Methods for producing recombinant Cutibacterium acnes and their uses

Abstract

The present invention relates to recombinant C. acnes and methods for its production. The present invention provides a recombinant bacterially derived human neutrophil gelatinase-associated lipocalin (NGAL) protein and uses thereof. In particular, the present invention relates to the use of NGAL protein obtained from recombinant bacteria in the treatment of acne vulgaris or as a cosmetic composition. Also provided herein are recombinant bacteria expressing NGAL protein and uses thereof.

Description

[Technical field]

[0001] The present invention relates to recombinant C. acnes and methods for its production. The present invention relates to the field of skin disorders. In particular, the present invention relates to the use of bacterially produced neutrophil gelatinase-associated lipocalin (NGAL) protein in subjects in need of treatment for retinoid-responsive conditions, particularly acne vulgaris. [Background technology]

[0002] Retinoids (RA) have been used as treatments for a number of skin disorders, from psoriasis to acne and wrinkles. Acne vulgaris is a common chronic skin disorder involving blockage and / or inflammation of the pilosebaceous unit (hair follicles and their associated sebaceous glands). Acne can exist as non-inflammatory lesions, inflammatory lesions, or a mixture of both, and it primarily affects the face, but also the back and chest.

[0003] Acne vulgaris affects nearly 10% of people worldwide. One of the pathogenic factors for the development of acne is the overgrowth of Cutibacterium acnes (C. acnes (also called Propionibacterium acnes or P. acnes)), the most abundant commensal of human skin, which resides in the pilosebaceous unit within the hair follicle.

[0004] Treatments include topical agents, hormonal treatments, or oral treatments. In severe cases of acne, the only effective treatment is isotretinoin, a systemic drug also known as 13-cis retinoic acid (13-cis RA). However, this drug is associated with serious side effects, including depression, psychosis, anemia, and teratogenicity. Approximately 20% of acne patients develop severe acne, and due to the amount and severity of side effects resulting from isotretinoin treatment, replacement is still required, which can lead to release of acne symptoms.

[0005] It has been shown that patients treated with isotretinoin have a seven-fold increase in neutrophil gelatinase-associated lipocalin 2 (NGAL) protein, and this increase leads to a decrease in sebum production, ultimately resulting in a reduction in the number of C. acnes. Sebocytes treated with C. acnes increased NGAL expression and secretion via a TLR-2-dependent mechanism. Thus, NGAL has been shown to mediate the apoptotic response of isotretinoin in human sebocytes. Furthermore, NGAL is not only an apoptotic agent but also has antibacterial functions, as it can bind bacterial siderophores and prevent bacteria from receiving the iron required for cellular processes. With this in mind, the most widely accepted hypothesis for the efficacy observed upon isotretinoin treatment is that the drug causes an increase in NGAL protein, resulting in both sebum reduction and antibacterial activity, reducing the proliferation of P. acnes in the skin of affected individuals.

[0006] To use NGAL as an alternative to isotretinoin in the treatment of acne, recombinant protein production is required. Protein can be produced in eukaryotic or prokaryotic cells. However, eukaryotic cell-based production systems are more expensive and not industrially scalable, while prokaryotic protein synthesis can be much faster and cheaper, since bacterial cells are easier to produce, grow and maintain. Importantly, when a condition affects millions of people around the world, such as acne or acne-related diseases, the scalability and cost-effectiveness of treatment are important factors to be considered.

[0007] Several reports indicate that human recombinant NGAL (rhNGAL) produced in eukaryotic cells can induce apoptosis in SEB-1 sebaceous gland cells (Nelson et al. Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid-induced apoptosis of human sebaceous gland cells. J Clin Invest. 2008 Apr;118(4):1468-78. doi:10.1172 / JCI33869). At the same time, other reports have shown that recombinant NGAL from prokaryotic sources (produced in E. coli) does not induce apoptosis in TSS-1 sebaceous cells (Kimberly Ruth Lumsden. The innate immune protein neutrophil gelatinase-associated lipocalin is involved in the early therapeutic response to 13-cis retinoic acid in acne patients. Doctor of Philosophy, May 2011), indicating that post-translational modifications of NGAL (such as glycosylation patterns) inherent to the production method may affect the functionality of the protein. Furthermore, due to the antibacterial activity shown by NGAL in prokaryotic cells such as E. coli, P. acnes or Mycobacterium tuberculosis (Goetz, Holmes, Borregaard et al. 2002; Martineau, Newton, Wilkinson et al. 2007), strategies aimed at producing this protein using prokaryotic cells proved impossible. Overall, the current state of the art indicates that recombinant NGAL produced in bacteria does not have the same function or expression pattern as endogenous NGAL produced in eukaryotic cells, and therefore, recombinant NGAL produced in eukaryotic cells represents the most promising NGAL-based treatment for acne, albeit more expensive and less scalable.

[0008] The present invention aims to provide an industrially scalable and cost-effective system for producing NGAL protein based on the use of prokaryotic cells without interfering with the effectiveness of NGAL protein in reducing sebum production.

[0009] Meanwhile, the second objective of the present invention is to provide an efficient method for introducing nucleic acids into C. acnes, which has a highly efficient restriction-modification system (RM system) that functions as a form of bacterial immune system that can protect the host bacterium from foreign DNA invasion. The RM system consists of two enzyme components, DNA methyltransferase (MTase) and restriction endonuclease (REase). MTase methylates specific sites in DNA sequences, whereas REase cleaves DNA in a sequence-specific manner. The presence of both the restriction and modification components of the RM system allows C. acnes to essentially distinguish between "self" and "non-self" genetic material. However, this RM methylation system efficiently degrades foreign nucleic acids introduced into this bacterium, thus presenting an obstacle to the efficient production of recombinant C. acnes.

[0010] The present invention solves the above problems, overcomes the RM system of C. acnes, and provides an improved method for producing recombinant C. acnes. Summary of the Invention

[0011] In one aspect, the present invention provides a method for introducing a nucleic acid into C. acnes, comprising the steps of: a) providing an unmethylated nucleic acid, provided that if it contains a C. acnes methylation motif, said methylation motif is methylated; and b) introducing the nucleic acid from step a) into C. acnes.

[0012] Preferably, the C. acnes methylation motif which is methylated in step a) is AGC(m)AGY and the unmethylated C. acnes methylation motif is AGCAGY.

[0013] Preferably, the C. acnes in step b) is obtained by i) having previously been cultured in the presence of at least one agent for permeabilizing the bacterial cell wall and / or ii) having previously been frozen and thawed.

[0014] Preferably, the nucleic acid sequence of step a) comprises one or more methylated C. acnes methylation motifs in its sequence, said nucleic acid being obtained by introducing a nucleic acid comprising one or more C. acnes methylation motifs in its sequence into a non-C. acnes bacterium, said non-C. acnes bacterium being characterised in that it comprises or expresses a C. acnes methylase and does not comprise or express other methylases, from which said nucleic acid is then harvested for proceeding with step b).Preferably, the C. acnes methylase is the C. acnes IIIB methylase.

[0015] Preferably, the nucleic acid sequence of step a) is unmethylated and does not comprise a C. acnes methylation motif in its sequence, said nucleic acid sequence being obtained by introducing a nucleic acid that does not comprise a C. acnes methylation motif in its sequence into a non-C. acnes bacterium, which is characterized in that it does not express other methylases, from which said nucleic acid is then harvested in order to proceed with step b).

[0016] Preferably, the non-C. acnes bacterium is the dam-dcm-hsdMS-E. coli strain, most preferably the E. coli strain deposited at the Spanish Collection of Type Cultures under CECT No. 30749.

[0017] Preferably, the nucleic acid comprises a C. acnes integration element leading to integration of said target nucleic acid or at least a part thereof in the genome of C. acnes. Preferably, the nucleic acid encodes the human neutrophil gelatinase-associated lipocalin (NGAL) gene, and the recombinant C. acnes is characterized in that it expresses, and preferably secretes, the NGAL protein.

[0018] Preferably, transformed C. acnes are selected, the selection being based on at least two bacterial selectable markers.

[0019] In another aspect, the present invention relates to a recombinant C. acnes obtainable by the method of the preceding aspect. Preferably, said recombinant C. acnes expresses the NGAL protein.

[0020] In another aspect, the present invention relates to the therapeutic, diagnostic and cosmetic use of the recombinant C. acnes of the preceding aspect. Preferably, the use comprises topical administration of the recombinant C. acnes to the skin.

[0021] In another aspect, the present invention refers to a recombinant Cutibacterium acnes characterized in that it expresses human neutrophil gelatinase-associated lipocalin (NGAL) protein. Preferably, the NGAL protein has at least 85% amino acid sequence identity over its entire length with SEQ ID NO: 1. Preferably, said NGAL is operably linked to a promoter selected from the group consisting of camp2 promoter, camp1 promoter or roxP promoter. In a preferred aspect, C. acnes expressing NGAL is used in therapy or as a medicament or in medicine, preferably for the treatment of acne vulgaris, urticaria, eczema, rosacea, hidradenitis suppurativa and / or psoriasis. Another possible use is in cosmetics, preferably for preventing, reducing and / or improving skin aging. Preferably, the use comprises topical administration of recombinant C. acnes to the skin.

[0022] In a further aspect, the present invention relates to a method for producing an active ingredient of a pharmaceutical composition, wherein said active ingredient is human neutrophil gelatinase-associated lipocalin (NGAL) protein, the method comprising culturing a recombinant Cutibacterium acnes of the preceding aspect. [Brief description of the drawings]

[0023] [Figure 1]Cytotoxicity assay of rhNGAL on human SZ95 sebaceous cells incubated with different concentrations from 0, 50, 100, 500, 1000ng / mL. After 24 hours, cell viability was measured by MTT assay. The average of four independent experiments is shown. Untreated cells were used as negative control. [Diagram 2] NGAL toxicity study against C. acnes cultures. Data represent experimental duplicates treated with 0, 100, 500 or 1000 ng / mL of rhNGAL protein. Proteins used were expressed and purified from E. coli BL21 cells. [Diagram 3] Western blot of cytosolic (pellet) and secreted (supernatant) fractions of C. acnes pBR13, pBR14 and pBR16 compared to 40ug (pBR13), 10ug (pBR14) and 25ug (pBR16) purified protein. [Figure 4] To induce sebum production, sebocytes PCi-SEB Cau were treated with 5 uM arachidonic acid (AA5) and either 10 uM isotretinoin, rhNGAL produced in (E. coli), or NGAL pBR13 (produced in C. acnes) for 48 hours. Lipids were stained by using BODIPY dye and visualized by confocal microscopy. Data are presented as normalized BODIPY intensity after treatment with NGAL from C. acnes pBR13 compared to isotretinoin (10 uM) and recombinant NGAL protein (50 ng / mL). Results are shown as the mean standard deviation (SD) of triplicate measurements (p<0.05) relative to vehicle control. [Diagram 5] TDK test (see Shao X, et al. A markerless gene deletion and integration system for Thermoanaerobacter ethanolicus. Biotechnol Biofuels. 2016;9:100. Published 2016 May 4. doi:10.1186 / s13068-016-0514-1). [Figure 6]Western blot of NGAL protein produced in either HEK293, E. coli or C. acnes. HEK293-produced NGAL is glycosylated and therefore of higher molecular weight than the E. coli-produced protein. [Figure 7] Functional testing of KO strains on selective agar plates containing no antibiotic / inhibitor, 50 μg / mL FUDR (inhibitor) or 10 μg / mL erythromycin. [Figure 8] Viable C. acnes cells incubated with HEK293 produced NGAL. [Figure 9] The viable fraction of C. acnes incubated with E. coli produced NGAL. [Figure 10] Viable PCi-SEB_Cau sebocytes incubated for 24 h with HEK293 produced human NGAL. [Figure 11] Viable HEK293 and SZ95 sebocyte cells incubated for 24 hours produced human NGAL. [Figure 12] TUNEL assay of PCi-SEB_Cau incubated with NGAL produced in HEK293 (human), E. coli (rh), or C. acnes (pBR13, 14, 16) at concentrations ranging from 0 μg / mL to 1 μg / mL. The percentage of TUNEL-positive cells was determined by fluorescence microscopy. [Figure 13] TUNEL assay of SZ95 incubated with NGAL produced in HEK293 (human), E. coli (rh), or C. acnes (pBR13, 14, 16) at concentrations ranging from 0 μg / mL to 1 μg / mL. The percentage of TUNEL-positive cells was determined by fluorescence microscopy. [Figure 14]Fluorescence measurements of competent cells prepared with different electroporation buffers (EPB) and transformed with fluorescently labeled Cy3 oligonucleotides. DNA internalization is measured by fluorescence intensity. EPB-1: 0.5 M sucrose, 1 mM KOAc, pH 5.8, EPB-2: 30% PEB6000, 6.6% sucrose, EPB-3: 272 mM sucrose, 7 mM NaPi, 1 mM MgCl2, EPB-4: 272 mM sucrose, EPB-5: H20. [Figure 15] Fluorescence measurements of cells after transfection with Cy3 oligonucleotides compared with 1 hour recovery. [Figure 16] Comparison of fresh versus frozen cell transformation efficiencies measured in the resulting transformants. [Figure 17] Transformants obtained with DNA mimicking the C. acnes methylation pattern (extracted from E. coli CECT No. 30749) compared with DNA containing the hsdMS methylation pattern (extracted from E. coli dam-dcm- cells). [Figure 18] Fold change in transformation efficiency compared to no pretreatment with various cell wall weakening agents. Results are representative of three independent experiments. [Figure 19] Fold change in transformation efficiency using MC strains (ZYCY, JMC2, JMC3) with (ermE) or without (ermE RM) the AGCAGY motif compared to the original pMW535 suicide vector. [Figure 20] Number of transformants as a function of the optical density of the competent cell preparation. [Figure 21] Number of transformants according to different amounts of DNA transformed. [Figure 22] Agarose gel of skin samples swabbed followed by DNA extraction and PCR. Junction PCR is shown and compared to control (vehicle) or wild type KPA171202. Only the modified strain shows amplification. [Figure 23] Gram staining of frozen sections from control versus C. acnes-colonized mice. [Figure 24]qPCR of the LCN2 gene in skin biopsies compared to control and wild-type treated skin (control and wild-type samples were taken together in the plot). [Diagram 25] SLST and 16S PCR SLST primers for mouse bacterial flora on days 0 and 1 after C. acnes application are specific for C. acnes and amplify only when C. acnes is present. [Figure 26] qPCR targeting inflammatory markers TNFα, IL-1β and IL6 in skin biopsies compared to control, wild-type or engineered C. acnes treated skin. [Figure 27] A) Junction PCR of successful replacement of the tdk gene with genes encoding LCN2 and ermE. B) Representative diagram of tdk gene replacement with genes encoding NGAL and an erythromycin resistance cassette. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Description of the Invention General definition It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element of the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0025] The term "about" with respect to a given amount or quantity is meant to include a deviation of ±5 percent.

[0026] As used herein, the conjunctive term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning and thus meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more options is also understood to be within the meaning and thus meet the requirements of the term "and / or".

[0027] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are understood to mean the inclusion of a recited integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced with the terms "containing" or "including", or, as sometimes used herein, with the term "having". Any of the foregoing terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the invention, may be replaced, but is less preferred, with the term "consisting of".

[0028] As used herein, "consisting of" excludes elements, steps, or ingredients not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0029] As used herein, the term "NGAL" has its general meaning in the art and refers to neutrophil gelatinase-associated lipocalin as described in Schmidt-Ott K M. et al. (2007) (Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Barasch J. Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol. 2007 February;18(2):407-13. Epub 2007 Jan.17. Review.). NGAL has been shown to exist as both a 25 kDa monomer and a 45 kDa disulfide-linked homodimer, and can also be covalently complexed with neutrophil gelatinase (also known as matrix metalloproteinase 9, MMP-9) via an intermolecular disulfide bridge as a 135 kDa heterodimeric form.

[0030] As used herein, the term "derivative" or "analog" refers to any substance that is sufficiently structurally similar to the material to be identified as a derivative such that when the substance is used in place of the material, it has a substantially similar function or activity, e.g., therapeutic efficacy, as the material.

[0031] As used herein, "endogenous gene" or "endogenous promoter" refers to a naturally occurring nucleic acid sequence that originates within an organism, i.e., has no external factors. The opposite of "endogenous gene" or "endogenous promoter" is "exogenous gene" or "exogenous promoter", or "heterologous promoter" or "heterologous gene", which refers to a nucleotide sequence that has an external origin, i.e., is not found naturally or in nature in the organism. An example of an exogenous gene in the context of the present invention is a nucleic acid encoding an NGAL protein.

[0032] As used herein, the term "retinoid-responsive condition" refers to some pathological conditions that are currently treated using retinoids or that the administration of one or more retinoids has a beneficial effect, including those characterized by the overproduction of sebum by the skin, such as acne vulgaris. Other retinoid-responsive conditions include rosacea, urticaria, eczema and psoriasis. Retinoids are well known in the art and include retinol, retinal, tretinoin, isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene and adapalene. Preferably, the term "skin" includes the skin of the face or body as well as the lips.

[0033] As used herein, the expression "non-therapeutic use in cosmetics" refers to a method used to improve the appearance of a person, i.e. to beautify the appearance.Preferably, the use in cosmetics includes stopping, reversing, improving, reducing, and / or reducing skin defects, imperfections, and / or aesthetically unpleasant conditions, including but not limited to age spots, sun spots, age spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, pimples, skin blemishes, skin fragility, dryness, spots, rough texture, cracks, sagging, thinning, enlarged pores, cellulite formation, acne formation, rosacea, psoriasis, and eczema.The use in cosmetics does not include surgical or therapeutic treatment.

[0034] The term "isolated" as used herein refers to a material that has been separated from contaminating cellular components associated with the material that are not intended to be associated with the material in nature, preventing the use of the material in therapeutic, prophylactic, diagnostic or other uses. Generally, an isolated material as described herein is at least about 80% pure, at least about 90% pure, at least about 95% pure, or greater than about 99% pure. Purification is accomplished using well-known standard methodologies such as fractionation and / or chromatography.

[0035] The term "recombinant" refers to a protein or bacterium that is not wild-type, i.e., not found in nature, and thus is formed by laboratory methods. A "recombinant bacterium" refers to a bacterium that has been altered, modified or engineered (e.g., genetically engineered) to exhibit an altered, modified or different genotype or phenotype (e.g., when the genetic modification affects a coding nucleic acid sequence of the microorganism) compared to the naturally occurring or starting microorganism from which it is derived.

[0036] The terms "treat" and "treatment" as used to refer to the treatment of a retinoid responsive condition in a subject include preventing, inhibiting, or ameliorating a retinoid responsive condition in a subject, e.g., slowing the progression of the condition, and / or reducing or ameliorating the signs or symptoms of the condition.

[0037] The term "therapeutically effective amount" refers to an amount that produces a desired physiological or pharmacological effect in a subject and prevents or ameliorates the condition being treated in the subject. For example, a therapeutically effective amount is an amount that reduces or eliminates the signs or symptoms of the retinoid-responsive condition being treated in the subject.

[0038] As used herein, the term "subject" refers to a living multi-cellular vertebrate organism, including, for example, humans, non-human mammals, and (non-human) primates.

[0039] explanation As mentioned above, there is scientific evidence supporting the use of NGAL as an alternative to isotretinoin in the treatment of acne. However, to date, NGAL produced in bacteria did not induce apoptosis in the TSS-1 sebaceous cell model, so the production of NGAL protein can only be based on eukaryotic systems. One possible reason why NGAL produced by bacteria does not function as expected may be due to the specific glycosylation pattern provided by eukaryotic cells. Klausen et al. showed that recombinant NGAL produced in E. coli cannot induce apoptosis in human bone marrow cells (Klausen, Niemann, Cowland et al. 2005), and NGAL has been reported to be an N-glycosylated protein when expressed in eukaryotic cells. However, prokaryotic cells do not glycosylate proteins in the same manner as mammalian cells, and it is possible that the glycosylation of NGAL in eukaryotic cells aids in the recognition of the protein by its receptor.

[0040] Bacteria have long been the preferred expression system for recombinant protein production, however, in the case of NGAL production, it is also known that NGAL has antibacterial activity, meaning that its production in a bacterial-based system is probably not possible.

[0041] NGAL Production The inventors surprisingly found that recombinant bacteria (e.g., P. acnes or E. coli) could indeed be grown and used to produce NGAL, although NGAL has been reported to have antibacterial activity against C. acnes. Moreover, and most importantly, NGAL produced by C. acnes and E. coli and secreted into the supernatant of the bacterial culture was found to be effective in reducing sebum in sebocytes PCi-SEB Cau (see FIG. 4). These results provide evidence that functional NGAL can be recombinantly produced in a bacterial system and that it maintains its inhibitory effect, paving the way for the production of NGAL at industrially scalable levels and the use of this protein to treat acne worldwide.

[0042] In view of this, a first aspect of the present invention relates to a neutrophil gelatinase-associated lipocalin (NGAL) protein derived from a bacterial culture for use in therapy or as a medicament. In one embodiment of the first aspect, the NGAL protein derived from a bacterial culture is used to inhibit sebum production in epithelial cells. In a preferred embodiment, the neutrophil gelatinase-associated lipocalin (NGAL) protein derived from a bacterial culture is used to treat a disease characterized by excessive sebum production, preferably acne vulgaris. As used herein, "acne vulgaris" refers to a skin condition that occurs when hair follicles become blocked with sebum and dead skin cells, usually causing bacterial overgrowth. Acne vulgaris includes the presence of whiteheads, blackheads or pimples, or a combination thereof. More specifically, acne vulgaris includes the presence of small red tender bumps, papules, plugged pores, open pores, pustules, nodules, and cystic lesions, or any combination thereof. The acne vulgaris condition may be present on the face, forehead, chest, upper back, and shoulders of the subject being treated, or a combination thereof. The acne vulgaris may be severe or mild acne vulgaris.

[0043] Retinoids are a large family of compounds derived from vitamin A, known to regulate epithelial cell proliferation and inhibit sebaceous gland differentiation. As shown in the following examples, it has been found that recombinant bacterially produced NGAL can inhibit sebum production in sebaceous gland cells, making it plausible that NGAL according to the first aspect may also be useful in the treatment of other retinoid-responsive diseases, such as urticaria, eczema, dermatitis, rosacea, and / or psoriasis.

[0044] Preferably, NGAL derived from recombinant bacterial culture is used to inhibit sebum production in epithelial cells. Preferably, the epithelial cells are human skin cells, more preferably human sebocytes. Sebocytes are highly specialized sebum-producing epithelial cells that release their contents by cell membrane destruction and cell disintegration. Sebocytes are terminally differentiated epithelial cells that produce and accumulate lipids (sebum). These cells are most commonly found in skin in association with hair follicles (forming pilosebaceous units) and arise from follicular keratinocytes, although sebaceous glands (SGs) that are not associated with hair follicles also exist. Sebum synthesis by sebocytes is strongly regulated by hormones, especially androgens. Excessive sebum production is seen in acne vulgaris, one of the most common skin diseases. Histologically, sebocytes can generally be identified by lipophilic dyes such as Oil Red O, Nile Red and Sudan IV, or by immunostaining for lipid droplet-associated proteins such as perilipin and adipophilin, the enzyme fatty acid synthase, keratin 7 and other specific differentiation markers. Methods for measuring the sebum inhibitory effect of NGAL provided herein are known in the art and are also illustrated in the Examples below. In one embodiment, NGAL from a recombinant bacterial culture can cause a reduction in sebum production in NGAL-treated cells, preferably sebocytes, which is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 4, 6, 8, 10-fold or more than 10-fold reduction compared to sebum production in untreated cells. In one embodiment, the reduction in sebum production caused by NGAL provided herein is a statistically significant reduction compared to sebum production in untreated cells, preferably untreated sebocytes.

[0045] In a second aspect, the present invention provides a non-therapeutic indication or use of neutrophil gelatinase-associated lipocalin (NGAL) protein derived from bacterial culture in the cosmetic field. Preferably, the NGAL protein obtained or obtainable from bacterial culture is used for the non-therapeutic purpose of cleaning, beautifying, adding attractiveness, changing the appearance, or maintaining or promoting good condition of the skin or hair. The purpose may also be whitening, minimizing the appearance of facial and body wrinkles, protection from sunlight and sunburn. In one embodiment, the non-therapeutic use is to prevent, reduce and / or ameliorate skin aging.

[0046] In some embodiments, the bacterium according to the first or second aspect is a skin bacterium that is part of the skin flora or skin microbiota. Skin flora is usually non-pathogenic, either commensal (not harmful to the host) or mutualistic (beneficial) bacteria, and does not cause any skin disease under normal circumstances. In one embodiment, the bacterium is a member of the microbiota (collection of microorganisms) present on the skin, typically human skin. Preferably, the bacterium is selected from the group consisting of Cutibacterium, Escherichia, Staphylococcus, Corynebacterium, or Micrococcus, or any combination thereof.

[0047] In some embodiments, the bacterium according to the first or second aspect is from the genus Cutibacterium or Escherichia. More preferably, the bacterium is Cutibacterium acnes (C. acnes) or Escherichia coli. It should be noted that Cutibacterium acnes was previously known as Propionibacterium acnes (P. acnes), and for the purposes of the present invention, both names are considered synonymous and interchangeable. Cutibacterium acnes is a gram-positive anaerobic bacterium that normally occupies hair follicles and sebaceous glands. The Cutibacterium acnes used in the present invention can be any as long as it allows the introduction of a nucleic acid encoding an NGAL protein and its expression. Preferably, C. acnes is strain KPA171202. Meanwhile, Escherichia coli, also known as E. coli, is a gram-negative, facultative anaerobic, rod-shaped coliform bacterium of the genus Escherichia commonly found in the environment, food, and intestines of humans and animals. In the present invention, any E. coli may be used to produce the NGAL protein, so long as it allows the introduction of a nucleic acid encoding the NGAL protein and its expression. Preferably, the E. coli is the BL21 strain.

[0048] The recombinant bacterium, more preferably C. acnes or E. coli, is characterized in that it comprises a nucleic acid molecule encoding an NGAL protein. Thus, the recombinant bacterium described herein is capable of expressing the NGAL protein. In one embodiment, the nucleic acid molecule encoding the NGAL protein comprises or consists of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length with SEQ ID NO: 2. Preferably, the NGAL protein expressed by a recombinant bacterium, preferably a recombinant C. acnes or E. coli according to the invention, is encoded by a nucleic acid consisting of SEQ ID NO: 2. In one embodiment, the nucleic acid encoding the NGAL protein is codon-optimized, preferably codon-optimized for the bacterium in which it is produced, e.g. codon-optimized for E. coli or C. acnes.

[0049] In one embodiment, the NGAL protein derived from a bacterial culture for use according to the first or second aspect is of human origin. Preferably, the NGAL protein expressed by the recombinant bacterium, preferably a recombinant C. acnes or E. coli, comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 1. Preferably, the NGAL protein expressed by the recombinant bacterium, preferably a recombinant C. acnes or E. coli, consists of SEQ ID NO: 1.

[0050] Techniques for determining sequence identity between nucleic acids and amino acids are known in the art. Two or more sequences can be compared by determining their "percent identity". The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence multiplied by 100. "Percent (%) amino acid sequence identity" with respect to proteins, polypeptides, antigenic protein fragments, antigens and epitopes described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference sequence (i.e., the protein, polypeptide, antigenic protein fragment, antigen or epitope from which it is derived) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared, which also applies mutatis mutandis to "percent (%) nucleotide sequence identity."

[0051] The gene encoding the NGAL protein is preferably designed to facilitate secretion of the protein from the bacterium or to facilitate expression of the NGAL protein directly on the surface of the bacterium. In some embodiments, the NGAL protein produced by the bacteria defined herein is released into the extracellular medium when the bacteria are growing and thus metabolically active. Thus, in one embodiment, the NGAL protein from a culture of a recombinant bacterium for use according to the first or second aspect is obtained from the supernatant medium collected from a culture of said recombinant bacterium, preferably a recombinant C.acnes or E.coli. Preferably, the NGAL protein is obtained from the supernatant collected after a period of growth under suitable conditions. The period may be 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 48, 72 hours or longer. In order to recover the protein from the supernatant, the protein should contain a signal peptide in its amino acid sequence that leads to its secretion into the extracellular medium. A signal peptide is a small amino acid sequence that functions to direct the protein outside the cytoplasm. Several signal peptides, such as PelB, TorA, OmpA, represent secretory pathways commonly used for extracellular expression in bacteria. Preferably, the signal peptide is located at the N-terminus of the NGAL protein. In a preferred embodiment, the signal peptide has an amino acid sequence comprising or consisting of SEQ ID NO:6, 7, or 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of SEQ ID NO:6, 7, or 8. In a preferred embodiment, the signal peptide has an amino acid sequence that includes or consists solely of SEQ ID NO:18-33, or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of SEQ ID NO:18-33.

[0052] Thus, in a preferred embodiment, the NGAL protein obtained or obtainable from a culture of bacteria, preferably recombinant C. acnes or E. coli, for use according to the first or second aspect, is secreted into the extracellular medium and comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 9, 10 or 11. Preferably, the NGAL protein expressed by the recombinant bacteria, preferably recombinant C. acnes or E. coli, consists solely of SEQ ID NO: 9, 10 or 11.

[0053] Furthermore, any recombinant NGAL protein, such as a derivative or analog, can be used to practice the invention, so long as it is obtained from a bacterium. A "derivative" or "analog" of NGAL includes truncated forms of the protein, where the function of NGAL is not altered by the truncation. A "derivative" or "analog" also includes fusion proteins that are longer than the wild-type NGAL protein due to the addition of extra sequences, such as a signal peptide, leader peptide, etc.

[0054] To obtain a recombinant bacterium expressing an NGAL protein as defined herein, a nucleotide sequence encoding said protein must be introduced into the bacterium to provide a recombinant bacterium expressing NGAL. Various means and methods for introducing an exogenous nucleotide sequence and thus producing a recombinant bacterium are known in the art, such as conventional transformation methods, electroporation, conjugation or protoplast transformation.

[0055] Introduction of DNA encoding a heterologous antigen into a bacterium to produce a recombinant bacterium can be accomplished, for example, by the creation of a recombinant bacterium in which the DNA encoding the NGAL protein is carried on a vector, such as a plasmid. Alternatively, the DNA encoding the NGAL protein can be stably integrated into the bacterial chromosome, for example, by using transposon mutagenesis, or by homologous recombination. A preferred method for producing a recombinant bacterium containing nucleotides encoding a chromosomally integrated NGAL protein is the induction of homologous recombination between a plasmid transfer vector containing the DNA encoding the NGAL protein and the bacterial chromosomal DNA. In some embodiments, a nucleotide sequence encoding an NGAL protein, preferably a human NGAL protein, is located in a plasmid transfer vector that is transformed into a bacterium, preferably C. acnes or E. coli. Integration of the nucleic acid encoding the human NGAL protein is preferentially achieved by homologous recombination. A homologous recombination event is the exchange between DNA molecules (in this case, between the bacterial genome and the plasmid transfer vector) that share sufficient sequence identity to trigger a homologous recombination mechanism between them. Techniques that promote homologous recombination are known in the art and result in the introduction of the desired portion of the plasmid transfer vector into the bacterial genome. To produce a recombinant bacterium, preferably C. acnes or E. coli, that expresses NGAL by homologous recombination, one must assemble a plasmid transfer vector that contains an NGAL-encoding nucleic acid flanked by two regions of at least 50 base pairs that are homologous to regions in the bacterial genome, one upstream and one downstream of the NGAL coding sequence. Once the plasmid transfer vector is provided, it is transformed into a bacterium and a homologous recombination event between the bacterial genome and the plasmid transfer vector results in a recombinant bacterium that contains the NGAL gene in its genome and thus expresses the NGAL protein.Methods for producing recombinant bacteria are known in the art, see, for example, Sorensen et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants, Journal of Microbiological Methods, Volume 83, Issue 2, 2010, Pages 211-216, ISSN 0167-7012, https: / / doi.org / 10.1016 / j.mimet.2010.09.008. Examples of transfer vectors for causing homologous recombination are provided in the Examples section below.

[0056] In a preferred embodiment, the nucleic acid encoding the NGAL protein is integrated into a non-essential region of the bacterial genome. Preferably, the nucleic acid sequence encoding the human NGAL protein is inserted into a gene selected from the group consisting of camp2 (PPA0687), camp1 (PPA1340), roxP (PPA1939), thymidine kinase (tdk) (PPA1049) or the restriction modified IIIB locus (PPA1610, 1611, 1612). The genes encode camp2, camp1, RoxP, Tdk, and restriction modified IIIB proteins, respectively, and therefore the insertion of the NGAL-encoding nucleic acid leads to the interruption or even replacement of the gene. As shown in FIG. 5, the interruption or replacement of the tdk locus represents a promising tool for knockout C. acnes expressing NGAL, which can grow on Brucella plates using FUDR as negative selection.

[0057] In order for the homologous recombination event to occur specifically at the gene in the bacterial genome, the plasmid transfer vector used should contain flanking regions that show homology to the gene, said flanking regions being located upstream and downstream of the NGAL-encoding nucleic acid. Thus, in a preferred embodiment, a plasmid transfer vector that guides the insertion of a nucleic acid sequence encoding an NGAL protein into a bacterial genome contains regions that include the following, in the following order: i) a nucleotide region of at least 50, 100 or 150 base pairs, preferably 200, 250, 300, 350, 400, 452, 500 or more than 500 base pairs, which is at least 70%, 80%, 85%, 90%, 95% or 100% identical to a nucleotide region of the same length contained in the bacterial genes camp2 (PPA0687), camp1 (PPA1340), roxP (PPA1939), tdk (PPA1049) or the restriction modified IIIB locus (PPA1610, 1611, 1612); ii) an NGAL nucleic acid encoding a human NGAL protein, preferably an NGAL nucleic acid of SEQ ID NO: 2 or a nucleic acid having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 2; and iii) a nucleotide region of at least 50, 100 or 150, preferably more than 200, 250, 300, 350, 400, 452, 500 or 500 base pairs, which is at least 70%, 80%, 85%, 90%, 95% or 100% identical to a nucleotide region of the same length contained in the bacterial genes camp2 (PPA0687), camp1 (PPA1340), roxP (PPA1939), tdk (PPA1049) or the restriction modified IIIB locus (PPA1610, 1611, 1612); As a result, the NGAL nucleic acid is inserted into the bacterial genome by homologous recombination, interrupting the genes camp2 (PPA0687), camp1 (PPA1340), roxP (PPA1939), tdk (PPA1049) or the restriction modified IIIB locus (PPA1610, 1611, 1612) from the bacterial genome, preferably the C. acnes or E. coli genome. An example of such a vector for targeting the tdk locus is defined in SEQ ID NO: 15 and in the Examples section below.

[0058] Other methods known in the art of genetic engineering useful for producing recombinant bacteria can be used, such as CRISPR, phage integration, or transposon insertion. In one embodiment, the nucleic acid encoding the NGAL protein is contained in a plasmid that is transformed into the bacterium. In another embodiment, the nucleic acid construct is a shuttle plasmid. In another embodiment, the nucleic acid construct is an integration vector that includes an integration site. In another embodiment, the nucleic acid construct is a site-specific integration vector. In another embodiment, the nucleic acid construct is any other type of nucleic acid construct known in the art.

[0059] It should be noted that the nucleotide sequence introduced into the recombinant bacterium can include not only the gene encoding the NGAL protein, but also other elements such as regulatory sequences or selectable markers, in one embodiment the regulatory sequence is a promoter, and in another embodiment the regulatory sequence is an enhancer.

[0060] In some embodiments, the nucleic acid contained in the recombinant bacterium comprises an expression cassette comprising a nucleic acid encoding an NGAL protein operably linked to a promoter. The promoter driving the expression of the NGAL protein can be an endogenous promoter, i.e., a promoter naturally found in the bacterium in which the NGAL protein is being expressed, preferably C.acnes or E.coli. In this case, the promoter does not need to be included in the plasmid transfer vector, since it is already present in the bacterial genome. In some embodiments, the nucleic acid encoding the NGAL protein is operably linked to the endogenous camp2, camp1, roxP, Tdk or restricted modified IIIB promoter, respectively, by inserting the nucleic acid into the camp2 (PPA0687), camp1 (PPA1340), roxP (PPA1939), tdk (PPA1049) or restricted modified IIIB locus (PPA1610, 1611, 1612) gene, thereby the endogenous promoter controls the expression of the NGAL protein.

[0061] In some other embodiments, the promoter is an artificial promoter or an exogenous promoter. In this case, the promoter must be provided, for example, by the transfer plasmid vector, together with the NGAL coding gene. In some other embodiments, the camp2, camp1, roxP, Tdk or restricted modified IIIB promoter, or any other suitable promoter, is also included in the transfer plasmid vector, flanked by regions that provide for homologous recombination, so that the NGAL nucleotide sequence is not operably linked to an endogenous promoter, but its expression is driven by a promoter, preferably camp2, camp1, roxP, Tdk or restricted modified IIIB promoter, included in the transfer plasmid vector, and is also inserted into the bacterial genome upon homologous recombination events.

[0062] In one embodiment, the NGAL protein for use according to the first or second aspect of the invention, obtained or obtainable from a bacterial culture, is encoded by a nucleic acid operably linked to a promoter selected from the list of camp2, camp1, roxP, Tdk, or restricted modified IIIB promoter. Preferably, the NGAL protein for use according to the first or second aspect of the invention, obtained or obtainable from a bacterial culture, is encoded by a nucleic acid operably linked to a promoter comprising or consisting of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 12 (camp1 promoter), 13 (RoxP promoter) or 14 (camp2 promoter).

[0063] As described in the Examples, several nucleotides comprising the NGAL nucleic acid operably linked to one of the promoters camp2, camp1 or roxP were produced, and the produced NGAL proteins further comprised a signal peptide (designated PPA1939, PPA0687, PPA1340) at their N-terminus (see Table 1). Thus, in one embodiment, the NGAL protein for use according to the first or second aspect of the invention, obtained or obtainable from a bacterial culture, comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 1, 9, 10 or 11, and the NGAL protein is The AL protein is encoded by a nucleic acid operably linked to a promoter comprising or consisting of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 12 (camp1), 13 (RoxP promoter) or 14 (camp2 promoter). Preferably, the NGAL protein for use according to the first or second aspect of the invention obtained or obtainable from a bacterial culture comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO:10, and the NGAL protein is encoded by a nucleic acid operably linked to a promoter comprising or consisting of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO:12.Preferably, the NGAL protein for use according to the first or second aspect of the invention obtained or obtainable from a bacterial culture comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO:11, and the NGAL protein is encoded by a nucleic acid operably linked to a promoter comprising or consisting of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO:12. Most preferably, the NGAL protein for use according to the first or second aspect of the invention obtained or obtainable from a bacterial culture comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO:9, and the NGAL protein is encoded by a nucleic acid operably linked to a promoter comprising or consisting of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO:12.

[0064] As described above, the nucleic acid transformed in the bacterium may include a nucleic acid sequence encoding a selectable marker. The selectable marker may confer resistance to one or more antibiotics. For example, the selectable marker may include genes for ampicillin resistance, streptomycin resistance, kanamycin resistance, tetracycline resistance, chloramphenicol resistance, sulfonamide resistance, erythromycin resistance, lincomycin resistance, or a combination of these markers. Typically, the selectable marker is operably linked to a promoter that promotes the expression of the marker. Plasmids and other cloning vectors that include selectable markers are known in the art.

[0065] As defined above, in order to obtain a recombinant bacterium expressing an NGAL protein, once the step of introducing a nucleic acid encoding the NGAL protein into a bacterium has been carried out, the NGAL protein used according to the first and second aspects can be obtained by carrying out the following steps: a) culturing a recombinant bacterium in a suitable medium and under suitable conditions for protein expression, said recombinant bacterium being characterized in that it expresses a human neutrophil gelatinase-associated lipocalin (NGAL) protein, preferably a secreted NGAL protein as defined above, b) harvesting the NGAL protein from the culture of a), preferably from the supernatant of the culture of a); c) optionally isolating the NGAL collected in b). The present invention is obtained or obtainable from a method comprising the steps of:

[0066] Suitable culture media include any growth medium in a solid, liquid or semi-solid state that allows for the growth of recombinant bacteria and the expression of NGAL protein. Thus, suitable culture media contain all the elements necessary for bacteria to grow and be metabolically active, including at least one carbon source, water, salts, and at least one amino acid source and nitrogen source. As will be appreciated by those skilled in the art armed with the present disclosure, several approaches can be used to express heterologous antigens in bacteria.

[0067] The produced NGAL protein may be obtained from inside the bacteria, for example by cell lysis and optionally purification, or from the extracellular medium if the NGAL protein is designed to be a secreted protein. In the latter case, the NGAL protein is obtained directly from the extracellular medium or after centrifuging the medium to discard the bacterial cells. Optionally, the NGAL is purified before being used. Protein purification includes a series of processes intended to isolate one or a few proteins from a complex mixture, such as the culture medium in which the producing recombinant bacteria is growing. The purification process separates the NGAL protein from all other proteins and compounds present in the medium. The separation steps usually exploit differences in protein size, physicochemical properties, binding affinity and biological activity known to those skilled in the art. Optionally, the purified protein is further concentrated before use. Stable transformants expressing NGAL can be isolated and characterized as described herein in the experimental examples.

[0068] The NGAL protein derived from bacterial culture for use according to the first or second aspect of the invention may be administered to a subject in a single dose or multiple (i.e., 2, 3, 4, etc.) doses. The appropriate schedule for administration of the doses depends on several factors, including the age, weight, sex, medical history and health of the subject, the type of composition used and the route of administration. The skilled artisan can easily determine the dosage and administration schedule for a particular subject. The appropriate dosage range depends on various factors, such as the age of the subject, the severity and type of condition being treated in the subject, the general condition of the subject, the route and form of administration of the composition administered, and the particular composition administered. For therapeutic use according to the first aspect, the skilled artisan will be able to ascertain a therapeutically effective amount without undue experimentation given the state of the art. In some embodiments, administration may be at the onset of symptoms of the acne vulgaris condition, for example, at the time of the first hair follicle clogging.

[0069] In certain embodiments, the subject in need thereof is a human. In certain embodiments, the subject is a teenager, preferably between 10 and 19 years of age. In other embodiments, the subject to be treated or in need thereof is an adult. In some embodiments, the subject has acne.

[0070] In certain embodiments of the invention, there is provided a method of treating and / or preventing acne in a subject, comprising administering a therapeutically effective amount of NGAL, an NGAL analog, a supernatant containing NGAL, or C. acnes or E. coli expressing NGAL as defined above.

[0071] The subject treated according to the use of the present invention may be a mammal or a non-mammal. Although humans are the preferred subjects, the mammalian subject may be any mammal, including, but not limited to, a non-human primate; a rodent such as a mouse, rat, or guinea pig; a domestic pet such as a cat or dog; a horse, cow, pig, sheep, goat, or rabbit.

[0072] Since the human NGAL protein from a recombinant bacterium according to the first or second aspect can be secreted into the extracellular medium, in a third aspect the invention relates to a supernatant medium obtained or obtainable from a culture of a recombinant bacterium, preferably C. acnes or E. coli, characterized in that the bacterium expresses an NGAL protein, said NGAL protein comprising a signal peptide leading to its export into the extracellular medium. In some embodiments the supernatant further comprises bacteria, preferably recombinant C. acnes or E. coli, in suspension. In some embodiments the supernatant has been treated, for example by centrifugation or filtration, to remove remaining bacteria. Optionally the NGAL obtained from the supernatant is purified before use. The uses defined in the first and second aspects also apply to the supernatant of the third aspect. Thus, the therapeutic uses and non-therapeutic cosmetic uses defined above are also uses of the supernatant according to the third aspect.

[0073] It is important to note that although the NGAL protein (purified or contained in the supernatant) can be used as defined in the above aspects, it is also very interesting to provide recombinant vectors that can efficiently produce and deliver said protein, preferably in its niche of action, i.e. in the hair follicle surrounded by sebaceous cells. As shown in Paetzhold et al. 2019, C. acnes can colonize and persist in human skin and is a promising producer and transport therapeutic NGAL protein directly on human skin. Thus, in a fourth aspect, the present invention relates to a recombinant bacterium, preferably C. acnes or E. coli, characterized in that it contains an NGAL-encoding nucleic acid and expresses the neutrophil gelatinase-associated lipocalin (NGAL) protein, preferably human NGAL. Preferably, the recombinant bacterium, preferably C. acnes or E. coli, is characterized in that it expresses an NGAL protein comprising or consisting of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 1, 9, 10 or 11. Preferably, the NGAL protein expressed by the recombinant bacterium, preferably recombinant C. acnes or E. coli, consists solely of SEQ ID NO: 1, 9, 10 or 11. Preferably, the recombinant bacterium, preferably C. acnes or E. coli, is characterized in that it expresses an NGAL protein encoded by a nucleic acid comprising or consisting of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 2. Preferably, the recombinant bacterium has been transformed with a nucleic acid molecule comprising a nucleic acid encoding an NGAL protein operably linked to a transcription promoter, preferably the camp1, camp2 or roxP promoter.Optionally, the NGAL protein is encoded by a nucleic acid sequence operably linked under the control of a promoter comprising or consisting of a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 12 (camp1 promoter), 13 (RoxP promoter) or 14 (camp2 promoter).

[0074] The embodiments relating to the recombinant bacterium, preferably recombinant C. acnes or E. coli, and methods for producing said recombinant bacterium are described above and also apply here. The embodiments relating to the NGAL protein and its sequences described in the preceding aspects also apply here.

[0075] Furthermore, in a fifth aspect, the present invention provides a recombinant bacterium, preferably C. acnes or E. coli, as defined in the fourth aspect, characterized in that it expresses an NGAL protein, preferably human NGAL, for use in therapy or as a medicament, said NGAL being defined in any of the preceding aspects. Preferably, the recombinant bacterium is used to inhibit sebum production in human skin cells or to treat diseases characterized by excessive sebum production, such as acne vulgaris. Other uses of the recombinant bacterium according to the fifth aspect include the treatment of other retinoid-responsive diseases, such as urticaria, eczema, dermatitis, rosacea, and / or psoriasis. Preferably, the recombinant bacterium for use in therapy or as a medicament is C. acnes, as this bacterium is a natural commensal in hair follicles and is therefore in direct contact with human cells such as sebocytes, thus offering the benefits of targeted delivery and increased protein penetration.

[0076] In a sixth aspect, the present invention provides a recombinant bacterium, preferably C. acnes or E. coli, characterized in that it expresses NGAL, preferably human NGAL, as defined in any of the preceding aspects, for non-therapeutic use in the cosmetic field or as a cosmetic composition. In a preferred embodiment, the recombinant bacterium for non-therapeutic cosmetic use is C. acnes expressing NGAL, preferably human NGAL. Preferably, the recombinant bacterium, preferably C. acnes or E. coli, characterized in that it expresses an NGAL protein, is used for the non-therapeutic purpose of cleaning, beautifying, adding attractiveness, changing the appearance, or maintaining or promoting good condition of the skin or hair. The purpose may also be whitening, minimizing the appearance of facial and body wrinkles, protection from sunlight and sunburn. In one embodiment, the non-therapeutic cosmetic use is to prevent, reduce and / or ameliorate skin aging. Preferably, the recombinant bacterium, when used according to the fifth and sixth aspects, is administered alive.

[0077] In a seventh aspect, the present invention relates to a composition, preferably a pharmaceutical or cosmetic composition, comprising human NGAL derived from a culture with a recombinant bacterium, preferably C. acnes or E. coli, as defined above, or a supernatant obtained from a culture of said bacterium, and a pharma- ceutical or cosmetically acceptable carrier. Since C. acnes is a common commensal of the skin microbiome and can therefore be administered at least topically to a subject, the seventh aspect of the present invention also includes a composition, preferably a pharmaceutical or cosmetic composition, comprising a recombinant C. acnes according to any of the preceding aspects, characterized in that it expresses an NGAL protein, the pharmaceutical or cosmetic composition further comprising a pharma- ceutical or cosmetically acceptable carrier. The seventh aspect of the present invention also includes a composition, preferably a pharmaceutical or cosmetic composition, comprising a recombinant E. coli according to any of the preceding aspects, characterized in that it expresses an NGAL protein, the pharmaceutical or cosmetic composition further comprising a pharma- ceutical or cosmetically acceptable carrier.

[0078] In some embodiments, the composition comprises a substantially pure, preferably up to 80%, 85%, 90%, 95% or 100% pure, preparation of isolated NGAL protein derived from a culture of recombinant bacteria expressing NGAL. In some other embodiments, the composition, preferably a pharmaceutical or cosmetic composition, comprises a substantially pure, preferably up to 80%, 85%, 90%, 95% or 100% pure, preparation of recombinant C. acnes or E. coli expressing NGAL protein.

[0079] Preferably, the pharmaceutical composition comprises a therapeutically effective amount of an NGAL protein, preferably an isolated NGAL protein, or a therapeutically effective amount of C. acnes, or a combination thereof.

[0080] The term "pharmaceutical acceptable carrier" refers to a carrier that is suitable for use in a subject without undue toxicity or irritation to the subject and is compatible with other ingredients contained in the pharmaceutical composition. Pharmaceutically acceptable carriers, methods for making pharmaceutical compositions and various dosage forms, and modes of administration are well known in the art. Other substances that may be included in the composition include approved additives, antibiotics, preservatives, adjuvants, diluents and / or stabilizers. Such auxiliary substances may be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, and the like. Suitable carriers are typically large slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.

[0081] In some embodiments, the pharmaceutical composition is formulated for local, topical and / or systemic administration to a subject. Formulations for administering the pharmaceutical composition according to the seventh aspect include those suitable for oral, rectal, nasal, pulmonary, ophthalmic, aural, intra-arterial, intradermal, intravenous, intramuscular, intraosseous, subcutaneous, topical, transdermal, and transmucosal, such as sublingual, buccal, vaginal, and inhalation routes of administration.

[0082] The pharmaceutical or cosmetic compositions according to embodiments of the present invention may be in any dosage form suitable for administration to a subject, illustratively including solid, semi-solid and liquid dosage forms such as tablets, capsules, powders, granules, suppositories, pills, liquids, suspensions, ointments, lotions, creams, gels, pastes, sprays and aerosols. For topical administration, the compositions may be formulated for administration to the skin for localized effect and / or as "patch" formulations for transdermal delivery. Pharmaceutical formulations suitable for topical administration include, for example, ointments, lotions, creams, gels, pastes, sprays and powders. Ointments, lotions, creams, gels and pastes may include, in addition to one or more active agents, a base such as an absorbent base, a water-removable base, a water-soluble base or an oily base, and excipients such as a thickening agent, a gelling agent, a coloring agent, a stabilizer, an emulsifier, a suspending agent, a sweetener, a flavoring or a fragrance. Powders and sprays for topical administration of one or more active agents can include excipients such as talc, lactose, and one or more silicic acids. Sprays can include pharmaceutical propellants such as fluorinated hydrocarbon propellants, carbon dioxide, or suitable gases. Alternatively, sprays can be delivered from pump-type spray devices that do not require propellants. The spray device delivers a metered dose of the composition contained therein, for example, using a valve to regulate the amount delivered. Transdermal formulations can include transdermal absorption enhancers such as acetone, azone, dimethylacetamide, dimethylformamide, dimethylsulfoxide, ethanol, oleic acid, polyethylene glycol, propylene glycol, and sodium lauryl sulfate. Ionophoresis and / or sonophoresis can be used to enhance transdermal delivery.

[0083] Liposomes and emulsions are well-known types of pharmaceutical formulations that can be used to deliver pharmaceuticals, particularly hydrophobic pharmaceuticals. Liposomes can contain any type of amphiphilic material that is compatible with the composition to be delivered, illustratively including naturally occurring lipids, synthetic lipids, and combinations thereof.

[0084] The term "cosmetically acceptable carrier" refers to a carrier suitable for non-therapeutic use in a subject without undue toxicity or irritation. A cosmetically acceptable carrier may comprise or consist of a cream, gel, serum, balm, sunscreen cream, after-sun cream, foundation, tinted cream, tinted sun cream, greenish soothing redness control cream, scalp serum, solution, suspension, emulsion, ointment, foam, paste, lotion, powder, soap, surfactant-containing cleansing oil or spray. Dosage forms and modes of administration are well known in the art. The step of applying the cosmetic composition to the skin may be repeated once or twice daily for up to 8 days. The step of applying the cosmetic composition to an area of ​​the skin may be repeated for at least 2 days, at least 3 days, at least 4 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, or at least 8 weeks.

[0085] In some embodiments, the compositions may be provided in a kit for single or multiple administration, hi some embodiments, the compositions may be applied topically to the skin.

[0086] In an eighth aspect, the present invention relates to a method for producing an active ingredient of a pharmaceutical composition, said active ingredient being an NGAL protein capable of inhibiting sebum production in epithelial cells, preferably sebaceous cells, said method comprising the steps of: a) introducing a nucleic acid encoding an NGAL protein into a bacterium to obtain a recombinant bacterium expressing an NGAL protein, preferably a secreted NGAL protein; b) culturing the recombinant bacterium in a suitable medium and collecting the NGAL protein from the culture of a), preferably from the supernatant of the culture of a); c) optionally isolating the NGAL collected in b). The present invention is obtained or obtainable from a method comprising the steps of:

[0087] In some embodiments, the recombinant bacteria according to any of the above aspects is a skin bacterium that is part of the skin flora or skin microbiota. Skin flora is usually non-pathogenic, either commensal (not harmful to the host) or mutualistic (beneficial) bacteria, and does not cause any skin disease under normal circumstances. In one embodiment, the bacteria is a member of the microbiota (collection of microorganisms) present on the skin, typically human skin. Preferably, the bacteria is selected from the group consisting of Cutibacterium, preferably C. acnes, Escherichia, preferably E. coli, Staphylococcus, Corynebacterium, or Micrococcus, or any combination thereof.

[0088] Since the eighth aspect refers to an NGAL protein and a bacterium producing said protein, all embodiments disclosed above relating to an NGAL protein and a recombinant bacterium expressing said NGAL protein are also included in the eighth aspect.

[0089] Methods for Producing Recombinant C. acnes It is known that C. acnes has a highly efficient restriction modification system (RM) that can eliminate any foreign methylated DNA introduced into the bacterium, thereby preventing DNA delivery. This RM system is the reason why an efficient transformation method has not yet been established for C. acnes. Genome manipulation is considered to be a fundamental tool for functional research in C. acnes, and therefore there is an urgent need to develop an efficient transformation method that allows the introduction of DNA into this bacterium. The present invention also aims to overcome this obstacle, and in its ninth aspect, provides an efficient method for producing recombinant C. acnes.

[0090] The present inventors have found that a way to increase the transformation efficiency of C. acnes is to provide a nucleic acid that can completely circumvent the restriction-modification system (RM) of C. acnes. This is possible if the nucleic acid mimics the methylation pattern of C. acnes so that it is not recognized as a foreign nucleic acid, or if the nucleic acid is completely unmethylated and does not contain the C. acnes methylation motif, so that the C. acnes RM system does not recognize and degrade it. As used herein, "methylation" refers to the process of adding methyl groups to specific regions of a nucleic acid, preferably to specific nucleobases.

[0091] Both options were tested in the examples provided below: First, we generated a ΔdamΔdcmΔhsdMS E. coli strain that harbors the C. acnes IIIB methylase. This strain, called EC-24, lacks E. coli specific methylation but instead produces DNA that mimics the methylation pattern of C. acnes. Therefore, shuttling the plasmid through EC-24 before delivery to C. acnes should contribute to RM evasion. While there were few transformants in the absence of C. acnes specific methylation, transformation efficiency increased up to 200-fold when plasmid methylation mimicked that of the host bacterium (Figure 17).

[0092] Furthermore, DNA lacking methylation was also produced using E. coli JMC3 strain, which lacks endogenous RM systems (Δdam, Δdcm, ΔhsdMS) and produces completely unmethylated minicircles (suicide vectors of reduced size). It was observed that vectors from strains lacking RM systems (JMC3 strain) increased the number of transformants compared to strains containing one or more endogenous RM systems (JMC2 strain: dam+, Δdcm, ΔhsdMS; ZYCY strain: dam+, dcm+, hsdMs+), indicating that DNA delivery to C. acnes is also improved when the DNA does not contain methylation patterns or motifs that C. acnes can recognize as exogenous.

[0093] In view of the above results, the ninth aspect provides a method for introducing nucleic acid into C. acnes. As used herein, "introducing nucleic acid" refers to any method for delivering nucleic acid into bacteria, such as electroporation, transduction (e.g., injection of nucleic acid by bacteriophage), microinjection, introduction of chemical competence (e.g., by adding alkaline cation, cesium, lithium, polyethylene glycol, or by osmotic shock), heat shock, conjugation, etc. It should be understood that "introducing nucleic acid" refers to stable incorporation of nucleic acid into bacteria, not just introduction, i.e., nucleic acid is not immediately degraded in bacteria. In particular, "introducing nucleic acid into C. acnes" refers to any method for delivering nucleic acid into C. acnes, and the nucleic acid introduced into C. acnes is not recognized as exogenous by C. acnes RM system, preferably by C. acnes REase, and therefore is not degraded upon delivery.

[0094] A first embodiment of this aspect is a method for introducing a nucleic acid into C. acnes, comprising the steps of: a) providing a nucleic acid sequence suitable for introduction into C. acnes; and b) introducing the nucleic acid sequence of a) into C. acnes The present invention includes a method comprising the steps of:

[0095] Step a) comprises or consists solely of providing a nucleic acid sequence suitable for introduction into C. acnes.

[0096] A suitable nucleic acid sequence for introduction into C. acnes is a polynucleotide that is not recognized as exogenous or "non-self" by the C. acnes RM system, and therefore is not degraded by the C. acnes REase. In one embodiment, the nucleic acid sequence of step a) is suitable for introduction into C. acnes if it does not contain a motif recognized by the C. acnes methylase, and therefore is not methylated (i.e., is an unmethylated nucleic acid sequence), and if it contains one or more C. acnes methylation motifs, they are methylated and the motifs are the only methylation motifs present in the sequence of the nucleic acid. Thus, a suitable nucleic acid sequence for introduction into C. acnes is an unmethylated nucleic acid sequence, provided that if it contains a C. acnes methylation motif, the methylation motif is methylated. The nucleic acid is recognized as "self" by the C. acnes RM system, and therefore is not degraded when delivered to C. acnes. However, if the nucleic acid contains a methylation motif from another bacterium or organism, or contains an unmethylated C. acnes methylation motif, it will be recognised by the C. acnes RM system, in particular the REase, which then degrades it. - is unmethylated and does not contain the C. acnes methylation motif, or -contains one or more C. acnes methylation motifs that are methylated and does not contain any other methylated nucleobases other than the methylation motif(s).

[0097] As used herein, in the context of nucleic acids, a "methylation motif" refers to a short nucleotide sequence of about 3-15, preferably 5-10 nucleotides, that is included in a longer nucleic acid in the form of a repeating pattern and that is associated with nucleic acid methylation events. A "C. acnes methylation motif" refers to a methylation motif in nucleic acids that is recognized by and methylated by C. acnes methylase (MTase). The most important methylation pattern in bacteria occurs at adenine, particularly at position N6. Other methylation can occur at the C5 carbon or the N4 amino group of cytosine residues. C. acnes methylation motifs are known in the art and can be retrieved from open databases such as REBASE. In one embodiment, the C. acnes methylation motif is BNNDCNNNNNNGTCCCC (SEQ ID NO: 40), where B represents C or G or T; D represents A or G or T; and N represents any of A, G, C, T, U.

[0098] In a preferred embodiment, the C. acnes methylation motif is AGCAGY (SEQ ID NO: 38), which is methylated at the second adenine in the 5' to 3' direction by a C. acnes MTase, thereby resulting in the methylated C. acnes methylation motif AGC(m)AGY (SEQ ID NO: 39). - is unmethylated and does not contain the C. acnes methylation motif AGCAGY, or It comprises one or more C. acnes methylation motifs AGCAGY that are methylated at the -2 adenine (AGC(m)AGY), and the nucleic acid sequence contains no other methylated nucleobases other than the C. acnes methylation motif AGC(m)AGY.

[0099] In one embodiment the nucleic acid sequence provided in step a) comprises one or more methylated C. acnes methylation motifs, wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the C. acnes methylation motifs are methylated, preferably the methylation motif is AGC(m)AGY.In one embodiment the nucleic acid sequence provided in step a) comprises less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, preferably less than 1 or 0 unmethylated C. acnes methylation motifs, preferably the unmethylated C. acnes methylation motif is AGCAGY.

[0100] As explained above, the nucleic acid sequence of step a) is either completely unmethylated and does not contain any C. acnes methylation motifs, or is unmethylated and contains one or more C. acnes methylation motifs that are methylated. In a preferred embodiment, the nucleic acid sequence of step a) is characterized in that it contains one or more C. acnes methylation motifs AGCAGY, which are methylated and the methylation is located at the second adenine (or at the fourth nucleobase in the 5' to 3' direction), and the rest of the nucleic acid sequence is unmethylated. Thus, the nucleic acid sequence of step a) does not contain unmethylated AGCAGY motifs, because they are degraded by REases of C. acnes. Methods for determining whether a nucleic acid sequence contains methylated C. acnes methylation motifs are known in the art and include Oxford Nanopore Sequencing Technologies, SMRT Sequencing, immunoprecipitation or bisulfite sequencing (for methylated C).

[0101] The nucleic acid sequence of step a) can be obtained by introducing it into a non-C. acnes bacterium, also referred to herein as a shuttle bacterium, and then harvesting the nucleic acid sequence from said bacterium for proceeding to step b). Methods for harvesting nucleic acids from bacteria or bacterial cultures, such as E. coli cultures, are known in the art and include standard protocols for purifying DNA or purifying plasmids.

[0102] The shuttle bacterium may be a bacterium engineered or modified to not express or contain a functional methylase, or may be a bacterium engineered to contain or express only a functional C. acnes methylase, or a methylase that can reproduce the methylation pattern of the C. acnes methylase. "Functional methylase" as used herein refers to a methylase that is capable of methylating nucleobases, i.e., is an active methylase. A functional C. acnes methylase is capable of methylating a C. acnes methylation motif, such as AGCAGY.

[0103] A nucleic acid sequence lacking methylated nucleobases can be obtained, for example, by introducing the nucleic acid into a non-C. acnes bacterium characterized by not containing or expressing a methylase. The bacterium not expressing a methylase does not have the ability to methylate nucleic acids and therefore lacks methylated nucleobases or motifs when the nucleic acid sequence is collected. A non-C. acnes bacterium not containing or expressing a methylase can be produced by silencing or inactivating its endogenous methylase or the endogenous gene encoding the methylase, deviating from a non-C. acnes wild-type bacterium, such as E. coli. When this shuttle bacterium is used, it is important that the nucleic acid sequence is designed to not contain C. acnes methylation motifs such as AGCAGY, because the shuttle bacterium cannot methylate them, and if they are not methylated, they will be degraded by the C. acnes RM system, as explained above. The nucleic acid sequence can be designed to lack the unmethylated AGCAGY motif, for example by replacing the second A with either G or C or T, or possibly by removing or recoding the entire motif. When the nucleic acid sequence is harvested from the shuttle bacterium, for example by purifying it after growing it for 24 hours, it lacks methylated nucleobases and unmethylated C. acnes methylation motifs and is therefore suitable for introduction into C. acnes, as explained above. Preferably, the non-C. acnes bacterium that is modified not to express methylase is E. coli, most preferably the dam-dcm-hsdMS-E. coli strain, in which the E. coli endogenous genes dam, dcm, and hsdMS are inactivated or their encoded proteins are inactivated or silenced.

[0104] The nucleic acid of step a) may be obtained by introducing into a non-C. acnes bacterium that has been modified so that the only methylase it expresses is a functional C. acnes methylase or a methylase that can reproduce the methylation pattern of the C. acnes methylase. This is particularly important when the nucleic acid sequence contains a C. acnes methylation motif that needs to be methylated before introducing the nucleic acid sequence into C. acnes. Shuttle bacteria can be produced from shuttle bacteria with a silenced or inactivated methylation system, such as dam-dcm-hsdMS-E. coli, as described in the paragraph above, and the bacteria can be further modified to express or contain a functional C. acnes methylase. Thus, a non-C. acnes shuttle bacterium not only contains a functional C. acnes methylase, but also does not contain any other active methylases other than C. acnes. "Does not contain any other active methylases other than C. acnes" refers herein to the C. acnes methylase being the only methylase that is functional in the non-C. acnes bacterium. In other words, the shuttle bacteria used to produce and provide the nucleic acid of step a) preferably lack their endogenous RM system but are functional for C. acnes methylase. Preferably, the shuttle bacteria have silenced or inactivated their endogenous adenine methyltransferase (dam), cytosine methyltransferase (dcm) and / or methylase subunit of EcoKI restriction enzyme (hsdMS), or any combination thereof, genes or proteins. When a shuttle bacterium containing a functional C. acnes methylase is used, it does not matter if the nucleic acid contains one or more C. acnes methylation motifs in its sequence, such as AGCAGY, since the shuttle bacterium contains a functional C. acnes methylase that methylates them, thereby providing methylated C. acnes methylation motifs, preferably AGC(m)AGY, that are not degraded by the C. acnes RM system.When the nucleic acid is harvested from the shuttle bacterium, for example by purification after growing the shuttle bacterium for 24 hours, the nucleic acid will be methylated only at the C. acnes methylation motifs, if any, present in its sequence and will therefore be suitable for introduction into C. acnes, as described above.

[0105] Preferably, the functional C. acnes methylase expressed by the shuttle bacterium is C. acnes IIIB methylase (MIIIB). Preferably, the functional C. acnes IIIB methylase protein comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over its entire length to SEQ ID NO: 17. Preferably, the C. acnes IIIB methylase protein contained in or expressed by a non-C. acnes bacterium consists solely of SEQ ID NO: 17.

[0106] Preferably, the bacterium used as a non-C. acnes shuttle is E. coli, preferably a recombinant E. coli modified to express a C. acnes methylase and not to express an endogenous E. coli methylase. Preferably, the E. coli is a dam-dcm-hsdMS-E. coli strain in which the E. coli endogenous genes dam (DNA cytosine methyltransferase), dcm (DNA cytosine methyltransferase), and hsdMS (methylase subunit of EcoKI restriction enzyme) are inactivated or their encoded proteins are inactivated or silenced, and the E. coli contains at least a functional C. acnes methylase, preferably MIIIB. Preferably, the dam-dcm-hsdMS-E. coli strain expressing the C. acnes methylase is a strain deposited in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Purposes under the accession number CECT No. 30749 at the Spanish Collection of Type Cultures (Coleccion Espanola de Cultivos Tipo, Edificio 3 CUE. Parc Cientific Universitat de Valencia, Catedratico Augustin Escardino 9, 46980 Paterna, Valencia, Spain) on November 11, 2022 by Universitat Pompeu Fabra (Universitat Pompeu Fabra, C. de la Merce, 12, 08002, Barcelona, ​​Spain). To generate CECT No. 30749, a nucleic acid cassette containing C. acnes methylase and a gene encoding kanamycin resistance (KanR) was produced and introduced into the dam-dcm-E. coli strain genome, specifically into the hsdMS locus, thereby replacing the E. coli endogenous RM system and obtaining the dam-dcm-hsdMS-E. coli strain expressing C. acnes methylase.

[0107] As shown in the examples (Figures 17 and 19), the efficiency of the method was improved when short nucleic acids, i.e. minicircles, were transformed into C. acnes. Thus, the nucleic acids of step a) are preferably obtained by introduction into a shuttle bacterium that is a minicircle-producing bacterial strain, most preferably the dam-dcm-hsdMS-E. coli strain that produces minicircles. "Minicircle-producing bacterial strain" is herein referred to as a bacterium that expresses a set of inducible minicircle assembly enzymes, PhiC31 integrase and I-SceI homing endonuclease, thereby producing minicircles. Minicircles are small circular plasmids formed by site-specific recombination of parental plasmids that causes the removal of sequences involved in plasmid replication in the producing strain. Preferably, the minicircle-producing strains are the CY10P3S2T (Kay et al. 2010) (System Biosciences), JMC2 and JMC3 (Johnston et al. 2019) E. coli strains.

[0108] Therefore, the nucleic acid sequence of step a) is preferably a short nucleic acid, since minicircle (MC) nucleic acid is delivered to C. acnes more efficiently than larger nucleic acid. Minicircle is a plasmid with reduced size, which can be in the range of 3.000 base pairs or less, 2000 base pairs or less, 1000 base pairs or less. Thus, minicircle is a plasmid derived from the original plasmid reduced to a minimum size. In a preferred embodiment, the nucleic acid has a length of up to 10000, 8000, 7000, 6000, 5000, or 4000 nucleotides, preferably up to 3000 nucleotides long. Preferably, the nucleic acid is produced by collecting it from a minicircle-producing bacterial strain.

[0109] Alternatively, the nucleic acid lacking a methylated nucleobase in step a) may be obtained by in vitro synthesis or assembly of said nucleic acid or PCR using classical molecular biology techniques such as restriction digestion and ligation. Both nucleotide synthesis methods and molecular biology techniques are known in the art.

[0110] Alternatively, the nucleic acid sequence of step a) may be produced by incubating unmethylated nucleic acid with a C. acnes methylase, preferably MIIIB, which may be a purified isolated methylase or may be contained in a C. acnes cell lysate.

[0111] The nucleic acid sequence of step a) can be any nucleotide sequence or any polynucleotide, preferably DNA. Preferably, the nucleic acid sequence of step a) is a plasmid or is comprised in a vector, such as a plasmid vector. Preferably, the nucleic acid sequence or polynucleotide is a nucleic acid sequence comprising a gene encoding a protein of interest, such as NGAL, such that the recombinant C. acnes obtained from the method of the ninth aspect expresses the protein of interest. In one embodiment, the nucleic acid sequence comprises a C. acnes origin of replication, such that it resides in the cytoplasm of C. acnes as a plasmid. In some embodiments, the nucleic acid is obtained by harvesting it from a bacterium that is not C. acnes, and the nucleic acid also comprises a replication origin of the bacterium that is not C. acnes.

[0112] In one embodiment, the nucleic acid sequence is a nucleic acid that can be integrated into the C. acnes genome, preferably a suicide vector. In one embodiment, the integration is performed by CRISPR methodology. In a further embodiment, the integration is performed by using a transposase, a phage, a recombinase, an integrase, or any combination thereof. Preferably, the integration is performed by homologous recombination between the nucleic acid or a part thereof and a non-essential region of the genome of C. acnes. A non-essential region of the genome of C. acnes is a region that is not required for the growth and / or survival of C. acnes. In one embodiment, the integration of the nucleic acid sequence or a part thereof into the genome of C. acnes is performed by homologous recombination, and the nucleic acid introduced into the genome of C. acnes is flanked by sequences X and Y that are identical to sequences X' and Y' contained in the genome of C. acnes.

[0113] The method of designing nucleic acid that can be homologous recombined with the genome of a target bacterium is known and described above.In brief, in order for homologous recombination event to occur specifically at the target region of the bacterial genome, the nucleic acid sequence should contain flanking regions that show homology with the target region.When the nucleic acid sequence contains the gene of interest that is introduced into the genome of C. acnes by homologous recombination, the gene should be located in the nucleic acid between the flanking regions that show homology with the target region of the genome of C. acnes.The length of the flanking region for homologous recombination is defined above and applies here.

[0114] The nucleic acid sequence of step a) may comprise a gene encoding a specific protein that performs a specific function in C. acnes after expression by transcription and translation. A gene may comprise a segment of DNA involved in the production of a polypeptide chain, and may comprise regions before and after the coding region (e.g. an open reading frame) involved in the transcription / translation of the gene product and in the regulation of transcription / translation.

[0115] In one embodiment, the nucleic acid sequence of step a) is designed to create a gene knockout in C. acnes when inserted into an endogenous gene of C. acnes. As used herein, "gene knockout" refers to a combination of genetic techniques that can render a particular gene inoperable or inactive. In some embodiments, the gene knockout reduces or eliminates the expression of a polypeptide from a gene. In certain embodiments, the expression of the gene is substantially reduced or eliminated. By substantially reduced, it is meant that the expression of the gene is reduced by at least 80%, at least 90%, at least 95% or at least 98% when compared to the endogenous expression level of the gene.

[0116] In one embodiment, the nucleic acid sequence of step a) is designed to create a knock-in in C. acnes by inserting an exogenous gene or nucleotide sequence into C. acnes. As used herein, "knock-in" refers to a combination of genetic techniques that result in the introduction of a nucleic acid, preferably a gene, into the C. acnes genome. In some embodiments, the gene knock-in results in the expression of a polypeptide from a gene integrated into the C. acnes genome. In some embodiments, the nucleic acid comprises a gene of interest and a promoter configured to direct the expression of the gene of interest. Preferably, the gene of interest encodes NGAL, as described above.

[0117] In some embodiments, the nucleic acid introduced into C. acnes is designed to make it a C. acnes auxotroph. C. acnes auxotrophs require additional supplements / nutrients to grow. These nutrients, such as amino acids, are necessary because they cannot be synthesized by themselves as a result of the mutations caused by inserting the nucleic acid according to the method of the ninth aspect. The amount of C. acnes in the microenvironment can be controlled or the population of C. acnes can be eliminated by depleting the environment of the required nutrients. In some embodiments, the C. acnes comprises a genome with a mutation or knockout in a gene encoding glutamine synthetase. In some embodiments, the C. acnes comprises a genome with a mutation or knockout in a gene encoding asparagine synthetase, diaminopimelic acid (DAP), alanine racemase, d-alanine aminotransferase, thymidylate synthase, diaminopimelic acid decarboxylase, thiamine monophosphate kinase, superoxidase dismutase, pyridoxamine 5'-phosphate oxidase, or any combination thereof.

[0118] As described above, the nucleic acid introduced into, preferably transformed into, the bacterium may include a nucleic acid sequence encoding a selectable marker. The selectable marker may confer resistance to one or more antibiotics. For example, the selectable marker may include genes for ampicillin resistance, streptomycin resistance, kanamycin resistance, tetracycline resistance, chloramphenicol resistance, sulfonamide resistance, erythromycin resistance, lincomycin resistance, clindamycin resistance, or a combination of these markers. Typically, the selectable marker is operably linked to a promoter that promotes the expression of the marker. Plasmids and other cloning vectors that include selectable markers are known in the art.

[0119] Preferably, the nucleic acid is designed to be inserted into a gene selected from the group consisting of camp2 (PPA0687), camp1 (PPA1340), roxP (PPA1939), thymidine kinase (tdk) (PPA1049) or the restriction modified IIIB locus (PPA1610, 1611, 1612).

[0120] Step b) comprises or consists solely of introducing the nucleic acid sequence of step a) into C. acnes.

[0121] As explained above, "introducing a nucleic acid into C. acnes" refers to any method for delivering a nucleic acid into C. acnes, which is not recognized by the C. acnes RM system and is therefore not degraded by the C. acnes RM system. The nucleic acid can be introduced into C. acnes, for example, in the form of a linear or circular plasmid. Once the nucleic acid sequence of step a) is introduced into C. acnes, the C. acnes becomes recombinant C. acnes.

[0122] Preferably, C. acnes is electroporated to introduce the nucleic acid sequence of step a). In the context of the present invention, step b) is understood to be the stable integration of the nucleic acid into the recipient C. acnes, either in its genome or as a replication-independent or self-replicating plasmid. Thus, step b) can also be defined as the introduction of the nucleic acid from step a) into C. acnes, whereby the nucleic acid sequence of step a) is integrated into C. acnes without being degraded. Methods for knowing whether the nucleic acid sequence has been introduced into C. acnes without being degraded include growth under selective conditions, colony PCR, sequencing, or the use of a reporter, such as a fluorescent or luminescent reporter, encoded by the introduced nucleic acid.

[0123] In one embodiment, C. acnes is wild-type C. acnes.Preferably, C. acnes is C. acnes KPA171202 strain or KPA171202 RM knockout strain lacking functional IIIB methylase.As is easily clear in the context of the present invention, C. acnes as used herein is competent C. acnes, i.e., bacterial cells that have been treated to be able to receive extrachromosomal DNA or plasmid (naked DNA) from the environment.Methods for preparing competent C. acnes are known to those skilled in the art.

[0124] A further optimization developed herein was to culture C. acnes in the presence of at least one cell wall permeabilizing agent, such as L-glycine and penicillin G, to facilitate entry of nucleic acids into the cells. L-glycine pretreatment increased efficiency by 60-fold, while 2-10ug / mL penicillin G pretreatment increased transformation efficiency up to 1200-fold compared to the non-pretreated control (Figure 18). Thus, in one embodiment, the method of the eighth aspect comprises culturing C. acnes in the presence of at least one permeabilizing agent of the bacterial cell wall prior to introduction of nucleic acids. "Bacterial cell wall permeabilizing agent" as used herein refers to an agent that increases the permeability of the outer membrane, preferably by altering the molecular interactions of lipopolysaccharide components of the outer membrane. Thus, the method of the ninth aspect comprises: a) providing an unmethylated nucleic acid sequence, provided that if it contains a C. acnes methylation motif, said methylation motif is methylated as defined above; and b) culturing C. acnes in the presence of at least one agent for permeabilizing the bacterial cell wall, followed by introducing the nucleic acid sequence of step a) into said C. acnes.

[0125] In one embodiment, the bacterial cell wall permeabilizing agent is selected from the group consisting of antibiotics (beta-lactams and non-beta-lactams), enzymes (mutanolysin, lysozyme, lysostaphin, endolysin, etc.), chemicals (SDS, Triton X-100, Tween-20, EDTA). Most preferably, the at least one bacterial cell wall permeabilizing agent is glycine or penicillin, preferably L-glycine or penicillin G. In one embodiment, the bacterial cell wall permeabilizing agent is glycine, used in a concentration range of 0,01% to 50%, preferably 1 to 10%, 1 to 5%, most preferably 1,25% (v / v%). In one embodiment, the bacterial cell wall permeabilizing agent is penicillin, used in a concentration range of 1 ng / mL to 1 mg / mL, preferably 1 ng / mL to 0.5 mg / mL, 1 μg / mL to 10 μg / mL, most preferably 5 μg / mL.

[0126] In one embodiment, the step of culturing C. acnes in the presence of the agent for permeabilizing the bacterial cell wall prior to introduction of the nucleic acid sequence is carried out by culturing C. acnes for at least 1 to 48 hours, preferably 1 to 24 hours or 1 to 6 hours, more preferably 4 hours.

[0127] In one embodiment, the step of culturing C. acnes in the presence of said agent for permeabilizing the bacterial cell wall prior to introducing the nucleic acid sequence is carried out by culturing C. acnes at a temperature of 37° C., in a medium suitable for C. acnes culture, under anaerobic conditions, preferably in the presence of sucrose for osmotic stabilization. Suitable culture media include any growth medium, solid, liquid, or semi-solid, that allows for the growth of C. acnes.

[0128] Furthermore, as shown in Figure 14, DNA penetration of fluorescent oligos was increased by about 50-fold using a buffer containing 272 mM sucrose compared to other electroporation buffers tested (Figure 14). Thus, in a preferred embodiment, the nucleic acid sequence provided in step a) is introduced into C. acnes by electroporation, and the electroporation buffer contains 100 µM to 1 M sucrose, preferably 100 to 500 mM sucrose, and most preferably 272 mM sucrose.

[0129] Thus, in one embodiment, the method of the ninth aspect comprises: a) providing an unmethylated nucleic acid sequence, provided that if it contains a C. acnes methylation motif, the methylation motif is methylated; and b) introducing the nucleic acid sequence of step a) into C. acnes by electroporation, wherein the electroporation buffer comprises 100 μM to 1 M sucrose, preferably 100 to 500 mM sucrose, most preferably 272 mM sucrose.

[0130] Furthermore, transformation efficiency can also be increased by using previously frozen C. acnes. Freezing competent cells improved transformation efficiency by up to 12-fold over freshly prepared ones (Figure 16). Thus, in one embodiment, the method of the eighth aspect includes freezing C. acnes prior to the introduction of the nucleic acid sequence. Thus, in one embodiment, the method of the ninth aspect includes: a) providing an unmethylated nucleic acid sequence, provided that if it contains a C. acnes methylation motif, the methylation motif is methylated; and b) freezing and thawing C. acnes cells, and then introducing the nucleic acid sequence of step a) into the C. acnes cells, preferably by electroporation.

[0131] Other factors influencing transformation efficiency were the optical density (OD) of the cells at the time of competent cell preparation and the amount of transformed DNA. A smaller amount of DNA and interestingly a higher OD favored the results (Figures 20 and 21). Thus, in a preferred embodiment of the method of the ninth aspect, C. acnes is cultured before the introduction of the nucleic acid sequence until an optical density (OD) of about 0.1 to 2.5, preferably 1.7, is reached. Methods for measuring OD while culturing bacteria, such as spectrophotometry, are known.

[0132] In a further embodiment, C. acnes is cultured prior to introduction of the nucleic acid sequence until it reaches an optical density (OD) of about 0.1 to 2.5, preferably 1.7, and the nucleic acid sequence is prepared at a concentration of 50 ng to 8 ug, preferably 500 ng, to be introduced into C. acnes.

[0133] A further step after step b) involves selecting recombinant C. acnes in which the introduction of the nucleic acid sequence was successful. Preferably, recombinant C. acnes is selected by using an appropriate selection method, preferably at least one selectable marker. The selection or selectable marker can be positive or negative. A positive selection marker is a selectable marker that confers a selective advantage to the host organism. An example is antibiotic resistance, which allows the host organism to withstand antibiotic selection. Positive selection markers include 23S rRNA (adenine (2058)-N(6))-methyltransferase (ermE), beta-lactamase (bla), neomycin phosphotransferase (npt) or chloramphenicol acetyltransferase (cat), chloramphenicol resistance gene (cml). A negative or counterselectable marker is a selectable marker that eliminates or inhibits the growth of the host organism upon selection. An example would be thymidine kinase, which renders the host sensitive to ganciclovir or FUDR (5-fluoro-2'-deoxyuridine) selection. Negative selection markers include thymidine synthase (thyA), uracil phosphoribosyltransferase (upp), orotidine 5'-phosphate decarboxylase (pyrF), orotate phosphoribosyltransferase (pyrE), and uracil phosphoribosyltransferase (pyrR).

[0134] In a preferred embodiment, the selection of recombinant C. acnes is performed by using at least two selectable markers, preferably at least one positive and one negative marker. Combining a positive and a negative selectable marker is advantageous as it provides a stronger and more efficient selection of the engineered bacterial clone. Preferably, the selectable markers are 23S rRNA (adenine (2058)-N(6))-methyltransferase (ermE) and thymidine kinase (tdk).

[0135] Preferred embodiments of the eight aspects include the following:

[0136] In one embodiment, the method of the ninth aspect comprises: a) providing an unmethylated nucleic acid sequence, provided that if it contains a C. acnes methylation motif, the C. acnes methylation motif is methylated; and b) introducing the nucleic acid sequence of step a) into C. acnes, The nucleic acid sequence of a) is obtained by introducing a nucleic acid sequence into a bacterium that is not C. acnes, the bacterium containing or expressing a functional C. acnes methylase and not containing or expressing other functional methylases, and the nucleic acid sequence is harvested from said bacterium to provide the nucleic acid sequence of step a), preferably the C. acnes methylation motif is AGCAGY.

[0137] In one embodiment, the method of the ninth aspect comprises: a) providing an unmethylated nucleic acid sequence, provided that if it contains a C. acnes methylation motif, the methylation motif is methylated; and b) introducing the nucleic acid sequence of step a) into C. acnes, The nucleic acid sequence of a) is obtained by introducing a nucleic acid sequence into a non-C. acnes bacterium, preferably a dam-dcm-hsdMS-E. coli modified to express a C. acnes methylase, most preferably a bacterium of CECT No. 30749, from which the nucleic acid sequence is harvested to provide the nucleic acid sequence of step a).

[0138] In one embodiment, the method of the ninth aspect comprises: a) providing a nucleic acid sequence, said nucleic acid sequence comprising: i. is unmethylated and does not contain the C. acnes methylation motif, or ii. providing a nucleic acid sequence comprising one or more C. acnes methylation motifs, wherein the nucleic acid sequence is methylated only at said C. acnes methylation motifs; b) introducing the nucleic acid from step a) into C. acnes; The nucleic acid sequence of a) is obtained by introducing a nucleic acid sequence into a non-C. acnes bacterium, preferably a dam-dcm-hsdMS-E. coli modified to express a C. acnes methylase, most preferably a bacterium of CECT No. 30749, from which the nucleic acid sequence is harvested to provide the nucleic acid sequence of step a).

[0139] In one embodiment, the method of the ninth aspect comprises: a) providing a nucleic acid sequence comprising one or more C. acnes methylation motifs, wherein the one or more methylation motifs are methylated and the remainder of the nucleic acid sequence is unmethylated; b) introducing the nucleic acid sequence of step a) into C. acnes, which has previously been cultured in the presence of at least one permeabilizing agent of the bacterial cell wall, preferably L-glycine or penicillin G, The nucleic acid sequence of step a) is obtained by introducing a nucleic acid sequence comprising one or more C. acnes methylation motifs into a non-C. acnes bacterium, which is characterised in that it contains or expresses a functional C. acnes methylase, preferably a C. acnes IIIB methylase, and does not contain or express other functional methylases, and the nucleic acid sequence is harvested from said bacterium to provide the nucleic acid sequence of step a).

[0140] In one embodiment, the method of the ninth aspect comprises: a) providing an unmethylated nucleic acid sequence lacking a C. acnes methylation motif in its sequence; and b) introducing the nucleic acid sequence of step a) into C. acnes, which has previously been cultured in the presence of at least one permeabilizing agent of the bacterial cell wall, preferably L-glycine or penicillin G, The nucleic acid sequence of a) is obtained by designing a nucleic acid that lacks a C. acnes methylation motif in its sequence, preferably lacking AGCAGY, and introducing it into a non-C. acnes bacterium, which is characterised in that it does not contain or express a functional methylase and is therefore unable to methylate the nucleic acid, and a nucleic acid sequence is harvested from the bacterium to provide the nucleic acid sequence of step a).

[0141] In one embodiment, the method of the ninth aspect comprises: a) providing a nucleic acid sequence comprising one or more C. acnes methylation motifs defined by the sequence AGCAGY, wherein the one or more methylation motifs are methylated at the second adenine of the sequence (AGC(m)AGY), and the remainder of the nucleic acid sequence (i.e. the remaining nucleobases) are unmethylated; b) introducing the nucleic acid sequence of step a) into C. acnes, which has previously been cultured in the presence of at least one permeabilizing agent of the bacterial cell wall, preferably L-glycine or penicillin G, The nucleic acid sequence of step a) is obtained by introducing a nucleic acid sequence comprising one or more C. acnes methylation motifs defined by the sequence AGCAGY into a non-C. acnes bacterium, said bacterium being characterised in that it contains or expresses a functional C. acnes methylase, preferably a C. acnes IIIB methylase, and does not contain or express any other functional methylases, and from which the nucleic acid sequence is harvested to provide the nucleic acid sequence of step a).

[0142] In a tenth aspect, the present invention provides a recombinant C. acnes obtained or obtainable from the method of the ninth aspect or any of its embodiments, characterized in that it comprises a nucleic acid sequence as defined in step a) of the ninth aspect or any of its embodiments. Preferably, the nucleic acid encodes NGAL and the recombinant C. acnes is a recombinant C. acnes of the fourth aspect expressing NGAL, preferably obtained by the method of the ninth aspect.

[0143] A preferred embodiment of the tenth aspect refers to a composition, preferably a cosmetic or pharmaceutical composition, comprising recombinant C. acnes obtained or obtainable from the method of the ninth aspect or any of its embodiments. The composition may further comprise a carrier or additive, or auxiliary substance, for formulating the composition or for administering the composition in a desired manner, as described above in the seventh aspect or any of its embodiments. In some embodiments, the composition is formulated, for example, as a topical (e.g., dermal) formulation. In some embodiments, the composition is formulated, for example, for topical administration to a mammal. Topical formulations include, for example, gel formulations, cream formulations, lotion formulations, paste formulations, ointment formulations, oil formulations, foam formulations, and the like. The composition may further comprise, for example, an absorbent emollient. Further examples of compositions may optionally be formulated to be delivered to a mucosa, or by inhalation, respiration, intranasal, oral, buccal or sublingual administration. Chelating agents, buffering agents, co-solvents, preservatives, antioxidants, and antimicrobial agents may be added. Supplementary compounds (e.g. biocides and biological agents, such as antibacterial, antiviral and antifungal agents) can also be added. Compositions comprising recombinant C. acnes are described in the seventh aspect, and therefore that embodiment also applies herein. C. acnes is a common commensal of the skin microbiota and therefore can be administered at least topically to a subject. The tenth aspect of the invention also includes a composition, preferably a pharmaceutical or cosmetic composition, comprising recombinant C. acnes obtained from the method of the ninth aspect or any of its embodiments.

[0144] In an eleventh aspect, the present invention relates to different uses of recombinant C. acnes as defined in the tenth aspect, or of a composition comprising said C. acnes. The uses of said recombinant C. acnes may differ depending on the characteristics of the nucleic acid introduced into said recombinant C. acnes. Recombinant C. acnes carrying nucleic acids may be used for controlled expression of peptides, since it migrates on the skin and propagates deep into pores and hair follicles, allowing the absorption of secreted biomolecules. All uses include topical application of said recombinant C. acnes, including when the recombinant C. acnes is alive.

[0145] In one embodiment, recombinant C. acnes may be used in therapy or as a pharmaceutical composition. The recombinant C. acnes may be used to secrete a biological molecule (e.g., a protein) to treat a disorder in a subject in need thereof. Preferably, the recombinant C. acnes obtained or obtainable by the method of the ninth aspect or any of its embodiments is used to treat a skin disease, preferably a disease associated with excessive seborrheic secretion. Preferably, the use is in the treatment of acne vulgaris, urticaria, eczema, rosacea, hidradenitis suppurativa and / or psoriasis, as described above. Other uses of recombinant C. acnes include in diagnostic methods.

[0146] The recombinant C. acnes of the tenth aspect may be used in cosmetics or as a cosmetic composition. Preferably, the use in cosmetics includes halting, reversing, improving, diminishing, and / or reducing skin imperfections, defects, and / or aesthetically unpleasing conditions, including but not limited to age spots, sun spots, age spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, pimples, skin blemishes, skin fragility, dryness, spots, roughness to the touch, cracks, sagging, thinning, enlarged pores, cellulite formation, acne formation, rosacea, psoriasis, and eczema.

[0147] In a twelfth aspect, the present invention provides a kit comprising a recombinant C. acnes of the tenth aspect or any of its embodiments. The kit may comprise a composition and a pharmaceutical formulation thereof packaged in a suitable packaging material. The kit may be used in a variety of in vitro, ex vivo and in vivo methods and uses, such as the treatment methods or uses disclosed herein.

[0148] The kit typically includes a label or packaging insert that includes a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein. The kit can contain a collection of such components, e.g., C. acnes in combination with a non-C. acnes bacterium. The kit can include a label or insert. The label can include the identity of one or more components therein, the dose, the clinical pharmacology, pharmacokinetics, and pharmacodynamics of the active ingredient(s) including mechanism of action. The label or insert can include information identifying the manufacturer, lot number, manufacturer's location, and date.

[0149] The label or insert may include information regarding the condition, disorder, disease, or symptom for which the kit components may be used. The label or insert may include instructions for a clinician or subject to use one or more of the kit components in a method, use, treatment protocol, or therapeutic regimen. The instructions may include dosage, frequency, or duration, and instructions for carrying out any of the methods and uses, treatment protocols, or therapeutic regimens described herein.

[0150] In a thirteenth aspect, the present invention also relates to a nucleic acid sequence provided in step a), which is an unmethylated nucleic acid, with the proviso that, if it comprises a C. acnes methylation motif, said methylation motif is methylated as defined under the eighth aspect. Thus, all embodiments of the eight aspects relating to the nucleic acid sequence of step a), in particular how to obtain it, are included in the thirteenth aspect.

[0151] The following provisions are also included in the present invention:

[0152] 1. A human neutrophil gelatinase-associated lipocalin (NGAL) protein derived from a recombinant bacterial culture, the NGAL protein being produced by the following steps: a) culturing in a suitable medium a recombinant bacterium characterized in that it expresses a human neutrophil gelatinase-associated lipocalin (NGAL) protein, preferably a secreted NGAL protein; b) harvesting the NGAL protein from the culture of a), preferably from the supernatant of the culture of a); c) optionally isolating the NGAL collected in b).

[0023] In one embodiment, the method comprises the steps of: A human neutrophil gelatinase-associated lipocalin (NGAL) protein for use in inhibiting sebum production in human epithelial cells, preferably sebocytes.

[0153] 2. Human neutrophil gelatinase-associated lipocalin (NGAL) protein from a recombinant bacterial culture, the NGAL protein being produced by the following steps: a) culturing in a suitable medium a recombinant bacterium characterized in that it expresses a human neutrophil gelatinase-associated lipocalin (NGAL) protein, preferably a secreted NGAL protein; b) harvesting the NGAL protein from the culture of a), preferably from the supernatant of the culture of a); c) optionally isolating the NGAL collected in b).

[0023] In one embodiment, the method comprises the steps of: 1. A human neutrophil gelatinase-associated lipocalin (NGAL) protein for use in the treatment of acne vulgaris, urticaria, eczema, rosacea and / or psoriasis.

[0154] 3. A non-therapeutic cosmetic use of human neutrophil gelatinase-associated lipocalin (NGAL) protein derived from a recombinant bacterial culture, the NGAL protein being produced by the following steps: a) culturing in a suitable medium a recombinant bacterium characterized in that it expresses a human neutrophil gelatinase-associated lipocalin (NGAL) protein, preferably a secreted NGAL protein; b) harvesting the NGAL protein from the culture of a), preferably from the supernatant of the culture of a); c) optionally isolating the NGAL collected in b).

[0023] In one embodiment, the method comprises the steps of: Non-therapeutic cosmetic uses, preferably for preventing, reducing and / or ameliorating skin aging.

[0155] 4. The use according to any of clauses 1 or 2 or the method according to clause 3, wherein the recombinant bacterium is Cutibacterium acnes or Escherichia coli.

[0156] 5. The use according to any of clauses 1 or 2 or the method according to clause 3, wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length with SEQ ID NO:1.

[0157] 6. The use according to any of clauses 1 or 2 or the method according to clause 3, wherein the recombinant bacterium is Cutibacterium acnes and the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length to SEQ ID NO:1.

[0158] 7. A method for producing an active ingredient of a pharmaceutical composition, the active ingredient being a human neutrophil gelatinase-associated lipocalin (NGAL) protein capable of inhibiting sebum production in human epithelial cells, preferably sebocytes, the method comprising the steps of: a) culturing in a suitable medium a recombinant bacterium characterized in that it expresses a human neutrophil gelatinase-associated lipocalin (NGAL) protein, preferably a secreted NGAL protein; b) harvesting the NGAL protein from the culture of a), preferably from the supernatant of the culture of a); c) optionally isolating the NGAL collected in b).

[0023] The present invention is obtained or obtainable from a method comprising the steps of:

[0159] 8. The method according to clause 7, wherein the recombinant bacterium is Cutibacterium acnes or Escherichia coli.

[0160] 9. The method of clause 7 or 8, wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length with SEQ ID NO:1.

[0161] 10. The method of any of clauses 7 to 9, wherein the recombinant bacterium is Cutibacterium acnes and the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length to SEQ ID NO:1.

[0162] 11. A recombinant Cutibacterium acnes characterized by expressing the human neutrophil gelatinase-associated lipocalin (NGAL) protein.

[0163] 12. The recombinant Cutibacterium acnes according to clause 11, wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length with SEQ ID NO:1.

[0164] 13. The recombinant Cutibacterium acnes according to any of clauses 11 or 12, wherein the nucleic acid encoding human neutrophil gelatinase-associated lipocalin (NGAL) is operably linked to a promoter selected from the group consisting of a camp2 promoter, a camp1 promoter or a roxP promoter.

[0165] Sequence Listing NGAL amino acid sequence without the signaling peptide (SEQ ID NO:1): QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG* The nucleotide sequence of NGAL without the signal peptide (SEQ ID NO:2): * PPA1939 secreted peptide nucleotide sequence: (roxP) (SEQ ID NO:3): atgttcgtccaaatcgctgccagcctggcagccgcatcgtccattgcactcggcataccaggagctgcc PPA0687 secreted peptide nucleotide sequence: (Camp2) (SEQ ID NO:4): atgaagaagacccatcttgtagctcccctccttgtcggcgcaatgctcgtaccagcggcgctgtcagctcccagtgctcatgct PPA1340 secreted peptide nucleotide sequence: (camp1) (SEQ ID NO:5): atgaaggttaagttcttagcagcgccgctggttgttggtgccttgatggcgccggcagctttctctggagcgacagctcatgct PPA1939 secreted peptide amino acid sequence: (roxP) (SEQ ID NO:6): MFVQIAASLAAASSIALGIPGAA PPA0687 secreted peptide amino acid sequence: (Camp2) (SEQ ID NO:7): MKKTHLVAPLLVGAMLVPAALSAPSAHA PPA1340 secreted peptide amino acid sequence: (camp1) (SEQ ID NO:8): MKVKFLAAPLVVGALMAPAAFSGATAHA PPA0532 secreted peptide amino acid sequence: (SEQ ID NO:18): MWPIRTVRAYCAGTVATAVALAAFVTSGAHA PPA0533 secreted peptide amino acid sequence: (SEQ ID NO:19): MIRSLVATLAVGAIVTAGAQMPAQA PPA0534 secreted peptide amino acid sequence: (SEQ ID NO:20): MLRRAVVGAVTAAVTSTTVLSVVPAGA PPA0598 secreted peptide amino acid sequence: (SEQ ID NO:21): MALRRTLTLMLCAITGASLVATAVPSSA PPA0644 secreted peptide amino acid sequence: (SEQ ID NO:22): MYHHSWHSPDARRRGVTRWATTFIAALTAA PPA0721 secreted peptide amino acid sequence: (SEQ ID NO:23): MEHRYGASQVSGSAPRRGRRGVAFAAITGAILLGTVASVDPGAQA PPA1498 secreted peptide amino acid sequence: (SEQ ID NO:24): MMPENEDELTRSVDKAARNTLGRRTFLGGVTLVAAAAVTGTTPALAHA PPA1662 secreted peptide amino acid sequence: (SEQ ID NO:25): MNCSFNRRQRSWLAAMVSGMTVITLVPAAVAHA PPA1715 secreted peptide amino acid sequence: (SEQ ID NO:26): MKQRFKSPTTAVLTGVLIAGSALLLPPLPAKA PPA2097 secreted peptide amino acid sequence: (SEQ ID NO:27): MNHRRRARIAGLSVLCMAAGSFVAKPAFA PPA2105 secreted peptide amino acid sequence: (SEQ ID NO:28): MKINARFAVMAASVAVLMAAAPIAQA PPA2106 secreted peptide amino acid sequence: (SEQ ID NO:29): MRRKSALGFVALSLFATGMGVAAATPATA PPA2142 secreted peptide amino acid sequence: (SEQ ID NO:30): MTFTKKLSALAIAGTMAITGASIATVPAVA PPA2175 secreted peptide amino acid sequence: (SEQ ID NO:31): MTNTSTNAGTTLKRTVALAAAASLAVMGTIAEEAHA PPA2239 secreted peptide amino acid sequence: (SEQ ID NO:32): MSKVVASAIAGALSLTSAGGLTMVQA PPA2164 secreted peptide amino acid sequence (SEQ ID NO:33): MRTDLLRLVSLSRWHNSRREHNMRRSTVKRRTVMAAAAGAFAASALPLSRAAA NGAL amino acid sequence with signal peptide PPA1939 (SEQ ID NO:9): TIFF2025504502000001.tif26170 NGAL amino acid sequence with signal peptide PPA0687 (SEQ ID NO:10): TIFF2025504502000002.tif25170 NGAL amino acid sequence with signal peptide PPA1340 (SEQ ID NO:11): TIFF2025504502000003.tif26170PPA1340 Promoter sequence: (Camp1) (SEQ ID NO: 12): TAGGTTGACTGGAAAGGGGATATGACACCCATCGTCCTGGGTCTGACAGGGGTAGACAATATAGCTTAGAAATGTGTATAAGTAAGATGTTTCTCGCAATGAAAGACGATATGTCACGAGTTTTCTGGTG CTGTTTCCCGACACGGAACCGTTAGTTTGAATCAATTTTGAACTGAGGACACCAAGTTGACCCCCTGGTGTGCAACGATGCTTCTTGCTTGCAGTTGCGAGCAATTGTTCCCGATGAAAGGAACCCACAA PPA1939 promoter sequence: (RoxP) (SEQ ID NO: 13): ACACCGACGATCTGCACGGGCGAGTCTAGGGGTTATCGGCTACATGCGCGTCGCCGCATCAGGTTCACGAAACCGAAAGGCAGACCCACCCCCTACCGGGCATACCGTTGCCGCCGACACTTCGATGACACTATGCAGTGCTACACTTCAACTGAAAATCTATATGATTTTCGTCAATTCCCATACGTACGACACATCGGGAGATCGTC PPA0687 promoter sequence: (Camp2): (SEQ ID NO:14): gcaacaagcctagacgggtctatgcaccgtcgtagccatacaccacacgggcccctgtcggaatgacgcacgcctgatcacagctccaacaaatgttggc ggcgtacacgccgttcaccacaccgacccccatccaaaatatccgatacacttcgcttgcctcgaccggggcatccgcaaccaataaacggagaaccttt pBR13 complete plasmid sequence: Suicide vector for targeting the tdk locus (5139 bp): (SEQ ID NO: 15): Bold capital letters: PPA1340 promoter sequence: (Camp1) (SEQ ID NO: 12) Lowercase bold: roxP secretory peptide (SEQ ID NO:3) Underlined: LCN2 gene encoding NGAL (SEQ ID NO:2) TIFF2025504502000004.tif22170TIFF2025504502000005.tif249170TIFF2025504502000006.tif174170pBR13 complete plasmid sequence: replicative vector derivative of pBRESP36A (9215 bp): (SEQ ID NO: 16) Bold capital letters: PPA1340 promoter sequence: (Camp1) (SEQ ID NO: 12) Lowercase bold: roxP secretory peptide (SEQ ID NO:3) Underlined: LCN2 gene encoding NGAL (SEQ ID NO:2) TIFF2025504502000007.tif21170TIFF2025504502000008.tif251170TIFF2025504502000009.tif250170TIFF2025504502000010.tif250170TIFF2025504502000011.tif248170TIFF2025504502000012.tif53170C.acnes IIIB methylase protein (SEQ ID NO: 17) MTAQRLQLTWYNKDKALIPTETGKYGYTWVDPSDPRYCETHTLVLDDYVQGSQTPKSDEFAYSERADLEPQDDNLLILGESGDVLEALTRVPELAEKYVGKVKLIYIDPPFNTAQTFASYEDNLEHSIWLTMMRDRLHHMKKLLADDGSIWVHLDYAENHRMRLL LDEVFGCSNFIAEFVWQKADSPRGDAQRVSVDQDVILCYAASGSTVMNRMERTAADNARFSNPDGDSKGVWFSDNRSAPTNVMSWQHPSTFAIQHPISGEMIYPAKGGCWRFGRERLLESLNEYAEYASGDVDIAARVANTSLRSDQVRSDIPDLVLVDPASAAQC ARTRIDDGNWPEFFVTATSFGRKSYPPNEGQPARSWWPNDQVGHNREAKSEIKALFSGATPFSTPKPERLLERIIHIGSNPGDIVLDVFAGSGTTAAVAQKMGRRWVTCELLESTFTTFTRPRLEKVLNDQDPGGITRTKGERVDATEDGLPDGVSPEDAAKFTSV LNKLIKDDPELKKSIEVKTLKAASKTRRTKEVVNWRGGGGFQVAHLSPACFDYAPELDRVMLTAAATGQTLIESVTANLGFTLLHPDDDYIFDARRGNALLKVVEGVATTEIVDWLASQIQPGETIVLAATTVMDGVRQHLRKLVKGSRVVALPDDVFRYSEGGDQ EXAMPLES

[0166] Example 1: Methods bacterial culture C. acnes KPA171202 was grown on Brucella agar plates from glycerol and incubated anaerobically at 37 °C for 3 days using a GasPak EZ anaerobic pouch system (BD). When grown in liquid, C. acnes KPA171202 reached an OD of 0.1. 600and grown anaerobically in Brain-Heart Infusion broth (BHI) (Sigma) at 37°C and 110 rpm using the AnaeroGen system (Thermo Scientific) until the desired optical density was reached. E. coli DH5a (NZYTech) was used for routine cloning, and E. coli dam-dcm- (New England Biolabs) was used to generate C. acnes methylase-competent shuttle strains. E. coli strains were grown in LB medium supplemented with 50 μg / mL ampicillin or 50 μg / mL kanamycin and grown at 37°C and 225 rpm.

[0167] Cloning of different variations of promoters and signaling peptides: Six different constructs with two promoters and three secretory peptides were cloned into the pGEM-Teasy vector with 500bp of homology upstream and downstream of the genes to be replaced (camp2, camp1, roxP, tdk, etc.) (Meike Sorensen, et al. (https: / / doi.org / 10.1016 / j.mimet.2010.09.008), Nazipi S, et al. (The Skin Bacterium Propionibacterium acnes Employs Two Variants of Hyaluronate Lyase with Distinct Properties. Microorganisms. 2017 Sep 12;5(3):57. Doi:10.3390 / microorganisms5030057). and Allhorn, M et al. (A novel enzyme with antioxidant capacity produced by the ubiquitous skin colonizer Propionibacterium acnes. Sci Rep 6,36412 (2016). https: / / doi.org / 10.1038 / srep36412) or cloned into pBRESP36A plasmid, which is a replicative plasmid in C. acnes, and the transformation efficiency with this vector (or derivatives) is much higher than that with suicide vectors for homologous recombination, making it more suitable for protein production studies (see Jore JP,et al. Efficient transformation system for Propionibacterium freudenreichii based on a novel vector. Appl Environ Microbiol. 2001;67(2):499-503. doi:10.1128 / AEM.67.2.499-503.2001). All gene constructs were fused to a C-terminal histidine tag for purification or Western blot analysis.

[0168] [Table 1]

[0169] Recombinant NGAL protein purification from C. acnes supernatant Proteins from 100 mL of filtered supernatant were analyzed using HiTrap Ni 2+ After column purification, followed by desalting and size exclusion, total protein was quantified using the Qubit protein assay kit (Invitrogen) to determine the concentration and final protein yield of pBR13 (0.46 mg), pBR14 (0.14 mg), and pBR16 (0.50 mg).

[0170] Cultivation of C. acnes for protein production and secretion Transformed C. acnes KPA171202 was inoculated into 50mL BHI medium supplemented with 10μg / mL erythromycin to a starting OD600 of 0.1 and grown anaerobically for 48 hours until an OD of 1.2 was reached. Cells were spun down at 5000rpm for 10 minutes. The pellet was resuspended in 1mL S30 buffer and lysed by FastPrep FP120 for 25 seconds at speed 6.5 for 2 cycles and centrifuged at full speed for 30 minutes at 4 degrees. The supernatant was filtered through 0.22um. Both the filtered supernatant and the lysate were used for TCA precipitation of secreted proteins.

[0171] Deoxycholic acid-trichloroacetic acid (TCA) precipitation of C. acnes produced NGAL protein Proteins from the lysate and 50 mL of the filtered supernatant were TCA precipitated. Briefly, protein-containing supernatants were treated with deoxycholic acid (DOC) for 10 min at room temperature and then precipitated with 10% trichloroacetic acid (TCA) (Sigma Aldrich) for 30 min on ice. Samples were centrifuged at 10000xg, 4°C for 15 min, and protein pellets were washed twice with pure ice-cold acetone, air-dried, and resuspended in 100 μl of 25 mM Tris-HCl buffer (pH 8.0).

[0172] Western blotting 20 μl of TCA-precipitated proteins were mixed with SDS-PAGE sample loading buffer, boiled at 95°C for 5 min, and electrophoresed on NuPAGE 4-12% Bis-Tris SDS-gels (Invitrogen). Proteins were transferred to Hybond-P polyvinylidene difluoride membranes using an Invitrogen Mini Transblot apparatus. Membranes were blocked in PBS-0.05% Tween-20 and 5% skim milk (blocking solution) for 1 h at room temperature, incubated with a specific antibody against the histidine tag (BioRad), diluted 1:800 in blocking solution overnight at 4°C, and then incubated with an anti-mouse IgG antibody conjugated to peroxidase (1:10000) for 1 h at room temperature. Antibody-protein complexes were visualized using the ECL Plus Western blotting detection system (GeHealthcare).

[0173] Recombinant NGAL protein production in E. coli BL21 The LCN2 gene encoding NGAL protein was cloned into pETM14 N-terminal his-tagged vector and transformed into E. coli BL21 cells. Recombinant human (rhNGAL) protein was expressed overnight in autoinduction LB medium at 25°C. Cells were lysed by French press and protein was purified by HiTrap Ni2+ column followed by desalting and size exclusion. 98 mg of NGAL protein was obtained from 1 L culture.

[0174] Toxicity testing of human recombinant NGAL produced in E. coli (rhNGAL) or HEK293 cells against C. acnes cultures C. acnes KPA171202 was inoculated to a starting OD600 of 0.05. rhNGAL protein produced in E. coli BL21 or HEK293 cells (stock 1.35 mg / mL) was diluted and added to the cultures at final concentrations of 0, 100, 500 and 1000 ng / mL. Bacteria were incubated with rhNGAL or human NGAL protein for 24 hours at 37°C and 110 rpm. After 24 hours, toxicity to the cultures was measured by their OD600.

[0175] Sebaceous cell culture Immortalized human sebaceous cells SZ95 were seeded at a density of 2x10^5 cells / well and supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U / mL Pen / Strep, 1mM CaCl 2 Cells were maintained in DMEM / F12 (Gibco) supplemented with 0.1% ethanol and 5 ng / ml human epidermal growth factor (EGF; Sigma-Aldrich). Cells were incubated at 37°C in a humidified 5% CO2 incubator.

[0176] PCi-SEB_Cau (Phenocell) were seeded and maintained in PhenoCULT-SEB basal medium supplemented with Supplement A (1 / 1000) for 3 days prior to exposure and before the start of the assay, according to the manufacturer's protocol.

[0177] MTT viability assay of human recombinant NGAL produced in E. coli (rhNGAL) on human SZ95 sebaceous cells Human SZ95 sebocytes were seeded in 12-well plates at a density of 2x10^5 cells / well and incubated in a humidified atmosphere (37°C, 5% CO2) until 80% confluency in Sebomed medium supplemented with 10% heat-inactivated FBS, 100U / mL Pen / Strep, 1mM CaCl2, and 5ng / ml human epidermal growth factor (EGF; Sigma-Aldrich Co.). PCi-SEB_Cau (Phenocell) were seeded and incubated in a humidified atmosphere (37°C, 5% CO2) until 80% confluency, 2x10^5 cells / well in PhenoCULT-SEB basal medium supplemented with 1 / 1000 supplement A for 3 days before treatment. SZ95 was then incubated with purified rhNGAL protein produced in E. coli BL21 cells at concentrations ranging from 0, 50, 100, 500 or 1000ng / mL for 24 hours, followed by incubation with fresh medium and 10% MTT (5mg / mL in phosphate buffered saline) for 2 hours at 37°C. Furthermore, SZ95 and PCi-SEB_Cau were incubated with human NGAL protein produced in HEK293 cells (LC2-H5222, ACROBiosystems) at concentrations of 0, 100 and 1000ng / mL for 96 hours, followed by incubation with fresh medium and 10% MTT (5mg / mL in phosphate buffered saline) for 2 hours at 37°C. Afterwards, the medium was carefully removed and 500μL of dimethylsulfoxide (DMSO) 99% purity was added to dissolve the cells and dissolve the purple insoluble crystals of MTT. The cell lysates were transferred to a new 96-well plate and the absorbance was then read using a Microplate Autoreader (Tecan) at excitation / emission of 540 / 630 nm. The absorbance value was considered to be directly proportional to cell viability.

[0178] Tunel assay SZ95 and PCi-SEB_Cau sebocytes were cultured in 96-well plates as described above. Wells were rinsed with PBS and treated with vehicle control, 50 ng / ml, 100 ng / ml, or 1 μg / ml of NGAL (E. coli, HEK293, or C. acnes) protein in triplicate for 24 h. Samples were prepared according to the manufacturer's instructions for the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics). DNase I treatment of positive and negative controls (without terminal transferase) was included as assay controls according to the manufacturer's instructions. Digital images from Operetta HCS fluorescence microscopy experiments were processed using the Harmony high content analysis version 4.9 software package (Perkin-Elmer). When performed, image processing was limited to changes in brightness and / or contrast and was applied equally across images of the same experiment, including controls, and across different images. At least three fields of view per biological replicate were randomly selected for image analysis. To measure staining intensity, the Harmony high content analysis software package was used with a custom algorithm. The DAPI channel was used to detect nuclei. Cytoplasmic area was determined as a ring of constant size around each nucleus and used to measure the mean fluorescence intensity of the stain of interest.

[0179] Sebum regulation assay Recombinant NGAL incubation with Pci SEB-Cau sebocytes to measure sebum reduction. Pci-SEB_Cau human iPSC-derived sebocytes (Phenocell) were incubated at 25000 viable cells / cm in PhenoCULT SEB medium supplemented with 1 / 1000 supplement A. 2Cells were seeded in 24-well plates at a cell density of 1000 x 1000 cells / well. Cells were grown in a humidified incubator (37°C, 5% CO2) for 3 days before exposure and assay initiation. Experiments were performed in triplicate with treatment with either vehicle, 5μM AA (arachidonic acid), 5μM AA + 10μM ISO (13-cis retinoic acid), 5μM AA + 50ng / mL rhNGAL (from E. coli BL21), 5uM AA + 50ng / mL pBR13 NGAL (from C. acnes KPA171202, see Table 1), 5μM AA + 50ng / mL pBR14 NGAL (C. acnes KPA171202, see Table 1), or 5μM AA + 50ng / mL pBR16 NGAL (C. acnes KPA171202, see Table 1). Cells were exposed for 48 hours, then cellular lipid content was measured by BODIPY dye 493 / 503 (Sigma Aldrich) according to the manufacturer's recommendations. Nuclei were stained with DAPI. Samples were analyzed by confocal fluorescence microscopy, and fluorescence was measured and normalized.

[0180] Bacterial engraftment on mouse skin Bacteria (WT and KI) were cultured to an OD of 1 in 20 mL. 600The cells were grown to 100°C, spun down at 1700×g for 10 min, washed three times with 0.5% peptone / PBS, and resuspended in 0.5% peptone / PBS to a final volume of 500 ul. Mice (C57BL / 6J) were shaved over a 2 cm×5 cm area on their backs. Wild-type, knockout, or vehicle was applied to the shaved area with a cotton swab and repeated for three consecutive days. Before application, samples were taken with a moistened cotton swab each day, dissolved in 0.5 mL of QuickExtract™ DNA Extraction Solution (Lucigen), and treated at 65°C for 6 min, followed by 95°C for 2 min, vortexing for 15 sec between steps. Samples were taken every 24 hours for the entire assay period of 7 days. Five microliters of DNA extract was used for amplification of the SLST region or the 16S V3-V4 region linked to the illumination adapter using KAPA HiFi HotStart Readymix (Roche) (initial denaturation at 95°C for 5 min, followed by 35 cycles of 98°C for 20 s, 62°C for 25 s, and 72°C for 30 s; and a final extension at 72°C for 1 min).

[0181] SLST_illumina_fwd: SEQ ID NO: 34 TIFF2025504502000014.tif5170 and SLST_illumina_rev:SEQ ID NO:35 TIFF2025504502000015.tif517016S_illumina_fwd:SEQ ID NO:36 TIFF2025504502000016.tif4170 and 16S_illumina_rev: SEQ ID NO: 37 TIFF2025504502000017.tif11170

[0182] Junctions were PCR amplified using the primer pairs Junction1_fwd and rev and Junction2_fwd and rev using KAPA HiFi HotStart Readymix (Roche) (initial denaturation at 95°C for 3 min, followed by 25 cycles of 98°C for 20 s, 62°C for 15 s, and 72°C for 30 s; and a final extension at 72°C for 1 min) (see Supplementary Table 1). The PCR products were loaded onto a 1% agarose gel and visualized by transillumination.

[0183] Skin biopsy Bacteria were applied to the shaved backs of mice for three consecutive days. 24 hours after the last application, mice were sacrificed and skin biopsies were taken. Samples for RNA extraction were stored in RNAlater solution, and samples for cryosectioning were frozen and embedded in OCD solution.

[0184] RNA extraction and qPCR from skin biopsies Total RNA was extracted from skin (NucleoSpin RNA Extraction Kit, Macherey-Nagel) according to the manufacturer's instructions. RNA was then used for cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific). Quantitative PCR amplification reactions were performed on a QuantStudio 7 Pro Real-Time PCR System (Applied Biosystems) using TaqMan and Sybr Green Gene Expression Master Mix. Values ​​were normalized to Sdha. The following TaqMan assays (Thermo Fisher Scientific) were used to quantify mRNA expression of mouse Sdha (Mm01352366_m1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1) and Tnfα (Mm00443258_m1) as well as primers LCN_qPCR_fwd and rev to detect human NGAL.

[0185] Gram staining of skin sections Cryosections from frozen tissues embedded in OCT (Optimal Cutting Temperature (OCT) compound Tissue-Tek) were cut at a thickness of 7 um using a cryotome. Frozen skin sections were air-dried for 15 min at room temperature, fixed in 4% paraformaldehyde for 15 min, and stained with Gram following standard procedures. Crystal violet was applied for 1 min after a water wash, then iodine was applied for 1 min, followed by a water wash, rapid decolorization was achieved using acetone, and a final incubation with safranin for 1 min and subsequent washes. After the samples were dried, DPX (Sigma Aldrich) mounting medium was applied. Conventional images were captured using a Zeiss apoptome microscope.

[0186] Plasmid cloning Plasmids for homologous recombination were cloned as previously described (Sorensen et al. 2010). Briefly, homologous arms were amplified from genomic DNA containing restriction sites for NcoI, SpeI and Acc65I. They were digested with Acc65I, ligated and finally cloned into the pGEM-T-easy vector by restriction enzyme-mediated cloning. In a second step, the gene of interest was cloned between the homologous arms. The replicative plasmid was based on the P. freudenreichii vector pBRESP36A (Jore et al. 2001) and optimized by Lood et al. for protein production in C. acnes (Lood 2011). The selected secretory peptides and the LCN2 gene were cloned downstream of the p1340 promoter using the NheI and SpeI restriction sites.

[0187] Mini Circle Production Minicircle strains ZYCY10P3S2T (Kay et al. 2010) (Systems Biosciences), JMC2 and JMC3 (Johnston et al. 2019) were used to produce minicircles that were methylated or lacked endogenous methylation. Gene-flanking homology arms were cloned into the parent plasmid pMC BESPx MCS1 (Systems Biosciences) and transformed into ZYCY10P3S2T (ZYCY) (Systems Biosciences), JMC2 or JMC3 minicircle production strains. MC production was performed according to the manufacturer's protocol.

[0188] Construction of dam-dcm-hsdMS-E. coli strain with normal MIIIB function To generate E. coli strains producing plasmids with the C. acnes methylation pattern, lambda-Red recombineering was used as described by Datsenko & Wanner (Datsenko et al. 2000). Briefly, recipient dam- / dcm- strains (NEB) were first transformed with the pKD46 plasmid and selected with 100 μg / ml ampicillin at 30°C. Transformants were grown at 30°C and induced with 0.4% arabinose for 1 h before being transformed with linear DNA fragments to recombine into the chromosome (see below). Transformants were selected with 25 μg / ml kanamycin at 37°C and successful recombination was confirmed by colony PCR and Sanger sequencing. Loss of temperature dependence of pKD46 was confirmed by the absence of growth on Amp 100 μg / ml plates.

[0189] A linear DNA fragment to be recombined into the E. coli chromosome was prepared as follows: First, the MIIIB methylase gene was amplified from the genome of C. acnes KPA171202 and cloned together with the Tn5 neomycin phosphotransferase (KanR) gene amplified from pKD13 (Datsenko et al. 2000) under the Bba_J23100 promoter and Bba_B0034 RBS in the pJET1.2 vector. The region containing the MIIIB methylase and KanR genes was then amplified with primers PP-19 and PP20, which provide a 50 bp homologous region in the E. coli genome. In particular, the primers were designed to cause recombination at the hsdMS locus, thereby inactivating the hsdMS methylation system upon insertion of the C. acnes methylase gene. To ensure that only the linear PCR product (and not the template plasmid) was transformed, the template plasmid was digested with XhoI and AatII for 1 h at 37° C. prior to PCR, the PCR product was treated with DpnI for 1 h at 37° C., and the correct band was purified from a 1% agarose gel. The purified template-free PCR product was transformed into dam- / dcm- cells harboring pKD46 as described above.

[0190] Transformation of C. acnes: C. acnes competent cells are prepared as previously described (Meike Sorensen, et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants, Journal of Microbiological Methods, Volume 83, Issue 2, 2010, Pages 211-216, ISSN 0167-7012, https: / / doi.org / 10.1016 / j.mimet.2010.09.008.). Briefly, C. acnes cells are inoculated to a starting OD600 of 0.1 and grown anaerobically at 110 rpm at 37°C for 24 h. The bacterial culture is centrifuged at 4200 rpm for 10 min and washed a total of 7 times with ice-cold 272 mM sucrose buffer. 10 μl of the bacterial pellet is diluted in 30 μl of 272 mM sucrose buffer and mixed with 8 μg of methylated plasmid DNA. Methylated plasmid DNA was produced by shuttling the plasmid with a IIIB methylation-competent E. coli dam-dcm- strain (see above). This E. coli strain was engineered to produce C. acnes IIIB methylase. Bacteria were transformed with plasmid DNA by electroporation using a Biorad gene pulser instrument (BioRad) at 1500V, 400 ohms, 25uF, 1mm. Cells were allowed to recover anaerobically on Brucella agar plates for 24 hours and then selected on Brucella plates supplemented with 10μg / mL erythromycin. Plates were incubated anaerobically at 37°C for 7 days. Positive clones were confirmed by junction PCR and WGS for positive gene insertion / plasmid transformation.

[0191] To improve electroporation efficiency, different pretreatments were tested. For glycine or penicillin G pretreatment, cells were grown in the presence or absence of 1.25% glycine or 2, 5, 10 ug / mL penicillin G for 4 hours prior to electroporation. For osmotic stabilization, 0.4 M sucrose was also added 4 hours prior.

[0192] Inhibition of thymidine kinase (tdk) in wild-type C. acnes for potential negative selection To test whether tdk can be used as a negative selection in C. acnes, we tested its functionality using the known inhibitor 5-fluoro-2'-deoxyuridine (FUDR). We plated serial dilutions of C. acnes wild type KPA171202 from 10^-1 to 10^-8 in 10 μl drops on Brucella agar plates containing 0 or 50 μg / mL FUDR. The plates were incubated anaerobically at 37°C for 7 days and checked for potential growth. If no growth was observed, the inhibitor FUDR is functional for the tdk gene.

[0193] Thymidine kinase knockout functional assay The tdk knockout was made as previously described in Sorensen et al. 2010, targeting the thymidine kinase locus and cloning a pMW535 plasmid with homology arms into the region. For simplicity, an antibiotic resistance cassette encoding erythromycin (ermE) was cloned between the homology arms to replace the tdk gene with the ermE cassette. To evaluate the functionality of the tdk double selection marker (Shao et al. 2016; Norville et al. 2016), we tested the knockin sensitivity to FUDR (50ug / mL) and erythromycin (10ug / mL) and compared it to the wild type. Therefore, we plated the bacteria on Brucella agar plates containing ery (10ug / mL), FUDR (50ug / mL), or neither component. Only the knockin strain was able to grow on both ery- and FUDR-containing plates, whereas the wild type was sensitive to both.

[0194] Example 2: Results Survival study of SZ95 and PCi-Seb_Cau human sebocytes and C. acnes treated with rhNGAL To investigate the effect of rhNGAL protein produced in E. coli or HEK293 cells on the proliferation of human sebocytes, we used human SZ95 and PCi-Seb_Cau sebocyte cell lines. In this study, the anti-proliferative effect of NGAL on sebocytes was determined. Thus, cells were treated with various concentrations of human NGAL (produced in HEK293 cells) and cell proliferation and viability were evaluated by assaying MTT (a colorimetric assay for evaluating cellular metabolic activity). No significant effect on the viability and proliferation of SZ95 and PCi-Seb_Cau cells was found in a dose-dependent manner after 24 hours (Figures 1, 10 and 11).

[0195] In another test, we evaluated rhNGAL (E. coli) and human NGAL (HEK293) toxicity against C. acnes cultures using several doses. No toxic effect was observed against C. acnes bacteria as measured by OD600 after 24 hours of incubation and growth compared to untreated controls (Figures 2, 8, 9). The rhNGAL protein used was expressed and purified from E. coli BL21 cells (see Example 1 above), and human NGAL was produced in HEK293 cells and commercially obtained from ACROBiosystems (see Figures 8 and 9).

[0196] Protein production and secretion of NGAL in C. acnes Total protein production of NGAL protein was measured for three different constructs pBR13, pBR14 and pBR16 (see Table 1) in the secreted versus cytosolic fractions of TCA-precipitated protein. Secreted and cytosolic fractions were visualized by Western blot and compared with 40 μg (pBR13), 10 μg (pBR14) and 25 μg (pBR16) of purified protein. Western blot showed a higher amount of protein in the secreted fraction (Figure 3).

[0197] Western blot of NGAL produced in E. coli, C. acnes or HEK293 Equal amounts of purified proteins were loaded onto SDS gels, followed by Western blotting and detection with anti-histidine antibodies. HEK293 cells show a higher molecular weight due to glycosylation of the human protein. NGAL from E. coli has the lowest molecular weight due to the lack of glycosylation. C. acnes-produced NGAL appears to have some post-translational modifications, as it has an increased molecular weight compared to the E. coli-produced protein (Figure 6).

[0198] rhNGAL reduces sebum production in PCi-SEB Cau sebocytes in vitro To test the ability of purified rhNGAL produced in E. coli or C. acnes to reduce sebum in PCi-SEB_Cau, we induced sebum production with 5 μM arachidonic acid (AA) and treated them with rhNGAL and isotretinoin simultaneously for 48 h. It could be seen that all NGAL proteins, i.e. rhNGAL produced in E. coli (1.7-fold), pBR13 (2.5-fold), pBR14 (1.5-fold), pBR16 (1.6-fold) and isotretinoin (1.8-fold), induced a significant decrease in sebum production compared to AA-treated cells (Figure 4). Interestingly, NGAL protein produced in C. acnes (pBR13) significantly reduced sebum by 1.3-fold compared to the positive control isotretinoin (Figure 4).

[0199] TDK Testing We confirmed the growth ability of the C. acnes KPA171202 wild-type strain on Brucella agar plates supplemented with or without 50 μg / mL FUDR (5-fluoro-2'-deoxyuridine). The results (Figure 5) show that the bacteria can grow only in the absence of thymidylate synthase inhibitor (FUDR), indicating that the inhibitor is functional for thymidine kinase. These results showed that using a thymidine kinase (tdk) knockout, we can select for our LCN2 insertion by using an inhibitor, and therefore do not require antibiotic resistance. The absence of antibiotic resistance is important for the application of the bacteria to human skin.

[0200] After generating the tdk knockout by homologous recombination (Figure 27a and b), we tested its functionality by measuring its sensitivity or resistance to FUDR. Wild-type bacteria were unable to grow on FUDR (50ug / mL), whereas the tdk knockout survived and showed no growth inhibitory effect (Figure 7).

[0201] Bacterial engraftment in mice Finally, we wanted to test whether our sebum-modulating strains could efficiently engraft on the skin. Therefore, we applied both wild-type and engineered C. acnes to the backs of mice for three consecutive days and continued sampling until day 7. We followed engraftment by junction PCR and confirmed the presence of the engineered strains until the assay endpoint of day 7 (Figure 22). We performed Gram staining of frozen sections of the skin and observed an increase in Gram-positive bacteria in the skin and hair follicles of treated mice. Gram staining observed in control mice was quantitatively much less abundant than in treated mice, likely revealing a native bacterial flora (Figure 23).

[0202] In vivo protein production Since the bacteria appeared to colonize the skin of mice, in vivo protein production was evaluated. qPCR analysis revealed increased expression of NGAL in samples treated with engineered bacteria, while wild-type and vehicle-treated mouse skin showed limited expression (Figure 24). To examine potential inflammation resulting from C. acnes treatment, we evaluated the expression levels of the inflammatory cytokines IL1-β, IL-6, and TNFα. No differential expression of inflammatory cytokines was observed when comparing treated and untreated samples (Figure 26).

[0203] Improved transformation efficiency We screened various transformation buffer compositions and conditions that have been successfully used in other non-model organisms. Since the electrotransformation efficiency generally seemed very low, we used fluorescently labeled oligonucleotides to evaluate DNA entry and stability in C. acnes cells. DNA entry of fluorescent oligos was increased approximately 50-fold using a buffer containing 272 mM sucrose compared to other electroporation buffers tested (Figure 14). We observed a significant reduction in fluorescence after 1 hour of recovery electroporation, concluding that the fluorescent oligos must have been partially degraded (Figure 15).

[0204] Discussion of results regarding prokaryotes expressing NGAL In this study, the effect of bacterial NGAL on human sebocytes was evaluated. The tested proteins were produced in E. coli BL21 or C. acnes KPA171202. Three different plasmid versions of NGAL with combinations of regulatory parts (pBR13, pBR14 and pBR16) were transformed in C. acnes and tested for their ability to produce and secrete NGAL protein.

[0205] We verified the presence of NGAL protein in C. acnes supernatants by protein purification and Western blot and showed that pBR13, pBR14 and pBR16 were capable of secreting NGAL, but increased production was observed with pBR13 and pBR16.

[0206] Furthermore, NGAL protein had no effect on the viability of C. acnes in vitro and on human SZ95 sebocytes, which was contrary to what had been shown previously (Klausen et al. 2005. On mouse and man: neutrophil gelatinase associated lipocalin is not involved in apoptosis or acute response. Eur J Haematol: 75: 332-340 and Kimberly Ruth Lumsden. 2011. The Innate Immune Protein Neutrophil Gelatinase-Associated Lipocalin Is Involved in The Early Therapeutic Response To 13-Cis Retinoic Acid In Acne Patients. PhD Dissertation).

[0207] We also measured the ability of bacterial NGAL to reduce sebum from stimulated PCi SEB-Cau sebocytes in vitro. A significant reduction in sebum was observed after 48 hours, which was comparable to isotretinoin treatment. C. acnes-producing NGAL (pBR13) showed a 1.3-fold reduction in sebum compared to isotretinoin treatment.

[0208] In this study, we demonstrate that C. acnes can produce therapeutic amounts of NGAL and secrete the protein into the supernatant. The filtered supernatant and purified protein of C. acnes were able to reduce sebum equivalent to isotretinoin in an in vitro sebaceous cell model. This data suggests beneficial uses of NGAL produced in bacteria (E. coli and C. acnes) along with potential therapeutic uses of NGAL-producing C. acnes as a shuttle for cosmetic purposes or to reduce sebum and treat acne vulgaris in subjects in need thereof. This treatment may be a safe and efficient alternative to isotretinoin, which exhibits many severe side effects.

[0209] Furthermore, when applied to the skin, the skin microbes colonize hair follicles and exhibit persistence throughout the study period while producing and secreting therapeutics that can target and interact with sebaceous glands to regulate sebum production. This study shows evidence of the controlled use of skin commensals by dual selection based on the thymidine kinase gene. The basis of this strain will support future clinical studies and its use in humans while providing the basis for regulatory measures.

[0210] Discussion of results regarding transformation methods: The use of live biotherapeutics as a new class of medicine has both great potential and advantages over conventional medicine and can fill unmet needs. Multiple engineered biotherapeutics are currently in development and have entered clinical development, primarily focused on the gut.

[0211] Considering its easy accessibility and important functions for human health, skin is another great potential target for the application and development of live probiotics. Skin is the main barrier to the human body and is exposed to many external harsh conditions, such as UV radiation, pollution and pathogens. Therefore, the development of skin probiotics can protect humans from harmful environmental conditions and sense the early onset of damage and disease.

[0212] The present inventors demonstrate the development and efficient engineering of the skin commensal Cutibacterium acnes by improving various steps in bacterial electroporation. The present inventors demonstrate the importance of mimicking the host's own methylation pattern to avoid induced endogenous degradation of nucleic acids transformed into C. acnes. In this method, the nucleic acid introduced into C. acnes is characterized by a complete lack of methylated nucleic acid bases and recording REase recognition patterns or presenting only methylated nucleic acid bases that are methylated by C. acnes methylase and thus recognized by "self" when delivered to C. acnes. A shuttle bacterium such as E. coli can be used to provide the nucleic acid before introduction into C. acnes. However, the E. coli needs to be modified to completely silence its endogenous system (dam-dcm-hsdMS-E. coli), preferably to also express the C. acnes methylase. This differs from other previously described methods in which the shuttle E. coli only silenced one gene, such as dam-E. coli, or did not express the C. acnes methylase. Complete removal of the E. coli endogenous methylation system improves the efficiency of delivery of the plasmid into C. acnes, and expression of its own C. acnes methylation system further improves the efficiency.

[0213] Therefore, we developed an E. coli strain that lacks its own endogenous methylation system but mimics C. acnes methylation by expressing C. acnes IIIB methylase. This prevents foreign nucleic acids from being detected by C. acnes and is maintained by the cells. Furthermore, treatment of cells during competent cell preparation with cell wall permeabilizing agents such as penicillin G or L-glycine increased transformation efficiency. Also, lower DNA amounts, higher ODs and the use of frozen competent cells were favorable for transformation outcomes. Homologous recombination can also be increased by using minicircle vectors of the gene of interest recoded to remove C. acnes recognizable patterns, or by using minicircle-producing strains with silenced endogenous methylation systems.

Claims

1. 1. A method for introducing nucleic acid into Cutibacterium acnes (C. acnes), comprising: a) providing an unmethylated nucleic acid, provided that if it contains a C. acnes methylation motif, the methylation motif is methylated; and b) introducing the nucleic acid from step a) into C. acnes. A method comprising:

2. 2. The method of claim 1, wherein the C. acnes methylation motif methylated in step a) is AGC(m)AGY.

3. The C. acnes of step b) i) have been previously cultured in the presence of at least one agent for permeabilizing the bacterial cell wall, and / or ii) have been previously frozen and thawed 3. The method according to claim 1, wherein the compound is obtained by

4. 4. The method of claim 3, wherein the permeabilizing agent of bacterial cell walls is L-glycine or penicillin G.

5. 3. The method of claim 1 or 2, wherein the nucleic acid sequence of step a) comprises one or more methylated C. acnes methylation motifs in its sequence, the nucleic acid being obtained by introducing a nucleic acid comprising one or more C. acnes methylation motifs in its sequence into a non-C. acnes bacterium, wherein the non-C. acnes bacterium contains or expresses a C. acnes methylase and does not contain or express other methylases, and the nucleic acid is then collected from the bacterium for proceeding with step b).

6. 6. The method of claim 5, wherein the C. acnes methylase is a C. acnes IIIB methylase.

7. 3. The method of claim 1 or 2, wherein the nucleic acid sequence of step a) is unmethylated and does not contain a C. acnes methylation motif in its sequence, and wherein the nucleic acid sequence is obtained by introducing a nucleic acid that does not contain a C. acnes methylation motif in its sequence into a non-C. acnes bacterium, wherein the non-C. acnes bacterium does not express other methylases, and wherein the nucleic acid is then collected from the bacterium for proceeding with step b).

8. 6. The method of claim 5, wherein the bacterium that is not C. acnes is a dam-dcm-hsdMS-E. coli strain.

9. 6. The method of claim 5, wherein the bacterium that is not C. acnes is the E. coli strain deposited at the Spanish Collection of Type Cultures under CECT No. 30749.

10. 3. The method of claim 1 or 2, wherein the nucleic acid comprises a C. acnes integration element that results in integration of the target nucleic acid or at least a portion thereof in the genome of C. acnes.

11. 3. The method of claim 1 or 2, wherein the transformed C. acnes is selected, and the selection is based on at least two bacterial selectable markers.

12. 3. Recombinant C. acnes obtained by the method of claim 1 or 2.

13. 13. The recombinant C. acnes of claim 12, wherein the nucleic acid encodes the human neutrophil gelatinase-associated lipocalin (NGAL) gene, and the recombinant C. acnes expresses the NGAL protein.

14. A therapeutic composition comprising the recombinant C. acnes of claim 12.

15. A diagnostic composition comprising the recombinant C. acnes of claim 12.

16. 13. Use of C. acnes according to claim 12 in cosmetics.

17. A recombinant Cutibacterium acnes characterized by expressing the human neutrophil gelatinase-associated lipocalin (NGAL) protein.

18. 18. The recombinant Cutibacterium acnes of claim 17, wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length with SEQ ID NO:

1.

19. The recombinant Cutibacterium acnes of claim 17 or 18, wherein the nucleic acid encoding the human neutrophil gelatinase-associated lipocalin (NGAL) is operably linked to a promoter selected from the group consisting of a camp2 promoter, a camp1 promoter, or a roxP promoter.

20. A composition for treating acne vulgaris, urticaria, eczema, rosacea, hidradenitis suppurativa and / or psoriasis, comprising the recombinant Cutibacterium acnes of claim 17 or 18.

21. 19. Non-therapeutic cosmetic use of the recombinant Cutibacterium acnes according to claim 17 or 18.

22. 21. The recombinant Cutibacterium acnes for use according to claim 20, wherein said use comprises topical administration of said recombinant C. acnes to the skin.

23. 20. A method for producing an active ingredient of a pharmaceutical composition, wherein the active ingredient is human neutrophil gelatinase-associated lipocalin (NGAL) protein, the method comprising culturing the recombinant Cutibacterium acnes of claim 17 or 18.

24. 24. The method of claim 23, wherein the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length with SEQ ID NO:

1.

25. 24. The method of claim 23, wherein the recombinant bacterium is Cutibacterium acnes and the human neutrophil gelatinase-associated lipocalin (NGAL) protein has at least 85% amino acid sequence identity over its entire length to SEQ ID NO:1.

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