Compositions and methods for treatment of netherton syndrome with lekti expressing recombinant microbes

Abstract

To provide novel therapeutic agents to treat or ameliorate the symptoms of skin diseases, in particular, Netherton Syndrome.SOLUTION: A composition for the treatment of a skin disease comprises a microbe genetically modified to express and provide one or more LEKTI protein domains on the skin of a mammal, where the LEKTI protein domains are effective to penetrate one or more layers of the mammal's skin and effective to inhibit serine protease activity of at least one serine protease in or on the mammal's skin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 521,050, filed on 16 June 2017, the entirety of which is incorporated herein by reference.

[0003] Technical field This disclosure relates to methods, kits, and compositions for treating Netherton syndrome or improving its effects using one or more recombinant microorganisms genetically modified to express one or more therapeutic LEKTI domains on the skin of a subject. [Background technology]

[0004] The epidermis, or stratified squamous epithelium of the skin, consists of multiple sublayers and is one of the body's most important barriers to the outside world. The stratum corneum is the outermost layer of the epidermis and develops as a result of the final anucleated step in keratinocyte differentiation from cells in the nucleated epidermal layer. While the stratum corneum is recognized as the most important physical barrier, the nucleated epidermal layer is also important in barrier function (Proksch, Brandner et al., 2008). At the same time, the skin barrier protects against widespread water loss in one direction (internal-external barrier) and against the intrusion of harmful substances from the environment (external-internal barrier) (Proksch, Brandner et al., 2008). Maintaining the barrier is also important for balanced proliferation of the basal layer and the maintenance of the calcium ion gradient, and thus for proper epidermal differentiation (Lee, Jeong et al., 2006).

[0005] There are currently several limitations to skin treatments. Many treatments, such as topical corticosteroids or biopharmaceuticals, do not address the fundamental problems of endogenous protein deficiencies in the epidermis or imbalances in microbial diversity in the skin. Recombinant proteins are a promising group of therapeutic agents in the treatment of skin diseases, but their use in relation to skin presents several challenges.

[0006] Traditional methods involve purifying and concentrating recombinant proteins extracted from bacterial systems, and then incorporating such preparations into delivery systems. Recombinant protein purification is often a very expensive method for obtaining the protein. Furthermore, several problems are associated with these traditional methods, including proteolytic degradation, inefficient delivery, and the need for long-term, repeated application to achieve therapeutic effects.

[0007] One skin condition that would benefit from improved treatment modalities is Netherton syndrome (NS). NS is a rare autosomal cutaneous disorder that manifests as severe skin inflammation and scaling, hair shaft defects, persistent allergic symptoms, and immune system problems. Newborns with NS often have red, scaly skin that can leak fluid, leading to a risk of dehydration and infection of the skin or system. Affected children are also unable to grow at a normal rate. While the health of older children and adults with NS typically improves, these individuals are often underweight and short. Many people with NS also have immune system problems such as food allergies, hay fever, asthma, or eczema.

[0008] NS is caused by a loss-of-function defect in the SPINK5 gene (a Cazal-type 5 serine protease inhibitor) that encodes the lymphoepithelial Cazal-type 5 associated inhibitor type 5 (LEKTI) protein. LEKTI is a multi-domain serine protease inhibitor normally expressed in all stratified epithelial cells and Hassall bodies of the thymus. Among the cluster of other SPINK genes (e.g., SPINK6 and SPINKI9), the SPINK5 gene encoding LEKTI is located on chromosome 5 and contains 33 exons encoding 15 inhibitory domains separated by a linker region. SPINK5 stands out among the other SPINK genes for the large number of inhibitory domains it encodes. Furthermore, the SPINK5 gene is transcribed into three different transcripts, resulting in three different LEKTI proteins with different 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 elongation linker region 13.

[0009] The LEKTI protein is a Khazar-type associated inhibitor. The Khazar motif is defined by the presence of six cysteine ​​residues located at specific distances that allow for the formation of three disulfide bonds in 1-5, 2-4, and 3-6 patterns. Two domains of LEKTI (D2 and D5) form this six-cysteine ​​motif, while the other domain shares four cysteine ​​residues, forming a rigid inhibitory loop that is thought to mimic the substrate of the target protease and inactivate the target protease catalytic site.

[0010] LEKTI proteins require proteolytic cleavage to activate their inhibitory function against many proteases. Specifically, the full-length protein is cleaved into domains D1–D5 and D6–D15. The D6–D15 domain is then further cleaved in multiple steps into D6–D9 and D10–D15, →D6 and D7–D9→D7 and D8–D9→D8. This process results in LEKTI proteins containing one and six inhibitory domains, each protein having a different inhibitory function. For example, various LEKTI inhibitory fragments can inhibit various kallikrein-related peptidases (KLKs), such as KLK5, KLK7, and KLK14.

[0011] Deficited LEKTI proteins can result from substitution, insertion, or deletion mutations in the SPINK5 gene, often causing nonsense or frameshift mutations that result in premature stop codons. Other mutations in the splice site bases can lead to abnormal splicing events in the transcribed SPINK5 gene. Thus, many SPINK5 mutations result in the complete absence of LEKTI domain synthesis. LEKTI deficiency or LEKTI loss can lead to disorientation of protease activity, causing skin desquamation and epidermal permeability due to impaired epidermal differentiation and lipid metabolism, resulting in a compromised skin barrier. Furthermore, disorientation of the activity of some KLK proteins can lead to desmosome cleavage and stratum corneum separation.

[0012] Netherton syndrome is a rare disease for which no specific treatment is available. Given the above, there is a need for novel therapeutic agents for the treatment of NS. This application aims to address these and other needs. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Proksch, E., JM Brandner and JM Jensen (2008). "The skin: an indispensable barrier." Exp Dermatol 17(12): 1063-1072 [Overview of the project]

[0014] In one embodiment, the present disclosure provides a composition for the treatment of a skin disease, comprising a microorganism genetically modified to express and result in one or more LEKTI protein domains on mammalian skin, wherein the LEKTI protein domains are effective in penetrating one or more layers of mammalian skin and are effective in inhibiting the serine protease activity of at least one serine protease in or on mammalian skin.

[0015] According to some embodiments, microorganisms are adapted to survive on the surface of mammalian skin for a controlled period to provide a continuous supply of LEKTI protein domains. According to some embodiments, LEKTI protein domains are effective in improving the symptoms of Netherton syndrome. In one embodiment, the LEKTI domain is domain 6.

[0016] According to some embodiments, microorganisms are genetically modified by transfection / transformation using recombinant DNA plasmids encoding LEKTI protein domains. In some embodiments, the LEKTI domain is functionally ligated to one or more recombinant protein domains that are effective in enhancing secretion from the microorganism and / or permeability through mammalian skin. According to some embodiments, at least one LEKTI domain is functionally ligated to a SecA domain. According to some embodiments, at least one LEKTI domain is functionally ligated to an RMR domain.

[0017] According to some embodiments, at least one LEKTI domain comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0018] According to some embodiments, the microorganism is adapted to grow (propagate) on mammalian skin.

[0019] According to some embodiments, the expression of at least one LEKTI domain is controlled by an operon, and the amount of LEKTI provided to mammalian skin is proportional to the availability of external factors. In some embodiments, the expression of at least one LEKTI domain is controlled by a promoter that is constitutively active.

[0020] According to some embodiments, the microorganism is genetically modified by transfection / transformation with a recombinant DNA plasmid encoding a LEKTI protein domain and one or more antibiotic resistance genes.

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

[0022] According to one aspect, the present disclosure includes providing a microorganism genetically modified to express one or more LEKTI protein domains on the surface of the skin of a mammal, for treating or ameliorating the effects of a skin disease in a mammal that requires it, wherein the LEKTI protein domain is effective to penetrate one or more layers of the skin of the mammal and is effective to inhibit the activity of at least one serine protease in or on the skin of the mammal, a method is provided.

[0023] According to some embodiments, the microorganism is adapted to survive for a controlled period of time on the surface of the skin of the mammal to provide a continuous supply of the LEKTI protein domain.

[0024] According to another aspect, the present disclosure provides a kit for treating or ameliorating the effects of a skin disease in a mammal that requires it, comprising (1) a composition comprising a microorganism genetically modified to express one or more LEKTI protein domains, wherein the LEKTI protein domain is effective to penetrate one or more layers of the skin of the mammal and is effective to inhibit the serine protease activity of at least one serine protease in or on the skin of the mammal, and (2) a reagent for applying the composition to the skin of the mammal.

[0025] According to some embodiments, the microorganism is adapted to survive for a controlled period of time on the surface of the skin of the mammal to provide a continuous supply of the LEKTI protein domain.

[0026] According to one aspect, the present disclosure provides a composition for treating skin diseases comprising a microorganism comprising a pJB38-LEKTI complete plasmid construct.

[0027] According to some embodiments, the microorganisms are selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof.

[0028] According to one embodiment, the present disclosure provides a composition comprising a pJB38-LEKTI complete plasmid construct. According to several embodiments, the pJB38-LEKTI complete plasmid construct is expressed in microorganisms selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or mixtures thereof. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 shows a vector construct containing the therapeutic LEKTI domain of the present invention. The protein-coding regions of the plasmid are ligated together in a functional manner and contain SecA, 6xHis tags, LEKTI D8-11, and RMR tags, under the control of the CmR promoter. [Figure 2] Figure 2 shows vector constructs of the pJB38 plasmid according to several embodiments of the present invention. [Figure 3] Figure 3 shows the domains of the full-length LEKTI polypeptide. [Figure 4] Figure 4 shows the SDS-PAGE results indicating that LEKTId6 is highly soluble in E. coli BL21(De3). [Figure 5] Figure 5 shows the SDS-PAGE results demonstrating the success of affinity purification of H6-LEKTId6 (8.8 kDa). [Figure 6] Figure 6 shows the SDS-PAGE results indicating that LEKTId6-H6 (8.8kDa) is potentially N-terminally truncated. [Figure 7] Figures 7A and 7B show that recombinantly produced LEKTI domain 6 inhibits trypsin in vitro. Figure 7A is a schematic diagram of the experiment performed. Figure 7B is a graph showing trypsin activity. [Figure 8] Figures 8A and 8B show that recombinant LEKTI domain 6 (ct His6 tag) inhibits trypsin in vitro compared to LEKTI domains 10-15. Figure 8A is a schematic diagram of the experiment performed. Figure 8B is a graph showing trypsin activity. [Figure 9] Figures 9A and 9B show that recombinant LEKTI domain 6 inhibits KLK7 in vitro, similar to the inhibition of KLK7 by LEKTI domains 10-15. Figure 9A is a schematic diagram of the experiment performed. Figure 9B is a graph showing KLK7 activity. [Figure 10] Figures 10A and 10B show that recombinantly produced LEKTI domain 6 inhibits KLK5 in vitro at nanomolar concentrations. Figure 10A is a schematic diagram of the experiment performed. Figure 10B is a graph showing KLK5 activity. [Modes for carrying out the invention]

[0030] One aspect of this disclosure provides a skin-resident bacterium that has been genetically modified to express a recombinant protein for treating or improving Netherton syndrome. The genetically modified protein-producing bacterium can treat NS by expressing and, if necessary, secreting a therapeutic protein that addresses the underlying cause of the disease or its symptoms. According to some embodiments, the therapeutic protein comprises one or more LEKTI domains that are effective in inhibiting serine proteases in or on mammalian skin. According to some embodiments, the recombinant LEKTI domain compensates for a deficiency in the endogenous LEKTI protein naturally produced by the skin in mammals. According to some embodiments, the genetically modified bacterium can self-replicate while retaining the ability to produce recombinant proteins, thereby resulting in a continuous supply of the therapeutic agent.

[0031] According to some embodiments, the present disclosure provides compositions for the treatment of skin diseases, comprising a microorganism genetically modified to express and result in one or more LEKTI protein domains on mammalian skin, wherein the LEKTI protein domains are effective in penetrating one or more layers of mammalian skin and are effective in inhibiting the serine protease activity of at least one serine protease in or on mammalian skin.

[0032] As used herein, the term “skin disease” and its grammatical variations mean a skin condition or state of human skin that is generally undesirable or harmful compared to a normal or baseline state. Examples of abnormal skin conditions include, but are not limited to, Netherton syndrome, psoriasis, acne, atopic dermatitis, allergic contact dermatitis, exfoliative hyperkeratosis, seborrheic dermatitis, eczema, dry skin, allergies, rashes, UV-irritated skin, detergent-irritated skin (including irritation caused by molecules used in enzymes and detergents and sodium lauryl sulfate), skin thinning (e.g., skin of the elderly and children), bullous pemphigoid, pemphigus vulgaris, impetigo, vitiligo, alopecia, and hirsutism.

[0033] As used herein, the terms “genetically modified” and their grammatical variations are used to describe microorganisms (e.g., bacteria) that have been genetically modified or manipulated by the introduction of DNA prepared outside the microorganism. For example, by introducing plasmid DNA containing new genes into bacteria, the bacteria can express these genes. Alternatively, DNA containing new genes can be introduced into bacteria and then incorporated into the bacterial genome, where the bacteria will express these genes.

[0034] As used herein, the terms “to treat,” “to treat,” “treatment,” and their grammatical variations mean providing a subject, e.g., a patient, with a protocol, regimen, process, or treatment that is desirable for obtaining a physiological response or outcome in that subject. In particular, the methods and compositions of the present invention can be used to slow the development of disease symptoms, delay the onset of a disease or condition, or halt the progression of disease development. However, since not all treated subjects may respond to a particular treatment protocol, regimen, process, or treatment, a treatment does not require that the desired physiological response or outcome be achieved in all subjects or subject groups, e.g., patient populations. Thus, a given subject or subject group, e.g., patient populations, may not respond to a treatment, or may respond inadequately.

[0035] In the present invention, the subject may be a mammal. As used herein, “mammal” and its grammatical variations mean any category of mammal. In the present invention, examples of mammals include humans, agricultural animals, livestock, and laboratory animals. Some examples of agricultural animals include cattle, pigs, horses, and goats. Some examples of livestock include dogs and cats. Some examples of laboratory animals include primates, rats, mice, rabbits, and guinea pigs. Preferably, the mammal is a human.

[0036] Where used herein, the terms “effective amount” or “therapeutably effective amount” of a compound or composition disclosed herein means the amount of such compound or composition that, when administered to a subject, is sufficient to produce the beneficial or desirable results described herein. Effective dosage forms, modes of administration, and doses can be determined empirically, and such determinations are within the scope of the art of those skilled in the art. Those skilled in the art will understand that doses vary depending on the route of administration, elimination rate, duration of treatment, identity of any other drugs administered, the mammal, e.g., age, size, and species of the human patient, and similar factors well known in the fields of medicine and veterinary medicine. Generally, a preferred dose of a composition according to the present invention would be the amount of the composition that is the minimum dose effective to produce the desired effect. An effective dose of a composition of the present invention may be administered as two, three, four, five, six or more subdoses, administered separately at appropriate intervals throughout the day.

[0037] Microbial Compositions: According to several embodiments, the present disclosure provides microbial compositions comprising one or more diverse bacteria suitable for use on mammalian skin. Examples, but not limited to, include non-pathogenic bacteria and commensal bacteria. Suitable bacteria for use in the present invention include, but not limited to, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus (e.g., S. epidermidis and / or S. hominis), Lactobacillus (e.g., L. acidophilus), Pediococcus, Leuconostoc, or Oenococcus. According to some embodiments, the microbial composition includes one or more of the following: Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes, Corynebacterium spp., Acinetobacter johnsonii, and Pseudomonas aeruginosa. According to some embodiments, other relevant or similar species found on the skin are used.

[0038] Certain embodiments involve the use of Staphylococcus epidermidis bacteria. According to some embodiments, the strains of S. epidermidis used are unable to produce biofilms. Examples of these include S. epidermidis strains ATCC 12228 or NRRL B-4268.

[0039] According to some embodiments, recombinant microorganisms are adapted to survive on the surface of mammalian skin for an unlimited or controlled period to provide a continuous supply of the LEKTI protein domain. In some embodiments, recombinant microorganisms survive alongside naturally occurring commensal microorganisms on mammalian skin. In some embodiments, recombinant microorganisms survive excluding naturally occurring commensal microorganisms on mammalian skin. According to some embodiments, recombinant microorganisms are adapted to grow on mammalian skin. In other embodiments, recombinant microorganisms are no longer viable but contain an effective amount of therapeutic polypeptide, e.g., LEKTI or its therapeutically effective domain. Such cells may or may not be damaged, depending on the details of delivering the therapeutic peptide (or its domain) to the target site.

[0040] As used herein, the term “recombinant” and its grammatical variations mean relating to or referring to an organism, protein, or genetic material formed by or using recombinant DNA, which contains DNA fragments derived from different sources or from different parts of the same source. For example, the term “recombinant DNA” means a DNA molecule formed by a recombination method for splicing DNA fragments derived from different sources or from different parts of the same source. In some embodiments, two or more different sources of DNA are cut using restriction enzymes and joined together using ligases. As another example, the term “recombinant protein” or “recombinant domain” and its grammatical variations mean a protein molecule formed by a recombination method originating from spliced ​​DNA fragments derived from different sources or from different parts of the same source. As yet another example, the term “recombinant microorganism” or “recombinant bacterium” and its grammatical variations mean a microorganism / bacterium containing one or more recombinant DNA / protein molecules.

[0041] According to some embodiments, the microorganisms are 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.

[0042] LEKTI gene: According to some embodiments, recombinant microorganisms are engineered to express a mammalian gene encoding the LEKTI protein. The LEKTI gene can be obtained from any mammal, such as a mouse, rat, rabbit, goat, sheep, horse, cattle, dog, primate, or human gene sequence. According to some embodiments, the LEKTI gene sequence is a human gene sequence. According to some embodiments, recombinant microorganisms are engineered to contain a fragment of the LEKTI gene.

[0043] According to some embodiments, the recombinant protein expressed by the engineered microorganism includes the peptide sequence (LEKTI D8-D11) described in SEQ ID NO: 1. According to some embodiments, the recombinant protein expressed by the engineered microorganism includes the peptide sequence described in SEQ ID NO: 2. According to some embodiments, one or more fragments of the peptide sequence described in SEQ ID NO: 2 are expressed by the engineered microorganism. In one embodiment, the fragment includes one or more LEKTI domains. In one embodiment, the LEKTI domain is domain 6.

[0044] According to some embodiments, recombinant microorganisms include sequences disclosed herein that have at least about 75% identity, or 80% identity, or 85% identity, or 90% identity, or 95% identity to one or more of the sequence numbers listed herein. The term “identity” as used herein and its grammatical variations mean the degree to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. The percentage of 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.

[0045] According to some embodiments, the recombinant protein expressed by the engineered microorganism contains one or more protease inhibitory domains of the LEKTI protein. Some non-limiting examples include one or more of domains D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, and D15. According to some embodiments, the recombinant protein expressed by the engineered microorganism contains LEKTI inhibitory domain 6 or domains D8-D11.

[0046] According to several embodiments, the LEKTI protein domain is effective in improving the symptoms of Netherton syndrome. As used herein, the terms “improve,” “to improve,” and their grammatical variations mean reducing the severity of the symptoms of the disease in the subject. In some embodiments, the LEKTI protein domain acts as a competitive or non-competitive inhibitor of one or more proteases present in or on the skin of mammals. In some embodiments, the LEKTI protein domain acts as a serine protease inhibitor. As used herein, the terms “protease” and “proteinase” are used interchangeably, and both terms refer to enzymes that perform protein lysis.

[0047] According to some embodiments, microorganisms are genetically modified by transfection / transformation using recombinant DNA plasmids encoding the LEKTI protein domain. Other conventional or discoverable methods for introducing DNA into microorganisms may also be used in the present invention. According to some embodiments, the recombinant DNA plasmid comprises a sequence encoding the LEKTI protein domain and one or more secretory peptides and / or cell permeability peptides. According to some embodiments, the LEKTI domain is functionally ligated to one or more recombinant protein domains that are effective in enhancing secretion from microorganisms and / or permeability through mammalian skin.

[0048] The term "functionally linked" refers to the linking of nucleic acid sequences on a single nucleic acid fragment in such a way that the function of one is neither regulated nor inhibited by the other. For example, a promoter is functionally linked to a coding sequence if it can regulate the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be functionally linked to a regulatory sequence in either sense or antisense orientation. In another example, two proteins can be functionally linked so that the function of either protein is not impaired. In general, functionally linked means that the nucleic acid sequences to be linked are contiguous, and if two protein coding regions need to be linked, they must be contiguous and in the same reading frame.

[0049] As used herein, the terms “secretory peptide,” “secretory sequence,” “secretory tag,” “signal peptide,” or “transition signal,” and their grammatical variations, mean any peptide sequence capable of targeting a synthesized protein to a cellular secretory pathway. In some embodiments, the secretory peptide can be positioned on the N-terminus of a recombinant protein to target a tagged protein for secretion, either concurrently with or after translation. According to some embodiments, at least one LEKTI domain is functionally ligated to a SecA domain (SEQ ID NO: 3).

[0050] Secretory Peptides: According to several embodiments, the therapeutic LEKTI domain is ligated in a manner that can function as one or more secretory or transit signals that tag the protein for transport via the secretory pathway. Any secretory signal that facilitates the exit of the LEKTI protein from bacterial cells can be used as a secretory peptide. Non-limiting examples of secretory peptide signals are listed in Table 1 below.

[0051] [Table 1] JPEG2023138994000002.jpg36167

[0052] According to some embodiments, the therapeutic LEKTI domain is functionally ligated to one or more signal sequences derived from endogenous proteins of Staphylococcus epidermidis. Non-limiting examples of secretory signal peptides derived from endogenous proteins of Staphylococcus epidermidis are listed in Table 2 below.

[0053] [Table 2] JPEG2023138994000004.jpg238170JPEG2023138994000005.jpg247170JPEG20231389940 00006.jpg227170JPEG2023138994000007.jpg230170JPEG2023138994000008.jpg233170

[0054] According to several embodiments, the therapeutic LEKTI domain is functionally ligated to one or more secretory signal sequences derived from endogenous proteins of other bacteria. A non-limiting list of secretory signal peptides derived from various bacterial endogenous proteins is provided in Appendix A.

[0055] According to some embodiments, recombinant LEKTI domains are ligated in a manner that can function with a cell permeable peptide sequence that enhances the ability of the LEKTI domain to cross the cell membrane. The term “enhance” used to describe the cell permeable peptide / LEKTI means that the cell permeable sequence improves the passage of the recombinant LEKTI domain across the cell membrane compared to recombinant LEKTI domains lacking the cell permeable sequence.

[0056] Cell-permeable peptides: According to some embodiments, one or more cell-permeable peptides 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, one or more cell-permeable peptides are ligated to therapeutic proteins in a manner that allows them to function in order to facilitate entry into skin cells (e.g., keratinocytes). Non-limiting examples are listed in Table 3 below.

[0057] [Table 3] JPEG2023138994000010.jpg96170

[0058] According to some embodiments, the cell-penetrating peptide comprises 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 with 6-aminocaproic acid residues in between (RAca) (where there are 2 to 6 arginine repeats); arginine repeats with 4-aminobutyric acid in between (RAbu) (where there are 2 to 6 arginine repeats); arginine repeats with methionine in between (where there are 2 to 6 arginine repeats); arginine repeats with threonine in between (where there are 2 to 6 arginine repeats); arginine repeats with serine in between (where there are 2 to 6 arginine repeats); and arginine repeats with alanine in between (where there are 2 to 6 arginine repeats).

[0059] According to some embodiments, the LEKTI domain is operably linked to the RMR domain (SEQ ID NO: 4).

[0060] According to some embodiments, the expression of the LEKTI domain is controlled by an operon, and the amount of LEKTI provided to mammalian skin is proportional to the availability of external factors. For example, in some embodiments, the recombinant LEKTI gene may be under the control of a xylose-inducible promoter (e.g., the xylose isomerase gene (xylA) containing the xylose repressor (xylR), the xylose operator (xylO), and the cis-acting catabolite-responsive element (CRE)), and the amount of recombinant LEKTI protein made available to mammalian skin is controlled by the amount of exogenous xylose available to the recombinant microorganism. According to some embodiments, the expression of the LEKTI domain is controlled by a promoter that is constitutively active. According to some embodiments, the expression of the LEKTI domain is controlled by the CmR promoter set forth in SEQ ID NO: 8.

[0061] According to several embodiments, microorganisms are genetically modified by transfection / transformation using recombinant DNA plasmids encoding the LEKTI protein domain and one or more antibiotic resistance genes. For example, some embodiments of recombinant DNA plasmids include kanamycin resistance genes and / or trimethoprim resistance genes; e.g., dfrA (SEQ ID NO: 5). According to several embodiments, treatment of mammalian skin with antibiotics (to which the recombinant microorganisms are resistant) can be used to bias the commensal microbial population toward a larger proportion of LEKTI-producing microorganisms. Other elements that may be present in the recombinant DNA plasmid include, but are not limited to, replication protein genes, such as members of the Rep superfamily of replication proteins. For example, in some embodiments, the recombinant DNA plasmid includes the repF gene (SEQ ID NO: 6).

[0062] According to some embodiments, the recombinant DNA plasmid comprises one or more sequences of the pJB38 vector. In some embodiments, recombinant LEKTI is ligated in a manner that allows it to function as an inducible promoter, ribosome binding site, transition signal, and / or cell permeable peptide in the pJB38 vector. As used herein, the term “pJB38-LEKTI-complete” means a recombinant DNA plasmid construct comprising the pJB38 vector and one or more LEKTI domains. According to some embodiments, the recombinant DNA plasmid comprises the pJB38 vector described in SEQ ID NO: 1542. According to some embodiments, the LEKTI domain described in SEQ ID NO: 1 is ligated in a manner that allows it to function in the pJB38 vector described in SEQ ID NO: 1542.

[0063] According to some embodiments, the recombinant DNA plasmid comprises the pKK30-LEKTI complete sequence described in Sequence ID No. 7 (Appendix B). According to some embodiments, the present disclosure provides compositions for the treatment of skin diseases comprising a microorganism comprising a pKK30-LEKTI complete plasmid construct. According to some such embodiments, the microorganism 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.

[0064] According to some embodiments, the amount or duration of therapeutic LEKTI protein available is controlled by the stability of the LEKTI-carrying vector in the microorganism. For example, the persistence of a recombinant vector can be controlled by one or more elements of the plasmid, such as host-beneficial genes, plasmid stability mechanisms, and those that provide plasmid co-fitting. For example, some plasmids can provide stable replication, active distribution mechanisms, and mechanisms that ensure reliable inheritance of the plasmid to daughter cells over several generations (see, for example, JC Baxter, BE Funnell, Plasmid partition mechanisms, Microbiol. Spectr., 2 (2014) PLAS-0023-2014 and Nils Hulter et al., An evolutionary perspective on plasmid lifestyle modes, Current Opinion in Microbiology, Volume 38, August 2017, Pages 74-80, respectively, the entirety of which is incorporated herein by reference). According to some embodiments, the present invention includes the use of all conventional selection and stabilization methods known to those skilled in the art.

[0065] In one embodiment, the present disclosure provides a method for treating or improving the effects of a mammalian skin disease requiring such treatment, comprising providing a microorganism genetically modified to express one or more LEKTI protein domains on the surface of mammalian skin, wherein the LEKTI protein domains are effective in penetrating one or more layers of mammalian skin and are effective in inhibiting the activity of at least one serine protease in or on mammalian skin. According to some embodiments, the microorganism is adapted to survive on the surface of mammalian skin for a controlled period of time and to provide a continuous supply of LEKTI protein domains.

[0066] In another embodiment, the Disclosure provides (1) a composition comprising a microorganism genetically modified to express one or more LEKTI protein domains, wherein the LEKTI protein domains are effective in penetrating one or more layers of mammalian skin and are effective in inhibiting the serine protease activity of at least one serine protease in or on mammalian skin; and (2) a kit for treating or improving the effects of a mammalian skin disease requiring it, comprising reagents for applying the composition to mammalian skin. According to some embodiments, the microorganism is adapted to survive on the surface of mammalian skin for a controlled period of time and to result in a continuous supply of LEKTI protein domains.

[0067] In addition to the components described above, the kit in question will further include instructions for the use of the components and / or for carrying out the methods in question. These instructions may be present in the kit in question in various forms, and one or more of these forms may be present in the kit. One form in which these instructions may be present is printed information relating to a preferred medium or substrate, such as a piece of paper or multiple pieces of paper on which the information is printed, in the kit packaging or in the accompanying documentation. Yet another means is a computer-readable medium, such as a diskette or CD, on which the information is recorded. Yet another means in which the instructions may be present is the address of a website used via the Internet to access the information at a transferred site. Any conventional means may be present in the kit.

[0068] The components of the kit can be packaged in an aqueous medium or in a lyophilized form. The kit will generally be packaged to include at least one vial, test tube, flask, bottle, syringe or other container means that can contain and, preferably, suitably ali-coat the described reagents. If additional components are provided, the kit will also generally include a second, third, or other additional container that can contain such components.

[0069] The kits of this disclosure will typically also include means for containing tightly sealed reagent containers for commercial sale. Such containers may include injection or blow-molded plastic containers that hold the desired vials.

[0070] formulation According to several embodiments, a formulation for use according to the present invention comprises a pharmaceutically effective amount of recombinant bacteria producing a therapeutically effective amount of a desired polypeptide or its therapeutically effective domain, for example, at least about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.2% by weight, about 0.3% by weight, about 0.4% by weight, about 0.5% by weight, about 0.6% by weight, about 0.7% by weight, about 0.8% by weight, about 0.9% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 3.0% by weight, about 4.0% by weight, and about 5.0% by weight. It may contain recombinant bacteria in amounts of approximately 6.0% by weight, 7.0% by weight, 8.0% by weight, 9.0% by weight, 10.0% by weight, 11.0% by weight, 12.0% by weight, 13.0% by weight, 14.0% by weight, 15.0% by weight, 16.0% by weight, 17.0% by weight, 18.0% by weight, 19.0% by weight, 20.0% by weight, 25.0% by weight, 30.0% by weight, 35.0% by weight, 40.0% by weight, 45.0% by weight, or 50.0% by weight or more, with the upper limit being approximately 90.0% by weight of recombinant bacteria.

[0071] According to some embodiments, formulations for use according to the present invention may contain, 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, and about 1% to about 5% by weight or more of recombinant bacteria.

[0072] According to some embodiments, topical formulations may be in any form suitable for application to the body surface, such as creams, lotions, sprays, solutions, gels, ointments, pastes, plasters, coatings, bioadhesives, suspensions, and emulsions, and / or may be prepared to contain liposomes, micelles, and / or microspheres. Such formulations can be used in combination with a closed coating layer such that moisture evaporating from the body surface is maintained in the formulation during and after application to the body surface. According to some embodiments, the formulation may comprise a composition of living cell cultures, and may comprise at least one engineered bacterial strain that produces a therapeutically effective recombinant polypeptide or a therapeutically effective domain thereof. This engineered living cell culture composition can deliver polypeptides directly to the skin to treat or prevent abnormal skin conditions.

[0073] Topical formulations include any other active ingredients 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 a vehicle may include water, aqueous buffer solutions, non-aqueous solvents (ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycoflor, or glycerol), oils (e.g., mineral oils such as liquid paraffin, natural or synthetic triglycerides such as Miglyol®, or silicone oils such as dimethicone). In particular, depending on the properties of the formulation and its intended use and application site, the dermatological vehicle used may contain one or more components selected from the following list: solubilizers or solvents (e.g., β-cyclodextrins such as hydroxypropyl β-cyclodextrin, or alcohols or polyols such as ethanol, propylene glycol or glycerol); thickeners (e.g., hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose or carbomer); gelling agents (e.g., polyoxyethylene-polyoxypropylene copolymer); preservatives (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, benzoates, potassium sorbate or EDTA or salts thereof); and pH buffers (e.g., a mixture of dihydrogen phosphate and hydrogen phosphate, or a mixture of citric acid and hydrogen phosphate) (for example, components added to water if the formulation is an aqueous gel).

[0074] Pharmaceutically acceptable carriers may be included in the formulations of the present invention, and these may be any carriers conventionally used in the art. Examples include water, lower alcohols, higher alcohols, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based and emulsion-based mixtures of such carriers. As used herein, the terms “pharmaceutically acceptable” or “pharmaceutically acceptable carrier” refer to a compound or composition that can be included in a pharmaceutical formulation without causing undesirable biological effects or undesirable interactions with other components of the formulation, and as used herein, “carrier” or “vehicle” refer to a carrier material suitable for incorporation into a topically applied composition. Useful carriers and vehicles as used herein include any such material known in the art that is non-toxic and does not interact in a harmful manner with other components of the formulation in which it is contained. The term “aqueous” refers to a formulation that contains water or becomes water-containing after application to skin or mucous membrane tissue.

[0075] A film-forming agent forms a protective film on the application site as it dries. This film inhibits the removal of the active ingredient and maintains contact between it and the treated site. An example of a film-forming agent suitable for use in the present invention is Flexible Collodion, US P., described on page 1530 of Remington: The Science and Practice of Pharmacy, 19th edition (Easton, PA: Mack Publishing Co., 1995), where collodion is an ethyl ether / ethanol solution containing pyroxylin (nitrocellulose) which evaporates to release a pyroxylin film. Film-forming agents can also act as carriers. Solutions that dry to form a film are sometimes called coatings. A cream, as is well known in the pharmaceutical industry, is a viscous liquid or semi-solid emulsion of oil in water or water in oil.

[0076] The cream base is washable with water and contains an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the “internal” phase, typically contains petrolatum and fatty alcohols such as cetyl or stearyl alcohol. The aqueous phase is usually not essential, but it typically exceeds the volume of the oil phase and generally contains a humectant. The emulsifier in the cream formulation is typically a nonionic, anionic, cationic, or amphoteric surfactant.

[0077] A lotion is a preparation applied to the skin surface without friction, and is typically a liquid or semi-liquid preparation in which particles containing an active agent are present in a water or alcohol base. Lotions are usually suspensions of solids, preferably containing an oil-in-water liquid emulsion. Because lotions are easier to apply in larger quantities, they are the preferred formulations herein for treating larger body areas. Insoluble substances in lotions generally need to be finely fragmented.

[0078] The lotion will typically contain suspending agents to provide better dispersion, as well as compounds useful for localizing and retaining active ingredients in contact with the skin, such as methylcellulose, ethoxymethylcellulose sodium, etc.

[0079] A solution is a homogeneous mixture prepared by dissolving one or more chemical substances (solutes) in a liquid such that the molecules of the dissolved substance are dispersed among the molecules of the solvent. A solution may contain other pharmaceutically or cosmetically acceptable chemical substances to buffer, stabilize, or retain the solute. Common examples of solvents used in preparing a solution are ethanol, water, propylene glycol, or any other acceptable vehicle. As is well known, gels are semi-solid suspension systems. Single-phase gels typically contain organic polymers substantially uniformly distributed through a carrier liquid, which is typically aqueous, but preferably alcohol, and optionally oil as well. Preferred “organic polymers,” i.e., gelling agents, are polymers of the “carbomer” family, such as cross-linked acrylic polymers like carboxypolyalkylenes, commercially available under the Carbopol trademark. Also preferred are hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer and polyvinyl alcohol; cellulosic polymers such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate and methylcellulose; rubbers such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a uniform gel, a dispersant such as alcohol or glycerin may be added, or the gelling agent may be dispersed by grinding, mechanical mixing or stirring, or a combination thereof. Ointments are, as is also well known in the art, typically semi-solid preparations based on petrolatum or other petroleum derivatives. The specific ointment base used provides several desirable characteristics, such as emollient properties, as will be understood by those skilled in the art. Like other carriers or vehicles, the ointment base should be inert, stable, non-irritating, and non-sensitizing.As described in Remington: The Science and Practice of Pharmacy, 19th edition (Easton, PA: Mack Publishing Co., 1995), pages 1399–1404, ointment bases can be classified into four classes: fatty bases; emulsifying bases; emulsion bases; and water-soluble bases. Examples of fatty ointment bases include vegetable oils, animal fats, and semi-solid hydrocarbons derived from petroleum.

[0080] Emulsifying ointment bases, also known as absorbent ointment bases, contain little to no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum.

[0081] Emulsion ointment bases are water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, and include, for example, acetyl alcohol, stearyl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycol with varying molecular weights; for further information, see Remington: The Science and Practice of Pharmacy.

[0082] A paste is a semi-solid dosage form in which an active agent is suspended in a suitable base. Depending on the properties of the base, pastes are divided into those made from fatty pastes and those made from monophase aqueous gels. The base in fatty pastes is generally petrolatum or hydrophilic petrolatum. Pastes made from monophase aqueous gels generally contain carboxymethylcellulose as the base.

[0083] The accelerator is typically a lipophilic co-accelerator called a “plasticity” accelerator, i.e., an accelerator having a molecular weight in the range of about 150 to 1000, aqueous solubility of less than about 1 wt%, preferably less than about 0.5 wt%, and most preferably less than about 0.2 wt%. The Hildebrandt solubility parameter δ of the plasticity accelerator is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilic accelerators are fatty esters, fatty alcohols, and fatty ethers. Examples of particular most preferred fatty acid esters include methyl laurate, ethyl oleate, propylene glycol monolaurate, propylene glycol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Examples of fatty alcohols include stearyl alcohol and oleyl alcohol, while examples of fatty ethers include diols or triols, preferably compounds in which a C2-C4 alkanediol or triol is substituted with one or two fatty ether substituents.

[0084] Further penetration enhancers are known to those skilled in the art of topical drug delivery and / or described in relevant textbooks and literature. See, for example, Percutaneous Penetration Enhancers, Smith et al. (eds.) (CRC Press, 1995) (incorporated herein by reference).

[0085] In addition to those identified above, various other additives may be included in the compositions of the present invention. These include, but are not limited to, antioxidants, astringents, fragrances, preservatives, emollients, pigments, dyes, humectants, propellants, and sunscreens, as well as other classes of materials whose presence is pharmaceutically desirable or otherwise desirable. Typical examples of additives as needed for inclusion in the formulation of the present invention are as follows: preservatives such as sorbates; solvents such as isopropanol and propylene glycol; astringents such as menthol and ethanol; emollients such as polyalkylene methyl glucoside; humectants such as glycerin; emulsifiers such as glyceryl stearate, PEG-100 stearic acid, polyglyceryl-3-hydroxylauryl ether, and polysorbate 60; other polyhydroxy alcohols such as sorbitol and polyethylene glycol; sunscreens such as octyl methoxyl cinnamate (commercially available as Parsol MCX) and butyl methoxybenzoylmethane (available under the trademark Parsol 1789); ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζ ι - Tocopherol, Ζ ΛAntioxidants such as -tocopherol, η-tocopherol, and retinol (vitamin A); essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g., soybean oil, coconut oil, liquid fraction of shea butter, sunflower oil), animal oils (e.g., perhydrosqualene), synthetic oils, silicone oils or waxes (e.g., cyclomethicone and dimethicone), fluorinated oils (generally perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes (e.g., beeswax, carnauba wax, and paraffin wax); skin texture modifiers; and thickeners and structuring agents such as cross-linked carboxypolyalkylenes, which can be commercially obtained under the trademarks of Swelling Clay and Carbopol. Other additives include beneficial agents such as materials that tone the skin (especially the upper layers of skin in the stratum corneum), keep it soft by delaying the decrease in its moisture content, and / or protect the skin. Examples of such conditioners and moisturizers include pyrrolidinecarboxylic acid 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 glycylretinic acid; anti-seborrheic agents such as retinoic acid; vasodilators such as nicotinic acid; melanin-forming inhibitors such as kojic acid; and mixtures thereof. Further additional active agents include, for example, alpha hydroxy acids, alpha keto acids, polymeric hydroxy acids, moisturizers, collagen, marine extracts, and ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζ ιAntioxidants such as α-tocopherol, ζ2-tocopherol, η-tocopherol, and retinol (vitamin A), as well as / or pharmaceutically acceptable salts, esters, amides, or other derivatives thereof. A preferred tocopherol compound is α-tocopherol. Additional agents include, for example, those that can improve oxygen supply to skin tissue, as described in Gross et al. WO94 / 00098 and Gross et al. WO94 / 00109 (incorporated herein by reference), both assigned to Lancaster Group AG. Sunscreens and UV-absorbing compounds may also be included. Non-limiting examples of such sunscreens and UV-absorbing compounds include aminobenzoic acid (PABA), avobenzone, cinoxate, dioxybenzone, homosalate, menthyl anthranilate, octocrylene, octyl methoxycinnamate, octyl salicylate, oxybenzone, padimate O, phenylbenzimidazole sulfate, surisobenzone, titanium dioxide, trolamine salicylate, zinc oxide, ensulizole, melazilate, octinoxate, octisalate, and octocrylene. See Title 21, Chapter 1, Subchapter D, Part 352, “Sunscreen drug products for over-the-counter human use,” which is incorporated herein in its entirety.

[0086] Other embodiments may include a variety of non-carcinogenic, non-irritating healing materials to facilitate treatment with the formulations of the present invention. Such healing materials may include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, glandular extracts or animal extracts, or safe therapeutic agents that can be added to the formulations to facilitate the healing of skin disorders.

[0087] The amounts of these various additives are those conventionally used in the field of cosmetics, ranging, for example, from approximately 0.01% to approximately 20% of the total weight of the topical formulation.

[0088] The formulations of the present invention may also contain conventional additives such as opacifiers, fragrances, colorants, stabilizers, and surfactants. In certain embodiments, other agents such as antimicrobial agents may be added to prevent spoilage during storage, i.e., to inhibit the growth of microorganisms such as yeast and mold.

[0089] Suitable antimicrobial agents are typically selected from the group consisting of methyl and propyl esters of p-hydroxybenzoic acid (i.e., methyl and propylparaben), sodium benzoate, sorbic acid, imidourea, and combinations thereof. In other embodiments, repressors and inducers, i.e., other agents to inhibit (i.e., glycose) or induce (i.e., xylose) the production of the polypeptide of interest, may also be added. Such additives may be used on the condition that they are compatible with the function of the formulation and do not inhibit it.

[0090] The formulation may also contain irritation-reducing additives to minimize or eliminate the potential for skin irritation or skin damage resulting from the administered chemical or other components of the composition.

[0091] Suitable irritation-reducing additives include, for example, α-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcohols such as 2-phenyl-1-ethanol; salicylates; ascorbic acid salts; ionophores such as monensin; amphoteric amines; ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine. If present, irritation-reducing additives can be included in the composition at concentrations effective in reducing irritation or skin damage, typically accounting for about 20 wt% or less of the formulation, and more typically about 5 wt% or less.

[0092] Further preferred pharmacologically active substances that can be incorporated into the formulation in certain embodiments and thus applied topically together with the active agent include, but are not limited to, the following: agents that improve or eliminate pigmented or non-pigmented age spots, keratin, and wrinkles; antimicrobial agents; antibacterial agents; antipruritic and anti-drying agents; anti-inflammatory agents; local anesthetics and analgesics; corticosteroids; retinoids; vitamins; hormones; and antimetabolites.

[0093] Some examples of topical pharmacologically active substances include acyclovir, amphotericin, chlorhexidine, clotrimazole, ketoconazole, econazole, miconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin, phenytoin, para-aminobenzoate, octyl methoxycinnamate, octyl salicylate, oxybenzone, dioxybenzone, tocopherol, tocopherol acetate, selen sulfate, zinc pyrithione, diphenylhydramine, pramoxin, lidocaine, procaine, erythromycin, tetracycline, clindamycin, crotamiton, and hydro Examples include quinones and their monomethyl and benzyl ethers, naproxen, ibuprofen, cromolyn, retinol, retinyl palmitate, retinyl acetate, coal tar, griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetic acid, hydrocortisone 17-valeric acid, hydrocortisone 17-butyric acid, progesterone, betamethasone valerate, betamethasone dipropionate, triamcinolone acetonide, fluocinonide, clobetasol propionate, minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide, and 5-fluorouracil.

[0094] Creams, lotions, gels, ointments, and pastes can be spread and gently rubbed onto the affected surface. Solutions can be applied in a similar manner, but more typically, they are applied carefully to the affected area using a dropper, swab, or similar tool.

[0095] The application regimen will depend on several easily determinable factors, such as the severity of the condition and its response to initial treatment, but will typically involve continuous application at least once per day. Those skilled in the art will be able to easily determine the optimal dose, method of administration, and rate of repetition of the formulation to be administered. Generally, the formulation of the present invention is intended to be administered at a rate ranging from once or twice per week to a maximum of once or twice per day.

[0096] The pharmaceutical compositions of the present invention comprise one or more active ingredients, e.g., therapeutic agents, in a mixture of one or more pharmaceutically acceptable diluents or carriers and, optionally, one or more other compounds, drugs, components, and / or materials. Regardless of the chosen route of administration, the drugs / compounds of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).

[0097] Pharmaceutically acceptable diluents or carriers are well known in the industry (see, for example, Remington, The Science and Practice of Pharmacy (21st edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, DC)), sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starch, cellulose preparations, calcium phosphate (e.g., dicalcium phosphate, tricalcium phosphate, and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline solution, 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). Examples include polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoester), and poly(anhydride)), elastomer 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 waxes), paraffins, silicones, talc, salicylates, and the like. Each pharmaceutically acceptable diluent or carrier used in the pharmaceutical composition of the present invention must be "acceptable" in the sense that it is compatible with the other components of the formulation and is not harmful to the subject. Suitable diluents or carriers for the selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for the selected dosage form and method of administration can be determined using ordinary knowledge in the art.

[0098] The pharmaceutical composition of the present invention may optionally contain additional components and / or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the industry 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 acacia; (3) humectants such as glycerol; (4) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethylcellulose, and sodium carbonate; (5) dissolution retarders such as paraffin; (6) absorption enhancers such as quaternary ammonium compounds; (7) humectants 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 glycol, and sodium lauryl sulfate; (10) ethoxylated isostearyl (11) Buffering agents such as alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methhydroxyl, bentonite, agar and tragacanth; (12) Lactose, lactose, polyethylene glycol, animal fats and vegetable fats, oils, waxes, paraffins, cocoa butter, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylates, zinc oxide, agar hydroxide Excipients such as luminium, calcium silicates, and polyamide powders; (13) Inert diluents such as water or other solvents; (14) Preservatives; (15) Surfactants; (16) Dispersants; (17) Controlled release agents or absorption retarders such as hydroxypropyl methylcellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) Emulsifiers; (19) Adjuvants; (20) Wetting agents; (21) Emulsifiers and suspending agents;(22) Solubilizers and emulsifiers 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) Propellantizers such as chlorofluoro hydrocarbons and volatile unsubstituted hydrocarbons, e.g., butane and propane; (24) Antioxidants; (25) Substances that make the preparation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) Thickeners; (27) Coating materials such as lecithin; and (28) Sweeteners, flavorings, colorants, fragrances, and preservatives. Each such component or material must be “acceptable” in the sense that it is compatible with the other components of the preparation and is not harmful to the subject. Suitable ingredients and materials for the selected dosage form and intended route of administration are well known in the industry, and acceptable formulations and materials for the selected dosage form and method of administration can be determined using ordinary knowledge in the industry.

[0099] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, infusions, and inhalations. The active agent / compound can be mixed with a suitable pharmaceutically acceptable diluent or carrier under sterile conditions. Ointments, pastes, creams, and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0100] Pharmaceutical compositions of the present invention, suitable for parenteral administration, may contain one or more agents / compounds together with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersants, suspensions or emulsions, or sterile powders, which can be reconstituted into a sterile injectable solution or dispersant immediately before use, which may contain suitable antioxidants, buffers, formulations, and solutes, or suspending agents or thickeners that make the mixture isotonic with the intended recipient's blood. Appropriate fluidity can be maintained, for example, by the use of coating materials, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These pharmaceutical compositions may also contain suitable adjuvants, such as wetting agents, emulsifiers, and dispersants. It is also desirable to include isotonic agents. Furthermore, extension of absorption of the injectable pharmaceutical form can be achieved by including substances that delay absorption.

[0101] The following embodiments are provided to further illustrate the methods of the present invention. These embodiments are illustrative and are not intended to limit the scope of the present invention in any way. [Examples]

[0102] bacteria In some embodiments, the bacterium Staphylococcus aureus RN4220 strain can be used in vector preparation (Kreiswirth, BN et al., 1983). In some such embodiments, a stock solution of the strain is stored in LB or TS broth in 50% glycerol at -20°C.

[0103] According to some embodiments, the bacterium Staphylococcus epidermidis strain ATCC 12228 or NRRL B-4268 can be used (Zhang, YQ. et al., 2003). In some such embodiments, a stock solution of the strain is stored at -20°C in LB broth or TS broth in 50% glycerol. The bacteria are cultured in LB broth or TS broth. After 16 hours of incubation, the bacteria are harvested by centrifugation and 2 x 10⁶ 9 Concentrate 10-fold in LB broth or TS broth at a concentration of 1 bacteria / 100 μl. Prepare a bacterial stock preparation by inoculating 5 mL of broth with S. epidermidis and grow it overnight at 30°C. Then, add 3 mL of the fully grown culture to 1 mL of 60% glycerol and store at -80°C.

[0104] Expression vector According to some embodiments, the plasmid construct pKK30-LEKTI-complete may include a pKK30 vector along with a LEKTI domain insertion. According to some embodiments, the LEKTI domain can be functionally ligated to a SecA secretion signal, a 6xHis tag, and / or an RMR cell permeable sequence and expressed under the control of a chloramphenicol resistance (CmR) promoter sequence (derived from pDB114E). In some embodiments, the pKK30 vector includes a dihydrofolate reductase (dfrA) selector gene.

[0105] Transformation According to some embodiments, a vector carrying the LEKTI sequence can be used to transform S. epidermidis strains. A vector carrying the LEKTI sequence can be prepared / transformed by a method comprising the steps of preparing competent S. aureus bacterial cells, transforming S. aureus, isolating plasmid DNA from S. aureus, preparing competent S. epidermidis bacterial cells, transforming S. epidermidis, growing the transformed S. epidermidis bacteria, and preserving the transformed S. epidermidis.

[0106] In some embodiments, alternative intermediate strains may also be used for the transformation and isolation of plasmid DNA in the preparation for transformation into S. epidermidis. These strains may include, but are not limited to, other bacteria, including methylation-deficient strains, particularly E. coli strains.

[0107] According to some embodiments, S. aureus RN4220 cells can be made electrocompetent by growing a 50 ml culture overnight in LB or TS medium at 37°C, and then inoculating 10 ml of the overnight culture into 100 ml of fresh LB or TS medium. OD 600 When the level reaches 0.2-0.3, allow the cells to settle and resuspend them in 1x volume of 10% sucrose at 4°C. Repeat this process three times, then resuspend the cells in 0.1x volume of 10% sucrose at 4°C, allow them to settle, and resuspend them in 1 ml of 10% sucrose.

[0108] For the transformation of RN4220, 200-500 μg of LEKTI plasmid (e.g., pKK30-LEKTI-complete) can be mixed with electrocompetent cells and transformed using electroporation at 2.5 kV at room temperature with a MicroPulser Electroporator (Bio-Rad, Hercules, CA). The transformed cells are seeded overnight on selective LB or TB medium at 28°C, grown overnight in selective LB or TB medium, and then used to isolate the DNA.

[0109] According to several embodiments, electrocompetent S. epidermidis ATCC 12228 or NRRL B-4268 is prepared using the following method. First, a 50 ml overnight culture of ATCC 12228 or NRRL B-4268 from a -80°C stock is grown at 37°C in B2 medium (1.0% tryptone, 2.5% yeast extract, 0.5% glucose, 2.5% NaCl, 0.1% K2PO4, pH 7.5). A 10 ml overnight culture is diluted in fresh, pre-warmed B2 medium and OD 600 Shake until the volume reaches 0.5-0.6, then allow to settle at 4°C for 10 minutes. Next, wash the cells with 1, 1 / 2, 1 / 20, and 1 / 50 volumes of cold 10% glycerol, allowing to settle at 4°C between washes. Resuspend the final pellet in 700 μl of cold 10% glycerol.

[0110] According to some embodiments, electrocompetent ATCC 12228 or NRRL B-4268 are transformed using pKK30-LEKTI-complete isolated from S. aureus by electroporation at 2.5kV, 25μF, and 100Ω (a normal reading is 4.5–5 msec using a Micropulser Electroporator (Bio-Rad, Hercules, CA)). The cells are then seeded on selective LB or TB medium at 28°C. In some embodiments, bacterial transformation can also be carried out by alternative transformation methods, including, but not limited to, alternative intermediate strains, bacteriophage transduction, and heat shock.

[0111] Analysis of protein expression According to several embodiments, transformed cells are fractionated and analyzed by SDS-PAGE electrophoresis and Western blotting. Bacterial cells expressing recombinant LEKTI and bacterial control cells are precipitated and lysed with CelLytic B Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO). The supernatant derived from the induced samples is collected and concentrated. The samples are resuspended in reducing sample buffer and then electrophoresed on a 4-15% Tris-acrylimide gel using Tris-HCl electrophoresis buffer. After electrophoresis, the gel is transferred to a PVDF membrane and sequentially scanned for LEKTI domains 8-11 using primary goat monoclonal antibodies or His tags. Subsequently, horseradish peroxidase conjugate donkey anti-goat antibodies (sc-2020) are scanned, and secondary antibodies are detected by autoradiography (Syngene GeneGnome Bio Imaging System) using an enhanced chemiluminescent substrate (SuperSignal West Pico, Thermo Scientific).

[0112] Analysis of the supernatant and cell lysates demonstrates the successful expression and secretion of the therapeutic polypeptide during transformation using a plasmid containing the target protein. Detection of protein expression and secretion may also be possible using alternative methods, and this example should not be construed as a limitation to the present invention.

[0113] Treatments for Humans According to some embodiments, 1 x 10 9 Colony-forming units (CFUs) of S. epidermidis can be added to a pharmaceutically acceptable carrier. The composition is useful for treating or preventing abnormal skin conditions resulting from Netherton syndrome in subjects requiring it. The composition can be applied at least once a day, for example, up to about 3-4 times a day, or as needed, or as prescribed. In some embodiments, only one application is required to achieve a therapeutic effect. The composition can be used for as long as necessary to ensure treatment of the condition or to continue preventing the condition. The duration of treatment may vary from about 1 day to a maximum of about 10-14 days, or longer. In certain cases, long-term or chronic treatment may be administered. [Examples]

[0114] Testing of the serine protease inhibitory activity of recombinant LEKTI According to several embodiments, the protease inhibitory activity of recombinant LEKTI is tested for differences achieved when it is functionally linked to various secretory and cell-permeable peptides. According to some embodiments, certain combinations of secretory and cell-permeable peptides may have unpredictable effects on the protease inhibitory function of the LEKTI domain and may therefore be determined empirically.

[0115] In some embodiments, LEKTI domains D8–D11, ligated in a manner capable of functioning as secretory tags, 6xHis tags, and / or cell permeability tags, are cloned into insect expression vectors for large-scale production of purified recombinant proteins and evaluated for their inhibitory activity against one or more proteases (e.g., plasmin, cathepsin G, elastase, and trypsin).

[0116] Insect cells and reagents The following reagents can be commercially obtained as indicated: fall armyworm cell line Spodoptera frugiperda (Sf9), low-melting point agarose, cellFECTIN, pFASTBAC1, pCRII-TOPO, competent Escherichia coli DH10BAC, cabbage looper egg cell line Trichoplusia ni 5B1-4 (High Five), and ultimate serum-free insect medium from Invitrogen (Carlsbad, CA); restriction endonuclease from New England Biolabs (Beverly, MA); TALON Superflow from Clontech Laboratory (Palo Alto, CA); Insect-XPRESS medium and fetal bovine serum from BioWhittaker (Walkersville, MD); YM10 Centriplus from Millipore Corp. (Bedford, MA); precast SDS-PAGE gels, protein assay kits, SEC-250 size columns, and Bio-Rad Pre-staining markers from Hercules (CA); BSA from Kabi Pharmacia (Franklin, OH); DTT and glycerol from Boehringer Mannheim Biochemicals (Indianapolis, IN); and 5xHis mAb and 6xHis-tagged protein ladder from QIAGEN Inc. (Valencia, CA).

[0117] Cloning and expression of LEKTI D8-D11 A 6xHis-tagged LEKTI domain (e.g., Sequence ID No. 1), ligated in a manner capable of functioning with various permutations of secretory and cell-permeable peptides, can be cloned into the pFASTBAC1 vector according to the manufacturer's instructions. Recombinant LEKTI-complex viruses are then generated, as previously described by Gao, M. et al., (1996) J. Biol. Chem. 271, 27782-27787, which is incorporated herein by reference in its entirety. To test the recombinant LEKTI-complex viruses for recombinant LEKTI expression, Sf9 cells can be infected with the recombinant virus at various infection multiplicities, and cell lysates and media can be collected every 24–96 hours. The presence of histidine-tagged proteins can be confirmed by Western blotting analysis using a 5xHis mAb against a 6x histidine tag, according to the manufacturer's recommendations. The LEKTI-complex viruses showing the highest levels of expression can be selected for further experiments and spinner flasks.

[0118] Recombinant LEKTI protein can be produced on a large scale by infecting spinner cultures of Sf9 cells (1.6 billion cells) in Insect-XPRESS medium containing 10% serum with an infection multiplicity of 8 plaque-forming units (PFUs). Three days after infection, the cell pellet is harvested as previously described in Jayakumar, A. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 8695-8699, and Co 2+ Recombinant LEKTI can be selectively purified from cell lysates using a charged Sepharose affinity column (TALON), followed by SEC-250 size column chromatography. Fractions containing homogeneous LEKTI can be pooled and concentrated by ultrafiltration. The protein can then be quantified using the Bio-Rad Protein Assay Kit II.

[0119] Reagents and protocols for protease inhibition assays The following enzymes, chromogenic substrates, and reagents can be commercially obtained as indicated: human plasmin, human cathepsin L, human cathepsin S, human trypsin, human cathepsin G, human chymotrypsin, and human neutrophil elastase (HNE) from Athens Research & Technology, Inc. (Athens, GA); subtilisin A from Calbiome-Novabiochem (San Diego, CA); papain from Roche Molecular Biochemicals (Indianapolis, IN); furin from New England BioLabs; succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Succ-AAPF-pNA), succinyl-Ala-Ala-Val-pNA (Succ-AAVpNA), and D-Val-Leu-Lys-pNA (VLK-pNA) from Sigma Chemical Co. (St. Louis, MO); and Bachem Bioscience, Inc. (King of H-Glu-Gly-Arg-pNA (EGRpNA) and benzyloxycarbonyl-Phe-Arg-pNA (Z-FR-pNA) from Prussia, PA; and methoxy-Succ-Arg-Pro-Tyr-pNA (MeO-Succ-RPY-pNA) from Chromogenix Instrumentation Laboratory SpA (Milan, Italy). PBS reaction buffer (137 mM NaCl, 27 mM KCl, and 10 mM phosphate buffer (pH 7.4)) can be used with trypsin, plasmin, cathepsin G, HNE, and chymotrypsin. Cathepsin reaction buffer (0.1% CHAPS, 50 mM sodium acetate (pH 5.5), 1 mM EDTA) can be used with cathepsin K, L, and S, as well as papain. A unique reaction buffer can be used with subtilisin A (PBS and 0.1% Tween 20).

[0120] The ability of recombinant LEKTI to block the cleavage of small chromogenic peptide substrates, determined by the spectroscopic techniques previously described in Schick, C. et al. (1998) Biochemistry 37, 5258-5266, which is incorporated herein by reference in its entirety, allows for the detection of protease inhibitory activity. The protease can be pre-incubated with recombinant LEKTI for 2 minutes at 25°C in 100 μL of assay buffer, after which the inhibition of the protease can be evaluated. This mixture can be added to 890 or 880 μL of assay buffer in a 1 mL quartz cuvette. Protease activity can be initiated by adding 10 - 20 μL of the appropriate pNA substrate. The change in absorbance at 405 nm (A 405 =8.8 10 -3 M -1 cm -1 ) can be followed over 10 minutes using a spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). The rate changes (ΔA405 / min) of the inhibited and control reactions can be determined from the rate plots.

[0121] According to some embodiments, different combinations of secretion tags and cell-penetrating tags can elicit different LEKTI protease activities against each of the proteases being tested (e.g., trypsin, plasmin, cathepsin G, HNE, subtilisin A, and chymotrypsin). Furthermore, individual combinations of secretion tags and cell-penetrating tags can elicit different LEKTI protease activities among individual proteases.

Example

[0122] Transduction Peptide-Mediated Delivery According to some embodiments, various combinations of secretion tags and cell-penetrating tags can affect, more or less, the ability of recombinant LEKTI proteins to cross the cell membrane. Thus, various recombinant LEKTI products can be tested in cell culture to evaluate the effects of various combinations of secretion tags and cell-penetrating tags.

[0123] According to some embodiments, adherent fibroblast cell lines HS-68, NIH-3T3, 293, Jurkat T, or Cos-7 can be cultured at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% (vol / vol) 200 mM glutamine, 1% (vol / vol) antibiotics (streptomycin, 10,000 μg / ml; penicillin, 10,000 IU / ml), and 10% (wt / vol) FBS. For peptide-mediated delivery of recombinant LEKTI protein, purified recombinant LEKTI product (obtained above) can be loaded into DMEM or PBS (500 μl of DMEM containing 0.25 μg of protein) and incubated at 37°C for 30 minutes. Cells then grown to a 75% concentration density are coated with these recombinant LEKTI protein media. After incubation at 37°C for 30 minutes, without removing the recombinant LEKTI protein overlay, 1 ml of fresh DMEM supplemented with 10% FBS is added to the cells, and the cells are returned to the incubator for another 30 minutes. The cells are then thoroughly washed with PBS and examined for recombinant LEKTI protein. The cells can be observed by immunofluorescence by first fixing with 2% formalin (Sigma), permeabilizing, and then incubating with primary anti-6x His-tagged antibody and secondary antibody according to the manufacturer's instructions. Alternatively, cell lysates can be obtained as described above, and the presence of His-tagged recombinant LEKTI can be observed by Western blotting.

[0124] According to some embodiments, specific combinations of secretory proteins and permeable peptides have different effects on the ability of recombinant LEKTI proteins to cross the cell membrane. [Examples]

[0125] The LEKTI protein requires proteolytic cleavage to activate its inhibitory function against many 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 into D6–D9 and D10–D15, →D6 and D7–D9→D7 and D8–D9→D8. A schematic diagram of the full-length LEKTI polypeptide, domains, and native cleavage products is shown in Figure 3. In selecting the specific domain to express, the following criteria were considered: (1) activity against various kallikrein-related peptidases (KLK) such as KLK5 and KLK7; (2) protease resistance; (3) small size (not a metabolic burden); and (4) minimal disulfide bond content. Domain 6 was selected as the LEKTI fragment to express. The amino acid sequence of the full-length LEKTI protein is given as SEQ ID NO: 103, and each of the following 15 individual domains is described in fasta format.

[0126] LEKTI amino acid sequence residues 1-1064 (SEQ ID NO: 103): JPEG2023138994000011.jpg91165JPEG2023138994000012.jpg55164

[0127] The LEKTI domains are listed below: JPEG2023138994000013.jpg176166JPEG2023138994000014.jpg243166JPEG2023138994000015.jpg31166

[0128] The LEKTI nucleic acid sequence is described below as sequence number 119. LEKTI full-length nucleic acid sequence (SEQ ID NO: 119)

[0129] JPEG2023138994000016.jpg191168JPEG2023138994000017.jpg245166

[0130] Solubility in E. coli BL21(De3) Prokaryotes produce soluble and inclusion body-binding proteins. Solubility is influenced by temperature, protein charge, and protein structure and size. Insoluble inclusion body-binding proteins are often misfolded, are typically inactive, and are isolated within very pure, insoluble inclusion bodies. Inclusion body-binding proteins are isolated in vitro, refolded, and then purified. Soluble proteins are in their folded structure, are often functional, and reside in the cytoplasm along with the rest of the proteome.

[0131] To determine whether domain 6 is reliably produced in E. coli, a first set of experiments was performed. Soluble proteins were isolated and purified by affinity purification and buffer exchange. Solubility assays were used to determine the distribution between inclusion body proteins and the soluble protein fraction. Briefly, cells expressing domain 6 protein (E. coli BL21(De3)) were lysed with aqueous buffer. The soluble and inclusion body fractions were isolated using high-speed centrifugation and inclusion body purification. The isolated fractions were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 4 shows the SDS-PAGE results indicating that LEKTId6 (8.8 kDa) is highly soluble in E. coli BL21(De3). E. coli GFP (33.8 kDa) was used as a positive control, and no vector was used as a negative control. Experiments were performed at three different induction temperatures: 18°C, 30°C, and 37°C. As shown in Figure 4, an 8.8 kDa band was detected in the soluble fraction of the His6 LEKTId6 experimental group. The arrow indicates the 8.8 kDa band.

[0132] Figure 5 shows the SDS-PAGE results indicating that affinity purification was successfully performed for H6-LEKTId6 (8.8 kDa). The arrow indicates the 8.8 kDa band. Figure 6 shows that LEKTId6-H6 (8.8 kDa) can be N-terminally truncated. In both Figures 5 and 6, the following abbreviations were used for the experimental group: SN = Cleared cell lysate (supernatant). FT = Non-Ni2+ binding protein (leachate). W1-4 = Eluents from a series of washes (1-4). Note some contamination from neighboring ladders in W4. L = SDS-PAGE protein ladder (SeeBlue Plus2, ThermoFisher Scientific). E1-6 = Eluents from the column after imidazole treatment (i.e., the resulting affinity-purified proteins). Different eluent fractions (1-6) were collected when the column was treated. [Examples]

[0133] We evaluated the ability of purified recombinant LEKTI domain 6 (LEKTId6) fragments to function in vitro as serine protease inhibitors.

[0134] First, the ability of recombinantly produced LEKTId6 to inhibit trypsin in vitro was determined. Enzyme activity was measured using BApNA (Nα-benzoyl-1-arginine-p-nitroanilide) as a trypsin-specific substrate. Figure 7A shows a schematic diagram of the assay. The assay was performed by mixing 80 μL of LEKTId6 at concentrations of 0.25, 2.5, and 25 μM with 20 μL of trypsin (35 μg / mL) and 100 μL of 2x trypsin assay buffer (100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 100 mM CaCl2, 0.02% Triton X-100, 500 μM L-BAPNA). In the reaction mixture, the final concentrations were LEKTId6 (0.1, 1, 10 μM); trypsin (3.5 μg / mL); assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 50 mM CaCl2, 0.01% Triton X-100); and L-BAPNA (250 μM). The reaction was allowed to proceed at 37°C for 15 minutes. Leupeptin, a trypsin inhibitor, was used as a positive control. Product formation was measured at 405 nm using a microplate reader. A blank control was used. Trypsin activity was defined as the rate of change in absorbance at 405 nm per minute (an indicator of L-BAPNA cleavage) under established conditions. As shown in Figure 7, LEKTI domain 6 inhibited trypsin activity in vitro.

[0135] Next, the effect of LEKTI domain 6 (ct Hist tag) on ​​trypsin inhibition was determined and compared with the effect of LEKTI domains 10-15 on trypsin inhibition. A trypsin inhibition assay was performed as described above, measuring enzyme activity using L-BAPNA (Nα-benzoyl-1-arginine-p-nitroanilide) as a trypsin-specific substrate. Figure 8 shows a schematic diagram of the assay. LEKTId6 (10, 30, 100, 1000 nm) or LEKTI domains 10-15 (10, 30, 100 nm) were mixed with L-BAPNA (final concentration 250 μM) at 25°C for 10 minutes. Leupeptin, a trypsin inhibitor, was used as a positive control. The ability of recombinantly produced LEKTId6 to inhibit kallikrein 7 and 5 (KRK7 and KRK5) in vitro was determined. Briefly, proteinases KLR7 and KLK5 were incubated with increasing concentrations of LEKTId6 at 25°C for 5 minutes, followed by the addition of their respective optimal peptide substrates: Suc-Arg-Pro-Tyr-p-nitro-anilide for KLK7 and D-Ile-Pro-Arg-p-nitro-anilide for KLK5. Product formation was measured at 405 nm using a microplate reader. A blank control was used. Schematic diagrams of the KLK7 and KLK5 assays are shown in Figures 9A and 10A, respectively. For KLK7, increasing concentrations of LEKTId6 (10, 30, 100, 300, 1000 nm) and increasing concentrations of LEKTId10-15 (10, 30, 100 nm) were used. The trypsin inhibitor leupeptin was used as a negative control. For KLK5, increasing concentrations of LEKTId6 (10, 30, 100, 300, 1000 nm) were used. As shown in Figure 9B, recombinant LEKTI domain 6 inhibits KLK7 in vitro, much like LEKTI domains 10-15. As shown in Figure 10B, recombinant LEKTId6 inhibits KLK5 in vitro at nanomolar concentrations.High concentrations of LETKId6 have been shown to be irritating, but this is not theoretically constrained, and may be due to the buffer components of the assay, particularly excess imidazole remaining in the LETKId6 sample after affinity purification. [Examples]

[0136] The efficacy of the therapeutic LEKTId6 S. epidermidis strain will be evaluated in a conditioned Netherton mouse model. Briefly, the inventors will verify the absence of LEKTI in the skin of Netherton syndrome mice (conditional SPINK5- / -) created by CRISPR after Cre recombination induction at weeks 1, 2, and 4. Mice with verified Netherton syndrome phenotype will be treated with topical application of recombinant LEKTI to resolve the skin condition present in the Spink5 conditional mutants. Firstly, the rationale for using purified LEKTI is to avoid dependence on the S. epidermidis strain construct, allowing the inventors to rapidly demonstrate the efficacy of topical application in vivo. Secondly, the inventors will evaluate the ability of purified S. epidermidis or LEKTI to demonstrate the value of probiotic encombination for sustained improvement. As a control, the inventors will topically encombine pre-Cre induction of SPINK5 conditional mutants in the same mice. To evaluate the effects of LEKTId6 in a mouse model, the inventors performed long-term assays (1x / week) and endpoint assays (3 weeks after fixation), where possible, to test whether the application of therapeutic S. Epidermidis resulted in (1) in vivo detection of LEKTI as measured by immunohistochemical analysis of the skin (endpoint), (2) reduced severity of skin disease as measured by DASI (long-term and endpoint), (3) improvement of TEWL (long-term) and permeability scores (endpoint), (4) improvement of skin morphology as measured by histological analysis (endpoint), and (5) changes in protein lysis activity as measured using colorimetric assays targeting KLK5 and KLK7 (endpoint).

[0137] Embedding by reference The entire disclosure of each patent document, including patent application documents, scientific papers, government reports, websites, and other references listed herein, is incorporated herein by reference in its entirety for any purpose. Any inconsistencies in terminology shall be governed by this Specified Publication. All sequence listings or sequence numbers disclosed herein are incorporated herein by reference in their entirety.

[0138] The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement those described herein.

[0139] Armengot-Carbo, M. et al. (2014) "The role of filaggrin in the skin barrier and disease development." Actas Dermosifiliogr Mar; 106 (2):86-95. Brachkova, MI, P. Marques, J. Rocha, B. Sepodes, MA Duarte and JF Pinto (2011). "Alginate films containing Lactobacillus plantarum as wound dressing for prevention of burn infection." J Hosp Infect 79(4): 375-377. Brown, SJ., & McLean, WH. (2012) J. Invest. Dermatol. 132, 751-62 Chen, YE., & Tsao, H. (2013) J. Am. Acad. Dermatol. 69, 143-155 Cheung AL, et al. (2004) "Regulation of virulence determinants in vitro and in vivo in Staphylococcus aureus." FEMS Immunological Medical Microbiology 40(1): 1-9 "DNA Recombination." Methods in Molecular Biology 745(XIV): 1-565. Gross, et al, WO 94 / 00098, assigned to Lancaster Group AG Gross, et al, WO 94 / 00109, assigned to Lancaster Group AG Gueniche, A., P. Bastien, J. M. Ovigne, M. Kermici, G. Courchay, V. Chevalier, L. Breton and I. Castiel-Higounenc (2010). "Bifidobacterium longum lysate, a new ingredient for reactive skin." Exp Dermatol 19(8): 1-8. Jeong JG et al. (2011). A Tat- grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA. Biochem Biophys Res Commun. Mar 18;406(3):403-7. Kreiswirth, BN., et al. (1983). The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305:709-712. Lauderdale, et al. (2010). Biofilm dispersal of community-associated methicillin-resistant Staphylococcus aureus on orthopedic implant material. J. Orthop. Research. 28:55-61 Lee, SH., Jeong, SK. and Ahn, SK. (2006). "An update of the defensive barrier function of skin." Yonsei Med J 47(3): 293-306. Lin, YT., Wang, CT., and Chiang, BL. (2007). "Role of bacterial pathogens in atopic dermatitis." Clin Rev Allergy Immunol 33(3): 167-177. Ma, J., et a / .(2014) Cell-penetrating peptides mediated protein cross -membrane delivery and its use in bacterial vector vaccine. Fish & Shellfish Immunology 39 8-16 McAleer, MA., & Irvine, AD. (2013) J. Allergy Clin. Immunol. 131, 280-91. Mitsudo K. et al., (2003) “Inhibition of Serine Proteinases Plasmin, Trypsin, Subtilisin A, Cathepsin G, and Elastase by LEKTI: A Kinetic Analysis”, Biochemistry, 42, 3874-3881 Monk, I., et al. (2012) Direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. mBio. Muizzuddin, N., Maher, W., Sullivan, M., Schnittger, S., and Mammone, T. (2012). "Physiological effect of a probiotic on skin." J Cosmet Sci 63(6): 385-395. Nakanishi, N., T. Oshida, S. Yano, K. Takeda, T. Yamaguchi and Y. Ito (1986). "Construction and characterization of new cloning vectors derived from Streptomyces griseobrunneus plasmid pBTl and containing amikacin and sulfomycin resistance genes." Plasmid 15(3): 217-229. Nakatsuji, T. and R. L. Gallo (2014). "Dermatological therapy by topical application of non-pathogenic bacteria." J Invest Dermatol 134(1): 11-14. Oehike J et al. (1998).Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically. Biochim Biophys Acta. Nov 11; 1414(1-2): 127-39. Ostenson CG et al. (1997).Galparan: a powerful insulin-releasing chimeric peptide acting at a novel site. Endocrinology . Aug;138(8):3308-13. Otsuka, A., et al. (2014) J. Allergy Clin. Immunol. 133, 139-46.el-10 (2014). Peral, M. C, M. A. Martinez and J. C. Valdez (2009). "Bacteriotherapy with Lactobacillus plantarum in burns." Int Wound J 6(1): 73-81. Peral, M. C, M. M. Rachid, N. M. Gobbato, M. A. Huaman Martinez and J. C. Valdez (2010). "Interleukin-8 production by polymorphonuclear leukocytes from patients with chronic infected leg ulcers treated with Lactobacillus plantarum." Clin Microbiol Infect 16(3): 281-286 Powers, ME., et al. (2011). J Bacteriol, 193:340-348 Proksch, E., J. M. Brandner and J. M. Jensen (2008). "The skin: an indispensable barrier." Exp Dermatol 17(12): 1063-1072 Remington: The Science and Practice of Pharmacy, 19th edition. Easton, PA: Mack Publishing Co., 1995 Sambrook J, et al. (1989). Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Press, New York. Sambrook, JF., and Russell, DW., ed. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Vols 1, 2 and 3. Cold Spring Harbor Laboratory Press Simonen, M. and I. Palva (1993). "Protein secretion in Bacillus species." Microbiol Rev 57(1): 109-137 Smith, EW., & Maibach, HI., (1995) Percutaneous Penetration Enhancers, CRC Press ISBN 9780849321528 Stout, TE., et al .(2014) / Invest Dermatol. 134, 423-9 The Science and Practice of Pharmacy (1995), 19th Ed. Easton, PA: Mack Publishing Co. Volz, T., Y. Skabytska, E. Guenova, K. M. Chen, J. S. Frick, C. J. Kirschning, S. Kaesler, M. Rocken and T. Biedermann (2014). "Nonpathogenic bacteria alleviating atopic dermatitis inflammation induce IL- 10 -producing dendritic cells and regulatory Trl cells." J Invest Dermatol 134(1): 96-104 Webb, TR., & Hsu, CPS. US Patent. No. 4,659,774 assigned to American Hoechst Corporation Wyman TB, et al. (1997) Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry. March 11;36(10):3008-17 Zhang, YQ., et al. (2003). Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC12228). Molecular Microbiology 49(6), 1577-1593

[0140] While exemplary embodiments of the present invention have been described herein, it should be understood that the present invention is not limited thereto and that various other modifications or alterations can be made without departing from the scope or spirit of the invention by those skilled in the art.

[0141] This disclosure includes the following sequence information. SEQUENCE LISTING <110> AZITRA INC <120> COMPOSITIONS AND METHODS FOR TREATMENT OF NETHERTON SYNDROME WITH LEKTI EXPRESSING RECOMBINANT MICROBES <130> PA23-337 <140> JP 2023-107989 <141> 2018-06-15 <150> US 62 / 521,050 <151> 2017-06-16 <160> 125 <170> PatentIn version 3.5 <210> 1 <400> 1 000 <210> 2 <400> 2 000 <210> 3 <400> 3 000 <210> 4 <400> 4 000 <210> 5 <400> 5 000 <210> 6 <400> 6 000 <210> 7 <400> 7 000 <210> 8 <400> 8 000 <210> 9 <211> 25 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 9 Met Lys Lys Leu Ala Phe Ala Ile Thr Ala Ala Ser Gly Ala Ala Ala 1 5 10 15 Val Leu Ser His His Asp Ala Glu Ala 20 25 <210> 10 <211> 30 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 10 Trp Leu Asp Asn Arg Ala Phe Ser Lys Lys Phe Val Pro Val Val Met 1 5 10 15 Ala Thr Ser Val Ala Leu Phe Phe Leu Asn Leu Ala Phe Ala 20 25 30 <210> 11 <211> 29 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 11 Met Ala Lys Lys Phe Asn Tyr Lys Leu Pro Ser Met Val Ala Leu Thr 1 5 10 15 Leu Phe Gly Thr Ala Phe Thr Ala His Gln Ala Asn Ala 20 25 <210> 12 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 12 Met Lys Lys Arg Phe Leu Ser Ile Cys Thr Met Thr Ile Ala Ala Leu 1 5 10 15 Ala Thr Thr Thr Met Val Asn Thr Ser Tyr Ala 20 25 <210> 13 <211> 30 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 13 Asn Leu Lys Lys Gln Ser Lys Leu Ile Leu Ile Phe Ile Cys Ile Phe 1 5 10 15 Thr Phe Phe Ile Met Ile Ile Gln Ser Gln Phe Leu Met Gly 20 25 30 <210> 14 <211> 26 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 14 Met Lys Ile Phe Lys Leu Thr Ser Leu Thr Leu Ala Ala Leu Thr Leu 1 5 10 15 Path Phe Pro Phe Ser His Val Path Gln Path 20 25 <210> 15 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 15 Met Lys Lys Thr Val Ile Ala Ser Thr Leu Ala Val Ser Leu Gly Ile 1 5 10 15 Ala Gly Tyr Gly Leu Ser Gly His Glu Ala His 20 25 <210> 16 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 16 Met Lys Lys Asn Lys Phe Leu Val Tyr Leu Leu Ser Thr Ala Leu Ile 1 5 10 15 Thr Pro Thr Phe Ala Thr Gln Thr Ala Phe Ala 20 25 <210> 17 <211> 35 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 17 Met Lys Thr Arg Gln Asn Lys Tyr Ser Ile Arg Lys Phe Ser Val Gly 1 5 10 15 Ala Ser Ser Ile Leu Ile Ala Ala Leu Leu Phe Met Gly Gly Gly Ser 20 25 30 Ala Gln Ala 35 <210> 18 <211> 37 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 18 Met Lys Asn Asn Asn Glu Thr Arg Arg Phe Ser Ile Arg Lys Tyr Thr 1 5 10 15 Val Gly Val Val Ser Ile Ile Thr Gly Ile Thr Ile Phe Val Ser Gly 20 25 30 Gln His Ala Gln Ala 35 <210> 19 <211> 30 <212> PRT <213> Unknown <220> <223> Description of Unknown: Secretion peptide <400> 19 Met Lys Lys Lys Leu Ser Met Ile Thr And Met Leu Ala Phe Thr 1 5 10 15 Leu Ser Leu Ala Leu Gly Leu Phe Phe Asn Ser Ala His Ala 20 25 30 <210> 20 <211> 45 <212> PRT <213> Staphylococcus epidermidis <400> 20 Met Lys Arg Gln Gly Pro Ile Asn Lys Arg Val Asp Phe Leu 1 5 10 15 Ser Asn Lys Val Asn Lys Tyr Ser Ile Arg Lys Phe Thr Val Gly Thr 20 25 30 Ala Ser Ile Leu Val Gly Ala Thr Leu Met Phe Gly Ala 35 40 45 <210> 21 <211> 27 <212> PRT <213> Staphylococcus epidermidis <400> 21 Met Lys Lys Arg Phe Leu Ser Ile Cys Thr Met Thr Ile Ala Ala Leu 1 5 10 15 Ala Thr Thr Thr Met Val Asn Thr Ser Tyr Ala 20 25 <210> 22 <211> 29 <212> PRT <213> Staphylococcus epidermidis <400> 22 Met Ala Lys Lys Phe Asn Tyr Lys Leu Pro Ser Met Val Ala Leu Thr 1 5 10 15 Leu Phe Gly Thr Ala Phe Thr Ala His Gln Ala Asn Ala 20 25 <210> 23 <211> 50 <212> PRT <213> Staphylococcus epidermidis <400> 23 Met Ile Lys Lys Asn Asn Leu Leu Thr Lys Lys Lys Pro Ile Ala Asn 1 5 10 15 Lys Ser Asn Lys Tyr Ala Ile Arg Lys Phe Thr Val Gly Thr Ala Ser 20 25 30 Ile Val Ile Gly Ala Ala Leu Leu Phe Gly Leu Gly His Asn Glu Ala 35 40 45 Lys Ala 50 <210> 24 <211> 30 <212> PRT <213> Staphylococcus epidermidis <400> 24 Met Lys Pro Phe Lys Leu Ile Phe Ile Ser Ala Leu Met Ile Leu Ile 1 5 10 15 Met Thr Asn Ala Thr Pro Ile Ser His Leu Asn Ala Gln Ala 20 25 30 <210> 25 <211> 35 <212> PRT <213> Staphylococcus epidermidis <400> 25 Met Lys Thr Arg Gln Asn Lys Tyr Ser Ile Arg Lys Phe Ser Val Gly 1 5 10 15 Ala Ser Ser Ile Leu Ile Ala Ala Leu Leu Phe Met Gly Gly Gly Ser 20 25 30 Ala Gln Ala 35 <210> 26 <211> 23 <212> PRT <213> Staphylococcus epidermidis <400> 26 Met Asn Lys Phe Lys Phe Phe Ile Val Phe Leu Ile Leu Ser Leu Val 1 5 10 15 Phe Leu Gln Asn Glu Tyr Ala 20 <210> 27 <211> 51 <212> PRT <213> Staphylococcus epidermidis <400> 27 Met Ile Asn Lys Lys Asn Leu Leu Thr Lys Lys Pro Ile Ala 1 5 10 15 Asn Lys Ser Asn Lys Tyr Ala Ile Arg Lys Phe Thr Val Gly Thr Ala 20 25 30 Ser Ile Val Ile Gly Ala Thr Leu Leu Phe Gly Leu Gly His Asn Glu 35 40 45 Only Lys Only 50 <210> 28 <211> 26 <212> PRT <213> Staphylococcus epidermidis <400> 28 Met Lys Lys Ile Ala Thr Ala Thr Ile Ala Thr Ala Gly Ile Ala Thr 1 5 10 15 Phe Ala Phe Ala His His Asp Ala Gln Ala 20 25 <210> 29 <211> 28 <212> PRT <213> Staphylococcus epidermidis <400> 29 Met Lys Asn Phe Ser Lys Phe Ala Leu Thr Ser Ile Ala Ala Leu Thr 1 5 10 15 Val Ala Ser Pro Leu Val Asn Thr Glu Val Asp Ala 20 25 <210> 30 <211> 37 <212> PRT <213> Staphylococcus epidermidis <400> 30 Met Lys Asn Asn Asn Glu Thr Arg Arg Phe Ser Ile Arg Lys Tyr Thr 1 5 10 15 Val Gly Val Val Ser Ile Ile Thr Gly Ile Thr Ile Phe Val Ser Gly 20 25 30 Gln His Ala Gln Ala 35 <210> 31 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 31 Met Arg Tyr Leu Lys Arg Ile Thr Ile Tyr Ile Ser Leu Leu Ile Leu 1 5 10 15 Val Ser Gly <210> 32 <211> 20 <212> PRT <213> Staphylococcus epidermidis <400> 32 Met Lys Leu Met Asn Lys Ile Ile Val Pro Val Thr Ala Ser Ala Leu 1 5 10 15 Leu Leu Gly Ala 20 <210> 33 <211> 40 <212> PRT <213> Staphylococcus epidermidis <400> 33 Met Lys Lys Ile Asp Ser Trp Leu Thr Lys His Gly Leu Lys Asn Arg 1 5 10 15 Leu Thr Leu Val Val Ile Val Ile Phe Ile Ile Phe Leu Ile Leu Leu 20 25 30 Phe Met Phe Val Asn Leu Ser Asp 35 40 <210> 34 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 34 Met Lys Lys Lys Ala Leu Leu Pro Leu Phe Leu Gly Ile Met Ile Phe 1 5 10 15 Leu Ala Gly <210> 35 <211> 28 <212> PRT <213> Staphylococcus epidermidis <400> 35 Met Lys Lys Thr Val Ile Ala Ser Thr Leu Ala Val Ser Leu Gly Ile 1 5 10 15 Ala Gly Tyr Gly Leu Ser Gly His Glu Ala His Ala 20 25 <210> 36 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 36 Met Ser Lys Phe Lys Ser Leu Leu Leu Leu Phe Gly Thr Leu Ile Leu 1 5 10 15 Leu Ser Gly <210> 37 <211> 27 <212> PRT <213> Staphylococcus epidermidis <400> 37 Met Lys Lys Thr Leu Val Ala Ser Ser Leu Ala Ile Gly Leu Gly Val 1 5 10 15 Val Ala Gly Asn Ala Gly His Asp Ala His Ala 20 25 <210> 38 <211> 29 <212> PRT <213> Staphylococcus epidermidis <400> 38 Met Ala Lys Lys Phe Asn Tyr Lys Leu Pro Ser Met Val Ala Leu Thr 1 5 10 15 Leu Phe Gly Thr Ala Phe Thr Ala His Gln Ala Asn Ala 20 25 <210> 39 <211> 30 <212> PRT <213> Staphylococcus epidermidis <400> 39 Met Lys Lys Lys Leu Ser Tyr Met Ile Thr Ile Met Leu Ala Phe Thr 1 5 10 15 Leu Ser Leu Ala Leu Gly Leu Phe Phe Asn Ser Ala His Ala 20 25 30 <210> 40 <211> 18 <212> PRT <213> Staphylococcus epidermidis <400> 40 Met His Lys Arg Leu Phe Ile Thr Leu Leu Gly Phe Ile Ile Leu Leu 1 5 10 15 Gly Road <210> 41 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 41 Met Arg Tyr Leu Lys Arg Ile Thr Ile Tyr Ile Ser Leu Leu Ile Leu 1 5 10 15 Val Ser Gly <210> 42 <211> 25 <212> PRT <213> Staphylococcus epidermidis <400> 42 Met Gln Lys Lys Tyr Ile Thr Ala Ile Ile Gly Thr Thr Ala Leu Ser 1 5 10 15 Ala Leu Ala Ser Thr His Ala Gln Ala 20 25 <210> 43 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 43 Met Lys His Ser Ser Lys Ile Ile Val Phe Val Ser Phe Leu Ile Leu 1 5 10 15 Thr Ile Phe Ile Gly Gly 20 <210> 44 <211> 20 <212> PRT <213> Staphylococcus epidermidis <400> 44 Met Lys Lys Trp Gln Leu Val Gly Thr Thr Val Leu Gly Ala Ser Val 1 5 10 15 Leu Leu Gly Ala 20 <210> 45 <211> 52 <212> PRT <213> Staphylococcus epidermidis <400> 45 Met Gly Lys Arg Arg Gln Gly Pro Ile Asn Lys Lys Val Asp Phe Leu 1 5 10 15 Pro Asn Lys Leu Asn Lys Tyr Ser Ile Arg Lys Phe Thr Val Gly Thr 20 25 30 Ala Ser Ile Leu Leu Gly Ser Thr Leu Ile Phe Gly Ser Ser Ser His 35 40 45 Glu Ala Lys Ala 50 <210> 46 <211> 26 <212> PRT <213> Staphylococcus epidermidis <400> 46 Met Lys Lys Ile Ala Thr Ala Thr Ile Ala Thr Ala Gly Ile Ala Thr 1 5 10 15 Phe Ala Phe Ala His His Asp Ala Gln Ala 20 25 <210> 47 <211> 45 <212> PRT <213> Staphylococcus epidermidis <400> 47 Met Lys Lys Arg Arg Gln Gly Pro Ile Asn Lys Arg Val Asp Phe Leu 1 5 10 15 Ser Asn Lys Val Asn Lys Tyr Ser Ile Arg Lys Phe Thr Val Gly Thr 20 25 30 Ala Ser Ile Leu Val Gly Ala Thr Leu Met Phe Gly Ala 35 40 45 <210> 48 <211> 27 <212> PRT <213> Staphylococcus epidermidis <400> 48 Met Lys Lys Arg Phe Leu Ser Ile Cys Thr Met Thr Ile Ala Ala Leu 1 5 10 15 Ala Thr Thr Thr Met Val Asn Thr Ser Tyr Ala 20 25 <210> 49 <211> 35 <212> PRT <213> Staphylococcus epidermidis <400> 49 Met Lys Thr Arg Gln Asn Lys Tyr Ser Ile Arg Lys Phe Ser Val Gly 1 5 10 15 Ala Ser Ser Ile Leu Ile Ala Ala Leu Leu Phe Met Gly Gly Gly Ser 20 25 30 Ala Gln Ala 35 <210> 50 <211> 28 <212> PRT <213> Staphylococcus epidermidis <400> 50 Met Lys Asn Phe Ser Lys Phe Ala Leu Thr Ser Ile Ala Ala Leu Thr 1 5 10 15 Val Ala Ser Pro Leu Val Asn Thr Glu Val Asp Ala 20 25 <210> 51 <211> 17 <212> PRT <213> Staphylococcus epidermidis <400> 51 Met Lys Lys Val Leu Ala Ser Ala Thr Ile Leu Ser Leu Met Leu Val 1 5 10 15 Gly <210> 52 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 52 Met Lys Tyr Tyr Gly Lys Cys Ile Ser Tyr Ile Ser Ile Leu Ile Leu 1 5 10 15 Thr Phe Phe Ile Gly Gly 20 <210> 53 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 53 Met Lys His Ser Ser Lys Ile Ile Val Phe Val Ser Phe Leu Ile Leu 1 5 10 15 Thr Ile Phe Ile Gly Gly 20 <210> 54 <211> 30 <212> PRT <213> Staphylococcus epidermidis <400> 54 Met Lys Pro Phe Lys Leu Ile Phe Ile Ser Ala Leu Met Ile Leu Ile 1 5 10 15 Met Thr Asn Ala Thr Pro Ile Ser His Leu Asn Ala Gln Ala 20 25 30 <210> 55 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 55 Met Ser Lys Phe Lys Ser Leu Leu Leu Leu Phe Gly Thr Leu Ile Leu 1 5 10 15 Leu Ser Gly <210> 56 <211> 27 <212> PRT <213> Staphylococcus epidermidis <400> 56 Met Lys Lys Thr Leu Val Ala Ser Ser Leu Ala Ile Gly Leu Gly Val 1 5 10 15 Val Ala Gly Asn Ala Gly His Asp Ala His Ala 20 25 <210> 57 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 57 Met His Tyr Leu Lys Lys Val Thr Ile Tyr Ile Ser Leu Leu Ile Leu 1 5 10 15 Val Ser Gly <210> 58 <211> 25 <212> PRT <213> Staphylococcus epidermidis <400> 58 Met Gln Lys Lys Tyr Ile Thr Ala Ile Ile Gly Thr Thr Ala Leu Ser 1 5 10 15 Ala Leu Ala Ser Thr His Ala Gln Ala 20 25 <210> 59 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 59 Met Lys His Ser Lys Lys Leu Leu Leu Cys Ile Ser Phe Leu Leu Ile 1 5 10 15 Thr Phe Phe Ile Gly Gly 20 <210> 60 <211> 26 <212> PRT <213> Staphylococcus epidermidis <400> 60 Met Lys Lys Ile Ala Thr Ala Thr Ile Ala Thr Ala Gly Ile Ala Thr 1 5 10 15 Phe Ala Phe Ala His His Asp Ala Gln Ala 20 25 <210> 61 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 61 Met Arg Tyr Leu Lys Lys Val Thr Ile Tyr Ile Ser Leu Leu Ile Leu 1 5 10 15 Val Ser Gly <210> 62 <211> 27 <212> PRT <213> Staphylococcus epidermidis <400> 62 Met Lys Lys Arg Phe Leu Ser Ile Cys Thr Met Thr Ile Ala Ala Leu 1 5 10 15 Ala Thr Thr Thr Met Val Asn Thr Ser Tyr Ala 20 25 <210> 63 <211> 20 <212> PRT <213> Staphylococcus epidermidis <400> 63 Met Lys Lys Trp Gln Leu Val Gly Thr Thr Val Leu Gly Ala Ser Val 1 5 10 15 Leu Leu Gly Ala 20 <210> 64 <211> 29 <212> PRT <213> Staphylococcus epidermidis <400> 64 Met Ala Lys Lys Phe Asn Tyr Lys Leu Pro Ser Met Val Ala Leu Thr 1 5 10 15 Leu Phe Gly Thr Ala Phe Thr Ala His Gln Ala Asn Ala 20 25 <210> 65 <211> 30 <212> PRT <213> Staphylococcus epidermidis <400> 65 Met Lys Lys Lys Leu Ser Tyr Met Ile Thr Ile Met Leu Ala Phe Thr 1 5 10 15 Leu Ser Leu Ala Leu Gly Leu Phe Phe Asn Ser Ala His Ala 20 25 30 <210> 66 <211> 18 <212> PRT <213> Staphylococcus epidermidis <400> 66 Met His Lys Arg Leu Phe Ile Thr Leu Leu Gly Phe Ile Ile Leu Leu 1 5 10 15 Gly Road <210> 67 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 67 Met Arg Tyr Leu Lys Lys Val Thr Ile Tyr Ile Ser Leu Leu Ile Leu 1 5 10 15 Thr Ile Phe Ile Gly Gly 20 <210> 68 <211> 17 <212> PRT <213> Staphylococcus epidermidis <400> 68 Met Lys Lys Val Leu Ala Ser Ala Thr Ile Leu Ser Leu Met Leu Val 1 5 10 15 Gly <210> 69 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 69 Met Lys His Ser Lys Lys Leu Leu Leu Cys Ile Ser Phe Leu Leu Ile 1 5 10 15 Thr Val Phe Ile Ser Gly 20 <210> 70 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 70 Met Lys His Ser Lys Lys Leu Leu Leu Cys Ile Ser Phe Leu Leu Ile 1 5 10 15 Thr Phe Phe Ile Ser Gly 20 <210> 71 <211> 28 <212> PRT <213> Staphylococcus epidermidis <400> 71 Met Lys Lys Thr Val Ile Ala Ser Thr Leu Ala Val Ser Leu Gly Ile 1 5 10 15 Ala Gly Tyr Gly Leu Ser Gly His Glu Ala His Ala 20 25 <210> 72 <211> 22 <212> PRT <213> Staphylococcus epidermidis <400> 72 Met Lys His Ser Lys Lys Leu Leu Leu Cys Ile Ser Phe Leu Leu Ile 1 5 10 15 Thr Ile Phe Ile Ser Gly 20 <210> 73 <211> 40 <212> PRT <213> Staphylococcus epidermidis <400> 73 Met Lys Lys Ile Asp Ser Trp Leu Thr Lys His Gly Leu Lys Asn Arg 1 5 10 15 Leu Thr Leu Val Val Ile Val Ile Phe Ile Ile Phe Leu Ile Leu Leu 20 25 30 Phe Met Phe Val Asn Leu Ser Asp 35 40 <210> 74 <211> 19 <212> PRT <213> Staphylococcus epidermidis <400> 74 Met Lys Lys Lys - Pro Leu Phe Leu Gly And Met Leu Phe 1 5 10 15 Leu Ala Gly <210> 75 <211> 20 <212> PRT <213> Staphylococcus epidermidis <400> 75 Met Lys Leu Met Asn Lys Ile Ile Val Pro Val Thr Ala Ser Ala Leu 1 5 10 15 Leu Leu Gly Ala 20 <210> 76 <211> 35 <212> PRT <213> Staphylococcus epidermidis <400> 76 Met Lys Thr Arg Gln Asn Lys Tyr Ser Ile Arg Lys Phe Ser Val Gly 1 5 10 15 Wing Ser Ser With Leu Wing Wing Leu Leu Phe Met Gly Gly Gly Ser 20 25 30 Ala Gln Ala 35 <210> 77 <211> 13 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 77 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 78 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 78 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 <210> 79 <211> 18 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 79 Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala <210> 80 <211> 30 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 80 Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His 1 5 10 15 Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala 20 25 30 <210> 81 <211> 21 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 81 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 82 <211> 9 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 82 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 83 <211> 24 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 83 Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln 1 5 10 15 Thr Ala Ile Gly Val Gly Ala Pro 20 <210> 84 <211> 15 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 84 Arg Gln Ile Lys Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 85 <211> 11 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 85 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 86 <211> 18 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 86 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Thr Ser Thr 1 5 10 15 Gly Arg <210> 87 <211> 10 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 87 Arg Arg Leu Ser Tyr Ser Arg Arg Arg Phe 1 5 10 <210> 88 <211> 12 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 88 Pro Ile Arg Arg Arg Lys Lys Leu Arg Arg Leu Lys 1 5 10 <210> 89 <211> 12 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 89 Arg Arg Gln Arg Arg Thr Ser Lys Leu Met Lys Arg 1 5 10 <210> 90 <211> 15 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 90 Arg Arg Arg Arg Asn Arg Thr Arg Arg Asn Arg Arg Arg Val Arg 1 5 10 15 <210> 91 <211> 19 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 91 Lys Met Thr Arg Ala Gln Arg Arg Ala Ala Ala Arg Arg Asn Arg Trp 1 5 10 15 Thr Ala Arg <210> 92 <211> 13 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 92 Thr Arg Arg Gln Arg Thr Arg Arg Ala Arg Arg Asn Arg 1 5 10 <210> 93 <211> 13 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 93 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 94 <211> 13 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 94 Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Gln 1 5 10 <210> 95 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 95 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 <210> 96 <211> 17 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 96 Lys Leu Way Leu Lys Leu Way Leu Lys Leu Way Leu Way Leu Lys Leu 1 5 10 15 Way <210> 97 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 97 Met Gly Leu Gly Leu His Leu Leu Val Leu Ala Ala Ala Leu Gln Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25 <210> 98 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 98 Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25 <210> 99 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 99 Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25 <210> 100 <211> 27 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 100 Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val 20 25 <210> 101 <211> 21 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 101 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 102 <211> 21 <212> PRT <213> Unknown <220> <223> Description of Unknown: Cell penetrating peptide <400> 102 Lys Glu Thr Trp Phe Glu Thr Trp Phe Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 103 <211> 1064 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 103 Met Lys Ile Ala Thr Val Ser Val Leu Leu Pro Leu Ala Leu Cys Leu 1 5 10 15 Ile Gln Asp Ala Ala Ser Lys Asn Glu Asp Gln Glu Met Cys His Glu 20 25 30 Phe Gln Ala Phe Met Lys Asn Gly Lys Leu Phe Cys Pro Gln Asp Lys 35 40 45 Lys Phe Phe Gln Ser Leu Asp Gly Ile Met Phe Ile Asn Lys Cys Ala 50 55 60 Thr Cys Lys Met Ile Leu Glu Lys Glu Ala Lys Ser Gln Lys Arg Ala 65 70 75 80 Arg His Leu Ala Arg Ala Pro Lys Ala Thr Ala Pro Thr Glu Leu Asn 85 90 95 Cys Asp Asp Phe Lys Lys Gly Glu Arg Asp Gly Asp Phe Ile Cys Pro 100 105 110 Asp Tyr Tyr Glu Ala Val Cys Gly Thr Asp Gly Lys Thr Tyr Asp Asn 115 120 125 Arg Cys Ala Leu Cys Ala Glu Asn Ala Lys Thr Gly Ser Gln Ile Gly 130 135 140 Val Lys Ser Glu Gly Glu Cys Lys Ser Ser Asn Pro Glu Gln Asp Val 145 150 155 160 Cys Ser Ala Phe Arg Pro Phe Val Arg Asp Gly Arg Leu Gly Cys Thr 165 170 175 Arg Glu Asn Asp Pro Val Leu Gly Pro Asp Gly Lys Thr His Gly Asn 180 185 190 Lys Cys Ala Met Cys Ala Glu Leu Phe Leu Lys Glu Ala Glu Asn Ala 195 200 205 Lys Arg Glu Gly Glu Thr Arg Ile Arg Arg Asn Ala Glu Lys Asp Phe 210 215 220 Cys Lys Glu Tyr Glu Lys Gln Val Arg Asn Gly Arg Leu Phe Cys Thr 225 230 235 240 Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly Arg Met His Gly Asn 245 250 255 Lys Cys Ala Leu Cys Ala Glu Ile Phe Lys Gln Arg Phe Ser Glu Glu 260 265 270 Asn Ser Lys Thr Asp Gln Asn Leu Gly Lys Ala Glu Glu Lys Thr Lys 275 280 285 Val Lys Arg Glu Ile Val Lys Leu Cys Ser Gln Tyr Gln Asn Gln Ala 290 295 300 Lys Asn Gly Ile Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Arg Gly 305 310 315 320 Pro Asp Gly Lys Met His Gly Asn Leu Cys Ser Met Cys Gln Ala Tyr 325 330 335 Phe Gln Ala Glu Asn Glu Glu Lys Lys Lys Ala Glu Ala Arg Ala Arg 340 345 350 Asn Lys Arg Glu Ser Gly Lys Ala Thr Ser Tyr Ala Glu Leu Cys Ser 355 360 365 Glu Tyr Arg Lys Leu Val Arg Asn Gly Lys Leu Ala Cys Thr Arg Glu 370 375 380 Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys Val His Gly Asn Thr Cys 385 390 395 400 Ser Met Cys Glu Val Phe Phe Gln Ala Glu Glu Glu Glu Lys Lys Lys 405 410 415 Lys Glu Gly Lys Ser Arg Asn Lys Arg Gln Ser Lys Ser Thr Ala Ser 420 425 430 Phe Glu Glu Leu Cys Ser Glu Tyr Arg Lys Ser Arg Lys Asn Gly Arg 435 440 445 Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys 450 455 460 Met His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln Gln Glu 465 470 475 480 Glu Arg Ala Arg Ala Lys Ala Lys Arg Glu Ala Ala Lys Glu Ile Cys 485 490 495 Ser Glu Phe Arg Asp Gln Val Arg Asn Gly Thr Leu Ile Cys Thr Arg 500 505 510 Glu His Asn Pro Val Arg Gly Pro Asp Gly Lys Met His Gly Asn Lys 515 520 525 Cys Ala Met Cys Ala Ser Val Phe Lys Leu Glu Glu Glu Glu Lys Lys 530 535 540 Asn Asp Lys Glu Glu Lys Gly Lys Val Glu Ala Glu Lys Val Lys Arg 545 550 555 560 Glu Ala Val Gln Glu Leu Cys Ser Glu Tyr Arg His Tyr Val Arg Asn 565 570 575 Gly Arg Leu Pro Cys Thr Arg Glu Asn Asp Pro Ile Glu Gly Leu Asp 580 585 590 Gly Lys Ile His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 595 600 605 Gln Glu Ala Lys Glu Lys Glu Arg Ala Glu Pro Arg Ala Lys Val Lys 610 615 620 Arg Glu Ala Glu Lys Glu Thr Cys Asp Glu Phe Arg Arg Leu Leu Gln 625 630 635 640 Asn Gly Lys Leu Phe Cys Thr Arg Glu Asn Asp Pro Val Arg Gly Pro 645 650 655 Asp Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Lys Ala Val Phe 660 665 670 Gln Lys Glu Asn Glu Glu Arg Lys Arg Lys Glu Glu Glu Asp Gln Arg 675 680 685 Asn Ala Ala Gly His Gly Ser Ser Gly Gly Gly Gly Gly Asn Thr Gln 690 695 700 Asp Glu Cys Ala Glu Tyr Arg Glu Gln Met Lys Asn Gly Arg Leu Ser 705 710 715 720 Cys Thr Arg Glu Ser Asp Pro Val Arg Asp Ala Asp Gly Lys Ser Tyr 725 730 735 Asn Asn Gln Cys Thr Met Cys Lys Ala Lys Leu Glu Arg Glu Ala Glu 740 745 750 Arg Lys Asn Glu Tyr Ser Arg Ser Arg Ser Asn Gly Thr Gly Ser Glu 755 760 765 Ser Gly Lys Asp Thr Cys Asp Glu Phe Arg Ser Gln Met Lys Asn Gly 770 775 780 Lys Leu Ile Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly 785 790 795 800 Lys Thr His Gly Asn Lys Cys Thr Met Cys Lys Glu Lys Leu Glu Arg 805 810 815 Glu Ala Ala Glu Lys Lys Lys Lys Glu Asp Glu Asp Arg Ser Asn Thr 820 825 830 Gly Glu Arg Ser Asn Thr Gly Glu Arg Ser Asn Asp Lys Glu Asp Leu 835 840 845 Cys Arg Glu Phe Arg Ser Met Gln Arg Asn Gly Lys Leu Ile Cys Thr 850 855 860 Arg Glu Asn Asn Pro Val Arg Gly Pro Tyr Gly Lys Met His Ile Asn 865 870 875 880 Lys Cys Ala Met Cys Gln Ser Ile Phe Asp Arg Glu Ala Asn Glu Arg 885 890 895 Lys Lys Lys Asp Glu Glu Lys Ser Ser Ser Lys Pro Ser Asn Asn Ala 900 905 910 Lys Asp Glu Cys Ser Glu Phe Arg Asn Tyr Ile Arg Asn Asn Glu Leu 915 920 925 Ile Cys Pro Arg Glu Asn Asp Pro Val His Gly Ala Asp Gly Lys Phe 930 935 940 Tyr Thr Asn Lys Cys Tyr Met Cys Arg Ala Val Phe Leu Thr Glu Ala 945 950 955 960 Leu Glu Arg Ala Lys Leu Gln Glu Lys Pro Ser His Val Arg Ala Ser 965 970 975 Gln Glu Glu Asp Ser Pro Asp Ser Phe Ser Ser Leu Asp Ser Glu Met 980 985 990 Cys Lys Asp Tyr Arg Val Leu Pro Arg Ile Gly Tyr Leu Cys Pro Lys 995 1000 1005 Asp Leu Lys Pro Val Cys Gly Asp Asp Gly Gln Thr Tyr Asn Asn 1010 1015 1020 Pro Cys Met Leu Cys His Glu Asn Leu Ile Arg Gln Thr Asn Thr 1025 1030 1035 His Ile Arg Ser Thr Gly Lys Cys Glu Glu Ser Ser Thr Pro Gly 1040 1045 1050 Thr Thr Ala Ala Ser Met Pro Pro Ser Asp Glu 1055 1060 <210> 104 <211> 55 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 104 Lys Asn Glu Asp Gln Glu Met Cys His Glu Phe Gln Ala Phe Met Lys 1 5 10 15 Asn Gly Lys Leu Phe Cys Pro Gln Asp Lys Lys Phe Phe Gln Ser Leu 20 25 30 Asp Gly Ile Met Phe Ile Asn Lys Cys Ala Thr Cys Lys Met Ile Leu 35 40 45 Glu Lys Glu Ala Lys Ser Gln 50 55 <210> 105 <211> 63 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 105 Ala Pro Thr Glu Leu Asn Cys Asp Asp Phe Lys Lys Gly Glu Arg Asp 1 5 10 15 Gly Asp Phe Ile Cys Pro Asp Tyr Tyr Glu Ala Val Cys Gly Thr Asp 20 25 30 Gly Lys Thr Tyr Asp Asn Arg Cys Ala Leu Cys Ala Glu Asn Ala Lys 35 40 45 Thr Gly Ser Gln Ile Gly Val Lys Ser Glu Gly Glu Cys Lys Ser 50 55 60 <210> 106 <211> 62 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 106 Asn Pro Glu Gln Asp Val Cys Ser Ala Phe Arg Pro Phe Val Arg Asp 1 5 10 15 Gly Arg Leu Gly Cys Thr Arg Glu Asn Asp Pro Val Leu Gly Pro Asp 20 25 30 Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Ala Glu Leu Phe Leu 35 40 45 Lys Glu Ala Glu Asn Ala Lys Arg Glu Gly Glu Thr Arg Ile 50 55 60 <210> 107 <211> 67 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 107 Asn Ala Glu Lys Asp Phe Cys Lys Glu Tyr Glu Lys Gln Val Arg Asn 1 5 10 15 Gly Arg Leu Phe Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp 20 25 30 Gly Arg Met His Gly Asn Lys Cys Ala Leu Cys Ala Glu Ile Phe Lys 35 40 45 Gln Arg Phe Ser Glu Glu Asn Ser Lys Thr Asp Gln Asn Leu Gly Lys 50 55 60 Ala Glu Glu 65 <210> 108 <211> 62 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 108 Arg Glu Ile Val Lys Leu Cys Ser Gln Tyr Gln Asn Gln Ala Lys Asn 1 5 10 15 Gly Ile Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Arg Gly Pro Asp 20 25 30 Gly Lys Met His Gly Asn Leu Cys Ser Met Cys Gln Ala Tyr Phe Gln 35 40 45 Ala Glu Asn Glu Glu Lys Lys Lys Ala Glu Ala Arg Ala Arg 50 55 60 <210> 109 <211> 68 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 109 Glu Ser Gly Lys Ala Thr Ser Tyr Ala Glu Leu Cys Ser Glu Tyr Arg 1 5 10 15 Lys Leu Val Arg Asn Gly Lys Leu Ala Cys Thr Arg Glu Asn Asp Pro 20 25 30 Ile Gln Gly Pro Asp Gly Lys Val His Gly Asn Thr Cys Ser Met Cys 35 40 45 Glu Val Phe Phe Gln Ala Glu Glu Glu Glu Lys Lys Lys Lys Glu Gly 50 55 60 Lys Ser Arg Asn 65 <210> 110 <211> 59 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 110 Ala Ser Phe Glu Glu Leu Cys Ser Glu Tyr Arg Lys Ser Arg Lys Asn 1 5 10 15 Gly Arg Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Gln Gly Pro Asp 20 25 30 Gly Lys Met His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 35 40 45 Gln Glu Glu Arg Ala Arg Ala Lys Ala Lys Arg 50 55 <210> 111 <211> 62 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 111 Glu Ala Ala Lys Glu Ile Cys Ser Glu Phe Arg Asp Gln Val Arg Asn 1 5 10 15 Gly Thr Leu Ile Cys Thr Arg Glu His Asn Pro Val Arg Gly Pro Asp 20 25 30 Gly Lys Met His Gly Asn Lys Cys Ala Met Cys Ala Ser Val Phe Lys 35 40 45 Leu Glu Glu Glu Glu Lys Lys Asn Asp Lys Glu Glu Lys Gly 50 55 60 <210> 112 <211> 62 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 112 Glu Ala Val Gln Glu Leu Cys Ser Glu Tyr Arg His Tyr Val Arg Asn 1 5 10 15 Gly Arg Leu Pro Cys Thr Arg Glu Asn Asp Pro Ile Glu Gly Leu Asp 20 25 30 Gly Lys Ile His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 35 40 45 Gln Glu Ala Lys Glu Lys Glu Arg Ala Glu Pro Arg Ala Lys 50 55 60 <210> 113 <211> 63 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 113 Glu Ala Glu Lys Glu Thr Cys Asp Glu Phe Arg Arg Leu Leu Gln Asn 1 5 10 15 Gly Lys Leu Phe Cys Thr Arg Glu Asn Asp Pro Val Arg Gly Pro Asp 20 25 30 Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Lys Ala Val Phe Gln 35 40 45 Lys Glu Asn Glu Glu Arg Lys Arg Lys Glu Glu Glu Asp Gln Arg 50 55 60 <210> 114 <211> 57 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 114 Gly Asn Thr Gln Asp Glu Cys Ala Glu Tyr Arg Glu Gln Met Lys Asn 1 5 10 15 Gly Arg Leu Ser Cys Thr Arg Glu Ser Asp Pro Val Arg Asp Ala Asp 20 25 30 Gly Lys Ser Tyr Asn Asn Gln Cys Thr Met Cys Lys Ala Lys Leu Glu 35 40 45 Arg Glu Ala Glu Arg Lys Asn Glu Tyr 50 55 <210> 115 <211> 63 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 115 Glu Ser Gly Lys Asp Thr Cys Asp Glu Phe Arg Ser Gln Met Lys Asn 1 5 10 15 Gly Lys Leu Ile Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp 20 25 30 Gly Lys Thr His Gly Asn Lys Cys Thr Met Cys Lys Glu Lys Leu Glu 35 40 45 Arg Glu Ala Ala Glu Lys Lys Lys Lys Glu Asp Glu Asp Arg Ser 50 55 60 <210> 116 <211> 63 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 116 Asn Asp Lys Glu Asp Leu Cys Arg Glu Phe Arg Ser Met Gln Arg Asn 1 5 10 15 Gly Lys Leu Ile Cys Thr Arg Glu Asn Asn Pro Val Arg Gly Pro Tyr 20 25 30 Gly Lys Met His Ile Asn Lys Cys Ala Met Cys Gln Ser Ile Phe Asp 35 40 45 Arg Glu Ala Asn Glu Arg Lys Lys Lys Asp Glu Glu Lys Ser Ser 50 55 60 <210> 117 <211> 62 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 117 Asn Asn Ala Lys Asp Glu Cys Ser Glu Phe Arg Asn Tyr Ile Arg Asn 1 5 10 15 Asn Glu Leu Ile Cys Pro Arg Glu Asn Asp Pro Val His Gly Ala Asp 20 25 30 Gly Lys Phe Tyr Thr Asn Lys Cys Tyr Met Cys Arg Ala Val Phe Leu 35 40 45 Thr Glu Ala Leu Glu Arg Ala Lys Leu Gln Glu Lys Pro Ser 50 55 60 <210> 118 <211> 62 <212> PRT <213> Unknown <220> <223> Description of Unknown: sequence <400> 118 Ser Leu Asp Ser Glu Met Cys Lys Asp Tyr Arg Val Leu Pro Arg Ile 1 5 10 15 Gly Tyr Leu Cys Pro Lys Asp Leu Lys Pro Val Cys Gly Asp Asp Gly 20 25 30 Gln Thr Tyr Asn Asn Pro Cys Met Leu Cys His Glu Asn Leu Ile Arg 35 40 45 Gln Thr Asn Thr His Ile Arg Ser Thr Gly Lys Cys Glu Glu 50 55 60 <210> 119 <211> 3192 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 119 atgaagatag ccacagtgtc agtgcttctg cccttggctc tttgcctcat acaagatgct 60 gccagtaaga atgaagatca ggaaatgtgc catgaatttc aggcatttat gaaaaatgga 120 aaactgttct gtccccagga taagaaattt tttcaaagtc ttgatggaat aatgttcatc 180 aataaatgtg ccacgtgcaa aatgatactg gaaaaagaag caaaatcaca gaagagggcc 240 aggcatttag caagagctcc caaggctact gccccaacag agctgaattg tgatgatttt 300 aaaaaaggag aaagagatgg ggattttatc tgtcctgatt attatgaagc tgtttgtggc 360 420 tcccaaattg gtgtaaaaag tgaagggaa tgtaagagca gtaatccaga gcaggatgta 480 tgcagtgctt ttcggccctt tgttagagat ggaagaacttg gatgcacaag ggaaaatgat 540 cctgttcttg gtcctgatgg gaagacgcat ggcaataagt gtgcaatgtg tgctgagctg 600 tttttaaaag aagctgaaaa tgccaagcga gagggtgaaa ctagaattcg acgaaatgct 660 gaaaggatt tttgcaagga atatgaaaaa gaagagagaa atggaaggct ttttgtaca 720 cgggagagtg atccagtccg tggccctgac ggcaggatgc atggcaacaa atgtgccctg 780 tgtgctgaaa ttttcaagca gcgtttttca gaggaaaaca gtaaaacaga tcaaaatttg 840 ggaaaagctg aagaaaaaac taaagttaaa agaaattg tgaaactctg cagtcaatat 900 caaaatcagg caaagaatgg aatacttttc tgtaccagag aaaatgaccc tattcgtggt 960 ccagatggga aaatgcatgg caacttgtgt tccatgtgtc aagcctactt ccaagcagaa 1020 aatgaagaaa agaaaaaggc tgaagcacga gctagaaaca aaagagaatc tggaaaagca 1080 acctcatatg cagagctttg cagtgaatat cgaaagcttg tgaggaacgg aaaacttgct 1140 tgcaccagag agaacgatcc tatccagggc ccagatggga aagtgcatgg caacacctgc 1200 tccatgtgtg aggtcttctt ccaagcagaa gaagagaaa agaaaaagaa ggaaggtaa 1260 tcaagaaaca aaagacaatc taagagtaca gcttcctttg aggagttgtg tagtgaatac 1320 cgcaaatcca ggaaaaacgg acggcttttt tgcaccagag agaatgaccc catccagggc 1380 ccagatggaa aaatgcatgg caacacctgc tccatgtgtg aggccttctt tcaacaagaa 1440 gaagagcaa gagcaaaggc taaaagagaa gctgcaaagg aaatctgcag tgaattcgg 1500 1560 gatggcaaaa tgcatggaaa caagtgtgcc atgtgtgcca gtgtgttcaa acttgaagaa 1620 gaagagaaga aaaatgataa agaagaaaaa gggaaagtcg aggctgaaaa agttaagaga 1680 gaagcagttc aggagctgtg cagtgaatat cgtcattatg tgaggaatgg acgactcccc 1740 tgtaccagag agaatgatcc tattgagggt ctagatggga aaatccacgg caacacctgc 1800 tccatgtgtg aagccttctt ccagcaagaa gcaaaagaaa aagaaagagc tgaacccaga 1860 gcaaaagtca aaagagaagc tgaaaaggag acatgcgatg aatttcggag acttttgcaa 1920 aatggaaaac ttttctgcac aagagaaaat gatcctgtgc gtggcccaga tggcaagacc 1980 catggcaaca agtgtgccat gtgtaaggca gtcttccaga aagaaaatga ggaaagaaag 2040 aggaaagaag aggaagatca gagaaatgct gcaggacatg gttccagtgg tggtggagga 2100 ggaaacactc aggacgaatg tgctgagtat cgggaacaaa tgaaaaatgg aagactcagc 2160 tgtactcggg agagtgatcc tgtacgtgat gctgatggca aatcgtacaa caatcagtgt 2220 accatgtgta aagcaaaatt ggaaagagaa gcagagagaa aaaatgagta ttctcgctcc 2280 agatcaaatg ggactggatc agaatcaggg aaggatacat gtgatgagtt tagaagccaa 2340 atgaaaaatg gaaaactcat ctgcactcga gaaagtgacc ctgtccgggg tccagatggc 2400 aagacacatg gcaataagtg tactatgtgt aaggaaaaac tggaaaggga agcagctgaa 2460 aaaaaaaga aagaggatga agacaggagc aatacaggag aaaggagcaa tacaggagaa 2520 aggagcaatg acaaagagga tctgtgtcgt gaatttcgaa gcatgcagag aaatggaaag 2580 cttatctgca ccagagaaaa taaccctgtt cgaggcccat atggcaagat gcacatcaat 2640 aaatgtgcta tgtgtcagag catctttgat cgagaagcta atgaaagaaa aaagaaagat 2700 gaagagaaat caagtagcaa gccctcaaat aatgcaaagg atgagtgcag tgaatttcga 2760 actatataa ggaacaatga actcatctgc cctagagaga atgacccagt gcacggtgct gatggaagt tctatacaaa caagtgctac atgtgcagag ctgtctttct aacagaagct ttggaaaggg caaagcttca agaaaagcca tcccatgtta gagcttctca agaggaagac agcccagact ctttcagttc tctggattct gagatgtgca aagactaccg agtattgccc aggataggtt atctttgtcc aaaggattta aagcctgtct gtggtgacga tggccaaacc 3060. 3120. cttgcatgct ctgtcatgaa aacctgatac gccaaacaaa tacacacatc cgcagtacag ggagtgtga ggagagcagc accccagga ccaccgcagc cagcatgccc ccgtctgacg aa <210> 120 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 120 His His His His His His 1 5 <210> 121 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 121 Ala Ala Pro Phe 1 <210> 122 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 122 atgcgtcgta tgcgtcgtat g 21 <210> 123 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 123 gtggtggtgg tggtggtg 18 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 124 ttgatatgcc tcctaaattt tt 22 <210> 125 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 125 taggcgcgcc tattctaatg c 21

Claims

1. A composition for the treatment of a skin disease, wherein the skin disease is Netherton syndrome, and the composition is This includes microorganisms that have been genetically modified to express one or more LEKTI protein domains on mammalian skin, The aforementioned microorganisms are selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof. The one or more LEKTI protein domains include the LEKTI domain 6, The one or more LEKTI protein domains described above are effective in penetrating one or more layers of mammalian skin and are effective in inhibiting the serine protease activity of at least one serine protease in or on mammalian skin. The composition wherein the LEKTI protein domain is effective in improving the symptoms of Netherton syndrome.

2. The composition according to claim 1, wherein the microorganism is adapted to survive on the surface of mammalian skin for a controlled period of time to provide a continuous supply of LEKTI protein domains.

3. The composition according to claim 1, wherein the microorganism is genetically modified by transfection / transformation using a recombinant DNA plasmid encoding the LEKTI protein domain.

4. The composition according to claim 1, wherein the LEKTI domain is functionally linked to one or more recombinant protein domains that are effective in enhancing secretion from microorganisms and / or permeability through mammalian skin.

5. The composition according to claim 1, wherein at least one LEKTI domain is functionally linked to a SecA domain.

6. The composition according to claim 1, wherein at least one LEKTI domain is functionally linked to an RMR domain.

7. The composition according to claim 1, wherein at least one LEKTI domain comprises the amino acid sequence described in SEQ ID NO:

1.

8. The composition according to claim 1, wherein the microorganisms are adapted to grow on the skin of mammals.

9. The composition according to claim 1, wherein the expression of at least one LEKTI domain is controlled by an operon, and the amount of LEKTI delivered to mammalian skin is proportional to the availability of external factors.

10. The composition according to claim 1, wherein the expression of at least one LEKTI domain is controlled by a constitutively active promoter.

11. The composition according to claim 1, wherein the microorganism is genetically modified by transfection / transformation using a recombinant DNA plasmid encoding one or more LEKTI protein domains and one or more antibiotic resistance genes.

12. A composition according to claim 1 for use in a method for treating a skin disease in a mammal requiring treatment for a skin disease, or for improving the effects thereof, The method comprises providing the composition according to claim 1, which comprises a microorganism genetically modified to express one or more LEKTI protein domains on the surface of mammalian skin, wherein the LEKTI protein domains are effective in penetrating one or more layers of mammalian skin and are effective in inhibiting the activity of at least one serine protease in or on mammalian skin. The aforementioned composition.

13. The composition according to claim 12, wherein the microorganism is adapted to survive on the surface of mammalian skin for a controlled period of time to provide a continuous supply of LEKTI protein domains.

14. A kit for treating or improving the effects of skin disease in mammals requiring treatment or improvement of the effects of skin disease, (1) A composition comprising a microorganism genetically modified to express one or more LEKTI protein domains, wherein the LEKTI protein domains are effective in penetrating one or more layers of mammalian skin and are effective in inhibiting the serine protease activity of at least one serine protease in or on mammalian skin; and (2) Reagent for applying the composition to the skin of a mammal A kit that includes this.

15. The kit according to claim 14, wherein the microorganism is adapted to survive on the surface of mammalian skin for a controlled period of time to provide a continuous supply of LEKTI protein domains.

16. A composition for the treatment of a skin disease comprising a microorganism containing a pJB38-LEKTI domain 6 complete plasmid construct, wherein domain 6 has the amino acid sequence of SEQ ID NO:

109.

17. The composition according to claim 16, wherein the microorganism is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof.

18. A composition containing the complete pJB38-LEKTI plasmid construct.

19. The composition according to claim 18, wherein the pJB38-LEKTI complete plasmid construct is expressed in a microorganism selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus and mixtures thereof.

20. A recombinant microorganism capable of secreting polypeptides, comprising an expression vector comprising a first coding sequence containing a gene capable of expressing polypeptides and a second coding sequence containing a gene capable of expressing cell-permeable peptides, The polypeptide is LEKTI domain 6, The composition wherein the cell-permeable peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, and 102.

Images