Compositions and methods for regulated protein expression in gut

Abstract

The invention provides compositions and methods useful for treating disorders treatable by producing a protein in a regulatable manner in a mucosal cell or tissue of an animal. The treatment methods include in vivo and ex vivo methods, including transplanting in vitro transformed cells that secrete the protein into a mammalian subject.

1 . A method of generating a mucosal cell that produces a protein in response to a nutrient, comprising: 
 (a) contacting a mucosal cell with a polynucleotide comprising an expression control element in operable linkage with a nucleic acid encoding a protein under conditions allowing transformation of the cell; and    (b) identifying a cell transformant that produces the protein in a nutrient-regulatable manner, thereby generating a mucosal cell that produces a protein in response to a nutrient.    
     
     
         2 . An isolated or cultured mucosal cell that produces a protein regulatable by a nutrient, wherein expression of the protein is conferred by a transgene comprising an expression control element in operable linkage with a nucleic acid encoding the protein.  
     
     
         3 . The mucosal cell of  claim 2 , wherein the nutrient increases expression or secretion of the protein.  
     
     
         4 . The mucosal cell of  claim 2 , wherein the nutrient comprises a sugar, a fat, a carbohydrate or starch, an amino acid or polypeptide, a triglyceride, a vitamin, a mineral, or cellulose.  
     
     
         5 . The mucosal cell of  claim 2 , wherein the expression control element comprises a nutrient-regulatable element.  
     
     
         6 . The mucosal cell of  claim 5 , wherein the nutrient-regulatable element comprises a gut endocrine promoter.  
     
     
         7 . The mucosal cell of  claim 6 , wherein the gut endocrine promoter comprises a glucose-dependent insulinotropic polypeptide (GIP) promoter.  
     
     
         8 . The mucosal cell of  claim 2 , wherein the nucleic acid encodes insulin.  
     
     
         9 . The mucosal cell of  claim 2 , wherein the nucleic acid encodes leptin, GLP-1, GLP-2, cholecystokinin, a glucagon antagonist, a growth hormone, a clotting factor, or an antibody.  
     
     
         10 . The mucosal cell of  claim 2 , wherein the mucosal cell is obtained from a subject.  
     
     
         11 . The mucosal cell of  claim 11 , wherein the subject is human.  
     
     
         12 . The mucosal cell of  claim 2 , wherein the mucosal cell is obtained from a tissue or organ of the gastrointestinal tract or derived from a cell line of gut origin.  
     
     
         13 . The mucosal cell of  claim 12 , wherein the tissue is the stomach.  
     
     
         14 . The mucosal cell of  claim 12 , wherein the tissue is the duodenum.  
     
     
         15 . The mucosal cell of  claim 2 , wherein the mucosal cell is an endocrine cell.  
     
     
         16 . The mucosal cell of  claim 15 , wherein the endocrine cell is a K-cell.  
     
     
         17 . The mucosal cell of  claim 2 , wherein the mucosal cell is a stem cell.  
     
     
         18 . The mucosal cell of  claim 2 , wherein the mucosal cell is a non-endocrine cell.  
     
     
         19 . The mucosal cell of  claim 2 , wherein the expression control element in operable linkage with a nucleic acid further comprises a vector.  
     
     
         20 . The mucosal cell of  claim 19 , wherein the vector comprises a viral vector.  
     
     
         21 . A method of treating a subject having, or at risk of having, a disorder treatable by producing a protein in a tissue, comprising implanting one or more mucosal cells of  claim 2  into the tissue in an amount effective for treating the disorder.  
     
     
         22 . The method of  claim 21 , wherein the disorder comprises a hyperglycemic condition.  
     
     
         23 . The method of  claim 22 , wherein the hyperglycemic condition comprises diabetes.  
     
     
         24 . The method of  claim 21 , where the subject has a fasting plasma glucose level greater than 110 mg/dl.  
     
     
         25 . The method of  claim 21 , wherein the disorder comprises obesity or an undesirable body mass.  
     
     
         26 . The method of  claim 21 , wherein the mucosal cell expresses insulin.  
     
     
         27 . The method of  claim 21 , wherein the mucosal cell expresses leptin, GLP-1, GLP-2, cholecystokinin, a glucagon antagonist, a growth hormone, a clotting factor, or an antibody.  
     
     
         28 . The method of  claim 21 , wherein the tissue is a mucosal tissue.  
     
     
         29 . The method of  claim 21 , wherein the tissue is a non-mucosal tissue.  
     
     
         30 . The method of  claim 29 , wherein the non-mucosal tissue is liver, pancreas or muscle.  
     
     
         31 . A method of treating a subject having, or at risk of having, a disorder treatable by producing a therapeutic protein in a mucosal tissue, comprising contacting mucosal tissue cells in the subject transformed with a polynucleotide comprising an expression control element in operable linkage with a nucleic acid encoding the therapeutic protein with a nutrient that induces production of the protein in an amount effective to treat the disorder.  
     
     
         32 . The method of  claim 31 , wherein the disorder comprises a hyperglycemic condition.  
     
     
         33 . The method of  claim 32 , wherein the hyperglycemic condition comprises diabetes.  
     
     
         34 . The method of  claim 33 , wherein the diabetes comprises type I diabetes.  
     
     
         35 . The method of  claim 31 , wherein the subject has a fasting plasma glucose level greater than 110 mg/dl.  
     
     
         36 . The method of  claim 33 , wherein the diabetes comprises insulin-dependent diabetes.  
     
     
         37 . The method of  claim 31 , wherein the disorder comprises obesity or an undesirable body mass.  
     
     
         38 . The method of claims  1  or  31 , wherein the nutrient increases expression or secretion of the protein.  
     
     
         39 . The method of  claim 38 , wherein expression of the protein is increased in non-endocrine cells.  
     
     
         40 . The method of  claim 38 , wherein secretion of the protein is increased in endocrine cells.  
     
     
         41 . The method of claims  1  or  31 , wherein the nutrient comprises a sugar, a fat, a carbohydrate or starch, an amino acid or polypeptide, a triglyceride, a vitamin, a mineral, or cellulose.  
     
     
         42 . The method of claims  1  or  31 , wherein the expression control element comprises a nutrient-regulatable element.  
     
     
         43 . The method of  claim 42 , wherein the nutrient-regulatable element comprises a gut endocrine promoter, a functional variant thereof, or a functional subsequence thereof.  
     
     
         44 . The method of  claim 43 , wherein the gut endocrine promoter comprises a glucose-dependent insulinotropic polypeptide (GIP) promoter.  
     
     
         45 . The method of claims  1  or  31 , wherein the nucleic acid encodes insulin.  
     
     
         46 . The method of claims  1  or  31 , wherein the nucleic acid encodes leptin, GLP-1, GLP-2, cholecystokinin, a growth hormone, a clotting factor, or an antibody.  
     
     
         47 . The method of  claim 31 , wherein the mucosal cell is present in a tissue or organ of the gastrointestinal tract of a subject.  
     
     
         48 . The method of  claim 47 , wherein the tissue is the intestine.  
     
     
         49 . The method of  claim 47 , wherein the tissue is the gut.  
     
     
         50 . The method of  claim 31 , wherein the mucosal cell is an endocrine cell.  
     
     
         51 . The method of  claim 50 , wherein the endocrine cell is a K-cell.  
     
     
         52 . The method of  claim 50 , wherein the mucosal cell is a stem cell.  
     
     
         53 . The method of  claim 31 , wherein the mucosal cell is a non-endocrine cell.  
     
     
         54 . The method of claims  1  or  31 , wherein the expression control element in operable linkage with a nucleic acid further comprises a vector.  
     
     
         55 . The method of  claim 54 , wherein the vector comprises a viral vector.  
     
     
         56 . A non-human transgenic animal that produces insulin in a mucosal tissue, insulin production not naturally occurring in the mucosal tissue of the animal, insulin production conferred by a transgene present in mucosal tissue cells, wherein the transgene comprises a polynucleotide including an expression control element in operable linkage with a nucleic acid encoding insulin, and wherein production of the insulin in the mucosal tissue of the animal is responsive to the nutrient.  
     
     
         57 . The transgenic animal of  claim 56 , wherein the animal is a mouse.  
     
     
         58 . The transgenic animal of  claim 56 , wherein the expression control element comprises a nutrient-regulatable element.  
     
     
         59 . The transgenic animal of  claim 56 , wherein the nutrient-regulatable element comprises a glucose-inducible promoter, a functional variant thereof, or a functional subsequence thereof.  
     
     
         60 . The transgenic animal of  claim 59 , wherein the glucose-inducible promoter comprises a glucose-dependent insulinotropic polypeptide (GIP) promoter.  
     
     
         61 . The transgenic animal of  claim 56 , wherein the nucleic acid encoding insulin encodes a functional subsequence of insulin.  
     
     
         62 . The transgenic animal of  claim 56 , wherein the mucosal tissue is a tissue or organ of the gut.  
     
     
         63 . The transgenic animal of  claim 61 , wherein the mucosal tissue is the stomach.  
     
     
         64 . The transgenic animal of  claim 61 , wherein the mucosal tissue is the duodenum.  
     
     
         65 . The transgenic animal of  claim 57 , wherein the mucosal tissue includes endocrine cells.  
     
     
         66 . The transgenic animal of  claim 65 , wherein the endocrine cell is a K cell.  
     
     
         67 . The transgenic animal of  claim 65 , wherein the mucosal cell is a stem cell.  
     
     
         68 . The transgenic animal of  claim 56 , wherein the animal is resistant to developing a hyperglycemic condition.  
     
     
         69 . The transgenic animal of  claim 68 , wherein the hyperglycemic condition comprises diabetes.  
     
     
         70 . An isolated cell of the transgenic animal of  claim 56  that produces insulin in response to the nutrient.