Claims for Patent: 5,968,502

Summary for Patent: 5,968,502

Title:	Protein production and protein delivery
Abstract:	The present invention relates to transfected primary, secondary, and immortalized cells of vertebrate origin particularly mammalian origin, transfected with exogenous genetic material (DNA) which encodes a desired (e.g., a therapeutic) product or is itself a desired (e.g., therapeutic) product, methods by which primary, secondary and immortalized cells are transfected to include exogenous genetic material, including DNA targeting by homologous recombination, methods for the activation and amplification of endogenous cellular genes, methods by which cells useful for large-scale protein production can be obtained, methods of producing clonal cell strains or heterogenous cell strains, and methods of gene therapy in which transfected primary, secondary or immortalized cells are used. The present invention includes primary, secondary, and immortalized cells, such as fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, and other cells which can be cultured.
Inventor(s):	Treco; Douglas (Arlington, MA), Heartlein; Michael W. (Boxborough, MA), Selden; Richard F (Wellesley, MA)
Assignee:	Transkaryotic Therapies, Inc. (Cambridge, MA)
Application Number:	08/451,894
Patent Claims:	1. A method of providing a protein to a mammal, comprising introducing into the mammal a homologously recombinant cell which produces the protein, the homologously recombinant cell being generated by an in vitro process comprising: (a) providing a cell that is autologous to the mammal, the genomic DNA of which comprises an endogenous gene; (b) providing a DNA construct comprising: (1) a targeting sequence homologous to a target site within or upstream of the endogenous gene, (2) an exogenous regulatory sequence, (3) an exon, and (4) an unpaired splice-donor site at the 3' end of the exon, wherein the exogenous regulatory sequence is operatively linked to the exon; and (c) transfecting the cell that is autologous to the mammal with the DNA construct, thereby generating a homologously recombinant cell in which the splice-donor site is operatively linked to the second exon of the endogenous gene, and the exogenous regulatory sequence controls transcription of the construct-derived exon, the endogenous gene, and any sequence lying between the construct-derived exon and the endogenous gene, to produce an RNA transcript that encodes the protein, so that the homologously recombinant cell produces the protein, wherein the protein is provided to the mammal under appropriate conditions such that the protein has a desired effect. 2. The method of claim 1, wherein the cell that is autologous to the mammal is a primary or secondary cell. 3. The method of claim 2, wherein the mammal is human. 4. The method of claim 1, wherein the cell that is autologous to the mammal is an immortalized cell. 5. The method of claim 4, wherein the immortalized cell is of human origin. 6. The method of claim 1, wherein prior to introduction into the mammal, the homologously recombinant cell is cultured under in vitro conditions which permit proliferation of the homologously recombinant cell, thereby generating a plurality of homologously recombinant cells which produce the protein. 7. The method of claim 6, wherein the protein is a protein or glycoprotein that is secreted by the plurality of homologously recombinant cells. 8. The method of claim 1, wherein the construct-derived exon comprises a CAP site. 9. The method of claim 8, wherein the construct-derived exon further comprises the nucleotide sequence ATG at a site suitable for directing translation initiation of the transcript in the homologously recombinant cell. 10. The method of claim 9, wherein the construct-derived exon comprises a coding sequence, and, following said transcription, the portion of the transcript corresponding to the splice-donor site is spliced to a splice-acceptor site of the endogenous gene, such that the coding sequence of the construct-derived exon is in-frame with coding sequence of the endogenous gene. 11. The method of claim 10, wherein the coding sequence of the construct-derived exon is identical to the endogenous coding sequence in the first exon of the endogenous gene in the cell that is autologous to the mammal. 12. The method of claim 10, wherein the coding sequence of the construct-derived exon is different from the endogenous coding sequence in the first exon of the endogenous gene in the cell that is autologous to the mammal. 13. The method of claim 1, wherein the protein is a hormone. 14. The method of claim 1, wherein the protein is a cytokine. 15. The method of claim 1, wherein the protein is an enzyme. 16. The method of claim 1, wherein the protein is a clotting factor. 17. The method of claim 1, wherein the protein is selected from the group consisting of antigens, antibodies, transport proteins, receptors, regulatory proteins, structural proteins, and transcription factors. 18. The method of claim 1, wherein the protein is selected from the group consisting of calcitonin, insulinotropin, insulin-like growth factors, parathyroid hormone, nerve growth factors, immunoglobulins, catalytic antibodies, superoxide dismutase, tissue plasminogen activator, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, and immune response modifiers. 19. The method of claim 1, wherein the protein is an interferon. 20. The method, of claim 1, wherein the protein is a colony stimulating factor. 21. The method of claim 1, wherein the protein is glucocerebrosidase. 22. The method of claim 1, wherein the protein is blood clotting factor VIII. 23. The method of claim 1, wherein the protein is blood clotting factor IX. 24. The method of claim 1, wherein the protein is growth hormone. 25. The method of claim 1, wherein the protein is erythropoietin. 26. The method of claim 1, wherein the protein is insulin. 27. The method of claim 1, wherein the cell that is autologous to the mammal is a fibroblast. 28. The method of claim 1, wherein the cell that is autologous to the mammal is a primary or secondary fibroblast. 29. The method of claim 28, wherein the protein is human growth hormone (hGH). 30. The method of claim 28, wherein the protein is human erythropoietin (hEPO). 31. The method of claim 28, wherein the protein is human insulin. 32. The method of claim 25, wherein the mA produced by said transcription encodes a fusion protein, the sequence of which comprises amino acids 1-3 of human growth hormone signal peptide. 33. The method of claim 1, wherein the exogenous regulatory sequence comprises a constitutively active promoter. 34. The method of claim 33, wherein the constitutively active promoter is a mouse metallothionein-1 promoter. 35. The method of claim 1, wherein the mRNA homologously recombinant cell is, prior to introduction into the mammal, enclosed within a barrier device which permits passage of the protein from the interior of the barrier device to the exterior of the barrier device. 36. The method of claim 35, wherein the barrier device prevents the homologously recombinant cell from escaping the barrier device. 37. The method of claim 33, wherein (1) the DNA construct further comprises a sequence encoding an amplifiable marker permitting selection of cells containing multiple copies of the sequence encoding the amplifiable marker; and (2) prior to introduction into the mammal, the homologously recombinant cell is cultured under conditions which select for cells having multiple copies of the sequence encoding the amplifiable marker. 38. The method of claim 37, wherein the amplifiable marker is selected from the group consisting of dihydrofolate reductase, adenosine deaminase, and the trifunctional enzyme carbamoyl phosphate synthase-aspartate transcarbamylase-dihydroorotase (CAD). 39. The method of claim 37, wherein the vertebrate cell that is autologous to the mammal is of human origin. 40. The method of claim 37, wherein the cell that is autologous to the mammal is a primary or secondary cell. 41. The method of claim 37, wherein the cell that is autologous to the mammal is an immortalized cell. 42. A method of providing a protein to a mammal, comprising introducing into the mammal a homologously recombinant cell which produces the protein, the homologously recombinant cell being generated by an in vitro process comprising: (a) providing a vertebrate cell obtained from an organism other than the mammal, the genomic DNA of which comprises an endogenous gene; (b) providing a DNA construct comprising: (1) a targeting sequence homologous to a target site within or upstream of the endogenous gene, (2) an exogenous regulatory sequence, (3) an exon, and (4) an unpaired splice-donor site at the 3' end of the exon, wherein the exogenous regulatory sequence is operatively linked to the exon; and (c) transfecting the vertebrate cell with the DNA construct, thereby generating a homologously recombinant cell in which the splice-donor site is operatively linked to the second exon of the endogenous gene and the exogenous regulatory sequence controls transcription of the construct-derived exon, the endogenous gene, and any sequence lying between the construct-derived exon and the endogenous gene, to produce an RNA transcript that encodes the protein, so that the homologously recombinant cell produces the protein, wherein the protein is provided to the mammal under appropriate conditions such that the protein has a desired effect. 43. The method of claim 42, wherein the vertebrate cell is a primary or secondary cell. 44. The method of claim 43, wherein the primary or secondary cell is a mammalian cell. 45. The method of claim 43, wherein the primary or secondary cell is a human cell. 46. The method of claim 43, wherein the vertebrate cell is an immortalized cell. 47. The method of claim 46, wherein the immortalized cell is of mammalian origin. 48. The method of claim 46, wherein the immortalized cell is of human origin. 49. The method of claim 42, wherein prior to introduction into the mammal, the homologously recombinant cell is cultured under in vitro conditions which permit proliferation of the homologously recombinant cell, thereby generating a plurality of homologously recombinant cells which produce the protein. 50. The method of claim 49, wherein the protein is a protein or glycoprotein that is secreted by the plurality of homologously recombinant cells. 51. The method of claim 42, wherein the construct-derived exon comprises a CAP site. 52. The method of claim 51, wherein the exon further comprises the nucleotide sequence ATG at a site suitable for directing translation initiation of the endogenous gene in the homologously recombinant cell. 53. The method of claim 52, wherein the exon comprises a coding sequence, and, following said transcription, the portion of the transcript corresponding to the splice-donor site is spliced to a splice-acceptor site of the endogenous gene, such that the coding sequence of the construct-derived exon is in-frame with coding sequence of the endogenous gene. 54. The method of claim 53, wherein the coding sequence of the construct-derived exon is identical to the endogenous coding sequence in the first exon of the endogenous gene in the vertebrate cell. 55. The method of claim 53, wherein the coding sequence of the construct-derived exon is different from the endogenous coding sequence in the first exon of the endogenous gene in the vertebrate cell. 56. The method of claim 42, wherein the protein is a hormone. 57. The method of claim 42, wherein the protein is a cytokine. 58. The method of claim 42, wherein the protein is an enzyme. 59. The method of claim 42, wherein the protein is a clotting factor. 60. The method of claim 42, wherein the protein is selected from the group consisting of antigens, antibodies, transport proteins, receptors, regulatory proteins, structural proteins, and transcription factors. 61. The method of claim 42, wherein the protein is selected from the group consisting of calcitonin, insulinotropin, insulin-like growth factors, parathyroid hormone, nerve growth factors, immunoglobulins, catalytic antibodies, superoxide dismutase, tissue plasminogen activator, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, and immune response modifiers. 62. The method of claim 42, wherein the protein is an interferon. 63. The method of claim 42, wherein the protein is a colony stimulating factor. 64. The method of claim 42, wherein the protein is glucocerebrosidase. 65. The method of claim 42, wherein the protein is blood clotting factor VIII. 66. The method of claim 42, wherein the protein is blood clotting factor IX. 67. The method of claim 42, wherein the protein is growth hormone. 68. The method of claim 42, wherein the protein is erythropoietin. 69. The method of claim 42, wherein the protein is insulin. 70. The method of claim 42, wherein the vertebrate cell is a mammalian fibroblast. 71. The method of claim 42, wherein the vertebrate cell is a primary or secondary cell of human fibroblast origin. 72. The method of claim 71, wherein the protein is human growth hormone (hGH). 73. The method of claim 71, wherein the protein is human erythropoietin (hEPO). 74. The method of claim 71, wherein the protein is human insulin. 75. The method of claim 68, wherein the mRNA produced by said transcription encodes a fusion protein, the sequence of which comprises amino acids 1-3 of human growth hormone signal peptide. 76. The method of claim 42, wherein the exogenous regulatory sequence comprises a constitutively active promoter. 77. The method of claim 76, wherein the constitutively active promoter is a mouse metallothionein-1 promoter. 78. The method of claim 42, wherein the homologously recombinant cell is, prior to introduction into the mammal, enclosed within a barrier device which permits passage of the protein from the interior of the barrier device to the exterior of the barrier device. 79. The method of claim 78 wherein the barrier device prevents the homologously recombinant cell from escaping the barrier device. 80. The method of claim 76, wherein (1) the DNA construct further comprises a sequence encoding an amplifiable marker permitting selection of cells containing multiple copies of the sequence encoding the amplifiable marker; and (2) prior to introduction into the mammal, the homologously recombinant cell is cultured under conditions which select for cells having multiple copies of the sequence encoding the amplifiable marker. 81. The method of claim 80, wherein the amplifiable marker is selected from the group consisting of dihydrofolate reductase, adenosine deaminase, and the trifunctional enzyme carbamoyl phosphate synthase-aspartate transcarbamylase-dihydroorotase (CAD). 82. The method of claim 80, wherein the vertebrate cell is of human origin. 83. The method of claim 80, wherein the vertebrate cell is a primary or secondary cell. 84. The method of claim 80, wherein the vertebrate cell is an immortalized cell.

Details for Patent 5,968,502

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2016-10-19