eNOS mutations useful for gene therapy and therapeutic screening

Abstract

The present invention relates to new NOS variants or mutants which contain structural alterations in the site of Akt dependent phosphorylation. The altered NOS proteins or peptides, especially the human eNOS proteins or peptides, Akt proteins or polypeptides and their encoding nucleic acid molecules are useful as gene therapy agents for the treatment of diseases including post angioplasty restenosis, hypertension, atherosclerosis, heart failure, diabetes and diseases with defective angiogenesis. NOS proteins and peptides are also useful in methods of screening for agents which modulate NOS activity.

1 - 43  (Canceled)  
     
     
         44 . An isolated nucleic acid molecule encoding a nitric oxide synthetase (NOS) polypeptide selected from the group consisting of a constitutively active NOS polypeptide having an increased activity of NO production, a NOS polypeptide having an increased rate of NO production and a NOS polypeptide having an increased reductase activity when compared with the wild-type NOS polypeptide, wherein amino acid S/T of the consensus sequence motif RXRXXS/T is mutated.  
     
     
         45 . An isolated nucleic acid molecule comprising the sequence as set forth in SEQ ID NO: 8 or a variant thereof, wherein said nucleic acid molecule encodes a nitric oxide synthetase (NOS) polypeptide selected from the group consisting of a constitutively active NOS polypeptide having an increased activity of NO production, a NOS polypeptide having an increased rate of NO production and a NOS polypeptide having an increased reductase activity when compared with the wild-type NOS polypeptide, wherein amino acid S/T of the consensus sequence motif RXRXXS/T is mutated.  
     
     
         46 . The isolated nucleic acid molecule of  claim 45 , wherein the NOS polypeptide comprises a substituted amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 of human nNOS.  
     
     
         47 . The isolated nucleic acid molecule of  claim 46 , wherein the substituted amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 of human nNOS, mimics a phosphoserine.  
     
     
         48 . The isolated nucleic acid molecule of  claim 46 , wherein a serine corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 of human nNOS, has been substituted with an aspartic acid residue or a glutamic acid residue.  
     
     
         49 . The isolated nucleic acid molecule of  claim 46 , wherein the substituted amino acid comprises an R group that mimics a phosphate group.  
     
     
         50 . The isolated nucleic acid molecule of  claim 46 , wherein the substituted amino acid comprises a negatively charged R group.  
     
     
         51 . An isolated nucleic acid molecule encoding a human eNOS polypeptide as set forth in SEQ ID NO. 3, wherein the eNOS polypeptide is selected from the group consisting of a constitutively active NOS polypeptide having an increased activity of NO production, a NOS polypeptide having an increased rate of NO production and a NOS polypeptide having an increased reductase activity when compared with the wild-type NOS polypeptide, wherein amino acid S/T of the consensus sequence motif RXRXXS/T is mutated.  
     
     
         52 . An isolated nucleic acid molecule encoding a human eNOS polypeptide which comprises a substituted amino acid residue corresponding to residue 1177 of human eNOS as set forth in SEQ ID NO. 5.  
     
     
         53 . The isolated nucleic acid molecule of  claim 52 , wherein the substituted amino acid is an aspartic acid residue as set forth in SEQ ID NO. 6 or a glutamic acid residue as set forth in SEQ ID NO. 7.