In Vivo Production and Delivery of Erythropoietin or Insulinotropin for Gene Therapy

Abstract

The present invention relates to transfected primary and secondary somatic cells of vertebrate origin, particularly mammalian origin, transfected with exogenous genetic material (DNA) which encodes erythropoietin or an insulinotropin [e.g., derivatives of glucagon-like peptide 1 (GLP-1)], methods by which primary and secondary cells are transfected to include exogenous genetic material encoding erythropoietin or an insulinotropin, methods of producing clonal cell strains or heterogenous cell strains which express erythropoietin or an insulinotropin, methods of gene therapy in which the transfected primary or secondary cells are used, and methods of producing antibodies using the transfected primary or secondary cells. The present invention also includes primary and secondary somatic cells, such as fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, other somatic cells, which can be cultured and somatic cell precursors, which have been transfected with exogenous DNA encoding EPO or an insulinotropin, which is stably integrated into their genomes or is expressed in the cells episomally.

1 . A transfected primary or secondary cell of vertebrate origin having stably integrated into its genome:
 a) exogenous DNA which encodes erythropoietin and   b) DNA sequences, sufficient for expression of the exogenous DNA in the transfected primary or secondary cell, the primary or secondary cell capable of expressing erythropoietin.   
     
     
         2 - 9 . (canceled) 
     
     
         10 . A primary or secondary cell of vertebrate origin transfected with:
 a) exogenous DNA which encodes erythropoietin; and   b) DNA sequences, sufficient for expression of the exogenous DNA in the primary or secondary cell, the sequences of (a) and (b) present in the cell in an episome.   
     
     
         11 - 13 . (canceled) 
     
     
         14 . A clonal cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding erythropoietin incorporated therein. 
     
     
         15 - 19 . (canceled) 
     
     
         20 . A heterogenous cell strain of transfected secondary cells of vertebrate origin having stably incorporated into their genomes:
 a) exogenous DNA encoding erythropoietin and   b) DNA sequences sufficient for expression of the exogenous DNA in the transfected primary or secondary cell, the heterogenous cell strain capable of expressing erythropoietin.   
     
     
         21 - 23 . (canceled) 
     
     
         24 . A mixture of cells consisting essentially of transfected primary or secondary cells of  claim 1  and untransfected primary or secondary cells. 
     
     
         25 . (canceled) 
     
     
         26 . A method of producing a clonal cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding erythropoietin incorporated therein, comprising the steps of:
 a) producing a mixture of cells of vertebrate origin containing primary cells;   b) transfecting primary cells produced in (a) with a DNA construct comprising exogenous DNA encoding erythropoietin and additional DNA sequences sufficient for expression of the exogenous DNA in the primary cells, thereby producing transfected primary cells which express the exogenous DNA encoding erythropoietin;   c) culturing a transfected primary cell which expresses the exogenous DNA encoding erythropoietin produced in (b), under conditions appropriate for propagating the transfected primary cell which expresses the exogenous DNA encoding erythropoietin, thereby producing a clonal cell strain of transfected secondary cells from the transfected primary cell.   
     
     
         27 - 33 . (canceled) 
     
     
         34 . A method of producing a clonal cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding erythropoietin incorporated therein, comprising the steps of:
 a) providing a mixture of cells of vertebrate origin containing primary cells;   b) producing a population of secondary cells from the primary cells provided in (a);   c) transfecting secondary cells produced in (b) with a DNA construct comprising exogenous DNA encoding erythropoietin and additional DNA sequences sufficient for expression of the exogenous DNA in the secondary cells, thereby producing transfected secondary cells which express the exogenous DNA encoding erythropoietin; and   d) culturing a transfected secondary cell which expresses the DNA encoding erythropoietin, produced in (c), under conditions appropriate for propagating the transfected secondary cell which expresses the exogenous DNA encoding erythropoietin, thereby producing a clonal cell strain of transfected secondary cells from the transfected secondary cell of (d).   
     
     
         35 - 47 . (canceled) 
     
     
         48 . A method of producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding erythropoietin stably incorporated into the genome of said secondary cells, comprising the steps of:
 a) producing a mixture of cells of vertebrate origin containing primary cells;   b) transfecting primary cells produced in (a) with exogenous DNA encoding erythropoietin and operatively linked to DNA sequences of nonretroviral origin sufficient for expression of the exogenous DNA in transfected secondary cells, thereby producing a mixture of primary cells which includes transfected primary cells which express the exogenous DNA encoding erythropoietin;   c) culturing the product of (b) under conditions appropriate for propagation of transfected primary cells which express the exogenous DNA encoding erythropoietin, thereby producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express the exogenous DNA encoding erythropoietin.   
     
     
         49 - 55 . (canceled) 
     
     
         56 . A method of producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding erythropoietin stably incorporated into the genome of said secondary cells, comprising the steps of
 a) providing a mixture of cells of vertebrate origin containing primary cells;   b) producing a population of secondary cells from the primary cells provided in (a);   c) transfecting secondary cells produced in (b) with exogenous DNA encoding erythropoietin and operatively linked to DNA sequences of nonretroviral origin sufficient for expression of the exogenous DNA in transfected secondary cells, thereby producing a mixture of secondary cells which includes transfected secondary cells which express the exogenous DNA encoding erythropoietin;   d) culturing the product of (c) under conditions appropriate for propagation of transfected secondary cells which express the exogenous DNA encoding a therapeutic product, thereby producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express the exogenous DNA encoding erythropoietin.   
     
     
         57 - 64 . (canceled) 
     
     
         65 . A method of producing a clonal cell strain of secondary fibroblasts of mammalian origin which express exogenous DNA encoding erythropoietin upon introduction into a mammal, comprising the steps of:
 a) providing primary fibroblasts of mammalian origin;   b) producing a population of secondary fibroblasts from the primary fibroblasts provided in (a);   c) combining the secondary fibroblasts of mammalian origin with a DNA construct comprising:
 i) exogenous DNA encoding erythropoietin to be expressed in the fibroblasts; and 
 ii) additional DNA sequences of non-retroviral origin sufficient for expression of the exogenous DNA in the fibroblasts; 
   d) subjecting the combination produced in (c) to electroporation under conditions which result in transfection of the vector into the secondary fibroblasts of mammalian origin, thereby producing a mixture of transfected secondary fibroblasts of mammalian origin and nontransfected secondary fibroblasts of mammalian origin;   e) isolating a transfected secondary fibroblast of mammalian origin produced in (d); and   f) culturing the transfected secondary fibroblast of mammalian origin isolated in (e) under conditions appropriate for production of a clonal population consisting essentially of transfected secondary fibroblasts of mammalian origin which express the exogenous DNA encoding erythropoietin.   
     
     
         66 - 67 . (canceled) 
     
     
         68 . A method of providing erythropoietin in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal;   b) transfecting primary cells obtained in (a) with DNA construct comprising exogenous DNA encoding erythropoietin and additional DNA sequences sufficient for expression of the exogenous DNA in the primary cells, thereby producing transfected primary cells which express the exogenous DNA encoding the therapeutic product;   c) culturing a transfected primary cell which expresses the exogenous DNA encoding erythropoietin produced in (b), under conditions appropriate for propagating the transfected primary cell which expresses the exogenous DNA encoding erythropoietin, thereby producing a clonal cell strain of transfected secondary cells from the transfected primary cell;   d) culturing the clonal cell strain of transfected secondary cells produced in (c) under conditions appropriate for and sufficient time for the clonal cell strain of transfected secondary cells to undergo a sufficient number of doublings to provide a sufficient number of transfected secondary cells to produce an effective amount of erythropoietin; and   e) introducing transfected secondary cells produced in (d) into the mammal in sufficient number to produce an effective amount of erythropoietin in the mammal.   
     
     
         69 . (canceled) 
     
     
         70 . A method of providing erythropoietin in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal;   b) producing a population of secondary cells from the primary cells provided in (a);   c) transfecting secondary cells produced in (b) with a DNA construct comprising exogenous DNA encoding erythropoietin and additional DNA sequences sufficient for expression of the exogenous DNA in the primary cells, thereby producing transfected secondary cells which express the exogenous DNA encoding erythropoietin;   d) culturing a transfected secondary cell which expresses the exogenous DNA encoding erythropoietin produced in (c), under conditions appropriate for propagating the transfected secondary cell which expresses the exogenous DNA encoding erythropoietin, thereby producing a clonal cell strain of transfected secondary cells from the transfected secondary cell;   e) culturing the clonal cell strain of transfected secondary cells produced in (d) under conditions appropriate for and sufficient time for the clonal cell strain of transfected secondary cells to undergo a sufficient number of doublings to provide a sufficient number of transfected secondary cells to produce an effective amount of erythropoietin; and   f) introducing transfected secondary cells produced in (e) into the mammal in sufficient number to produce an effective amount of erythropoietin.   
     
     
         71 . (canceled) 
     
     
         72 . A method of producing erythropoietin in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal;   b) transfecting primary cells obtained in (a) with exogenous DNA encoding erythropoietin and operatively linked to DNA sequences of nonretroviral origin sufficient for expression of the exogenous DNA in transfected secondary cells, thereby producing a mixture of primary cells which includes transfected primary cells which express the exogenous DNA encoding erythropoietin;   c) culturing the product of (b) under conditions appropriate for propagation of transfected primary cells which express the exogenous DNA encoding erythropoietin, thereby producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express the exogenous DNA encoding erythropoietin; and   d) introducing transfected secondary cells produced in (c) into the mammal in sufficient number to produce an effective amount of erythropoietin in the mammal.   
     
     
         73 . (canceled) 
     
     
         74 . A method of providing erythropoietin in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal,   b) producing a population of secondary cells from the primary cells provided in (a);   c) transfecting secondary cells produced in (b) with exogenous DNA encoding erythropoietin and operatively linked to DNA sequences of nonretroviral origin sufficient for expression of the exogenous DNA in transfected secondary cells, thereby producing a mixture of secondary cells which includes transfected secondary cells which express the exogenous DNA encoding erythropoietin;   d) culturing the product of (c) under conditions appropriate for propagation of transfected secondary cells which express the exogenous DNA encoding erythropoietin, thereby producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express the exogenous DNA encoding erythropoietin; and   e) introducing transfected secondary cells produced in (c) into the mammal in sufficient number to produce an effective amount of erythropoietin in the mammal.   
     
     
         75 . (canceled) 
     
     
         76 . A method of providing erythropoietin to a mammal at biologically significant levels, comprising administering to the mammal transfected primary or secondary cells of mammalian origin which express erythropoietin in sufficient quantity to produce physiologically relevant levels in the mammal. 
     
     
         77 . (canceled) 
     
     
         78 . A transfected primary or secondary cell of vertebrate origin having stably integrated into its genome:
 a) exogenous DNA which encodes a glucagon-like peptide 1 related peptide with insulinotropin activity, and   b) DNA sequences, sufficient for expression of the exogenous DNA in the transfected primary or secondary cell, the primary or secondary cell capable of expressing the glucagon-like peptide 1 related peptide.   
     
     
         79 - 85 . (canceled) 
     
     
         86 . A primary or secondary cell of vertebrate origin transfected with:
 a) exogenous DNA which encodes a glucagon-like peptide 1 related peptide with insulinotropin activity; and   b) DNA sequences, sufficient for expression of the exogenous DNA in the primary or secondary cell, the sequences of (a) and (b) present in the cell in an episome.   
     
     
         87 . A clonal cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding a glucagon-like peptide 1 related peptide incorporated therein. 
     
     
         88 . (canceled) 
     
     
         89 . A heterogenous cell strain of transfected secondary cells of vertebrate origin having stably incorporated into their genomes:
 a) exogenous DNA encoding a glucagon-like peptide 1 related peptide with insulinotropin activity, and   b) DNA sequences sufficient for expression of the exogenous DNA in the transfected primary or secondary cell, the heterogenous cell strain capable of expressing a glucagon-like peptide 1 related peptide.   
     
     
         90 . (canceled) 
     
     
         91 . A method of producing a clonal cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding a glucagon-like peptide 1 related peptide incorporated therein, comprising the steps of:
 a) producing a mixture of cells of vertebrate origin containing primary cells;   b) transfecting primary cells produced in (a) with a DNA construct comprising exogenous DNA encoding a glucagon-like peptide 1 related peptide and additional DNA sequences sufficient for expression of the exogenous DNA in the primary cells, thereby producing transfected primary cells which express the exogenous DNA, encoding a glucagon-like peptide 1 related peptide; and   c) culturing a transfected primary cell which expresses the exogenous DNA encoding a glucagon-like peptide 1 related peptide produced in (b), under conditions appropriate for propagating the transfected primary cell which expresses the exogenous DNA encoding a glucagon-like peptide 1 related peptide, thereby producing a clonal cell strain of transfected secondary cells from the transfected primary cell.   
     
     
         92 . A method of producing a clonal cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding a glucagon-like peptide 1 related peptide incorporated therein, comprising the steps of:
 a) providing a mixture of cells of vertebrate origin containing primary cells;   b) producing a population of secondary cells from the primary cells provided in (a);   c) transfecting secondary cells produced in (b) with a DNA construct comprising exogenous DNA encoding a glucagon-like peptide 1 related peptide and additional DNA sequences sufficient for expression of the exogenous DNA in the secondary cells, thereby producing transfected secondary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide; and   d) culturing a transfected secondary cell which expresses the DNA encoding a glucagon-like peptide 1 related peptide produced in (c), under conditions appropriate for propagating the transfected secondary cell which expresses the exogenous DNA encoding a glucagon-like peptide 1 related peptide, thereby producing a clonal cell strain of transfected secondary cells from the transfected secondary cell of (d).   
     
     
         93 . A method of producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express exogenous DNA encoding a glucagon-like peptide 1 related peptide stably incorporated into the genome of said secondary cells, comprising the steps of:
 a) producing a mixture of cells of vertebrate origin containing primary cells;   b) transfecting primary cells produced in (a) with exogenous DNA encoding a glucagon-like peptide 1 related peptide and operatively linked to DNA sequences of non-retroviral origin sufficient for expression of the exogenous DNA in transfected secondary cells, thereby producing a mixture of primary cells which includes transfected primary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide;   c) culturing the product of (b) under conditions appropriate for propagation of transfected primary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide, thereby producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express the exogenous DNA encoding a glucagonlike peptide 1 related peptide.   
     
     
         94 . A method of producing a clonal cell strain of secondary fibroblasts of mammalian origin which express exogenous DNA encoding a glucagon-like peptide 1 related peptide upon incorporation into the genome of the secondary fibroblast, comprising the steps of:
 a) providing primary fibroblasts of mammalian origin;   b) producing a population of secondary fibroblasts from the primary fibroblasts provided in (a);   c) combining the secondary fibroblasts of mammalian origin with a DNA construct comprising:
 i) exogenous DNA encoding a glucagon-like peptide 1 related peptide to be expressed in the fibroblasts; and 
 ii) additional DNA sequences of non-retroviral origin sufficient for expression of the exogenous DNA in the fibroblasts; 
   d) subjecting the combination produced in (c) to electroporation under conditions which result in transfection of the vector into the secondary fibroblasts of mammalian origin, thereby producing a mixture of transfected secondary fibroblasts of mammalian origin and nontransfected secondary fibroblasts of mammalian origin;   e) isolating a transfected secondary fibroblast of mammalian origin produced in (d); and   f) culturing the transfected secondary fibroblast of mammalian origin isolated in (e) under conditions appropriate for production of a clonal population consisting essentially of transfected secondary fibroblasts of mammalian origin which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide.   
     
     
         95 . (canceled) 
     
     
         96 . A method of providing a glucagon-like peptide 1 related peptide in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal;   b) transfecting primary cells obtained in (a) with a DNA construct comprising exogenous DNA encoding a glucagon-like peptide 1 related peptide and additional DNA sequences sufficient for expression of the exogenous DNA in the primary cells, thereby producing transfected primary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide;   c) culturing a transfected primary cell which expresses the exogenous DNA encoding a glucagon-like peptide 1 related peptide produced in (b), under conditions appropriate for propagating the transfected primary cell which expresses the exogenous DNA encoding a glucagon-like peptide 1 related peptide, thereby producing a clonal cell strain of transfected secondary cells from the transfected primary cell;   d) culturing the clonal cell strain of transfected secondary cells produced in (c) under conditions appropriate for and sufficient time for the clonal cell strain of transfected secondary cells to undergo a sufficient number of doublings to provide a sufficient number of transfected secondary cells to produce an effective amount of a glucagon-like peptide 1 related peptide; and   e) introducing transfected secondary cells produced in (d) into the mammal in sufficient number to produce an effective amount of a glucagon-like peptide 1 related peptide in the mammal.   
     
     
         97 - 98 . (canceled) 
     
     
         99 . A method of providing a glucagon-like peptide 1 related peptide in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal;   b) producing a population of secondary cells from the primary cells provided in (a);   c) transfecting secondary cells produced in (b) with a DNA construct comprising exogenous DNA encoding a glucagon-like peptide 1 related peptide and additional DNA sequences sufficient for expression of the exogenous DNA in the primary cells, thereby producing transfected secondary cells which express the exogenous DNA encoding glucagon-like peptide;   d) culturing a transfected secondary cell which expresses the exogenous DNA encoding glucagon-like peptide produced in (c), under conditions appropriate for propagating the transfected secondary cell which expresses the exogenous DNA encoding a glucagon-like peptide 1 related peptide, thereby producing a clonal cell strain of transfected secondary cells from the transfected secondary cell;   e) culturing the clonal cell strain of transfected secondary cells produced in (c) under conditions appropriate for and sufficient time for the clonal cell strain of transfected secondary cells to undergo a sufficient number of doublings to provide a sufficient number of transfected secondary cells to produce an effective amount of a glucagon-like peptide 1 related peptide; and   f) introducing transfected secondary cells produced in (e) into the mammal in sufficient number to produce an effective amount of a glucagon-like peptide 1 related peptide.   
     
     
         100 . (canceled) 
     
     
         101 . A method of producing a glucagon-like peptide 1 related peptide in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal;   b) transfecting primary cells obtained in (a) with exogenous DNA encoding a glucagon-like peptide 1 related peptide and operatively linked to DNA sequences of non-retroviral origin sufficient for expression of the exogenous DNA in transfected secondary cells, thereby producing a mixture of primary cells which includes transfected primary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide;   c) culturing the product of (b) under conditions appropriate for propagation of transfected primary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide, thereby producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide; and   d) introducing transfected secondary cells produced in (c) into the mammal in sufficient number to produce an effective amount of a glucagon-like peptide 1 related peptide in the mammal.   
     
     
         102 . (canceled) 
     
     
         103 . A method of providing a glucagon-like peptide 1 related peptide in an effective amount to a mammal, comprising the steps of:
 a) obtaining a source of primary cells from the mammal;   b) producing a population of secondary cells from the primary cells provided in (a);   c) transfecting secondary cells produced in (b) with exogenous DNA encoding a glucagon-like peptide 1 related peptide and operatively linked to DNA sequences of non-retroviral origin sufficient for expression of the exogenous DNA in transfected secondary cells, thereby producing a mixture of secondary cells which includes transfected secondary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide;   d) culturing the product of (c) under conditions appropriate for propagation of transfected secondary cells which express the exogenous DNA encoding a glucagon-like peptide 1 related peptide, thereby producing a heterogenous cell strain of transfected secondary cells of vertebrate origin which express the exogenous DNA encoding glucagon-like peptide; and   e) introducing transfected secondary cells produced in (c) into the mammal in sufficient number to produce an effective amount of a glucagon-like peptide 1 related peptide in the mammal.   
     
     
         104 . (canceled) 
     
     
         105 . A method of providing erythropoietin in an effective amount to a mammal, comprising introducing into the mammal a barrier device containing:
 a) transfected primary cells expressing exogenous DNA encoding erythropoietin,   b) transfected secondary cells expressing exogenous DNA encoding erythropoietin,   c) or both a) and b), wherein the barrier device is made of a material which permits passage of erythropoietin into the circulation or tissues of the mammal and prevents contact between the immune system of the mammal and the transfected cells contained within the barrier device to a sufficient extent to prevent a deleterious immune response by the mammal.   
     
     
         106 . A method of providing erythropoietin in an effective amount to a mammal, comprising introducing into the mammal a DNA construct comprising exogenous DNA encoding erythropoietin and regulatory sequences sufficient for expression of erythropoietin in cells of the mammal, wherein the DNA construct is taken up by cells of the mammal and is expressed therein. 
     
     
         107 . (canceled)