Genetically modified bacteria and methods for genetic modification of bacteria

• US 10,584,344 B2
• Filed: 06/17/2015
• Issued: 03/10/2020
• Est. Priority Date: 06/17/2014
• Status: Active Grant

• Current Assignee: Crown Laboratories, Inc.
• Original Assignee: Crown Laboratories, Inc.
• Inventors: Hitchcock, Thomas, Rhee, Mun Su
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US14/742,492
• Publication Number:
  US 20150361436A1
• Time in Patent Office: 1,728 Days
• Field of Search: None
• CPC Class Codes: A61K 48/0025 wherein the non-active part clearly interacts with the delivered nucleic acid
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 17/08 Antiseborrheics
• CPC Class Codes: A61P 17/10 Anti-acne agents
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/715 for cytokines; for lymphokines; for interferons
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)

Abstract

Microbes can be genetically modified to express biomolecules that are beneficial to mammals and/or to reduce, or eliminate, expression of harmful virulence factors. The growth and viability of such genetically modified microbes can optionally be controlled by inducible promoters that regulate the expression of proteins that are essential to their growth and survival. Compositions comprising such genetically modified microbes as well as methods of making and using the same are disclosed herein.

Claims

1. A pharmaceutical composition formulated for administration to a mammal, the composition comprising a genetically modified Propionibacterium acnes (P. acnes) strain comprising a regulatory element operably linked to a nucleic acid encoding a mammalian growth factor or a mammalian cytokine, wherein the strain expresses the growth factor or the mammalian cytokine, the P. acnes strain comprises a CRISPR array, and the regulatory element and the nucleic acid are integrated into the genome of the P. acnes strain.

2. The pharmaceutical composition of claim 1, wherein the growth factor is a hormone.

3. The pharmaceutical composition of claim 1, wherein the growth factor is a human or a bovine hormone.

4. The pharmaceutical composition of claim 1, wherein the growth factor is a somatotropin.

5. The pharmaceutical composition of claim 4, wherein the somatotropin comprises the amino acid sequence of any one of SEQ ID NO:

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48 or SEQ ID NO;

49.

6. The pharmaceutical composition of claim 1, wherein the growth factor is a secreted growth factor.

7. The pharmaceutical composition of claim 1, wherein the growth factor is a human growth factor.

8. The pharmaceutical composition of claim 1, wherein the growth factor is selected from the group consisting of a transforming growth factor beta (TGF-β

), a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF), a fibroblast growth factor (FGF), an insulin-like growth factor-1 (IGF1), a platelet derived growth factor (PDGF), a granulocyte monocyte colony stimulating factor (GMCSF) and an epidermal growth factor (EGF).

9. The pharmaceutical composition of claim 1, wherein the growth factor is a transforming growth factor comprising the amino acid sequence of any one of SEQ ID NO:

63 to SEQ ID NO;

69, or a hepatocyte growth factor comprising the amino acid sequence of SEQ ID NO;

70.

10. The pharmaceutical composition of claim 1, wherein the growth factor is a VEGF comprising the amino acid sequence of any one of SEQ ID NO:

71 to SEQ ID NO;

91.

11. The pharmaceutical composition of claim 1, wherein the growth factor is a PDGF comprising the amino acid sequence of any one of SEQ ID NO:

93 to SEQ ID NO;

102.

12. The pharmaceutical composition of claim 1, wherein the growth factor is an EGF comprising the amino acid sequence of any one of SEQ ID NO:

103 to SEQ ID NO;

106.

13. The pharmaceutical composition of claim 1, wherein the growth factor is a FGF comprising the amino acid sequence of any one of SEQ ID NO:

107 to SEQ ID NO;

144.

14. The pharmaceutical composition of claim 1, wherein the mammalian cytokine is a secreted mammalian cytokine.

15. The pharmaceutical composition of claim 14, wherein the cytokine comprises the amino acid sequence of any one of SEQ ID NO:

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27 or SEQ ID NO;

28.

16. The pharmaceutical composition of claim 1, wherein the mammalian cytokine is an immunosuppressive cytokine.

17. The pharmaceutical composition of claim 1, wherein the mammalian cytokine is a human cytokine.

18. The pharmaceutical composition of claim 1, wherein the cytokine is selected from the group consisting of interleukin-10 (IL-10), interleukin-6 (IL-6), interleukin-7 (IL-7) and interleukin-8 (IL-8).

19. The pharmaceutical composition of claim 1, wherein the mammalian cytokine comprises IL-10.

20. The pharmaceutical composition of claim 19, wherein the IL-10 comprises the amino acid sequence of SEQ ID NO:

25.

22. The pharmaceutical composition of claim 1 or 21, wherein expression of a pathogenic protein within the P. acnes is substantially reduced or eliminated.

23. The pharmaceutical composition of claim 22, wherein the pathogenic protein comprises an endotoxin or an exotoxin.

24. The pharmaceutical composition of claim 22, wherein the pathogenic protein comprises a glyceraldehyde 3-phosphate dehydrogenase (GADPH) protein or a CAMP protein.

25. The pharmaceutical composition of claim 1, wherein the P. acnes further comprises an inducible promoter operably linked to a gene expressing an essential protein, wherein the gene is endogenous to the P. acnes strain and the essential protein is essential for growth or viability of the P. acnes strain.

26. The pharmaceutical composition of claim 25, wherein the essential protein is selected from the group consisting of DnaA, FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ, ZipA, aroE, atpD, gmk, guaA, lepA, recA, and sodA.

27. The pharmaceutical composition of claim 25, wherein the inducible promoter is regulated by a sugar.

28. The pharmaceutical composition of claim 27, wherein the sugar is lactose or arabinose.

29. The pharmaceutical composition of claim 25, wherein the inducible promoter is regulated by an amino acid.

30. The pharmaceutical composition of claim 25, wherein the inducible promoter is regulated by a synthetic amino acid.

31. The pharmaceutical composition of claim 25, wherein the essential protein is a chromosome replication initiator protein.

32. The pharmaceutical composition of claim 1, comprising a plurality of the genetically modified P. acnes strain, wherein each of the P. acnes have a further genetic modification comprising an inducible promoter operably linked to an endogenous P. acnes gene that encodes a protein essential for growth or viability of the P. acnes strain.

54. The pharmaceutical composition of claim 1 or 21, wherein the regulatory element is an inducible promoter.

55. The pharmaceutical composition of claim 1 or 21, wherein the regulatory element is a bacterial promoter that is endogenous to the genetically modified bacteria.

56. The pharmaceutical composition of claim 1 or 21, wherein the regulatory element comprises a lacZ promoter, lacZ operon, or an ara operon promoter.

57. The pharmaceutical composition of claim 1 or 21, wherein the nucleic acid encoding the mammalian growth factor or the mammalian cytokine is inserted into all or a part of a P. acnes gene that encodes a pathogenic protein, wherein the P. acnes gene is endogenous to the genetically modified P. acnes.

58. The pharmaceutical composition of claim 1 or 21, wherein expression of an endogenous P. acnes protein is substantially reduced or eliminated.

59. The pharmaceutical composition of claim 1 or 21, wherein the genetically modified P. acnes strain further comprises multiple genetic modifications comprising at least one of at least one additional nucleic acid operably linked to a regulatory element wherein the at least one additional nucleic acid encodes a mammalian growth factor or a mammalian cytokine, an elimination of a pathogenic protein endogenous to the P. acnes or a reduction in expression of a pathogenic protein endogenous to the P. acnes.

60. The pharmaceutical composition of claim 1 or 21, comprising a plurality of the genetically modified bacteria of the P. acnes strain, wherein each of the bacteria have multiple genetic modifications comprising at least one of at least one additional nucleic acid operably linked to a regulatory element wherein the at least one additional nucleic acid encodes a mammalian growth factor or a mammalian cytokine, an elimination of a pathogenic protein endogenous to the P. acnes or a reduction in expression of a pathogenic protein endogenous to the P. acnes.

64. A kit comprising the pharmaceutical composition of claim 1, 61 or 62, and instructions for use.

21. A pharmaceutical composition formulated for administration to a mammal, the composition comprising a genetically modified acnes (P. acnes) strain comprising a regulatory element operably linked to a nucleic acid encoding a mammalian growth factor or a mammalian cytokine, wherein the P. acnes strain expresses the growth factor or the mammalian cytokine, and wherein the P. acnes strain is P. acnes, type II, ribotype 6.

33. The pharmaceutical composition of claim 21, wherein the growth factor is a hormone.

34. The pharmaceutical composition of claim 21, wherein the growth factor is a human or a bovine hormone.

35. The pharmaceutical composition of claim 21, wherein the growth factor is a somatotropin.

36. The pharmaceutical composition of claim 35, wherein the somatotropin comprises the amino acid sequence of any one of SEQ ID NO:

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48 or SEQ ID NO;

49.

37. The pharmaceutical composition of claim 21, wherein the growth factor is a secreted growth factor.

38. The pharmaceutical composition of claim 21, wherein the growth factor is a human growth factor.

39. The pharmaceutical composition of claim 21, wherein the growth factor is selected from the group consisting of a T GF-fβ

, a VEGF, a HGF, a FGF, an IGF1, a PDGF, a GMCSF and an EGF.

40. The pharmaceutical composition of claim 21, wherein the growth factor is a TGF comprising the amino acid sequence of any one of SEQ ID NO:

63 to SEQ ID NO;

69, or a HGF comprising the amino acid sequence of SEQ ID NO;

70.

41. The pharmaceutical composition of claim 21, wherein the growth factor is a VEGF comprising the amino acid sequence of any one of SEQ ID NO:

71 to SEQ ID NO;

91.

42. The pharmaceutical composition of claim 21, wherein the growth factor is a PDGF comprising the amino acid sequence of any one of SEQ ID NO:

93 to SEQ ID NO;

102.

43. The pharmaceutical composition of claim 21, wherein the growth factor is an EGF comprising the amino acid sequence of any one of SEQ ID NO:

103 to SEQ ID NO;

106.

44. The pharmaceutical composition of claim 21, wherein the growth factor is a FGF comprising the amino acid sequence of any one of SEQ ID NO:

107 to SEQ ID NO;

144.

45. The pharmaceutical composition of claim 21, wherein the mammalian cytokine is a secreted mammalian cytokine.

46. The pharmaceutical composition of claim 21, wherein the mammalian cytokine is an immuno suppressive cytokine.

47. The pharmaceutical composition of claim 21, wherein the mammalian cytokine is a human cytokine.

48. The pharmaceutical composition of claim 21, wherein the cytokine is selected from the group consisting of IL-10, IL-6, IL-7 and IL-8.

49. The pharmaceutical composition of claim 45, wherein the cytokine comprises the amino acid sequence of any one of SEQ ID NO:

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27 or SEQ ID NO;

28.

50. The pharmaceutical composition of claim 21, wherein the mammalian cytokine comprises IL-10.

51. The pharmaceutical composition of claim 50, wherein the IL-10 comprises the amino acid sequence of SEQ ID NO:

25.

52. The pharmaceutical composition of claim 21, wherein the P. acnes strain further comprises an inducible promoter operably linked to a gene expressing an essential protein, wherein the gene is endogenous to the P. acnes strain and the essential protein is essential for growth or viability of the P. acnes strain.

53. The pharmaceutical composition of claim 21, comprising a plurality of the genetically modified P. acnes strain, wherein each of the P. acnes have a further genetic modification comprising an inducible promoter operably linked to an endogenous P. acnes gene that encodes a protein essential for growth or viability of the P. acnes strain.

61. A pharmaceutical composition formulated for topical administration comprising genetically modified P. acnes, the genetically modified P. acnes comprising:

a) a mammalian growth factor, wherein the mammalian growth factor is secreted; and

b) an inducible promoter directing the expression of an essential protein of the genetically modified P. acnes selected from the group consisting of DnaA, FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ, ZipA, aroE, atpD, gmk, guaA, lepA, recA, and soda and wherein the expression of CAMP2 protein or GADPH protein is substantially reduced or eliminated in the genetically modified bacteria, the CAMP2 protein or the GADPH protein is endogenous to the genetically modified P. acnes and the genetically modified P. acnes comprises a CRISPR array.

63. The pharmaceutical composition of claim 61 or 62, wherein the composition comprises a sugar or an amino acid, and wherein the inducible promoter is induced or stimulated by the presence of the sugar or the amino acid.

62. A pharmaceutical composition formulated for topical administration comprising genetically modified P. acnes, the genetically modified P. acnes comprising:

a) a human IL10, wherein the IL10 is secreted; and

b) an inducible promoter directing the expression of an essential protein of the genetically modified P. acnes selected from the group consisting of DnaA, FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ, ZipA, aroE, atpD, gmk, guaA, lepA, recA, and soda and wherein the expression of CAMP2 protein or GADPH protein is substantially reduced or eliminated in the genetically modified bacteria, the CAMP2 protein or the GADPH protein is endogenous to the genetically modified P. acnes and the genetically modified P. acnes comprises a CRISPR array.