells, or mammalian cells, such as COS-I, NIH 3T3, or HeLa cells). These cells are commercially available from, for example, the American Type Culture Collection, Manassas, VA (see also Ausubel et al., supra). The method of transformation and the choice of expression vehicle (e.g., expression vector) will depend upon the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al., supra, and expression vehicles can be chosen from those provided, e.g., in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987. Specific examples of expression systems that can be used in the invention are described further as follows.

For protein expression, eukaryotic or prokaryotic expression systems can be generated in which san or vtn gene sequences are introduced into a plasmid or other vector, which is then used to transform living cells. Constructs in which full-length san or vtn cDNAs, containing the entire open reading frame, inserted in the correct orientation into an expression plasmid, can be used for protein expression.

Alternatively, portions of san or vtn gene sequences, including wild type or mutant san or vtn sequences, can be inserted. Prokaryotic and eukaryotic expression systems allow various important functional domains of san or vtn proteins to be recovered, if desired, as fusion proteins, and then used for binding, structural, and functional studies, and also for the generation of antibodies.

Typical expression vectors contain promoters that direct synthesis of large amounts of mRNA corresponding to a nucleic acid molecule that has been inserted into the vector. They can also include a eukaryotic or prokaryotic origin of replication, allowing for autonomous replication within a host cell, sequences that confer resistance to an otherwise toxic drug, thus allowing vector-containing cells to be selected in the presence of the drug, and sequences that increase the efficiency with which the synthesized mRNA is translated. Stable, long-term vectors can be maintained as freely replicating entities by using regulatory elements of, for example, viruses (e.g., the OnP sequences from the Epstein Barr Virus genome). Cell lines can also be produced that have the vector integrated into genomic DNA of the cells and, in this manner, the gene product can be produced in the cells on a continuous basis.

Expression of foreign molecules in bacteria, such as Escherichia coli, requires the insertion of a foreign nucleic acid molecule, e.g., a san or vtn nucleic acid molecule, into a bacterial expression vector. Such plasmid vectors include several elements required for the propagation of the plasmid in bacteria, and for expression of foreign DNA contained within the plasmid. Propagation of only plasmid-bearing bacteria is achieved by introducing, into the plasmid, a selectable marker-encoding gene that allows plasmid-bearing bacteria to grow in the presence of an otherwise toxic drug. The plasmid also contains a transcriptional promoter capable of directing synthesis of large amounts of mRN A from the foreign DNA. Such promoters can be, but are not necessarily, inducible promoters that initiate transcription upon induction by culture under appropriate conditions (e.g., in the presence of a drug that activates the promoter). The plasmid also, preferably, contains a polylinker to simplify insertion of the gene in the correct orientation within the vector. Once an appropriate expression vector containing a san or vtn gene, or a fragment, fusion, or mutant thereof, is constructed, it can be introduced into an appropriate host cell using a transformation technique, such as, for example, calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection. Host cells that can be transfected with the vectors of the invention can include, but are not limited to, E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression), or cells derived from mice, humans, or other animals. Mammalian cells can also be used to express san or vtn proteins using a virus expression system (e.g., a vaccinia virus expression system) described, for example, in Ausubel et al., supra. In vitro expression of san or vtn proteins, fusions, polypeptide fragments, or mutants encoded by cloned DNA can also be carried out using the T7 late-promoter expression system. This system depends on the regulated expression of T7 RNA polymerase, an enzyme encoded in the DNA of bacteriophage T7. The T7 RNA polymerase initiates transcription at a specific 23 base pair promoter sequence called the T7 late promoter. Copies of the T7 late promoter are located at several sites on the T7 genome, but none are present in E. coli chromosomal DNA. As a result, in T7- infected E. coli, T7 RNA polymerase catalyzes transcription of viral genes, but not E. coli genes. In this expression system, recombinant E. coli cells are first engineered to carry the gene encoding T7 RNA polymerase next to the lac promoter. In the presence of IPTG, these cells transcribe the T7 polymerase gene at a high rate and synthesize abundant amounts of T7 RNA polymerase. These cells are then transformed with plasmid vectors that carry a copy of the T7 late promoter protein. When IPTG is added to the culture medium containing these transformed E. coli cells, large amounts of T7 RNA polymerase are produced. The polymerase then binds to the T7 late promoter on the plasmid expression vectors, catalyzing transcription of the inserted cDNA at a high rate. Since each E. coli cell contains many copies of the expression vector, large amounts of mRNA corresponding to the cloned cDNA can be produced in this system and the resulting protein can be radioactively labeled. Plasmid vectors containing late promoters and the corresponding RNA polymerases from related bacteriophages, such as T3, T5, and SP6, can also be used for in vitro production of proteins from cloned DNA. E. coli can also be used for expression using an M 13 phage, such as mGPI-2. Furthermore, vectors that contain phage lambda regulatory sequences, or vectors that direct the expression of fusion proteins, for example, a maltose-binding protein fusion protein or a glutathione-S- transferase fusion protein, also can be used for expression in E. coli.

Eukaryotic expression systems are useful for obtaining appropriate post- translational modification of expressed proteins. Transient transfection of a eukaryotic expression plasmid containing a san or vtn gene into a eukaryotic host cell allows the transient production of a san or vtn protein by the transfected host cell. San or vtn proteins can also be produced by a stably-transfected eukaryotic (e.g., mammalian) cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public (see, e.g., Pouwels et al., supra), as are methods for constructing lines including such cells (see, e.g., Ausubel et al., supra). In one example, cDNA encoding a san or vtn protein, fusion, mutant, or polypeptide fragment is cloned into an expression vector that includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, integration of the san or vtn protein-encoding gene, into the host cell chromosome is selected for by inclusion of 0.01-300 μM methotrexate in the cell culture medium (Ausubel et al., supra). This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods tor selecting cell lines bearing gene amplifications are described in Ausubel et al., supra. These methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. The most commonly used DHFR-containing expression vectors are pCVSEII-DHFR and pAdD26SV(A) (described, for example, in Ausubel et al., supra). The host cells described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR- cells, ATCC Accession No. CRL 9096) are among those that are most preferred for DHFR selection of a stably transfected cell line or DHFR-mediated gene amplification. Another preferred eukaryotic expression system is the baculovirus system using, for example, the vector pBacPAK.9, which is available from Clontech (Palo Alto, CA). If desired, this system can be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Molecular and Cellular Biology 5:3610-3616, 1985). Once a recombinant protein is expressed, it can be isolated from the expressing cells by cell lysis followed by protein purification techniques, such as affinity chromatography. In this example, an anti-san or anti-vtn antibody, which can be produced by the methods described herein, can be attached to a column and used to isolate the recombinant san or vtn. Lysis and fractionation of san or vtn-harboring cells prior to affinity chromatography can be performed by standard methods (see, e.g., Ausubel et al., supra). Once isolated, the recombinant protein can, if desired, be purified further by, e.g., high performance liquid chromatography (HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, Work and Burdon, Eds., Elsevier, 1980). Polypeptides of the invention, particularly short san or vtn fragments and longer fragments of the N-terminus and C-terminus of san or vtn, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2^nd ed., 1984, The Pierce Chemical Co., Rockford, IL). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful san or vtn fragments or analogs, as described herein. San or Vtn Protein Fragments

Polypeptide fragments that include various portions of san or vtn proteins are useful in identifying the domains of san or vtn that are important for its biological activities. Methods for generating such fragments are well known in the art (see, for example, Ausubel et al., supra), and can be carried out using the nucleotide sequences provided herein. For example, a san or vtn protein fragment can be generated by PCR amplifying a desired san or vtn nucleic acid molecule fragment using oligonucleotide primers designed based upon san or vtn nucleic acid sequences. Preferably, the oligonucleotide primers include unique restriction enzyme sites that facilitate insertion of the amplified fragment into the cloning site of an expression vector (e.g., a mammalian expression vector, see above). This vector can then be introduced into a cell (e.g., a mammalian cell; see above) by artifice, using any of the various techniques that are known in the art, such as those described herein, resulting in the production of a san or vtn protein fragment in the cell containing the expression vector. San or vtn protein fragments (e.g., chimeric fusion proteins) can also be used to raise antibodies specific for various regions of the san or vtn protein using, for example, the methods described below. In some instances, it maybe desirable to include conserved domains in fragments, while in others, less conserved domains can be included.

San or Vtn Protein Antibodies

To prepare polyclonal antibodies, san or vtn proteins, fragments of san or vtn proteins, or fusion proteins containing defined portions of san or vtn proteins can be synthesized in, e.g., bacteria, by expression of corresponding DNA sequences contained in a suitable cloning vehicle. Fusion proteins are commonly used as a source of antigen for producing antibodies. Two widely used expression systems for E. coli are lacZ fusions using the pUR series of vectors and trpE fusions using the pATH vectors. The proteins can be purified, coupled to a carrier protein, mixed with Freund's adjuvant to enhance stimulation of the antigenic response in an inoculated animal, and injected into rabbits or other laboratory animals. Alternatively, protein can be isolated from san or vtn-expressing cultured cells. Following booster injections at bi-weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated. The sera can be used directly or can be purified prior to use by various methods, including affinity chromatography employing reagents such as Protein A-Sepharose, antigen-Sepharose, and anti-mouse-Ig-Sepharose. The sera can then be used to probe protein extracts from san or vtn-expressing tissue fractionated by polyacrylamide gel electrophoresis to identify san or vtn proteins. Alternatively, synthetic peptides can be made that correspond to antigenic portions of the protein and used to inoculate the animals. To generate peptide or full-length protein for use in making, for example, san or vtn-specific antibodies, a san or vtn coding sequence can be expressed as a C- terminal or N-terminal fusion with glutathione S-transferase (GST; Smith et al., Gene 67:31-40, 1988). The fusion protein can be purified on glutathione-Sepharose beads, eluted with glutathione, cleaved with a protease, such as thrombin or Factor-Xa (at the engineered cleavage site), and purified to the degree required to successfully immunize rabbits. Primary immunizations can be carried out with Freund's complete adjuvant and subsequent immunizations performed with Freund's incomplete adjuvant. Antibody titers can be monitored by Western blot and immunoprecipitation analyses using the protease-cleaved san or vtn fragment of the GST-san or GST- vtn protein. Immune sera can be affinity purified using CNBr-Sepharose-coupled san or vtn. Antiserum specificity can be determined using a panel of unrelated GST fusion proteins.

Alternatively, monoclonal san or vtn antibodies can be produced by using, as an antigen, san or vtn isolated from san or vtn expressing cultured cells or san or vtn protein isolated from tissues. The cell extracts, or recombinant protein extracts containing san or vtn, can, for example, be injected with Freund's adjuvant into mice. Several days after being injected, the mouse spleens can be removed, the tissues disaggregated, and the spleen cells suspended in phosphate buffered saline (PBS). The spleen cells serve as a source of lymphocytes, some of which would be producing antibody of the appropriate specificity. These can then be fused with permanently growing myeloma partner cells, and the products of the fusion plated into a number of tissue culture wells in the presence of selective agents, such as hypoxanthine, aminopterine, and thymidine (HAT). The wells can then be screened by ELISA to identify those containing cells making antibodies capable of binding to san or vtn, polypeptide fragment, or mutant thereof. These cells can then be re-plated and, after a period of growth, the wells containing these cells can be screened again to identify antibody-producing cells. Several cloning procedures can be carried out until over 90% of the wells contain single clones that are positive for specific antibody production. From this procedure, a stable line of clones that produce the antibody can be established. The monoclonal antibody can then be purified by affinity chromatography using Protein A Sepharose and ion exchange chromatography, as well as variations and combinations of these techniques. Once produced, monoclonal antibodies are also tested for specific san or vtn recognition by Western blot or immunoprecipitation analysis (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., European Journal of Immunology 6:51 1, 1976; Kohler et al., European Journal of Immunology 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York, NY, 1981 ; Ausubel et al., supra).

As an alternate or adjunct immunogen to GST fusion proteins, peptides corresponding to relatively unique hydrophilic regions of san or vtn can be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced C-terminal lysine. Antiserum to each of these peptides can be similarly affinity-purified on peptides conjugated to BSA, and specificity tested by ELISA and Western blotting using peptide conjugates, and by Western blotting and immunoprecipitation using san or vtn, for example, expressed as a GST fusion protein. Antibodies of the invention can be produced using san or vtn amino acid sequences that do not reside within highly conserved regions, and that appear likely to be antigenic, as analyzed by criteria such as those provided by the Peptide Structure Program (Genetics Computer Group Sequence Analysis Package, Program Manual for the GCG Package, Version 7, 1991) using the algorithm of Jameson et al., CABIOS 4:181 , 1988. These fragments can be generated by standard techniques, e.g., by PCR, and cloned into the pGEX expression vector. GST fusion proteins can be expressed in E. coli and purified using a glutathione-agarose affinity matrix (Ausubel et al., supra). To generate rabbit polyclonal antibodies, and to minimize the potential for obtaining antisera that is non-specific, or exhibits low-affinity binding to san or vtn, two or three fusions are generated for each protein, and each fusion is injected into at least two rabbits. Antisera are raised by injections in series, preferably including at least three booster injections.

In addition to intact monoclonal and polyclonal anti-san or anti-vtn antibodies, the invention features various genetically engineered antibodies, humanized antibodies, and antibody fragments, including F(ab')2, Fab", Fab, Fv, and sFv fragments. Truncated versions of monoclonal antibodies, for example, can be produced by recombinant methods in which plasmids are generated that express the desired monoclonal antibody fragment(s) in a suitable host. Antibodies can be humanized by methods known in the art, e.g., monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA). Fully human antibodies, such as those expressed in transgenic animals, are also included in the invention (Green et al., Nature Genetics 7:13-21, 1994).

Ladner (U.S. Patent Nos. 4,946,778 and 4,704,692) describes methods for preparing single polypeptide chain antibodies. Ward et al., Nature 341:544-546, 1989, describes the preparation of heavy chain variable domains, which they term "single domain antibodies," and which have high antigen-binding affinities. McCafferty et al., Nature 348:552-554, 1990, shows that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen, and that rare phage (one in a million) can be isolated after affinity chromatography. Boss et al., U.S. Patent No. 4,816,397, describes various methods for producing immunoglobulins, and immunologically functional fragments thereof, that include at least the variable domains of the heavy and light chains in a single host cell. Cabilly et al., U.S. Patent No. 4,816,567, describes methods for preparing chimeric antibodies. Any of these types of antibodies made with respect to san or vtn are also included in the invention.

Use of San or Vtn Antibodies Antibodies to san or vtn can be used, as noted above, to detect san or vtn or to inhibit the biological activities of san or vtn. For example, a nucleic acid molecule encoding an antibody or portion of an antibody can be expressed within a cell to inhibit san or vtn function. In addition, the antibodies can be coupled to compounds, such as radionuclides and liposomes, for diagnostic or therapeutic uses. Antibodies that inhibit the activity of a san or vtn polypeptide described herein can also be useful in preventing or slowing the development of a disease caused by inappropriate expression of a wild type or mutant san or vtn gene.

Detection of San or Vtn Gene Expression As noted, the antibodies described above can be used to monitor san or vtn gene expression. In situ hybridization of RNA can be used to detect the expression of san or vtn genes. RNA in situ hybridization techniques rely upon the hybridization of a specifically labeled nucleic acid probe to the cellular RNA in individual cells or tissues. Therefore, RNA in situ hybridization is a powerful approach for studying tissue- and temporal-specific gene expression. In this method, oligonucleotides, cloned DNA fragments, or antisense RNA transcripts of cloned DNA fragments corresponding to unique portions of san or vtn genes are used to detect specific mRNA species, e.g., in the tissues of animals, such as mice, at various developmental stages. mRNA expression can also be measured through reverse transcription followed by quantitative PCR. Other gene expression detection techniques are known to those of skill in the art and can be employed for detection of san or vtn gene expression. Identification of Additional San or Vtn Genes

Standard techniques, such as the polymerase chain reaction (PCR) and DNA hybridization, can be used to clone san or vtn gene homologues in other species and san or vtn -related genes in humans. San or vtn -related genes and homologues can be readily identified using low-stringency DNA hybridization or low-stringency PCR with human san or vtn probes or primers. Degenerate primers encoding human san or vtn or human san or vtn-related amino acid sequences can be used to clone additional san or vtn-related genes and homologues by RT-PCR.

Construction of Transgenic Animals and Knockout Animals

Characterization of san or vtn genes provides information that allows san or vtn knockout animal models to be developed by homologous recombination. Preferably, a san or vtn knockout animal is a mammal, most preferably a mouse. Similarly, animal models of san or vtn overproduction can be generated by integrating one or more san or vtn sequences into the genome of an animal, according to standard transgenic techniques. Moreover, the effect of san or vtn mutations (e.g., dominant gene mutations) can be studied using transgenic mice carrying mutated san or vtn transgenes or by introducing such mutations into the endogenous san or vtn gene, using standard homologous recombination techniques. A replacement-type targeting vector, which can be used to create a knockout model, can be constructed using an isogenic genomic clone, for example, from a mouse strain such as 129/Sv (Stratagene Inc., LaJoIIa, CA). The targeting vector can be introduced into a suitably derived line of embryonic stem (ES) cells by electroporation to generate ES cell lines that carry a profoundly truncated form of a san or vtn gene. To generate chimeric founder mice, the targeted cell lines are injected into a mouse blastula-stage embryo. Heterozygous offspring can be interbred to homozygosity. San or vtn knockout mice provide a tool for studying the role of san or vtn in embryonic development and in disease. Moreover, such mice provide the means, in vivo, for testing therapeutic compounds for amelioration of diseases or conditions involving san or vtn-dependent or a san or vtn-effected pathway. Use of san or vtn as a Marker for Stem Cells of the Heart

As san or vtn is expressed in cells that give rise to the heart during the course of development, it can be used as a marker for stem cells of the heart. For example, san or vtn can be used to identify, sort, or target such stem cells. A pool of candidate cells, for example, can be analyzed for san or vtn expression, to facilitate the identification of heart stem cells, which, based on this identification can be separated from the pool. The isolated stem cells can be used for many purposes that are known to those of skill in this art. For example, the stem cells can be used in the production of new organs, in organ culture, or to fortify damaged or transplanted organs.

Experimental Results

A central question of developmental biology is how vertebrate organs acquire their form. For the heart, part of such morphogenesis reflects control of overall cell number (Rottbauer et al., Dev. Cell 1 :265, 2001 ; Rottbauer et al., Cell 111 :661, 2002). Others control development of cells along two axes: anterior-posterior (Stainier and Fishman, Dev. Biol. 153:91, 1992) and concentric (Mably et al., Curr. Biol. 13:2138, 2003). During the first stage of primitive heart tube formation, the heart grows in essentially an anterior-posterior direction, with each of the two chambers constituted by a single-layered myocardium around a single layer of endocardium. The onset of concentric growth is marked by the addition of new cells in the myocardium in a direction perpendicular to the lumen, an outward growth that thickens the wall in a concentric direction, especially in the ventricle.

We have identified three mutations that block concentric growth without affecting overall cell number or the organization of anterior-posterior growth. We recently cloned one of these, heart of glass (heg), which turned out to be a novel gene, expressed in the endocardium (see, e.g., PCT/US02/03558). Here we focus on the other two genes, santa (san) and valentine (vtn). By positional cloning, we identify san as the zebrafish homolog of human CCMl (kritl, (Laberge-le Couteulx et al., Nat. Genet. 23:189, 1999; Sahoo et al., Hum. MoI. Genet. 8:2325, 1999)) and vtn, the zebrafish homolog of human CCM2 (Denier et al., Am. J. Hum. Genet. 74:326, 2004). Mutations in these genes in humans have been implicated in the autosomal dominant disease, Cerebral Cavernous Malformations (CCM).

Evidence from potential interacting protein motifs, and from cross- sensitization of phenotype though morpholino injection, suggests that san, vtn, and heg may interact. This suggests that concentric growth of the ventricle is an essential element of cardiac patterning, controlled at least in part by signals from the endocardium, and involving a pathway comprised of san, vtn, and heg.

Materials and methods Histological sectioning and cell counting

Histology was performed on paraformaldehyde fixed embryos embedded in plastic (JB-4, Polysciences, Inc.). Sectioning was performed using a Jung supercut 2065 at 5 μm setting.

The zebrafish cardiac myosin light chain-2 (cmlc2) promoter-DsRed (red fluorescent protein; RPP) line has been described previously (Mably et al., Curr. Biol. 13:2138, 2003). Transgenic cmlc2:DsRed2-nuc zebrafish were bred with san heterozygotes. The progeny were raised and incrossed to identify san heterozygotes expressing RFP. The embryos from these clutches were scored for the san phenotype. Wild-type siblings and mutant embryos were raised at 28.5°C until 48 or 72 hpf (hours post fertilization), at which time the embryos were flat-mounted and RFP- positive myocardial cells were counted (Mably et al., Curr. Biol. 13:2138, 2003; Shu et al., Development 130:6165, 2003). Morpholino injected transgenic cmlc2:DsRed2- nuc embryos were analyzed in a similar manner. The same flat-mount technique was used to determine endocardial cell number in morpholino injected transgenic (flil :nEGFP)^y7 embryos (Roman et al., Development 129:3009, 2002). The nuclear localization of each fluorescent protein facilitates easier determination of cell number. Positional cloning

Embryos were separated into mutant and wild-type pools based on phenotypic analysis. Genomic DNA was isolated from individual embryos by incubation in DNA isolation buffer overnight at 50⁰C (DNA isolation buffer: 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 0.3% Tween-20; 0.3% Nonidet P40; 0.5 mg/ml proteinase K).

Proteinase K was inactivated prior to PCR setup by heating samples to 98°C for 10 min. PCR reactions were performed using diluted genomic DNA as described (Knapik et al., Development 123:451, 1996). Bulked segregant analysis (Michelmore et al., Proc. Natl. Acad. Sci. U.S.A. 88:9828, 1991) and identification of the critical genetic interval was performed essentially as described previously (Mably et al., Curr. Biol. 13:2138, 2003). To identify the san gene, BACs 92il2 and 184dO7 (Fig. 4) were sequenced by shotgun cloning of partial AIuI and Sau3AI digested fragments subcloned into pBluescript. Sequence analysis was performed on an ABI3700 to generate approximately fivefold coverage. The sequence was assembled using the Phred/Phrap/Consed programs (Ewing and Green, Genome Res. 8:186, 1998; Ewing et al., Genome Res. 8:175, 1998; Gordon et al., Genome Res. 8:195, 1998). The vtn gene was identified through morpholino analysis of genes identified as candidates by position through synteny with the Takifugu rubripes (fugu) genome, followed by sequencing of cDNA and genomic DNA from mutant and wild-type embryos.

RNA isolation and real-time PCR analysis

RNA was isolated using trizol (Invitrogen) or RNeasy columns (Qiagen) as instructed by the manufacturer. For determination of the mRNA transcript variants induced by splice site blocking morpholinos, the QIAGEN® OneStep RT-PCR (reverse transcriptase) Kit was used with primers designed from exons on either side of the morpholino target. The splice variants induced by the various morpholinos are summarized below:

Analysis of mRNA levels in the san wt (wild-type) and -/- (mutant) embryos was performed using the Qiagen QuantiTect® S YBR® Green RT-PCR kit, as described by the manufacturer (Qiagen). The primers used for this analysis were designed to exon 13 (5'-GAGCAAAGCACATCACTGGA-S', SEQ ID NO:26) and exon 14 (S'-ATCACCTTGTGTGTGCTGGA-S', SEQ ID NO:27).

DNA cloning and RNA rescue For RT-PCR analysis, RNA was isolated (RNeasy columns, Qiagen) from pools of wild-type and mutant embryos. cDNA was amplified using RACE (SMART RACE cDNA amplification kit, Clontech). Fragments were then subcloned into PCRII-TOPO (Invitrogen). The 5' end of the san cDNA was amplified by 5' RACE with a primer designed from the full-length cDNA predicted by Genscan (Burge and Karlin, Curr. Opin. Struct. Biol. 8:346, 1998)

(5'-TTCAGCAGGTTGGGGTTACAGTTGC-S', SEQ ID NO:28). A 3' RACE product was generated using a primer

(5'-TCTCAGTCAAACAGCTGGACAGCGAC-S', SEQ ID NO:29) designed to amplify an overlying fragment of the san cDNA. Both cDNA fragments were digested with Sphl (a unique restriction site within the overlapping region) and EcoRI, and then were ligated into an EcoRI-digested pCS2 vector (Turner and Weintraub, Genes Dev. 8:1434, 1994) to create the full-length san cDNA construct. Clones with the correct orientation were identified by sequencing. The ftill-length vtn cDNA was amplified using primers designed to the 5 'and 3'UTR sequences within the Genscan predicted cDNA (5'UTR Fl : AATACAGCGAAAATGAAGAGCA, SEQ ID NO:30, 3'UTR_R1 : CAGCATCCAAACTTTCAGCA, SEQ ID NO:31). The PCR product was subcloned into pCRII-TOPO (Invitrogen). The vtn cDNA was excised from pCRII-TOPO by digestion with EcoRI, and then subcloned into EcoRI-digested pCS2 (Turner and Weintraub, Genes Dev. 8:1434, 1994). Clones with the correct orientation were identified by sequencing.

Injection RNA was generated from the full-length pCS2 san and vtn constructs using the Ambion mMESSAGE mMACHINE® kit (digested with Notl followed by transcription with SP6 polymerase).

Wholemount in situ and antibody staining

For wholemount in situ hybridization and immunohistochemistry, embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline, and then stored in 100% methanol at -20⁰C. Digoxigenin-labeled antisense RNA probes were generated by in vitro transcription (Roche) and in situ hybridization was carried out as previously described (Jowett and Lettice, Trends Genet. 10:73, 1994; Mably et al., Curr. Biol. 13:2138, 2003). The san probe used was derived from a partial EST in pSPORTl , fb36fO7 (GenBank accession: AI415912, digested with EcoRI, followed by transcription with SP6 polymerase). The vtn probe was derived from the full- length pCS2 construct described previously (digested with BamHI followed by transcription with T7 polymerase). Embryos were allowed to develop in BM purple (Roche) at 28°C, and then were stopped by several rinses in Ix PBT and stored at 4°C. Antibody staining with the S46 (Developmental Studies Hybridoma Bank,

University of Iowa) and MF20 (DSHB) antibodies was performed as previously described (Mably et al., Curr. Biol. 13:2138, 2003; Yelon et al., Dev. Biol. 214:23, 1999). Morpholino analysis

The antisense morpholino oligonucleotide designed over the san exon 1 donor site (5'-GCTTTATTTCACCTCAC, SEQ ID NO:32(intron-exon)CTCATAGG-3', SEQ ID NO:33, GeneTools, LLC) was dissolved at a concentration of 200 μM in IX Danieau's buffer (5 mM Hepes pH 7.6, 58 mM NaCl, 0.7 mM KCl, 0.6 mM Ca(NO₃J₂ , 0.4 mM MgSU4 ). 1 nL of this solution or IX Danieau's buffer was injected into each 1-4 cell embryo before allowing the embryos to develop at 28.5 ⁰C. The analysis with the san exon 14 donor site morpholino (5'-TTGAAGTCTCAC, SEQ ID NO:34 (intron-exonJTTTTGTCTCCATG-S¹, SEQ ID NO:35, GeneTools, LLC) and vtn exon 2 donor site morpholino (5'-GAAGCTGAGTAATAC, SEQ ID NO:36 (intron- exon)CTTAACTTCC-3', SEQ ID NO:37, GeneTools, LLC) were performed in the same manner.

Results Santa and valentine exhibit defects of the myocardium and cardiac cushions

Both santa and valentine were identified from a large-scale zebrafish genetic screen as ENU (N-ethyl-N-nitrosourea)-induced recessive embryonic lethal mutations with massively dilated hearts (Chen et al., Development 123:293, 1996; Driever et al., Development 123:37, 1996; Stainier et al., Development 123:285, 1996). These mutants are unable to generate blood circulation, despite the presence of cardiac contractions. They are distinguishable from their wild-type siblings by 28-30 hpf on this basis, and later by the onset of chamber dilation. The cardiac chambers are dramatically enlarged by 48 hpf.

In contrast to wild-type (wt) controls, in which the myocardial wall is 2-3 cells thick (Fig. IA), san and vtn mutants both have only a single layer, in both chambers of the heart (Fig. 1C,E). Cells within the myocardium of both chambers of the heart are differentiated cardiac cells, as indicated by labeling with molecular markers for the atrium (S46 antibody) and ventricle (ventricular myosin heavy chain vMHC, Fig. 1B,D,F) (Yelon et al., Dev. Biol. 214:23, 1999). Both the endocardial and myocardial layers of the heart are present and individual myocardial cells are thinner than in wild- type controls (Fig. 1C',E').

By 48 hpf, wild-type zebrafish embryos develop valvular precursors and endocardial cushions at the atrioventricular junction (Hu et al., Anat. Rec. 260:148, s 2000). Endocardial cushions are absent in san and vtn mutant embryos and the myocardial and endocardial layers, which are very thin, are closely juxtaposed (Figs. 1 and 2). Electron microscopic (EM) analysis of the structure of cardiomyocytes within the ventricles of san and vtn hearts reveals the presence of sarcomeres, consistent with the ability of the hearts to contract (Fig. 3). The absence of endocardial cushions and0 the variations in cellular morphology detected by EM could both be secondary effects of the severe cardiac dilation.

Myocardial and endocardial cell number is normal

The massive enlargement of the san and vtn hearts could be suggestive of an 5 increase in cell number. To determine cell number, we specifically labeled cardiomyocytes in vivo, using transgenic zebrafish with a red fluorescent protein (DsRed2-nuc) expressed under the control of the cardiac myosin light chain-2 (cmlc- 2) promoter (Mably et al., Curr. Biol. 13:2138, 2003; Rottbauer et al., Cell 111:661, 2002). This assay indicated that the number of myocardial cells is indistinguishable0 between wild-type and san mutant embryos and between mock injected and vtn morphant embyros at 2 dpf (days post fertilization, Table 1). Similarly, we determined the number of endocardial cells in mock injected and san and vtn morphant embryos at 2 dpf (Table 1). Morpholinos were injected into the progeny of transgenic (flil :nEGFP)^y7 crossed with transgenic cmlc2:DsRed2-nuc zebrafish. The5 number of eGFP expressing cells in the hearts of progeny expressing both the myocardial RFP and endothelial/endocardial eGFP was determined. As noted for the myocardial cell counts, endocardial cell number did not vary from that determined for wild-type. Hence, the dilated heart is not caused by an increase in the number of cardiac cells but, rather, in the manner in which they are assembled. The myocardial0 cells stretch in a single layer along the circumference of the cardiac chambers, rather than intercalating to form a thick myocardial wall, resulting in the observed chamber dilation.

Characterization of the san and vtn genes san

We positionally cloned both san and vtn (Fig. 4). The complete san gene is comprised of 16 coding exons with a deduced amino acid sequence of 741 amino acids. The human homolog is kritl (CCMl, Fig. 5A). The protein is characterized by the presence of several protein domains including two NPxY motifs (residues 191-194 and 229-232), 3 ankyrin repeats (residues 285-317, 318-351, and 352-385), and a C- terminal B41/FERM domain (residues 414-638).

The N-terminal NPxY motif is conserved in vertebrate species examined and also in the C. elegans ortholog (ZK265.1). This motif has been shown to interact with the PTB-containing protein ICAP let (Zawistowski et al., Hum. MoI. Genet. 11 :389, 2002; Zhang et al., Hum. MoI. Genet. 10:2953, 2001). In C. elegans, this sequence is NPXF (Fig. 6A), similar to the elements within the intracellular domain of β2- integrins (Calderwood et al., Proc. Natl. Acad. Sci. U.S.A. 100:2272, 2003). The Y -→ F substitution results in a motif that can still interact with a PTB-domain, but is not subject to regulation by phosphorylation state (Calderwood et al., Proc. Natl Acad. Sci. U.S.A. 100:2272, 2003). The three ankyrin repeats, believed to be sites of protein-protein interactions (reviewed in Mosavi et al., Protein Sci. 13:1435, 2004), and the FERM domain (reviewed in Bretscher et al., Nat. Rev. MoI. Cell. Biol. 3:586, 2002), implicated in the association of proteins with the cell membrane, are conserved across species, although the sequence of the FERM domain in the C. elegans homolog is poorly conserved and truncated (Fig. 5A).

Sequencing of the m775 (Stainier et al., Development 123:285, 1996) san cDNA predicts a splicing defect with a consequent in- frame deletion of exon 14. This results from a splice acceptor mutation within the intron at the start of exon 14 (agAG -> aaAG, Fig. 5A). The effect on the level of full-length san message was confirmed by real-time PCR amplification of m775 mRNA, showing significantly decreased levels of RNA transcripts containing exon 14 (Fig. 5B). In addition we sequenced another san allele, ty219c (Chen et al., Development 123:293, 1996). This mutation is a C to A transversion within codon 694 of exon 15 (TAC -^ TAA) that predicts a tyrosine change to a stop codon (Y -> stop, Fig. 5A). Both mutations would be predicted to cause loss of a C-terminal portion of the santa protein, possibly disrupting function of the FERM domain.

vtn

The complete vtn gene is comprised of 10 coding exons with a deduced amino acid sequence of 455 amino acids. The predicted protein is cytosolic with a molecular weight of 50 kDa. The protein has one recognizable protein motif, a PTB domain (amino acids 61-229). The vtn protein is highly conserved across vertebrates and encodes the zebrafish homolog of the human gene associated with CCM2 (MGC4607, malcalverin; Fig. 5C). The m201 allele of vtn (Stainier et al., Development 123:285, 1996) is defined by a C to A transversion within codon 119 of exon 4 (TAC → TAA). This results in a tyrosine change to a stop codon (Y- »stop, Fig. 5C), and the formation of a truncated protein with an incomplete PTB domain.

Morpholino confirmation and RNA rescue of genetic lesions san

We employed an antisense morpholino designed to block splicing at the donor site at the end of exon 14 of the san mRNA (Draper et al., Genesis 30:154, 2001 ; Ekker, Yeast 17:302, 2000; Mably et al., Curr. Biol. 13:2138, 2003). Injection of this morpholino at the 1-cell stage resulted in a complete copy of the mutant phenotype (>95% of embryos show the phenotype, n>1000). Analysis by RT-PCR using primers within exons flanking exon 14 revealed a partial in- frame loss of exon 14 sequence, confirming that loss of part of the FERM domain is sufficient to reproduce the mutant phenotype. Mock-injected controls reveal no effect on the san PCR products.

We also designed a morpholino to block splicing of the donor site at the end of exon 1 , in an attempt to create a truncated protein (through the excision of 5' exon sequence and disruption of the reading frame). However, this morpholino causes deletion of 75 bp, predicting an in-frame loss of 25 amino acids, due to the recruitment of a splice donor site within exon 1. Loss of these 25 amino acids was sufficient to produce a complete phenocopy, implicating the N-terminus of the protein as essential for normal function. Injection of mRNA derived from the predicted full- length san cDNA is unable to rescue the mutant phenotype. At high levels, expression of san mRNA in wild-type embryos impaired early development probably due to misexpression of san in all cells.

vtn

A morpholino was designed to the donor site at the end of exon 2 of the vtn gene. Injection of this morpholino at the 1 -cell stage results in a complete phenocopy of the vtn mutation (>95% phenocopy with n>1000). Analysis by RT-PCR using primers within exons flanking exon 2 and sequence analysis of the products revealed transcripts predicting both a partial and complete loss of exon 2 sequence. Injection of predicted vtn mRNA into progeny of vtn heterozygote matings rescues embryos completely (Table 2).

San and vtn mRNA expression Analysis of san mRNA by wholemount in situ at 28 hpf reveals staining in the ventricular zone of the brain (Fig. 6A,B), with weaker staining throughout the entire brain, and in the posterior cardinal vein (Fig. 6C,D). The venous expression is maintained at 48 hpf although expression is patchy (Fig. 6E,F). At 48 hpf there is also prominent notochord staining. Similarly, strong expression of vtn mRNA at 28 hpf is detected in the brain ventricular zone (Fig. 7A-D) with weaker expression in the vein (Fig. 7E,G). vtn mRNA is also obvious in the posterior intermediate cell mass at this stage (Fig. 7A,E,F). By 48 hpf, expression of vtn is weaker, but is detectable in vein (Fig. 7H- J). At both 28 hpf and 48 hpf, a low level of vtn mRNA is detectable in a region near the dorsal aorta (Fig. 7G,J). Co-morpholino evidence of interaction of san, vtn. and hep

The similarity of phenotype between san, vtn, and heg, along with the predicted interactive ability of protein motifs (PTB domain of vtn and NPxY motif of san/NPxF motif of heg), suggested that these proteins might be part of a pathway that controls concentric growth of the heart. To examine this, we lowered amounts of each protein by morpholino injection to a level where less than 10% of embryos demonstrated a complete phenocopy. Combinations of morpholinos were then injected at these doses to determine if the effects of these morpholinos are additive. Injection of san and vtn together at these doses resulted in a dramatic increase in the percentage of embryos exhibiting complete phenocopy (Fig. 8 and Table 3). When injected together with either the san or vtn morpholino at low doses, the heg combinations also produce a significant increase in phenocopy level (Fig. 8). These results suggest that san, vtn, and heg interact.

Tables

Table 1. The number of cells in san mutant hearts and vtn morphants is similar to wild-type hearts at day 2 of development. The myocardial cell counts were determined from flatmounts of progeny generated from san/cmlc2:DsRed2-nuc transgenic heterozygotes and from cmlc2:DsRed2-nuc transgenic wild-types that were either mock injected or injected with the vtn exon 2 donor morpholino. The number of myocardial cells was based on the number of RFP-expressing cells present in the hearts of individual animals. (The slight difference between the san and vtn groups reflects variation in the time during day 2 of development when the counts were made.) The endocardial cell counts were determined from flatmounts of progeny generated from (flil :nEGFP)^y7 crossed with transgenic cmlc2:DsRed2-nuc, which were mock injected or injected with the san exon 1 donor or vtn exon 2 donor morpholinos. The number of endocardial cells was based on the number of GFP- expressing cells present within the heart domain outlined by expression of the RFP transgene. n = number of hearts counted, wt sibs, san wild-type siblings, san -/- = san mutants, mock inj. = mock injected, vtn mo = vtn exon 2 donor morpholino injected, 200 μM, san mo = san exon 1 donor morpholino injected, 200 μM.

Table 2. Rescue ofvtn mutant phenotype by mRN A injection. Injection of vtn mRNA, but not san mRNA, into the progeny of m201 heterozygotes is able to rescue the vtn mutant phenotype. Neither san or vtn mRNA injection is able to rescue the san mutant phenotype.

Table 3. Morpholino co-injections reveal evidence of interactions between san, vtn, and heg. Injection of heg, san, or vtn morpholinos alone at 10 μM or 15 μM is unable to produce a complete phenocopy. However, injection of combinations of any two of these morpholinos produces a dramatic increase in the level of phenocopy. A number of embryos display a phenotype intermediate between wild-type and mutant, characterized by a less dramatic dilation of the heart and some with weak circulation. These results are displayed graphically in Fig. 6.

What is claimed is:

Claims

1. A method of determining whether a test subject has, or is at risk of developing, a heart disease or condition related to san or vtn, said method comprising analyzing a nucleic acid molecule of a sample from the test subject to determine whether the test subject has a mutation in a gene encoding said san or vtn, wherein the presence of a mutation indicates that said test subject has, or is at risk of developing, a heart disease or condition related to san or vtn.

2. The method of claim 1 , wherein said test subject is a mammal.

3. The method of claim 1, wherein said test subject is a human.

4. The method of claim 1, wherein said heart disease or condition is heart failure.

5. The method of claim 1, wherein said analyzing is carried out with respect to san.

6. The method of claim 1, wherein said analyzing is carried out with respect to vtn.

7. A method for identifying a compound that modulates the activity of san, said method comprising the steps of: (a) incubating san, or a gene encoding san, with a candidate compound; (b) comparing the activity of san in the presence of said candidate compound with the activity of san absent said candidate compound.

8. A method for identifying a compound that modulates the activity of vtn, said method comprising the steps of: (a) incubating vtn, or a gene encoding vtn, with a candidate compound; (b) comparing the activity of vtn in the presence of said candidate compound with the activity of vtn absent said candidate compound.

9. The method of claim 7 or 8, wherein said incubating is carried out in a cell- free mixture.

10. The method of claim 7 or 8, wherein said incubating is carried out in a cell-based mixture.

1 1. The method of claim 7 or 8, wherein said incubating is carried out in a recombinant cell.

12. The method of claim 7 or 8, wherein said incubating is carried out in an animal.

13. The method of claim 7 or 8, wherein said animal is a zebrafish, mouse, or human.

14. The method of claim 7, wherein said activity of san is the onset of concentric growth in a developing heart.

15. The method of claim 8, wherein said activity of vtn is the onset of concentric growth in a developing heart.

16. A method of treating or preventing a disease or condition associated with san or vtn in a patient, said method comprising administering to said patient an expression vector encoding san or vtn.

17. The method of claim 16, wherein said vector is an adeno-associated virus (AAV).

18. The method of claim 16, wherein said san is human san or said vtn is human vtn.

19. The method of claim 16, wherein said disease or condition is heart disease.

20. The method of claim 19, wherein said heart disease is heart failure.

21. A method of treating or preventing a disease or condition associated with san or vtn in a patient, said method comprising administering to said patient a protein preparation of san or vtn.

22. The method of claim 21 , wherein said preparation is a protein transduction domain-san or vtn fusion.

23. The method of claim 22, wherein said protein transduction domain comprises Tat, Antp, or VP22.

24. The method of claim 21 , wherein said san is human san or said vtn is human vtn. •

25. An isolated nucleic acid molecule encoding zebrafish san.

26. The nucleic acid molecule of claim 25, comprising the sequence of SEQ ID NO:6.

27. An isolated polypeptide comprising the sequence of SEQ ID NO:3.

28. An isolated nucleic acid molecule encoding the polypeptide of claim 27.

29. The isolated nucleic acid molecule of claim 28, wherein said nucleic acid molecule is operatively linked to an expression control sequence.

30. A vector comprising the nucleic acid molecule of claim 29.

31. A host cell comprising the vector of claim 30.

32. A non-human transgenic animal comprising a nucleic acid molecule encoding san.

33. The non-human transgenic animal of claim 32, wherein said animal is a zebrafish or mouse.

34. A non-human animal having a knockout mutation in one or both alleles encoding a san polypeptide.

35. A cell from the non-human knockout animal of claim 34.

36. An isolated nucleic acid molecule encoding zebrafish vtn.

37. The nucleic acid molecule of claim 36, comprising the sequence of SEQ ID NO: 12.

38. An isolated polypeptide comprising the sequence of SEQ ID NO:9.

39. An isolated nucleic acid molecule encoding the polypeptide of claim 38.

40. The isolated nucleic acid molecule of claim 39, wherein said nucleic acid molecule is operatively linked to an expression control sequence.

41. A vector comprising the nucleic acid molecule of claim 40.

42. A host cell comprising the vector of claim 41.

43. A non-human transgenic animal comprising a nucleic acid molecule encoding vtn.

44. The non-human transgenic animal of claim 43, wherein said animal is a zebrafish or mouse.

45. A non-human animal having a knockout mutation in one or both alleles encoding a vtn polypeptide.

46. A cell from the non-human knockout animal of claim 45.

47. A method of treating or preventing a disease or condition associated with san or vtn in a patient, said method comprising administering to said patient a host cell expressing san or vtn.

48. The method of claim 47, wherein said host cell is autologous.

49. An isolated nucleic acid molecule comprising an siRNA that inhibits expression of san.

50. An isolated nucleic acid molecule comprising an siRNA that inhibits expression of vtn.

51. The isolated nucleic acid molecule of claim 49 or 50, wherein said nucleic acid molecule is operatively linked to an expression control sequence.

52. A vector comprising the nucleic acid molecule of claim 51.

53. A method of treating or preventing a disease or condition associated with san or vtn in a patient, said method comprising administering to said patient the vector of claim 52.

54. A host cell comprising the vector of claim 52.

55. A method of treating or preventing a disease or condition associated with san or vtn in a patient, said method comprising administering to said patient the host cell of claim 54.

56. An antibody that specifically binds san or vtn.