Chimeric receptor binding proteins for use in bacterial delivery vehicles

Patent metadata

Field	Value
Publication number	US-11236133-B2
Application number	US-202016816675-A
Country	US
Kind code	B2
Filing date	Mar 12, 2020
Priority date	Nov 27, 2018
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

Abstract

The present disclosure relates generally to bacterial delivery vehicles for use in efficient transfer of a desired payload into a target bacterial cell. More specifically, the present disclosure relates to bacterial delivery vehicles with desired host ranges based on the presence of a chimeric receptor binding protein (RBP) composed of a fusion between the N-terminal region of a RBP derived from a lambda-like bacteriophage and the C-terminal region of a different RBP.

First claim

What is claimed is:

1. A chimeric receptor binding protein (RBP) wherein the chimeric RBP comprises a fusion between the N-terminal domain of a RBP from a lambda-like or lambda bacteriophage, wherein said lambda-like bacteriophage comprises amino acid sequence homology of around 35% identity for 45 amino acids or more, around 50% identity for 30 amino acids or more, or around 90% identity for 18 amino acids or more in one or more of three amino acid regions ranging from positions 1-150, 320-460, and 495-560 with reference to the lambda bacteriophage stf sequence of SEQ ID NO: 1, and the C-terminal domain of a different RBP and wherein said N-terminal domain is fused to said C-terminal domain within one of the amino acid regions selected from positions 1-150, 320-460 or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1), wherein said region of fusion within the N-terminal RBP from positions 1-150, 320-460 or 495-560 comprises amino acid sequence homology of around 35% identity for 45 amino acids or more, around 50% identity for 30 amino acids or more, or around 90% identity for 18 amino acids or more with reference to the lambda bacteriophage stf sequence of SEQ ID NO:1.

2. The chimeric RBP of claim 1 , wherein said different RBP is derived from any bacteriophage or bacteriocin.

3. The chimeric receptor binding protein (RBP) of claim 2 , wherein the different RBP comprises amino acids sequence homology of around 35% identity for 45 amino acids or more, around 50% identify for 30 amino acids or more, or around 90% identity for 18 amino acids or more in one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 with reference to the lambda bacteriophage stf sequence of SEQ ID NO: 1.

4. The chimeric RBP of claim 1 , wherein said N-terminal domain of the chimeric RBP is fused to said C-terminal domain within one of the amino acid regions selected from positions 80-150, 320-460, or 495-560 of the N-terminal RBP.

5. The chimeric RBP of claim 1 , wherein the N-terminal domain and the C-terminal domain are fused within said region at an insertion site having at least 80% identity with insertion site selected from the group consisting of amino acids SAGDAS (SEQ ID NO:248), ADAKKS (SEQ ID NO: 249), MDETNR (SEQ ID NO: 250), SASAAA (SEQ ID NO: 251), and GAGENS (SEQ ID NO: 252).

6. The chimeric RBP of claim 1 , wherein the chimeric RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56, 59, 123-129, 130, 131, 132, 135, 138, 139, 142, 145, 148, 151, 216, 219, 221, 223, 227, 230, 232, 234, 236, 238, 240, 243, 245 or 246.

7. The chimeric RBP of claim 1 , wherein the C-terminal domain of the different RBP has a depolymerase activity against an encapsulated bacterial strain.

8. A bacterial delivery vehicle comprising the chimeric RBP of claim 1 .

9. The bacterial delivery vehicle of claim 8 wherein said different RBP is derived from any bacteriophage or bacteriocin.

10. The bacterial delivery vector of claim 9 , wherein the different RBP comprises amino acids sequence homology of around 35% identity for 45 amino acids or more, around 50% identify for 30 amino acids or more, or around 90% identity for 18 amino acids or more in one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 with reference to the lambda bacteriophage stf sequence of SEQ ID NO: 1.

11. The bacterial delivery vehicle of claim 8 , wherein said N-terminal domain of the chimeric RBP is fused to said C-terminal domain within one of the amino acid regions selected from positions 80-150, 320-460 or 495-560 of the N-terminal RBP with reference to the lambda stf sequence.

12. The bacterial delivery vehicle of claim 8 , wherein the N-terminal domain and the C-terminal domain of the chimeric RBP are fused within said region at an insertion site having at least 80% identity with insertion site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 248), ADAKKS (SEQ ID NO: 249), MDETNR (SEQ ID NO: 250), SASAAA (SEQ ID NO: 251), and GAGENS (SEQ ID NO: 252).

13. The bacterial delivery vehicle of claim 8 , wherein the chimeric RBP comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56, 59, 123-129, 130, 131, 132, 135, 138, 139, 142, 145, 148, 151, 216, 219, 221, 223, 227, 230, 232, 234, 236, 238, 240, 243, 245 or 246.

14. The bacterial delivery vehicle of claim 8 , wherein the C-terminal domain of the different RBP has a depolymerase activity against an encapsulated bacterial strain.

15. The bacterial delivery vehicle of claim 8 , further comprising a nucleic acid payload encoding a protein of interest or a nucleic acid of interest.

16. The bacterial delivery vehicle of claim 15 , wherein the nucleic acid of interest is selected from the group consisting of a Cas nuclease gene, a Cas9 nuclease gene, a guide RNA, a CRISPR locus, a toxin gene, a gene expressing an enzyme such as a nuclease, a kinase, a TALEN, a ZFN, a meganuclease, or a recombinase, a bacterial receptor gene, a membrane protein gene, a structural protein gene, a secreted protein gene, a gene expressing resistance to an antibiotic or resistance to a drug in general, a gene expressing a toxic protein or a toxic factor, and a gene expressing a virulence protein or a virulence factor, or any combination thereof.

17. The bacterial delivery vehicle of claim 15 , wherein the protein of interest is a nuclease that targets cleavage of a host bacterial cell genome or a host bacterial cell plasmid.

18. The bacterial delivery vehicle of claim 17 , wherein the cleavage occurs in an antibiotic resistant gene.

19. The bacterial delivery vehicle of claim 15 , wherein the nucleic acid payload encodes a therapeutic protein.

20. The bacterial delivery vehicle of claim 15 , wherein the nucleic acid payload encodes an anti-sense nucleic acid molecule.

21. A pharmaceutical or veterinary composition comprising one or more bacterial delivery vehicles of claim 8 and a pharmaceutically-acceptable carrier.

Assignees

• Eligo Bioscience

Inventors

• Fernandez Rodriguez Jesus

Classifications

• C12N2795/10022

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N2795/10152

  relating to complementing cells and packaging systems for producing virus or viral particles · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2795/10322

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title