The invention provides isolated nucleic acids molecules, designated COCH5B2 nucleic acid molecules, which encode polypeptides involved in inner ear biology. The invention also provides antisense nucleic acid molecules, expression vectors containing COCH5b2 nucleic acid molecules, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a COCH5b2 gene has been introduced or disrupted. The invention still further provides isolated COCH5B2 polypeptides, fusion polypeptides, antigenic peptides, and anti-COCH5B2 antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery of a nucleic acid and corresponding protein molecule, referred to herein as COCH5B2 ("COCH5B2") molecules. The COCH5B2 molecules of the present invention are useful in diagnosing and treating hearing disorders, e.g., human nonsyndromic sensorineural deafness with vestibular involvement (DFNA9).

Accordingly, in one aspect, the invention features an isolated nucleic acid molecule (e.g., cDNAs) comprising a nucleotide sequence encoding a COCH5B2 protein or a biologically active portion thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of COCH5B2-encoding nucleic acid (e.g., mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, or the coding region or a complement of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention includes a nucleotide sequence which hybridizes to or has at least about 60-65%, preferably at least about 70-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:6, or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:7. The preferred COCH5B2 nucleic acid encodes a protein which also preferably possesses at least one of the COCH5B2 activities described herein.

In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:7, e.g., sufficiently homologous to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:7 such that the protein or portion thereof maintains a COCH5B2 activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to play a role in inner ear biology. In one embodiment, the protein encoded by the nucleic acid molecule has at least about 60-70%, preferably at least about 80-85%, and more preferably at least about 86, 88, 90%, and most preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 (e.g., the entire amino acid sequence of SEQ ID NO:2) or SEQ ID NO:7. In another preferred embodiment, the protein is a full length human protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2 (encoded by the open reading frame shown in SEQ ID NO:3).

In yet another embodiment, the isolated nucleic acid molecule is derived from a human and encodes a portion of a protein which includes at least one or two von Willebrand factor (vWF) type A-like domains. Preferably, the vWF type A-like domain encoded by the human nucleic acid molecule has at least about 75%, preferably at least about 80-85%, and most preferably at least about 80-90% or more sequence identity to the vWF type A-like domain (i.e., about amino acid residues 165 to 309 and about amino acid residues 366 to 514) of SEQ ID NO:2 which are shown as separate sequences designated SEQ ID NO:4 and SEQ ID NO: 5, respectively. In stPill another embodiment, the nucleic acid molecule is a nonmammalian molecule which encodes at least one or two vWF type A-like domains. Preferably, the vWF type A-like domain encoded by the nonmammalian nucleic acid has at least about 55%, more preferably at least about 60-65%, even more preferably at least about 70%-75% and most preferably at least about 80-950% or more sequence identity to SEQ ID NO:4 or SEQ ID NO:5.

In yet another embodiment, the isolated nucleic acid molecule is derived from a human and encodes a portion of a protein which includes at least one factor C homologous domain. Preferably, the factor C homologous domain encoded by the human nucleic acid molecule has at least about 75%, preferably at least about 80-85%, and most preferably at least about 80-90% or more sequence identity to the factor C homologous domain (i.e., about amino acid residues 32-136) of SEQ ID NO:2 which is shown as a separate sequence designated SEQ ID NO: 1. In still another embodiment, the nucleic acid molecule is a nonmammalian molecule which encodes at least one factor C homologous domain. Preferably, the factor C homologous domain encoded by the nonmammalian nucleic acid has at least about 55%, more preferably at least about 60-65%, even more preferably at least about 70%-75% and most preferably at least about 80-950% or more sequence identity to SEQ ID NO: 11.

In another preferred embodiment, the isolated nucleic acid molecule is derived from a human and encodes a protein (e.g., a COCH5B2 fusion protein) which includes a vWF type A-like domain which has at least about 55% or more sequence identity to SEQ ID NO:4 and/or SEQ ID NO:5 and has one or more of the following activities involved with inner ear biology:

• 1) it can interact, e.g., bind, with components of extracellular matrix (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); 2) it can modulate cell/extracellular matrix interactions; 3) it can modulate cell—cell adhesions; 4) it can interact, e.g., bind, with glycoproteins and/or proteoglycans for clearing them; 5) it can provide scaffolding by interacting with other extracellular matrix components (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); and 6) it can modulate an inner ear secretory pathway (e.g., it can modulate production of acidophillic deposits).

In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:6. Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes naturally-occurring human COCH5B2 or a biologically active portion thereof. Preferably, the biologically active portion is preferably encoded by a nucleotide sequence greater than 200 base pairs in length and/or excludes nucleotides 1457-1654. In yet another preferred embodiment, the biologically active portion is preferably encoded by a nucleotide sequence greater than 400, 500 or 600 base pairs in length, e.g., it is at least 700 or 1000 base pairs in length. Moreover, given the disclosure herein of COCH5B2-encoding cDNA sequences (e.g., SEQ ID NO: 1 and SEQ ID NO:6), antisense nucleic acid molecules (i.e., molecules which are complementary to the coding strand of the COCH5B2 cDNA sequence) are also provided by the invention.

In a preferred embodiment, the encoded COCH5B2 protein differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:2 or SEQ ID NO:7. The differences, however, are such that: the COCH5B2 encoded protein exhibits a COCH5B2 biological activity, e.g., the encoded COCH5B2 protein retains a biological activity of a naturally occurring COCH5B2, e.g., the COCH5B2 protein of SEQ ID NO:2 or SEQ ID NO:7.

In preferred embodiments, the encoded polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:2 or SEQ ID NO:7, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5′ to the genomic DNA which encodes a sequence from SEQ ID NO:2 or SEQ ID NO:7.

In preferred embodiments the encoded COCH5B2 protein includes a COCH5B2 sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.

In another aspect, the invention features vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce COCH5B2 protein by culturing the host cell in a suitable medium. The COCH5B2 protein can be then isolated from the medium or the host cell.

In yet another aspect, the invention features transgenic nonhuman animals in which a COCH5B2 gene has been introduced or altered. In one embodiment, the genome of the nonhuman animal has been altered by introduction of a nucleic acid molecule of the invention encoding COCH5B2 as a transgene. In another embodiment, an endogenous COCH5B2 gene within the genome of the nonhuman animal has been altered, e.g., functionally disrupted, by homologous recombination.

In still another aspect, the invention features an isolated COCH5B2 protein or a portion, e.g., a biologically active portion, thereof. In a preferred embodiment, the isolated COCH5B2 protein or portion thereof plays a role in inner ear biology. In another preferred embodiment, the isolated COCH5B2 protein or portion thereof is sufficiently homologous to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:7 such that the protein or portion thereof maintains one or more COCH5B2 activities.

In one embodiment, the biologically active portion of the COCH5B2 protein includes a domain or motif, preferably a domain or motif which has a COCH5B2 activity. The domain can be, e.g., a vWF type A-like domain. If the active portion of the protein which includes one or two vWF type A-like domains is isolated or derived from a human, it is preferred that the vWF type A-like domain have at least about 75%, preferably at least about 80-85%, and most preferably about 90-95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4 and/or SEQ ID NO:5. If the active portion of the protein which includes the vWF type A-like domain is isolated or derived from an animal which is not a mammal, it is preferred that the vWF type A-like domain have at least about 55%, preferably at least about 60-65%, even more preferably at least about 70%-75% and most preferably at least about 80-90% 90-95% 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4 and/or SEQ ID NO:5. The domain can also be, e.g., a factor C homologous domain. If the active portion of the protein which includes one factor C homologous domain is isolated or derived from a human, it is preferred that the factor C homologous domain have at least about 75%, preferably at least about 80-85%, and most preferably about 90-95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11. If the active portion of the protein which includes the factor C homologous domain is isolated or derived from an animal which is not a mammal, it is preferred that the factor C homologous domain have at least about 55%, preferably at least about 60-65%, even more preferably at least about 70%-75% and most preferably at least about 80-90% 90-95% 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:11. Preferably, the biologically active portion of the COCH5B21 protein which includes one or two domains also has one of the following activities: 1) it can interact, e.g., bind, with components of extracellular matrix (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); 2) it can modulate cell/extracellular matrix interactions; 3) it can modulate cell—cell adhesions; 4) it can interact, e.g., bind, with glycoproteins and/or proteoglycans for clearing them; 5) it can provide scaffolding by interacting with other extracellular matrix components (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); and 6) it can modulate an inner ear secretory pathway (e.g., it can modulate production of acidophillic deposits).

The invention also provides an isolated preparation of a COCH5B2 protein. In preferred embodiments, the COCH5B2 protein includes the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:7. In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2 (encoded by the open reading frame shown in SEQ ID NO:3) or SEQ ID NO:7. In yet another embodiment, the protein has at least about 60-70%, preferably at least about 80-85%, and more preferably at least about 86, 88, 90%, and most preferably at least about 90-95%96%, 97%, 98% or 99% sequence identity to the entire amino acid sequence of SEQ ID NO:2 or SEQ ID NO:7. In other embodiments, the isolated COCH5B2 protein includes an amino acid sequence which has at least about 60-70% or more sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:7 and has an one or more of the following activities: 1) it can interact, e.g., bind, with components of extracellular matrix (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); 2) it can modulate cell/extracellular matrix interactions; 3) it can modulate cell—cell adhesions; 4) it can interact, e.g., bind, with glycoproteins and/or proteoglycans for clearing them; 5) it can provide scaffolding by interacting with other extracellular matrix components (e.g., fibrillar collagen, e.g., COL1A2, COL3A1); and 6) it can modulate an inner ear secretory pathway (e.g., it can modulate production of acidophillic deposits).

Alternatively, the isolated COCH5B2 protein can include an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, or has at least about 60-65%, preferably at least about 70-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95%96%, 97%, 98% or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:6. It is also preferred that the preferred forms of COCH5B2 also have one or more of the COCH5B2 activities described herein.

In a preferred embodiment, the COCH5B2 protein differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 2 or SEQ ID NO:7. In other preferred embodiments, the COCH5B2 protein differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO: 2 or SEQ ID NO:7. Preferably, the differences are such that: the COCH5B2 protein exhibits a COCH5B2 biological activity, e.g., the COCH5B2 protein retains a biological activity of a naturally occurring COCH5B2.

In preferred embodiments, the COCH5B2 polypeptide includes a COCH5B2 sequence described herein as well as other N-terminal, and/or a C-terminal amino acid sequence.

The COCH5B2 protein (or polypeptide) or a biologically active portion thereof can be operatively linked to a non-COCH5B2 polypeptide to form a fusion protein. The COCH5B2 protein or a biologically active portion thereof can be incorporated into a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier.

The COCH5B2 protein of the invention, or portions or fragments thereof, can be used to prepare anti-COCH5B2 antibodies. Accordingly, the invention also provides an antigenic peptide of COCH5B2 which includes at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:7 and encompasses an epitope of COCH5B2 such that an antibody raised against the peptide forms a specific immune complex with COCH5B2. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30, 50, 70, 80 amino acid residues. The invention further provides an antibody that specifically binds COCH5B2. In one embodiment, the antibody is monoclonal. In another embodiment, the antibody is coupled to a detectable substance. In yet another embodiment, the antibody is incorporated into a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.

Another aspect of the invention features methods for modulating a cell associated activity, e.g., cell—cell adhesion, e.g., in vitro or in vivo. Such methods include contacting the cell with an agent which modulates COCH5B2 protein activity or nucleic acid expression such that a cell associated activity is altered relative to a cell associated activity (e.g., the same cell associated activity) of the cell in the absence of the agent. The agent which modulates COCH5B2 activity can be an agent which stimulates COCH5B2 protein activity or COCH5B2 nucleic acid expression. Examples of agents which stimulate COCH5B2 protein activity or COCH5B2 nucleic acid expression include small molecules, active COCH5B2 proteins, and nucleic acids encoding COCH5B2 that have been introduced into the cell. Examples of agents which inhibit COCH5B2 activity or expression include small molecules, antisense COCH5B2 nucleic acid molecules, and antibodies that specifically bind to COCH5B2. In a preferred embodiment, the cell is present within a subject and the agent is administered to the subject.

The present invention also features methods for treating subjects having a hearing disorder. For example, the invention pertains to methods for treating a subject having a disorder characterized by aberrant COCH5B2 protein activity or nucleic acid expression such as a hearing disorder, e.g., a nonsyndromic sensorineural deafness with vestibular involvement (DFNA9). These methods include administering to the subject a COCH5B2 modulator (e.g., a small molecule) such that treatment of the subject occurs.

In other embodiments, the invention pertains to methods for treating a subject having a hearing disorder, e.g., a nonsyndromic sensorineural deafness with vestibular involvement (DFNA9), comprising administering to the subject a COCH5B2 protein or portion thereof such that treatment occurs. Hearing disorders can also be treated according to the invention by administering to the subject having the disorder a nucleic acid encoding a COCH5B2 protein or portion thereof such that treatment occurs.

The invention also features methods for detecting genetic lesions in a COCH5B2 gene, thereby determining if a subject with the lesioned gene is at risk for (or is predisposed to have) a disorder characterized by aberrant or abnormal COCH5B2 nucleic acid expression or COCH5B2 protein activity, e.g., a hearing disorder, e.g., DFNA9. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by an alteration affecting the integrity of the gene encoding a COCH5B2 protein, or the misexpression of the COCH5B2 gene. Genetic lesions can be detected, e.g., by contacting the sample with a nucleic acid probe capable of hybridizing to COCH5B2 mRNA, e.g., a labeled probe, or an antibody capable of binding to COCH5B2 protein, e.g., a labeled antibody. In a preferred embodiment, the method can also be used in fetal or neonatal diagnosis.

Another aspect of the invention features methods for detecting the presence of COCH5B2 nucleic acid or protein in a biological sample. In a preferred embodiment, the methods involve contacting a biological sample (e.g., a cell sample) with a compound or an agent capable of detecting COCH5B2 protein or COCH5B2 nucleic acid, e.g., mRNA, such that the presence of COCH5B2 nucleic acid or protein is detected in the biological sample. The compound or agent can be, for example, a labeled or labelable nucleic acid probe capable of hybridizing to COCH5B2 mRNA or a labeled or labelable antibody capable of binding to COCH5B2 protein. The invention further provides methods for diagnosis of a subject with, for example, a hearing disorder, e.g., DFNA9, based on detection of COCH5B2 protein or mRNA. In one embodiment, the method involves contacting a cell or tissue sample (e.g., a biopsy sample) from the subject with an agent capable of detecting COCH5B2 protein or mRNA, determining the amount of COCH5B2 protein or mRNA expressed in the cell or tissue sample, comparing the amount of COCH5B2 protein or mRNA expressed in the cell or tissue sample to a control sample and forming a diagnosis based on the amount of COCH5B2 protein or mRNA expressed in the cell or tissue sample as compared to the control sample. Specific diagnostic tests are described in greater detail below. Kits for detecting COCH5B2 nucleic acid or protein in a biological sample are also within the scope of the invention and are described in greater detail below.

Still another aspect of the invention features methods, e.g., screening assays, for identifying a compound for treating a disorder characterized by aberrant COCH5B2 nucleic acid expression or protein activity, e.g., a hearing disorder, e.g., DNA9. These methods typically include assaying the ability of the compound or agent to modulate the expression of the COCH5B2 gene or the activity of the COCH5B2 protein thereby identifying a compound for treating a disorder characterized by aberrant COCH5B2 nucleic acid expression or protein activity. In a preferred embodiment, the method involves contacting a biological sample, e.g., a cell or tissue sample, obtained from a subject having the disorder with the compound or agent, determining the amount of COCH5B2 protein expressed and/or measuring the activity of the COCH5B2 protein in the biological sample, comparing the amount of COCH5B2 protein expressed in the biological sample and/or the measurable COCH5B2 biological activity in the cell to that of a control sample. An alteration in the amount of COCH5B2 protein expression or COCH5B2 activity in the cell exposed to the compound or agent in comparison to the control is indicative of a modulation of COCH5B2 expression and/or COCH5B2 activity.

The invention also pertains to methods for identifying a compound or agent which interacts with (e.g., binds to) a COCH5B2 protein. These methods can include the steps of contacting the COCH5B2 protein with the compound or agent under conditions which allow binding of the compound to the COCH5B2 protein to form a complex and detecting the formation of a complex of the COCH5B2 protein and the compound in which the ability of the compound to bind to the COCH5B2 protein is indicated by the presence of the compound in the complex.

The invention further pertains to methods for identifying a compound or agent which modulates, e.g., stimulates or inhibits, the interaction of the COCH5B2 protein with a target molecule, e.g., a component of the extracellular matrix, e.g., fibrillar collagens, or a protein involved in a secretory pathway, e.g., a protein involved in the secretion of acidophillic deposits. In these methods, the COCH5B2 protein is contacted, in the presence of the compound or agent, with the target molecule under conditions which allow binding of the target molecule to the COCH5B2 protein to form a complex. An alteration, e.g., an increase or decrease, in complex formation between the COCH5B2 protein and the target molecule as compared to the amount of complex formed in the absence of the compound or agent is indicative of the ability of the compound or agent to modulate the interaction of the COCH5B2 protein with a target molecule.
 

Claim 1 of 33 Claims

1. A nucleic acid primer for diagnosing a hearing disorder which hybridizes under stringent conditions to a portion of the nucleic acid sequence of SEQ ID NO: 1 or complement thereof, wherein the primer amplifies all or a portion of exons 4 and 5 of SEQ ID NO: 1 such that one or more nucleotides encoding one or more of an amino acid at residue 51, an amino acid at residue 66, an amino acid at residue 88 and an amino acid at residue 117 of SEQ ID NO:2 is amplified.
 

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