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EP-4739777-A1 - TRANSMISSIBLE ELEMENTS COMPRISING PATHWAY MODIFICATION SYSTEMS AND EXOGENOUS NUCLEIC ACIDS FOR THE PRODUCTION OF MOLECULES OF INTEREST

EP4739777A1EP 4739777 A1EP4739777 A1EP 4739777A1EP-4739777-A1

Abstract

The present invention relates to transmissible elements (e.g. conjugative plasmids, phage and phagemid) which are engineered to express molecules of interest (MOIs) and a pathway modification system (PMS) which is able to introduce mutations in target endogenous genes in a recipient bacterium, or is able to produce nucleic acids which inhibit or increase expression of target endogenous genes in a recipient bacterium, or is able to express peptide inhibitor molecules which inhibit a target endogenous protein in a recipient bacterium. The invention relates to host cells containing the transmissible elements, to pharmaceutical compositions containing such transmissible elements or host cells, and their use in the treatment of various diseases.

Inventors

  • LE MIÈRE, Jennifer
  • SØNDBERG, Emilie
  • SEMSEY, Szabolcs
  • MOUGIAKOS, Ioannis

Assignees

  • SNIPR Biome ApS

Dates

Publication Date
20260513
Application Date
20240703

Claims (20)

  1. Claims 1. A transmissible element (e.g. a plasmid, conjugative plasmid, phage or phagemid) for transmission to a recipient bacterium, or a bacterium comprising said transmissible element, wherein the transmissible element comprises: A) at least one exogenous nucleic acid sequence to the recipient bacterium for the production of a first molecule of interest (MOI) in the recipient bacterium; and B) a Pathway Modulation System (PMS) which encodes a nucleic acid modifier or a peptide molecule to modulate a target endogenous nucleic acid or protein for the production or consumption (or degradation) of the first MOI in the recipient bacterium.
  2. 2. The transmissible element or bacterium according to claim 1 for use as medicament.
  3. 3. The transmissible element or bacterium according to claim 1 or claim 2, wherein the exogenous nucleic acid (G1) of A) encodes the first MOI (A).
  4. 4. The transmissible element or bacterium according to any preceding claim, wherein the first MOI is a therapeutic molecule, such as a peptide molecule (e.g. an antibody fragment, a hormone such as GLP-1, an interleukin, a cytokine, a chemokine, an eukaryotic growth factor, or an enzyme) or a small molecule (e.g. a metabolite such as L-DOPA, indole-3-acetic acid, butyrate etc).
  5. 5. The transmissible element or bacterium according to any preceding claim, wherein the exogenous nucleic acid of A) encodes one or more (e.g. one) exporter(s) of the first MOI from the recipient bacterium.
  6. 6. The transmissible element or bacterium according to any preceding claim, wherein the exogenous nucleic acid of A) further encodes a sequence encoding a signal peptide for the secretion of the first MOI to the periplasm of the recipient bacterium, to the cell surface of the recipient bacterium, or to the extracellular space outside of the recipient bacterium.
  7. 7. The transmissible element or bacterium according to any preceding claim, wherein the exogenous nucleic acid of A) encodes, in 5’ to 3’ direction, a promoter, a sequence encoding a signal peptide, at least one nucleic acid for the expression of the first MOI and optionally a further nucleic acid sequence encoding one or more (e.g. one) exporter(s) of the first MOI from the recipient bacterium.
  8. 8. The transmissible element or bacterium according to claim 1 or claim 2, wherein the exogenous nucleic acid of A) encodes one or more protein(s) (G1, G2… GX) for the conversion of a second MOI (B) to the first MOI (A).
  9. 9. The transmissible element or bacterium according to claim 8, wherein the exogenous nucleic acid of A) encoding one or more protein(s) (G1, G2… GX) is comprised by one or more operons (e.g. by one operon).
  10. 10. The transmissible element or bacterium according to claim 9, wherein the one or more operons are under the control of a constitutive promoter.
  11. 11. The transmissible element or bacterium according to any one of claims 8 to 10, wherein the first MOI is selected from a therapeutic molecule (e.g. a peptide molecule) and a beneficial or therapeutic metabolite, in particular a beneficial or therapeutic metabolite, or alternatively, the second MOI is a detrimental metabolite.
  12. 12. The transmissible element or bacterium according to any one of claims 9 to 11, wherein the exogenous nucleic acid of A) further encodes one or more (e.g. one) exporter(s) of the first MOI, and/or one or more (e.g. one) importer(s) of the second MOI, and/or one or more (e.g. one) importer(s) of any further substrate which is converted by one of the protein(s) (G1, G2… GX) for the conversion of the second MOI (B) to the first MOI (A).
  13. 13. The transmissible element or bacterium according to claim 11 or claim 12, wherein the first MOI is a sugar which can be used by the recipient bacterium as a carbon source and the second MOI is rare carbohydrate, optionally wherein the rare carbohydrate is selected from the group consisting of porphyran, agarose, carrageenan, and any combination thereof and the exogenous nucleic acid of A) encodes one or more protein(s) selected from the group consisting of porphyranase, glycoside hydrolase, sulfatase, galactosidase, and any combination thereof.
  14. 14. The transmissible element or bacterium according to claim 13, wherein the exogenous nucleic acid of A) further encodes an importer for the importation of the rare carbohydrate.
  15. 15. The transmissible element or bacterium according to any preceding claim, wherein the nucleic acid modifier expressed by the PMS of B) reduces expression (or prevents expression) of the target endogenous nucleic acid, and wherein the reduction in expression of the target endogenous nucleic acid (i) increases or maintains the production of the first MOI, or (ii) reduces consumption or degradation of the first MOI.
  16. 16. The transmissible element or bacterium according to claim 15, wherein: the target endogenous nucleic acid encodes an enzyme (D1) which degrades the first MOI (A), and the reduction of expression of the enzyme increases or maintains (e.g. increases) production of the first MOI by reducing consumption or degradation of the first MOI.
  17. 17. The transmissible element or bacterium according to claim 15, wherein: the exogenous nucleic acid of A) encodes one or more protein(s) (G1, G2… GX) for the conversion of a second MOI (B) to the first MOI (A), and wherein the recipient bacterium comprises an endogenous pathway for the conversion of one or more substrates (S, S1, S2… etc) into the second MOI (B), and wherein the target endogenous nucleic acid encodes: (a) an enzyme (E3) in the endogenous pathway which converts the second MOI (B) into a substrate (S1) which is earlier in the endogenous pathway, and the reduction of expression of the enzyme (E3) increases or maintains (e.g. increases) production of the first MOI by reducing degradation of said second MOI (B) to said substrate (S1) in the recipient bacterium; or (b) an enzyme (E4) in the endogenous pathway which converts a first substrate (S1) used in the production of the second MOI (B) into a second substrate (M1) which is not used in the production of said second MOI, and the reduction of expression of the enzyme (E4) increases or maintains (e.g. increases) production of the first MOI by increasing the amount of said first substrate (S1) in the recipient bacterium; or (c) an enzyme (E5) in the endogenous pathway which converts a first substrate (S1) of the endogenous pathway into a second substrate (S) which is earlier in the endogenous pathway, and the reduction of expression of the enzyme (E5) increases or maintains (e.g. increases) production of the first MOI by reducing degradation of said first substrate (S1) in the recipient bacterium; or (d) an exporter (EXP2) of a substrate in the endogenous pathway (S1), and the reduction of expression of the exporter (EXP2) increases or maintains (e.g. increases) production of the first MOI by increasing the amount of said substrate (S1) in the recipient bacterium.
  18. 18. The transmissible element or bacterium according to claim 15, wherein: the exogenous nucleic acid of A) encodes one or more protein(s) (G1, G2… GX) for the conversion of a second MOI (B) to the first MOI (A); and wherein the recipient bacterium comprises: a first endogenous pathway comprising at least one enzyme for the conversion of one or more substrates (S, S1, S2… etc) into the second MOI (B), and a second endogenous pathway comprising at least one enzyme (E1) which is common to the first and second endogenous pathways, and the at least one common enzyme (E1) can convert a first substrate of the first endogenous pathway (S) and a second, different substrate of the second endogenous pathway (X); and wherein the target endogenous nucleic acid encodes: (a) an importer (IMP2) which imports the second, different substrate of the second endogenous pathway (X), and the reduction of expression of the importer (IMP2) increases or maintains (e.g. increases) production of the first MOI by increasing the availability and/or activity of said common enzyme (E1) to convert said first substrate (S) in the first endogenous pathway in the recipient bacterium; or (b) an enzyme which is part of the second endogenous pathway and is for the production of the second, different substrate of the second endogenous pathway (X), for example is an endogenous enzyme (P1) which is part of the second endogenous pathway and is for the direct production of the second, different substrate of the second endogenous pathway (X), and the reduction of expression of the enzyme (P1) increases or maintains (e.g. increases) production of the first MOI by reducing the amount of the second, different substrate of the second endogenous pathway (X), thereby increasing the availability and/or activity of said common enzyme (E1) to convert said first substrate (S) in the first endogenous pathway in the recipient bacterium.
  19. 19. The transmissible element or bacterium according to any one of claims 15 to 18, wherein the nucleic acid modifier expressed by the PMS of B) reduces expression (or prevents expression) of the target endogenous nucleic acid by (i) introducing a modification to one or more nucleotides in the endogenous DNA (e.g. comprised by the chromosome or a plasmid) of the recipient bacterium, for example by one or more point mutations in one or more codons of the target endogenous nucleic acid, or (ii) producing a nucleic acid inhibitor molecule, for example a small regulatory RNA (sRNA).
  20. 20. The transmissible element or bacterium according to claim 19(i), wherein: a. the target endogenous nucleic acid encodes a protein (such as an enzyme, importer or exporter) and the modification introduces a stop codon in the target endogenous nucleic acid, thereby preventing expression of said protein, or truncating expression such that any expressed protein is non-functional; or b. the target endogenous nucleic acid comprises a promoter to control expression of a protein expressed from the target endogenous nucleic acid (such as an enzyme, importer or exporter) and the modification modifies the promoter, thereby reducing or preventing expression of said protein; or c. the target endogenous nucleic acid comprises a ribosome binding site or a translational start site to control expression of a protein expressed from the target endogenous nucleic acid (such as an enzyme, importer or exporter) and the modification modifies the ribosome binding site or the translational start site, thereby reducing or preventing expression of said protein.

Description

TRANSMISSIBLE ELEMENTS COMPRISING PATHWAY MODIFICATION SYSTEMS AND EXOGENOUS NUCLEIC ACIDS FOR THE PRODUCTION OF MOLECULES OF INTEREST Background to invention There have been many attempts to engineer bacteria to produce molecules of interest (MOIs) in situ in local environments, such as the gut microbiome of animals, such as humans, or in soil, water and other natural microbiomes (see for example, Omer et al., Frontiers Bioengineering & Biotechnology, 10, 870675, 2022, doi:10.3389/fbioe.2022.870675, and Bai et al., Nat. Rev. Bioeng., 1, 665–679, 2023, doi:https://doi.org/10.1038/s44222-023-00072-2, for human medical uses; Li et al., AIChE J., 69(12), e18228, 2023, doi:10.1002/aic.18228 and Zhang et al., Environmental Science: Water Research & Technology, 6, 1967-1985, 2020, doi https://doi.org/10.1039/D0EW00393J for water treatment; and Ribello et al., Bioengineered, 12(2), 12839–12853, 2021, doi: 10.1080/ 21655979.2021.2004645 for a review on bacteria-based soil treatments). For example, it has been proposed that engineered bacteria can be used to deliver several different categories of molecule for therapeutic uses. For example, Lactococcus lactis has been engineered to deliver anti-tumor necrosis factor (TNF)-α antibody for the treatment of chronic inflammatory bowel disease (IBD), see Vandenbroucke et al., Mucosal Immunol., 3, 2010, 49-56; and Bacteroides ovatus has been engineered to produce human keratinocyte growth factor-2 (KGF-2) to treat IBD, see Hamady et al., Gut, 59, 2010, 461-469. In other fields, Bacillus subtilis has been engineered to express a S- adenosylmethionine methyltransferase gene (CmarsM) which converts most of the inorganic arsenic contained in organic manure into dimethylarsenate and trimethylarsine oxide, as a bioremediation strategy, see Huang et al., Appl. Environ. Microbiol., 81 (19), 2015, 6718–6724. In water treatment applications, Synechococcus elongatus has been engineered to produce an oxidative laccase enzyme which can degrade a number of different water contaminants, such as dyes and other chemical pollutants, see Datta et al., Nature Communications, 14, 4742, 2023. One ongoing problem which is yet to be addressed is how to effectively introduce such modified bacteria in a way that they reach a sufficient colonisation level to allow them to produce quantities of the MOI to produce the desired effect. In human applications, often the administration of antibiotics precedes the administration of the modified bacteria, killing the existing bacteria in the microbiome to create a niche, into which the modified bacteria can grow. However, there are a number of problems associated with this approach: 1. The use of antibiotics tends to remove bacteria indiscriminately, and may remove helpful and useful bacteria from the microbiome of interest; 2. The remaining bacteria may comprise a higher amount of antibiotic resistant bacteria, which may subsequently be more difficult to remove; 2. The engineered bacteria must compete for colonisation of the niche with the bacteria remaining in the microbiome of interest. This may result in fewer of the engineered bacteria than desired and/or an increase in undesirable (e.g. antimicrobial resistant) bacteria filling the niche if those undesirable bacteria have a competitive advantage over the engineered bacteria. It has been proposed to use conjugative plasmids (see, for example, WO2020/010452A1 (Société de Commercialisation des Produits de la Recherche Appliquée Socpra Sciences Santé et Humaines S.E.C.), WO2021/037732A1 (SNIPR Biome, Aps), WO2020/229372A1 (Folium Food Science, Limited), WO2015/069682A2 (President and Fellows of Harvard College), and Hamilton et al., Nature Comms, 10:4544, 2019, doi: https://doi.org/10.1038/s41467-019-12448-3), or phage (see, for example, WO2021/250284A1 (Eligo Bioscience), WO2021/092210A1 and WO2022/098916A1 (Locus Biosciences, Inc) and WO2016/205276A1 (North Carolina State University)) to deliver exogenous genes to target recipient bacteria. The main purpose of these disclosures is killing of the recipient bacteria, re-sensitisation to antibiotics or delivery of genetic cargo providing a competitive advantage to the recipient bacteria. Some disclose the addition of the exogenous genes and circuitry for destroying the delivered plasmids (known as kill switches), which target and cut the plasmids comprised by the recipient bacteria. WO2021/204967A1 (Eligo Biosciences) proposes to use phage to deliver a base editor to a target recipient cell which edits a target sequence in the recipient bacteria The present invention aims to solve some of the above-mentioned problems. Summary The present invention, instead of trying to introduce a modified bacterium directly into the microbiome of interest uses transmissible elements, which may be comprised within a donor bacterium, a phage or as part of a packaged phagemid. These transmissible elements provide the recipient bacteria, i.e. those which already exist within