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

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).

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.