Treatment of diabetes with synthetic beta cells

Abstract

Disclosed is a method for obtaining glucose-regulated expression of active insulin in the cells of a mammalian subject. The method involves delivering into the subject a genetic construct comprising a coding sequence for a human proinsulin operably connected a promoter functional in the host cells. The construct includes a glucose responsive regulatory module having at least one glucose inducible regulatory element comprising a pair of CACGTG motifs linked by a five base nucleotide sequence, which confers glucose inducible expression of the proinsulin coding sequence. To ensure proper processing of the proinsulin to active insulin, the coding sequence was modified to direct the synthesis of a mutant proinsulin polypeptide having amino acid sequences that can be cleaved to mature insulin in suitable host cells, such as hepatocytes.

1 . A genetic construct for glucose-regulated synthesis of active insulin in a host cell in which insulin is not natively produced, comprising 
 a promoter functional in the host cell;    a coding sequence for a human proinsulin, the coding sequence operably connected to the promoter, wherein the protein encoded by the coding sequence comprises amino acid sequences corresponding to B-C and C-A junctions of native human proinsulin that are cleavable in the host cell;    a glucose responsive regulatory module located 5′ of the promoter, the module having at least one glucose inducible regulatory element comprising a pair of CACGTG motifs linked by a five base nucleotide sequence; and    a 5′ untranslated region not natively associated with the coding sequence for human proinsulin, wherein the 5′ untranslated region is located 5′ of the coding sequence and 3′ of the promoter.    
     
     
         2 . The genetic construct of  claim 1 , wherein the 5′ untranslated region is characterized by reduced formation of secondary structures by intramolecular base pairing, relative to the formation of secondary structures by the 5′ untranslated region natively associated with the coding sequence for human insulin.  
     
     
         3 . The genetic construct of  claim 1 , wherein the regulatory module comprises at least two glucose inducible regulatory element.  
     
     
         4 . The genetic construct of  claim 1 , wherein the proinsulin encoded by the coding sequence comprises a modified C-A junction and a modified B-C junction, the modified C-A junction corresponding to a conversion of the amino acid sequence LQKR to RQKR and the modified B-C junction corresponding to the conversion of the amino acid sequence KTRR to RTKR  
     
     
         5 . A host cell comprising the genetic construct of  claim 1 .  
     
     
         6 . The genetic construct of  claim 1 , wherein the module comprises a sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.  
     
     
         7 . A virus infective for a host cell, the virus comprising 
 a promoter functional in the host cell;    a coding sequence for a human proinsulin, the coding sequence operably connected to the promoter, wherein the protein encoded by the coding sequence comprises amino acid sequences corresponding to the B-C and C-A junctions of native human proinsulin that are cleavable in the host;    a glucose responsive regulatory module located 5′ of said promoter, the module having at least one glucose inducible regulatory element comprising a pair of CACGTG motifs linked by a five base nucleotide sequence; and    a 5′ untranslated region not natively associated with the coding sequence for human proinsulin, wherein the 5′ untranslated region is located 5′ of the coding sequence and 3′ of the promoter.    
     
     
         8 . The virus of  claim 7 , wherein the host cell is a hepatocyte.  
     
     
         9 . A method for obtaining glucose regulated expression of insulin in a suitable host cell, comprising 
 delivering into the cell a genetic construct cording to  claim 1 .    
     
     
         10 . The method of  claim 9 , wherein the host cell is a hepatocyte.  
     
     
         11 . The method of  claim 9 , wherein the genetic construct is delivered by exposing the cells to a virus infective for the cells, the virus comprising the genetic construct, under suitable conditions and at a multiplicity of infection sufficient to allow at least a portion of the cells to be infected by virus.  
     
     
         12 . A method for obtaining glucose-regulated expression of active insulin in a mammalian subject, comprising the step of delivering into the subject a genetic construct according to  claim 1  in an amount and under conditions effective to allow at least a portion the subject's cells to take up the construct.  
     
     
         13 . The method of  claim 12 , wherein the cells are hepatocytes.  
     
     
         14 . The method of  claim 12 , wherein the delivering step comprises exposing the subject to a virus infective for the subject, the virus comprising the genetic construct, under conditions that allow at least a portion of the subject's cells to be infected with the virus.  
     
     
         15 . The method of  claim 12 , wherein the subject is diabetic.  
     
     
         16 . The method of  claim 12 , wherein glucose-induced expression of insulin correlates with a reduction in blood glucose levels, relative to an similar control organism that does not receive the genetic construct.  
     
     
         17 . A method for regulating blood glucose levels in a mammalian subject in need thereof, comprising the step of delivering into the subject a genetic construct according to  claim 1  in an amount and under conditions effective to allow at least a portion the subject's cells to take up the construct.  
     
     
         18 . A genetic construct comprising a promoter capable of being recognized by an RNA polymerase of a host cell; a nucleotide sequence, operably connected to the promoter, for a preproinsulin cDNA encoding a preproinsulin cleavable into active insulin; a glucose responsive regulatory module located 5′ of the promoter, the module having at least two glucose inducible regulatory elements, each element comprising a pair of CACGTG motifs linked by a five base nucleotide sequence; and a 5′ untranslated region not natively associated with the coding sequence for human proinsulin, wherein the 5′ untranslated region is located 5′ of the coding sequence and 3′ of the promoter.  
     
     
         19 . The genetic construct of  claim 18  wherein the glucose responsive regulatory module has at least three glucose inducible regulatory elements.