Modified Nucleotide Sequence Encoding Glucagon-Like Peptide-1 (GLP-1), Nucleic Acid Construct Comprising Same for Production of Glucagon-Like Peptide-1 (GLP-1), Human Cells Comprising Said Construct and Insulin-Producing Constructs, and Method of Use Thereof

Abstract

An isolated chimeric GLP-1 nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1), and a furin cleavable site between the human pro-insulin leader sequence and the GLP-1 is provided. Also provided is an isolated modified chimeric GLP-1 nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1), and a furin cleavable site between the human pro-insulin leader sequence and the GLP-1. Recombinant expression vectors comprising the chimeric GLP-1 nucleic acid sequences, which produce GLP-1 constitutively are provided, as are human cells transfected with such an expression vector in combination with an expression vector comprising a proinsulin nucleic acid sequence and an expression vector comprising a furin and a glucose-regulatable TGF-alpha promoter. Methods of producing human GLP-1 constitutively are provided as are method of producing GLP-1 and insulin or in a glucose-dependent manner using such transfected cells. Methods of treating a subject having Type II diabetes and methods of treating a subject prone to hyperglycemia or suffering from hyperglycemia are provided in which transfected cells produce human GLP-1 and insulin in a glucose-dependent manner. Also provided are methods of reducing weight in a subject by implanting into the subject transfected cells which produce human GLP-1 and insulin in a glucose-dependent manner.

1 - 3 . (canceled) 
     
     
         4 . An isolated modified nucleic acid encoding GLP-1 human GLP-1(7-37) whose sequence is shown in SEQ ID NO:11 or encoding GLP-1(7-36), wherein SEQ ID NO:11 lacks the codon GGA encoding the final glycine residue (G). 
     
     
         5 - 6 . (canceled) 
     
     
         7 . A recombinant expression vector comprising an isolated modified chimeric nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1)(7-37), a furin cleavable site between the human pro-insulin leader and the GLP-1(7-37), and a constitutive promoter which drives the expression of the nucleic acid sequence encoding GLP-1 constitutively, wherein the modified chimeric nucleic acid sequence is shown in SEQ ID NO:8. 
     
     
         8 . A human cell stably transfected with (a) the recombinant expression vector of  claim 7 , (b) a second expression vector comprising an isolated nucleic acid sequence encoding human furin and a glucose-regulatable TGF-alpha promoter, wherein said promoter drives expression of the nucleotide sequence encoding human furin in a glucose-regulated manner, and (c) a third expression vector comprising an isolated nucleic acid sequence encoding proinsulin, a furin cleavable site between the human pro-insulin leader sequence and insulin sequence and a constitutive promoter which drives the expression of the nucleic acid sequence encoding proinsulin constitutively. 
     
     
         9 . (canceled) 
     
     
         10 . A human cell stably transfected with (a) a recombinant expression vector comprising a constitutive promoter which drives the expression of the nucleic acid sequence of SEQ ID NO: 20 encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1)(7-37), and a furin cleavable site between the human pro-insulin leader and the (GLP-1)(7-37), (b) a second expression vector comprising an isolated nucleic acid sequence encoding human furin and a glucose-regulatable TGF-alpha promoter, wherein said promoter drives expression of the nucleic acid sequence encoding human furin in a glucose-regulated manner, and (c) a third expression vector comprising an isolated nucleic acid sequence encoding proinsulin, wherein the proinsulin has a proinsulin leader sequence and an insulin sequence, and a furin cleavable site between the human pro-insulin leader sequence and the insulin sequence; and a constitutive promoter which drives the expression of the nucleic acid sequence encoding proinsulin. 
     
     
         11 - 13 . (canceled) 
     
     
         14 . A method of producing human GLP-1 and insulin in a glucose-dependent manner in an isolated human cell, said method comprising:
 (a) providing a stably transfected human cell of  claim 10 ; and   (b) stimulating said cell with a glucose, wherein the concentration of the glucose is higher than an ambient concentration of glucose.   
     
     
         15 . A method of treating a subject having Type II diabetes, said method comprising:
 implanting into the subject an immunoisolatory device, wherein said device comprises isolated human cells of  claim 10 ;   wherein the isolated stably transfected human cells express a therapeutically effective amount of GLP-1 and insulin in a glucose-dependent manner GLP-1 upon stimulation with a concentration of glucose in the blood of the subject, wherein the stimulating concentration of glucose is higher than an ambient concentration of glucose.   
     
     
         16 . A method of treating a subject prone to hyperglycemia or suffering from hyperglycemia, said method comprising:
 implanting into the subject an immunoisolatory device, wherein said device comprises isolated human cells of  claim 10 ;   wherein the isolated stably transfected human cells express a therapeutically effective amount of GLP-1 and insulin in a glucose-dependent manner GLP-1 upon stimulation with a concentration of glucose in the blood of the subject, wherein the stimulating concentration of glucose is higher than an ambient concentration of glucose, thereby reducing glucose blood level.   
     
     
         17 . A method of reducing weight in a subject, said method comprising:
 implanting into the subject an immunoisolatory device, wherein said device comprises isolated human cells of  claim 10 ;   wherein the isolated stably transfected cells express a therapeutically effective amount of GLP-1 and insulin in a glucose-dependent manner upon stimulation with a concentration of glucose in the blood of the subject, wherein the stimulating concentration of glucose is higher than an ambient concentration of glucose, wherein the GLP-1 and insulin expression reduces the weight of the subject.   
     
     
         18 - 20 . (canceled)