Biosynthetic amyloid-based materials displaying functional protein sequences

Patent metadata

Field	Value
Publication number	US-2020248190-A1
Application number	US-202016785874-A
Country	US
Kind code	A1
Filing date	Feb 10, 2020
Priority date	Apr 6, 2015
Publication date	Aug 6, 2020
Grant date	—

Abstract

Methods of making biofilms having non-native functional polypeptides attached thereto are provided.

First claim

1 . A method of making a biofilm comprising
proliferating a bacteria cell including a nucleic acid sequence encoding a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker to produce a population of bacteria cells expressing the fusion and forming a biofilm having the non-native functional polypeptide attached thereto, wherein the linker is a polypeptide comprising 7 or more amino acids attached to either the C terminus or the N terminus of the CsgA protein, wherein the non-native functional polypeptide is selected from the group consisting of TFF1, TFF2 and TFF3.

2 . The method of claim 1 , wherein the nucleic acid sequence is introduced into the bacteria cell.

3 . The method of claim 1 , wherein the bacteria cell is E. coli.

4 - 5 . (canceled)

6 . The method of claim 1 , wherein the bacteria cell includes a genomic deletion of the CsgA gene.

7 - 11 . (canceled)

12 . The method of claim 1 , wherein the linker:
is between 7 and 250 amino acids; is a flexible linker or a rigid linker; is hydrophilic or hydrophobic; comprises a repeating amino acid subunit; comprises 3 repeating amino acid subunits or more; comprises [GGGS] n wherein n is an integer from 2 to 20 (SEQ ID NO:12); comprises [GGGS] n wherein n is an integer being 3, 6, or 12 (SEQ ID NO:5-7); comprises [P] n wherein n is an integer from 1 to 30 (SEQ ID NO:13); comprises [P] n wherein n is an integer being 12 or 24 (SEQ ID NO:8-9); comprises [EAAAK] n wherein n is an integer from 1 to 15 (SEQ ID NO:15); comprises [EAAAK] n wherein n is an integer being 3 or 9 (SEQ ID NO:10-11); comprises one or more of glycine, serine, alanine or leucine; comprises one or more of [GGGS] n wherein n is an integer from 2 to 20 (SEQ ID NO:12), [P] n wherein n is an integer from 1 to 30 (SEQ ID NO:13) or [EAAAK] n wherein n is an integer from 1 to 15 (SEQ ID NO:15); is cleavable; is cleavable by an enzyme; or is GGGSGGGSGGGS, GGGSGGGSGGGSGGGSGGGSGGGS, GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO:5-7), PPPPPPPPPPPP, PPPPPPPPPPPPPPPPPPPPPPPP (SEQ ID NO:8-9), EAAAKEAAAKEAAAK, or EAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO:10-11).

13 - 27 . (canceled)

28 . The method of claim 1 , wherein the non-native functional polypeptide is releasable.

29 - 30 . (canceled)

31 . The method of claim 1 , wherein the bacteria cell is Nissle strain 1917 (EcN) and the linker is [GGGS] n wherein n is an integer being 3, 6, or 12 (SEQ ID NO:5-7)

32 . A non-naturally occurring fusion of a CsgA protein linked to a non-native functional polypeptide by a linker, wherein the linker comprises [GGGS] n wherein n is an integer from 2 to 20 (SEQ ID NO:12),
wherein the non-native functional polypeptide is selected from the group consisting of TFF1, TFF2 and TFF3.

33 . A nucleic acid sequence encoding the fusion of claim 32 .

34 . A vector comprising the nucleic acid sequence of claim 33 .

35 . A bacteria cell comprising the nucleic acid sequence of claim 33 .

36 . (canceled)

37 . A bacteria cell expressing the fusion of claim 32 .

38 . A biofilm comprising the bacteria cell of claim 35 .

39 . A method of delivering a bacteria cell to a tissue within an organism comprising
introducing into the organism the bacteria cell of claim 35 , wherein the bacteria cell attaches to the tissue by the non-native functional polypeptide, thereby localizing the bacteria cell to the tissue.

40 . The method of claim 39 , wherein the bacteria cell proliferates and a biofilm including a population of bacteria cells is formed that is attached to the tissue.

41 - 42 . (canceled)

43 . The method of claim 39 , wherein the tissue within the organism is gastrointestinal tract epithelial tissue, Peyer's Patches, an infection site, an injured area of epithelium, tumor tissue, a site of inflammation, colon carcinoma cells, gut mucosa, or M cells.

44 - 51 . (canceled)

52 . A method of treating an organism with inflammation of the gastrointestinal tract comprising
introducing into the organism the bacteria cell of claim 35 , and wherein the bacteria cell is an E. coli bacterial cell; wherein the bacteria cell attaches to tissue of the gastrointestinal tract by the polypeptide, thereby localizing the bacteria cell to the tissue in a manner to reduce the inflammation.

53 - 54 . (canceled)

55 . The method of claim 52 , wherein the inflammation results from inflammatory bowel disease, Crohn's disease, or ulcerative colitis.

56 - 145 . (canceled)

Assignees

• Harvard College

Inventors

• Joshi Neel Satish
• Nguyen Peter Q
• Duraj-Thatte Anna M
• Praveschotinunt Pichet

Classifications

• A61K35/74

  Bacteria (therapeutic use of a bacterial protein A61K38/00) · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• A61P1/00

  Drugs for disorders of the alimentary tract or the digestive system · CPC title

• C07K14/245

  Escherichia (G) · CPC title

• C12N15/62 Primary

  DNA sequences coding for fusion proteins · CPC title