Methods for Identifying and Compounds Useful for Increasing the Functional Activity and Cell Surface Expression of CF-Associated Mutant Cystic Fibrosis Transmembrance Conductance Regulator

Abstract

The present invention relates to agents, and methods for identifying compounds, which agents and compounds result in the modulation of cellular trafficking of proteins in particular that of CF-associated mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In addition, the invention relates to compositions and methods for the use thereof in treating conditions that are characterized by an ER-associated protein misfolding and abnormal cellular trafficking of disease-associated proteins, including cystic fibrosis (CF).

1 . A method for identifying a compound that increases the functional activity of CF-associated mutant CFTR, comprising:
 (a) contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 45, 30, 32-44, and 46-55 and fragments thereof; and   (b) measuring a compound-polypeptide property related to CF-associated mutant CFTR activity.   
     
     
         2 . The method according to  claim 1 , wherein said polypeptide is in an in vitro cell-free preparation. 
     
     
         3 . The method according to  claim 1 , wherein said polypeptide is present in a mammalian cell. 
     
     
         4 . The method of  claim 2 , wherein said property is a binding affinity of said compound to said polypeptide. 
     
     
         5 . The method of  claim 1 , wherein the method is used to identify compounds that promote migration of ΔF508-CFTR to the plasma membrane. 
     
     
         6 . The method of  claim 4 , which additionally comprises the steps of:
 c) contacting a population of mammalian cells expressing said polypeptide with the compound that exhibits a binding affinity of at least 10 micromolar; and   d) identifying a compound that increases the functional activity of CF-associated mutant CFTR.   
     
     
         7 . The method of  claim 1 , wherein said property is increased activity of ΔF508-CFTR or CFTR. 
     
     
         8 . The method according to  claim 1 , wherein said property is the activity of said polypeptide. 
     
     
         9 . The method according to  claim 1 , wherein said property is the expression of said polypeptide. 
     
     
         10 . The method according to  claim 8 , which additionally comprises the steps of:
 c) contacting a population of mammalian cells expressing said polypeptide with the compound that significantly inhibits the expression or activity of the polypeptide; and   d) identifying the compound that increases the functional activity of CF-associated mutant CFTR.   
     
     
         11 . The method according to  claim 1 , which additionally comprises the step of comparing the compound to be tested to a control. 
     
     
         12 . The method according to  claim 11 , wherein said control is where the polypeptide has not been contacted with said compound. 
     
     
         13 . The method according to  claim 6 , which additionally comprises the step of comparing the compound to a control, wherein said control is a population of mammalian cells that does not express said polypeptide. 
     
     
         14 . The method according to  claim 1 , wherein said compound is selected from the group consisting of compounds of a commercially available screening library and compounds having binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 45, 30, 32-44, and 46-55. 
     
     
         15 . The method according to  claim 1 , wherein said compound is a peptide in a phage display library or an antibody fragment library. 
     
     
         16 . An agent effective in increasing the functional activity of CF-associated mutant CFTR, selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 19, 1, 3-18 and 20-29. 
     
     
         17 . The agent according to  claim 16 , wherein a vector in a mammalian cell expresses said agent. 
     
     
         18 . The agent according to  claim 16 , which increases the functional activity of CF-associated mutant CFTR. 
     
     
         19 . The agent according to  claim 17 , wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector. 
     
     
         20 . The agent according to  claim 16 , wherein said antisense polynucleotide and said siRNA comprise an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 19, 1, 3-18 and 20-29. 
     
     
         21 . The agent according to  claim 20 , wherein said siRNA further comprises said sense strand. 
     
     
         22 . The agent according to  claim 21 , wherein said sense strand is selected from the group consisting of SEQ ID NO: 56, 57, 81, 82, 58-80 and 83-99. 
     
     
         23 . The agent according to  claim 20 , wherein said siRNA further comprises a loop region connecting said sense and said antisense strand. 
     
     
         24 . The agent according to  claim 23 , wherein said loop region comprises a nucleic acid sequence selected from the group consisting of UUGCUAUA or GUUUGCUAUAAC (SEQ ID NO: 100). 
     
     
         25 . The agent according to  claim 16 , wherein said agent is an antisense polynucleotide, ribozyme, or siRNA comprising a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 56, 57, 81, 82, 58-80 and 83-99. 
     
     
         26 . A pharmaceutical composition comprising a therapeutically effective amount of an agent according to  claim 16  in admixture with a pharmaceutically acceptable carrier. 
     
     
         27 . A method for the treatment and/or prevention of a disease involving ER-associated protein misfolding in a subject suffering from or susceptible to the disease, comprising administering a therapeutically effective amount of a pharmaceutical composition according to  claim 26 . 
     
     
         28 . The method according to  claim 27  wherein the disease is selected from Cystic Fibrosis, Parkinson's disease, Gaucher's disease, nephrogenic diabetes insipidus, emphysema and liver disease (alpha-1-antitrypsin deficiency), Maple syrup urine disease, Fabry's disease, hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia, beta-galactosidosis, Wilson's disease, long QT syndrome, retinitis pigmentosa, transthyretin-linked amyloidosis, Alzheimer's disease, prion disease, and inclusion body myositis. 
     
     
         29 . The method according to  claim 28 , wherein the disease is Cystic Fibrosis. 
     
     
         30 . A method for the treatment and/or prevention of a disease involving ER-associated protein misfolding, or the treatment or prevention of a condition characterized by ER-associated protein misfolding, comprising administering a therapeutically effective amount of an agent according to  claim 16 . 
     
     
         31 . The method according to  claim 30 , wherein the disease is selected from the group consisting of Cystic Fibrosis, Parkinson's disease, Gaucher's disease, nephrogenic diabetes insipidus, emphysema and liver disease (alpha-1-antitrypsin deficiency), Maple syrup urine disease, Fabry's disease, hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia, beta-galactosidosis, Wilson's disease, long QT syndrome, retinitis pimentosa, transthyretin-linked amyloidosis, Alzheimer's disease, prion disease, and inclusion body myositis. 
     
     
         32 . The method according to  claim 30 , wherein the disease is Cystic Fibrosis. 
     
     
         33 . (canceled) 
     
     
         34 . A method for diagnosing a pathological condition in a subject involving ER-associated protein misfolding, or a pathological condition involving inflammation, comprising determining a first amount or activity of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 45, 30, 32-44, and 46-55 present in a biological sample obtained from said subject, and comparing said first amount or activity with the ranges of amounts or activities of the polypeptide determined in a population of healthy subjects, wherein an increase of the amount or activity of polypeptide in said biological sample compared to the range of amounts or activities determined for healthy subjects is indicative of the presence of the pathological condition. 
     
     
         35 . A method for the treatment and/or prevention of a disease involving inflammation in a subject suffering from or susceptible to the disease, comprising administering a therapeutically effective amount of a pharmaceutical composition according to  claim 26 . 
     
     
         36 . The method according to  claim 35  wherein the disease is selected from Cystic Fibrosis, COPD and asthma. 
     
     
         37 . A method for the treatment and/or prevention of a disease involving or characterized by inflammation, comprising administering a therapeutically effective amount of an agent according to  claim 16 . 
     
     
         38 . The method according to  claim 37 , wherein the disease is selected from the group consisting of Cystic Fibrosis, COPD and asthma. 
     
     
         39 . (canceled) 
     
     
         40 . (canceled)