MN/CA9 Splice Variants
Herein disclosed is an alternatively-spliced [AS] variant of MN/CA9 mRNA and its related protein—AS MN/CA IX. Unlike the tumor-associated, full-length [FL] MN/CA9 mRNA and FL MN/CA IX, which in most tissues signify oncogenesis and/or hypoxia, the AS MN/CA9 mRNA is constitutively-expressed under normoxia and is not stimulated by hypoxia, and the AS MN/CA IX is not confined to the cell membrane. Provided herein are diagnostic/prognostic methods for preneoplastic/neoplastic disease to differentiate between AS and FL MN/CA9 expression, and probes, primers, and antibodies useful in such methods. Also disclosed are methods to treat pre-neoplastic/neoplastic disease involving the MN gene and protein, which methods are based on the ability of AS MN protein (AS MN/CA IX) to interfere with the catalytic activity of FL MN protein (FL MN/CA IX); such methods may also use AS MN protein fragments that have that interference capability. Such methods may comprise increasing the levels of AS MN/CA IX relative to the levels of FL MN/CA IX. Exemplary therapeutic methods may comprise the administration of agents, such as, AS MN/CA IX itself, a vector expressing AS MN/CA9 mRNA, an antisense oligonucleotide that blocks expression of FL MN/CA IX but not that of AS MN/CA IX, a vector expressing such an antisense oligonucleotide, a FL MN/CA9 isoform-specific siRNA, or a vector expressing such FL MN/CA9 isoform-specific siRNA. Further disclosed are methods to identify agents capable of modulating levels of AS MN/CA IX.
1 . A diagnostic and/or prognostic method for a preneoplastic/neoplastic disease associated with abnormal MN/CA IX expression in a mammal, comprising differentiating between full-length [FL] and alternatively-spliced [AS] MN/CA9 mRNA or MN/CA IX expression.
2 . The method of claim 1 , comprising the use of one or more probes and/or primers to detect or detect and quantitate FL and/or AS MN/CA9 mRNA expression.
3 . The method of claim 2 , comprising the use of:
(a) probes and/or primers to detect full-length [FL] MN/CA9 mRNA but not alternatively-spliced [AS] MN/CA9 mRNA;
(b) probes and/or primers to detect AS MN/CA9 mRNA but not FL MN/CA9 mRNA; and/or
(c) probes and/or primers to detect both FL and AS MN/CA9 mRNA.
4 . The method of claim 2 , wherein said mammal is a human, and wherein said one or more probes and/or primers is/are selected from the group consisting of SEQ ID NOS: 97-101 and nucleic acid sequences that are at least 80% homologous to SEQ ID NOS: 97-101.
5 . The method of claim 2 , comprising the use of a nucleic acid amplification method.
6 . The method of claim 5 , wherein said nucleic acid amplification method comprises the use of PCR, RT-PCR, real-time PCR or quantitative real-time RT-PCR.
7 . The method of claim 2 , comprising the use of a microarray chip that comprises a probe that binds to full-length [FL] MN/CA9 mRNA but not to alternatively-spliced [AS] MN/CA9 mRNA, and/or a probe that binds to AS MN/CA9 mRNA but not FL MN/CA9 mRNA.
8 . The method of claim 2 , further comprising determining the ratio of FL:AS MN/CA9 mRNA.
9 . The method of claim 2 , wherein said AS MN/CA9 mRNA expression indicates normal MN/CA9 gene expression, and said FL MN/CA9 mRNA expression indicates abnormal MN/CA9 gene expression.
10 . The method of claim 2 , wherein said AS MN/CA9 mRNA expression indicates normoxic MN/CA9 gene expression, and said FL MN/CA9 mRNA expression indicates hypoxic MN/CA9 gene expression.
11 . The method of claim 1 , comprising the use of one or more antibodies to differentiate between FL and AS MN/CA IX expression in a preneoplastic/neoplastic tissue.
12 . The method of claim 11 , comprising detecting or detecting and quantitating AS MN/CA IX in said tissue.
13 . The method of claim 12 , further comprising determining the ratio of FL MN/CA IX levels to AS MN/CA IX levels in said tissue.
14 . The method of claim 13 , wherein said ratio indicates presence or degree of hypoxia in said tissue.
15 . The method of claim 11 , comprising detecting or detecting and quantitating FL MN/CA IX and AS MN/CA IX in a vertebrate tissue, comprising the steps of:
(a) contacting a sample of said vertebrate tissue synchronously or sequentially with at least two antibodies, at least two antigen-binding antibody fragments, or a mixture of antibodies and antigen-binding antibody fragments, wherein at least one antibody/antibody fragment specifically binds to FL MN/CA IX protein but not to AS MN/CA IX protein, and wherein at least one other antibody/antibody fragment specifically binds to both FL and AS MN/CA IX;
(b) detecting and quantifying the binding of said antibodies/antibody fragments in said sample; and
(c) comparing the binding of said differentially binding antibodies/antibody fragments to determine the relative levels of FL MN/CA IX and AS MN/CA IX.
16 . The method of claim 15 , wherein the antibody/antibody fragment, or antibodies/antibody fragments, that specifically bind(s) to FL MN/CA IX but not to AS MN/CA IX is/are specific for the carbonic anhydrase (CA) domain of MN/CA IX; and wherein the antibody/antibody fragment, or antibodies/antibody fragments, that specifically bind(s) both FL MN/CA IX and AS MN/CA IX is/are specific for the proteoglycan-like (PG) domain of MN/CA IX.
17 . The method of claim 16 , wherein said antibody specific for the CA domain of MN/CA IX is the V/10 monoclonal antibody which is produced by the hybridoma VU-V/10, deposited at BCCM™/LMBP in Ghent, Belgium under Accession No. LMBP 6009CB; and wherein the antibody specific for the PG domain of MN/CA IX is the M75 monoclonal antibody which is produced by the hybridoma VU-M75 deposited at the American Type Culture Collection (ATCC) under the ATCC designation No. HB 11128.
18 . A diagnostic and/or prognostic method for a preneoplastic/neoplastic disease associated with abnormal MN/CA IX expression in a vertebrate, comprising detecting or detecting and quantitating full-length [FL] MN/CA IX protein but not alternatively-spliced [AS] MN/CA IX protein in a vertebrate tissue, comprising the steps of:
(a) contacting a sample of said vertebrate tissue with an antibody or antibody fragment, wherein said antibody or antibody fragment specifically binds to FL MN/CA IX but not to AS MN/CA IX; and
(b) detecting and quantifying binding of said antibody/antibody fragment in said sample.
19 - 20 . (canceled)
21 . A diagnostic and/or prognostic method for a preneoplastic/neoplastic disease associated with abnormal MN/CA IX expression in a mammal, comprising detecting or detecting and quantitating full-length [FL] MN/CA9 mRNA but not alternatively-spliced [AS] MN/CA9 mRNA in a mammalian preneoplastic/neoplastic sample, comprising contacting mRNA from said sample with a primer or a probe that specifically binds to FL MN/CA9 mRNA but not to AS MN/CA9 mRNA.
22 - 29 . (canceled)
30 . A pair of probes and/or primers used to differentiate between alternatively-spliced [AS] MN/CA9 mRNA and full-length [FL] MN/CA9 mRNA expression in a mammal.
31 - 35 . (canceled)
36 . An isolated nucleic acid encoding a mammalian alternatively-spliced [AS] MN/CA IX, wherein said AS MN/CA IX has a molecular weight of from about 43 to about 48 kilodaltons.
37 - 43 . (canceled)
44 . An antibody or antigen-binding antibody fragment that binds specifically to the AS MN/CA IX of claim 36 , but does not bind specifically to FL MN/CA IX.
45 . An antibody or antigen binding antibody fragment that binds specifically to the AS MN/CA IX of claim 36 , but does not bind specifically to soluble MN/CA IX (s-CA IX).
46 . A method for treating preneoplastic/neoplastic disease in a mammal, wherein said disease is associated with abnormal expression of MN/CA IX, the method comprising administering to said mammal a therapeutically effective amount of a composition comprising an agent that increases levels of alternatively-spliced [AS] MN/CA IX relative to levels of full-length [FL] MN/CA IX.
47 - 50 . (canceled)
51 . An oligonucleotide that increases levels of alternatively-spliced [AS] MN/CA IX relative to levels of full-length [FL] MN/CA IX, wherein said oligonucleotide is used in treatment of a preneoplastic/neoplastic disease associated with abnormal MN/CA IX expression.
52 - 54 . (canceled)
55 . An in vitro method of identifying agents capable of modulating levels of alternatively-spliced [AS] MN/CA IX, comprising contacting cells expressing AS MN/CA IX with an agent suspected of modulating the level of said AS MN/CA IX in the cells, and detecting and quantitating changes in levels of said AS MN/CA IX.