EXPRESSION OF THE HUMAN LEKTI GENE FROM THE CHROMOSOMES OF STAPHYLOCOCCUS EPIDERMIDIS

Abstract

The present disclosure provides, inter alia, engineered microbes that express one or more therapeutic proteins from chromosomally integrated nucleotide sequences, which are effective for treating or ameliorating diseases or disorders, such as skin diseases. In certain embodiments, compositions, methods and kits are provided that include a microbe that expresses one or more therapeutic proteins from chromosomally integrated nucleotide sequences. According to one aspect, the present disclosure provides a recombinant microbe that can secrete one or more therapeutic polypeptides.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 305,458, filed February 1, 2022, the entire contents of which are incorporated herein by reference.

[0002] Field of the Disclosure The present disclosure relates to methods, kits and compositions for treating or ameliorating the effects of a disease or disorder, e.g., a skin disease, using one or more recombinant microorganisms that have been genetically modified to express one or more therapeutic polypeptides, e.g., LEKTI domains, in the skin of a subject. [Background technology]

[0003] 2. Background of the Invention Proteases or proteolytic enzymes are essential in organisms, from bacteria and viruses to mammals. Proteases digest and break down proteins by hydrolyzing peptide bonds. Serine proteases (EC.3.4.21) share common characteristics in the active site, mainly the active serine residue. There are two main types of serine proteases; chymotrypsin / trypsin / elastase-like and subtilisin-like, which have identical spatial arrangements of catalytic His, Asp and Ser, but in completely different protein scaffolds. More than 20 families of serine proteases (S1-S27) have been identified, grouped into six clans, SA, SB, SC, SE, SF and SG, based on structural similarities and other functional evidence. The family of chymotrypsin / trypsin / elastase-like serine proteases has been subdivided into two classes: The "large" class (ca230 residues) includes primarily mammalian enzymes such as trypsin, chymotrypsin, elastase, kallikrein, and thrombin. The "small" class (ca190 residues) includes bacterial enzymes. Examples of serine proteases include trypsin, tryptase, chymotrypsin, elastase, thrombin, plasmin, kallikrein, complement Cl, acrosomal proteases, lysosomal proteases, cocoonase, a-lytic proteases, protease A, protease B, serine carboxypeptidase, subtilisin, urokinase (uPA), Vila factor, factor IXa, and factor Xa. Serine proteases have been extensively investigated for many years and are a major focus of research as drug targets due to their role in controlling a wide variety of physiological processes.

[0004] One division of the family of serine protease inhibitors is that of the Kazal-type (SPINK) genes, including SPINK1, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINK13 and SPINK14. Lymphoid epithelial Kazal-type inhibitor (LEKTI) is encoded by SPINK5 (serine proteinase inhibitor Kazal-type 5) (Magert et al. (1999) J Biol. Chem. 274; 21499-21502). The SPINK5 gene is located on chromosome 5q32 in a cluster of other SPINK genes (e.g., SPINK1, SPINK6, SPINK7, SPINK9 and SPINK13) and contains 33 exons that code for 15 inhibitory domains separated by linker regions. SPINK5 has been shown to be expressed in skin, oral mucosa, tonsils, parathyroid gland, thymus and lung (Magert et al., Int J Biochem Cell Biol. 2002;34(6):573-6; Magert et al., Eur J Med Res. 2002;7(2):49-56).

[0005] SPINK5 stands out among other SPINK genes for the large number of inhibitory domains it encodes. The SPINK5 gene is transcribed into three different transcripts, giving rise to three different LEKTI proteins that differ in their C-terminal regions: a 145 kDa full-length protein with inhibitory domains D1-D15, a 125 kDa (short) protein with inhibitory domains D1-D12, and a 148 kDa (long) protein with an extended linker region 13. LEKTI is expressed as a high molecular weight precursor that is rapidly processed into several proteolytic fragments that are secreted into the intercellular space (Bitoun et al. (2003) Hum. Mol. Genet. 12:2417-2430). The Kazal motif of LEKTI is defined by the presence of six cysteine ​​residues positioned at specific distances that allow the formation of three disulfide bonds in the 1-5, 2-4, and 3-6 patterns. Two of the domains of LEKTI (D2 and D5) form this six cysteine ​​motif, while the other domain shares four cysteine ​​residues, producing a tight inhibitory loop that is thought to mimic the substrate of the target protease and inactivate the target protease catalytic site. The LEKTI protein requires proteolytic cleavage for activation of its inhibitory function against a number of proteases. The full-length protein is cleaved into domains D1-D5 and D6-D15. The D6-D15 domain is then further cleaved in multiple steps to D6-D9 and D10-D15, →D6 and D7-D9 →D7 and D8-D9 →D8. This process results in LEKTI proteins that contain between one and six inhibitory domains, with each protein having a different protease target. For example, it has been shown that LEKTI fragments can efficiently and specifically inhibit epithelial kallikrein (KLK)5, KLK7 and KLK14 (DeRaison et al. (2007) Mol. Biol. Cell. 18:3607-3619).

[0006] There are currently some limitations in diseases, such as skin diseases or disorders. Many treatments, such as topical corticosteroids or biologics, do not treat the underlying problem, such as the lack of endogenous proteins in the epidermis or the imbalance in microbial diversity in the skin. While recombinant proteins represent a promising group of therapeutic agents in the treatment of skin diseases, some problems are associated with their use in skin conditions.

[0007] Traditional methods purify and concentrate recombinant proteins extracted from bacterial systems, and then incorporate such preparations into delivery systems. Often, purification of recombinant proteins is a very expensive method to obtain proteins. Furthermore, several problems accompany these traditional methods, including proteolytic degradation, inefficient delivery, and the need for repeated application over time to achieve therapeutic effects. In view of the foregoing, there is a need for new therapeutic agents for the treatment of diseases or disorders in subjects. This application is directed to meeting these and other needs. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Magert et al. (1999) J Biol. Chem. 274; 21499 -21502 [Non-Patent Document 2] Magert et al., Int J Biochem Cell Biol. 2002;34(6):573-6;Magert et al., Eur J Med Res. 2002;7(2):49-56 [Non-Patent Document 3] Bitoun et al. (2003) Hum. Mol. Genet. 12:2417-2430 [Non-Patent Document 4] DeRaison et al. (2007) Mol. Biol. Cell. 18:3607-3619 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention According to one aspect, the disclosure provides a recombinant microorganism capable of secreting one or more therapeutic polypeptides, the recombinant microorganism comprising: (i) a first coding sequence comprising a nucleic acid sequence encoding one or more therapeutic polypeptides; (ii) a second coding sequence comprising a nucleic acid sequence encoding one or more secretory sequences that are not essentially related to the one or more therapeutic polypeptides; and (iii) a third coding sequence comprising a propeptide, wherein the first coding sequence, the second coding sequence and the third coding sequence are in frame, and the one or more therapeutic polypeptides are encoded by one or more SPINK genes or one or more protein domains thereof.

[0010] According to some embodiments, the first coding sequence, the second coding sequence and the third coding sequence are integrated into a chromosome.

[0011] According to some embodiments, the first coding sequence, the second coding sequence and the third coding sequence are chromosomally integrated at the hld gene.

[0012] According to some embodiments, the recombinant microorganism is attenuated by an auxotrophy.

[0013] According to some embodiments, the recombinant microorganism is a D-alanine auxotroph.

[0014] According to some embodiments, expression of the first coding sequence, the second coding sequence and the third coding sequence are operably linked to a promoter.

[0015] According to some embodiments, the promoter is P3.

[0016] According to some embodiments, the recombinant microorganism comprises a first coding sequence, a second coding sequence, and a third coding sequence, the expression of which is regulated by a promoter P yxiE According to some embodiments, the P yxiE comprises SEQ ID NO:120.

[0017] In some embodiments, the secretory peptide is derived from the Bacillus subtilis yfhK gene. In some embodiments, the secretory peptide comprises SEQ ID NO:122.

[0018] According to some embodiments, the propeptide comprises SEQ ID NO:123.

[0019] In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the microorganism is selected from the group consisting of Moraxella, Corynebacteria, Pasteurella, Haemophilus, Streptococcus, or Staphylococcus. In some embodiments, the recombinant microorganism is Staphylococcus epidermidis.

[0020] According to some embodiments, the one or more SPINK genes are selected from the group consisting of SPINK1, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINK13 and SPINK14. According to some embodiments, the one or more SPINK genes are SPINK5. According to some embodiments, the one or more SPINK genes encode a LEKTI protein or one or more protein domains thereof.

[0021] According to some embodiments, the recombinant microorganism secretes one or more therapeutic polypeptides.

[0022] According to some embodiments, the LEKTI protein domain is selected from the group consisting of D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 and D15.

[0023] According to some embodiments, the LEKTI protein domain is D6.

[0024] According to another aspect, the disclosure provides a method for producing a live bacterial biological therapeutic composition, comprising the steps of: (i) transfecting a cell with a nucleic acid sequence comprising one or more LEKTI protein domains, (ii) a secretory peptide essentially unrelated to one or more therapeutic polypeptides, (iii) a propeptide, and (iv) a promoter; and integrating the nucleic acid sequence into a chromosome of the cell; and obtaining a live bacterial biological therapeutic.

[0025] In some embodiments, the secretory peptide is selected from the Bacillus subtilis yfhK gene. In some embodiments, the secretory peptide comprises SEQ ID NO:122.

[0026] According to some embodiments, the propeptide comprises SEQ ID NO:123.

[0027] According to some embodiments, the nucleic acid sequence is integrated into the hld gene.

[0028] According to some embodiments, the promoter is P yxiE According to some embodiments, P yxiE comprises SEQ ID NO:120.

[0029] According to some embodiments, the nucleic acid sequence is under the control of an endogenous promoter.

[0030] According to some embodiments, the endogenous promoter is P3.

[0031] According to some embodiments, the arrangement of the LEKTI protein domain, the secretory peptide and the propeptide is in-frame.

[0032] In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the recombinant microorganism is selected from the group consisting of Moraxella, Corynebacteria, Pasteurella, Haemophilus, Streptococcus, or Staphylococcus. In some embodiments, the recombinant microorganism is Staphylococcus epidermidis.

[0033] According to some embodiments, the recombinant microorganism secretes one or more LEKTI protein domains or variants thereof.

[0034] According to another aspect, the present disclosure provides a composition obtained by any one of the methods disclosed herein.

[0035] According to some embodiments, the composition comprises a pharma- ceutically acceptable carrier, the pharma- ceutically acceptable carrier being selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, and an ointment.

[0036] According to another aspect, the disclosure provides a live bacterial biological therapy composition comprising a recombinant microorganism, wherein the recombinant microorganism comprises a nucleic acid sequence comprising: (i) one or more LEKTI protein domains, (ii) one or more secretory peptides, (iii) a propeptide, and (iv) a promoter, wherein the recombinant microorganism is capable of secreting the one or more LEKTI protein domains or variants thereof.

[0037] According to some embodiments, the nucleic acid sequence is integrated into a chromosome of the cell.

[0038] According to some embodiments, the nucleic acid sequence is integrated into the hld gene.

[0039] In some embodiments, the secretory peptide is derived from the Bacillus subtilis yfhK gene. In some embodiments, the secretory peptide comprises SEQ ID NO:122.

[0040] According to some embodiments, the propeptide comprises SEQ ID NO:123.

[0041] According to some embodiments, the promoter is P yxiE According to some embodiments, P yxiE comprises SEQ ID NO:120.

[0042] According to some embodiments, one or more LEKTI protein domains, one or more secretory peptides and propeptides are under the control of an endogenous promoter.

[0043] According to some embodiments, the endogenous promoter is P3.

[0044] According to some embodiments, the arrangement of one or more LEKTI protein domains, one or more secretory peptides and propeptides is in-frame.

[0045] In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the recombinant microorganism is selected from the group consisting of Moraxella, Corynebacteria, Pasteurella, Haemophilus, Streptococcus, or Staphylococcus. In some embodiments, the recombinant microorganism is Staphylococcus epidermidis.

[0046] According to some embodiments, the composition comprises a pharma- ceutically acceptable carrier, wherein the pharma- ceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, and an ointment.

[0047] According to another aspect, the present disclosure provides a kit comprising any one of the compositions disclosed herein and instructions for use.

[0048] According to another aspect, the present disclosure provides a method of treating a disease or disorder comprising administering to a subject in need thereof any one of the compositions disclosed herein.

[0049] According to some embodiments, the disease or disorder is selected from a skin disease, a pain-related disease or disorder, cancer, and a viral infection.

[0050] According to some embodiments, the skin disease is selected from the group consisting of pruritus, rosacea, psoriasis, atopic dermatitis, ichthyosis vulgaris, and Netherton syndrome.

[0051] According to some embodiments, the pain-related disease or disorder is selected from the group consisting of acute pain, chronic pain, nociceptive pain, neuropathic pain, traumatic pain, inflammatory pain, post-operative incision pain, cancer-related pain, fracture pain, osteoporotic pain, bone cancer pain and gouty joint pain.

[0052] 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 cancer, and oral cancer.

[0053] 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. [Brief description of the drawings]

[0054] [Figure 1A] FIG. 1A shows the chromosomal hLEKTI-d6 expression cassette.

[0055] [Figure 1B]FIG. 1B shows the plasmid pLEKTI-27a-d6 in S. epidermidis strain 27a.

[0056] [Diagram 2] FIG. 2 shows Western blot characterization of the expression of hLEKTI-d6 from culture supernatants of S. epidermidis chromosomal hLEKTI-d6 strains.

[0057] [Figure 3A] Figure 3A shows a bar graph measuring active LEKTI expressed from an episomal plasmid compared to chromosomal expression. Values ​​are expressed as the average of technical replicates (%CV≦10%).

[0058] [Figure 3B] Figure 3B shows a bar graph measuring active LEKTI per CFU expressed from an episomal plasmid compared to chromosomal expression. Values ​​are expressed as the average of technical replicates (%CV≦10%).

[0059] [Figure 4A] FIG. 4A shows a graph comparing the expression of LEKTI during logarithmic and stationary growth phases of S. epidermidis strain 27a.

[0060] [Figure 4B] FIG. 4B shows a graph comparing the expression of LEKTI during logarithmic and stationary growth phases of strain SE352.

[0061] [Figure 4C] FIG. 4C shows a graph comparing LEKTI expression during logarithmic and stationary growth phase of strain SE355. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Detailed Description of the Invention One aspect of the disclosure provides skin-colonizing bacteria, e.g., Staphylococcus epidermidis, that are genetically modified to express a recombinant protein integrated into their chromosome to treat or ameliorate skin disease. The bacteria producing the genetically modified protein can treat skin disease by expressing and optionally secreting a therapeutic protein that treats the underlying cause of the skin disease or its symptoms. In some embodiments, the therapeutic protein comprises one or more LEKTI domains that are effective to inhibit serine proteases in or on the skin of a mammal. In some embodiments, the recombinant LEKTI domain compensates for a missing endogenous LEKTI protein that is naturally produced by the skin in a mammal. In some embodiments, the genetically modified bacteria can self-replicate while retaining the ability to produce the recombinant protein, thereby providing a continuous supply of the therapeutic agent.

[0063] According to some embodiments, the present disclosure provides a composition for the treatment of skin diseases comprising a microbe genetically modified to express and provide one or more LEKTI protein domains in mammalian skin, where the LEKTI protein domains are effective to penetrate one or more layers of the mammalian skin and are effective to inhibit serine protease activity of at least one serine protease on or in the mammalian skin.

[0064] In some embodiments, the present disclosure provides a recombinant Staphylococcus epidermidis strain that contains in its chromosome a DNA cassette 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 SE. ΔΔΔstrain, which is defective in 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)], and the strain is auxotrophic for this amino acid.

[0065] In some embodiments, the S. epidermidis S. serovar expresses the secreted hLEKTI-d6 protein from a plasmid construct that also contains the alrA gene, which complements D-alanine auxotrophy. ΔΔΔ Compared to another strain, S. epidermidis 27a, the chromosomal hLEKTI-d6 construct strain possesses several distinguishing characteristics. These characteristics include: 1) the SE ΔΔΔ 2) unexpected enhanced production of secreted hLEKTI-d6 protein, possibly influenced by the genetic environment at the chromosomal site of integration. Enhanced levels of secreted hLEKTI-d6 protein may be due to synergistic effects of promoter combinations driving hLEKTI-d6 gene expression (1;2), activation of transcription triggered by quorum sensing during high-density culture (3;4), and / or unique DNA or RNA structures associated with the regulation of the delta-toxin gene, hld (4); and 3) potentially enhanced stability of the expression cassette due to its chromosomally integrated nature versus being carried on an extrachromosomal genetic element (plasmid), and reduced likelihood of lateral spread to commensal bacterial flora.

[0066] As used herein, the term "skin disease" and its grammatical variations refer to a skin condition or state that is generally undesirable or harmful compared to the normal or baseline state of human skin.Examples of abnormal skin conditions include, but are not limited to, Netherton syndrome, psoriasis, acne, atopic dermatitis, allergic contact dermatitis, epidermolytic keratosis, seborrheic dermatitis, eczema, dry skin, allergies, rashes, UV-irritated skin, detergent-irritated skin (including the irritation caused by enzymes and molecules used in detergents and sodium lauryl sulfate), skin thinning (e.g., elderly and pediatric skin), bullous pemphigoid, pemphigus vulgaris, impetigo, vitiligo, alopecia and hirsutism.

[0067] As used herein, the term "genetically modified" and grammatical variations thereof are used to describe a microbial organism (e.g., a bacterium) that has been genetically modified or engineered by the introduction of DNA prepared outside the microbe. For example, the introduction of plasmid DNA containing novel genes into the bacterium will enable the bacterium to express these genes. Alternatively, DNA containing novel genes may be introduced into the bacterium and then integrated into the bacterial genome, where the bacterium will express these genes.

[0068] 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 natural state. Thus, "recombinant bacterial cell" or "recombinant bacterium" refers to a bacterial cell or bacterium that has been genetically modified from their natural state. For example, recombinant bacterial cells may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacterium or bacterial cell. Recombinant bacterial cells may contain exogenous nucleotide sequences stably integrated into their chromosome.

[0069] As used herein, the terms "treat", "treating", "treatment" and grammatical variations thereof refer to providing a subject with a protocol, regimen, process or therapeutic agent that is desired to obtain a physiological response or result in a subject, e.g., a patient. In particular, the methods and compositions of the present invention can be used to slow the development of disease symptoms, or to delay the onset of a disease or condition, or to stop the progression of disease development. However, treating does not require that a desired physiological response or result be achieved in each and every subject or subject population, e.g., a patient population, since all subjects treated may not respond to a particular treatment protocol, regimen, process or therapeutic agent. Thus, a given subject or subject population, e.g., a patient population, may not respond to treatment or may respond inappropriately.

[0070] In the present invention, the subject may be a mammal. As used herein, "mammal" and its grammatical variations refer to any category of mammals. In the present invention, mammals include, for example, humans, livestock, farm animals, laboratory animals, etc. Some examples of livestock include cows, pigs, horses, goats, etc. Some examples of farm animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. Preferably, the mammal is a human.

[0071] 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 the beneficial or desired results described herein when administered to a subject. Effective dosage forms, modes of administration and dosages may be empirically determined, and making such determinations is within the ability of one of ordinary skill in the art. It is understood by those skilled in the art that dosages vary according to 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 factors well known in the medical and veterinary fields. In general, a suitable dose of a composition according to the present invention is the 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 two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[0072] Microbial composition: In some embodiments, the present disclosure provides a microbial composition that includes one or more of a wide variety of bacteria suitable for use on mammalian skin. Examples include, but are not limited to, non-pathogenic 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. In some embodiments, the microbial composition comprises one or more of Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes, Corynebacterium spp., Acinetobacter johnsonii, Pseudomonas aeruginosa. In some embodiments, other related or similar species found on the skin are used.

[0073] Certain embodiments include the use of the bacterium Staphylococcus epidermidis. In some embodiments, the strain of S. epidermidis used is not capable of producing a biofilm. Examples of this are the S. epidermidis strains ATCC 12228 or NRRL B-4268.

[0074] In some embodiments, the recombinant microbes are adapted to survive indefinitely or for a controlled duration on the surface of the mammalian skin to provide a sustained supply of the LEKTI protein domain. In some embodiments, the recombinant microbes survive with symbiotic microbes naturally present on the mammalian skin. In some embodiments, the recombinant microbes survive to eliminate symbiotic microbes naturally present on the mammalian skin. In some embodiments, the recombinant microbes are adapted to grow on the mammalian skin. In other embodiments, the recombinant microbes are no longer alive but contain an effective amount of a therapeutic polypeptide, e.g., LEKTI or a therapeutically effective domain(s) thereof. Such cells may be intact or not relying on the feature of delivering the therapeutic peptide (or domain(s) thereof) to a target site.

[0075] As used herein, the term "recombinant" and grammatical variations thereof refer to or describe an organism, protein, or genetic material formed by or using recombinant DNA that contains pieces of DNA from different sources or from different parts of the same source. For example, the term "recombinant DNA" refers to a DNA molecule formed through recombinant methods that splice together pieces of DNA from different sources or from different parts of the same source. In some embodiments, DNA from two or more different sources is cut using a restriction enzyme and joined together using a ligase. As another example, the term "recombinant protein" or "recombinant domain" and grammatical variations thereof refer to a protein molecule formed through recombinant methods originating from spliced ​​fragments of DNA from different sources or from different parts of the same source. As another example, the term "recombinant microbe" or "recombinant bacterium" and grammatical variations thereof refer to a microbe / bacterium that contains one or more recombinant DNA / protein molecules.

[0076] LEKTI gene: In some embodiments, the recombinant microbe is engineered to express a mammalian gene encoding a LEKTI protein. The LEKTI gene can be obtained from any mammal, for example, mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate, or human gene sequence. In some embodiments, the LEKTI gene sequence is a human gene sequence. In some embodiments, the recombinant microbe is engineered to include a fragment of the LEKTI gene.

[0077] In some embodiments, the recombinant protein expressed by the engineered microbe 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. In some embodiments, the recombinant protein expressed by the engineered microbe comprises LEKTI inhibitor domain 6 or domains D8 to D11.

[0078] In some embodiments, the recombinant protein expressed by the engineered microbe comprises LEKTI D8-11. In some embodiments, the recombinant protein expressed by the engineered microbe comprises LEKTI-D6. In some embodiments, one or more fragments of LEKTI are expressed by the engineered microbe. In one embodiment, the fragment comprises one or more LEKTI domains. In a particular embodiment, the LEKTI domain is domain 6.

[0079] The present disclosure also relates to allelic variants of LEKTI or parts 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 mutated genes of SPINK (e.g., SPINK5) modified to, for example, alter recombinant protein expression or activity. It is also noted that the degeneracy of the nucleic acid code can be considered as a variation in 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, dog, sheep, rat, hamster, chicken and pig. Accordingly, another embodiment of the invention relates to a SPINK (e.g., SPINK5) nucleic acid encoding a polypeptide having at least about 70% to 80% identity, preferably 90% to 95% identity, and more preferably 98% to 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).

[0080] According to some embodiments, the LEKTI domain is selected from the non-limiting examples, Tables 1 and 2 below. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0081] In some embodiments, the recombinant microbe 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. Percentage (%) 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.

[0082] In some embodiments, the recombinant protein expressed by the engineered microbe 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. In some embodiments, the recombinant protein expressed by the engineered microbe comprises LEKTI inhibitor domain 6 or domains D8 to D11.

[0083] In some embodiments, the LEKTI protein domain is effective for ameliorating symptoms of Netherton syndrome. As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof refer to reducing the severity of symptoms of 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 on or in mammalian skin. In some embodiments, the LEKTI protein domain acts as a serine protease inhibitor. As used herein, the terms "protease" and "proteinase" are used interchangeably, with both terms referring to enzymes that perform protein degradation.

[0084] In some embodiments, the microbe is genetically modified by transfection / transformation with a recombinant DNA plasmid encoding a LEKTI protein domain. Other conventional or discovered methods for introducing DNA into the microbe may also be used in the present invention. In some embodiments, the recombinant DNA plasmid comprises a sequence encoding a LEKTI protein domain and one or more secretory and / or cell-penetrating peptides. In some embodiments, the LEKTI domain is operably linked to one or more recombinant protein domains that are effective for enhancing secretion from the microbe and / or penetration through mammalian skin.

[0085] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is controlled by or not hindered by the other. For example, a promoter is operably linked to a coding sequence if it can control the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a control sequence in a sense or antisense orientation. In another example, two proteins can be operably linked such that the function of either protein is not impaired. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to connect two protein coding regions, contiguous and in the same reading frame.

[0086] As used herein, the term "secretory peptide" or "secretory sequence" or "secretion tag" or "signal peptide" or "export signal" and grammatical variations thereof refer to any peptide sequence capable of targeting a synthesized protein to the secretory pathway of a cell. In some embodiments, the secretory peptide may be positioned at the N-terminus of a recombinant protein and can co-translationally or post-translationally target the tagged protein for secretion. In some embodiments, at least one LEKTI domain is operably linked to a SecA domain (SEQ ID NO: 3).

[0087] Secretory peptide: In some embodiments, the therapeutic LEKTI domain is operably linked to one or more secretory or export signals that tag the protein for transport through the secretory pathway. Any secretory signal that facilitates export of the LEKTI protein out of the bacterial cell can be used as a secretory peptide. Non-limiting examples of secretory peptide signals are listed in Table 1 below: [Table 3]

[0088] In some embodiments, the therapeutic LEKTI domain is operably linked to one or more signal sequences from endogenous proteins of Staphylococcus epidermidis. Non-limiting examples of secretory signal peptides from endogenous proteins of Staphylococcus epidermidis are listed in Table 2 below: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0089] In some embodiments, the therapeutic LEKTI domain is operably linked to one or more secretory signal sequences from endogenous proteins of other bacteria. Non-limiting examples of secretory signal peptides from endogenous proteins of various bacteria are listed in Appendix A.

[0090] According to some embodiments, the recombinant LEKTI domain is operably linked to a cell-penetrating peptide sequence that enhances the ability of the LEKTI domain to cross the cell membrane. When used to describe a cell-penetrating peptide / LEKTI, the term "enhances" means that the cell-penetrating sequence improves the passage of the recombinant LEKTI domain through the cell membrane as compared to the recombinant LEKTI domain lacking the cell-penetrating sequence.

[0091] Cell-penetrating peptides: According to some embodiments, peptides that penetrate one or more cells are used to mediate the delivery of therapeutic proteins in vivo without using cell surface receptors and without causing significant membrane damage. According to some embodiments, peptides that penetrate one or more cells are operably linked to a therapeutic protein to facilitate entry into skin cells (e.g., keratinocytes). Non-limiting examples are described in Table 3 below:

Table 5

[0092] According to some embodiments, the peptide that penetrates cells contains a periodic amino acid sequence. Non-limiting examples of periodic cell-penetrating sequences include: polyarginine, Rxn (where 4 < n < 17); polylysine, Kxn (where 4 < n < 17); arginine repeats containing 6-aminocaproic acid residues (RAca) in between, where there are 2 to 6 arginine repeats; arginine repeats containing 4-aminobutyric acid (RAbu) in between, where there are 2 to 6 arginine repeats; arginine repeats containing methionine in between, where there are 2 to 6 arginine repeats; arginine repeats containing threonine in between, where there are 2 to 6 arginine repeats; arginine repeats containing serine in between, where there are 2 to 6 arginine repeats; and arginine repeats containing alanine in between, where there are 2 to 6 arginine repeats.

[0093] In some embodiments, the expression of the LEKTI domain is regulated by an operon, and the amount of LEKTI delivered to the skin of a mammal is proportional to the availability of an exogenous factor. For example, in some embodiments, the recombinant LEKTI gene may be under the control of a xylose-inducible promoter (e.g., a xylose repressor (xylR), a xylose operator (xylO), a xylose isomerase gene (xylA) containing a cis-acting catabolite response element (CRE)), and the amount of recombinant LEKTI protein available to the skin of a mammal is regulated by the amount of exogenous xylose available to the recombinant microbe. In some embodiments, the expression of the LEKTI domain is regulated by a constitutively active promoter. In some embodiments, the expression of the LEKTI domain is regulated by a CmR promoter according to SEQ ID NO:8.

[0094] In some embodiments, the microbe is genetically modified by chromosomal integration of a DNA plasmid encoding a LEKTI protein domain. In some embodiments, the DNA plasmid integrated into the bacterial chromosome comprises one or more sequences of a pJB38 vector. In some embodiments, the recombinant LEKTI protein is operably linked to an inducible promoter, a ribosome binding site, an export signal, and / or a cell-permeable peptide in the pJB38 vector. In some embodiments, the recombinant LEKTI protein is fused in frame to a secretory peptide and a synthetic propeptide. In some embodiments, the secretory peptide is a yfhK secretory peptide from Bacillus subtilis. In some embodiments, the secretory peptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:78. In some embodiments, the secretory peptide comprises or consists of SEQ ID NO:78.

[0095] According to some embodiments, the DNA plasmid encoding the recombinant LEKTI protein fused in-frame to the secretory peptide and propeptide is integrated into the bacterial chromosome. In some embodiments, the DNA plasmid encoding the recombinant LEKTI protein fused in-frame to the secretory peptide and propeptide is integrated into the hld gene (delta toxin gene). In some embodiments, the integrated nucleotide sequence encoding the LEKTI protein fused in-frame to the secretory peptide and propeptide is operably linked to one or more promoters. In some embodiments, the one or more promoters are endogenous, e.g., derived from the bacterial chromosome. In some embodiments, the one or more promoters are P yxiE and P3. yxiE The promoter is located immediately upstream of the hld gene, and the P3 promoter is part of the adjacent accessory gene control (agr) operon. In some such embodiments, the microbe is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus (e.g., S. epidermidis and / or S. hominis), Lactobacillus (e.g., L. acidophilus), Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof.

[0096] According to one aspect, the disclosure provides a method of treating or ameliorating the effects of a skin disease in a mammal in need thereof, comprising providing to the surface of the mammalian skin a microbe genetically modified to express one or more LEKTI protein domains, wherein the LEKTI protein domains are effective to penetrate one or more layers of the mammalian skin and are effective to inhibit the activity of at least one serine protease in or on the mammalian skin. According to some embodiments, the microbe is adapted to survive on the surface of the mammalian skin for a controlled duration and provide a continuous supply of the LEKTI protein domains.

[0097] According to another aspect, the disclosure provides: (1) a composition comprising a microbe genetically modified to express one or more LEKTI protein domains, the LEKTI protein domains being effective to penetrate one or more layers of a mammalian skin and inhibiting serine protease activity of at least one serine protease in or on the mammalian skin; and (2) a kit for treating or ameliorating the effects of a skin disease in a mammal in need thereof, the kit comprising a reagent for applying the composition to the mammalian skin. According to some embodiments, the microbe is adapted to survive for a controlled duration on the surface of the mammalian skin and provide a continuous supply of the LEKTI protein domains.

[0098] In addition to the above components, the subject kit further comprises instructions for using the components and / or for carrying out the subject method. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, in the kit's packaging or in a package insert. Yet another means is a computer readable medium, such as a diskette, or a CD on which the information is recorded. Yet another means in which the instructions may be present is a website address that is used via the Internet to access the information at a remote location. Any convenient means may be present in the kit.

[0099] The components of the kit may be packaged either in aqueous media or in lyophilized form. The kit is generally packaged to include at least one vial, test tube, flask, bottle, syringe or other container means containing the described reagents, preferably appropriately aliquoted. If additional components are provided, the kit will also generally include a second, third or other additional container into which such components can be placed.

[0100] Kits of the present disclosure will also typically include a means for containing the reagent containers in close confinement for commercial sale. Such containers may include syringes or blow-molded plastic containers into which the desired vials are retained. formulation

[0101] According to some embodiments, a formulation for use according to the present invention comprises any pharma- ceutical effective amount of the recombinant bacteria, e.g., at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 21.0%, about 22.0%, about 23.0%, about 24.0%, about 25.0%, about 26.0%, about 27.0%, about 28.0%, about 29.0%, about 30.0%, about 31.0%, about 32.0%, about 33.0%, about 34.0%, about 35.0%, about 36.0%, about 37.0%, about 38.0%, about 39.0%, about 40.0%, about 41.0%, about 42.0%, about 43.0%, about 44.0%, about 45.0%, about 46.0%, about 47.0%, about 48.0%, about 49.0%, about 50.0%, about 51.0%, about 52.0%, about 53.0%, about 54.0%, about 55.0%, about 56.0%, about 57.0%, about 58.0%, about The composition may contain about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% or more by weight of recombinant bacteria, up to an upper limit of about 90.0% by weight of recombinant bacteria.

[0102] According to some embodiments, a formulation 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 bacteria.

[0103] According to some embodiments, the topical formulation may be in any form suitable for application to a body surface, such as a cream, lotion, spray, solution, gel, ointment, paste, cataplasm, paint, bioadhesive, suspension, emulsion, etc., and / or may be prepared to contain liposomes, micelles, and / or microspheres. Such formulations may be used in combination with an occlusive covering layer, whereby moisture that evaporates from the body surface is retained within the formulation during and after application to the body surface. According to some embodiments, the formulation may include a live cell culture composition, which may include at least one engineered bacterial strain that produces a therapeutically effective recombinant polypeptide or a therapeutically effective domain(s) thereof. This engineered live cell culture composition may deliver the polypeptide directly to the skin to treat or prevent abnormal skin conditions.

[0104] The topical formulations include formulations in which any other active ingredient(s) are 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).The components of such vehicles include water, aqueous buffers, non-aqueous solvents (e.g., ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycofurol or glycerol), oils (e.g., mineral oils, e.g., liquid paraffin, natural or synthetic triglycerides, e.g., Miglyol™, or silicone oils, e.g., 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 components (e.g., components added to water if the formulation is an aqueous gel) selected from the following list: solubilizers or solvents (e.g., β-cyclodextrin, e.g., bydroxypropyl β-cyclodextrin, or alcohols or polyols, e.g., ethanol, propylene glycol or glycerol); thickeners (e.g., hydroxyethylceliulose, hydroxypropylcellulose, carboxymethylcellulose or carbomer); gelling agents (e.g., polyoxyethylene-polyoxypropylene copolymers); preservatives (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorobutol, benzoic acid, potassium sorbate or EDTA or a salt thereof); and pH buffer(s) (e.g., a mixture of dihydrogen phosphate and hydrogen phosphate, or a mixture of citric acid and hydrogen phosphate).

[0105] Pharmaceutically acceptable carriers may also be incorporated into the formulations of the present invention and may be any carrier conventionally used in the art. Examples of these 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 "pharmaceutically acceptable" or "pharmaceutically acceptable carrier" is used herein to refer to a compound or composition that can be incorporated into a pharmaceutical formulation without causing undesirable biological effects or undesirable interactions with other components of the formulation. As used herein, "carrier" or "vehicle" refers to a carrier material that is suitable for incorporation in a composition that is applied topically. Carriers and vehicles useful herein include any such materials known in the art that are non-toxic and do not interact in a deleterious manner with other components of the formulation in which they are contained. The term "aqueous" refers to a formulation that contains water or becomes water-containing after application to skin or mucosal tissue.

[0106] When the film former dries, it forms a protective film that covers the site of application. The film prevents the active ingredient from being removed and keeps it in contact with the site to be treated. An example of a film former suitable for use in the present invention is Flexible Collodion USP. As described in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, PA: Mack Publishing Co., 1995), at page 1530, collodion is an ethyl ether / ethanol solution containing pyroxylin (nitrocellulose) that evaporates leaving a film of pyroxylin. The film former can additionally act as a carrier. Solutions that dry to form a film are sometimes referred to as paints. Creams are viscous liquids or semi-fluid emulsions, either oil-in-water or water-in-oil, as is well known in the art of pharmaceutical formulations.

[0107] Cream bases are water-washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the "internal" phase, is generally composed of mineral oil and a fatty alcohol, such as cetyl or stearyl alcohol. The aqueous phase usually, but not necessarily, exceeds the oil phase in volume and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic, or amphoteric surfactant.

[0108] Lotion is a preparation that is applied to the skin surface without friction, and is typically a liquid or semi-fluid preparation, in which particles containing active agent are present in water or alcohol base.Lotion is usually a suspension of solids, and preferably contains a liquid oily emulsion of oil-in-water type.Lotion is the preferred formulation herein for treating large body surface, because it is easy to apply the more fluid composition.The insoluble matter in lotion generally needs to be granulated.

[0109] Lotions typically contain suspending agents to produce better dispersion, as well as compounds that are useful for localizing and maintaining the active agent in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose, and the like.

[0110] 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 in the solvent. The solution may contain other pharma- ceutically or cosmetically acceptable chemicals to buffer, stabilize and preserve the solute. Common examples of solvents used in preparing a solution are ethanol, water, propylene glycol or any other acceptable vehicle. As is of course well known, a gel is a semi-fluid, suspension-type system. Single-phase gels contain organic macromolecules dispersed substantially uniformly throughout the carrier liquid, typically aqueous, but preferably also containing alcohol and optionally oil. The preferred "organic macromolecules", i.e., gelling agents, are crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes, which can be obtained commercially under the trademark Carbopol. Also preferred are hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulosic polymers such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthaiate and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a homogeneous gel, a dispersing agent such as alcohol or glycerin may be added, or the gelling agent may be dispersed by trituration, mechanical mixing or stirring, or a combination thereof. As is also well known in the art, ointments are semi-fluid preparations that are typically based on mineral oil or other mineral oil derivatives. The specific ointment base used is one that provides some desirable characteristics, such as skin emolliency, as recognized by those skilled in the art. As with other carriers or vehicles, the ointment base should be inert, stable, non-irritating and non-sensitizing.As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, PA: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases are classified into four types: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, semi-solid hydrocarbons derived from vegetable oils, fats obtained from animals, and mineral oils.

[0111] Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic mineral oil.

[0112] Emulsion ointment bases are either water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, and include, for example, acetyl alcohol, glyceryl monostearate, lanolin and stearic acid.Preferred water-soluble ointment bases are prepared from polyethylene glycols of various molecular weights; for more information, see Remington: The Science and Practice of Pharmacy.

[0113] Pastes are semi-solid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided into fatty pastes and those made from single-phase aqueous gels. The base in fatty pastes is generally mineral oil or aqueous mineral oil, etc. Pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose, etc., as a base material.

[0114] The enhancer is typically a lipophilic co-enhancer, referred to as a "plasticizing" enhancer, i.e., an enhancer having a molecular weight in the range of about 150 to 1000, and a water solubility of less than about 1% by weight, preferably less than about 0.5% by weight, and most preferably less than about 0.2% by weight. The Hildebrand solubility parameter δ of the plasticizing enhancer is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilic enhancers are fatty esters, fatty alcohols, and fatty ethers. Specific examples of the most preferred fatty acid esters include methyl laurate, ethyl oleate, propylene glycol nionolaurace, propylene glycerol 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 the alkanediol or triol is substituted with one or two fatty ether substituents.

[0115] Additional penetration enhancers will be known to those skilled in the art of topical drug delivery and / or are described in the relevant texts and literature, see, for example, Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, 1995), incorporated herein by reference.

[0116] Various other additives in addition to those specified above may be included in the compositions of the present invention, including, but not limited to, antioxidants, astringents, fragrances, preservatives, emollients, pigments, dyes, humectants, propellants and sunscreens, as well as other types of materials whose presence is medicamentally or otherwise desirable. Typical examples of optional additives that may be included in the formulations of the invention are as follows: preservatives, such as sorbates; solvents, such as isopropanol and propylene glycol; astringents, such as methanol and ethanol; emollients, such as polyalkylene methyl glucosides; humectants, such as glycerin; emulsifiers, such as glycerol stearate, PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol and other polyhydroxy alcohols, such as polyethylene glycol; sunscreens, such as octyl methoxyl cinnamate (commercially available as Parsol MCX) and butyl methoxybenzoylmethane (available under the trade name Parsol 1789); antioxidants, such as ascorbic acid (vitamin C), a-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζι-tocopherol, Z Λ-tocopherol, η-tocopherol, and retinol (vitamin A); essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g., soybean oil, palm 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 (generally perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes (e.g., beeswax, carnauba wax, and paraffin wax); skin-feel modifiers; and thickeners and structurants, 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, the stratum corneum), keep it soft by slowing the loss of its moisture content, and / or protect the skin. Such conditioners and moisturizers include, by way of 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; anti-seborrhoeic agents such as retinoic acid; vasodilators such as nicotinic acid; inhibitors of melanogenesis such as kojic acid; and combinations thereof. Further additional active agents include, for example, alpha hydroxy acids, alpha keto acids, polymeric hydroxy acids, moisturizers, collagen, marine extracts, and antioxidants such as ascorbic acid (vitamin C), a-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, 6-tocopherol, ε-tocopherol, ζι-tocopherol, ζ 2The tocopherol compound includes α-tocopherol, η-tocopherol and retinol (vitamin A) and / or their pharma- ceutically acceptable salts, esters, amides or other derivatives.Preferred tocopherol compound is a-tocopherol.Additional agents include those that can improve oxygen supply in skin tissue, for example, as described in Gross et al., WO94 / 00098 and Gross et al., WO94 / 00109, both of which are assigned to Lancaster Group AG (incorporated by reference).Sunscreen and UV absorbing compounds may also be included. Non-limiting examples of such sunscreen and UV absorbing compounds include aminobenzoic acid (PABA), avobenzone, cinoxate, dioxybenzone, homosalate, methyl anthranilate, oxtocrylene, octyl methoxycinnamate, octyl salicylate, oxybenzone, padirnate O, phenylbenzimidazole sulfonic acid, sulisobenzone, titanium dioxide, trolamine salicylate, zinc oxide, ensulizole, meradiraate, 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.

[0117] Other embodiments may also include various non-carcinogenic, non-irritating healing materials that facilitate treatment with the formulations of the present invention, including 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.

[0118] 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 preparation.

[0119] The formulations of the present invention may also contain conventional additives such as opacifiers, flavorings, colorants, stabilizers, surfactants, etc. In certain embodiments, other agents may also be added, such as antimicrobial agents to prevent spoilage during storage, i.e., to inhibit the growth of microbes such as yeasts and molds.

[0120] 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, other agents, such as repressors and inducers, may also be added, i.e., to inhibit (i.e., glycose) or induce (i.e., xylose) the production of the polypeptide of interest. Such additives may be used provided that they are compatible and do not interfere with the function of the formulation.

[0121] The formulation may also include additives that reduce irritation to minimize or eliminate the possibility of skin irritation or damage resulting from the administered chemicals or other components of the composition.

[0122] Suitable irritation-reducing additives include, for example: a-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcohols, such as 2-phenyl-1-ethanol; glycerin; salicylates; ascorbates; ionophores, such as monensin; amphiphilic amines; ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine. When present, the irritation-reducing additive may be incorporated into the composition at a concentration effective to reduce irritation or skin damage, typically corresponding to about 20% or less by weight of the formulation, more typically about 5% or less by weight.

[0123] Additional suitable pharmacologically active agents that may be incorporated into 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 eliminate pigmented or non-pigmented age spots, keratoses and wrinkles; antibacterial agents; antibacterial agents; antipruritic and antisiccotic agents; anti-inflammatory agents; local anesthetics and analgesics; corticosteroids; retinoids; vitamins; hormones; and antimetabolites.

[0124] Some examples of topical pharmacologically active agents include acyclovir, amphotericin, chlorhexidine, clotrimazole, ketoconazole, econazole, miconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin, phenytoin, para-aminobenzoic acid esters, octyl methoxycinnamate, octyl salicylate, oxybenzone, dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide, zinc pyrithione, diphenhydramine, pramoxine, lidocaine, procaine, erythromycin, tetracycline, clindamycin, crotamiton, and hydroquinone. and its 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.

[0125] Creams, lotions, gels, ointments, pastes, and the like may be spread onto the affected surface and gently rubbed in. Liquids may be applied similarly, but more typically with a dropper, swab, or the like, and carefully applied to the affected area.

[0126] The application regimen depends on several factors that can be easily determined, such as the severity of the condition and its response to initial treatment, but usually includes one or more applications per day on an ongoing basis.Those skilled in the art can easily determine the optimal amount of the formulation to be administered, the method of administration and the number of repetitions.In general, it is contemplated that the formulation of the present invention is applied in the range of once or twice a week to once or twice a day.

[0127] The pharmaceutical compositions of the present invention comprise one or more active ingredients, e.g., therapeutic agents, in admixture with one or more pharma- ceutically acceptable diluents or carriers and, if necessary, 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- ceutically acceptable dosage forms by conventional methods known to those skilled in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).

[0128] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, DC)) 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), polyols (e.g., glycerol, glycerol, tert-butyl ether ... Examples of suitable pharmacopoeitic diluents include glycerol, propylene glycol and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn oil, germ oil, olive oil, castor oil, sesame oil, cottonseed oil and peanut oil), cocoa butter, waxes (e.g., suppository wax), paraffin, silicone, 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 injurious 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 skill in the art.

[0129] The pharmaceutical composition of the present invention may contain additional ingredients and / or materials commonly used in pharmaceutical compositions, if necessary. These ingredients and materials are well known in the art and include: (1) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethylcellulose, sucrose and gum arabic; (3) humectants, such as glycerol; (4) disintegrants, 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. (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) suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugar, polysaccharides ... (13) inert diluents, e.g., water or other solvents; (14) preservatives; (15) surface active agents; (16) dispersing agents; (17) sustained release or absorption retarding agents, e.g., hydroxypropyl methylcellulose, other polymeric materials, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents;(21) emulsifying and suspending agents; (22) solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which 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 agents, flavoring agents, coloring agents, fragrances, and preservatives. Each such component or material must be "acceptable" in the sense of being compatible with the other components of the formulation and not toxic to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a selected dosage form and method of administration can be determined using ordinary skill in the art;

[0130] The dosage form for external or transdermal administration includes powder, spray, ointment, paste, cream, lotion, gel, liquid, patch, drop and inhalant.Active agent(s) / compound(s) can be mixed with suitable pharmaceutically acceptable diluent or carrier under aseptic condition.Ointment, paste, cream and gel can contain excipient.Powder and spray can contain excipient and propellant.

[0131] Pharmaceutical compositions of the present invention suitable for parenteral administration may contain one or more drug(s) / compound(s) in combination with one or more pharma- ceutically acceptable sterile, isotonic aqueous or sterile, isotonic non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, and 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 the maintenance of 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 may also be desirable to include an isotonic agent. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption.

[0132] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any manner. EXAMPLES

[0133] Example 1 Method for integration of the hLEKTI-d6 cassette into the chromosome of S. epidermidis SEΔΔΔ A chromosomal hLEKTI-d6 expression strain was constructed as follows. An expression cassette from plasmid pLEKTI-27a-d6 (Figures 1A and 1B), containing the human hLEKTI-d6 domain fused in frame to the Bacillus subtilis yfhK secretion peptide (SP) and a synthetic propeptide (pro9; (5)), was exchanged by homologous recombination into the delta-toxin gene (hld, SE1634, based on Genbank accession AE015929.1 of S. epidermidis ATCC 12228). Transcription of the hLEKTI-d6 gene from the integrated cassette was initiated by the expression of the P yxiE P in the promoter (6) and chromosome yxiEThe SE1634 gene was designed to be driven by the native promoter, P3, located immediately upstream of the promoter. Promoter P3 is part of / controlled by the quorum sensing of the adjacent accessory gene regulator (agr) operon (7). It is activated by the agrA protein (Figures 1A and 1B) and recognized by the RNAP III transcription complex. The coding sequence of SE1634 between the start and stop codons was transduced into the P3 promoter, leaving the ribosome binding site (RBS) and the 3' untranslated end of the SE1634 mRNA intact. yxiE -yfhK Replaced with SP-pro9-LEKTI-d6 cassette.

[0134] For chromosomal integration, the exchange plasmid, pJB38-SE1634-KO-P yxiE -d6-KI, was designed using Benchling (an online sequence design tool, www.benchling.com). A set of eight overlapping oligonucleotide primers was designed to amplify four DNA fragments by PCR using a high-fidelity DNA polymerase (Table 1). Four PCR reactions using Q5® High-Fidelity 2X Master Mix (New England Biolabs (NEB)) were performed on the following DNA templates: 1) pJB38 plasmid ((8); one reaction, yielding approximately 7 kb), 2) S. epidermidis NRRL B-4268 strain genomic DNA (two reactions, each yielding approximately 1 kb fragments, one flanking the 5' and one flanking the 3' region of the SE1634 gene), and 3) pLEKTI-27a-d6 (one reaction, yielding a 690 bp fragment encoding the LEKTI-d6 expression cassette). All reactions were checked by agarose gel electrophoresis for the presence and correct size of the amplified DNA fragments. [Table 6]

[0135] One microliter of each PCR reaction was combined and assembled using NEBuilder® HiFi DNA Assembly Master Mix (NEB). A portion of the assembled reaction was transformed into competent Escherichia coli NEB 5-α cells. Plasmids from the transformants were analyzed by agarose gel electrophoresis and by DNA sequencing using the PCR primers. The sequence-verified plasmids were transformed into an E. coli dcm- strain to obtain unmethylated plasmid DNA. Plasmid DNA from the dcm- strain was also sequence-verified and identified as a methylated plasmid for S. epidermidis SE. ΔΔΔ Cells were transformed by electroporation. ΔΔΔ Plasmids were also isolated from the strains and sequence confirmed.

[0136] Plasmid pJB38-SE1634-KO-P yxiE -d6-KI was isolated by growing cells at 44 °C, the nonpermissive temperature for plasmid replication. ΔΔΔ pJB38-SE1634-KO-P yxiE -d6-KI-containing SE ΔΔΔFour colonies of were streaked onto tryptic soy agar (TSA) containing 100 μg / mL D-alanine (to complement auxotrophy) and 10 μg / mL chloramphenicol (to select for the plasmid) and incubated at 44 °C for 24-36 h. Resulting colonies were analyzed by PCR with primers anchored in the 5' and 3' flanking regions outside the integration and plasmid specific primers. To obtain a second integration event, confirmed integration colonies were incubated at 30 °C, the permissive temperature for plasmid replication, in tryptic soy broth (TSB) containing 100 μg / mL D-alanine but no chloramphenicol. After one or two passages in TSB + D-ala / no chloramphenicol, cells were plated on TSA + D-ala for single colonies. Colonies were assayed for plasmid loss by patching with TSA + / - chloramphenicol. Chloramphenicol-susceptible (Cam-S) colonies were analyzed by PCR using primer pairs that mapped outside and inside the allelic exchange region. Eight colonies that were both Cam-S and PCR positive were identified.

[0137] The chromosomal cassettes in all eight strains were PCR amplified and sequence verified. All eight strains were also confirmed for D-ala auxotrophy by their inability to grow without supplementation of D-ala in the growth medium. The chromosomal constructs were conserved and designated: SE350, SE351, SE352, SE353, SE354, SE355, SE356 and SE357.

[0138] Each strain was engineered with a single copy of [-P ] inserted into the delta toxin gene encoded by S. epidermidis gene SE1634, located downstream of the P3 promoter just outside the agrACDB operon, encoding a quorum-sensing regulatory protein. yxiEThe plasmid pLEKTI-27a-d6 was confirmed to contain the -RBSsod-SPyfhK-pro9-LEKTI-d6- cassette (Figures 1A and 1B). The same cassette is located downstream of the pUBTR-114 hpaII promoter (9) in plasmid pLEKTI-27a-d6. Plasmid pUBTR-114 is a derivative of the B. subtilis plasmid, pUB110, and is known to have a low to moderate copy number (approximately 10 to 50; (10)). The exact copy number of pLEKTI-27a-d6 has not been experimentally determined.

[0139] In both chromosomal and plasmid constructs, hLEKTI-d6 is constitutively expressed and the protein is secreted into the culture medium. However, several important properties distinguish the chromosomal and plasmid construct strains: 1) the chromosomal construct is single copy, while the plasmid construct is predicted to be multicopy, which may affect the overall expression level of the hLEKTI-d6 gene; 2) the chromosomal construct strain has a deletion of the delta-toxin gene due to the insertion of the hLEKTI-d6 expression cassette; 3) the chromosomal construct strain, unlike the 27a strain, does not express SE because its auxotrophy is not complemented by the alrA gene of the plasmid. ΔΔΔ (dependent on D-alanine supplementation); and 4) the chromosomal construct may have enhanced intrinsic stability of the expression cassette due to its integration into the chromosome, as opposed to being carried on an extrachromosomal genetic element (plasmid). Example 2 The chromosomal construct strain requires D-alanine supplementation and constitutively expresses secreted hLEKTI-d6 at higher levels than the plasmid construct strain 27a.

[0140] Growth of eight independently isolated sequence-verified strains carrying the chromosomal hLEKTI-d6 expression cassette (SE350-SE357) was tested in TSB supplemented with or without D-alanine (100 μg / mL). Cultures were grown for 22 hours at 30° C. with aeration. No growth was observed for the chromosomal construct strains in the absence of D-alanine, but saturation growth was observed in the presence of D-alanine (Table 2). As expected, the plasmid-containing strain, 27a, was able to grow in the absence of D-alanine due to complementation of the auxotrophy by the alanine racemase gene present in plasmid pLEKTI-27a-d6. [Table 7] Growth in TSB; ND: not performed; D-ala: 100 μg / mL

[0141] Duplicate cultures of chromosomal construct strains SE350-SE357 and plasmid construct strain 27a were grown in TSB+D-alanine (100 μg / mL) for 22 hours at 30° C. with aeration, and culture supernatants were analyzed by Western blot. Culture supernatants were sampled and 12% SDS-PAGE gels were run. Electrophoresed proteins were transferred to PVDF membranes by Western blot, and hLEKTI-d6 was detected by a primary chicken IgY antibody (anti-LEKTI-d6 antibody #337) and a secondary goat anti-chicken IgY horseradish peroxidase (HRP) conjugate. The HRP reaction was detected by chemiluminescence. The results showed that the Western blot bands for the chromosomal construct strains were thicker overall than those of the plasmid construct strain 27a (FIG. 2). As expected, no bands were detected in the negative control strain expressing green fluorescent protein in pUBTR-114 (SE-GFP).

[0142] The levels of active hLEKTI-d6-related species in S. epidermidis culture supernatants analyzed by Western blot in Figure 2 were quantified. Briefly, broth was diluted 1:10 in assay buffer (50 mM Tris, 150 mM NaCl, 0.05% (w / v) Brij®-35, pH 8.0) containing 3 nM activated hKLK14, which was serially diluted in 2-fold steps in assay buffer containing 3 nM activated hKLK14 to reach a final volume of 25 μL of mixture per well. The reaction was initiated by the addition of 25 μL of assay buffer containing 30 μM Boc-VPR-AdMC and monitored at 1 min intervals for 10 min at 23°C in a Biotek Hybrid plate reader (Ex / Em=380 / 460 nm). The final concentrations of each component in each well were as follows: 1.5 nM activated hKLK14, 15 μM Boc-VPR-AdMC and 10 to 0.32% (v / v) of the media source used. Media control samples SE-GFP and TSB+ D-ala showed no substantial hKLK14 inhibition at concentrations up to 5%. As samples were split into three 96-well plates, each plate had its own calibration curve using purified His-tagged hLEKTI-d6 (lot DM29 / 24) ranging from 0 to 4 nM protein. This procedure was performed to avoid plate-to-plate bias in the calculated concentrations of hLEKTI-d6 species in broth. Each sample and its respective dilutions were measured in technical replicates. The velocity of the hKLK14 reaction was calculated using data points from 0 to 4 min of the reaction and the summarized results are shown in Figure 3.

[0143] All samples contained measurable levels of active hLEKTI-d6 species, ranging from 39 to 333 zmol / CFU. There was high variation in zmol / CFU hLEKTI-d6 levels among biological replicates of the same strain. Strains expressing chromosomal hLEKTI-d6 exhibited 2- to 5-fold higher levels of active hLEKTI-d6 species compared to strains expressing LEKTI-d6 on an episomal plasmid. This result was consistent with observations by Western blot, where the chromosomal construct strain expressed more hLEKTI-d6 protein, as detected using anti-LEKTI-d6 antibody #337.

[0144] The timing of hLEKTI-d6 expression was examined in growth curve experiments using the plasmid construct strain 27a and two chromosomal hLEKTI-d6 strains, SE352 and SE355. Cultures were grown in TSB with aeration at 30°C, and culture supernatants were sampled at the onset of stationary phase and at several time points during stationary phase. Results showed that while the optical density at 600 nm did not change between the 19 and 23.5 hour time points, hLEKTI-d6 production in the plasmid and chromosomal construct strains was enhanced at later time points as visualized by Western blot and quantified by KLK14 assay. Enhancement of hLEKTI-d6 production during stationary phase was due to the P-regulation of the hLEKTI-d6 gene in both the plasmid and chromosomal construct strains. yxiE Stimulation of promoter-driven expression by nutrient deprivation (phosphate deprivation) may be part of the reason (Figures 1A and 1B). The overall high levels of hLEKTI-d6 protein observed in chromosomal construct strain cultures suggests that additional factors are involved in hLEKTI-d6 protein production in these strains. [Table 8] References 1. Phanaksri T, Luxananil P, Panyim S, Tirasophon W. 2015. Synergism of regulatory elements in sigma(B)- and sigma(A)-dependent promoters enhances recombinant protein expression in Bacillus subtilis. J Biosci Bioeng 120:470-5 2. Ozturk S, Ergun BG, Calik P. 2017. Double promoter expression systems for recombinant protein production by industrial microorganisms. Appl Microbiol Biotechnol 101:7459-75 3. Kong KF, Vuong C, Otto M. 2006. Staphylococcus quorum sensing in biofilm formation and infection. Int J Med Microbiol 296:133-9 4. Tan L, Li SR, Jiang B, Hu XM, Li S. 2018. Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System. Front Microbiol 9:55 5. Le Loir Y, Gruss A, Ehrlich SD, Langella P. 1998. A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J Bacteriol 180:1895-903 6. Antelmann H, Scharf C, Hecker M. 2000. Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis. J Bacteriol 182:4478-90 7. Otto M. 2004. Quorum-sensing control in Staphylococci -- a target for antimicrobial drug therapy? FEMS Microbiol Lett 241:135-41 8. Bose JL, Fey PD, Bayles KW. 2013. Genetic tools to enhance the study of gene function and regulation in Staphylococcus aureus. Appl Environ Microbiol 79:2218-24 9. Zyprian E, Matzura H. 1986. Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system. DNA 5:219-25 10. Leonhardt H. 1990. Identification of a low-copy-number mutation within the pUB110 replicon and its effect on plasmid stability in Bacillus subtilis. Gene 94:121-4 Incorporation by reference

[0145] The entire disclosures of each of the patent documents, including patent applications, scientific papers, government reports, websites and other references referred to herein are incorporated herein by reference in their entirety for all purposes. In the event of a conflict in terms, the present specification controls. All sequence listings or SEQ ID NOs disclosed herein are incorporated herein in their entirety.

[0146] 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.

[0147] Although illustrative embodiments of the present invention have been described herein, it is to be understood that the invention is not limited to what has been described, and various other changes or modifications can be made by those skilled in the art without departing from the scope or spirit of the invention.

Claims

1. A recombinant microorganism capable of secreting one or more therapeutic polypeptides, comprising: (i) a first coding sequence comprising a nucleic acid sequence encoding one or more therapeutic polypeptides; (ii) a second coding sequence comprising a nucleic acid sequence encoding one or more secretory sequences that are not essentially associated with said one or more therapeutic polypeptides; and (iii) a third coding sequence comprising a propeptide; wherein the first coding sequence, the second coding sequence, and the third coding sequence are in frame, and the one or more therapeutic polypeptides are encoded by one or more SPINK genes or one or more protein domains thereof. Recombinant microorganisms.

2. 2. The recombinant microorganism of claim 1, wherein the first coding sequence, the second coding sequence, and the third coding sequence are chromosomally integrated into the hld gene.

3. 3. The recombinant microorganism of claim 1 or 2, which is auxotrophically attenuated and optionally is a D-alanine auxotroph.

4. 3. The recombinant microorganism of claim 1 or 2, wherein expression of the first coding sequence, the second coding sequence, and the third coding sequence are operably linked to a promoter, optionally the promoter is P3 and / or PyxiE, and optionally the PyxiE comprises SEQ ID NO:

120.

5. 3. The recombinant microorganism of claim 1 or 2, wherein the secretory peptide is derived from the Bacillus subtilis yfhK gene, and optionally the secretory peptide comprises SEQ ID NO: 122 and / or the propeptide comprises SEQ ID NO:

123.

6. 3. The recombinant microorganism of claim 1 or 2, which is a bacterium, optionally selected from the group consisting of Moraxella, Corynebacteria, Pasteurella, Haemophilus, Streptococcus or Staphylococcus, and optionally is Staphylococcus epidermidis.

7. 3. The recombinant microorganism of claim 1 or 2, wherein the one or more SPINK genes are selected from the group consisting of SPINK1, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINK13 and SPINK14, and optionally the one or more SPINK genes is SPINK5.

8. 8. The recombinant microorganism of claim 7, wherein the one or more SPINK genes encode a LEKTI protein or one or more protein domains thereof, optionally wherein the LEKTI protein domain is selected from the group consisting of D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 and D15, optionally wherein the LEKTI protein domain is D6.

9. 3. The recombinant microorganism of claim 1 or 2, which secretes the one or more therapeutic polypeptides.

10. A method for producing a live biological therapeutic composition comprising a recombinant microorganism, comprising: (a) transfecting a cell with a nucleic acid sequence comprising (i) one or more LEKTI protein domains, (ii) a secretory peptide that is essentially unrelated to said one or more therapeutic polypeptides, (iii) a propeptide, and (iv) a promoter; and (b) integrating the nucleic acid sequence into the chromosome of the cell; and (c) Obtaining the live bacterial biological therapeutic agent A method comprising:

11. 11. The method of claim 10, wherein the secretory peptide is selected from the Bacillus subtilis yfhK gene, and optionally the secretory peptide comprises SEQ ID NO: 122 and / or the propeptide comprises SEQ ID NO:

123.

12. the nucleic acid sequence is integrated into the hld gene, and / or the promoter is PyxiE, optionally wherein PyxiE comprises SEQ ID NO: 120; and / or 12. The method of claim 11, wherein the nucleic acid sequence is under the control of an endogenous promoter, optionally wherein the endogenous promoter is P3.

13. The method of claim 11, wherein the LEKTI protein domain, secretory peptide and propeptide are arranged in frame.

14. 12. The method of claim 11, wherein the recombinant microorganism is selected from the group consisting of Moraxella, Corynebacteria, Pasteurella, Haemophilus, Streptococcus, or Staphylococcus.

15. The method of claim 11 , wherein the recombinant microorganism secretes the one or more LEKTI protein domains or variants thereof.

16. A composition obtainable by the method of claim 11.

17. 17. The composition of claim 16, comprising a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, and an ointment.

18. 1. A live biological therapeutic composition comprising a recombinant microorganism, said recombinant microorganism comprising: (i) one or more LEKTI protein domains, (ii) one or more secretory peptides, (iii) a propeptide, and (iv) a nucleic acid sequence comprising a promoter. Including, the recombinant microorganism is capable of secreting the one or more LEKTI protein domains or variants thereof; Live bacteria biological therapeutic composition.

19. the nucleic acid sequence is integrated into the chromosome of the cell at the hld gene, and / or the secretory peptide is derived from the Bacillus subtilis yfhK gene, and optionally the secretory peptide comprises SEQ ID NO: 122; and / or the propeptide comprises SEQ ID NO: 123, and / or the promoter is PyxiE, optionally wherein PyxiE comprises SEQ ID NO: 120; and / or wherein the one or more LEKTI protein domains, one or more secretory peptides and propeptides are under the control of endogenous promoter P3; 19. The composition of claim 18.

20. 19. The composition of claim 18, wherein the recombinant microorganism is selected from the group consisting of Moraxella, Corynebacteria, Pasteurella, Haemophilus, Streptococcus, or Staphylococcus.

21. 20. The composition of claim 18, comprising a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel, and an ointment.

22. 20. A kit comprising the composition of claim 18 and instructions for use.

23. A composition for use in a method of treating a disease or disorder in a subject, the method comprising administering to the subject a composition described in claim 18.

24. the disease or disorder is selected from a skin disease, a pain-related disease or disorder, cancer, and a viral infection; Optionally, the skin disease is selected from the group consisting of pruritus, rosacea, psoriasis, atopic dermatitis, ichthyosis vulgaris, and Netherton syndrome. The pain-related disease or disorder is selected from the group consisting of acute pain, chronic pain, nociceptive pain, neuropathic pain, traumatic pain, inflammatory pain, post-operative incision pain, cancer-related pain, fracture pain, osteoporosis pain, bone cancer pain, and gouty joint pain. 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 cancer and oral cancer; or 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; 24. The composition of claim 23.

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