# US 2025/0146007 A1 · Methods to produce recombinant cutibacterium acnes and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0146007 A1 - Document ID: US20250146007A1 - Status: Published application - Kind: A1 - Application number: 18/832,444 - Filed: 2023-01-24 - Published: 2025-05-08 - Earliest claimed: 2022-01-24 - Applicants: Universitat Pompeu Fabra - Inventors: Marc GÜELL CARGOL; Nastassia KNÖDLSEDER; María José FÁBREGA FERNÁNDEZ; Javier SANTOS MORENO; Guillermo NEVOT SÁNCHEZ - Web page: https://patentgrep.com/patent/US20250146007A1 ## CPC - A61K 38/1709: {from mammals} - C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora - A61K 8/99: from microorganisms other than algae or fungi, e.g. protozoa or bacteria - A61K 31/203: Retinoic acids {; Salts thereof} - A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00) - A61P 17/06: Antipsoriatics - A61P 17/10: Anti-acne agents - A61Q 19/08: Anti-ageing preparations - C07K 14/47: from mammals - A61K 2035/11: {Medicinal preparations comprising living procariotic cells} - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - A61Q: SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to recombinant C. acnes and method of producing thereof. The present invention provides a human neutrophil gelatinase-associated lipocalin (NGAL) protein derived from a recombinant bacterium and its uses thereof. Particularly, the present invention relates to the use of NGAL protein obtained from a recombinant bacterium in the treatment of acne vulgaris or as a cosmetical composition. Provided herein are also recombinant bacterium expressing NGAL protein and their uses. ## Claims ### Claim 1 A method for introducing a nucleic acid into Cutibacterium acnes (C. acnes), the method comprising the steps of: a) providing an unmethylated nucleic acid with the proviso that if it comprises a C. acnes methylation motif, said methylated motif is methylated, and b) introducing said nucleic acid from step a) into C. acnes. ### Claim 2 (depends on 1) The method according to claim 1, wherein C. acnes methylation motif that is methylated in step a) is AGC(m)AGY. ### Claim 3 (depends on 1, 2) The method according to any one of claims 1 or 2, wherein the C. acnes of step b) is obtained by: i) having been previously cultured in the presence of at least one permeabilizing agent of bacterial cell wall, and/or ii) having been previously frozen and thawed. ### Claim 4 (depends on 3) The method according to claim 3, wherein the permeabilizing agent of bacterial cell wall is L-glycine or penicillinG. ### Claim 5 (depends on 1, 2, 3, 4) The method according to any of claims 1 to 4 wherein the nucleic acid sequence of step a) comprises one or more methylated C. acnes methylation motifs in its sequence, wherein said nucleic acid is obtained by introducing a nucleic acid comprising one or more C. acnes methylation motifs in its sequence into a bacterium that is not C. acnes, wherein the bacterium that is not C. acnes is characterized by comprising or expressing a C. acnes methylase and by not comprising or expressing any other methylase, and subsequently collecting said nucleic acid from said bacteria to proceed with step b). ### Claim 6 (depends on 5) The method according to claim 5, wherein the C. acnes methylase is a C. acnes IIIB methylase. ### Claim 7 (depends on 1, 2, 3, 4) The method according to any of claims 1 to 4 wherein the nucleic acid sequence of step a) is unmethylated and does not comprise any C. acnes methylation motifs in its sequence, wherein said nucleic acid sequence is obtained by introducing a nucleic acid that does not comprise any C. acnes methylation motifs in its sequence into a bacterium that is not C. acnes, wherein the bacterium that is not C. acnes is characterized by not expressing any other methylase, and subsequently collecting said nucleic acid from said bacterium to proceed with step b). ### Claim 8 (depends on 5, 6, 7) The method according to any one of the claims 5 to 7, wherein the bacterium that is not C. acnes is a dam− dcm− hsdMS− E. coli strain. ### Claim 9 (depends on 5, 6, 7, 8) The method according to any one of the claims 5 to 8, wherein the bacterium that is not C. acnes is the E. coli strain deposited in the Spanish Collection of Type Cultures with CECT No. 30749. ### Claim 10 The method according to any one of the previous claims, wherein the nucleic acid comprises C. acnes integration elements that lead the integration of said target nucleic acid or at least a portion thereof in the genome of C. acnes. ### Claim 11 The method according to any of the previous claims, wherein the C. acnes that have been transformed are selected, and wherein the selection is based on at least two bacterial selection markers. ### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) A recombinant C. acnes obtained by the method of claims 1 to 11. ### Claim 13 (depends on 12) The recombinant C. acnes according to claim 12, wherein the nucleic acid encodes for the human neutrophil gelatinase-associated lipocalin (NGAL) gene and wherein the recombinant C. acnes is characterized in that it expresses, and preferably secretes, NGAL protein. ### Claim 14 (depends on 12, 13) A recombinant C. acnes according to claims 12 or 13, for use in therapy. ### Claim 15 (depends on 12, 13) A recombinant C. acnes according to claims 12 or 13, for use in diagnosis. ### Claim 16 (depends on 12, 13) Use of the C. acnes of claims 12 or 13, in cosmetics. ### Claim 17 A recombinant Cutibacterium acnes characterized in that it expresses the human neutrophil gelatinase-associated lipocalin (NGAL) protein. ### Claim 18 (depends on 17) The recombinant Cutibacterium acnes according to claim 17, wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over the full length to SEQ ID NO: 1. ### Claim 19 (depends on 17, 18) The recombinant Cutibacterium acnes according to any one of the claims 17 or 18, wherein the nucleic acid encoding for the human neutrophil gelatinase-associated lipocalin (NGAL) is operably linked to a promoter selected from the list consisting of camp2 promoter, camp1 promoter or roxP promoter. ### Claim 20 (depends on 17, 18, 19) A recombinant Cutibacterium acnes according to any one of the claims 17 to 19, for use in the treatment of acne vulgaris, urticaria, eczema, rosacea, Hidradenitis suppurativa and/or psoriasis. ### Claim 21 (depends on 17, 18, 19) A non-therapeutical cosmetic use of the recombinant Cutibacterium acnes according to any one of claims 17 to 19, wherein the use is preferably to prevent, reduce and/or ameliorate skin aging. ### Claim 22 (depends on 20, 21) A recombinant Cutibacterium acnes for use according to claims 20 or 21, wherein the use comprises the topic administration of the recombinant C. acnes into the skin. ### Claim 23 (depends on 17, 18, 19) A method for producing the active ingredient of a pharmaceutical composition, wherein said active ingredient is a human neutrophil gelatinase-associated lipocalin (NGAL) protein, wherein the method comprises culturing the recombinant Cutibacterium acnes defined in any of claims 17 to 19. ### Claim 24 (depends on 23) The method according to claim 23, wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over the full length to SEQ ID NO: 1. ### Claim 25 (depends on 23, 24) The method according to any of claims 23 or 24, wherein the recombinant bacterium is Cutibacterium acnes and wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over the full length to SEQ ID NO: 1.