Methods and compositions for treating skin diseases using recombinant microorganisms

Abstract

The present disclosure provides isolated plasmids, recombinant microorganisms, kits, and methods for treating microbial infections, particularly MRSA. In a first aspect, the disclosure provides a recombinant microorganism comprising deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes, where the D-alanine biosynthesis genes are inactive, deletions or substitutions in one or more genes encoding naturally occurring antibiotic resistance genes, where the naturally occurring antibiotic resistance genes are inactive, and deletions or substitutions in one or more genes encoding naturally occurring lysogenic bacteriophage genes, where the naturally occurring lysogenic bacteriophage genes are inactive.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 442,855, filed February 2, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Viral, bacterial, and fungal infections of the skin are increasing in frequency and severity. For example, pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are rapidly increasing, limiting the antibiotics available for treating infections, especially those related to the skin. There is a great need for new therapeutic agents for both the prevention and acute care of viral, bacterial, and fungal infections of the skin. Summary of the Invention [Means for solving the problem]

[0003] overview The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements that confer antibiotic resistance would allow for the modulation of therapeutic exposure and improve safety. The studies described herein support the potential safety and utility of live biotherapeutic bacterial strains whose growth can be controlled by D-alanine for use in treating various skin diseases and disorders. In particular, the present disclosure features, in some embodiments, recombinant D-alanine auxotrophic strains engineered to secrete therapeutic proteins (e.g., bacterial and fungal cell wall hydrolases, optional defense expression—host immune proteins) extracellularly onto the surface of the skin and / or other suitable tissues, for use as preventative, post-exposure prophylactic, or therapeutic treatment for dysbiosis due to primary or secondary pathogenic bacterial or fungal overgrowth for superficial or severe skin diseases or disorders.

[0004] In a first aspect, the disclosure provides a recombinant microorganism comprising deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive; deletions or substitutions in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; and deletions or substitutions in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive.

[0005] In another aspect, the disclosure provides a recombinant microorganism comprising: 1) deletions or replacements in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; 2) deletions or replacements in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive; and 3) deletions or replacements in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive.

[0006] In some embodiments of the above aspects, the one or more naturally occurring antibiotic resistance genes are selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a cotrimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

[0007] In some embodiments of the above aspects and embodiments, the one or more naturally occurring antibiotic resistance genes are mupirocin resistance genes.

[0008] In some embodiments of the above aspects and embodiments, the one or more naturally occurring lysogenic bacteriophage genes encode bacteriophage capsid proteins.

[0009] In some embodiments of the above aspects and embodiments, the one or more D-alanine biosynthesis genes include the D-alanine aminotransferase gene (dat), the alanine racemase gene alr1, or alr2. In some embodiments of the above aspects and embodiments, the deletion or replacement in the one or more genes encoding D-alanine biosynthesis genes includes deletions in dat, alr1, and alr2.

[0010] In some of the above aspects and embodiments, the recombinant microorganism further comprises one or more genes encoding heterologous genes.

[0011] In some embodiments of the above aspects and embodiments, the heterologous gene is selected from the group consisting of a gene encoding an antimicrobial polypeptide or a variant thereof, a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, a gene encoding an enzyme or a variant thereof, a gene encoding an enzyme inhibitor or a variant thereof, a gene encoding an antigen or a variant thereof, and a gene encoding an immunomodulatory polypeptide or a variant thereof, or a combination thereof.

[0012] In some embodiments of the above aspects and embodiments, the heterologous gene encodes an antimicrobial polypeptide or a variant thereof.

[0013] In some embodiments of the above aspects and embodiments, the one or more antimicrobial polypeptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

[0014] In some of the above aspects and embodiments, the one or more microbial pathogens are selected from the group consisting of a bacterial pathogen, a fungal pathogen, or a viral pathogen.

[0015] In some embodiments of the above aspects and embodiments, the one or more bacterial pathogens are selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Cutibacterium species and Mycoplasma, or combinations thereof.

[0016] In some of the above aspects and embodiments, the fungal infection is caused by a fungus selected from the group consisting of Malassezia species, Candida species, Aspergillus, Cryptococcus, and Pneumocystis.

[0017] In some of the above aspects and embodiments, the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

[0018] In some embodiments of the above aspects and embodiments, the antimicrobial polypeptide is selected from the group consisting of epidermin-like lantibiotic, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviductal glycoprotein 1, and the like. 1), cartilage glycoprotein 1, chitotriosidase, mucin 9, cartilage glycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetyl esterase, acetyl xylan esterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, lysostaphin, zoocin A, millericin B, muraminidase Cpl-1, lysozyme, endolysin PlyC, endolysin PlyV12, enterolysin A A), C.difficile autolysin (Acd), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilin, epidermin, gallidermin, mutacin Ba Ny266, mutacin 1140, Pep5, epicidin 280, epilancin K7, lacticin 481, cytolysin, lacticin 3147, staphylococcin C55, salvaricin A, lactocin S, streptococcin A-FF2, sublancin 168, carnocin U149 U149, Variacin 8, Cypemycin, Cinnamycin, Duramycin, Ancovenin, Mersacidin, and Actagardine.

[0019] In some embodiments, the one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains, or variants thereof, and the LEKTI protein, one or more LEKTI protein domains, or variants thereof are secreted.

[0020] In some embodiments of the above aspects and embodiments, the heterologous gene encodes an antimicrobial biosynthetic enzyme or a variant thereof, wherein the antimicrobial biosynthetic enzyme is capable of producing 6-N-hydroxyaminopurine (6-HAP), and the 6-HAP is secreted.

[0021] In some embodiments of the above aspects and embodiments, the gene encoding the immunomodulatory polypeptide or variant thereof is a gene encoding a lipoteichoic acid (LTA) biosynthetic enzyme, and the recombinant microorganism is capable of producing and secreting LTA.

[0022] In some embodiments of the above aspects and embodiments, the recombinant microorganism is a bacterium or a combination of bacteria.

[0023] In some embodiments of the above aspects and embodiments, the recombinant microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or a combination thereof.

[0024] In some embodiments of the above aspects and embodiments, the recombinant microorganism is Staphylococcus epidermidis (S. epidermidis).

[0025] In some of the above aspects and embodiments, the recombinant microorganism secretes one or more therapeutic polypeptides or variants thereof.

[0026] Accordingly, in another aspect, the present disclosure provides a pharmaceutical composition comprising a cell culture composition comprising one or more recombinant microorganisms of the above aspect or any other aspect of the present disclosure set forth herein, and a pharmaceutically acceptable carrier.

[0027] In some embodiments of the above aspects and embodiments, the cell culture composition is a live cell culture composition.

[0028] In some embodiments, the cell culture composition comprises from 0% water to up to 90% water.

[0029] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, emulsion, cream, lotion, gel, or ointment.

[0030] In another aspect, the disclosure provides a method of treating a disease, disorder, or condition in a subject, the method comprising administering to a subject a recombinant microorganism comprising deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes, where the D-alanine biosynthesis genes are inactive, deletions or substitutions in one or more genes encoding naturally occurring antibiotic resistance genes, where the naturally occurring antibiotic resistance genes are inactive, and deletions or substitutions in one or more genes encoding naturally occurring lysogenic bacteriophage genes, where the naturally occurring lysogenic bacteriophage genes are inactive.

[0031] Accordingly, in another aspect, the disclosure provides a method of treating a disease, disorder, or condition in a subject, the method comprising administering to a subject a cell culture composition comprising a recombinant microorganism comprising: 1) deletions or substitutions in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; 2) deletions or substitutions in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive; and 3) deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive.

[0032] In some embodiments, the one or more naturally occurring antibiotic resistance genes are selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a cotrimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

[0033] In some embodiments of the above aspects and embodiments, the one or more naturally occurring antibiotic resistance genes are mupirocin resistance genes.

[0034] In some embodiments of the above aspects and embodiments, the one or more naturally occurring lysogenic bacteriophage genes encode bacteriophage capsid proteins.

[0035] In some embodiments of the above aspects and embodiments, the one or more D-alanine biosynthetic genes include a D-alanine aminotransferase gene (dat), an alanine racemase gene alr1 or alr2. In some embodiments of the above aspects and embodiments, the deletion or replacement in one or more genes encoding D-alanine biosynthetic genes comprises deletions in dat, alr1, and alr2.

[0036] In some of the above aspects and embodiments, the recombinant microorganism further comprises one or more genes encoding heterologous genes.

[0037] In some embodiments of the above aspects and embodiments, the heterologous gene is selected from the group consisting of a gene encoding an antimicrobial polypeptide or a variant thereof, a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, a gene encoding an enzyme or a variant thereof, a gene encoding an enzyme inhibitor or a variant thereof, a gene encoding an antigen or a variant thereof, and a gene encoding an immunomodulatory polypeptide or a variant thereof, or a combination thereof.

[0038] In some embodiments of the above aspects and embodiments, the heterologous gene encodes an antimicrobial polypeptide or a variant thereof.

[0039] In some embodiments of the above aspects and embodiments, the one or more antimicrobial polypeptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

[0040] In some of the above aspects and embodiments, the one or more microbial pathogens are selected from the group consisting of a bacterial pathogen, a fungal pathogen, or a viral pathogen.

[0041] In some embodiments of the above aspects and embodiments, the one or more bacterial pathogens are selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Cutibacterium, Propionibacterium species, and Mycoplasma, or combinations thereof.

[0042] In some of the above aspects and embodiments, the fungal infection is caused by a fungus selected from the group consisting of Malassezia species, Candida species, Aspergillus, Cryptococcus, and Pneumocystis.

[0043] In some of the above aspects and embodiments, the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

[0044] In some embodiments of the above aspects and embodiments, the antimicrobial polypeptide is selected from the group consisting of epidermin-like lantibiotic, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, cartilage glycoprotein 1, chitotriosidase, mucin 9, cartilage glycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronos ... amide, α-L-arabinofuranosidase, acetyl esterase, acetyl xylan esterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, lysostaphin, zucin A, millelicin B, muraminidase Cpl-1, lysozyme, endolysin PlyC, endolysin, PlyV12, enterolysin A, Clostridium difficile (C. difficile) autolysin (Acd), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilin, epidermin, gallidermin, mutacin Ba Ny266, mutacin 1140, Pep5, epicidin 280, epilancin K7, lacticin 481, cytolysin, lacticin 3147, staphylococcin C55, salvaricin A, lactocin S, streptococcin A-FF2, sublancin 168, carnosin U149, variacin 8, sipemycin, cinnamycin, duramycin, ancovenin, mersacidin, and actagardin.

[0045] In some embodiments of the above aspects and embodiments, the one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains, or variants thereof, and the LEKTI protein, one or more LEKTI protein domains, or variants thereof are secreted.

[0046] In some embodiments of the above aspects and embodiments, the heterologous gene encodes an antimicrobial biosynthetic enzyme or a variant thereof, wherein the antimicrobial biosynthetic enzyme is capable of producing 6-N-hydroxyaminopurine (6-HAP), and the 6-HAP is secreted.

[0047] In some embodiments of the above aspects and embodiments, the gene encoding the immunomodulatory polypeptide or variant thereof is a gene encoding a lipoteichoic acid (LTA) biosynthetic enzyme, and the recombinant microorganism is capable of producing and secreting LTA.

[0048] In some embodiments of the above aspects and embodiments, the recombinant microorganism is a bacterium or a combination of bacteria.

[0049] In some embodiments of the above aspects and embodiments, the recombinant microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or a combination thereof.

[0050] In some of the above aspects and embodiments, the recombinant microorganism is Staphylococcus epidermidis (S. epidermis). In some of the above aspects and embodiments, the recombinant microorganism secretes one or more therapeutic polypeptides or variants thereof.

[0051] In some of the above aspects and embodiments, the cell culture composition is a live cell culture composition. In some of the above aspects and embodiments, the cell culture composition comprises 0% water to up to 90% water.

[0052] In some embodiments of the above aspects and embodiments, the subject is a mammal. In some embodiments of the above aspects and embodiments, the mammal is a human.

[0053] In some embodiments of the above aspects and embodiments, the disease, disorder or condition is a microbial infection, an inflammatory disease or disorder, or a metabolic disease or disorder.

[0054] In some embodiments of the above aspects and embodiments, the microbial infection comprises one or more microbial pathogens selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Cutibacterium, Propionibacterium species, Mycoplasma, Malassezia species, Candida species, Aspergillus, Cryptococcus, Pneumocystis, respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus, or a combination thereof.

[0055] In some embodiments of the above aspects and embodiments, the skin disease or disorder is an inflammatory skin disease or disorder. In some embodiments of the above aspects and embodiments, the skin disease or disorder is a skin dysbiosis. In some embodiments of the above aspects and embodiments, the skin disease or disorder is skin toxicity. In some embodiments of the above aspects and embodiments, the skin disease or disorder is ichthyosis. In some embodiments of the above aspects and embodiments, the skin disease or disorder is a psoriatic disease or disorder. In some embodiments of the above aspects and embodiments, the skin disease or disorder is dermatitis. In some embodiments of the above aspects and embodiments, the skin disease or disorder is an autoimmune blistering disease. In some of the above aspects and embodiments, the skin disease or disorder is selected from the group consisting of Netherton syndrome, hidradenitis suppurativa, psoriasis, pustular psoriasis, plaque psoriasis, and palmoplantar psoriasis, atopic dermatitis, acne, acneiform rash, impetigo, folliculitis, acute suppurative paronychia, lymphangitis, necrotizing fasciitis, and cellulitis.

[0056] In another aspect, the disclosure provides a method for producing a bacterial strain comprising: selecting a bacterial strain and performing, in the following order: (1) deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive; (2) deletions or substitutions in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; and (3) deletions or substitutions in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive;

[0057] and (b) preparing a recombinant D-alanine auxotrophic bacterial strain. In further embodiments, the deletion or substitution in one or more genes encoding D-alanine biosynthesis genes comprises deletions in dat, alr1, and alr2. In some embodiments of the above aspects and embodiments, the recombinant microorganism is Staphylococcus epidermidis (S. epidermis). BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1A shows the inhibition screening of Staphylococcus aureus (S. aureus) from the Micromyx-50 library of S. epidermidis strains.

[0059] Figure 1B shows the inhibition of S. aureus by strain SE25 MM23.

[0060] [Figure 2] Figure 2 shows the inhibition of S. aureus SA25923, S. aureus SA29213, S. epidermidis NRRL, MRSA USA 300, Bacillus subtilis, and the gram-negative strains Pseudomonas aeruginosa and Escherichia coli (E. coli) by SE25.

[0061] [Figure 3] FIG. 3 shows a graph depicting the growth of S. aureus grown in conditioned cell-free SE25 medium or SE3 medium compared to TSB alone.

[0062] [Figure 4]Antimicrobial activity of SE25 cell-free supernatant against coagulase-negative Staphylococcus isolated from the skin of patients with ichthyosis. SE25 cell-free supernatant was tested against S. aureus ATCC 29213, S. aureus ATCC 25923, S. epidermidis NRRL B-4268, S. epidermidis 1457, S. epidermidis 25, B. subtilis, E. faecalis, E. coli, P. aeruginosa, and the ichthyosis CoNS isolates S. lugdunensis, S. auricularis, S. capitis, S. haemolyticus, S. hominis, S. pettenkoferi, S. saprophyticus, and S. warneri.

[0063] [Figure 5] FIG. 5A shows a graph demonstrating that SE25 cell-free supernatant has activity against S. aureus, reducing S. aureus growth by approximately 7 logs.

[0064] FIG. 5B shows a graph depicting the bactericidal activity of linezolid, levofloxacin, erythromycin, and vancomycin against S. aureus.

[0065] [Figure 6] Figure 6A shows biofilm formation on plastic using crystal violet indicator. The graph shows that SE25 does not form biofilm, while the positive control SE1457 does.

[0066] Figure 6B shows biofilm detection using the Congo Red assay. SE25 formed red colonies, indicating the absence of biofilm formation.

[0067] FIG. 6C shows a graph demonstrating that serial dilutions of SE25 cell-free supernatant disperse biofilms formed by S. aureus.

[0068] [Figure 7] FIG. 7 shows a graph demonstrating that SE25 can colonize reconstituted human epidermis (RHE) after 24 and 48 hours.

[0069] [Figure 8] FIG. 8 shows a graph demonstrating that RHE colonized with SE25 results in a 4-log reduction of S. aureus ATCC 29213.

[0070] [Figure 9] FIG. 9 shows a graph demonstrating that SE25 colonized on RHE induces human β-defensin 2 expression from keratinocytes.

[0071] [Figure 10-1] FIG. 10 shows the epidermin biosynthetic gene cluster in SE25. [Figure 10-2] Same as above.

[0072] [Figure 11-1] FIG. 11 shows the prophage phiSpy gene cluster in SE25. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0073] [Figure 12] Figure 12 shows auxotrophy testing on TSA plates with (Plate A) or without (Plate B) D-alanine ("DA100") supplementation. SE480: SE25 wild type, SE482: SE25 double deletion auxotroph, SE484: SE25 triple deletion auxotroph (all with ΔmupAΔcapsid), SE123: SEΔΔΔ triple deletion auxotroph of NRRL B-4268.

[0074] [Figure 13]FIG. 13A shows the screening assay of the Δalr1Δalr2Δdat SE25 strain.

[0075] FIG. 13B shows the screening assay of the Δalr1Δdat SE25 strain.

[0076] [Figure 14] Figure 14A shows the anti-S. aureus activity of the Δalr1Δdat SE25 strain in an agar overlay assay. The SE25 strain was grown on TSB at 37°C for 48 hours and overlaid with top agar containing S. aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity.

[0077] Figure 14B shows the anti-S. aureus activity of the Δalr1Δalr2Δdat SE25 strain in an agar overlay assay. The SE25 strain was grown on TSB at 37°C for 48 hours and overlaid with a top agar containing S. aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity.

[0078] [Figure 15] Figure 15A shows a screening assay for chloramphenicol (Cam-10)- and mupirocin (Mup-20)-sensitive colonies for wild-type SE25. Circles indicate positive Cam-10 / Mup-20-sensitive colonies.

[0079] Figure 15B shows a screening assay for chloramphenicol (Cam-10)- and mupirocin (Mup-20)-sensitive colonies for a DA auxotrophic strain of SE25. Circles indicate positive Cam-10 / Mup-20-sensitive colonies.

[0080] [Figure 16]Figure 16A shows the anti-S. aureus activity of strain SE25 in an agar overlay assay. Strain SE25 was grown on TSA for 24 hours at 37°C, then grown for an additional 24 hours at 30°C and overlaid with a top agar containing S. aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity. SE25 ΔmupA Δcapsid (SE480, SE481), SE25 ΔmupA Δcapsid Δalr1 Δdat (SE482, SE483), and SE25 ΔmupA Δcapsid Δalr1 Δalr2 Δdat (SE484, SE485).

[0081] Figure 16B shows the anti-S. aureus activity of strain SE25 in an agar overlay assay. The SE25 strain was grown on TSA at 37°C for 24 hours, then grown at 30°C for an additional 24 hours and overlaid with top agar containing S. aureus ATCC 29213. Zones of growth inhibition indicate anti-staphylococcal activity. Wild-type parent SE25 strain (SE398).

[0082] [Figure 17] FIG. 17 shows agar overlay assays of the SE25 ΔepiA mutant strain (plates 1-4) and S. aureus ATCC 29213, the negative wild-type control SE120 (NRRL B-4268, plate 5), and the positive control SE25 wild-type (plate 6).

[0083] [Figure 18] FIG. 18A shows strain SE25 screened for epiA knockout.

[0084] FIG. 18B shows PCR analysis of Cam-sensitive colonies of the ΔepiA mutant strain (colonies 5, 8, 11, 32).

[0085] [Figure 19] Figure 19A shows a screening assay for candidate colonies of D-alanine auxotrophic strain SE464 streaked onto tryptic soy agar (TSA) in the presence of 100 μg / mL D-alanine without chloramphenicol.

[0086] FIG. 19B shows a screening assay for candidate colonies of D-alanine auxotrophic strain SE464 streaked onto TSA supplemented with 10 μg / mL chloramphenicol in the absence of D-alanine.

[0087] FIG. 19C shows the identification of three clones (highlighted with red circles) that grew only on TSA supplemented with additional D-alanine.

[0088] [Figure 20-1] FIG. 20A is a plot showing the growth kinetics of strains SE25 and SE484.

[0089] Figure 20B shows the anti-S. aureus activity of strain SE25 in an agar overlay assay. Strain SE25 was streaked onto TSA, which was further supplemented with 100 mg / mL of D-alanine and incubated at 37°C. After 48 hours of incubation, soft agar containing S. aureus was poured onto the plate and then incubated at 37°C for an additional 24 hours.

[0090] Figure 20C shows the anti-S. aureus activity of strain SE484 in an agar overlay assay. Strain SE484 was streaked onto TSA, supplemented with 100 mg / mL of d-alanine, and incubated at 37°C. After 48 hours of incubation, soft agar containing S. aureus was poured onto the plate and then incubated at 37°C for an additional 24 hours.

[0091] [Figure 20-2] Figure 20D shows verification of D-alanine auxotrophy and mupirocin sensitivity of SE484. SE484 was streaked onto TSA + / - 100 μg / ml D-alanine and D-alanine-containing TSA + / - 20 μg / ml mupirocin. Plates were incubated at 37°C and data were recorded after 48 hours of incubation.

[0092] [Figure 21] Figure 21A shows the anti-S. aureus activity of SE123. Reconstructed human epidermis (RHE) was inoculated with SE123 and incubated for 4 hours in a tissue culture incubator at 37°C and 5% CO2 before being challenged with approximately 104 CFU of S. aureus USA300. After an additional 24 hours of incubation, USA300 cell numbers were determined by punch biopsy and dilution plating.

[0093] Figure 21B shows the anti-S. aureus activity of SE484. Reconstructed human epidermis (RHE) was inoculated with SE484 and incubated in a tissue culture incubator at 37°C and 5% CO for 4 hours. After incubation, approximately 10 4 CFU of S. aureus USA300 were challenged. After a further 24-hour incubation, USA300 cell numbers were determined by punch biopsy and dilution plating. DETAILED DESCRIPTION OF THE INVENTION

[0094] Detailed Description The present disclosure relates to the use of engineered microorganisms designed to deliver therapeutic agents to the skin of a subject, which offers significant advantages over the use of probiotic strains that secrete naturally occurring antimicrobial agents. For example, engineered symbionts can be modified to have desirable properties that improve their colonization, longevity, and antimicrobial properties, whereas probiotics have some inherent limitations and may have limited or differential colonization success in the skin. The present disclosure provides methods and compositions of engineered microorganisms that produce therapeutic agents that can be secreted, such as antimicrobial agents of different human or bacterial origin.

[0095] According to some embodiments, the recombinant microorganism comprises a deletion or substitution in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive or deleted. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or substitution in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive.

[0096] According to some embodiments, the recombinant microorganism comprises: 1) deletions or replacements in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; 2) deletions or replacements in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive; and 3) deletions or replacements in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive.

[0097] According to some embodiments, the recombinant microorganism, one or more naturally occurring antibiotic resistance genes are selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a cotrimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene. In some of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is located on a chromosome or a plasmid, or both, of the recombinant microorganism. In some of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is a mupirocin resistance gene.

[0098] In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is inactivated on the chromosome but not on the plasmid. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is inactivated on the plasmid but not on the chromosome. In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance gene is inactivated on both the plasmid and the chromosome.

[0099] In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance polypeptide has 90% identity to the entire sequence of SEQ ID NO: 152. Thus, in one embodiment, the naturally occurring antibiotic resistance polypeptide has at least about 95% identity to the entire sequence of SEQ ID NO: 152. Thus, in one embodiment, the naturally occurring antibiotic resistance polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the entire sequence of SEQ ID NO: 152. In another embodiment, the naturally occurring antibiotic resistance polypeptide comprises the sequence of SEQ ID NO: 152. In yet another embodiment, the naturally occurring antibiotic resistance polypeptide consists of the sequence of SEQ ID NO: 152.

[0100] In some embodiments of the above aspects and embodiments, the naturally occurring antibiotic resistance polypeptide has 90% identity to the entire sequence of SEQ ID NO: 153. Thus, in one embodiment, the naturally occurring antibiotic resistance polypeptide has at least about 95% identity to the entire sequence of SEQ ID NO: 153. Thus, in one embodiment, the naturally occurring antibiotic resistance polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the entire sequence of SEQ ID NO: 153. In another embodiment, the naturally occurring antibiotic resistance polypeptide comprises the sequence of SEQ ID NO: 153. In yet another embodiment, the naturally occurring antibiotic resistance polypeptide consists of the sequence of SEQ ID NO: 153.

[0101] According to some embodiments, the naturally occurring temperate bacteriophage genes encode structural proteins essential for the formation and cycle of phage particles. In some embodiments of the above aspects and embodiments, the naturally occurring temperate bacteriophage genes encode bacteriophage structural capsid proteins.

[0102] In some embodiments of the above aspects and embodiments, the naturally occurring lysogenic bacteriophage polypeptide has 90% identity to the entire sequence of SEQ ID NO: 154. Thus, in one embodiment, the naturally occurring lysogenic bacteriophage polypeptide has at least about 95% identity to the entire sequence of SEQ ID NO: 154. Thus, in one embodiment, the naturally occurring lysogenic bacteriophage polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the entire sequence of SEQ ID NO: 154. In another embodiment, the naturally occurring lysogenic bacteriophage polypeptide comprises the sequence of SEQ ID NO: 154. In yet another embodiment, the naturally occurring lysogenic bacteriophage polypeptide consists of the sequence of SEQ ID NO: 154.

[0103] According to some embodiments, the recombinant microorganism is auxotrophic for one or more essential genes. In some embodiments of the above aspects and embodiments, the recombinant microorganism is auxotrophic for D-alanine biosynthesis. In some embodiments, the D-alanine biosynthesis genes are deleted or mutated resulting in inactivation. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or replacement of the D-alanine aminotransferase (dat) gene, the alanine racemase gene alr1 or alr2. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or replacement in the dat, alr1, and alr2 genes. In some embodiments of the above aspects and embodiments, the recombinant microorganism comprises a deletion or replacement of the dat gene and the alr1 gene.

[0104] According to some embodiments, the present disclosure provides a composition for treating a bacterial infection in a subject, comprising a microorganism genetically engineered to express a therapeutic agent, e.g., an antimicrobial polypeptide, and providing the composition to the subject. According to some embodiments, the therapeutic agent is an antimicrobial polypeptide effective in treating bacterial infections and skin dysbiosis (e.g., atopic dermatitis, superficial fungal infections such as dandruff, tinea, candidiasis, etc.). In some of the above aspects and embodiments, the therapeutic agent is a LEKTI protein, one or more LEKTI protein domains, or variants thereof. In some of the above aspects and embodiments, the therapeutic agent is 6-N-hydroxyaminopurine (6-HAP).

[0105] As used herein, the term "genetically modified" and grammatical variations thereof are used to describe microbial organisms (e.g., bacteria) that have been genetically modified or engineered by the introduction of DNA prepared outside the microorganism. For example, plasmid DNA containing new genes can be introduced into bacteria, allowing the bacteria to express those genes. Alternatively, DNA containing new genes can be introduced into bacteria and integrated into the bacterial genome, either on a chromosome and / or a plasmid, whereupon the bacteria express those genes.

[0106] As used herein, the terms "treat," "treating," "treatment," and grammatical variations thereof refer to providing a subject, e.g., a patient, with a protocol, regimen, process, or therapy that is desirable to achieve a physiological response or outcome. In particular, the methods and compositions of the present disclosure can be used to delay pathogen infection, the onset of infectious symptoms, or delay the onset of an infectious disease, or stop the progression of an infectious disease. However, treating does not require that a desired physiological response or outcome be achieved in each and every subject or subject population, e.g., a patient population, since not all treated subjects may respond to a particular treatment protocol, regimen, process, or therapy. Thus, a given subject or subject population, e.g., a patient population, may not respond or respond poorly to treatment.

[0107] According to some embodiments, the recombinant microorganism secretes the antimicrobial polypeptide at a level effective to ameliorate symptoms of the bacterial pathogen. As used herein, the terms "ameliorate," "ameliorating," and grammatical variations thereof refer to reducing the severity of symptoms of an infection in a subject.

[0108] As used herein, the term "preventing" refers to completely or nearly completely arresting the onset of a disease or condition or symptoms associated with a disease, for example, when a patient or subject is susceptible to or at risk of contracting a respiratory disease. Preventing can also include inhibiting the onset of a respiratory disease, e.g., halting or post-exposure prophylaxis (e.g., preventing the onset of a disease or symptoms associated with a disease after initial exposure to a pathogen associated with the disease).

[0109] As used herein, the term "reducing risk" refers to lowering the likelihood or probability of a respiratory disease occurring, for example, when a patient or subject is susceptible to or at risk of contracting a respiratory disease.

[0110] As used herein, the term "adjuvant" refers to an agent that enhances the pharmaceutical effect of another agent. As used herein, an antimicrobial agent is expressed and secreted by an engineered microorganism by, for example, enhancing the level of effectiveness (i.e., the ability to kill pathogenic bacteria and / or fungi) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 35%, at least 50%, at least 60%, at least 90%, and all amounts therebetween, compared to the level of effectiveness of the same type of microorganism that has not been engineered to express and secrete the antimicrobial agent. Thus, the antimicrobial agent expressed and secreted by the engineered microorganism functions.

[0111] As used herein, the terms "antimicrobial agent," "antimicrobial protein," or "antimicrobial polypeptide" can be used interchangeably and refer to any entity having antimicrobial activity, i.e., the ability to inhibit the growth of and / or kill bacteria and / or fungi, e.g., gram-positive and gram-negative bacteria and fungi. An antimicrobial agent is any agent that results in an inhibition of bacterial and / or fungal growth or a reduction in viability by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or more than 70%, or any integer between 30% and 70% or more, compared to the absence of the antimicrobial agent. In other words, an antimicrobial agent is any agent that reduces the population of bacterial and / or fungal cells by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or more than 70%, or any integer between 30% and 70%, compared to the absence of the antimicrobial agent. In one embodiment, the antimicrobial agent is an agent that specifically targets bacterial cells. In another embodiment, the antimicrobial agent modifies (i.e., inhibits, activates, or increases) a pathway that is specifically expressed in bacterial cells. In some embodiments of the above aspects and embodiments, the antimicrobial agent is a polypeptide, i.e., a polypeptide that is expressed and secreted by the engineered microorganism.

[0112] The antimicrobial agent may include a chitinase, a glucanase, or a peptidoglycan hydrolase.

[0113] As used herein, the term "chitinase" refers to an enzyme capable of catalyzing the hydrolysis of β-1,4-linked N-acetylglucosamine polymers that form chitin chains, the major component of fungal cell walls. Chitinases are expressed in plants in response to pathogens.

[0114] As used herein, the term "glucanase" refers to an enzyme capable of catalyzing the degradation or depolymerization of complex carbohydrates. The glucanase in the composition can degrade one or more of cellooligosaccharides, lignocellulose, cellulose, hemicellulose, and pectin. Such enzyme activity can be, but is not limited to, endoglucanase, exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetyl esterase, acetyl xylan esterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, or pectate lyase activity. The glucanases of the composition are capable of degrading one or more of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside, and xylan.

[0115] As used herein, the term "peptidoglycan hydrolase" refers to an enzyme capable of degrading bacterial cell walls when exposed to the outside. Bacterial cell walls consist of glycan chains cross-linked by flexible peptide side chains, providing strength and rigidity to the bacterial cell wall. Peptidoglycan in both Gram-positive and Gram-negative bacteria is characterized by repeating units of N-acetylglucosamine (NAG) and β-(1-4)-N-acetylmuramic acid (NAM) cross-linked by peptide stem chains attached to NAM residues. So-called peptidoglycan hydrolases (PGHs) are enzymes responsible for cleaving bonds within peptidoglycan chains and side chain branches.

[0116] The terms "infection" or "microbial infection," as used interchangeably herein, refer in their broadest sense to any infection caused by a microorganism, including bacterial, fungal, yeast, and protozoal infections.

[0117] In the present disclosure, the subject may be a mammal. As used herein, "mammal" and grammatical variations thereof refer to any category of mammals. In the present disclosure, mammals include, for example, humans, livestock, domestic animals, laboratory animals, etc. Some examples of domestic animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. Preferably, the mammal is a human.

[0118] As used herein, the term "effective amount" or "therapeutically effective amount" of a compound or composition disclosed herein refers to an amount of such a compound or composition that, when administered to a subject, is sufficient to produce the beneficial or desired results described herein. Effective dosage forms, modes of administration, and dosages can be determined empirically, and making such determinations is within the skill of the art. It will be understood by those skilled in the art that dosages will vary depending on the route of administration, excretion rate, duration of treatment, the identity of any other drugs administered, the age, size, and species of the mammal, e.g., human patient, and similar factors well known in the medical and veterinary fields. In general, a suitable dose of a composition according to the present disclosure is the amount of the composition that is the minimum dose effective to produce the desired effect. An effective dose of a composition of the present disclosure may be administered as two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day.

[0119] As used herein, the term "administration" is meant to refer to the contact of a pharmaceutical, therapeutic, or diagnostic composition with a recipient, preferably a human. The therapeutic agents disclosed herein can be administered intranasally, topically, or to the nasal or nasopharyngeal cavity.

[0120] As used herein, the term "polypeptide" or "protein" refers to a biological molecule or macromolecule made up of amino acid residues linked together in a chain. The definition of polypeptide as used herein is intended to encompass proteins (generally of high molecular weight) made up of one or more long chains of amino acid residues, as well as small peptides (generally of low molecular weight) of a few amino acids. In other embodiments, single amino acids, although not technically polypeptides, are also considered within the scope of this disclosure.

[0121] As used herein, the articles "a" and "an" should be understood to mean "at least one," unless expressly indicated otherwise.

[0122] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0123] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0124] Microbial Compositions: According to some embodiments, the present disclosure provides microbial compositions comprising one or more of a wide range of bacteria suitable for use on mammalian skin. Examples include, but are not limited to, non-pathogenic bacteria and commensal bacteria. Bacteria suitable for use in the present disclosure include, but are not limited to, Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Lactococcus, Streptococcus, Staphylococcus (e.g., S. epidermidis and / or S. hominis), Lactobacillus (e.g., L. acidophilus), Pediococcus, Leuconostoc, and Oenococcus. According to some embodiments, the microbial composition comprises one or more of Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Cutibacterium acnes, Corynebacterium species, Acinetobacter johnsonii, Pseudomonas aeruginosa. According to some embodiments, other related or similar species found on the skin are used.

[0125] Particular embodiments involve the use of the bacterium Staphylococcus epidermidis. According to some embodiments, the strain of S. epidermidis used is incapable of producing a biofilm. Examples of this are S. epidermidis strains ATCC 12228, NRRL B-4268, or SE25.

[0126] According to some embodiments, the recombinant microorganism is adapted to survive indefinitely or for a controlled period of time on the surface of the mammalian skin to provide a controlled or continuous supply of a therapeutic polypeptide, e.g., an antimicrobial agent. In some embodiments of the above aspects and embodiments, the antimicrobial agent is a small molecule or polypeptide. In some embodiments of the above aspects and embodiments, the recombinant microorganism survives in conjunction with symbiotic microorganisms naturally present on the mammalian skin. In some embodiments of the above aspects and embodiments, the recombinant microorganism survives to the exclusion of symbiotic microorganisms naturally present on the mammalian skin. According to some embodiments, the recombinant microorganism is adapted to grow on the mammalian skin.

[0127] In other embodiments, the recombinant microorganism is no longer viable but contains an effective amount of an antimicrobial polypeptide, such as, for example, an epidermin-like lantibiotic, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, cartilage glycoprotein 1, chitotriosidase, mucin 9, cartilage glycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetyltransfer ... Xylan esterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, lysostaphin, zucin A, millericin B, muraminidase Cpl-1, lysozyme, endolysin PlyC, endolysin PlyV12, enterolysin A, C. difficile autolysin (Acd), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilin, epidermin, gallidermin, mutacin Ba Ny266, mutacin 1140, Pep5, epicidin 280, epilancin K7, lacticin 481, cytolysin, lacticin 3147, staphylococcin C55, salvaricin A, lactocin S, streptococcin A-FF2, sublancin 168, carnosine U149, variacin 8, sipemycin, cinnamycin, duramycin, ancovenin, mersacidin and actagardin or one or more therapeutically active domains thereof.

[0128] In some embodiments of the above aspects and embodiments, the recombinant microorganism is capable of producing 6-N-hydroxyaminopurine (6-HAP).

[0129] In some of the above aspects and embodiments, the antimicrobial agent is effective against bacterial, fungal, or viral pathogens. In some of the above aspects and embodiments, the antimicrobial agent is effective against Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Propionibacteria species, Mycoplasma, Malassezia species, Candida species, Aspergillus, Cryptococcus, Pneumocystis, respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus. In some embodiments of the above aspects and embodiments, the antimicrobial agent is effective against E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Streptococcus (Str.) pneumoniae, Str. pyogenes, Enterococcus (E.) faecalis, E. faecium, and Candida albicans.

[0130] According to some embodiments, the therapeutic protein comprises one or more LEKTI domains effective to inhibit serine proteases present in or on the skin, upper and lower respiratory tract tissues, other mammalian tissues, and systemic circulation. According to some embodiments, the recombinant LEKTI domains compensate for defective endogenous LEKTI proteins or other defective serine protease inhibitors naturally produced in or on the skin, upper and lower respiratory tract tissues, other mammalian tissues, and systemic circulation. According to some embodiments, the genetically modified bacteria are capable of self-replicating while retaining the ability to produce the recombinant protein, thereby providing a continuous supply of therapeutic agent.

[0131] According to some embodiments, the therapeutic protein comprises one or more proteins encoded by one or more SPINK genes. According to some embodiments, the one or more SPINK genes are selected from the group consisting of SPINK1, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINK13, and SPINK14. According to some embodiments, the SPINK protein is SPINK5. The SPINK gene can be obtained from any mammal, such as a mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate, or human gene sequence. According to some embodiments, the SPINK gene sequence is a human gene sequence.

[0132] SPINK5 gene: According to some embodiments, the recombinant microorganism is engineered to express the mammalian gene SPINK5, which encodes the LEKTI protein. The SPINK5 gene can be obtained from any mammal, such as a mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate, or human gene sequence. According to some embodiments, the SPINK5 gene sequence is a human gene sequence. According to some embodiments, the recombinant microorganism is engineered to include a fragment of the SPINK5 gene.

[0133] According to some embodiments, the recombinant protein expressed by the engineered microorganism comprises a peptide sequence according to SEQ ID NO: 109 (LEKTI D6). According to some embodiments, the recombinant protein expressed by the engineered microorganism comprises a peptide sequence according to SEQ ID NO: 109. According to some embodiments, one or more fragments of the peptide sequence according to SEQ ID NO: 103 are expressed by the engineered microorganism. In one embodiment, the fragment comprises one or more LEKTI domains. In a particular embodiment, the LEKTI domain is domain 6.

[0134] According to some embodiments, the recombinant microorganism comprises a sequence disclosed herein that has at least about 75% identity, or 80% identity, or 85% identity, or 90% identity, or 95% identity to any one or more of the SEQ ID NOs listed herein. As used herein, the term "identity" and grammatical variations thereof refer to the degree to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. Percent (%) identity is calculated by multiplying the number of matches in a sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps.

[0135] According to some embodiments, the recombinant protein expressed by the engineered microorganism comprises one or more protease inhibitor domains of the LEKTI protein. Some non-limiting examples include one or more of domains D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, and D15. According to some embodiments, the recombinant protein expressed by the engineered microorganism comprises LEKTI inhibitor domain 6 or domains D8-D11.

[0136] In some of the above aspects and embodiments, the LEKTI protein domain acts as a competitive or non-competitive inhibitor of one or more proteases present on or in mammalian skin. In some of the above aspects and embodiments, the LEKTI protein domain acts as a serine protease inhibitor. As used herein, the terms "protease" and "proteinase" are used interchangeably, and both terms refer to enzymes that perform proteolysis.

[0137] According to some embodiments, the microorganism is genetically modified by transfection / transformation with a recombinant DNA plasmid encoding a therapeutic polypeptide (e.g., a LEKTI protein domain), small molecule, or metabolite. Other conventional or future discovered methods for introducing DNA into a microorganism may also be used in the present disclosure. According to some embodiments, the recombinant DNA plasmid comprises a sequence encoding a LEKTI protein domain and one or more secretory and / or cell-penetrating peptides. According to some embodiments, the LEKTI domain is operably linked to one or more recombinant protein domains effective to enhance secretion from the microorganism and / or penetration into mammalian tissues.

[0138] According to some embodiments, the present disclosure provides compositions for preventing or treating respiratory bacterial, viral, fungal, and other microbial infections, oral / pharyngeal cancer, cancer of the respiratory tract, neuronal degeneration in spinal cord injury, multiple sclerosis, glioblastoma, and other oligodendrogliopathy, comprising a microorganism genetically modified to express and provide one or more LEKTI protein domains or LEKTI proteins in the nasal cavity of a mammal, wherein the LEKTI protein domains or LEKTI proteins are effective in inhibiting the serine protease activity of at least one serine protease in or on mammalian skin, upper and lower respiratory tract tissues, other mammalian tissues, or in the systemic circulation. According to some embodiments, the present disclosure provides compositions for preventing or treating respiratory diseases (e.g., chronic sinusitis). According to some embodiments, the present disclosure provides compositions for preventing or treating central nervous system disorders and diseases. As used herein, the term "recombinant" and grammatical variations thereof refer to or refer to an organism, protein, or genetic material formed by or using recombined DNA, including pieces of DNA from different sources or different parts of the same source. For example, the term "recombinant DNA" refers to a DNA molecule formed by recombinant methods to splice together fragments of DNA from different sources or different parts of the same source. In some embodiments, two or more different sources of DNA are cut using a restriction enzyme and joined together using a ligase. As another example, the term "recombinant protein" or "recombinant domain" and grammatical variations thereof refer to a protein molecule formed by recombinant methods that is derived from spliced ​​fragments of DNA from different sources or different parts of the same source. As another example, the terms "recombinant microorganism" or "recombinant bacterium" and grammatical variations thereof are used interchangeably and refer to a microorganism that contains one or more recombinant DNA / protein molecules.

[0139] According to some embodiments, the recombinant microorganism comprises a sequence disclosed herein that has at least about 75% identity, or 80% identity, or 85% identity, or 90% identity, or 95% identity to any one or more of the SEQ ID NOs listed herein. As used herein, the term "identity" and grammatical variations thereof refer to the degree to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. Percent (%) identity is calculated by multiplying the number of matches in a sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps.

[0140] According to some embodiments, the microorganism is genetically modified by transfection / transformation with one or more recombinant DNA plasmids encoding one or more therapeutic proteins (e.g., antimicrobial polypeptides, such as epiA, elafin, hiracin, lysostaphin, and LL-37). Other conventional or future discovered methods for introducing DNA into a microorganism may also be used in the present disclosure. According to some embodiments, the one or more recombinant DNA plasmids comprise sequences encoding the antimicrobial polypeptides.

[0141] In some embodiments of the above aspects and embodiments, the therapeutic protein has 90% identity to the entire sequence of SEQ ID NOs: 130-145. Thus, in one embodiment, the therapeutic protein has at least about 95% identity to the entire sequence of SEQ ID NOs: 130-145. Thus, in one embodiment, the therapeutic protein has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the entire sequence of SEQ ID NOs: 130-145. In another embodiment, the therapeutic protein comprises the sequence of SEQ ID NOs: 130-145. In yet another embodiment, the therapeutic protein consists of the sequence of SEQ ID NOs: 130-145.

[0142] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is not regulated by or interfered with by the other. For example, a promoter is operably linked to a coding sequence if it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. In another example, two proteins can be operably linked such that the function of either protein is not impaired. Generally, operably linked means that the linked nucleic acid sequences are contiguous and, where necessary to link two protein-coding regions, contiguous and in the same reading frame.

[0143] As used herein, the term "gene" refers to a genomic gene comprising transcriptional and / or translational regulatory sequences and / or coding regions and / or non-translated sequences (e.g., introns, 5' and 3' non-translated sequences and regulatory sequences). The coding region of a gene can be a nucleotide sequence encoding an amino acid sequence or functional RNA (such as tRNA, rRNA, catalytic RNA, siRNA, miRNA, and antisense RNA). A gene can also be an mRNA or cDNA corresponding to the coding region (e.g., exons and miRNA), optionally including linked 5' or 3' non-translated sequences. A gene can also be an in vitro-produced and amplified nucleic acid molecule comprising all or part of the coding region and / or linked 5' or 3' non-translated sequences.

[0144] As used herein, the term "gene product(s)" is intended to include RNA transcribed from a gene or a polypeptide encoded by a gene or translated from RNA.

[0145] As used herein, the terms "secretory peptide" or "secretory sequence" or "secretion tag" or "signal peptide" or "export signal" and grammatical variations thereof refer to any peptide sequence capable of targeting a synthesized protein to the secretory pathway of a cell. In some embodiments of the above aspects and embodiments, the secretory peptide may be positioned at the N-terminus of the recombinant protein and may co-translationally or post-translationally target the tagged protein for secretion. In some embodiments of the above aspects and embodiments, the secretory peptide is positioned at the C-terminus of the recombinant protein. In some embodiments, the antimicrobial polypeptide is fused to the secretory peptide. In some embodiments, the antimicrobial polypeptide is fused to the secretory peptide and the propeptide.

[0146] Secretory peptides: According to some embodiments, one or more therapeutic recombinant polypeptides and / or metabolites, such as LEKTI domains, are operably linked to one or more secretory or export signals that tag the proteins for transport through the secretory pathway. Any secretory signal that facilitates export of the LEKTI protein from the bacterial cell can be used as a secretory peptide. Non-limiting examples of secretory peptide signals are shown in Table 1 below: Table 1: List of secretory peptides [Table 1]

[0147] According to some embodiments, the therapeutic LEKTI domain is operably linked to one or more signal sequences derived from endogenous proteins of S. epidermidis. Non-limiting examples of secretory signal peptides derived from endogenous proteins of Staphylococcus epidermidis are shown in Table 2 below: Table 2: List of Staphylococcus epidermidis signal peptides [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0148] According to some embodiments, the recombinant therapeutic polypeptide, e.g., a LEKTI domain, is operably linked to one or more secretory signal sequences derived from other endogenous bacterial proteins. Non-limiting examples of secretory signal peptides derived from various endogenous bacterial proteins are shown in Tables 1 and 2.

[0149] According to some embodiments, a recombinant therapeutic polypeptide, e.g., a LEKTI domain, is operably linked to a cell-penetrating peptide sequence that enhances the ability of the recombinant therapeutic polypeptide to cross a cell membrane. The term "enhance" as used to describe a cell-penetrating peptide / recombinant therapeutic polypeptide means that the cell-penetrating sequence improves passage of the recombinant therapeutic polypeptide through a cell membrane compared to a recombinant therapeutic polypeptide lacking the cell-penetrating sequence.

[0150] Cell-penetrating peptides: According to some embodiments, one or more cell-penetrating peptides are used to mediate delivery of therapeutic proteins in vivo without using cell surface receptors and without causing significant membrane damage. According to some embodiments, one or more cell-penetrating peptides are operably linked to a therapeutic protein to facilitate entry into skin cells (e.g., keratinocytes). Non-limiting examples are shown in Table 3 below: Table 3: List of cell-penetrating peptides

Table 3-1

Table 3-2

[0151] According to some embodiments, the cell-penetrating peptide comprises a periodic amino acid sequence. Non-limiting examples of periodic cell-penetrating sequences include the following: polyarginine, R×n (where 4 < n < 17); polylysine, K×n (where 4 < n < 17); arginine repeats with 6-aminocaproic acid residues (RAca) interposed, with 2 to 6 arginine repeats present, arginine repeats; arginine repeats with 4-aminobutyric acid (RAbu) interposed, with 2 to 6 arginine repeats present, arginine repeats; arginine repeats with methionine interposed, with 2 to 6 arginine repeats present, arginine repeats; arginine repeats with threonine interposed, with 2 to 6 arginine repeats present, arginine repeats; arginine repeats with serine interposed, with 2 to 6 arginine repeats present, arginine repeats; arginine repeats with alanine interposed, with 2 to 6 arginine repeats present, arginine repeats.

[0152] According to some embodiments, one or more therapeutic recombinant polypeptides and / or metabolites are operably linked to a cell-penetrating peptide, such as the RMR domain (SEQ ID NO: 4).

[0153] According to some embodiments, the LEKTI domain is operably linked to the RMR domain (SEQ ID NO: 4).

[0154] According to some embodiments, expression of a recombinant therapeutic polypeptide, e.g., a LEKTI domain, is controlled by an operon, and the amount of LEKTI provided to mammalian skin is proportional to the availability of exogenous factors. For example, in some embodiments, the recombinant LEKTI gene can be under the control of a xylose-inducible promoter (e.g., a xylose repressor (xylR), a xylose operator (xylO), a xylose isomerase gene (xylA) (comprising a cis-acting catabolite response element (CRE))), and the amount of recombinant LEKTI protein made available to mammalian skin is controlled by the amount of exogenous xylose available to the recombinant microorganism. According to some embodiments, expression of a recombinant therapeutic polypeptide, e.g., a LEKTI domain, is controlled by a constitutively active promoter. According to some embodiments, expression of a recombinant therapeutic polypeptide, e.g., a LEKTI domain, is controlled by a CmR promoter.

[0155] According to some embodiments, the microorganism is genetically modified by transfection / transformation with a recombinant DNA plasmid encoding a recombinant therapeutic polypeptide, e.g., a LEKTI protein domain, and one or more antibiotic resistance genes. For example, some embodiments of the recombinant DNA plasmid include a kanamycin resistance gene and / or a trimethoprim resistance gene, e.g., dfrA. According to some embodiments, treatment of mammalian skin with an antibiotic (to which the recombinant microorganism is resistant) can be used to bias the population of commensal microorganisms toward a greater proportion of recombinant therapeutic polypeptide-producing microorganisms. Other elements that may be present in the recombinant DNA plasmid include, but are not limited to, replication protein genes, e.g., members of the Rep superfamily of replication proteins. For example, in some embodiments, the recombinant DNA plasmid includes the repF gene.

[0156] The present disclosure utilizes standard molecular biology techniques, such as those described in Sambrook et al. 2001. In some embodiments, the genetic construct used in the present disclosure is based on the plasmid pBT-2, an allelic exchange shuttle vector between E. coli and Staphylococcal species (Nakanishi, Oshida et al. 1986). In some embodiments, the gene for the recombinant therapeutic polypeptide is inserted into the plasmid. In some embodiments, the coding sequence is driven by a promoter, such as an inducible promoter or a constitutive promoter. Examples of inducible promoters include those activated by compounds such as alcohol, sugars, metals, or tetracycline, or by physical factors such as light or high temperature.

[0157] According to some embodiments, the recombinant DNA plasmid comprises one or more sequences of a pUBTR vector. According to some embodiments, the recombinant LEKTI is operably linked to an inducible promoter, a ribosome binding site, an export signal, and / or a cell-penetrating peptide in the pUBTR vector. According to some embodiments, the recombinant LEKTI is operably linked to an inducible promoter, a ribosome binding site, an export signal, and / or a cell-penetrating peptide in the pUBTR vector. According to some embodiments, the pUBTR vector is pUBTR119.

[0158] According to some embodiments, the recombinant DNA plasmid comprises the complete pKK30-LEKTI sequence. According to some embodiments, the present disclosure provides a composition for treating a skin disease comprising a microorganism comprising the complete pKK30-LEKTI plasmid construct.

[0159] According to some embodiments, the amount or duration of availability of a recombinant therapeutic polypeptide, e.g., a LEKTI protein, in a microorganism is controlled by the stability of the vector carrying the recombinant therapeutic polypeptide. For example, the persistence of a recombinant vector can be controlled by one or more elements of the plasmid, including those that provide host-beneficial genes, plasmid stability mechanisms, and plasmid co-adaptation. For example, some plasmids can provide stable replication, active partitioning mechanisms, and mechanisms that ensure reliable inheritance of the plasmid to daughter cells across generations. (See, e.g., J.C.Baxter, B.E.F. Unnell, Plasmid partition mechanisms, Microbiol. Spectr., 2 (2014) PLAS-0023-2014 and Nils Hülter et al., An evolutionary perspective on plasmid lifestyle modes, Current Opinion in Microbiology, Volume 38, August 2017, Pages 74-80, each of which is incorporated herein by reference in its entirety.) According to some embodiments, the present disclosure encompasses the use of all conventional selection and stability methods known to those skilled in the art.

[0160] Examples of proteins that can be administered in accordance with the present disclosure are primarily eukaryotic proteins, which can include, but are not limited to, single amino acids, small peptides, and large proteins. More specifically, genes encoding proteins useful in the present disclosure as recombinant therapeutic proteins include, but are not limited to, the following: genes encoding members of the interleukin family of genes and their receptor antagonists, including but not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15; genes encoding hematopoietic growth factors, including but not limited to erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, leukemia inhibitory factor, and thrombopoietin, are also contemplated in the present disclosure; genes encoding neurotrophic factors, including but not limited to nerve growth factor, brain-derived neurotrophic factor, and ciliary neurotrophic factor; and genes encoding interferons, including but not limited to IFN-alpha, IFN-beta, and IFN-gamma. Also contemplated in the present disclosure are genes encoding chemokines, such as the CC and CXC families of cytokines, genes encoding hormones, such as proinsulin and growth hormone, and genes encoding thrombolytic enzymes, including other enzymes, such as tissue plasminogen activator, streptokinase, urokinase, or trypsin inhibitor. The present disclosure also includes genes encoding tissue repair factors, growth factors, and regulatory factors, including, but not limited to, oncostatin M, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, bone morphogenetic protein, insulin-like growth factor, calcitonin, and transforming growth factor alpha and beta. Further contemplated genes include genes encoding structural proteins, such as filaggrin, actin, collagen, fibrillin, elastin, or scleroproteins.In some embodiments, the recombinant microorganism includes a gene encoding an antimicrobial polypeptide (e.g., a defensin, a bacterial or fungal cell wall hydrolase, elafin, hyracin, lysostaphin, LL-37, and epidermin, or a variant thereof, or a combination thereof).

[0161] The present disclosure also relates to allelic variants of LEKTI or portions thereof (one or more of domains D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, and D15), as well as synthetic or mutant genes of SPINK (e.g., SPINK5) modified to, for example, alter the expression or activity of recombinant proteins. It should also be noted that degeneracy in the nucleic acid code can be considered as variation in nucleotide sequences encoding the same amino acid residue. Thus, the present disclosure includes nucleic acid residues that can hybridize under moderately stringent conditions. Those skilled in the art can determine effective combinations of salt and temperature to constitute moderately stringent hybridization conditions. It is also envisioned that orthologs of LEKTI exist in other species, such as dogs, sheep, rats, hamsters, chickens, and pigs. Accordingly, another embodiment of the present disclosure relates to a SPINK (e.g., SPINK5) nucleic acid encoding a polypeptide having at least about 70%-80% identity, preferably 90%-95% identity, and more preferably 98%-99% identity to LEKTI or a portion thereof (one or more of domains D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, and D15) set forth in SEQ ID NO: 103. In a specific embodiment, the LEKTI domain is domain 6.

[0162] According to some embodiments, the LEKTI domain is selected from the following non-limiting examples, Tables 4 and 5. Table 4: LEKTI amino acid sequence [Table 4-1] [Table 4-2] Table 5: SPINK5 nucleotide sequence [Table 5-1] [Table 5-2] [Table 5-3]

[0163] As used herein, the terms "auxotrophic" or "auxotrophy" refer to the inability of an organism to synthesize a particular compound required for its growth. An auxotroph is an organism that exhibits this characteristic.

[0164] As used herein, the terms "alrA" and "alr" refer to D-alanine racemase genes, including normal alleles of the alrA gene. In some embodiments, the alr gene from S. epidermidis (UniProtKB-Q8CNK7(ALR_STAES) encodes a D-alanine racemase protein (EC 5.1.1.1). In some embodiments, the locus identifiers SE1674 (alr1) and SE1079 (alr2) refer to specific S. epidermidis D-alanine racemase genes.

[0165] As used herein, the term "dat" refers to a D-alanine aminotransferase gene, including a normal allele of the dat gene. In some embodiments, the dat gene of S. epidermidis (UniProtKB-Q8CS41(DAAA_STAES)) encodes a D-alanine aminotransferase protein (EC:2.6.1.21). In some embodiments, the locus identifier SE1423(dat) refers to the specific S. epidermidis D-alanine aminotransferase gene. As used herein, the term "murI" refers to a glutamate racemase gene, including a normal allele of the murI gene. In some embodiments, the murI gene from S. epidermidis (UniProtKB-Q8CPL0(MURI_STAES)) encodes a glutamate racemase protein (EC:5.1.1.3). In some embodiments, the locus identifier SE0843(murl) refers to the specific S. epidermidis glutamate racemase gene.

[0166] D-alanine auxotrophs of S. aureus have been produced for the purpose of producing a vaccine against MRSA (Moscoso M, et al. 27th ECCMID 22-25 April 2017, The Congress of ESCMID (P0473); Moscoso et al., Virulence (2018) Vol. 9(1):604-620, the contents of each of which are incorporated herein by reference in their entirety). In this case, it was found that only the alanine racemases alr1 and alr2, as well as the dat gene, needed to be knocked out.

[0167] According to some embodiments, the recombinant microorganism is engineered to express a therapeutic polypeptide, small molecule, or metabolite to treat, prevent, or ameliorate a disease, condition, or infection. In some embodiments, a composition comprises the engineered microorganism. In some embodiments, the composition can treat, prevent, or ameliorate bacterial and other microbial (i.e., viral) infections, oral cancer, CNS disease or injury, and non-infectious (i.e., chronic sinusitis) or infectious nasal, oral, or respiratory diseases.

[0168] According to some embodiments, non-infectious and infectious diseases may include, but are not limited to, non-allergic rhinitis, vasomotor rhinitis, non-allergic rhinitic eosinophilic syndrome, rhinitis medicamentosa, atrophic rhinitis, pemphigus, benign mucous membrane pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis, epidermolysis bullosa acquisita, erythema multiforme, lichen planus, asthma, allergic rhinitis, cough, chronic bronchitis chronic obstructive pulmonary disease (COPD), cystic fibrosis, acute bacterial sinusitis, chronic sinusitis, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, bronchitis, and pneumonia.

[0169] According to another aspect, the disclosure provides a kit for treating or ameliorating the effects of bacterial and fungal infections, e.g., S. aureus, in a mammal in need thereof, the kit comprising: (1) a composition comprising a microorganism genetically modified to express an antimicrobial polypeptide; and (2) a reagent for applying the composition to the skin of the mammal. According to some embodiments, the microorganism is adapted to survive on the surface of the mammalian skin for a controlled period of time and provide a controlled or continuous supply of the antimicrobial polypeptide.

[0170] According to another aspect, the disclosure provides a kit for treating or ameliorating the effects of a disease, e.g., RSV, in a mammal in need thereof, the kit comprising: (1) a composition comprising a microorganism genetically modified to express a therapeutic agent, e.g., a LEKTI protein or protein domain, or a variant thereof; and (2) a reagent for applying the composition to the skin or nose of the mammal. According to some embodiments, the microorganism is adapted to survive on the surface of the mammal's skin for a controlled period of time and provide a controlled or continuous supply of the antimicrobial polypeptide.

[0171] In some embodiments, the engineered microorganism is administered to the skin of a subject. In some embodiments, the engineered microorganism is administered to a subject with a skin disorder. In some embodiments, the skin disorder is a dermatological disease. In some embodiments, the skin disease or disorder is a skin dysbiosis. Dysbiosis in the skin and / or gut microbiome is associated with an altered immune response and promotes the development of the skin disease. In some embodiments, the skin disease or disorder is a skin inflammatory disease or disorder. In some embodiments, the skin disease is skin toxicity due to one or more chemotherapeutic agents (including but not limited to, epidermal growth factor receptor (EGFR) inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, RAS inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, extracellular signal-regulated kinase (ERK) inhibitors). In some embodiments, the skin disease is ichthyosis. In some embodiments, the skin disease is Netherton syndrome. In some embodiments, the skin disease is hidradenitis suppurativa. In some embodiments, the skin disease is a psoriatic disease (including, but not limited to, psoriasis, pustular psoriasis, plaque psoriasis, and palmoplantar psoriasis). In some embodiments, the skin disease is neutrophilic dermatosis. In some embodiments, the skin disease is atopic dermatitis. In some embodiments, the skin disease is an autoimmune bullous disease. In some embodiments, the skin disease is acne and / or acneiform rash. In some embodiments, the skin disease is impetigo. In some embodiments, the skin disease is folliculitis. In some embodiments, the skin disease is acute suppurative paronychia. In some embodiments, the skin disease is lymphangitis. In some embodiments, the skin disease is cellulitis. In some embodiments, the skin disease is necrotizing fasciitis.

[0172] As used herein, the term "cutaneous" (e.g., administered to the skin of a subject) refers to the outer layer of a subject's (e.g., a mammal or human) body.

[0173] As used herein, the term "skin dysbiosis" refers to a condition in which the microbiome, such as that of the skin, becomes imbalanced. As a result of skin dysbiosis, a subject may develop skin inflammatory diseases and disorders, such as atopic dermatitis, eczema, seborrheic dermatitis, psoriasis, acne vulgaris, dandruff, and even skin cancer. Atopic dermatitis may refer to a chronic inflammatory skin disorder.

[0174] Atopic dermatitis can be associated with scaly skin, itchy rashes, and lesions. Atopic dermatitis is sometimes called atopic eczema or eczema. A characteristic of atopic dermatitis is pruritus, which refers to the itchy condition that causes the rash. Any part of the skin can be affected by atopic dermatitis. Age can play a role in the localization of atopic dermatitis. For example, in infants, atopic dermatitis can affect the cheeks, scalp, trunk, and extremities; in early childhood, atopic dermatitis can be localized to the flexor regions; in adolescents and adults, atopic dermatitis can affect the hands and feet.

[0175] In addition to the above components, the subject kit further comprises instructions for using the components and / or implementing the subject method.These instructions can be present in the subject kit in various forms, and one or more of them can be present in the kit.One form in which these instructions can be present is as printed information on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, in the packaging of the kit or in the package insert.Another means is a computer-readable medium, such as a diskette or CD, on which the information is recorded.Another means in which the instructions can be present is a website address that is used via the Internet to access the information at a remote site.Any convenient means can be present in the kit.

[0176] The components of the kit can be packaged either in aqueous medium or in lyophilized form. The kit is generally packaged to include at least one vial, test tube, flask, bottle, syringe or other container means, into which the described reagents can be placed, preferably suitably aliquoted. If additional components are provided, the kit also generally includes a second, third or other additional container into which such components can be placed.

[0177] The kits of the present disclosure also typically include a means for close containment of the reagent containers for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials are retained.

[0178] formulation According to some embodiments, formulations for use in accordance with the present disclosure comprise any pharmaceutically effective amount of recombinant bacteria (e.g., at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 21.0%, about 22.0%, about 23.0%, about 24.0%, about 25.0%, about 26.0%, about 27.0%, about 28.0%, about 29.0%, about 30.0%, about 31.0%, about 32.0%, about 33.0%, about 34.0%, about 35.0%, about 36.0%, about 37.0%, about 38.0%, about 39.0%, about 40.0%, about 41.0%, about 42.0%, about 43.0%, about 44.0%, about 45.0%, about 46.0%, about 47.0%, about 48.0%, about 49.0%, about 50.0%, about 51.0%, about 52.0%, about 53.0%, about 54.0%, about 55.0%, about 56.0%, about 57.0%, about 58.0%, about 59.0%, about 0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% or more recombinant bacteria by weight (the upper limit being about 90.0% recombinant bacteria by weight).

[0179] According to some embodiments, a formulation for use in accordance with the present disclosure can include, for example, at least about 0.01% to about 30% by weight, about 0.01% to about 20% by weight, about 0.01% to about 5% by weight, about 0.1% to about 30% by weight, about 0.1% to about 20% by weight, about 0.1% to about 15% by weight, about 0.1% to about 10% by weight, about 0.1% to about 5% by weight, about 0.2% to about 5% by weight, about 0.3% to about 5% by weight, about 0.4% to about 5% by weight, about 0.5% to about 5% by weight, about 1% to about 5% by weight, or more, of the recombinant bacterium.

[0180] According to some embodiments, the topical formulation may be in any form suitable for application to a body surface, such as a cream, lotion, spray, solution, gel, ointment, paste, plaster, paint, bioadhesive, suspension, emulsion, etc., and / or may be prepared to include liposomes, micelles, and / or microspheres. Such formulations may be used in combination with an occlusive top layer to maintain moisture during application to the body surface and during subsequent administration of the formulation as it evaporates from the body surface. According to some embodiments, the formulation may include a live cell culture composition, which may include at least one engineered bacterial strain producing a therapeutically effective recombinant polypeptide or therapeutically effective domain(s) thereof. This engineered live cell culture composition may deliver the polypeptide directly to the skin to treat or prevent an abnormal skin condition.

[0181] Topical formulations include those in which any other active ingredient or ingredients are dissolved or dispersed in a dermatological vehicle known in the art (e.g., aqueous or non-aqueous gel, ointment, water-in-oil or oil-in-water emulsion). Components of such vehicles can include water, aqueous buffer solutions, non-aqueous solvents (e.g., ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycofurol, or glycerol), oils (e.g., mineral oils, such as liquid paraffin, natural or synthetic triglycerides, such as Miglyol™, or silicone oils, such as dimethicone). Depending, inter alia, on the nature of the formulation and its intended use and site of application, the dermatological vehicle used may contain one or more ingredients (e.g., ingredients in addition to water if the formulation is an aqueous gel) selected from the following list: solubilizers or solvents (e.g., β-cyclodextrin, e.g., hydroxypropyl β-cyclodextrin, or alcohols or polyols, e.g., ethanol, propylene glycol, or glycerol); thickeners (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, or carbomer); gelling agents (e.g., polyoxyethylene-polyoxypropylene copolymers); preservatives (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, benzoates, potassium sorbate, or EDTA, or salts thereof); and pH buffering agents (e.g., a mixture of dihydrogen phosphate and hydrogen phosphate, or a mixture of citric acid and hydrogen phosphate)).

[0182] Pharmaceutically acceptable carriers can also be incorporated into the formulations of the present disclosure and can be any carrier conventionally used in the art. Examples include water, lower alcohols, higher alcohols, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and aqueous and emulsion-based mixtures of these carriers. The term "pharmaceutically acceptable" or "pharmaceutically acceptable carrier" is used herein to refer to a compound or composition that can be incorporated into a pharmaceutical formulation without causing undesired biological effects or undesired interactions with other components of the formulation, and "carrier" or "vehicle" as used herein refers to a carrier material that is suitable for incorporation into a topically applied composition. Carriers and vehicles useful herein include any such materials known in the art that are non-toxic and do not interact with other components of the formulation in a harmful manner. The term "aqueous" refers to a formulation that contains water or becomes water-containing after application to the skin or mucosal tissue.

[0183] The film-forming agent, upon drying, forms a protective film over the application site. The film inhibits removal of the active ingredient and maintains it in contact with the treated area. An example of a film-forming agent suitable for use in the present disclosure is Flexible Collodion, US P. Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, PA: Mack Publishing Co., 1995) page 1530. Collodion is an ethyl ether / ethanol solution containing pyroxylin (nitrocellulose), which evaporates to leave a film of pyroxylin. The film-forming agent can also act as a carrier. Solutions that dry to form a film are sometimes called paints. Creams, as is well known in the field of pharmaceutical formulations, are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil.

[0184] Cream bases are water-washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the "internal" phase, is generally composed of petrolatum and a fatty alcohol such as cetyl alcohol or stearyl alcohol. The aqueous phase usually, but not necessarily, exceeds the oil phase in volume and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic, or amphoteric surfactant.

[0185] Lotion is a preparation that is applied to the skin surface without friction, and is typically a liquid or semi-liquid preparation that particles containing active agent are present in water or alcohol base.Lotion is usually a suspension of solid, and preferably comprises a liquid oily emulsion of oil-in-water type.Lotion is the preferred preparation herein for treating large body area, because it is easy to apply a more fluid composition.Generally, the insoluble material in lotion needs to be spread evenly.

[0186] Lotions typically contain suspending agents to produce better dispersion, as well as compounds useful for localizing the active agent and holding it in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose, and the like.

[0187] A solution is a homogeneous mixture prepared by dissolving one or more chemicals (solutes) in a liquid so that the molecules of the dissolved substance are dispersed among the molecules of the solvent. Solutions may contain other pharmaceutically or cosmetically acceptable chemicals to buffer, stabilize, or preserve the solute. Typical examples of solvents used to prepare solutions are ethanol, water, propylene glycol, or any other acceptable vehicle. Of course, as is well known, gels are semisolid suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout a carrier liquid, which is typically aqueous, but preferably also contains alcohol and, optionally, oil. Preferred "organic macromolecules," i.e., gelling agents, are crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes commercially available under the trademark Carbopol. Also preferred are hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulose-based polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a uniform gel, a dispersing agent such as alcohol or glycerin can be added, or the gelling agent can be dispersed by grinding, mechanical mixing, stirring, or a combination thereof. Similarly, ointments well known in the art are typically semi-solid preparations based on petrolatum or other petroleum derivatives. As will be understood by those skilled in the art, the specific ointment base used will provide some desirable characteristics, such as emollience. As with other carriers or vehicles, the ointment base must be inert, stable, non-irritating, and non-sensitizing.As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, PA: Mack Publishing Co., 1995), pages 1399-1404, ointment bases can be divided into four classes: oleaginous bases, emulsifiable bases, emulsion bases, and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semi-solid hydrocarbons obtained from petroleum.

[0188] Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum.

[0189] Emulsion ointment bases are either water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, and include, for example, acetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of various molecular weights. For more information, see Remington: The Science and Practice of Pharmacy.

[0190] Paste is a semi-solid dosage form in which active agent is suspended in a suitable base.Depending on the nature of the base, paste is divided into fatty paste or one made from single-phase aqueous gel.The base in fatty paste is generally petrolatum or hydrophilic petrolatum etc.Paste made from single-phase aqueous gel generally incorporates carboxymethylcellulose etc. as a base.

[0191] The enhancer is typically a lipophilic co-enhancer, also known as a "plasticizing" enhancer, i.e., an enhancer having a molecular weight in the range of about 150 to 1000 and a water solubility of less than about 1% by weight, preferably less than about 0.5% by weight, and most preferably less than about 0.2% by weight. The Hildebrand solubility parameter δ of the plasticizing enhancer is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilic enhancers are fatty esters, fatty alcohols, and fatty ethers. Examples of certain, most preferred, fatty acid esters include methyl laurate, ethyl oleate, propylene glycol monolaurate, propylene glycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol, and fatty ethers include compounds in which a diol or triol, preferably a C2-C4 alkane diol or triol, is substituted with one or two fatty ether substituents.

[0192] Additional penetration enhancers will be known to those skilled in the art of topical drug delivery and / or described in relevant texts and literature, see, e.g., Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, 1995) (incorporated herein by reference).

[0193] In addition to those identified above, various other additives may be included in the compositions of the present disclosure, including, but not limited to, antioxidants, astringents, perfumes, preservatives, emollients, pigments, dyes, humectants, propellants, and sunscreens, as well as other classes of materials whose presence may be pharmaceutically or otherwise desirable. Typical examples of optional additives for inclusion in the formulations of the present disclosure are as follows: preservatives, such as sorbates; solvents, such as isopropanol and propylene glycol; astringents, such as menthol and ethanol; emollients, such as polyalkylene methyl glucosides; humectants, such as glycerin; emulsifiers, such as glycerol stearate, PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60, sorbitol and other polyhydroxy alcohols, such as polyethylene glycol; sunscreens, such as octyl methoxyl cinnamate (commercially available as Parsol MCX) and butyl methoxybenzoylmethane (Parsol 1789), antioxidants such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζι-tocopherol, ζ2-tocopherol, η-tocopherol and retinol (vitamin A), essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g., soybean oil, palm oil, the liquid fraction of shea butter, sunflower oil), animal oils (e.g., perhydrosqualene), synthetic oils, silicone oils or waxes (e.g., cyclomethicone and dimethicone), fluorinated oils (commonly perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes (e.g., beeswax, carnauba wax, and paraffin wax), skin-feel modifiers modifier), thickeners and structurants such as swelling clays and crosslinked carboxypolyalkylenes available under the Carbopol trademark.Other additives include beneficial agents such as materials that condition the skin (especially the upper layer of the skin in the stratum corneum), slow the loss of its moisture content, thereby keeping the skin soft and / or protecting the skin. Examples of such conditioners and moisturizers include pyrrolidine carboxylic acids and amino acids, organic antimicrobial agents such as 2,4,4'-trichloro-2-hydroxydiphenyl ether (triclosan) and benzoic acid, anti-inflammatory agents such as acetylsalicylic acid and glycyrrhetinic acid, anti-seborrhoeic agents such as retinoic acid, vasodilators such as nicotinic acid, melanogenesis inhibitors such as kojic acid, and mixtures thereof. Further additional active agents include, for example, α-hydroxy acids, α-keto acids, polymeric hydroxy acids, moisturizers, collagen, marine extracts, and antioxidants, such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζι-tocopherol, ζ2-tocopherol, η-tocopherol, and retinol (vitamin A), and / or their pharmaceutically acceptable salts, esters, amides, or other derivatives.Preferred tocopherol compound is α-tocopherol.Additional active agents include those that can improve oxygen supply in skin tissue, as described, for example, in Gross, et al., International Publication No. 94 / 00098 and Gross, et al., WO94 / 00109, both of which are assigned to Lancaster Group AG (incorporated herein by reference).Sunscreens and UV-absorbing compounds can also be included.Non-limiting examples of such sunscreens and UV-absorbing compounds include aminobenzoic acid (PABA), avobenzone, cinoxate, dioxybenzone, homosalate, menthyl anthranilate, octocrylene, octyl methoxycinnamate, octyl salicylate, oxybenzone, padirnate O, phenylbenzimidazole sulfonic acid, sulisobenzone, titanium dioxide, trolamine salicylate, zinc oxide, ensulizole, meradiraate, octinoxate, octisalate, and octocrylene. See "Sunscreen drug products for over-the-counter human use" (Title 21, Chapter 1, Subchapter D, Part 352), which is incorporated herein in its entirety.

[0194] Other embodiments can include a variety of non-carcinogenic, non-irritating healing materials that facilitate treatment with the formulations of the present disclosure. Such healing materials can include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant, glandular or animal extracts, or safe therapeutic agents that can be added to the formulation to facilitate healing of skin disorders.

[0195] The amounts of these various additives are those conventionally used in the cosmetic field, for example, in the range of about 0.01% to about 20% of the total weight of the topical formulation.

[0196] The formulations of the present disclosure may also include conventional additives such as opacifiers, flavors, colorants, stabilizers, surfactants, etc. In certain embodiments, other agents such as antimicrobial agents may also be added to prevent spoilage during storage, i.e., to inhibit the growth of microorganisms such as yeast and mold.

[0197] Suitable antimicrobial agents are typically selected from the group consisting of methyl and propyl esters of p-hydroxybenzoic acid (i.e., methylparaben and propylparaben), sodium benzoate, sorbic acid, imidurea, and combinations thereof. In other embodiments, other agents such as repressors and inducers can also be added, i.e., inhibit (i.e., glycose) or induce (i.e., xylose) the production of the polypeptide of interest. Such additives can be used, provided they are compatible with the formulation and do not interfere with the function of the formulation.

[0198] The formulation may also include mild additives to minimize or eliminate the possibility of skin irritation or damage due to the administered chemical entity or other components of the composition.

[0199] Suitable irritation-reducing additives include, for example, α-tocopherol, monoamine oxidase inhibitors, particularly phenyl alcohols such as 2-phenyl-1-ethanol, glycerin, salicylates, ascorbates, ionophores such as monensin, amphoteric amines, ammonium chloride, N-acetylcysteine, capsaicin, and chloroquine. When present, irritation-reducing additives can be incorporated into the composition at a concentration effective to reduce irritation or skin damage, typically not more than about 20% by weight of the formulation, more usually not more than about 5% by weight.

[0200] Creams, lotions, gels, ointments, pastes, etc. can be spread on the affected surface and gently rubbed in. Solutions can be applied in the same manner, but are more typically applied with a dropper, swab, etc. and carefully applied to the affected area.

[0201] The application regimen depends on several factors that can be easily determined, such as the severity of the condition and its response to initial treatment, but usually involves continuous application once or more times a day.Those skilled in the art can easily determine the optimal dosage of the formulation to be administered, administration method and repetition rate.Generally, it is contemplated that the formulation of the present disclosure is applied in a range of once or twice a week to once or twice a day.

[0202] The pharmaceutical compositions of the present disclosure comprise one or more active ingredients, e.g., therapeutic agents, mixed with one or more pharmaceutically acceptable diluents or carriers, and optionally one or more other compounds, drugs, ingredients and / or materials. Regardless of the route of administration selected, the agents / compounds of the present disclosure are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).

[0203] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, DC)), sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starch, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate, and calcium hydrogen phosphate), sodium citrate, ectoine, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, oleyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn oil, germ oil, olive oil, castor oil, sesame oil, cottonseed oil, and peanut oil), cocoa butter, waxes (e.g., suppository wax), paraffin, silicones, talc, silicates, and the like. Each pharmaceutically acceptable diluent or carrier used in the pharmaceutical compositions of the present disclosure must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a selected dosage form and method of administration can be determined using ordinary skill in the art.

[0204] The pharmaceutical compositions of the present disclosure may optionally contain additional components and / or materials commonly used in pharmaceutical compositions. These components and materials are well known in the art and include (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, hydroxypropylmethylcellulose, sucrose, and acacia; (3) humectants, such as glycerol; and (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethylcellulose, and carbonate. sodium, (5) solution retarders, such as paraffin, (6) absorption enhancers, such as quaternary ammonium compounds, (7) wetting agents, such as cetyl alcohol and glycerol monostearate, (8) absorbents, such as kaolin, bentonite clay, (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate, (10) suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide,metahydroxide), bentonite, agar, and tragacanth, (11) buffering agents, (12) excipients, such as lactose, milk sugar, polyethylene glycol, animal and vegetable fats, oils, waxes, paraffin, cocoa butter, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, salicylates, zinc oxide, aluminum hydroxide, calcium silicate, and polyamide powder, (13) inert diluents, such as water or other solvents, (14) preservatives, (15) surface-active agents, (16) dispersing agents, (17) controlled-release or absorption-retarding agents, such as hydroxypropyl methylcellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes, (18) opacifying agents, (19) adjuvants, (20) ) wetting agents, (21) emulsifying and suspending agents, (22) solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane, (24) antioxidants, (25) agents that render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride, (26) thickening agents, (27) coating materials, such as lecithin, and (28) sweeteners, flavorings, colorings, fragrances, and preservatives. Each such component or material must be "acceptable" in the sense of being compatible with the other components of the formulation and not harmful to the subject. Components and materials that are suitable for a selected dosage form and intended route of administration are well known in the art, and components and materials that are acceptable for a selected dosage form and method of administration can be determined using ordinary skill in the art.

[0205] Dosage forms for topical or transdermal administration include powder, spray, ointment, paste, cream, lotion, gel, solution, patch, droplets and inhalant.One or more active agents / compounds can be mixed with suitable pharmaceutically acceptable diluents or carriers under sterile conditions.Ointment, paste, cream and gel can contain excipients.Powder and spray can contain excipients and propellants.

[0206] Pharmaceutical compositions of the present disclosure suitable for parenteral administration may contain one or more agents / compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain suitable antioxidants, buffers, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. These pharmaceutical compositions may also contain suitable adjuvants such as wetting agents, emulsifying agents, and dispersing agents. It may be desirable to include an isotonic agent. Additionally, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption.

[0207] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way. [Example]

[0208] Example The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.

[0209] Example 1 Phenotypic characterization of the secreted antimicrobial activity of SE25 SE25 was positive in the screening of the "Micromyx-50 library" Analysis of the Micromyx library of S. epidermidis strains for antimicrobial activity, particularly anti-S. aureus ATCC 29213 activity, revealed S. epidermidis strain SE25 (Figures 1A and 1B). The Micromyx library was obtained by obtaining samples from the forearms of human subjects. For further study, samples were cultured on selective plates for S. epidermidis and grown in TSB. The zone of inhibition indicated that SE25 could inhibit the growth of S. aureus when streaked onto TSA plates, grown for 48 hours, and then overlaid with S. aureus. The general experimental procedure involved inoculating the library of S. epidermidis strains into tryptic soy broth (TSB) and growing at 37°C for one day. On the second day, strains were copied onto TSA plates and grown overnight at 37°C and then at room temperature. The library of S. epidermidis strains was overlaid with the indicator strain S. aureus and grown at room temperature for 4 to 5 days.

[0210] Further analysis revealed that SE25 had antimicrobial activity against other Gram-positive bacteria, but not against Gram-negative bacteria (Figure 2). SE25 was overlaid with the Gram-positive bacteria S. aureus SA25923, S. aureus SA29213, MRSA USA300, S. epidermidis NRRL B-4268, and B. subtilis, as well as the Gram-negative strains P. aeruginosa and E. coli (Figure 2). Table 6 below summarizes the antimicrobial activity of SE25 against various pathogenic bacteria. Table 6: Antimicrobial activity of strain SE25 against the indicated bacteria [Table 6]

[0211] SE25 cell-free supernatant has bactericidal activity The anti-S. aureus activity of SE25 is secreted. S. aureus ATCC 29213 was grown in either conditioned growth medium from SE25 cultures, conditioned growth medium from SE3 cultures (negative control), or unconditioned growth medium (Figure 3). Conditioned medium from SE25 cultures inhibited the growth of S. aureus ATCC 29213. In contrast, neither conditioned medium nor unconditioned medium from SE3 cultures inhibited the growth of S. aureus ATCC 29213. Cell-free conditioned medium was prepared by growing S. epidermidis cultures in tryptic soy broth (TSB) for 24 hours at 30°C with shaking at 250 RPM. The conditioned medium, also known as cell-free supernatant (CFS), was sterilized by filtration (PES, 0.22 μm membrane) and the pH was adjusted to 6.5-7.5. The anti-S. aureus activity assay was performed by adding 90 μL of CFS to 10 μL of approximately 5 x 10 5 This was done by mixing CFUs with an indicator strain, e.g., S. aureus. The indicator strain was prepared in fresh TSB. Assay negative controls included fresh TSB medium and CFS without added bacteria. Cultures were grown overnight at 37°C, and S. aureus bacterial growth was enumerated after plating for CFU enumeration.

[0212] The spectrum of activity of SE25 CFS was assayed against S. aureus ATCC 25923, S. epidermidis NRRL B-4268, S. epidermidis 1457, S. epidermidis SE25, B. subtilis, E. faecalis, E. coli, P. aeruginosa, and the ichthyosis CoNS isolates S. lugdunensis, S. auricularis, S. capitis, S. haemolyticus, S. hominis, S. pettenkoferi, S. saprophyticus, and S. warneri. SE25 CFS had inhibitory activity against all strains tested, except for E. faecalis, E. coli, and P. aeruginosa.

[0213] Bactericidal activity was 3-log of the indicator strain within 24 hours of growth. 10 Bacteriostatic activity is defined as a reduction in the number of CFUs by + / - 1 log of the starting CFU within 24 hours. 10 Figure 5A shows that SE25 has bactericidal activity against S. aureus ATCC 29213. SE25 cell-free supernatant reduced the bacterial load of S. aureus from the starting inoculum by approximately 7-log 10 Similarly, the known antimicrobial agents levofloxacin and vancomycin reduced the CFU of S. aureus by approximately 6.75-log 10 It was reduced.

[0214] SE25 does not produce biofilms on plastic and disperses S. aureus biofilms Strains were tested for biofilm formation and S. aureus biofilm dispersal activity. Strain SE25, S. epidermidis NRRL B-4268, and S. epidermidis 1457 were incubated in polystyrene plastic wells to allow adhesion (Figure 6A). Unbound and loosely bound bacterial cells were removed, and the wells were washed. Crystal violet solution was used to stain adherent cells that had adhered to the polystyrene plastic wells and formed biofilms. The positive control, S. epidermidis 1457, demonstrated high biofilm formation by crystal violet staining. Strain SE25 cells did not form biofilms, as evidenced by the lack of crystal violet staining. S. epidermidis NRRL B-4268 also tested negative for biofilm formation (Figure 6A). This result was confirmed by a Conga Red assay, in which strain SE25 grew as red colonies (negative for biofilms) (Figure 6B).

[0215] The SE25 strain demonstrated the ability to disperse S. aureus biofilms (Figure 6C). First, S. aureus cultures were allowed to adhere to plastic wells and form biofilms. Then, serial dilutions of the SE25 culture were added. Untreated S. aureus cells remained in the biofilm, but the S. aureus biofilms were dispersed after treatment with dilutions of SE25 (Figure 6C).

[0216] SE25 is sensitive to antistaphylococcal antibiotics The susceptibility of strains SE25 and SE-27a was tested to different antibiotics to determine the MICs shown in Table 7 below.

[0217] Table 7: MICs of antibiotic resistance assays against SE25 and NRRL B-4268 derivative strains [Table 7] *Although there is no clinical resistance breakpoint for mupirocin, strain SE25 was identified as harboring the resistance gene mupA on a plasmid that may allow resistance to >500 μg / ml.

[0218] Reconstructed human epidermis The reconstructed human epidermis (RHE) experimental procedure involves inoculating SE into reconstructed human epidermal tissue prepared from cultured keratinocytes on an inert polycarbonate medium. After inoculation, SE was incubated for 24 or 48 hours (Figure 7). SE was incubated for 10 minutes. 3 , 10 5 and 10 7 The inoculum concentration colonized the RHE.

[0219] RHE competition assay was performed at 10 5 SE cells were inoculated into RHE and incubated for 48 hours. 3 Colonization with SE25 resulted in a 4-log reduction in SA29213 (Figure 8). After an additional 24 hours, the RHE was rinsed to remove unattached bacteria, and S. aureus was enumerated by CFU plating.

[0220] SE25 induced antimicrobial peptide production (AMP) in RHE cells 72 hours after inoculation (Figure 9). AMP is known to have S. aureus activity. SE25 and NRRL induced similar host responses.

[0221] Example 2 Whole genome sequence (WGS) analysis of strain SE25 and annotation of naturally occurring plasmids in the strain Analysis of the genome sequence of strain SE25 revealed the presence of four plasmids, p36.326 kb ("p36.3"; multiple variants are possible), p43.056 kb ("p43.1"), p24.273 kb ("p24.3"), and p9.793 kb ("p9.8"), and a chromosome of 2.402255 Mb in size. The determinant for high-level resistance to mupirocin was found on plasmid p24.3, which carries the mupA gene encoding the MupA protein (ileS2, isoleucyl-tRNA synthetase, EC 6.1.1.5). This plasmid-encoded gene can determine resistance to >64 μg / ml.

[0222] Subsystem features identified in plasmids include: cofactors, vitamins, prosthetic groups, pigments (97), cell wall and capsule (36), virulence, disease, and defense (43), potassium metabolism (3), photosynthesis (0), other (10), phages, prophage, transposable elements, and plasmids (13), membrane transport (26), iron acquisition and metabolism (24), RNA metabolism (36), nucleosides and nucleotides (78), and protein metabolism. Xie (157), cell division and cell cycle (5), motility and chemotaxis (0), regulation and cell signaling (28), secondary metabolism (5), DNa metabolism (54), fats, lipids, and isoprenoids (45), nitrogen metabolism (20), dormancy and sporulation (9), respiration (19), stress response (36), metabolism of aromatic compounds (3), amino acids and derivatives (224), sulfur metabolism (5), phosphorus metabolism (13), and carbohydrates (189).

[0223] See Tables 8-11 for an overview of the annotation of ORFs in the SE25 plasmid. Table 8. Features on the SE25-p36.326 kb plasmid: Epidermin BGC gene is in bold. [Table 8-1] [Table 8-2] [Table 8-3] Table 9. SE25-p43.056 kb characteristics of the plasmid "p43.1" [Table 9-1] [Table 9-2] Table 10. Features of the SE25-p24.273 kb plasmid "p24.3". The MupA gene is in bold. [Table 10-1] [Table 10-2] Table 11. SE25-p9.793 kb characteristics of the plasmid "p19.4" [Table 11]

[0224] The absence of mupirocin resistance genes, the epidermin lanthipeptide biosynthetic gene cluster (Fig. 10), siderophores, prophage (Fig. 11), and the ica operon was identified by genome analysis of strain SE25.

[0225] Example 3 Engineering of S. epidermidis strain SE25 Based on the overall positive results of the phenotypic tests, particularly with regard to its bactericidal activity against S. aureus on agar, reconstructed human epidermis (RHE) and in liquid medium, as well as its amenability to genetic manipulation, strain SE25 was selected as a promising strain for further modification(s).

[0226] Strain SE25 was engineered to be auxotrophic for D-alanine with either a two-gene deletion (Δalr1Δdat) or a three-gene deletion (Δalr1Δalr2Δdat) plus additional deletions of the mupirocin resistance gene (ΔmupA) and prophage gene (Δcapsid).

[0227] Engineering D-alanine auxotrophy. To engineer a D-alanine auxotroph in strain SE25, the chromosomal regions flanking the alr1, alr2, and dat genes were analyzed for homology to S. epidermidis NRRL B-4268. In the absence of single nucleotide polymorphisms, the D-alanine auxotrophic strain S. epidermidis SE25 was ΔΔΔ The same knockout plasmid construct used to engineer the SE25 auxotroph was found to be suitable for engineering the SE25 auxotroph. Primer sequences, their specific use, and PCR product sizes are listed in Table 12 below. Table 12. Primers for knockout of D-alanine aminotransferase (SE1423) [Table 12-1] [Table 12-2] Overlap PCR using primers 1423-5F / 1423-3R: 1.5 Kb PCR product from wild-type using primers 1423-5F / 1423-3R: 2.3 Kb F: forward primer R: reverse primer Restriction sites added for cloning purposes are underlined and in bold.

[0228] A 2018 study by Moscoso et al. (Moscoso, Garcia et al. 2018), describing the construction of S. aureus D-alanine auxotrophic strains, reported that "...the double ΔdatΔalr1 mutant and the triple ΔdatΔalr1Δalr2 mutant require exogenous D-alanine for growth." Similar to what was done for S. aureus, two different SE25 auxotrophic strains were constructed in parallel by sequential deletion of either the alr1, alr2, and dat genes ("triple mutant") or the alr1 and dat genes ("double mutant"). Both knockout variants were engineered and screened for the ability to grow on agar plates with and without D-alanine and retested in liquid medium with and without additional D-alanine supplementation (Figure 12). Both variants were significantly different from S. epidermidis SE25 auxotrophic strains. ΔΔΔ D-alanine was required at the same concentration (100 μg / mL) as required by

[0229] The pJB-1423KO plasmid isolated from the dam- / dcm- E. coli strain (NEB) was transformed into competent cells of SE25Δalr1Δalr2 using plates containing TSA + chloramphenicol (10 μg / mL). The presence of the pJB-1423KO plasmid in the transformants was confirmed by detecting a 1.5-kb PCR product using primers 1423-5F (EcoRI) and 1423-3R (SalGI). A 1.5-kb PCR product was observed, whereas a 2.3-kb PCR product was observed in the reaction containing cell lysate from wild-type SE host cells. Cells from two confirmed clones were streaked onto fresh plates containing TSA + Cm (10 μg / mL) + D-alanine (100 μg / mL). The plates were incubated at 43°C for 48 hours for plasmid integration by homologous recombination. Again, isolated colonies were streaked for purification at 43°C and grown for 48 hours. To loop out the plasmid backbone by a second round of homologous recombination, four isolated colonies were inoculated into 3 mL of TSB + D-alanine (100 μg / mL) in a 15 mL tube. The culture was shaken overnight at 30°C. An aliquot of 50 μl of the culture was transferred to a 15 mL tube containing 3 mL of fresh TSB, DA100 medium. Cells from the first and second 30°C cultures were plated onto TSB + anhydrotetracycline (ATC 2 μg / mL) + D-alanine (DA, 100 μg / mL). After 2 days of incubation at 30°C, approximately 100–200 colonies formed on the plated plates.

[0230] SE25Δalr1Δalr2Δdat (6 colonies) and SE25Δalr1Δdat (14 colonies) were screened and verified for Cm sensitivity on Cm10 TSA plates (Figures 13A and 13B). SE25Δalr1Δdat (Figure 14A) and SE25Δalr1Δalr2Δdat (Figure 14B) retained anti-S. aureus activity.

[0231] Manipulation of mupirocin susceptibility. 3063 bp of the 3072 bp ORF of the mupA gene on plasmid p24.3, which confers resistance to the topical skin antibiotic mupirocin, was deleted in three strain variants (wild-type strain and both auxotrophs) to engineer susceptibility to mupirocin, resulting in three strains: SE25ΔmupA, SE25Δalr1ΔdatΔmupA, and SE25Δalr1Δalr2ΔdatΔmupA.

[0232] To delete a portion of the mupA ORF, 968-bp and 966-bp flanking regions were PCR-amplified and assembled into the shuttle pJB38 plasmid, which contains a temperature-sensitive Gram-positive origin of replication. The assembled recombinant plasmid was transformed into the E. coli host NEB 5a maintenance strain. PCR-confirmed plasmid DNA was purified and sequenced to confirm correct assembly and the absence of mutations. Plasmids with confirmed expected sequences were transformed into the methylation-deficient E. coli strain NEB (dam- / dcm-). Plasmid DNA was purified and retransformed into strain SE25 by electroporation. Following a standard allelic exchange procedure, chloramphenicol-resistant colonies were screened for positive ΔmupA mutants on TSA plates with or without mupirocin (Figures 15A and 15B).

[0233] Deletion of the prophage capsid gene A putative prophage measuring 41,881 bp in size was identified on the chromosome, encoding over 50 structural, replication, and lysis genes, as well as a duplicated integration site at the 3' end of the ORF for the iron-sulfur cluster assembly protein SufB gene. As a preventative measure to inactivate potential release of mature lytic phage particles, the putative prophage gene encoding the structural capsid protein was deleted in all three ΔmupA derivative strains. The putative prophage is structurally similar to S. aureus phage phi11, Siphoviridae Staphylococcus phage phiMR25, Siphoviridae Staphylococcus phage phiMR11, Siphoviridae Staphylococcus phage phiSauS-IPLA88, bacteriophage 92, bacteriophage 88, 29, bacteriophage 55, and Siphoviridae Staphylococcus aureus phage phiNM2.

[0234] To delete 1338 bp of the 1353 bp capsid ORF, 1004 bp and 958 bp flanking regions were PCR amplified and assembled into the shuttle pJB38 plasmid, which contains a temperature-sensitive Gram-positive replication origin. The assembled recombinant plasmid was transformed into the E. coli host NEB 5α maintenance strain. PCR-confirmed plasmid DNA was purified and sequenced to confirm correct assembly and the absence of mutations. Plasmids with confirmed expected sequences were transformed into the methylation-deficient E. coli strain NEBa (dam- / dcm-), purified, and retransformed into strain SE25 by electroporation. Following standard allelic exchange procedures for chloramphenicol-resistant colonies, positive Δcapsid mutants were screened by PCR.

[0235] Confirmation of antistaphylococcal activity of engineered strains The resulting strains SE25 ΔmupAΔcapsid, SE25 ΔmupAΔcapsid Δalr1Δdat, and SE25 ΔmupAΔcapsid Δalr1Δalr2Δdat were assayed for retention of antimicrobial activity against S. aureus in an agar overlay assay (FIG. 15).

[0236] Identification of a plasmid-mediated biosynthetic gene cluster encoding the biosynthetic genes for the epidermin lantibiotic Gene annotation of the p36.3 plasmid revealed the presence of a biosynthetic gene cluster (BGC) involved in the synthesis of the epidermin lantibiotic peptide. The gene cluster shares 100% identity with the reported epidermin biosynthetic gene cluster. The approximately 15 kb BGC was annotated with the following genes: epiA, B, C, D, E, F, G, H, P, Q, T, signal peptidase I (EC 3.4.21.89), and several hypothetical proteins (Figure 10). To disrupt lantibiotic production in SE25, the open reading frame (ORF) of the epiA gene, encoding a 52-amino acid prepropeptide (MEAVKEKNDL FNLDVKVNAK ESNDSGAEPRIASKFICTPGCAKTGSFNSYCC*), was truncated to six amino acids (including MEA-YCC*) in the wild-type SE25 strain, and the function of the gene was confirmed by loss-of-function deletion.

[0237] To delete a portion of the epiA ORF, 994-bp and 974-bp flanking regions were PCR-amplified and assembled into the shuttle pJB38 plasmid, which contains a temperature-sensitive Gram-positive origin of replication. The assembled recombinant plasmid was transformed into the E. coli host NEB 5α maintenance strain. Transformants were analyzed by PCR, and confirmed plasmid DNA purified from positive clones was sequenced to verify correct assembly and the absence of mutations. Plasmids with confirmed expected sequences were transformed into the methylation-deficient E. coli strain NEB (dam- / dcm-), purified, and retransformed into strain SE25 by electroporation. Following a standard allelic exchange procedure for chloramphenicol-resistant colonies, PCR screening for ΔepiA mutant colonies was performed, and a final S. aureus overlay assay was performed on the positive ΔepiA mutant and wild-type strains (Figures 16A and 16B).

[0238] The results showed that in the S. aureus agar overlay assay, the SE25ΔepiA mutant strain exhibited a loss / significant reduction in the zone of growth inhibition, thereby confirming the role of epiA in conferring anti- S. aureus activity (Fig. 17).

[0239] The reduced anti-SA activity in the ΔepiA mutant strain was also confirmed by liquid culture assays (not shown). Example 4 Table 13. Exemplary sequences [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6]

[0240] Example 5 Preparation of S. epidermidis strain SE484 To construct S. epidermidis strain SE484, three D-alanine biosynthetic genes, alanine racemase 1 (alr1), alanine racemase 2 (alr2), and D-alanine aminotransferase (dat), were first deleted to generate strain SE464, a D-alanine auxotroph that does not grow on media without additional D-alanine supplementation. Subsequent deletion of the mupirocin resistance gene (mupA, also known as iles2) and the prophage capsid gene from SE464 created strain SE484.

[0241] Engineering D-alanine auxotrophy As described in Example 3, each of the three D-alanine biosynthetic genes and their flanking DNA sequences was individually cloned into a temperature-sensitive knockout plasmid. These plasmids were then transformed into the parent S. epidermidis strain SE25. The resulting colonies were analyzed by PCR to verify integration of the plasmid at the target locus. A second round of recombination was then performed to remove the plasmid from the genome, after which colonies were screened for excision of the plasmid from the genome and its subsequent loss during growth in the absence of chloramphenicol selection. The phenotype of potential candidates was screened by growth on tryptic soy agar (TSA) containing D-alanine and / or chloramphenicol (Figures 19A-C). Candidate colonies were initially streaked onto TSA in the presence of 100 μg / ml D-alanine without chloramphenicol (Figure 19A). The same colonies were then streaked onto TSA supplemented with 10 μg / ml chloramphenicol in the absence of D-alanine (Figure 19B). Candidate colonies that required additional D-alanine supplementation for growth were selected (Figure 19C). To verify successful deletions, approximately 2 kilobases of flanking regions around the alr1, alr2, and dat deletions were amplified by PCR and sequenced. All deletions were confirmed by sequencing, and deletion of the three D-alanine biosynthetic genes resulted in strain SE464, a D-alanine auxotroph that could not grow on medium without additional D-alanine supplementation.

[0242] Engineering mupirocin susceptibility and deletion of prophage capsid genes To generate strain SE484 from strain SE464, two subsequent genes were deleted: mupA (also called ilse2) and the gene encoding the major capsid protein of a putative prophage identified by the PhiSPY algorithm during whole-genome sequencing (WGS). Deletions were performed using the same allelic exchange strategy used to delete the alanine racemase gene. Transformants were screened for mupirocin-sensitive colonies and D-alanine auxotrophs.

[0243] Example 6 Characterization of S. epidermidis strain SE484 Whole genome sequencing of strain SE484 confirmed that it was identical to the parent strain SE25, except for the intended deletion.

[0244] Growth profile of strain SE484 The growth profile of strain SE484 in TSB at 30°C was observed to be comparable to that of the parent strain SE25, confirming that the modifications compared to the parent did not affect the physiology of SE484. The growth kinetics of both strains are compared in the plot shown in Figure 20A. Next, to compare the activity of both strains against S. aureus, a zone of inhibition plate assay was performed. Here, SE25 and SE484 were each streaked onto TSA supplemented with 100 μg / ml of D-alanine and incubated at 37°C. After 48 hours of incubation, soft agar containing S. aureus was poured onto both plates, which were then incubated for an additional 24 hours at 37°C. Both S. epidermidis strains showed comparable inhibition of S. aureus growth (Figures 20B-20C). These results indicated that the modifications of the five target genes resulting in strain SE484 did not affect its anti-S. aureus properties.

[0245] Phenotypic and genotypic stability of SE484 The phenotypic and genotypic stability of SE484 was assessed over a 5-day period with daily subcultures. To verify phenotypic stability, daily subcultures of SE484 were evaluated for D-alanine auxotrophy, mupirocin susceptibility, growth rate, CFU count, and antimicrobial activity against S. aureus. To verify genotypic stability, amplification of the alr1, alr2, dat, and epiA genes was performed by PCR on samples obtained from passages 1 and 5 of the SE484 culture.

[0246] Various phenotypic and genotypic characteristics of SE484 were evaluated over time for five passages and 179 generations. SE484 was streaked onto either TSA, TSA containing 100 μg / ml D-alanine, or TSA containing D-alanine and 20 μg / ml mupirocin, and plates were incubated at 37°C for 48 hours. As shown in Figure 20D, the growth profile and macroscopic colony morphology remained unchanged from passage 1 to passage 5, and SE484 cultures grew only when additional D-alanine was supplemented at both time points, thus verifying the maintenance of the D-alanine auxotrophic phenotype. Furthermore, mupirocin sensitivity was also tested through passage 5 and was found to be present in all passages.

[0247] To assess the stability of the anti-S. aureus properties of SE484 after serial passages, glycerol stocks prepared and harvested at each passage were plated and overlaid with S. aureus. These results did not indicate a loss of anti-S. aureus activity, as S. aureus clearance was observed near the heavy streak of SE484 in all five passages. Furthermore, the observed zone of clearance pattern was consistent from passage 1 to passage 5, indicating that this antimicrobial activity of SE484 is stable over at least five passages.

[0248] Example 7 S. epidermidis strain SE484 inhibits the growth of S. aureus The anti-S. aureus activity of strains SE484 and SE123 (SEΔΔΔ) was quantified and compared. A preventive approach was employed for the measurements. Reconstructed human epidermis (RHE) was inoculated with either SE123 or SE484 and incubated for 4 hours in a tissue culture incubator at 37°C and 5% CO2. RHE was then inoculated with approximately 10 4CFU of S. aureus MRSA strain USA300 were challenged. After an additional 24 h of incubation, USA300 cell numbers were determined by punch biopsy and dilution plating. These results revealed that SE123 did not exhibit significant ability to inhibit USA300 growth (Figure 21A), whereas SE484 inhibited USA300 growth by approximately 3 logs after 24 h of incubation (Figure 21B).

[0249] These results indicated that S. epidermidis SE484 has anti-S. aureus activity, whereas SE123 (SEΔΔΔ) does not.

[0250] Incorporation by Reference The entire disclosure of each patent document, including patent application documents, scientific papers, government reports, websites and other references referred to in this specification is incorporated herein by reference in its entirety for all purposes.If there is a conflict in terms, this specification shall prevail.All sequence listings or SEQ ID NOs disclosed herein are incorporated herein in their entirety.

[0251] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0252] Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the present disclosure is not limited to what has been described, and that various other changes or modifications may be made by those skilled in the art without departing from the scope or spirit of the present disclosure.

Claims

1. 1. A recombinant microorganism comprising: deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive; and deletions or substitutions in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; a deletion or substitution in one or more genes encoding naturally occurring temperate bacteriophage genes, wherein the naturally occurring temperate bacteriophage genes are inactive; and A recombinant microorganism comprising:

2. 2. The recombinant microorganism of claim 1, wherein the one or more naturally occurring antibiotic resistance genes are selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a cotrimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

3. 3. The recombinant microorganism of claim 1 or 2, wherein the one or more naturally occurring antibiotic resistance genes is a mupirocin resistance gene (mupA).

4. 4. The recombinant microorganism of claim 1, wherein the one or more naturally occurring lysogenic bacteriophage genes encode bacteriophage capsid proteins.

5. 5. The recombinant microorganism of claim 1, wherein the one or more D-alanine biosynthetic genes comprise a D-alanine aminotransferase (dat) gene, an alanine racemase gene alr1, or alr2.

6. 6. The recombinant microorganism of claim 1, wherein the deletion or substitution in one or more genes encoding D-alanine biosynthesis genes comprises deletions in dat, alr1, and alr2.

7. 6. The recombinant microorganism of any one of claims 1 to 5, further comprising one or more genes encoding heterologous genes.

8. 8. The recombinant microorganism of claim 7, wherein the heterologous gene is selected from the group consisting of a gene encoding an antimicrobial polypeptide or a variant thereof, a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, a gene encoding an enzyme or a variant thereof, a gene encoding an enzyme inhibitor or a variant thereof, a gene encoding an antigen or a variant thereof, and a gene encoding an immunomodulatory polypeptide or a variant thereof, or a combination thereof.

9. 10. The recombinant microorganism of any one of the preceding claims, wherein the one or more antimicrobial polypeptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

10. 10. The recombinant microorganism of claim 9, wherein the one or more microbial pathogens are selected from the group consisting of a bacterial pathogen, a fungal pathogen, or a viral pathogen.

11. 11. The recombinant microorganism of claim 10, wherein the one or more bacterial pathogens are selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Cutibacterium species and Mycoplasma, or a combination thereof.

12. 11. The recombinant microorganism of claim 10, wherein the fungal infection is caused by a fungus selected from the group consisting of Malassezia species, Candida species, Aspergillus, Cryptococcus, and Pneumocystis.

13. 11. The recombinant microorganism of claim 10, wherein the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

14. The antimicrobial polypeptide is selected from the group consisting of epidermin-like lantibiotic, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, cartilage glycoprotein 1, chitotriosidase, mucin 9, cartilage glycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α- L-arabinofuranosidase, acetyl esterase, acetyl xylan esterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, lysostaphin, zucin A, millelisin B, muraminidase Cpl-1, lysozyme, endolysin PlyC, endolysin, PlyV12, enterolysin A, C. The recombinant microorganism according to any one of claims 8 to 13, wherein the cytolysin is selected from the group consisting of C. difficile autolysin (Acd), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilin, epidermin, gallidermin, mutacin B-a Ny266, mutacin 1140, Pep5, epicidin 280, epilancin K7, lacticin 481, cytolysin, lacticin 3147, staphylococcin C55, salvaricin A, lactocin S, streptococcin A-FF2, sublancin 168, carnosin U149, variacin 8, sipemycin, cinnamycin, duramycin, ancovenin, mersacidin, and actagardin.

15. The recombinant microorganism of any one of claims 1 to 6, wherein the one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains, or variants thereof, and the LEKTI protein, one or more LEKTI protein domains, or variants thereof are secreted.

16. 8. The recombinant microorganism of claim 7, wherein the heterologous gene is a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, the antimicrobial biosynthetic enzyme being capable of producing 6-N-hydroxyaminopurine (6-HAP), and the 6-HAP is secreted.

17. The recombinant microorganism of claim 8, wherein the gene encoding an immunomodulatory polypeptide or a variant thereof is a gene encoding a lipoteichoic acid (LTA) biosynthetic enzyme, and the recombinant microorganism is capable of producing and secreting LTA.

18. The recombinant microorganism of any one of claims 1 to 17, which is a bacterium or a combination of bacteria.

19. 10. The recombinant microorganism of any one of the preceding claims, selected from the group consisting of Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or a combination thereof.

20. 20. The recombinant microorganism of claim 19, wherein the recombinant microorganism is Staphylococcus epidermidis.

21. 10. The recombinant microorganism of any one of the preceding claims, which secretes one or more therapeutic polypeptides or variants thereof.

22. 10. A pharmaceutical composition comprising a cell culture composition comprising one or more recombinant microorganisms according to any one of the preceding claims and a pharmaceutically acceptable carrier.

23. 23. The pharmaceutical composition of claim 22, wherein the cell culture composition is a live cell culture composition.

24. 24. The pharmaceutical composition of claim 22 or 23, wherein the cell culture composition comprises 0% water to up to 90% water.

25. 25. The pharmaceutical composition of any one of claims 22 to 24, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, emulsion, cream, lotion, gel, or ointment.

26. 1. A method of treating a disease, disorder, or condition in a subject, comprising: Administering to the subject a recombinant microorganism comprising deletions or replacements in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive; deletions or replacements in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; and deletions or replacements in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive.

27. 27. The method of claim 26, wherein the recombinant microorganism is in a cell culture composition.

28. 28. The method of claim 26 or 27, wherein the cell culture composition is a live cell culture composition.

29. 29. The method of any one of claims 26 to 28, wherein the one or more naturally occurring antibiotic resistance genes are selected from the group consisting of a mupirocin resistance gene, an ampicillin resistance gene, a cefotaxime resistance gene, a chloramphenicol resistance gene, a ciprofloxacin resistance gene, a cotrimoxazole resistance gene, a nalidixic acid resistance gene, an oxytetracycline resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, and a trimethoprim resistance gene.

30. 30. The method of any one of claims 26 to 29, wherein the one or more naturally occurring antibiotic resistance genes are mupirocin resistance genes.

31. 31. The method of any one of claims 26 to 30, wherein the one or more naturally occurring lysogenic bacteriophage genes encode a bacteriophage capsid protein.

32. 32. The method of any one of claims 26 to 31, wherein the deletion or substitution in one or more genes encoding D-alanine biosynthetic genes comprises deletions in dat, alr1 and alr2.

33. 33. The method of any one of claims 26 to 32, wherein the recombinant microorganism further comprises one or more genes encoding heterologous genes.

34. 34. The method of claim 33, wherein the heterologous gene is selected from the group consisting of a gene encoding an antimicrobial polypeptide or a variant thereof, a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, a gene encoding an enzyme or a variant thereof, a gene encoding an enzyme inhibitor or a variant thereof, a gene encoding an antigen or a variant thereof, and a gene encoding an immunomodulatory polypeptide or a variant thereof, or a combination thereof.

35. 35. The method of any one of claims 26 to 34, wherein the one or more antimicrobial polypeptides or variants thereof are capable of inhibiting or preventing the growth of one or more microbial pathogens.

36. 36. The method of claim 35, wherein the one or more microbial pathogens are selected from the group consisting of a bacterial pathogen, a fungal pathogen, or a viral pathogen.

37. 37. The method of claim 36, wherein the one or more bacterial pathogens are selected from the group consisting of Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Propionibacteria species, and Mycoplasma, or a combination thereof.

38. 37. The method of claim 36, wherein the fungal infection is caused by a fungus selected from the group consisting of Malassezia species, Candida species, Aspergillus, Cryptococcus, and Pneumocystis.

39. 37. The method of claim 36, wherein the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus.

40. The antimicrobial polypeptide is selected from the group consisting of epidermin-like lantibiotic, YM-1, YM-2, acidic mammalian chitinase (AMCase), oviduct glycoprotein 1, cartilage glycoprotein 1, chitotriosidase, mucin 9, cartilage glycoprotein-39, chondrocyte protein 39, endoglucanase, exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α- L-arabinofuranosidase, acetyl esterase, acetyl xylan esterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, pectate lyase, lysostaphin, zucin A, millelisin B, muraminidase Cpl-1, lysozyme, endolysin PlyC, endolysin, PlyV12, enterolysin A, C. The method of any one of claims 34 to 39, wherein the antimicrobial agent is selected from the group consisting of C. difficile autolysin (Acd), autolysin (LytA), PL-1 amidase hydrolase, nisin A, nisin Z, subtilin, epidermin, gallidermin, mutacin B-a Ny266, mutacin 1140, Pep5, epicidin 280, epilancin K7, lacticin 481, cytolysin, lacticin 3147, staphylococcin C55, salvaricin A, lactocin S, streptococcin A-FF2, sublancin 168, carnosine U149, variacin 8, sipemycin, cinnamycin, duramycin, ancovenin, mersacidin, and actagardin.

41. The method of any one of claims 26 to 33, wherein the one or more heterologous genes encode a LEKTI protein, one or more LEKTI protein domains, or variants thereof, and the LEKTI protein, one or more LEKTI protein domains, or variants thereof are secreted.

42. 34. The method of claim 33, wherein the heterologous gene is a gene encoding an antimicrobial biosynthetic enzyme or a variant thereof, the antimicrobial biosynthetic enzyme being capable of producing 6-N-hydroxyaminopurine (6-HAP), and the 6-HAP is secreted.

43. 35. The method of claim 34, wherein the gene encoding an immunomodulatory polypeptide or a variant thereof is a gene encoding a lipoteichoic acid (LTA) biosynthetic enzyme, and the recombinant microorganism is capable of producing and secreting LTA.

44. 44. The method of any one of claims 26 to 43, wherein the recombinant microorganism is a bacterium or a combination of bacteria.

45. 45. The method of any one of claims 26 to 44, wherein the recombinant microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Corynebacterium, Cutibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or a combination thereof.

46. 46. ​​The method of claim 45, wherein the recombinant microorganism is Staphylococcus epidermidis.

47. 47. The method of any one of claims 26 to 46, wherein the recombinant microorganism secretes one or more therapeutic polypeptides or variants thereof.

48. 48. The method of any one of claims 26 to 47, wherein the cell culture composition is a live cell culture composition.

49. 49. The method of claims 26-48, wherein the cell culture composition comprises from 0% water to up to 90% water.

50. 50. The method of any one of claims 26 to 49, wherein the subject is a mammal.

51. 51. The method of claim 50, wherein the mammal is a human.

52. 52. The method of any one of claims 26 to 51, wherein the disease, disorder or condition is selected from the group consisting of a microbial infection, a skin disease or disorder, an inflammatory disease or disorder, and a metabolic disease or disorder.

53. Microbial infections include Staphylococcus, Streptococcus, Haemophilus, Moraxella, Escherichia, Enterobacter, Proteus, Klebsiella, Pseudomonas, Legionella, Chlamydia, Propionibacteria species, Mycoplasma, Mala 52. The method of claim 51 , wherein the pathogen comprises one or more microbial pathogens selected from the group consisting of S. szezia species, Candida species, Aspergillus, Cryptococcus, Pneumocystis, respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, and rhinovirus, or a combination thereof.

54. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is an inflammatory skin disease or disorder.

55. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is a skin dysbiosis.

56. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is skin toxicity.

57. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is ichthyosis.

58. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is a psoriatic disease or disorder.

59. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is dermatitis.

60. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is an autoimmune blistering disease.

61. 52. The method of any one of claims 25 to 51, wherein the skin disease or disorder is selected from the group consisting of Netherton syndrome, hidradenitis suppurativa, psoriasis, pustular psoriasis, plaque psoriasis, and palmoplantar psoriasis, atopic dermatitis, acne, acneiform rash, impetigo, folliculitis, acute suppurative paronychia, lymphangitis, necrotizing fasciitis, and cellulitis.

62. Selecting a bacterial strain; In the following order: (1) deletions or replacements in one or more genes encoding D-alanine biosynthesis genes, wherein the D-alanine biosynthesis genes are inactive; (2) deletions or replacements in one or more genes encoding naturally occurring antibiotic resistance genes, wherein the naturally occurring antibiotic resistance genes are inactive; and (3) deletions or replacements in one or more genes encoding naturally occurring lysogenic bacteriophage genes, wherein the naturally occurring lysogenic bacteriophage genes are inactive. and Thereby, a recombinant D-alanine auxotrophic bacterial strain is prepared.

2. A recombinant D-alanine auxotrophic bacterial strain prepared by a process comprising:

63. 63. The recombinant D-alanine auxotrophic bacterial strain of claim 62, wherein the deletion or substitution in one or more genes encoding D-alanine biosynthetic genes comprises a deletion in dat, alr1, and alr2.

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