METHODS AND COMPOSITIONS FOR USE OF CRL 5803 CELLS FOR EXPRESSION OF BIOTHERAPEUTICS AND ENCAPSULATED CELL-BASED DELIVERY

• US 20090196854A1
• Filed: 02/04/2008
• Published: 08/06/2009
• Est. Priority Date: 02/04/2008
• Status: Abandoned Application

• Current Assignee: Kytos Biosystems S.A.
• Original Assignee: Kytos Biosystems S.A.
• Inventors: Clark, Samuel A., Costandy, Heba, Quaade, Christian
• Application Number:
  US12/025,392
• Publication Number:
  US 20090196854A1
• Time in Patent Office: Days
• US Class Current: 424/93.2
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/02 Cells for production

Abstract

The use of CRL 5803 cells as a robust cell substrate for transgene expression of polypeptides is contemplated. Furthermore, use of CRL 5803 cells in cell-based delivery and methods of making and using the same in encapsulated devices are contemplated.

Claims

1. A CRL 5803 cell genetically modified by transformation with one or more expression constructs, each construct comprising a nucleic acid encoding a transgene operatively linked to a promoter, wherein said cell expresses at least a first transgene, a second transgene, a third transgene and a fourth transgene,

wherein at least one of said first, second, third or fourth transgenes encodes a therapeutic polypeptide and

wherein said cell expresses said therapeutic polypeptide for at least 40 population doublings.

2. The cell of claim 1, further comprising at least one selectable marker.

3. The cell of claim 1, wherein said therapeutic polypeptide is selected from the group consisting of a of a therapeutic polypeptide growth factor, an enzyme, a cytokine, a tumor suppressor, an apoptosis inducer, a hormone, a hematopoietic factor, a hemostasis factor, a pressor molecule, a receptor, a transporter protein, and a channel protein.

4. The cell of claim 2, wherein said first transgene is a neurotrophic factor gene, said second transgene is secreted alkaline phosphatase (SEAP) gene, said third transgene is a wild type cytosine deaminase gene, and said fourth transgene is a neurotrophic factor gene, said marker gene is a neomycin phosphotransferase gene, and wherein said cell is further transformed with a hygromycin phosphotransferase gene.

5. The cell of claim 1, wherein said promoter is heterologous to said nucleic acid encoding said therapeutic polypeptide.

6. The cell of claim 1, wherein said promoter is homologous to said nucleic acid encoding said therapeutic polypeptide.

7. The cell of claim 1, wherein said promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, and a non-inducible promoter.

8. The cell of claim 1, wherein said promoter is selected from the group consisting of a human cytomegalovirus (CMV) immediate early gene promoter, a SV40 early promoter, a Rous sarcoma virus long terminal repeat, a rat insulin promoter, an EF-1 α

promoter, an Ubiquitin promoter, and a glyceraldehyde-3-phosphate dehydrogenase promoter.

10. A composition comprising the cell of claim 1 and a pharmaceutically acceptable carrier, excipient or diluent.

12. A method of treatment comprising the step of implanting in a subject the cell according to claim 1, wherein said cell expresses said therapeutic polypeptide in amount effective to treat a condition treatable with said therapeutic polypeptide.

13. The method of claim 12, wherein said cell is implanted in said subject'"'"'s central nervous system.

14. The method of claim 13, wherein said cell is implanted in said subject'"'"'s brain.

15. The method of claim 13, wherein said cell is implanted in said subject'"'"'s spinal cord.

16. The method of claim 13, wherein said cell is implanted in a tumor of said subject.

17. The method of claim 12, further comprising the step of administering an immunosuppressant.

18. The method of claim 12, further comprising the step of administering an anti-viral agent.

19. The method of claim 12, further comprising the step of administering an anti-bacterial agent.

20. The method of claim 12, wherein said subject is a human.

21. The method of claim 12, wherein said therapeutic polypeptide is selected from the group consisting of a growth factor, an enzyme, a cytokine, a tumor suppressor, an apoptosis inducer, a hormone, a hematopoietic factor, hemostasis factor, and a pressor molecule.

22. The method of claim 12, wherein said cell is implanted in said subject in a cell density of about 1×

10⁷ cells/ml to about 1×

10⁹ cells/ml.

23. A method of treating a central nervous system (CNS) disorder in a subject comprising implanting the cell according to claim 1 in said subject, wherein said cell expresses a therapeutic polypeptide in amount effective to treat said CNS disorder.

24. The method of claim 23, wherein said cell is implanted said subject'"'"'s brain.

25. The method of claim 23, wherein said cell is implanted in said subject'"'"'s spinal cord.

26. The method of claim 23, further comprising the step of administering an immunosuppressant.

27. The method of claim 23, further comprising the step of administering an anti-viral agent.

28. The method of claim 23, further comprising the step of administering an anti-bacterial agent.

29. The method of claim 23, wherein said subject is a human.

30. The method of claim 23, wherein said therapeutic polypeptide is selected from the group consisting of a growth factor, an enzyme, an antibody, a cytokine, a tumor suppressor, an apoptosis inducer, a hormone, a hematopoietic factor, hemostasis factor, and a pressor molecule.

31. The method of claim 23, wherein said cell is implanted in said subject in a cell density of about 1×

10⁷ cells/ml to about 1×

10⁹ cells/ml.

32. A method of delivering a therapeutic polypeptide to a site in a human body comprising implanting at said site the cell according to claim 1, wherein said cell expresses said therapeutic polypeptide.

33. The method of claim 32, wherein said site is a central nervous system site.

34. The method of claim 33, wherein said site is a brain.

35. The method of claim 33, wherein said site is a spinal cord site.

36. The method of claim 32, wherein said site is a tumor.

37. The method of claim 32, wherein said therapeutic agent is selected from the group consisting of a growth factor, an enzyme, a cytokine, a tumor suppressor, an apoptosis inducer, a hormone, a hematopoietic factor, hemostasis factor, and a pressor molecule.

38. The method of claim 32, wherein said cell is implanted in said body in a cell density of about 1×

10⁷ cells/ml to about 1×

10⁹ cells/ml.

39. A method for sustaining in vitro production of a therapeutic polypeptide comprising culturing the cell of claim 1 under conditions that allow production of said polypeptide for at least 40 population doublings.

43. An implantable system comprising the cell of claim 1, wherein said system is immobilized at an implantation site to maintain said cell at said implantation site and permit diffusion of an expressed and secreted therapeutic polypeptide from said implantation site.

44. The system of claim 43, wherein said therapeutic polypeptide is selected from the group of a therapeutic polypeptide growth factor, an enzyme, a cytokine, a tumor suppressor, an apoptosis inducer, a hormone, a hematopoietic factor, hemostasis factor, a pressor molecule.

45. The system of claim 43, wherein said site is a subject'"'"'s central nervous system

46. The system of claim 43, wherein said site is a subject'"'"'s brain.

47. The system of claim 43, wherein said site is a subject'"'"'s spinal cord.

48. The system of claim 43, wherein said site is a tumor.

49. The system of claim 43, wherein said system comprises a cell density between about 1×

10⁷ cells/ml to about 1×

10⁹ cells/ml device volume.

50. The system of claim 43, wherein said cell survives under culture conditions or in vivo in said system for at least a month with a functionality that represents at least 80% of the function expressed at the time the cells are/were introduced into said system.

51. The system of claim 43, wherein said cell in said system expand in said system to increase in cell density and/or cell function upon implantation of the system in vivo.

52. The system of claim 43, wherein said cell in said system have been genetically engineered by transformation with an expression construct that comprises a nucleic acid that encodes a therapeutic polypeptide operatively linked to a promoter, wherein said transformed cell expresses and secretes said therapeutic polypeptide.

53. The system of claim 43, wherein said promoter is heterologous to said nucleic acid encoding said therapeutic polypeptide.

54. The system of claim 43, wherein said promoter is homologous to said nucleic acid encoding said therapeutic polypeptide.

55. The system of claim 43, wherein said promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, and a non-inducible promoter.

56. The system of claim 43, wherein said promoter is selected from the group consisting of a human cytomegalovirus (CMV) immediate early gene promoter, a SV40 early promoter, a Rous sarcoma virus long terminal repeat, a rat insulin promoter, an EF-1 α

promoter, an Ubiquitin promoter, and a glyceraldehyde-3-phosphate dehydrogenase promoter.

9. A CRL 5803 cell deposited with DSMZ under accession number DSM ACC2730.

11. A composition comprising the cell of claim 9 and a pharmaceutically acceptable carrier, excipient or diluent.

40. A human cell engineered with a cytosine deaminase expression construct derived from a wild-type cytosine deaminase bacterial gene sequence operably linked to a promoter element, wherein the cytosine deaminase expression construct expresses a protein comprising the amino acid sequence set forth in SEQ ID NO:

11.

41. The cell of claim 40, wherein said cell is a CRL5803 cell.

42. A composition comprising the cell of claim 40 and a pharmaceutically acceptable carrier, excipient or diluent.