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1. (WO2017139366) METHODS AND COMPOSITIONS RELATING TO ENGINEERED MICROBIAL CELLS
Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

What is claimed herein is:

1. An engineered, non-pathogenic, gram negative microbial cell comprising:

a) a first nucleic acid sequence comprising genes encoding a type 3 secretion system (T3SS)-derived extracellular secretion system (TDESS); wherein the TDESS comprises at least virB; mxiG; mxiH; mxiI; mxiJ; mxiK; mxiN; mxiL; mxiM; mxiD; mxiA; spa47; spa13; spa32; spa33; spa24; spa9; spa29; and spa40; and

b) a second nucleic acid sequence encoding an T3SS-compatible payload polypeptide.

2. The microbial cell of claim 1, wherein the cell does not comprise or express at least one of:

a. IpaB;

b. IpaD; and

c. MxiC.

3. The microbial cell of claim 2, wherein the cell does not comprise or express at least one of:

a. IpaB and IpaD; and

b. MxiC.

4. The microbial cell of claim 3, wherein the cell does not comprise or express IpaB;

IpaD; and MxiC.

5. The microbial cell of claim 3, wherein the cell does not comprise or express IpaB;

IpaD; IpaC; and MxiC.

6. The microbial cell of any of claims 1-5, wherein the cell has a mutated MxiH. 7. The microbial cell of claim 6, wherein the cell has a D73A mutation in MxiH. 8. The microbial cell of any of claims 1-7, wherein the second nucleic acid sequence comprises 1) an inducible promoter sequence that is operably linked to 2) a sequence encoding an T3SS-compatible payload polypeptide.

9. The microbial cell of claim 8, wherein the inducible promoter sequence is regulated by a master T3SS transcriptional regulator.

10. The microbial cell of any of claims 8-9, wherein the inducible promoter sequence comprises a (T3SS)-associated promoter or promoter element.

11. The microbial cell of claim 10, wherein the T3SS-associated promoter or promoter element is a MxiE recognition sequence.

12. The microbial cell of any of claims 1-11, wherein the cell comprises a third nucleic acid sequence encoding a master T3SS transcriptional regulator.

13. The microbial cell of claim 12, wherein the master T3SS transcriptional regulator is selected from the group consisting of:

VirB and VirF.

14. The microbial cell of any of claims 12-13, wherein the third nucleic acid sequence comprises 1) an inducible promoter sequence that is operably linked to 2) a sequence encoding a master T3SS transcriptional regulator.

15. The microbial cell of claim 14, wherein the inducible promoter is selected from the group consisting of:

an arabinose-inducible promoter; pBAD arabinose-inducible promoter; an IPTG-inducible promoter; tumor-induced promoters; ansB promoter; pflE promoter; napF promoter; and an inflammation-induced promoter.

16. The microbial cell of any of claims 1-15, wherein the TDESS comprises at least: virB; acp; ipaA; ipgC; ipgB1; ipgA; icsB; ipgD; ipgE; ipgF; mxiG; mxiH; mxiI; mxiJ; mxiK; mxiN; mxiL; mxiM; mxiE; mxiD; mxiA; spa15; spa47; spa13; spa32; spa33; spa24; spa9; spa29; and spa40.

17. The microbial cell of any of claims 1-16, wherein the TDESS comprises polypeptides endogenous to a bacterium selected from the group consisting of:

Shigella spp; Salmonella spp; enteropathogenic E. coli; and Yersinia spp. 18. The microbial cell of any of claims 1-17, wherein the first nucleic acid sequence is located on a plasmid.

19. The engineered microbial cell of any of claims 1-17, wherein the first nucleic acid sequence is located on a chromosome.

20. The microbial cell of any of claims 1-19, wherein the second nucleic acid sequence is located on a plasmid.

21. The engineered microbial cell of any of claims 1-19, wherein the second nucleic acid sequence is located on a chromosome.

22. The engineered microbial cell of any of claims 1-21, wherein the first nucleic acid sequence comprising genes encoding a type 3 secretion system (T3SS)-derived extracellular secretion system (TDESS) and/or the genes encoding a type 3 secretion system (T3SS)-derived extracellular secretion system (TDESS) are exogenous to the microbial cell.

23. The engineered microbial cell of any of claims 1-22, wherein the first nucleic acid sequence is no greater than 3kb in size.

24. The engineered microbial cell of any of claims 1-22, wherein the first nucleic acid sequence and third nucleic acid sequence are cumulatively no greater than 3 kb in size.

25. The engineered microbial cell of any of claims 1-24, wherein the cell did not

comprise a T3SS prior to being engineered to comprise the first and second nucleic acid sequences.

26. The microbial cell of any of claims 1-25, wherein the T3SS-compatible payload polypeptide comprises a T3SS secretion sequence.

27. The microbial cell of any of claims 1-26, wherein the T3SS-compatible payload polypeptide comprises an N-terminal T3SS secretion sequence.

28. The microbial cell of any of claims 1-27, wherein the T3SS-compatible payload polypeptide comprises a OspC3 T3SS secretion sequence.

29. The microbial cell of any of claims 1-28, wherein the T3SS-compatible payload polypeptide comprises an anti-inflammatory polypeptide.

30. The microbial cell of claim 29, wherein the anti-inflammatory polypeptide is IL-10 or IL-27.

31. The microbial cell of any of claims 1-30, wherein the T3SS-compatible payload polypeptide comprises an antibody reagent.

32. The microbial cell of claim 31, wherein the antibody reagent is selected from the group consisting of:

a nanobody; a VNA; and a VHH.

33. The microbial cell of any of claims 31-32, wherein the cell comprises at least one further nucleic acid sequence encoding an additional T3SS-compatible payload polypeptide comprising an antibody reagent, VHH, or VNA.

34. The microbial cell of any of claims 31-32, wherein the one or more antibody reagents form a multimeric complex.

35. The microbial cell of claim 34, wherein the multimeric complex is multispecific.

36. The microbial cell of any of claims 31-35, wherein the antibody reagent specifically binds to a cancer cell marker.

37. The microbial cell of any of claims 31-35, wherein the antibody reagent specifically binds to a cancer checkpoint polypeptide.

38. The microbial cell of any of claims 31-37, wherein the antibody reagent is an anti-PD- L1; anti-PD-1; or anti-CTLA-4 reagent.

39. The microbial cell of any of claims 31-38, wherein the antibody reagent is an anti-PD- L1; anti-PD-1; or anti-CTLA-4 VNA or VHH.

40. The microbial cell of any of claims 31-39, wherein the antibody reagent specifically binds to an inflammatory cytokine receptor or an inflammatory cytokine.

41. The microbial cell of claim 40, wherein the antibody reagent binds to a molecule selected from the group consisting of:

TNFĮ, IL-8; IL-6, IL-18, IL-21, Il-33 and IL-13.

42. The microbial cell of any of claims 31-35, wherein the antibody reagent specifically binds to a bacterial toxin.

43. The microbial cell of claim 42, wherein the bacterial toxin is an E. coli or C. difficile toxin.

44. The microbial cell of any of claims 42-43, wherein the bacterial toxin is selected from the group consisting of:

shiga toxin; C. difficile toxin A (TcdA); C. difficile toxin B (TcdB); cholera toxin; anthrax toxin; and botulinum toxin.

45. The microbial cell of any of claims 1-28, wherein the T3SS-compatible payload polypeptide comprises a toxin.

46. The microbial cell of any of claims 1-28, wherein the T3SS-compatible payload polypeptide comprises an antigen.

47. The microbial cell of any of claims 1-46, wherein the microbial cell is engineered from a microbial cell selected from the group consisting of:

E. coli NISSLE 1917 (EcN); E. coli K12; MP; HS; and derivative strains thereof.

48. The microbial cell of claim 47, wherein the strain which is derivative of E. coli K12 is selected from the group consisting of:

E. coli DH10ȕ and E. coli DH5Į.

49. The microbial cell of any of claims 1-47, wherein the microbial cell is engineered from E. coli NISSLE 1917 (EcN).

50. The microbial cell of any of claims 1-49, wherein the microbial cell is engineered from a commensal intestinal microbial cell.

51. The microbial cell of claim 50, wherein the commensal intestinal microbial cell is E. coli NISSLE 1917 (EcN).

52. The microbial cell of any of claims 1-51, wherein the non-pathogenic microbial cell is engineered from a pathogenic microbial cell organism by deletion or mutation of one or more T3SS components.

53. The microbial cell of claim52, wherein the one or more T3SS components is selected from the group consisting of:

a toxin; a T3SS effector; a structural T3SS polypeptide; and a master transcriptional regulator of T3SS components.

54. The microbial cell of any of claims 52-53, wherein the pathogenic microbial cell is selected from the group consisting of:

Salmonella spp.; Shigella Spp; andYersinia spp.

55. The microbial cell of claim 52-54, wherein the pathogenic microbial cell is selected from the group consisting of:

Salmonella typhimurium SPI1and Shigella felxneri mxi-spa.

56. The microbial cell of any of claims 1-55, wherein cell has been:

a. contacted with a mutagenic treatment; and

b. selected for increased secretion.

57. The microbial cell of any of claims 1-56, wherein cell has been:

a. contacted with a mutagenic treatment; and

b. selected for increased secretion of the T3SS-compatible payload polypeptide.

58. The microbial cell of any of claims 1-57, wherein the cell further comprises a nucleic acid sequence encoding one or more polypeptides that increase adhesion to a target cell.

59. The microbial cell of claim 58, wherein the polypeptides that increase adhesion to the target cell comprise Tir and intimin.

60. The microbial cell of claim 59, wherein the polypeptide that increases adhesion to the target cell is selected from a group consisting of:

a bacterial adhesion; Afa1; AIDA; invasion; an antibody reagent specific for an extracellular epitope of a target cell polypeptide; and a single chain antibody specific for an extracellular epitope of a target cell polypeptide.

61. A method of introducing a polypeptide into a target tissue or organism, the method comprising contacting the target tissue or organism with a microbial cell of any of claims 1-60.

62. A method of reducing inflammation in a subject, the method comprising

administering an microbial cell of any of claims 29-30 or 40-41 to a subject in need thereof.

63. The method of claim 62, wherein the inflammation is inflammation of the

gastrointestinal tract.

64. The method of any of claims 62-63, wherein the subject is in need of treatment for a condition selected from the group consisting of:

asthma; inflammatory bowel disease; Crohn’s disease; obesity; and ulcerative colitis.

65. The method of claim 64, wherein the subject is a subject in need of treatment for inflammatory bowel disease.

66. The method of any of claims 62-65, wherein the microbial cell is administered orally. 67. A method of treating cancer in a subject, the method comprising administering an microbial cell of any of claims 30-39 to a subject in need thereof.

68. The method of claim 67, wherein the microbial cell is administered systemically. 69. The method of claim 67, wherein the microbial cell is administered intratumorally. 70. The method of claim 67, wherein the cancer is a cancer of the gastrointestinal tract and the microbial cell is administered orally.

71. The method of any of claims 67-70, wheiren the microbial cell is engineered from E. coli NISSLE 1917 (EcN).

72. A method of treating an intestinal infection in a subject, the method comprising administering a microbial cell of any of claims 42-43 to a subject in need thereof. 73. The method of claim 72, wherein the intestinal infection is EHEC and/or the subject has hemolytic uremic syndeom and the toxin is shiga toxin.

74. The method of claim 72, wherein the intestinal infection is a C. difficile infection and/or the subject has C. difficile colitis and the toxin is TcdA and/or TcdB.

75. The method of claim 72, wherein the intestinal infection is cholera and the toxin is cholera toxin.

76. The method of claim 72, wherein the intestinal infection is gastrointestinal anthrax and the toxin is anthrax toxin.

77. The method of claim 72, wherein the intestinal infection is botulism and the toxin is botulinum toxin.

78. The method of any of claims 72-77, wherein the microbial cell is administered orally. 79. The method of any of claims 61-78, wherein secretion of the T3SS-compatible

payload polypeptide is induced by further administering the subject a compound to induce expression of the T3SS-compatible payload polypeptide and/or the T3SS master transcriptional regulator.

80. The method of claim 79, wherein the compound is arabinose.

81. A method for delivering a polypeptide into a) the extracellular milieu of a subject’s gastrointestinal tract, b) the lumen of a tumor, or c) the extracellular milieu of a subject’s tumor , the method comprising contacting administering a microbial cell of any of claims 1-60 to the subject.

82. A kit comprising the microbial cell of any of claims 1-60.

83. The use of a microbial cell of any of claims 29-30 or 40-41 to reduce inflammation in a subject in need thereof.

84. The use of claim 83, wherein the inflammation is inflammation of the gastrointestinal tract.

85. The use of any of claims 83-84, wherein the subject is in need of treatment for a condition selected from the group consisting of:

asthma; inflammatory bowel disease; Crohn’s disease; obesity; and ulcerative colitis.

86. The use of claim 85, wherein the subject is a subject in need of treatment for

inflammatory bowel disease.

87. The use of any of claims 83-86, wherein the microbial cell is administered orally.

88. The use of an microbial cell of any of claims 30-39 treat cancer in a subject in need thereof.

89. The use of claim 88, wherein the microbial cell is administered systemically.

90. The use of claim 88, wherein the microbial cell is administered intratumorally.

91. The use of claim 88, wherein the cancer is a cancer of the gastrointestinal tract and the microbial cell is administered orally.

92. The use of any of claims 88-91, wheiren the microbial cell is engineered from E. coli NISSLE 1917 (EcN).

93. The use of a microbial cell of any of claims 42-4339 to treat an intestinal infection in a subject in need thereof.

94. The use of claim 93, wherein the intestinal infection is EHEC and/or the subject has hemolytic uremic syndeom and the toxin is shiga toxin.

95. The use of claim 93, wherein the intestinal infection is a C. difficile infection and/or the subject has C. difficile colitis and the toxin is TcdA and/or TcdB.

96. The use of claim 93, wherein the intestinal infection is cholera and the toxin is cholera toxin.

97. The use of claim 93, wherein the intestinal infection is gastrointestinal anthrax and the toxin is anthrax toxin.

98. The use of claim 93, wherein the intestinal infection is botulism and the toxin is botulinum toxin.

99. The use of any of claims 93-97, wherein the microbial cell is administered orally. 100. The use of any of claims 83-99, wherein secretion of the T3SS-compatible payload polypeptide is induced by further administering the subject a compound to induce expression of the T3SS-compatible payload polypeptide and/or the T3SS master transcriptional regulator.

101. The use of claim100, wherein the compound is arabinose.