Rath genes and polypeptides and methods for the treatment and diagnosis of immune disorders

Abstract

The present invention relates, first, to the identification of novel nucleic acid molecules, termed RATH genes and RATH gene products encoded by such nucleic acid molecules, or degenerate variants thereof, that participate in the regulation, control and/or modulation of G-protein-mediated signal transduction involved in T cell activation, including, but not limited to T helper (TH) cell and TH cell subpopulation activation. Specifically, the nucleic acid molecules of the present invention include the genes corresponding to the mammalian RATH genes, including the RATH1.1 genes. Sequence analysis indicates that the RATH genes are novel genes belonging to the RGS (“regulator of G-protein signalling”) gene family, a gene family which encodes gene products involved in G-protein-mediated signal transduction.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule comprising: 
 (a) nucleotide sequence that encodes a polypeptide having the amino acid sequence of FIG. 1 or FIG. 2;    (b) the complement of the nucleotide sequence of (a);    (c) a RATH gene or a complement of a RATH gene as contained in ATCC Deposit No. 98116; and    (d) a nucleic acid molecule capable of hybridizing to and which is at least 80% identical to a nucleic acid molecule of (a), (b) or (c).    
     
     
         2 . An isolated nucleic acid molecule comprising a nucleotide sequence that hybridize to the nucleic acid of  claim 1  and encodes a naturally occurring RATH polypeptide.  
     
     
         3 . An isolated nucleic acid molecule comprising the nucleotide sequence of FIG. 1.  
     
     
         4 . An isolated nucleic acid molecule of  claim 1 , which is genomic.  
     
     
         5 . An isolated nucleic acid molecule of  claim 1 , which is cDNA.  
     
     
         6 . An isolated nucleic acid molecule of  claim 1 , which is RNA.  
     
     
         7 . An isolated nucleic acid molecule of  claim 1 , which hybridizes to at least 6 consecutive nucleotides of the RATH1.1 gene as set forth in FIG. 1.  
     
     
         8 . An isolated nucleic acid molecule of  claim 7 , further comprising a label.  
     
     
         9 . A delivery complex comprising a nucleic acid molecule of  claim 2  and a targeting means.  
     
     
         10 . The delivery complex of  claim 9 , wherein the targeting means is selected from the group consisting of: a sterol, lipid, virus or target cell specific binding agent.  
     
     
         11 . An isolated nucleic acid molecule comprising a nucleotide sequence that hybridizes to the nucleic acid of  claim 1  and encodes a polypeptide with an activity of a RATH protein, or a fragment thereof, linked uninterrupted by stop codons to a nucleotide sequence that encodes a heterologous protein or peptide.  
     
     
         12 . A nucleic acid molecule comprising a nucleotide sequence encoding (a) a deletion mutant of RATH; or (b) the complement of the nucleotide sequence.  
     
     
         13 . The nucleic acid molecule of  claim 12  wherein the nucleic acid molecule is missing the RGS domain of RATH.  
     
     
         14 . A nucleic acid molecule comprising a nucleotide sequence encoding (a) a polypeptide corresponding to the RGS domain; or (b) the complement of the nucleotide sequence.  
     
     
         15 . A recombinant vector containing the nucleotide sequence of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7  or  11 .  
     
     
         16 . An expression vector containing the nucleotide sequence of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7  or  11  operatively associated with a regulatory nucleotide sequence containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in a host cell.  
     
     
         17 . A delivery complex comprising an expression vector of  claim 16  and a targeting means.  
     
     
         18 . A delivery complex of  claim 17 , wherein the targeting means is selected from the group consisting of: a sterol, lipid, virus or target cell specific binding agent.  
     
     
         19 . A genetically engineered host cell containing the nucleotide sequence of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7  or  11 .  
     
     
         20 . A genetically engineered host cell containing the nucleotide sequence of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7  or  11  operatively associated with a regulatory nucleotide sequence containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in a host cell.  
     
     
         21 . The genetically engineered host cell of  claim 20  in which the host cell is prokaryotic.  
     
     
         22 . The genetically engineered host cell of  claim 20  in which the host cell is eukaryotic.  
     
     
         23 . A method of making a RATH polypeptide comprising the steps of: 
 (a) culturing the cell of  claim 21  in an appropriate culture medium to produce RATH polypeptide; and    (b) isolating the RATH polypeptide.    
     
     
         24 . A method of making a RATH polypeptide comprising the steps of: 
 (a) culturing the cell of  claim 22  in an appropriate culture medium to produce RATH polypeptide; and    (b) isolating the RATH polypeptide.    
     
     
         25 . An antibody preparation, which is specifically reactive with an epitope of a RATH polypeptide.  
     
     
         26 . A transgenic animal comprising the nucleic acid of  claim 1 .  
     
     
         27 . A transgenic animal in which expression of a genomic sequence encoding a functional RATH polypeptide is prevented or suppressed.  
     
     
         28 . A substantially pure polypeptide encoded by the nucleic acid molecule of  claim 1 .  
     
     
         29 . A substantially pure mammalian RATH polypeptide, as depicted in FIG. 1.  
     
     
         30 . A polypeptide of  claim 29 , which is at least 90% identical to the polypeptide as set forth in FIG. 1.  
     
     
         31 . A fusion protein comprising a polypeptide of  claim 30  and a second polypeptide which is a detectable label or a matrix binding domain.  
     
     
         32 . A pharmaceutical preparation comprising a therapeutically effective amount of the polypeptide of  claim 28  and a pharmaceutically acceptable carrier.  
     
     
         33 . A test kit for detecting and/or quantitating a wild type or mutant RATH nucleic acid molecule in a sample, comprising the steps of contacting the sample with a nucleic acid molecule of  claim 1;  and detecting and/or quantitating the label as an indication of the presence or absence and/or amount of a wildtype or mutant RATH nucleic acid.  
     
     
         34 . A test kit for detecting and/or quantitating a wild type or mutant RATH polypeptide in a sample, comprising the steps of contacting the sample with the antibody of claim  25 ; and detecting and/or quantitating a polypeptide-antibody complex as an indication of the presence or absence and/or amount of a wildtype or mutant RATH nucleic acid.  
     
     
         35 . A method for identifying compounds that regulate immune disorders, comprising: 
 (a) contacting a test compound with a cell which expresses a nucleic acid of  claim 1 , and    (b) determining whether the test compound modulates RATH activity.    
     
     
         36 . A method for identifying compounds that regulate immune disorders comprising: 
 (a) contacting a test compound with a nucleic acid of  claim 1;  and    (b) determining whether the test compound interacts with the nucleic acid of  claim 1 .    
     
     
         37 . A method for identifying compounds that inhibit an interaction between RATH and its cognate G-α-I subunit comprising the steps of: 
 (a) combining the peptide of  claim 28 , or bioreactive fragment thereof, a G-α-I polypeptide and a test compound under conditions appropriate for formation of a peptide/G-α-I polypeptide complex; and  
 (b) quantitating the amount of peptide/G-α-I polypeptide complex and comparing the amount to the amount of peptide/G-α-I polypeptide complex formed under the same conditions, but in the absence of the test compound, wherein a lesser amount of peptide/G-α-I polypeptide complex formed in the presence of the test compound indicates that the test compound is an inhibitor of the interaction between a RATH protein and a G-α-I subunit.  
 
     
     
         38 . A method for identifying compounds that potentiate an interaction between RATH and its cognate G-α-I subunit comprising the steps of: 
 (a) combining a peptide of  claim 28 , or bioreactive fragment thereof, a G-α-I polypeptide and a test compound under conditions appropriate for formation of an peptide/G-α-I polypeptide complex; and  
 (b) quantitating the amount of peptide/G-α-I polypeptide complex and comparing the amount to the amount of peptide/G-α-I polypeptide complex formed under the same conditions, but in the absence of the test compound, wherein a greater amount of peptide/G-α-I polypeptide complex formed in the presence of the test compound indicates that the test compound is an potentiator of the interaction between a RATH protein and a G-α-I subunit.  
 
     
     
         39 . A method for identifying compounds that regulate immune disorders, comprising: 
 (a) contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a RATH regulatory element; and    (b) detecting expression of the reporter gene product.    
     
     
         40 . A method for identifying compounds that regulate immune disorders comprising: 
 (a) contacting a test compound with a cell or cell lysate containing RATH transcripts; and    (b) detecting the translation of the RATH transcript.    
     
     
         41 . A method for identification of compounds which modulate RATH activity comprising: 
 (a) contacting yeast cells containing a nucleic acid molecule of  claim 1  with pheromone and a test compound; and    (a) measuring the growth inhibition of said yeast cells in which test compounds which inhibit the growth of yeast cells inhibit or suppress RATH activity.    
     
     
         42 . A method for identification of compounds which modulate RATH activity comprising: 
 (a) contacting yeast cells containing a nucleic acid molecule of  claim 1  with pheromone and a test compound; and    (b) measuring the growth inhibition of said yeast cells in which test compounds which fail to inhibit the growth of yeast cells increase or enhance RATH activity.    
     
     
         43 . A method for identification of proteins that interact with RATH in which, total genomic or cDNA sequences fused to DNA encoding an activation domain is cotransformed into a yeast reporter strain with DNA encoding RATH fused to a DNA binding domain and the transformants are screened for expression of a reporter gene.  
     
     
         44 . A method for modulating immune disorders in a subject, comprising administering to the subject a therapeutically effective amount of RATH polypeptide.  
     
     
         45 . The method of  claim 44 , wherein the subject is a human.  
     
     
         46 . A method of gene therapy, comprising administering to a subject an effective amount of a delivery complex of  claim 17 .  
     
     
         47 . A method for the treatment of immune disorders, comprising modulating the activity of RATH.  
     
     
         48 . The method of  claim 47 , wherein the method comprises administering an effective amount of a compound that agonizes or antagonizes the activity of RATH.  
     
     
         49 . The method of  claim 47  in which the compound activates RATH and induces T helper cells activation.  
     
     
         50 . The method of  claim 48  in which the compound is an agonist that binds to and activates RATH.  
     
     
         51 . The method of  claim 48  in which the compound inhibits activation of RATH and inhibits T helper cell activation.  
     
     
         52 . A method for the treatment of immune disorders, comprising administering an effective amount of a compound that decreases expression of RATH.  
     
     
         53 . The method of  claim 52  in which the compound is an oligonucleotide encoding an antisense or ribozyme molecule that targets RATH transcripts and inhibits translation.  
     
     
         54 . The method of  claim 52  in which the compound is an oligonucleotide that forms a triple helix with the promoter of the RATH gene and inhibits transcription.  
     
     
         55 . A method for the treatment of immune disorders, comprising administering an effective amount of a compound that increases expression of RATH.  
     
     
         56 . A pharmaceutical formulation for the treatment of immune disorders, comprising a compound that activates or inhibits RATH, mixed with a pharmaceutically acceptable carrier.  
     
     
         57 . A method for identifying compounds that inhibit an interaction between RATH and calpactin, comprising the steps of: 
 (a) combining the peptide of  claim 28 , or bioreactive fragment thereof, a calpactin polypeptide and a test compound under conditions appropriate for formation of a peptide/calpactin polypeptide complex; and    (b) quantitating the amount of peptide/calpactin polypeptide complex and comparing the amount to the amount of peptide/calpactin polypeptide complex formed under the same conditions, but in the absence of the test compound, wherein a lesser amount of peptide/calpactin polypeptide complex formed in the presence of the test compound indicates that the test compound is an inhibitor of the interaction between a RATH protein and calpactin.