Methods and compositions relating to engineered microbial cells

Patent metadata

Field	Value
Publication number	US-10702559-B2
Application number	US-201716075410-A
Country	US
Kind code	B2
Filing date	Feb 8, 2017
Priority date	Feb 9, 2016
Publication date	Jul 7, 2020
Grant date	Jul 7, 2020

Abstract

Described herein are compositions and methods relating to engineered bacteria which have a modified Type 3 Secretion System (T3SS) which permits them to deliver proteins to the extracellular space (e.g., as opposed to the intracellular space of a target cell as done with a wild-type T3SS). In some embodiments, the engineered bacteria comprise a transgenic T3SS. In some embodiments, the delivered protein is non-native or transgenic with respect to the engineered bacteria.

First claim

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 virulence regulon transcriptional activator (vir) B (virB); membrane expression of invasion plasmid antigens (mxi) G (mxiG); mxiH; mxiI; mxiJ; mxiK; mxiN; mxiL; mxiM; mxiD; mxiA; surface presentation antigens (spa) 47 (spa47); spa13; spa32; spa33; spa24; spa9; spa29; and spa40;
b) a second nucleic acid sequence encoding an T3SS-compatible payload polypeptide; and not comprising or expressing at least one of
invasion plasmid antigen (Ipa) B (IpaB);
IpaD; or
MxiC 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.

2. The microbial cell of claim 1 , wherein the cell has a mutated MxiH.

3. The microbial cell of claim 1 , 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.

4. The microbial cell of claim 1 , wherein the cell comprises a third nucleic acid sequence encoding a master T3SS transcriptional regulator.

5. The microbial cell of claim 4 , wherein the master T3SS transcriptional regulator is selected from the group consisting of:
VirB and VirF.

6. The microbial cell of claim 1 , wherein the TDESS comprises at least: virB; acyl carrier protein (acp); ipaA; invasion plasmid gene (ipg) C (ipgC); ipgB1; ipgA; intra-inter-cellular spread (ics) B (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.

7. The engineered microbial cell of claim 1 , 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.

8. The engineered microbial cell of claim 1 , wherein the cell did not comprise a T3SS prior to being engineered to comprise the first and second nucleic acid sequences.

9. The microbial cell of claim 1 , wherein the T3SS-compatible payload polypeptide comprises an anti-inflammatory polypeptide.

10. The microbial cell of claim 1 , wherein the T3SS-compatible payload polypeptide comprises an antibody reagent; a nanobody; a VNA; or a VHH.

11. The microbial cell of claim 10 , wherein the antibody reagent specifically binds to a cancer checkpoint polypeptide.

12. The microbial cell of claim 11 , wherein the antibody reagent is an anti-PD-L1; anti-PD-1; or anti-CTLA-4 reagent.

13. The microbial cell of claim 10 , wherein the antibody reagent specifically binds to an inflammatory cytokine receptor or an inflammatory cytokine.

14. The microbial cell of claim 10 , wherein the antibody reagent specifically binds to a bacterial toxin.

15. The microbial cell of claim 1 , 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; E. coli DH1013 and E. coli DH5a.

16. The microbial cell of claim 1 , wherein the microbial cell is engineered from a commensal intestinal microbial cell.

17. 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 claim 1 .

18. 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 claim 1 to the subject.

Assignees

• Massachusetts Gen Hospital

Inventors

• Lesser Cammie
• Reeves Analise

Classifications

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C07K14/195

  from bacteria · CPC title

• A61K31/70

  Carbohydrates; Sugars; Derivatives thereof (sorbitol A61K31/047) · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C07K14/25

  Shigella (G) · CPC title