Nucleic acid constructs useful for glucose regulated production of human insulin in somatic cell lines

Abstract

Unique multifunctional nucleotide expression cassettes comprising a nucleotide sequence encoding a human protease driven by a glucose-regulatable promoter, a nucleotide sequence encoding human proinsulin having sites at its B-C and C-A junctions altered to permit cleavage by the human protease, and a CMV promoter driving the transcription of the nucleotide sequence encoding the human proinsulin, such cassettes optionally having polyadenylation sequences after each structural gene and the nucleotide sequence encoding human proinsulin optionally carrying an H10D variant, and cells transformed thereby.

1  to  19 . (canceled)  
     
     
         20 . A non-LTR containing recombinant vector for transforming cells to produce mature human insulin, said recombinant vector comprising: 
 a promoter;    a nucleotide sequence encoding human proinsulin having furin cleavable sites at its B-C and C-A junctions, transcription of said human proinsulin nucleotide sequence being driven by said promoter; and    a polyadenylation sequence linked 3′ to said nucleotide sequence encoding human proinsulin.    
     
     
         21 . The recombinant vector of  claim 20  wherein said promoter is selected from the group consisting of: cytomegalovirus (CMV), phosphoglycerate kinase (PGK), and elongation factor 1 (EF1).  
     
     
         22 . The recombinant vector of  claim 21  wherein said nucleotide sequence encoding human proinsulin encodes a human proinsulin selected from the group consisting of: wild-type human proinsulin and human proinsulin having an H10D variant as compared to wild-type human proinsulin.  
     
     
         23 . The recombinant vector of  claim 21  further comprising: 
 a glucose-regulatable promoter;    a nucleotide sequence encoding human furin, said human furin nucleotide sequence being driven by said glucose-regulatable promoter; and    a polyadenylation sequence linked 3′ to said nucleotide sequence encoding human furin.    
     
     
         24 . The recombinant vector of  claim 23  wherein the glucose-regulatable promoter is a TGF-α promoter.  
     
     
         25 . The recombinant vector of  claim 23  further comprising: 
 a constitutive promoter; and    a selectable marker gene operatively linked to said constitutive promoter.    
     
     
         26 . The recombinant vector of  claim 25  wherein said selectable marker gene is neomycin phosphotransferase.  
     
     
         27 . The recombinant vector of  claim 25  wherein said constitutive promoter is the SV40 promoter.  
     
     
         28 . The recombinant vector of  claim 25  further comprising a polyadenylation sequence linked 3′ to said nucleotide sequence encoding neomycin phosphotransferase.  
     
     
         29 . An expression system for inducing insulin production in a cell, said system comprising: 
 (a) a first expression cassette comprising: 
 1. a CMV promoter;  
 2. a nucleotide sequence encoding human proinsulin having furin cleavable sites at its B-C and C-A junctions, transcription of said human proinsulin nucleotide sequence being driven by said CMV promoter; and  
 3. a polyadenylation sequence 3′ to said nucleotide sequence encoding human proinsulin.  
   (b) a second expression cassette comprising: 
 1. a glucose-regulatable promoter; and  
 2. a nucleotide sequence encoding human furin, said human furin nucleotide sequence being driven by said glucose-regulatable promoter; and  
 3. a polyadenylation sequence 3′ to said nucleotide sequence encoding human furin.  
   (c) a third expression cassette comprising: 
 (1) a nucleotide sequence encoding a selectable marker gene;  
 (2) a constitutive promoter of said selectable marker gene operably linked to said selectable marker gene; and  
 (3) a polyadenylated sequence 3′ to said nucleotide sequence encoding said selectable marker gene.  
   
     
     
         30 . The expression system of  claim 29  wherein said nucleotide sequence encoding human proinsulin of said first expression cassette encodes a human proinsulin selected from the group consisting of: wild-type human proinsulin and human proinsulin having an H10D variant as compared to wild-type human pro insulin.  
     
     
         31 . The expression system of  claim 29  wherein said first and second expression cassettes are linked.  
     
     
         32 . The expression system of  claim 29  wherein said first and third expression cassettes are linked.  
     
     
         33 . The expression system of  claim 29  wherein said second and third expression cassettes are linked.  
     
     
         34 . The expression system of  claim 29  wherein said first, second and third expression cassettes are linked.  
     
     
         35 . The express system of  claim 29  wherein said first, second and third expression cassettes are packaged in separate transformation vectors.  
     
     
         36 . The expression system of  claim 29  wherein said glucose-regulatable promoter of said second expression cassette is the TGF-α promoter.  
     
     
         37 . The expression system of  claim 29  wherein said selectable marker gene of said third expression cassette is a nucleotide encoding for neomycin phosphotransferase.  
     
     
         38 . The expression system of  claim 29  wherein said constitutive promoter of said selectable marker gene of said third expression cassette is the SV40 promoter.  
     
     
         39  to  41 . (canceled)  
     
     
         42 . A cell transformed to produce insulin by way of the non-LTR containing recombinant vector of  claim 20 .  
     
     
         43 . A cell transformed to produce insulin by way of the non-LTR containing recombinant vector of  claim 25 .  
     
     
         44 . A cell transformed to produce insulin using the expression system of  claim 29 .  
     
     
         45 . An engineered cell comprising a gene encoding for human proinsulin having furin cleavable sites at its B-C and C-A junctions and a gene encoding for human furin, wherein both genes are recombinant genes and the cell secretes insulin in response to glucose when said genes have been introduced into the cell by means of a recombinant vector.  
     
     
         46 . The engineered cell of  claim 45  wherein the human proinsulin gene is cDNA.  
     
     
         47 . The engineered cell of  claim 45  wherein the recombinant human furin gene is cDNA.  
     
     
         48  canceled.  
     
     
         49 . A cell derived from said engineered cell of  claim 47 .  
     
     
         50 . A method for treating diabetes mellitus, said method comprising the step of administering to a patient in need thereof, genetically engineered cells in an immunoisolatory device, where the said cells are engineered by introducing a first DNA segment comprising a structural gene encoding for human proinsulin having furin cleavable sites at its B-C and C-A junctions and a constitutive promoter operably linked to said proinsulin structural gene, and a second DNA segment comprising a structural gene encoding for human furin and a glucose-regulatable promoter operably linked to said furin structural gene, wherein said first and second DNA segments are effective for the expression of a therapeutically effective amount of insulin in said patient.  
     
     
         51 . The method of  claim 50  wherein said DNA segments are an integral part of one or more vectors.  
     
     
         52 . The method of  claim 50  wherein said DNA segments are introduced to said patients in cells, said cells having been treated in vitro to incorporate said DNA segments.  
     
     
         53 . The method of  claim 52  wherein said cells are introduced into the patient by way of an immuno-privileged cell factory.  
     
     
         54 . The method of  claim 52  wherein said cells are directly injected into a tissue of said patient.  
     
     
         55 . The method of  claim 52  wherein said cells are introduced to said patient in a neo-organ.  
     
     
         56 . A method for treating an individual with diabetes mellitus, said method comprising the steps of: 
 (a) introducing a nucleic acid encoding a human proinsulin gene having furin cleavable sites at its B-C and C-A junctions and a constitutive promoter operably linked to said human proinsulin gene, and a nucleic acid encoding for human furin and a glucose-regulatable promoter operably linked to said human furin gene, into a cell;    (b) transplanting the cell of step (a) into the individual so as to ameliorate the individual's diabetes mellitus.    
     
     
         57 . A method for providing glucose-responsive insulin secreting capability to a mammal comprising implanting into a mammal in need of insulin secreting capability a therapeutically effective amount of a cell population positioned in a selectively permeable membrane, said cell population comprising engineered cells that secrete insulin in response to glucose, the engineered cells comprising a nucleic acid encoding a human proinsulin gene having furin cleavable sites at its B-C and C-A junctions, a constitutive promoter operably linked to said human proinsulin gene, a nucleic acid encoding for human furin, and a glucose-regulatable promoter operably linked to said human furin gene.  
     
     
         58 . A method for producing human insulin, the method comprising the steps of: 
 (a) culturing a cell that secretes human insulin, the cell comprising a gene encoding human proinsulin having furin cleavable sites at its B-C and C-A junctions and a gene encoding human furin, wherein at least both genes are recombinant genes introduced into the cell by means of a recombinant vector;    (b) stimulating said cell to secrete insulin;    (c) collecting the secreted insulin.    
     
     
         59 . A method for producing human insulin, the method comprising the steps of: 
 (a) obtaining a cell that secretes human insulin in response to glucose, the cell comprising a gene encoding human proinsulin having furin cleavable sites at its B-C and C-A junctions and a gene encoding human furin, wherein at least both genes are recombinant genes introduced into the cell by means of a recombinant vector;    (b) growing the cell of step (a) in culture medium;    (c) contacting said cell with a glucose-containing buffer for a period of time sufficient to allow said cell to respond;    (d) collecting the medium that has been in contact with said cell; and    (e) purifying human insulin from the collected media.