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 - 9 . (canceled) 
     
     
         10 . A method for stimulating coronary collateral vessel development in a patient, comprising the step of delivering a transgene coding for a NOS polypeptide or an Akt polypeptide in a manner effective to promote coronary collateral vessel development wherein said NOS polypeptide is selected from the group consisting of a constitutively active NOS polypeptide, a NOS polypeptide with increased rate of NO production, and a NOS polypeptide with increased reductase activity. 
     
     
         11 . The method of  claim 10 , wherein the transgene is expressed from a ventricular myocyte-specific promoter, smooth muscle cell specific promoter, or endothelial cell specific promoter. 
     
     
         12 . The method according to  claim 11 , wherein the ventricular myocyte-specific promoter has the sequences of ventricular myosin light chain-2 or myosin heavy chain promoter. 
     
     
         13 . A method for stimulating vessel development in a patient having peripheral-deficient vascular disease, comprising the step of delivering a transgene coding for a NOS peptide or an Akt polypeptide to the peripheral vascular system of the patient in a manner effective to promote peripheral vascular development, wherein said NOS polypeptide is selected from the group consisting of a constitutively active NOS polypeptide, a NOS of polypeptide with increased rate of NO production, and a NOS polypeptide with increased reductase activity. 
     
     
         14 . A method for treating myocardial ischemia, comprising the step of delivering a transgene coding for a NOS polypeptide or an Akt polypeptide to the myocardium in a manner effective to treat myocardial ischemia, wherein said NOS polypeptide is selected from the group consisting of a constitutively active NOS polypeptide, a NOS polypeptide with increased rate of NO production, and a NOS polypeptide with increased reductase activity. 
     
     
         15 . The method of  claim 14 , wherein expression of the transgene is limited to cardiac myocytes. 
     
     
         16 . The method of  claim 15 , wherein expression of the transgene is limited to ventricular cardiac myocytes. 
     
     
         17 . The method of  claim 10 , wherein the transgene is encoded by a replication-defective vector. 
     
     
         18 . The method of  claim 17 , wherein the replication-defective vector is an adenovirus. 
     
     
         19 . The method of  claim 10 , wherein the transgene is transfected into a cell in vitro. 
     
     
         20 . The method of  claim 19 , wherein the transfected cells are delivered to the patient. 
     
     
         21 . A non-human transgenic animal which express a NOS polypeptide, wherein said NOS polypeptide is selected from the group consisting of a constitutively active NOS polypeptide, a NOS polypeptide with increased rate of NO production, and a NOS polypeptide with increased reductase activity. 
     
     
         22 . A method of identifying an agent which modulates the Akt regulated activity of NOS, comprising the steps of: (a) exposing a cell which expresses NOS to an agent; and (b) measuring the Akt regulated activity of NOS, thereby identifying an agent which modulates the Akt regulated activity of NOS. 
     
     
         23 . The method of  claim 22 , wherein step (b) comprises the determination of the phosphorylation state of NOS at an amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 at human nNOS 
     
     
         24 . The method of  claim 23 , wherein NOS is activated by the agent. 
     
     
         25 . The method of  claim 24 , wherein the agent mimics Akt mediated phosphorylation of eNOS. 
     
     
         26 . The method of  claim 25 , wherein the agent inhibits the dephosphorylation of an amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 at human nNOS 
     
     
         27 . The method of  claim 22  wherein step (b) comprises the measurement of NO production. 
     
     
         28 . The method of  claim 27 , wherein NO is detected by measuring nitrite concentrations or the conversion of  3 H-L-arginine to  3 H-L-citrulline. 
     
     
         29 . An in vitro method of identifying an agent which modulates Akt regulated activity of eNOS, comprising the steps of: (a) exposing Akt and NOS protein or peptide to the agent; and (b) measuring the activity of NOS. 
     
     
         30 . The method of  claim 29 , wherein NOS activity is measured by determining the Akt dependent phosphorylation state of NOS. 
     
     
         31 . The method of  claim 30 , wherein the NOS peptide comprises SEQ ID NO:2. 
     
     
         32 . The method of  claim 30 , wherein in step (b) the activity of NOS is reductase activity. 
     
     
         33 . The method of  claim 30 , wherein in step (b), the activity of NOS is production of NO by NOS. 
     
     
         34 - 37 . (canceled) 
     
     
         38 . The method of  claim 11 , wherein the endothelial cell specific promoter is Tie-2 promoter, endothelin promoter, or eNOS promoter. 
     
     
         39 . The method of  claim 11 , wherein the smooth muscle cell promoter is SM22 promoter or SM alpha actin promoter. 
     
     
         40 . A method for treating a cardiovascular disease in a patient, comprising the step of delivering a transgene coding for a NOS polypeptide or an Akt polypeptide to the patient, wherein said NOS polypeptide is selected from the group consisting of a constitutively active NOS polypeptide, a NOS polypeptide with increased rate of NO production, and a NOS polypeptide with increased reductase activity. 
     
     
         41 . The method of  claim 40 , wherein the cardiovascular disease is selected from the group consisting of heart failure, myocardial infarction, restenosis, post-angioplasty stenosis, stent stenosis, and by-pass graft failure. 
     
     
         42 . A method for treating erectile dysfunction in a patient, comprising the step of delivering a transgene coding for a NOS polypeptide or an Akt polypeptide to the patient, wherein said NOS polypeptide is selected from the group consisting of a constitutively active NOS polypeptide, a NOS polypeptide with increased rate of NO production, and a NOS polypeptide with increased reductase activity. 
     
     
         43 . (canceled)