Methods and compositions for increasing protein expression in genetically engineered bacteria - Patents.com

Abstract

The present disclosure provides, inter alia, compositions and methods for making compositions comprising a microorganism expressing a therapeutic protein, one or more disaccharides, one or more oligosaccharides, one or more antioxidants, one or more fatty alcohols, and colloidal oatmeal and / or alkaline oatmeal extract.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 144,076, filed February 1, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Created on January 31, 2022, said ASCII copy is named 129062-02020_SL.txt and is 45,460 bytes in size.

[0003] Technical Field The present disclosure relates to methods, kits, and compositions for increasing protein expression in genetically engineered bacteria. [Background technology]

[0004] Recent improvements in the application of genetically engineered bacteria as targeted delivery systems for proteins or peptides have made them attractive as therapeutic agents. This type of genetically engineered bacteria is sometimes called recombinant probiotic bacteria (US 2018 / 0161380 A1, AM Munivar et al., US 2018 / 0135062 A1, T. Wirth et al., WO 2017 / 044836 A1, T. Hitchcock et al., WO 2017 / 147507 A1, T. Hitchcock et al.). Other examples include bacterial strains that produce natural products with therapeutic value (WO 2019 / 046801 A1, RL Gallo et al.).

[0005] The current state of the art formulations in this area involve lyophilized or freeze-dried bacteria placed into capsules or creams. Capsules dissolve in mucous membranes or fluids, while creams can be applied directly to the target area.

[0006] There remains a need to increase protein expression and to develop improved formulations of compositions comprising genetically engineered bacteria that continuously express a protein of interest. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US 2018 / 0161380 A1 [Patent Document 2] US 2018 / 0135062 A1 [Patent Document 3] WO 2017 / 044836 A1 [Patent Document 4] WO 2017 / 147507 A1 [Patent Document 5] WO 2019 / 046801 A1 Summary of the Invention

[0008] According to one aspect, the present disclosure provides a composition comprising one or more live microorganisms and a first mixture, wherein the one or more microorganisms produce a therapeutic agent, the first mixture comprising one or more disaccharides, one or more oligosaccharides, and one or more antioxidants, and wherein the first mixture comprises at least 1×10 10 The composition has a viability of 100-150 CFU / g.

[0009] According to some embodiments, the composition further comprises one or more fatty alcohols or alcohols.

[0010] According to some embodiments, the one or more disaccharides are selected from the group consisting of lactose, trehalose, sucrose, maltose, and cellobiose.

[0011] According to some embodiments, the one or more oligosaccharides are selected from the group consisting of fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), mannan-oligosaccharides (MOS), isomalto-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides, and raffinose.

[0012] According to some embodiments, the one or more antioxidants are selected from the group consisting of ascorbic acid, vitamin A, vitamin E, a-carotene, lycopene, lutein, zeaxanthin, and water-soluble derivatives of lipophilic antioxidants.

[0013] According to some embodiments, the water soluble derivative of a lipophilic antioxidant is alpha-tocopherol phosphate or alpha-tocopherol polyethylene glycol ester.

[0014] According to some embodiments, the one or more disaccharides are lactose, the one or more oligosaccharides are fructo-oligosaccharides (FOS), and the one or more antioxidants are ascorbic acid.

[0015] According to some embodiments, the one or more fatty alcohols are selected from the group consisting of saturated fatty alcohols and unsaturated fatty alcohols.

[0016] According to some embodiments, the saturated fatty or alcoholic acid is cetyl alcohol and the unsaturated fatty alcohol is oleyl alcohol. According to some embodiments, the mixture of oleyl alcohol and cetyl alcohol ranges from about 25% oleyl alcohol / 75% cetyl alcohol to about 50% oleyl alcohol / 50% cetyl alcohol (w / w).

[0017] According to some embodiments, the second mixture comprises oleyl alcohol and cetyl alcohol in about a 1:1 ratio.

[0018] According to some embodiments, the one or more disaccharides are from about 50% (w / w) to about 55% (w / w), the one or more oligosaccharides are from about 10% (w / w) to about 15% (w / w), the one or more antioxidants are from about 0.1% (w / w) to about 2% (w / w), and the one or more microorganisms are from about 30% (w / w) to about 35% (w / w).

[0019] According to some embodiments, the composition of the microorganism and the first mixture is in a ratio of about 10:1, and the second mixture comprises said one or more fatty alcohols.

[0020] According to some embodiments, the second mixture further comprises colloidal oatmeal and / or alkaline oatmeal extract. According to some embodiments, the second mixture comprises about 1% (wt / wt) colloidal oatmeal and about 0.5% (wt / wt) alkaline oatmeal extract when combined with the fatty alcohol.

[0021] According to some embodiments, the microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, Oenococcus, or Corynebacterium, and mixtures thereof. According to some embodiments, the microorganism is Staphylococcus epidermidis.

[0022] According to some embodiments, the therapeutic agent is naturally produced by the microorganism. According to some embodiments, the microorganism is genetically engineered to produce the therapeutic agent.

[0023] According to some embodiments, the therapeutic agent is selected from a polypeptide, a small molecule, and a metabolite.

[0024] According to some embodiments, the therapeutic agent is selected from the group consisting of one or more LEKTI protein domains, filaggrin, interferons, enkephalins, interleukins, and antimicrobial agents. According to some embodiments, the one or more LEKTI protein domains is LEKTI-d6.

[0025] According to some embodiments, the one or more antimicrobial agents are selected from the group consisting of chitinase, glucanase, or peptidoglycan hydrolase.

[0026] According to some embodiments, the microorganism is attenuated by auxotrophy. According to some embodiments, the microorganism is a D-alanine auxotroph. According to some embodiments, the microorganism comprises a deletion of one or more of the alr1, alr2, and dat genes.

[0027] According to some embodiments, the composition is administered to the skin of the mammal.

[0028] According to some embodiments, expression of the therapeutic agent is increased when compared to a composition without the second mixture.

[0029] According to some embodiments, expression of the therapeutic agent is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, or at least 13-fold greater after 8 hours than a composition that does not include the second mixture. According to some embodiments, expression of the therapeutic agent is at least 5-fold greater after 8 hours than a composition that does not include the second mixture. According to some embodiments, expression of the therapeutic agent is at least 10-fold greater after 8 hours than a composition that does not include the second mixture.

[0030] According to some embodiments, the composition comprises at least 2.5×10 10 CFU / g, at least 5x10 10 CFU / g, at least 1x10 11 CFU / g, at least 2.5x10 11 CFU / g, at least 5x10 11 CFU / g, at least 1x10 12 CFU / g, or at least 2.5x10 12 According to some embodiments, the composition has a viability of about 1x10 10 CFU / g ~ approx. 1x10 11 CFU / g, approx. 5x10 10 CFU / g ~ approx. 5x10 11 CFU / g, approximately 1x10 11 CFU / g ~ approx. 1x10 12 CFU / g, or approximately 5x10 11 CFU / g ~ approx. 5x10 12 It has a viability of CFU / g.

[0031] According to some embodiments, the antimicrobial activity is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times greater after 8 hours compared to a composition that does not include the second mixture. According to some embodiments, the antimicrobial activity is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, or at least 12 times greater after 8 hours compared to a composition that does not include the second mixture.

[0032] According to some embodiments, the LEKTI protein domain activity is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold greater after 8 hours compared to a composition that does not include the second mixture. According to some embodiments, the LEKTI protein domain activity is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, or at least 12-fold greater after 24 hours compared to a composition that does not include the second mixture.

[0033] According to another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the compositions disclosed herein and a pharma- ceutically acceptable carrier. According to some embodiments, the pharma-ceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, or an ointment.

[0034] According to another aspect, the disclosure provides a method for preparing any one of the compositions disclosed herein or any one of the pharmaceutical compositions disclosed herein, the method comprising the steps of: (a) combining one or more microorganisms with a first mixture, wherein the first mixture comprises one or more disaccharides, one or more oligosaccharides, and one or more antioxidants; (b) lyophilizing the composition resulting from step (a); and (c) combining a second mixture comprising one or more fatty alcohols with the lyophilized composition resulting from step (b).

[0035] According to another aspect, the present disclosure provides a method of treating a disease, disorder, or condition in a subject, comprising administering to the subject any one of the compositions disclosed herein or any one of the pharmaceutical compositions disclosed herein.

[0036] According to some embodiments, the disease, disorder, or condition is a skin disease or disorder, an inflammatory disease, and a cancer.

[0037] According to some embodiments, the skin disease or disorder is Netherton syndrome, psoriasis, acne, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, seborrheic dermatitis, eczema, dry skin, allergies, rash, UV-irritated skin, detergent-irritated skin, thinning skin, bullous pemphigoid, pemphigus vulgaris, impetigo, vitiligo, alopecia, and / or hirsutism.

[0038] According to some embodiments, the disease or disorder is associated with pain and is selected from the group consisting of acute pain, chronic pain, nociceptive pain, neuropathic pain, traumatic pain, inflammatory pain, post-operative amputation pain, pain associated with cancer, bone fracture pain, osteoporotic pain, osteosarcoma pain, and gouty joint pain.

[0039] According to some embodiments, the cancer is selected from the group consisting of malignant melanoma, colon cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, oral tongue squamous cell carcinoma, squamous cell carcinoma, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, gallbladder cancer, brain tumors, and oral cancer.

[0040] According to another aspect, the disclosure provides a method of increasing expression of a therapeutic agent by a microorganism, comprising the steps of: (a) combining one or more microorganisms with a first mixture comprising one or more disaccharides, one or more oligosaccharides, and one or more antioxidants; (b) lyophilizing the composition resulting from step (a); and (c) combining a second mixture comprising one or more fatty alcohols with the lyophilized composition resulting from step (b), to prepare any one of the compositions disclosed herein or any one of the pharmaceutical compositions disclosed herein. [Brief description of the drawings]

[0041] [Figure 1-1] Figure 1 shows graphs depicting the effect of media formulations on SE27a growth (Figures 1A-1D) and LEKTI-d6 production (Figures 1E-1H) in liquid culture. Growth of SE27a is shown on (A) ASM medium, (B) TSB medium, (C) ASM + 1% colloidal oatmeal (CO) and (D) ASM + 0.5% colloidal oatmeal extract (COE). Active LEKTI production is shown on (E) ASM medium, (F) TSB medium, (G) ASM + 1% CO and (H) ASM + 0.5% COE. [Figure 1-2] This is a continuation of Figure 1-1. [Diagram 2] FIG. 2 shows a graph illustrating the effect of addition of colloidal oatmeal (CO) and colloidal oatmeal extract (COE) on bacterial growth on agar plates. [Diagram 3] FIG. 3 shows a graph depicting the effect of addition of colloidal oatmeal (CO) and colloidal oatmeal extract (COE) on active LEKTI levels on agar plates. [Figure 4] FIG. 4 shows a graph depicting the growth of bacterial growth on ASM agar plates of SE-27 formulated in oleyl alcohol:cetyl alcohol (OA:CA), OA:CA + colloidal oatmeal (CO), and OA:CA + colloidal oatmeal extract (COE). [Diagram 5] FIG. 5 shows a graph depicting active LEKTI-d6 production from SE-27a formulated in oleyl alcohol:cetyl alcohol (OA:CA), OA:CA+colloidal oatmeal (CO), and OA:CA+colloidal oatmeal extract (COE) after growth on ASM agar plates for 8 hours. [Figure 6] FIG. 6 shows a graph depicting the bacterial content of placebo or SE-27a formulated in oleyl alcohol:cetyl alcohol (OA:CA)+colloidal oatmeal (CO) over 24 h of incubation on pig skin surfaces. [Figure 7] FIG. 7 shows a graph depicting active LEKTI-d6 levels of placebo or SE-27a formulated in oleyl alcohol:cetyl alcohol (OA:CA)+colloidal oatmeal (CO) over 24 h of incubation on pig skin surfaces. [Figure 8] Figure 8A shows a graph depicting the growth of SE484 in artificial sweat medium containing 2% D-alanine with or without 1% colloidal oatmeal (ASM). Figure 8B shows a graph depicting the antimicrobial activity of SE484 spent broth against Bacillus subtilis. The time points shown correspond to the time of growth at which the spent medium was collected, with higher MIC values ​​corresponding to stronger antimicrobial activity. [Figure 9]9A-9C show graphs showing growth and active LEKTI production by SE351 formulated in oleyl alcohol:cetyl alcohol 1:1 (w / w) supplemented with 1% colloidal oatmeal (w / w) and 2% D-alanine (w / w) and applied ex vivo onto pig skin at 10 (FIG. 9A), 10 (FIG. 9B) and 10 (FIG. 9C) CFU / cm. Times shown represent skin incubation periods at 30° C. Dotted lines represent the mean CFU / cm (top) and LEKTI activity (bottom) seen in placebo samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present disclosure relates to microbial products formulated in non-aqueous compositions designed to stabilize the bacterial product and increase protein expression upon administration. According to some embodiments, the dry version of the composition acts to stabilize the composition for storage. According to some embodiments, the composition increases protein expression after administration of the microbial product to a subject in the treatment of a disease or disorder.

[0043] One aspect of the present disclosure provides compositions that increase protein expression of recombinant proteins in Staphylococcus epidermidis that have been genetically altered to express recombinant proteins to treat or ameliorate a disease or disorder, such as a skin disease or disorder, an inflammatory disease, and cancer.

[0044] The term "skin disease" and its grammatical variants as used herein refer to a skin condition or condition that is generally undesirable or harmful compared to the normal or baseline condition of human skin. Examples of abnormal skin conditions include, but are not limited to, Netherton syndrome, psoriasis, acne, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, seborrheic dermatitis, eczema, dry skin, allergies, rash, UV-irritated skin, detergent-irritated skin (including irritation caused by enzymes and molecules used in cleaning detergents and sodium lauryl sulfate), skin thinning (e.g., skin from elderly people and children), bullous pemphigoid, pemphigus vulgaris, impetigo, vitiligo, alopecia, and hirsutism.

[0045] As used herein, the term "genetically modified" and grammatical variations thereof are used to describe microorganisms (e.g., bacteria) that have been genetically modified or engineered by the introduction of DNA prepared outside the microorganism. For example, the introduction of plasmid DNA containing new genes into the bacteria allows the bacteria to express these genes. Alternatively, the DNA containing new genes can be introduced into the bacteria and then integrated into the bacterial genome, where the bacteria will express these genes.

[0046] As used herein, the term "microorganism" or "recombinant microorganism" refers to a microorganism, e.g., a bacterial cell, that has been genetically modified from its native state. Thus, a "recombinant bacterial cell" or "recombinant bacterium" refers to a bacterial cell or bacterium that has been genetically modified from its native state. For example, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into its DNA. These genetic modifications may be present in the chromosome of the bacterium or bacterial cell. A recombinant bacterial cell may contain an exogenous nucleotide sequence stably integrated into its chromosome.

[0047] The terms "treat", "treating", "treatment" and grammatical variations thereof as used herein refer to providing a subject, e.g., a patient, with a protocol, regimen, process or treatment that is desirable to obtain a physiological response or outcome. In particular, the methods and compositions of the present invention can be used to slow down the development of disease symptoms, or to delay the onset of a disease or condition, or to stop the progression of disease development. However, treatment does not require that the desired physiological response or outcome is 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 treatment. Thus, a given subject or subject population, e.g., a patient population, may not respond or may respond poorly to treatment.

[0048] In the present invention, the subject may be a mammal. As used herein, "mammal" and grammatical variations thereof refer to any category of mammal. In the present invention, mammals include, for example, humans, agricultural animals, livestock, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of livestock include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. Preferably, the mammal is a human.

[0049] As used herein, the term "effective amount" or "therapeutically effective amount" of a compound or composition disclosed herein is an amount of such compound or composition that is sufficient to produce a beneficial or desired result as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosages can be determined empirically, and such determinations are within the skill of one of ordinary skill in the art. Those skilled in the art will understand that dosages vary depending on the route of administration, rate of excretion, duration of treatment, identity of any other drugs administered, age, size, and species of mammal, e.g., human patient, and similar factors well known in the medical and veterinary arts. In general, a suitable dose of a composition according to the present invention will be that amount of the composition that is the lowest dose effective to produce the desired effect. An effective dose of a composition of the present invention may be administered as 2, 3, 4, 5, 6 or more subdoses administered separately at appropriate intervals throughout the day.

[0050] As used herein, the term "about" generally refers to a range that may be 1.5% greater or less than the numerical value being described within the context of the particular use. For example, "about 5" would include a range of 3.5 to 6.5.

[0051] According to some embodiments, the composition comprises a mixture of live microorganisms and one or more disaccharides, one or more oligosaccharides, and one or more antioxidants. According to some embodiments, the composition further comprises one or more fatty alcohols. According to some embodiments, the composition further comprises colloidal oatmeal and / or alkaline oatmeal extract.

[0052] According to some embodiments, bacteria suitable for use in the present invention include, but are not limited to, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof. According to some embodiments, the microbial composition comprises one or more of Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes, Corynebacterium spp., Acinetobacter johnsonii, and Pseudomonas aeruginosa. According to some embodiments, other related or similar species found on the skin are used. Particular embodiments include the use of Staphylococcus epidermidis bacteria.

[0053] According to some embodiments, the composition comprises a mixture of one or more disaccharides selected from the group consisting of lactose, trehalose, sucrose, maltose, and cellobiose. According to some embodiments, the one or more oligosaccharides are selected from the group consisting of fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), mannan oligosaccharides (MOS), isomalto-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides, and raffinose. According to some embodiments, the composition comprises one or more antioxidants selected from the group consisting of ascorbic acid, vitamin A, vitamin E, a-carotene, lycopene, lutein, zeaxanthin, and water-soluble derivatives of lipophilic antioxidants, such as alpha-tocopherol phosphate, alpha-tocopherol polyethylene glycol ester. According to one embodiment, the disaccharide is lactose, the oligosaccharide is a fructo-oligosaccharide, and the antioxidant is ascorbic acid.

[0054] According to some embodiments, the microorganism is mixed with disaccharides, oligosaccharides, and antioxidants, and the composition further comprises one or more fatty alcohols selected from the group consisting of saturated fatty alcohols and unsaturated fatty alcohols. According to some embodiments, the saturated fatty alcohol is cetyl alcohol and the unsaturated fatty alcohol is oleyl alcohol. According to some embodiments, the cetyl alcohol and the oleyl alcohol are in a 1:1 ratio.

[0055] According to some embodiments, the one or more disaccharides is about 20% (w / w), about 25% (w / w), about 30% (w / w), about 35% (w / w), about 40% (w / w), about 45% (w / w), about 50% (w / w), about 55% (w / w), or about 60% (w / w). According to some embodiments, the one or more disaccharides is from about 20% (w / w) to about 30% (w / w), from about 25% (w / w) to about 35% (w / w), from about 30% (w / w) to about 40% (w / w), from about 35% (w / w) to about 45% (w / w), from about 40% (w / w) to about 50% (w / w), from about 45% (w / w) to about 55% (w / w), or from about 50% (w / w) to about 60% (w / w). According to some embodiments, the one or more disaccharides is at least 20% (w / w), at least 25% (w / w), at least 30% (w / w), at least 35% (w / w), at least 40% (w / w), at least 45% (w / w), at least 50% (w / w), at least 55% (w / w), or at least 60% (w / w). According to some embodiments, the one or more disaccharides is no more than 20% (w / w), no more than 25% (w / w), no more than 30% (w / w), no more than 35% (w / w), no more than 40% (w / w), no more than 45% (w / w), no more than 50% (w / w), no more than 55% (w / w), or no more than 60% (w / w).

[0056] According to some embodiments, the one or more oligosaccharides is about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 10% (w / w), about 11% (w / w), about 12% (w / w), about 13% (w / w), about 14% (w / w), about 15% (w / w), about 16% (w / w), about 17% (w / w), about 18% (w / w), about 19% (w / w), or about 20% (w / w). According to some embodiments, the one or more oligosaccharides is from about 5% (w / w) to about 20% (w / w), from about 5% (w / w) to about 15% (w / w), from about 5% (w / w) to about 10% (w / w), from about 10% (w / w) to about 20% (w / w), from about 10% (w / w) to about 15% (w / w), or from about 15% (w / w) to about 20% (w / w). According to some embodiments, the one or more oligosaccharides is at least 5% (w / w), at least 6% (w / w), at least 7% (w / w), at least 8% (w / w), at least 9% (w / w), at least 10% (w / w), at least 11% (w / w), at least 12% (w / w), at least 13% (w / w), at least 14% (w / w), at least 15% (w / w), at least 16% (w / w), at least 17% (w / w), at least 18% (w / w), at least 19% (w / w), or at least 20% (w / w). According to some embodiments, the one or more food source oligosaccharides is 5% (w / w) or less, 6% (w / w) or less, 7% (w / w) or less, 8% (w / w) or less, 9% (w / w) or less, 10% (w / w) or less, 11% (w / w) or less, 12% (w / w) or less, 13% (w / w) or less, 14% (w / w) or less, 15% (w / w) or less, 16% (w / w) or less, 17% (w / w) or less, 18% (w / w) or less, 19% (w / w) or less, or 20% (w / w) or less.

[0057] According to some embodiments, the one or more antioxidants are about 0.1% (w / w), about 0.2% (w / w), about 0.3% (w / w), about 0.4% (w / w), about 0.5% (w / w), about 0.6% (w / w), about 0.7% (w / w), about 0.8% (w / w), about 0.9% (w / w), about 1.0% (w / w), about 1.1% (w / w), about 1.2% (w / w), about 1.3% (w / w), about 1.4% (w / w), about 1.5% (w / w), about 1.6% (w / w), about 1.7% (w / w), about 1.8% (w / w), about 1.9% (w / w), or about 2.0% (w / w). According to some embodiments, the one or more antioxidants are present in an amount of from about 0.1% (w / w) to about 2.0% (w / w), from about 0.1% (w / w) to about 1.5% (w / w), from about 0.1% (w / w) to about 1.0% (w / w), from about 0.1% (w / w) to about 0.5% (w / w), from about 0.5% (w / w) to about 2.0% (w / w), from about 0.5% (w / w) to about 1.5% (w / w), from about 0.5% (w / w) to about 1.0% (w / w), from about 1.0% (w / w) to about 2.0% (w / w), or from about 1.0% (w / w) to about 1.5% (w / w). According to some embodiments, the one or more antioxidants are at least 0.1% (w / w), at least 0.2% (w / w), at least 0.3% (w / w), at least 0.4% (w / w), at least 0.5% (w / w), at least 0.6% (w / w), at least 0.7% (w / w), at least 0.8% (w / w), at least 0.9% (w / w), at least 1.0% (w / w), at least 1.1% (w / w), at least 1.2% (w / w), at least 1.3% (w / w), at least 1.4% (w / w), at least 1.5% (w / w), at least 1.6% (w / w), at least 1.7% (w / w), at least 1.8% (w / w), at least 1.9% (w / w), or at least 2.0% (w / w).According to some embodiments, the one or more antioxidants are present at 0.1% (w / w) or less, 0.2% (w / w) or less, 0.3% (w / w) or less, 0.4% (w / w) or less, 0.5% (w / w) or less, 0.6% (w / w) or less, 0.7% (w / w) or less, 0.8% (w / w) or less, 0.9% (w / w) or less, 1.0% (w / w) or less, 1.1% (w / w) or less, 1.2% (w / w) or less, 1.3% (w / w) or less, 1.4% (w / w) or less, 1.5% (w / w) or less, 1.6% (w / w) or less, 1.7% (w / w) or less, 1.8% (w / w) or less, 1.9% (w / w) or less, or 2.0% (w / w) or less.

[0058] According to some embodiments, the one or more microorganisms are about 20% (w / w), about 25% (w / w), about 30% (w / w), about 35% (w / w), about 40% (w / w), or about 45% (w / w). According to some embodiments, the one or more microorganisms are present in an amount of from about 20% (w / w) to about 45% (w / w), from about 20% (w / w) to about 40% (w / w), from about 20% (w / w) to about 35% (w / w), from about 20% (w / w) to about 30% (w / w), from about 20% (w / w) to about 25% (w / w), from about 25% (w / w) to about 45% (w / w), from about 25% (w / w) to about 40% (w / w), from about 25% (w / w) to about 35% (w / w), from about 25% (w / w) to about 30% (w / w), from about 30% (w / w) to about 45% (w / w), from about 30% (w / w) to about 40% (w / w), from about 30% (w / w) to about 35% (w / w). (w / w), about 35% (w / w) to about 45% (w / w), about 35% (w / w) to about 40% (w / w), or about 40% (w / w) to about 45% (w / w). According to some embodiments, the one or more microorganisms are at least 20% (w / w), at least 25% (w / w), at least 30% (w / w), at least 35% (w / w), at least 40% (w / w), or at least 45% (w / w). According to some embodiments, the one or more microorganisms are 20% (w / w) or less, 25% (w / w) or less, 30% (w / w) or less, 35% (w / w) or less, 40% (w / w) or less, 45% (w / w) or less.

[0059] According to some embodiments, the microorganisms of the composition are at least 2.5x10 10 CFU / g, at least 5x10 10 CFU / g, at least 1x10 11 CFU / g, at least 2.5x10 11 CFU / g, at least 5x10 11 CFU / g, at least 1x10 12 CFU / g, at least 2.5x10 12 CFU / g, or at least 5x10 12 According to some embodiments, the microorganisms of the composition have a viability of about 2.5x10 CFU / g. 10 CFU / g, approximately 5 x 10 10 CFU / g, approximately 1 x 10 11 CFU / g, approx. 2.5 x 10 11 CFU / g, approximately 5 x 10 11 CFU / g, approximately 1 x 10 12 CFU / g, approx. 2.5 x 10 12 CFU / g, or approximately 5 x 10 12 According to some embodiments, the microorganisms of the composition have a viability of about 2.5 x 10 CFU / g. 10 CFU / g ~ approx. 2.5 x 10 12 CFU / g, approx. 2.5 x 10 10 CFU / g ~ approx. 1 x 10 12 CFU / g, approx. 2.5 x 10 10 CFU / g ~ approx. 5 x 10 11 CFU / g, approx. 2.5 x 10 10 CFU / g ~ approx. 2.5 x 10 11 CFU / g, approx. 2.5 x 10 10 CFU / g ~ approx. 1 x 10 11 CFU / g, 2.5 x 10 10 CFU / g ~ approx. 5 x 10 10 CFU / g, approximately 5 x 10 10 CFU / g ~ approx. 2.5 x 10 12 CFU / g, approximately 5 x 10 10 CFU / g ~ approx. 1 x 10 12CFU / g, about 5 x 10 10 CFU / g to about 5 x 10 11 CFU / g, about 5 x 10 10 CFU / g to about 2.5 x 10 11 CFU / g, about 5 x 10 10 CFU / g to about 1 x 10 11 CFU / g, about 1 x 10 11 CFU / g to about 2.5 x 10 12 CFU / g, about 1 x 10 11 CFU / g to about 1 x 10 12 CFU / g, about 1 x 10 11 CFU / g to about 5 x 10 11 CFU / g, about 1 x 10 11 CFU / g to about 2.5 x 10 11 CFU / g, about 2.5 x 10 11 CFU / g to about 2.5 x 10 12 CFU / g, about 2.5 x 10 11 CFU / g to about 1 x 10 12 CFU / g, about 2.5 x 10 11 CFU / g to about 5 x 10 11 CFU / g, about 5 x 10 11 CFU / g to about 2.5 x 10 12 CFU / g, about 5 x 10 11 to about 1 x 10 12 CFU / g, about 1 x 10 12 CFU / g to about 2.5 x 10 12 CFU / g, about 2.5 x 10 10 CFU / g to about 5 x 10 12 CFU / g, about 5 x 10 10 CFU / g to about 5 x 10 12 CFU / g, about 1 x 10 11 CFU / g to about 5 x 10 12 CFU / g, about 2.5 x 10 11 CFU / g to about 5 x 10 12 CFU / g, about 5 x 10 11 CFU / g to about 5 x 10 12 CFU / g, or about 1 x 10 12CFU / g ~ approx. 5 x 10 12 It has a viability of CFU / g.

[0060] According to some embodiments, the mixture of microorganisms, disaccharides, oligosaccharides, and antioxidants is in a ratio of about 10: 1 to the mixture of fatty alcohols. According to some embodiments, the ratio of the mixture of microorganisms, disaccharides, oligosaccharides, and antioxidants to the mixture of fatty alcohols is about 8: 1, about 9: 1, about 10: 1, or about 11: 1.

[0061] According to some embodiments, the composition further comprises colloidal oatmeal and / or alkaline oatmeal extract. According to some embodiments, the colloidal oatmeal and / or alkaline oatmeal extract is mixed with fatty alcohol. According to some embodiments, the colloidal oatmeal is about 0.1% (wt / wt), about 0.5% (wt / wt), about 1.0% (wt / wt), about 1.5% (wt / wt), or about 2.0% (wt / wt). According to some embodiments, the colloidal oatmeal is from about 0.1% (wt / wt) to about 2.0% (wt / wt), from about 0.1% (wt / wt) to about 1.5% (wt / wt), from about 0.1% (wt / wt) to about 1.0% (wt / wt), from about 0.1% (wt / wt) to about 0.5% (wt / wt), from 0.5% (wt / wt) to about 2.0% (wt / wt), from about 0.5% (wt / wt) to about 1.5% (wt / wt), from 0.5% (wt / wt) to about 1.0% (wt / wt), from about 1.0% to about 2.0% (wt / wt), from about 1.0% (wt / wt) to about 1.5% (wt / wt), or about 1.5% The colloidal oatmeal is at least 0.1% (wt / wt), at least 0.5% (wt / wt), at least 1.0% (wt / wt), at least 1.5% (wt / wt), or at least 2.0% (wt / wt). According to some embodiments, the colloidal oatmeal is no more than 0.1% (wt / wt), no more than 0.5% (wt / wt), no more than 1.0% (wt / wt), no more than 1.5% (wt / wt), or no more than 2.0% (wt / wt).

[0062] According to some embodiments, the alkaline oatmeal extract is about 0.1% (wt / wt), about 0.5% (wt / wt), about 1.0% (wt / wt), about 1.5% (wt / wt), or about 2.0% (wt / wt). According to some embodiments, the alkaline oatmeal extract is from about 0.1% (wt / wt) to about 2.0% (wt / wt), from about 0.1% (wt / wt) to about 1.5% (wt / wt), from about 0.1% (wt / wt) to about 1.0% (wt / wt), from about 0.1% (wt / wt) to about 0.5% (wt / wt), from 0.5% (wt / wt) to about 2.0% (wt / wt), from about 0.5% (wt / wt) to about 1.5% (wt / wt), from 0.5% (wt / wt) to about 1.0% (wt / wt), from about 1.0% to about 2.0% (wt / wt), from about 1.0% (wt / wt) to about 1.5% (wt / wt), or about 1.5% (wt / wt) to about 2.0% (wt / wt). According to some embodiments, the alkaline oatmeal extract is at least 0.1% (wt / wt), at least 0.5% (wt / wt), at least 1.0% (wt / wt), at least 1.5% (wt / wt), or at least 2.0% (wt / wt). According to some embodiments, the alkaline oatmeal extract is 0.1% (wt / wt) or less, 0.5% (wt / wt) or less, 1.0% (wt / wt) or less, 1.5% (wt / wt) or less, or 2.0% (wt / wt) or less.

[0063] According to some embodiments, the composition is prepared by combining disaccharides, oligosaccharides, antioxidants and live microorganisms in a first mixture and freeze-drying the mixture. According to some embodiments, the freeze-dried first mixture containing disaccharides, oligosaccharides, antioxidants and live microorganisms is combined with fatty alcohols, such as oleyl alcohol and cetyl alcohol. According to some embodiments, the fatty alcohols further comprise colloidal oatmeal and / or alkaline oatmeal extract.

[0064] According to some embodiments, the composition provides increased protein expression when compared to a composition that does not contain fatty alcohol and / or colloidal oatmeal and / or alkaline oatmeal extract. According to some embodiments, the increase in protein expression when compared to a composition that does not contain fatty alcohol and / or colloidal oatmeal and / or alkaline oatmeal extract is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, or about 15-fold.

[0065] According to some embodiments, the composition provides an increase in the protein activity of the expressed protein when compared to a composition that does not contain fatty alcohol and / or colloidal oatmeal and / or alkaline oatmeal extract. According to some embodiments, the increase in the protein activity of the expressed protein when compared to a composition that does not contain fatty alcohol and / or colloidal oatmeal and / or alkaline oatmeal extract is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, or about 15-fold.

[0066] According to some embodiments, a composition for use according to the invention comprises a pharma- ceutical effective amount of a recombinant bacterium producing a therapeutically effective amount of a desired polypeptide or a therapeutically effective domain thereof, 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 ... , about 6.0% by weight, about 7.0% by weight, about 8.0% by weight, about 9.0% by weight, about 10.0% by weight, about 11.0% by weight, about 12.0% by weight, about 13.0% by weight, about 14.0% by weight, about 15.0% by weight, about 16.0% by weight, about 17.0% by weight, about 18.0% by weight, about 19.0% by weight, about 20.0% by weight, about 25.0% by weight, about 30.0% by weight, about 35.0% by weight, about 40.0% by weight, about 45.0% by weight, about 50.0% by weight or more of the recombinant bacteria, with the upper limit being about 90.0% by weight of the recombinant bacteria.

[0067] According to some embodiments, a composition for use according to the invention may comprise, 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.

[0068] Microorganisms and microbial compositions The genetically altered protein-producing microorganisms can treat a disease or disorder by expressing and, if necessary, secreting a therapeutic protein that treats the underlying cause of the disease or disorder or its symptoms. According to some embodiments, the therapeutic protein is a LEKTI protein domain, filaggrin, interferon, or interleukin.

[0069] According to some embodiments, the therapeutic protein comprises one or more LEKTI domains that are effective in inhibiting serine proteases in or on the skin of a mammal. According to some embodiments, the recombinant LEKTI domains compensate for a deficiency in the endogenous LEKTI protein that is naturally produced in the mammal. According to some embodiments, the genetically altered bacteria can self-replicate while retaining the ability to produce the recombinant protein, thereby providing a continuous supply of the therapeutic agent.

[0070] According to some embodiments, the present disclosure provides a composition for a microbial product for the treatment of a disease or disorder comprising a microorganism genetically modified to express and deliver one or more LEKTI protein domains at a target site in a mammal, wherein the LEKTI protein domains are effective to penetrate one or more layers of the mammal's skin and are effective to inhibit serine protease activity of at least one serine protease in or on the mammal's skin.

[0071] As used herein, the term "auxotrophic" or "auxotrophy" refers to the inability of an organism to synthesize a particular compound required for its growth. An auxotroph is an organism that exhibits this characteristic.

[0072] 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 the D-alanine racemase protein (EC 5.1.1.1). In some embodiments, the locus identifiers SE1674 (alrl) and SE1079 (alr2) refer to specific S. epidermidis D-alanine racemase genes.

[0073] As used herein, the term "dat" refers to the D-alanine aminotransferase gene, including the normal allele of the dat gene. In some embodiments, the dat gene from S. epidermidis (UniProtKB-Q8CS41 (DAAA_STAES)) encodes the 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 the glutamate racemase gene, including the normal allele of the murI gene. In some embodiments, the murI gene from S. epidermidis (UniProtKB - Q8CPL0 (MURI_STAES)) encodes the glutamate racemase protein (EC:5.1.1.3). In some embodiments, the locus identifier SE0843 (murI) refers to the specific S. epidermidis glutamate racemase gene.

[0074] D-alanine auxotrophic strains of S. aureus were produced for the purpose of producing MRSA vaccines (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 necessary to knock out not only the alanine racemase alr1, but also the dat gene.

[0075] According to some embodiments, the present disclosure provides a recombinant Staphylococcus epidermidis strain that contains a DNA cassette in its chromosome that expresses a secreted form of domain 6 of the LEKTI protein of the human SPINK5 gene (hLEKTI-d6). The host strain carrying this expression cassette in its chromosome is S. epidermidis strain SE, a strain that contains a deletion of three genes involved in the biosynthesis of the essential amino acid, D-alanine [two alanine racemase genes (alr1 and alr2) and the D-alanine aminotransferase gene (dat)]. ΔΔΔ , making the strain auxotrophic for this amino acid.

[0076] According to some embodiments, the chromosomal hLEKTI-d6 construct strain is a S. epidermidis SE strain that expresses a secreted hLEKTI-d6 protein derived from a plasmid construct that also contains the alrA gene that complements D-alanine auxotrophy. ΔΔΔ It has seven distinct characteristics compared to another strain, S. epidermidis 27a, which is a strain of S. epidermidis. These characteristics include: 1) providing a measure of growth control using D-alanine supplementation; ΔΔΔ 2) enhanced levels of secreted hLEKTI-d6 protein possibly due to synergistic effects of the genetic environment at the chromosomal integration site, the combination of promoters driving hLEKTI-d6 gene expression (1;2), activation of transcription triggered by quorum-sensing mechanisms in dense cultures (3;4), and / or unique DNA or RNA structures associated with regulation of the delta-toxin gene, hld (4); and 3) potential enhanced stability of the expression cassette and reduced potential for horizontal transfer to commensal flora due to the nature of the expression cassette integrated into the chromosome versus being carried on an extrachromosomal genetic element (plasmid).

[0077] According to some embodiments, the present disclosure provides a microbial composition comprising one or more different bacteria suitable for use on mammalian skin. Examples include, but are not limited to, non-pathogenic bacteria and commensal bacteria. Suitable bacteria for use in the present invention include, but are not limited to, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus (e.g., S. epidermidis and / or S. hominis), Lactobacillus (e.g., L. acidophilus), Pediococcus, Leuconostoc, or Oenococcus. According to some embodiments, the microbial composition comprises one or more of Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes, Corynebacterium spp., Acinetobacter johnsonii, Pseudomonas aeruginosa. According to some embodiments, other related or similar species found on the skin are used.

[0078] Particular embodiments include the use of Staphylococcus epidermidis bacteria. According to some embodiments, the strain of S. epidermidis used is not capable of producing a biofilm. Examples of this are S. epidermidis strains ATCC 12228 or NRRL B-4268.

[0079] 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 continuous supply of the LEKTI protein domain. In some embodiments, the recombinant microorganism survives together with the commensal microorganisms naturally present on the mammalian skin. In some embodiments, the recombinant microorganism survives to the exclusion of the commensal microorganisms naturally present on the mammalian skin. According to some embodiments, the recombinant microorganism is adapted to grow on the mammalian skin. In other embodiments, the recombinant microorganism is no longer alive but contains an effective amount of a therapeutic polypeptide, e.g., LEKTI or a therapeutically effective domain thereof. Such cells may or may not be intact, depending on the details of delivering the therapeutic peptide (or domain thereof) to the target site.

[0080] As used herein, the term "recombinant" and grammatical variations thereof means relating to or referring to an organism, protein, or genetic material formed by or using recombinant DNA that includes pieces of DNA from different sources or from different parts of the same source. For example, the term "recombinant DNA" means a DNA molecule formed by recombinant methods to splice together pieces of DNA from different sources or from 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 means a protein molecule formed by recombinant methods that originates from spliced ​​fragments of DNA from different sources or from different parts of the same source. As another example, the term "recombinant microorganism" or "recombinant bacterium" and grammatical variations thereof means a microorganism / bacterium that includes one or more recombinant DNA / protein molecules.

[0081] LEKTI gene: According to some embodiments, the recombinant microorganism is engineered to express a mammalian gene encoding a LEKTI protein. The LEKTI gene can be obtained from any mammal, such as mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate, or human gene sequence. According to some embodiments, the LEKTI gene sequence is a human gene sequence. According to some embodiments, the recombinant microorganism is engineered to include a fragment of the LEKTI gene.

[0082] 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. The percent identity (%) is calculated by multiplying the number of matches in the sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps.

[0083] 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.

[0084] According to some embodiments, the LEKTI protein domain is effective in improving symptoms of Netherton syndrome. As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof mean to reduce the severity of symptoms of a disease in a subject. In some embodiments, the LEKTI protein domain acts as a competitive or non-competitive inhibitor of one or more proteases present in or on the skin of a mammal. In some 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.

[0085] According to some embodiments, the microorganism is genetically modified by transfection / transformation with a recombinant DNA plasmid encoding the LEKTI protein domain. Other conventional or to be discovered methods for introducing DNA into the microorganism can also be used in the present invention. According to some embodiments, the recombinant DNA plasmid comprises a sequence encoding the 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 that are effective for enhancing secretion from the microorganism and / or penetration through mammalian skin.

[0086] 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) that have been modified to alter, for example, the expression or activity of recombinant proteins. It is also known that the degeneracy of the nucleic acid code may be considered to alter the nucleotide sequence that codes for 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 that constitute moderately stringent hybridization conditions. It is also envisioned that orthologs of LEKTI exist in other species, for example, dogs, sheep, rats, hamsters, chickens, and pigs. Accordingly, another embodiment of the invention 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 set forth in SEQ ID NO: 103 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).

[0087] According to some embodiments, the LEKTI domain is selected from the non-limiting examples in Tables 1 and 2 below.

[0088] [Table 1] TIFF2024505253000002.tif55162

[0089] [Table 2] TIFF2024505253000004.tif17163

[0090] According to some embodiments, the disease or disorder is selected from skin diseases, diseases or disorders associated with pain, cancer, and viral infections. According to some embodiments, the skin disease is selected from the group consisting of pruritus, rosacea, psoriasis, atopic dermatitis, ichthyosis vulgaris, and Netherton syndrome. According to some embodiments, the disease or disorder associated with pain is selected from the group consisting of acute pain, chronic pain, nociceptive pain, neuropathic pain, traumatic pain, inflammatory pain, postoperative amputation pain, cancer-related pain, fracture pain, osteoporosis pain, osteosarcoma pain, and gouty joint pain. According to some embodiments, the cancer is selected from the group consisting of malignant melanoma, colon cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, oral tongue squamous cell carcinoma, squamous cell carcinoma, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, gallbladder cancer, brain tumor, and oral cancer. According to some embodiments, the viral infection is selected from the group consisting of a respiratory infection, a skin infection, and a viral infection that causes cancer in the subject.

[0091] Antimicrobial agent: As used herein, the terms "antimicrobial agent", "antimicrobial protein" or "antimicrobial polypeptide" can be used interchangeably and refer to any entity that has 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 greater than 70%, or any integer between 30% and 70% or greater, 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 greater 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 or activates or increases) a pathway that is specifically expressed in bacterial cells. In some embodiments, the antimicrobial agent is a polypeptide, i.e., a polypeptide that is expressed and secreted by the engineered microorganism.

[0092] The antimicrobial agent may include a chitinase, a glucanase, or a peptidoglycan hydrolase.

[0093] The term "chitinase" as used herein refers to an enzyme capable of catalyzing the hydrolysis of β-1,4 linked N-acetylglucosamine polymers that form chitin chains, a major component of fungal cell walls. Chitinases are expressed in plants in response to pathogens.

[0094] The term "glucanase" as used herein refers to an enzyme that can catalyze 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 may 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.

[0095] As used herein, the term "peptidoglycan hydrolase" refers to an enzyme that can degrade 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 the peptidoglycan chains and side chain branches.

[0096] The terms "infection" or "microbial infection", as used interchangeably herein, refer in their broadest sense to any infection caused by a microorganism, and include bacterial infections, fungal infections, yeast infections and protozoal infections.

[0097] Formulations: According to some embodiments, topical formulations may be in any form suitable for application to body surfaces, such as creams, lotions, sprays, solutions, gels, ointments, pastes, salves, paints, bioadhesives, suspensions, emulsions, and / or may be prepared to contain liposomes, micelles, and / or microspheres. Such formulations may be used in combination with a closed covering layer, such that moisture that evaporates from the body surface is maintained in the formulation during and after application to the body surface. According to some embodiments, the formulation may include a live cell culture composition, and may include at least one engineered bacterial strain that produces a therapeutically effective recombinant polypeptide or a therapeutically effective domain thereof. The engineered live cell culture composition may deliver the polypeptide directly to the skin to treat or prevent abnormal skin conditions.

[0098] Topical formulations include any other active ingredient dissolved or dispersed in a dermatological vehicle known in the art (e.g., aqueous or non-aqueous gels, ointments, water-in-oil or oil-in-water emulsions). Constituents of such vehicles may include water, aqueous buffer solutions, non-aqueous solvents (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). In particular, depending 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-cyclodextrins such as hydroxypropyl β-cyclodextrin, or alcohols or polyols such as ethanol, propylene glycol or glycerol); thickeners (e.g., hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose 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 buffers (such as mixtures of dihydrogen phosphate and hydrogen phosphate, or mixtures of citric acid and hydrogen phosphate).

[0099] Pharmaceutically acceptable carriers may be included in the formulations of the present invention, and may 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, silicon oils, nonionic surfactants, ionic surfactants, silicon surfactants, and water-based and emulsion-based mixtures of such carriers. The term "pharmaceutical acceptable" or "pharmaceutical acceptable carrier" as used herein refers to a compound or composition that can be included in a pharmaceutical formulation without causing undesirable biological effects or undesirable interactions with other components of the formulation, and "carrier" or "vehicle" as used herein refers to a carrier material 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 in a harmful manner with other components of the formulation in which they are included. The term "aqueous" refers to a formulation that contains water or becomes water-containing after application to skin or mucosal tissue.

[0100] The film former forms a protective film over the application site when it dries. This film inhibits the removal of the active ingredient and maintains its contact with the site being treated. An example of a film former suitable for use in the present invention is Flexible Collodion, US P., described in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, PA: Mack Publishing Co., 1995), page 1530, where collodion is an ethyl ether / ethanol solution containing pyroxylin (nitrocellulose) that evaporates to release a film of pyroxylin. The film former can also act as a carrier. Solutions that dry to form a film are sometimes called paints. Creams are viscous liquids or semi-solid emulsions of oil-in-water or water-in-oil, as is well known in the art of pharmaceutical formulations.

[0101] Cream bases are water washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the "internal" phase, typically contains petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase is usually, but not necessarily, outnumbered in volume by the oil phase and typically contains a moisturizer. The emulsifier in a cream formulation is typically a nonionic, anionic, cationic, or amphoteric surfactant.

[0102] Lotion is a preparation that is applied to the skin surface without friction, and is typically a liquid or semi-liquid preparation in which particles containing active agents are present in water or alcohol base.Lotion is usually a suspension of solids, and preferably comprises a liquid oily emulsion of oil-in-water type.Lotion is the preferred formulation herein for treating large body areas, because it is easy to apply more liquid composition.The insoluble material in lotion generally needs to be finely divided.

[0103] Lotions will typically contain suspending agents to provide better dispersion as well as compounds useful for localizing and holding the active agent in contact with the skin, such as methylcellulose, sodium ethoxymethyl-cellulose, and the like.

[0104] A solution is a homogeneous mixture prepared by dissolving one or more chemicals (solutes) in a liquid such that the molecules of the dissolved substance are dispersed among the molecules of the solvent. A solution may contain other pharma- ceutically or cosmetically acceptable chemicals to buffer, stabilize, or hold the solute. Common examples of solvents used in preparing solutions are ethanol, water, propylene glycol, or any other acceptable vehicle. Of course, as is well known, gels are semi-solid suspension-type systems. Single-phase gels contain organic polymers substantially uniformly distributed throughout the carrier liquid, which is typically aqueous, but preferably also contains alcohol, and optionally, oil. A preferred "organic polymer", i.e., gelling agent, is a crosslinked acrylic acid polymer 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 hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a homogeneous gel, dispersants such as alcohol or glycerin can be added, or the gelling agent can be dispersed by grinding, mechanical mixing or stirring, or a combination thereof. Ointments, as also well known in the art, are typically semi-solid preparations based on petrolatum or other petroleum derivatives. The particular ointment base used will provide some desirable characteristics, such as emollience, as will be understood by those skilled in the art. As with other carriers or vehicles, ointment bases should be inert, stable, non-irritating and non-sensitizing.As explained in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, PA: Mack Publishing Co., 1995), pages 1399-1404, ointment bases can be divided into four classes: fatty bases, emulsifiable bases, emulsion bases, and water-soluble bases. Fatty ointment bases include, for example, vegetable oils, fats obtained from animals, and semi-solid hydrocarbons obtained from petroleum.

[0105] 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.

[0106] Emulsion ointment bases are water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions and include, for example, acetyl alcohol, stearyl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weights; see Remington: The Science and Practice of Pharmacy for further information.

[0107] Pastes are semi-solid dosage forms in which active agents are suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from single-phase aqueous gels. The base in fatty pastes is generally petrolatum or hydrophilic petrolatum, etc. Pastes made from single-phase aqueous gels generally contain carboxymethylcellulose, etc. as base.

[0108] The enhancers are typically lipophilic co-enhancers, referred to as "plasticity" enhancers, i.e., enhancers having a molecular weight in the range of about 150-1000 and an aqueous solubility of less than about 1 wt%, preferably less than about 0.5 wt%, and most preferably less than about 0.2 wt%. The Hildebrand solubility parameter δ of the plasticity 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 glycol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol, while fatty ethers include diols or triols, preferably C 2 ~C 4 Included are compounds in which an alkanediol or triol is substituted with one or two fatty ether substituents.

[0109] Additional penetration enhancers will be known to those skilled in the art of topical drug delivery and / or are described in the relevant textbooks and literature, see, e.g., Percutaneous Penetration Enhancers, Smith et al. (eds.) (CRC Press, 1995), incorporated herein by reference.

[0110] In addition to those identified above, various other additives may be included in the compositions of the present invention, including, but not limited to, antioxidants, astringents, fragrances, preservatives, emollients, pigments, dyes, moisturizers, propellants, and sunscreens, as well as other classes of materials whose presence may be pharma- ceutical or otherwise desirable. Representative examples of optional additives for inclusion in the formulations of the invention are: 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; other polyhydroxy alcohols such as sorbitol and polyethylene glycol; sunscreens such as octyl methoxyl cinnamate (commercially available as Parsol MCX) and butyl methoxybenzoylmethane (available under the trademark Parsol 1789); ascorbic acid (vitamin C), a-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζ ι -Tocopherol, Z Λ-Antioxidants such as tocopherol, η-tocopherol, and retinol (vitamin A);essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g. soybean oil, coconut oil, 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;and thickening and structuring agents such as swelling clays and crosslinked carboxypolyalkylenes, which can be obtained commercially under the trademark Carbopol.Other additives include beneficial agents such as materials that condition the skin (especially the upper layer of the skin in the stratum corneum), keep it soft by retarding the loss of its moisture content, and / or protect the skin. Such conditioners and moisturizers include, for example, 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; antiseborrheic agents such as retinoic acid; vasodilators such as nicotinic acid; inhibitors of melanogenesis such as kojic acid; and mixtures thereof. Further additional active agents include, for example, alpha hydroxy acids, alpha keto acids, polymeric hydroxy acids, moisturizers, collagen, marine extracts, and ascorbic acid (vitamin C), a-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζ ι -Tocopherol, ζ 2Antioxidants such as -tocopherol, η-tocopherol, and retinol (vitamin A), and / or pharma-ceutical acceptable salts, esters, amides, or other derivatives thereof.Preferred tocopherol compound is a-tocopherol.Additional agents include those that can improve oxygen supply to skin tissue, for example, as described in WO 94 / 00098 to Gross et al. and WO 94 / 00109 to Gross et al., both assigned to Lancaster Group AG (herein incorporated 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, padimate O, phenylbenzimidazole sulfate, sulisobenzone, titanium dioxide, trolamine salicylate, zinc oxide, ensulizole, melazilate, octinoxate, octisalate, and octocrylene. See Title 21. Chapter 1. Subchapter D. Part 352. "Sunscreen drug products for over-the-counter human use," which is incorporated herein in its entirety.

[0111] Other embodiments may include various non-carcinogenic, non-irritating healing materials that facilitate treatment with the formulations of the present invention. Such healing materials may include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, glandular or animal extracts, or safe therapeutic agents that can be added to the formulation to facilitate healing of skin disorders.

[0112] The amounts of these various additives are those conventionally used in the cosmetic field and range, for example, from about 0.01% to about 20% of the total weight of the topical formulation.

[0113] The formulations of the present invention may also contain 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 yeasts and molds.

[0114] Suitable antimicrobial agents are typically selected from the group consisting of methyl and propyl esters of p-hydroxybenzoic acid (i.e., methyl and propyl parabens), sodium benzoate, sorbic acid, imidurea, and combinations thereof. In other embodiments, repressors and inducers, i.e., other agents for inhibiting (i.e., glucose) or inducing (i.e., xylose) the production of the polypeptide of interest, can also be added. Such additives can be used, provided they are compatible with and do not inhibit the function of the formulation.

[0115] The formulations may also contain mild additives to minimize or eliminate the possibility of skin irritation or damage resulting from the administered chemicals or other components of the composition.

[0116] Suitable irritation-relieving additives include, for example, a-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcohols such as 2-phenyl-1-ethanol; salicylates; ascorbates; ionophores such as monensin; amphoteric amines; ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine. When present, the irritation-relieving additive can be included in the composition at a concentration effective for relieving irritation or skin damage, and typically accounts for about 20 wt% or less of the formulation, more typically about 5 wt% or less.

[0117] Additional suitable pharmacologically active substances that can be included in the formulation in certain embodiments and thus applied topically along with the active agent include, but are not limited to, the following: agents that improve or eradicate pigmented or non-pigmented age spots, keratinocytes, and wrinkles; antimicrobial agents; antibacterial agents; anti-itch and anti-drying agents; anti-inflammatory agents; local anesthetics and analgesics; corticosteroids; retinoids; vitamins; hormones; and antimetabolites.

[0118] Some examples of topical pharmacologically active substances include acyclovir, amphotericin, chlorhexidine, clotrimazole, ketoconazole, econazole, miconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin, phenytoin, paraaminobenzoic acid esters, octyl methoxycinnamate, octyl salicylate, oxybenzone, dioxybenzone, tocopherol, tocopherol acetate, selenium sulfide, zinc pyrithione, diphenylhydramine, pramoxine, lidocaine, procaine, erythromycin, tetracycline, clindamycin, crotamiton, hydrochloride, benzoic acid ... These include quinones and their monomethyl and benzyl ethers, naproxen, ibuprofen, cromolyn, retinol, retinyl palmitate, retinyl acetate, coal tar, griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetate, hydrocortisone 17-valerate, hydrocortisone 17-butyrate, progesterone, betamethasone valerate, betamethasone dipropionate, triamcinolone acetonide, fluocinonide, clobetasol propionate, minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide, and 5-fluorouracil.

[0119] Creams, lotions, gels, ointments, pastes, etc. can be spread onto 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.

[0120] 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 will usually include one or more applications per day on a continuous basis.Those skilled in the art can easily determine the optimal amount of formulation to be administered, the method of administration and the repetition rate.In general, it is contemplated that the formulation of the present invention is applied in a range of once or twice a week to once or twice a day at most.

[0121] The pharmaceutical composition of the present invention comprises one or more active ingredients, e.g., therapeutic agents, in a mixture of one or more pharma- ceutical 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 invention are formulated into pharma- ceutical acceptable dosage forms by conventional methods known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st ed., Lippincott Williams and Wilkins, Philadelphia, Pa.).

[0122] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st ed., Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, DC)) and include 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, 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, and benzyl alcohol), and the like. Examples of suitable pharmacopoeitic diluents include glycerol, 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), paraffins, silicones, talc, salicylates, and the like. Each pharmacopoeitic diluent or carrier used in the pharmaceutical compositions of the present invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Suitable diluents or carriers 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 knowledge in the art.

[0123] The pharmaceutical compositions of the present invention may optionally contain additional ingredients and / or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include, for example, (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose, and acacia; (3) humectants such as glycerol; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethylcellulose, and sodium carbonate; (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 and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) ethoxylated isostearyl alcohols, such as glyceryl stearate, ... (11) suspending agents, such as polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth; (12) buffering agents, such as lactose, milk sugar, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, salicylates, zinc oxide, alumina hydroxide. excipients, such as aluminum, calcium silicates, and polyamide powder;(13); inert diluents, such as water or other solvents;(14) preservatives;(15) surfactants;(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, e.g., butane and propane; (24) antioxidants (antioxidants); (25) substances 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) sweetening, flavoring, coloring, fragrance and preservatives. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Suitable ingredients and materials for a selected dosage form and intended route of administration are well known in the art, and acceptable formulations and materials for a selected dosage form and method of administration can be determined using ordinary knowledge in the art.

[0124] The dosage form for topical or transdermal administration includes powder, spray, ointment, paste, cream, lotion, gel, solution, patch, drops and inhalant.Active agent / compound can be mixed with suitable pharmacopoeia acceptable diluent or carrier under aseptic condition.Ointment, paste, cream and gel can contain excipient.Powder and spray can contain excipient and propellant.

[0125] The pharmaceutical compositions of the present invention suitable for parenteral administration may contain one or more drugs / compounds together with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted immediately before use into a sterile injectable solution or dispersion, 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, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These pharmaceutical compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents, and dispersing agents. It is also desirable to include an isotonic agent. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by including substances that delay absorption.

[0126] The following examples are provided to further illustrate the method of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way. (Example)

[0127] The following examples are provided to further illustrate the method of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way. EXAMPLES

[0128] bacteria Each LEKTI-D6-expressing strain was derived from S. epidermidis SE transformed with a different derivative of pUBTR119 expressing hLEKTI-D6 from a different set of promoters. ΔΔΔ The plasmid constructs were made as described below.

[0129] The original pUBTR119 plasmid contains the Kan-R gene and a triple promoter expression cassette including 1) the hpaII native promoter essential for plasmid replication, 2) the phosphate starvation-inducible yxiE promoter, and 3) the constitutive sarA promoter with the open reading frame (ORF) of the SsaA1 secretion signal fused downstream to the protein to be expressed.

[0130] To generate plasmid construct #27a, expressing hLEKT1-D6 under the control of the inducible xylA promoter, PxylA, a set of overlapping primers designed for the Gibson assembly protocol, SEQ ID NO:1 and SEQ ID NO:2 for the vector, and SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 for the insert (Table 3), designed and synthesized by IDT, Inc. (Gibson et al., Nature Methods 6:343-345 (2009)), were used. The backbone of pUBTR (Km-, without the kanamycin resistance gene) plasmid containing the xylR / PxylA promoter was used. yfhKSP-pro-LEKTI-D6-6xHis in pRKK-Blue vector was used as a template to amplify the ORF of the recombinant protein. Q5® High Fidelity (HiFi) Polymerase (NEB, Inc., Ipswich, MA) was used for all PCR reactions. The PCR product was purified from a 0.7% agarose gel by freeze-thaw method and used in NEBuilder® HiFi DNA assembly reaction. The entire HiFi reaction (20 μl) was incubated with 220 μl of B. subtilis competent cells for 90 min at 37° C. and plated on Luria-Bertani (LB) agar plates for overnight incubation at 37° C. Plasmid DNA was purified in 4 ml overnight cultures in LB inoculated from the resulting B. subtilis colonies. Constructs were sequence verified from the xylR repressor ORF to the stop codon of the LEKTI-D6 ORF. Stocks were made in 20% glycerol from the sequence verified cultures and stored at −80° C. Plasmid DNA purified from B. subtilis was transformed into the S. epidermidis SE.ΔΔΔ The four SE ΔΔΔ Transformants were fully sequence verified and deposited at -80°C.

[0131] [Table 3]

[0132] Constructs SE27a and SE27d were made from DNA of SE27e to delete the XylR / PxylA cassette (SE27a) or the PyxiE promoter (SE27d) by PCR amplification with primers SEQ ID NO:7 and SEQ ID NO:4, or SEQ ID NO:6 and SEQ ID NO:8 (Table 3). Linearized construct DNA was gel purified and then recircularized by site-directed mutagenesis in a KLD reaction (containing a blend of kinase, ligase and DpnI enzymes, NEB, Inc.) and transformed into B. subtilis cells. Plasmid DNA prepared from B. subtilis clones was verified by sequencing and identified as a nucleotide sequence of the SE27e clone. ΔΔΔ The plasmids from the transformants were verified by PCR and sequencing of DNA preparations. All confirmed clones were stored in 20% glycerol at -80°C. A summary of the ORF structures in SE27a, SE27d, and SE27d is presented in Table 4.

[0133] [Table 4] TIFF2024505253000007.tif242170TIFF2024505253000008.tif242170TIFF2024505253000009.tif200170 EXAMPLES

[0134] Preparation of lyophilized powder of SE27a The SE27a strain was inoculated into two 500 mL cultures in 2 L baffled Erlenmeyer flasks. The cultures were grown for 24 h at 30 °C and 250 rpm. After incubation, the cultures were pooled and centrifuged at 10,000 x g for 10 min at 4 °C and the supernatant was discarded. The pooled pellet was thoroughly suspended in 80 mL of autoclaved cryoprotectant solution (120 g / L lactose, 25 g / L fructooligosaccharides and 1 g / L ascorbic acid) under magnetic stirring (Chen et al., 2019, Artif. Cells Nanomed. Biotechnol.). Two 100 μL aliquots of the homogenate were saved for seeding and CFU assessment. The remaining bacterial suspension was placed into a UV-sterilized lyophilization flask and frozen in an ethanol-dry ice bath under constant manual spinning. The frozen paste was lyophilized for 48 h, scraped under sterile conditions, and the resulting powder was crushed by hand in a stomacher bag. Three samples of the powder were taken, weighed, and suspended in 10 mL of autoclaved 0.1xTSB (tryptic soy broth medium) supplemented with 12.5% ​​Tween 80 (v / v). After suspension, the homogenate was serially diluted and plated for CFU assessment.

[0135] This method yielded a total of 14.5 g of powder containing approximately 34% dry bacterial load as calculated by subtracting the mass of cryoprotectant from the total powder recovered. Prior to lyophilization, the bacterial suspension in the cryoprotectant contained a total of 9.1x10 12 Contains CFU, of which 6.7x10 12 CFU were recovered after freeze-drying. Thus, this process resulted in a recovery of 74% of the initial input, with a final bacterial density in the powder of 1.4x10 12 CFU / g of powder. Table 5 summarizes the ingredients and percentages (w / w).

[0136] [Table 5] EXAMPLES

[0137] method Screening for growth in solution and LEKTI expression was performed by growing SE27a in liquid cultures of 30 g / L TSB medium, artificial sweat medium (ASM, 20.9 g / L MOPS, 2 g / L NaCl, 1 g / L yeast extract, 0.1 g / L Tween 80 and 0.65 mg / L cod liver fatty acid methyl ester), ASM + 1% colloidal oatmeal (wt / v) or ASM + 0.5% colloidal oatmeal extract (wt / v). SE27a powder prepared in Example 2 was used to inoculate 50 mL liquid cultures in 250 mL flasks at a cell density of approximately 108 CFU / mL and the cultures were grown at 30° C. and 250 rpm. At each time point, 100 μL of culture was taken, serially diluted in 10-fold steps using 1xPBS, and plated onto 30 g / L TSB, 15 g / L NaCl, and 15 g / L agar media for CFU assessment after 24 h of growth at 37° C. Additionally, 1 mL aliquots of culture were centrifuged at 12,000×g for 10 min at 4° C. for collection of supernatant. These spent media samples were recentrifuged twice and subjected to active LEKTI determination using the assay described below.

[0138] Active LEKTI concentrations were tested using the KLK14 inhibition assay. To activate the pro-KLK14 form for the assay, 3.5 μM of the protein was incubated in 50 mM Tris, 10 mM CaCl 2KLK14 was activated using 0.3 μM thermolysin in a solution containing 150 mM NaCl, 0.05% (wt / v) Brij-35, pH 8.0, at 37° C. for 1 h. Reactions were quenched with 50 mM EDTA and samples were diluted 1:1 (v / v) in 50 mM Tris, 150 mM NaCl, 0.05% (wt / v) Brij-35 and 10% glycerol, pH 8.0. Aliquots were frozen at −80° C. and used in their entirety in each assay to avoid freeze-thaw cycles. The amount of active LEKTI in broths and extracts was assayed in black 96-well plates using a solution of 50 mM Tris, 150 mM NaCl and 0.05% (wt / v) Brij-35, pH 8.0, containing 1.5 nM activated KLK14 and 30 μM Boc-VPR-AdMC. Reactions were performed in serial dilutions of media and extracts (10-0.08% of final well volume) and followed over 10 min at 1 min intervals at 23 °C in a plate reader (Ex / Em = 380 / 460 nm). Inhibition of KLK14-mediated cleavage of Boc-VPR-AdMC was determined as percent reaction ± inhibitor. The concentration of active LEKTI in samples was determined using a calibration curve of percent reaction in the presence of 0.0-5.0 nM purified recombinant His-tagged LEKTI-d6.

[0139] Evaluation of added nutrients in solution S. cerevisiae was cultured in a flask at a defined volume in artificial sweat medium ("ASM" - 20.9 g / L MOPS, 2 g / L NaCl, 1 g / L yeast extract, 0.1 g / L Tween 80, and 0.65 mg / L cod liver fatty acid methyl esters (described by Oates and McBain, Biofouling 32(1): 25-33 (2016)) (Figure 1A), tryptic soy broth (TSB) (Figure 1B), ASM + 1% colloidal oatmeal (CO) (Figure 1C), and ASM + 0.5% colloidal oatmeal extract (COE) (Figure 1D). Liquid cultures of D. epidermidis protein-expressing strain SE27a. Samples were taken at selected time points representing the treatment timeline. Bacterial counts were measured (CFU / ml) using a standard dilution protocol, and "active protein concentrations" were measured using an in vitro KLK14 inhibition assay for ASM (Figure 1E), TSB (Figure 1F), ASM + 1% colloidal oatmeal (CO) (Figure 1G), and ASM + 0.5% colloidal oatmeal extract (COE) (Figure 1H).

[0140] The TSB control solution grew (Figure 1B) and expressed LEKTI (Figure 1F) as expected. For ASM alone (Figure 1A), the solution contained 6.0x10 of SE27a. 7 The inoculum contained 100 CFU / ml. At 3 hours, the bacterial count was 6.3x10 7 CFU / ml, representing 10% growth. The active LEKTI concentration was below the detection limit (Figure 1E). A solution containing ASM + 1% colloidal oatmeal produced 5.6x10 7 CFU / ml of inoculum (Figure 1C). At 3 hours, the bacterial count was 6.3x10 7 CFU / ml, or 71% growth. The active LEKTI concentration was 6.5 nM (Figure 1G). For the solution of ASM + 0.5% colloidal oatmeal extract, the inoculum of SE27a was 5.8 x 10 7 CFU / ml (Figure 1D). At 3 hours, the bacterial count was 6.2x10 7 CFU / ml, representing 7% proliferation. The active LEKTI concentration was 215 nM (FIG. 1C).

[0141] The addition of colloidal oatmeal or colloidal oatmeal extract increases LEKTI expression from below detection limits to significant levels at 3 hours. These results indicate that the addition of oatmeal products is useful in ensuring expression. EXAMPLES

[0142] method Growth and LEKTI expression in the different agar preparations were evaluated by the following procedure. Powder of the SE27a strain was diluted with 10 11 Lyse the cells at a cell density of CFU / mL and plate 100 μL aliquots of these suspensions onto the following agar plates (78.5 cm) containing 15 g / L bacterial agar: 2 The plates were spread on approximately 100 mm (0.1 inch) of the following media: 30 g / L TSB, ASM medium, ASM + 1% colloidal oatmeal (wt / v), and ASM + 0.5% colloidal oatmeal extract (wt / v). For each medium, the plates were spread on approximately 100 mm (0.1 inch) of the following media: 30 g / L TSB, ASM medium, ASM + 1% colloidal oatmeal (wt / v), and ASM + 0.5% colloidal oatmeal extract (wt / v). 8 CFU / cm 2 Four plates per experiment were inoculated containing 100% ethanol and 100% ethanol. The plates were incubated at 30° C. for 8 h, at the end of which time the four plates of the same media condition were pooled and resuspended in 200 mL of sterile HO. 2 The homogenate was homogenized using a kitchen blender containing 0.1 mL of 100 μL of the homogenate was collected for inoculation and CFU assessment, while the remainder was centrifuged at 12,000 × g for 20 min at 4 °C. The pellet containing the agar was discarded and the supernatant was recentrifuged under the same conditions. The final liquid phase was frozen in a dry ice-ethanol bath and lyophilized for 48 h. The resulting dry material was lyophilized using Milli-Q 0.1 mL of ... 2 2. The mixture was dissolved in HO, acidified with 1% trifluoroacetic acid (TFA, v / v), injected into a preparative HPLC cartridge, and purified according to the gradient described in Table 6: 2 Fractionation was performed in a mobile phase containing (A) O+0.1% TFA (v / v) and (B) acetonitrile (ACN)+0.1% TFA (v / v) at a flow rate of 10.9 mL / min.

[0143] [Table 6]

[0144] Eluate fractions eluting between 20.2 and 30.2 min were collected, pooled, and dried completely in a SpeedVac. The resulting pellet was dissolved in 100 μL of 0.5 M Tris, 75 mM NaCl, and 0.03% (wt / v) Brij-35 (pH 8.0) and subjected to active LEKTI determination as described above.

[0145] Evaluation of nutrients added to agar Colloidal oatmeal (CO) and colloidal oatmeal extract (COE) were mixed in ASM agar plates, plated with bacteria, and incubated for 8 hours at 30°C. The amount of bacteria (CFU / cm) was measured at T0 and 8 hours (T8). 2 ) and expressed active LEKTI-d6 protein (KLK14 inhibition assay) are presented on Figure 2. Figure 3 shows the active LEKTI-d6 levels for each condition.

[0146] The results showed that there was little bacterial growth in the colloidal oatmeal or colloidal oatmeal extract conditions (Figure 2). In the presence of colloidal oatmeal and colloidal oatmeal extract, there were high levels of active LEKTI (Figure 3). In the presence of colloidal oatmeal, there were 3 ng / cm 2 In the presence of LEKTI and colloidal oatmeal extract, 2 existed. Example 4

[0147] method Oleyl alcohol:cetyl alcohol 1:1 ± 1% colloidal oatmeal (wt / wt) or 0.5% colloidal oatmeal extract (wt / wt) was prepared by incubating all ingredients at 60°C until the cetyl alcohol was completely melted. The mixture was shaken manually until cooled and solidified. The final formulation was added to a lyophilized powder of SE27a in a ratio of 10:1 (wt / wt) using a stomacher bag. Briefly, the bacterial powder was placed in a stomacher bag and gently crushed using a glass rod. The formulation was then added and the bacterial powder and formulation were homogenized with a glass rod using 25 up and down cycles of an outward rolling motion against the bag. An aliquot of approximately 100 mg of formulation was plated on an ASM plate (78.5 cm) containing 15 g / L bacterial agar. 2 (area) and spread it over about 10 8 CFU / cm 2 The plates were incubated at 30°C for 8 h, at the end of which time four plates of the same media condition were pooled and resuspended in 200 mL of sterile HO. 2 The homogenates were homogenized using a kitchen blender containing 0. A 100 μL aliquot of the homogenate was collected for plating and CFU assessment, while the remainder was centrifuged at 12,000 x g for 20 min at 4 °C. The pellet containing the agar was discarded and the supernatant was recentrifuged under the same conditions. The final liquid phase was frozen in a dry ice-ethanol bath and lyophilized for 48 h. The resulting dry material was dissolved in Milli-Q HO, acidified with 1% trifluoroacetic acid (TFA, v / v), fractionated by HPLC, dried, and assayed for active LEKIT levels as described above.

[0148] An alkaline extract of colloidal oatmeal was prepared as follows: A lot of colloidal oatmeal was obtained from a commercial source and dissolved at 3% wt / v in aqueous 50 mM NaOH under magnetic stirring. The suspension was then incubated without stirring for 48 h at room temperature. At the end of this time, the suspension was neutralized with 50 mM HCl and the supernatant was carefully collected to avoid recovery of decanted material. The supernatant was centrifuged twice at 12,000×g for 20 min at 4° C., frozen in a dry ice bath, and lyophilized for 72 h. The dried material was scraped off, weighed, and stored in a sealed stomacher bag at room temperature until use.

[0149] The colloidal oatmeal used as starting material and its corresponding alkaline extract were characterized using an iodine test to determine its starch content. Briefly, colloidal oatmeal and alkaline colloidal oatmeal extract were dissolved in 6 mL of MilliQ water at a concentration of 10 mg / mL. 0.2 mL of a 10 mg / mL ethanolic solution of iodine was added to the mixture. A color change to black indicated the presence of starch in the suspension. The colloidal oatmeal turned black (positive for starch) while the alkaline colloidal oatmeal extract was clear (negative for starch).

[0150] Evaluation of nutrients added to the formulation SE27a was formulated in oleyl and cetyl alcohol 1:1 (OA:CA), OA:CA with colloidal oatmeal (CO) and OA:CA with colloidal oatmeal extract (COE) and plated onto ASM agar plates and incubated for 8 hours at 30° C. Bacterial growth under these conditions is presented in FIG. 4 and LEKTI expression is presented in FIG. 5.

[0151] SE27a in oleyl alcohol and cetyl alcohol 1:1 did not grow or express active LEKTI. As shown in Figure 4, there was no significant bacterial growth in the presence of colloidal oatmeal or colloidal oatmeal extract. However, we observed that SE27a formulated in OA:CA with 1% colloidal oatmeal (CO) or 0.5% colloidal oatmeal extract (COE) had the beneficial effect of expressing active LEKTI on the surface (agar containing ASM), as shown in Figure 5. EXAMPLES

[0152] method 27a containing placebo and formulations of oleyl alcohol:cetyl alcohol 1:1 ± 1% colloidal oatmeal (wt / wt) were prepared as described under Example 4. Powder of 27a was mixed with the formulations in a ratio of 10:1 (wt / wt) using a stomacher bag as described under Example 4. Briefly, the bacterial powder was placed in a stomacher bag and gently crushed using a glass rod. Aliquots of approximately 100-150 mg of formulation were applied to defatted, shaved pig skin (64 cm) from a local butcher. 2 (area) and spread it over about 10 8 CFU / cm 2A bacterial density of 10 ... This fraction was centrifuged twice under the same conditions and the first supernatant was used for the measurement of active LEKTI as described under Example 3.

[0153] Bacterial recovery and LEKTI activity from formulated 27a applied to pig skin SE27a was formulated with colloidal oatmeal (CO) in oleyl and cetyl alcohol 1:1 (OA:CA) and applied onto pig skin followed by incubation for 8 hours at 30° C. The bacterial content under these conditions is presented in FIG. 6 and the LEKTI activity is presented in FIG. 7.

[0154] The bacterial content of formulated SE27a was stable over 24 h on pig skin, as shown in Figure 7. No LEKTI activity was observed from the sample applied to pig skin at T=0. As shown in Figure 7, LEKTI activity was 60 ng / cm in the swab sample from formulated 27a at T=8. 2 After reaching a sharp peak of 34ng / cm at T=24, 2 Consistent with the observations from Example 5, 27a bacteria formulated in OA:CA containing CO can produce active LEKTI on pig skin surfaces without substantial expansion of its CFU content. EXAMPLES

[0155] Auxotrophic strain growth and production of natural antimicrobial agents Strain SE484 is a D-alanine Staphylococcus epidermidis auxotroph that produces and secretes antimicrobial compounds. In skin-like conditions, i.e., in artificial sweat medium (ASM), the growth of this strain is reduced (Figure 8A) and it is unable to produce and secrete antimicrobial agents (Figure 8B). Upon addition of colloidal oatmeal, SE484 shows a 10-fold increase in CFU / mL within 8 h (Figure 8A). Furthermore, spent medium of SE484 grown in medium supplemented with colloidal oatmeal shows significant antibacterial activity against Bacillus subtilis 168QC compared to plain ASM medium. EXAMPLES

[0156] Bacterial survival and active LEKTI production by formulated SE351 on ex vivo porcine skin SE351 is a D-alanine auxotroph of S. epidermidis carrying a chromosomally integrated construct expressing domain 6 of the protease inhibitor LEKTI. The strain was formulated in oleyl alcohol:cetyl alcohol 1:1 (w / w) supplemented with 1% colloidal oatmeal (w / w) and 2% D-alanine (w / w) and incubated for 10 min at 4°C. 6 (Figure 9A), 10 7 (Figure 9B) and 10 8 CFU / cm 2 When applied ex vivo on pig skin (Figure 9C), it increased CFU / cm within the first 6 h. 2 Over 24 h, bacterial populations increased by approximately 10 at all cell densities. 6 CFU / cm 2 (Figures 9A to 9C). 6 CFU / cm 2 Detectable levels of active LEKTI were observed from 10 to 24 h, with levels at approximately 200 ng / cm at 24 h. 2 gradually increases to a peak of

[0157] 10 7CFU / cm 2 In the present study, active LEKTI levels showed an initial peak at 6–8 h, with levels of 150 ng / cm 2 These levels were 100 ng / cm at 10–14 h (Figure 9B). 2 Although the levels dropped to 250 ng / cm within 20–24 h, 2 A similar trend was observed at 10 8 CFU / cm 2 (Fig. 9C), and LEKTI activity was increased to 150 ng / cm within 3 h. 2 It first peaked at 300 ng / cm, then declined and spiked at 24 h to 300 ng / cm 2 Despite the different kinetics of the active LEKTI levels present at these cell densities, SE351 formulated in an ointment containing colloidal oatmeal is able to deliver substantial levels of active recombinant LEKTI onto the skin surface. EXAMPLES

[0158] Preparation of lyophilized powders of auxotrophic strains SE351 and SE484 The SE351 and SE484 strains were inoculated into 500 mL of TSB+2% D-alanine (w / v) medium in a 2 L baffled Erlenmeyer flask. Cultures were grown at 37°C and 250 rpm for 6 h for SE351 and 16 h for SE484. After incubation, cultures were pooled and centrifuged at 10,000xg for 10 min at 4°C and the supernatant was discarded. The pooled pellet was thoroughly suspended in 80 mL of autoclaved cryoprotectant solution (120 g / L lactose, 25 g / L fructooligosaccharides and 1 g / L ascorbic acid) under magnetic stirring. Two 100 μL aliquots of the homogenate were saved for inoculation and CFU assessment. The remaining bacterial suspension was placed in a UV-sterilized lyophilization flask and frozen in an ethanol-dry ice bath under constant manual spinning. The frozen paste was lyophilized for 48 h, scraped under sterile conditions, and the resulting powder was crushed by hand in a stomacher bag. Three samples of powder were taken, weighed, and suspended in 10 mL of autoclaved 0.1xTSB supplemented with 12.5% ​​Tween 80 (v / v). After suspension, the homogenate was serially diluted and plated for CFU assessment. This method yielded a total of 8 g of powder per L of culture containing approximately 34% dry bacterial load, as calculated by subtracting the mass of cryoprotectant from the total powder recovered. The CFU / g content ranged from 1.0 to 2.5x10 11 Changed in CFU / g. EXAMPLES

[0159] Elucidation of auxotrophic growth and production of natural antimicrobial agents The screening was carried out by growing SE484 in liquid cultures of artificial sweat medium (ASM, 20.9 g / L MOPS, 2 g / L NaCl, 1 g / L yeast extract, 0.1 g / L Tween 80 and 0.65 mg / L cod liver fatty acid methyl esters) supplemented with 2% D-alanine (w / v) ± 1% colloidal oatmeal (w / v). 8A cell density of 1000 CFU / mL was used to inoculate 50 mL liquid cultures in 250 mL flasks and the cultures were grown at 30°C and 250 rpm. At 24 h, 100 μL of culture was taken and serially diluted in 10-fold steps using 1xPBS and plated on 30 g / L TSB, 15 g / L NaCl and 15 g / L agar media for CFU assessment after 24 h of growth at 37°C. Additionally, 1 mL aliquots of culture were centrifuged at 12,000xg for 10 min at 4°C for collection of supernatant. These spent media samples were placed in 96-well microplates and serially diluted in TSB media using 2-fold steps. Serial dilutions of spent broth were plated at 2x10 5 CFU / mL of B. subtilis 168QC was mixed with a 1:1 v / v bacterial suspension in TSB and the microplates were incubated at 37°C for 16 h. Plates were visually inspected for turbidity, indicating bacterial growth. Wells in which the medium was translucent were considered to contain high antibacterial activity, and the lowest concentration of spent broth, i.e., the highest dilution, that produced such an effect was considered the minimum inhibitory concentration (MIC) of the sample. EXAMPLES

[0160] Preparation of SE351 Formulated Product The placebo formulation contained oleyl alcohol:cetyl alcohol 1:1 (w / w) supplemented with 1% colloidal oatmeal (w / w) and 2% D-alanine (w / w) and was prepared by incubating all ingredients at 60°C until the cetyl alcohol was completely melted. The mixture was shaken manually until cooled and solidified. The final placebo formulation was added to SE351 lyophilized powder (prepared as in Example 8) in a ratio of 20:1 (w / w) using a stomacher bag. Briefly, the bacterial powder was placed in a stomacher bag and gently crushed using a glass rod. The formulation was then added and the bacterial powder and formulation were homogenized with a glass rod using 25 up-and-down cycles of an outward rolling motion against the bag. This process resulted in 10 10 CFU / g of the preparation was obtained. 1010 CFU / g of formulation was mixed with fresh placebo in a ratio of 1:10 and 1:100 w / w, respectively. 9 and 10 8 A formulation with a lower titer of CFU / g was obtained. Mixing was carried out in a stomacher bag with the aid of a glass rod as described above. EXAMPLES

[0161] Bacterial survival and active LEKTI production by formulated SE351 on ex vivo porcine skin Freshly prepared, shaved and degreased pig skin was obtained from a local butcher and used within 24 h of preparation. For each experiment, large pieces of pig skin were cut into three 36 cm 2 Each piece was divided into rectangular samples of approximately 10 mg / cm 2 The pigs were treated with placebo or SE351 formulations at 10 °C. Briefly, the formulations were applied to the skin and pressed with a gloved finger. The applied formulation was then rubbed 15 times in a horizontal motion to form a uniform thin film of ointment on the skin. The weight of the pig skin and the retained formulation was determined. Application of SE351 ointment resulted in a 10% reduction in the ointment concentration. 6 ~10 8 CFU / cm 2 The skin pieces were then incubated at 30°C and incubated for 4 h at 4 cm 2 The area was scrubbed with a polyester-tipped flocked swab using 10 strokes in a vertical orientation and 10 strokes in a horizontal orientation. For each treatment (placebo and SE351), two sets of swabs were collected per time point: CFU / cm 2 The set used for determination of was processed immediately after collection, whereas the set collected for protein extract preparation was placed in Precellys homogenization tubes and stored at -20°C until use.

[0162] CFU / cm 2For the determination of CFU, swabs collected from formulation-treated pig skin were immediately transferred to 15 mL Falcon tubes where they were treated with 0.5 mL of a sterile solution of 3 g / L TSB and 1% Triton X-100 (w / v). The tubes were vortexed for 1 min and aliquots of the homogenate were serially diluted in PBS using 10-fold increments. The diluted samples were plated on TSB agar plates supplemented with 15 g / L NaCl and grown at 37° C. for 24 h. CFU content was assessed and normalized by the swab area.

[0163] Protein extracts prepared for active LEKTI measurements were prepared from a second set of swabs derived from pig skin by an extensive cleaning procedure to remove the matrix interface in biochemical measurements. Briefly, the swabs in Precellys homogenization tubes were treated with 1 mL of homogenization solution (90% methanol v / v and 0.5% formic acid v / v). The tubes were capped and homogenized in a bead beater at 2,500 rpm for 30 s. The swabs were removed from the tubes and the homogenates were centrifuged at 12,000 x g for 5 min at 20 °C. The mixture supernatant was collected and placed in a SpeedVac for 3-4 h at room temperature until complete dryness. The dried pellet was resuspended in 250 μL of homogenization solution, sonicated for 1 min, and treated with 750 μL of 8 M guanidine-HCl solution. After mixing by inversion, 250 μL of pure hexane was added to each tube followed by 5 manual inversions. The tubes were centrifuged at 12,000×g for 5 min at 20° C. and the upper phase containing residual formulation components and the interface were discarded. The lower phase was transferred to a 3 kDa Amicon concentrator and washed six times with 500 μL of 50 mM Tris pH 8.0 according to the manufacturer's specifications. The final retentate was removed from the filter by a reverse spin with 250 μL of aqueous 50% acetonitrile (v / v), 0.025% Brij-35 (w / v) and 0.25% formic acid (v / v). The suspended retentate was placed in a SpeedVac for 24 h at room temperature and the final pellet was reconstituted in 50 μL of 0.25 M Tris pH 8.0. These samples were used for titration of KLK14 inhibitory activity and total trypsin-like activity as described in Example 2. Incorporation by Reference

[0164] The entire disclosure of each patent document, including patent application documents, scientific papers, government reports, websites, and other references described herein, is incorporated herein by reference in its entirety for all purposes.In case of conflict in terms, the present specification controls.All sequence listings or sequence numbers disclosed herein are incorporated herein in their entirety.

[0165] 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.

[0166] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention 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 invention.

Claims

1. 1. A composition comprising one or more live microorganisms, a first mixture, and a second mixture, wherein the one or more microorganisms produce a therapeutic agent, the first mixture comprising one or more disaccharides, one or more oligosaccharides, and one or more antioxidants, and the one or more microorganisms are at least 1×10 10 CFU / g viability, The composition, wherein the second mixture comprises 1) one or more fatty alcohols, and 2) colloidal oatmeal and / or alkaline oatmeal extract.

2. 2. The composition of claim 1, wherein the one or more disaccharides are selected from the group consisting of lactose, trehalose, sucrose, maltose, and cellobiose.

3. 3. The composition of claim 1, wherein the one or more oligosaccharides are selected from the group consisting of fructo-oligosaccharides (FOS), galactooligosaccharides (GOS), mannanoligosaccharides (MOS), isomalto-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides, and raffinose.

4. 4. The composition of claim 1, wherein the one or more antioxidants are selected from the group consisting of ascorbic acid, vitamin A, vitamin E, α-carotene, lycopene, lutein, zeaxanthin, and water-soluble derivatives of lipophilic antioxidants.

5. 5. The composition of claim 4, wherein the water-soluble derivative of a lipophilic antioxidant is selected from alpha-tocopherol phosphate or alpha-tocopherol polyethylene glycol ester.

6. 2. The composition of claim 1, wherein the one or more disaccharides are lactose, the one or more oligosaccharides are fructooligosaccharides (FOS), and the one or more antioxidants are ascorbic acid.

7. The composition according to any one of claims 1 to 6, wherein the one or more fatty alcohols are selected from the group consisting of saturated fatty alcohols and unsaturated fatty alcohols.

8. 8. The composition of claim 7, wherein the saturated fatty or alcoholic acid is cetyl alcohol and the unsaturated fatty alcohol is oleyl alcohol.

9. 9. The composition of claim 8, wherein the mixture of oleyl alcohol and cetyl alcohol ranges from about 25% oleyl alcohol / 75% cetyl alcohol to about 50% oleyl alcohol / 50% cetyl alcohol (w / w).

10. 9. The composition of claim 8, wherein the second mixture comprises oleyl alcohol and cetyl alcohol in a ratio of about 1:

1.

11. 11. The composition of any one of claims 1 to 10, wherein the one or more disaccharides are present in an amount of about 50% (w / w) to about 55% (w / w), the one or more oligosaccharides are present in an amount of about 10% (w / w) to about 15% (w / w), the one or more antioxidants are present in an amount of about 0.1% (w / w) to about 2% (w / w), and the one or more microorganisms are present in an amount of about 30% (w / w) to about 35% (w / w).

12. 12. The composition of any one of claims 1 to 11, wherein the composition of the microorganism and the first mixture is in a ratio of about 10:1, and the second mixture comprises one or more fatty alcohols.

13. 13. The composition of claim 12, wherein the second mixture comprises about 1% (wt / wt) colloidal oatmeal and about 0.5% (wt / wt) alkaline oatmeal extract when combined with a fatty alcohol.

14. 14. The composition of any one of claims 1 to 13, wherein the microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, Oenococcus, or Corynebacterium, and mixtures thereof.

15. 15. The composition of claim 14, wherein the microorganism is Staphylococcus epidermidis.

16. The composition of any one of claims 1 to 15, wherein the therapeutic agent is naturally produced by a microorganism.

17. The composition of any one of claims 1 to 15, wherein the microorganism is genetically engineered to produce a therapeutic agent.

18. The composition of any one of claims 1 to 17, wherein the therapeutic agent is selected from a polypeptide, a small molecule, and a metabolite.

19. The composition of any one of claims 1 to 18, wherein the therapeutic agent is selected from the group consisting of one or more LEKTI protein domains, filaggrin, interferons, enkephalins, interleukins, and antimicrobial agents.

20. The composition of claim 19, wherein the one or more LEKTI protein domains are LEKTI-d6.

21. 20. The composition of claim 19, wherein the one or more antimicrobial agents are selected from the group consisting of chitinase, glucanase, or peptidoglycan hydrolase.

22. The composition of any one of claims 1 to 21, wherein the microorganism is attenuated by auxotrophy.

23. 23. The composition of claim 22, wherein the microorganism is a D-alanine auxotroph.

24. 24. The composition of claim 23, wherein the microorganism comprises a deletion of one or more of the alr1, alr2, and dat genes.

25. The composition according to any one of claims 1 to 24, wherein the composition is administered to the skin of a mammal.

26. 26. The composition of any one of claims 1 to 25, wherein expression of the therapeutic agent is increased when compared to a composition not comprising the second mixture.

27. 27. The composition of any one of claims 1-26, wherein expression of the therapeutic agent is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, or at least 13-fold greater after 8 hours compared to a composition without the second mixture.

28. 28. The composition of claim 27, wherein expression of the therapeutic agent is at least 5-fold greater after 8 hours when compared to a composition without the second mixture.

29. 28. The composition of claim 27, wherein expression of the therapeutic agent is at least 10-fold greater after 8 hours when compared to a composition without the second mixture.

30. The one or more microorganisms are at least 2.5 x 10 10 CFU / g, at least 5x10 10 CFU / g, at least 1x10 11 CFU / g, at least 2.5x10 11 CFU / g, at least 5x10 11 CFU / g, at least 1x10 12 CFU / g, or at least 2.5x10 12 30. The composition of any one of claims 1 to 29, having a viability of CFU / g.

31. The one or more microorganisms are about 1 x 10 10 CFU / g ~ approx. 1x10 11 CFU / g, approximately 5x10 10 CFU / g ~ approx. 5x10 11 CFU / g, approximately 1x10 11 CFU / g ~ approx. 1x10 12 CFU / g, or approximately 5x10 11 CFU / g ~ approx. 5x10 12 31. The composition of any one of claims 1 to 30, having a viability of CFU / g.

32. 22. The composition of claim 19 or 21, wherein the activity of the antimicrobial agent is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times greater after 8 hours when compared to a composition without the second mixture.

33. 22. The composition of claim 19 or 21, wherein the activity of the antimicrobial agent is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, or at least 12 times greater after 24 hours when compared to a composition without the second mixture.

34. 21. The composition of claim 19 or 20, wherein the LEKTI protein domain activity is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold greater after 8 hours compared to a composition not containing the second mixture.

35. 21. The composition of claim 19 or 20, wherein the LEKTI protein domain activity is about 1.5 to about 12 times greater after 24 hours compared to a composition not containing the second mixture.

36. A pharmaceutical composition comprising any one of the compositions of claims 1 to 35 and a pharmaceutically acceptable carrier.

37. 37. The pharmaceutical composition of claim 36, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, or an ointment.

38. A method for preparing a composition according to any one of claims 1 to 35 or a pharmaceutical composition according to claim 36 or 37, comprising: (a) combining the one or more microorganisms with the first mixture comprising the one or more disaccharides, the one or more oligosaccharides, and the one or more antioxidants; (b) lyophilizing the composition resulting from step (a); and (c) combining the freeze-dried composition resulting from step (b) with the second mixture comprising 1) one or more fatty alcohols, and 2) colloidal oatmeal and / or alkaline oatmeal extract; The method.

39. A composition according to any one of claims 1 to 35, or a pharmaceutical composition according to claim 36 or 37, for treating a disease, disorder or condition in a subject.

40. 40. The composition or pharmaceutical composition of claim 39, wherein the disease, disorder, or condition is a skin disease or disorder, an inflammatory disease, or a cancer.

41. 41. The composition or pharmaceutical composition of claim 40, wherein the skin disease or disorder is Netherton syndrome, psoriasis, acne, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, seborrheic dermatitis, eczema, dry skin, allergies, rash, UV-irritated skin, detergent-irritated skin, skin thinning, bullous pemphigoid, pemphigus vulgaris, impetigo, vitiligo, alopecia, and / or hypertrichosis.

42. 40. The composition or pharmaceutical composition of claim 39, wherein the disease or disorder is associated with pain and is selected from the group consisting of acute pain, chronic pain, nociceptive pain, neuropathic pain, traumatic pain, inflammatory pain, post-operative amputation pain, pain associated with cancer, bone fracture pain, osteoporosis pain, osteosarcoma pain, and gouty joint pain.

43. 41. The composition or pharmaceutical composition of claim 40, wherein the cancer is selected from the group consisting of malignant melanoma, colon cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, oral tongue squamous cell carcinoma, squamous cell carcinoma, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, gallbladder cancer, brain tumor and oral cancer.

44. 1. A method for increasing expression of a therapeutic agent by a microorganism, comprising: (a) combining the one or more microorganisms with a first mixture comprising the one or more disaccharides, the one or more oligosaccharides, and the one or more antioxidants; (b) lyophilizing the composition resulting from step (a); and (c) combining the freeze-dried composition resulting from step (b) with the second mixture comprising 1) one or more fatty alcohols, and 2) colloidal oatmeal and / or alkaline oatmeal extract.

38. The method of claim 37, comprising preparing a composition according to any one of claims 1 to 35, or a pharmaceutical composition according to claim 36 or 37, comprising:

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