Neurogenin 3 promoter

Abstract

The present invention features polypeptides having activity of human neurogenin3 (hNgn3), and nucleic acid encoding such polypeptide. The invention also features use of islet transcription factors such as hNgn3 to facilitate production of pancreatic islet cells from progenitor cells, and to facilitate insulin delivery by production of islet cells so produced.

What is claimed is:  
     
         1 . A recombinant polypeptide comprising: 
 a basic helix-loop-helix (bHLH) domain comprising an amino acid sequence that is at least 90% identical to a contiguous amino acid sequence of residues 82 to 138 of SEQ ID NO:2; and    an overall amino acid sequence identity of at least 85% to a contiguous amino acid sequence of SEQ ID NO:2;    wherein the recombinant polypeptide induces insulin gene transcription when expressed in a mammalian cell.    
     
     
         2 . The recombinant polypeptide of  claim 1 , where the polypeptide comprises an activation domain comprising amino acid residues 190-214 of SEQ ID NO:2.  
     
     
         3 . The recombinant polypeptide of  claim 1 , wherein the polypeptide does not include a contiguous amino acid sequence of residues 1-75 of SEQ ID NO:2.  
     
     
         4 . The recombinant polypeptide of  claim 1 , wherein the polypeptide does not include a contiguous amino acid sequence of residues 139-189 of SEQ ID NO:2.  
     
     
         5 . A recombinant polypeptide comprising an activation domain comprising an amino acid sequence that is at least 80% identical to amino acid residues 190-214 of SEQ ID NO:2, wherein the polyeptide induces gene transcription when expressed in a mammalian cell.  
     
     
         6 . The recombinant polypeptide of  claim 5 , further comprising a bHLH domain.  
     
     
         7 . The recombinant polypeptide of  claim 6 , wherein the bHLH domain is heterologous to the activation domain.  
     
     
         8 . The recombinant polypeptide of  claim 5 , wherein the recombinant polypeptide has an overall amino acid sequence identity of at least 85% to a contiguous amino acid sequence of SEQ ID NO:2.  
     
     
         9 . The recombinant polypeptide of  claim 8 , wherein the polypeptide does not include a contiguous amino acid sequence of residues 1-75 of SEQ ID NO:2.  
     
     
         10 . A recombinant polypeptide comprising: 
 a basic helix-loop-helix (bHLH) domain comprising an amino acid sequence that is at least 90% identical to a contiguous amino acid sequence of residues 82 to 138 of SEQ ID NO:2; and    an activation domain comprising an amino acid sequence that is at least 80% identical to amino acid residues 190-214 of SEQ ID NO:2, wherein the polypeptide induces gene transcription when expressed in a mammalian cell.    
     
     
         11 . An isolated polypeptide comprising at least 85% amino-acid sequence identity to the contiguous amino acid sequence of SEQ ID NO:2.  
     
     
         12 . An isolated polypeptide comprising an amino acid sequence that is at least 85% identical to a contiguous amino acid sequence of residues 76-215 of SEQ ID NO:2.  
     
     
         13 . A recombinant polynucleotide encoding the polypeptide of any of claims  1 - 12 .  
     
     
         14 . An isolated polynucleotide encoding a neurogenin3 promoter, wherein the polynucleotide comprises a nucleotide sequence having an overall sequence identity of at least 80% to: 
 a) a contiguous nucleotide sequence of nucleotides positioned at −105 to −158 of SEQ ID NO:1 relative to the transcription start site at nucleotide residue 2643 of SEQ ID NO:1;    b) a contiguous nucleotide sequence of nucleotides positioned at-3728 to −3653 of SEQ ID NO:1 relative to the transcription start site at nucleotide residue 2643 of SEQ ID NO:1;    c) a contiguous nucleotide sequence of nucleotides 2435 to 2643 of SEQ ID NO:1;    d) a contiguous nucleotide sequence extending about 2.6 kb 5′ of nucleotide residue 2643 of SEQ ID NO:1;    e) a contiguous nucleotide sequence extending about 5.7 kb 5′ of nucleotide residue 2643 of SEQ ID NO:1; or    f) a contiguous nucleotide sequence of the sequence extending about 700 bp 5′ of nucleotide residue 719 of SEQ ID NO:3.    
     
     
         15 . The isolated polynucleotide of  claim 14 , which is operably linked to a heterologous nucleic acid in a manner suitable for expression of gene product encoded by the heterologous nucleic acid.  
     
     
         16 . An expression construct comprising, from 5′ to 3′: 
 a polynucleotide comprising a 5′ flanking sequence of an Ngn3 gene, where the 5′ flanking sequence comprises the polynucleotide of  claim 15;   
 a heterologous nucleic acid encoding a gene product of interest; and  
 a polynucleotide comprising a 3′ flanking sequence of an Ngn3 gene;  
 wherein introduction of the expression construct into a suitable mammalian cell provides for expression of the heterologous nucleic acid in an expression pattern similar to that of Ngn3.  
 
     
     
         17 . The expression construct of  claim 16 , wherein the gene product is a reporter polypeptide.  
     
     
         18 . A transgenic, non-human animal containing an expression construct of  claim 16 .  
     
     
         19 . A method for producing a mammalian islet cell, the method comprising the steps of: 
 introducing into a mammalian pancreatic cell a nucleic acid molecule encoding an islet transcription factor, which islet transcription factor is a neuroendocrine bHLH transcription factor, said introducing providing for expression of the islet transcription factor in the cell and for production of the islet cell phenotype in the cell.    
     
     
         20 . The method of  claim 19 , wherein the neuroendocrine bHLH transcription factor is neurogenin3 (Ngn3), NeuroD1, Mash1, neurogenin1, neurogenin2, neuroD2, math2, NeuroD4/Math3, math1/ATOH 1, or mash2.  
     
     
         21 . The method of  claim 19 , wherein the bHLH transcription factor is a positive regulator of a neurogenin3 (Ngn3) regulatory pathway.  
     
     
         22 . The method of  claim 21 , wherein the positive regulator is Ngn3, Mash1, NeuroD1, HNF1, HNF2, or HNF6.  
     
     
         23 . The method of  claim 19 , wherein the mammalian pancreatic cell is a human pancreatic cell.  
     
     
         24 . The method of  claim 19 , wherein the islet cell phenotype is that of a pancreatic beta cell.  
     
     
         25 . A method for delivering insulin to the bloodstream of a mammalian subject, the method comprising: 
 introducing an islet cell produced by the method of  claim 1  into a pancreas of a mammalian subject, said introducing providing for production of insulin by the islet cell and delivery of insulin to the bloodstream of the mammalian subject.    
     
     
         26 . A method for producing islet cells in a mammalian subject, the method comprising: 
 administering a formulation to a pancreatic cell of a mammalian subject, the formulation comprising a nucleic acid molecule encoding and adapted for expression of an islet transcription factor, which islet transcription factor is a neuroendocrine bHLH transcription factor, said administering being in an amount sufficient for production of the islet transcription factor and production of islet cells in the pancreas of the subject.    
     
     
         27 . The method of  claim 26 , wherein the wherein the neuroendocrine bHLH transcription factor is neurogenin3 (Ngn3), NeuroD1, Mash1, neurogenin1, neurogenin2, neuroD2, math2, NeuroD4/Math3, math1/ATOH1 or mash2.  
     
     
         28 . A method of delivering insulin to the bloodstream of a mammalian subject, the method comprising: 
 administering a formulation to a pancreatic cell of a mammalian subject, the formulation comprising a nucleic acid molecule encoding an islet transcription factor, which islet transcription factor is a neuroendocrine bHLH transcription factor, said administering being in an amount sufficient for production of the islet transcription factor at a level sufficient to induce production of beta cells, wherein the islet cells produce insulin, which insulin is delivered to the bloodstream of the subject.    
     
     
         29 . The method of  claim 28 , wherein the wherein the neuroendocrine bHLH transcription factor is neurogenin3 (Ngn3), NeuroD1, Mash1, neurogenin1, neurogenin2, neuroD2, math2, NeuroD4/Math3, math1/ATOH1 or mash2.