Methods and compositions for treating skin diseases using recombinant microorganisms

Abstract

The present disclosure provides methods and compositions for treating skin diseases, including engineered microorganisms expressing therapeutically relevant recombinant fusion polypeptides. In one embodiment, the present disclosure provides methods for treating skin diseases (e.g., atopic dermatitis), comprising administering to a subject in need thereof a composition comprising a live biotherapeutic product (LBP), the LBP comprising Staphylococcus epidermidis secreting human filaggrin. In a further aspect, the present invention provides a pharmaceutical composition comprising a filaggrin polypeptide as described herein. Still further, the present invention provides a kit comprising the compositions disclosed herein and instructions for use.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 653,021, filed April 5, 2018, the entire contents of which are incorporated herein by reference for all purposes. Background of the Invention

[0004] Ichthyosis vulgaris (IV) is a chronic, dry, scaly skin disease with an estimated incidence and prevalence of 1 in 250 1,2 , which results in a total patient population of 1.3 million in the United States. Clinical features of IV usually appear at about 2 months of age and include dryness and fine, white to gray scales all over the body, primarily on the abdomen, chest, and extensor surfaces of the limbs. 3 Some IV patients also experience hypohidrosis and heat intolerance 4 The pathogenesis of IV has long been identified as a decrease in the size or number of epidermal keratohyalin granules, or even their complete absence. 5-7 In addition, due to genetic factors, IV patients are at increased risk for atopic dermatitis (AD), asthma, and allergies. 8 .

[0005] Ichthyosis vulgaris is an autosomal semidominant disease caused by loss-of-function mutations in the gene encoding filaggrin. 9 Filaggrin is a basic structural protein derived from profilaggrin, which is broken down into monomeric filaggrin in the stratum corneum and strengthens the skin barrier by binding to keratin and other intermediate filament proteins in the keratinocyte cytoskeleton. 10 .

[0006] Many studies have identified IV and atopic dermatitis patients FLG Loss-of-function mutations 11-14 These mutations are associated with disordered keratin filaments in the stratum corneum, skin barrier defects, 15 Associated with microcracks, leading to enhanced transcutaneous allergen sensitization 16-19 In addition, filaggrin and its breakdown products have other important functions in the skin, including increasing skin moisture (via hygroscopic amino acids or "natural moisturizing factors") 20,21 , leading to the production of antimicrobial molecules (particularly against Staphylococcus aureus) 22 and maintain a favorable lipid profile in the skin 23,24 and pH 24-26 both.

[0007] Current treatment options for IV therapy primarily include topical evaporation inhibitors (e.g., sodium chloride, urea, lactic acid, salicylic acid) and moisturizers (e.g., glycerin, propylene glycol, dexpanthenol)4 Topical retinoids may also be prescribed to try to slow the body's production of skin cells; however, as vitamin A derivatives, long-term use is not ideal. Of note, many IV patients experience a significant reduction in quality of life due to self-consciousness and social distress. 27-29 , and see negative impacts on family life, education / professional life, and even leisure / sports activities 28,29 It's clear that IV is a huge unmet need.

[0008] A diverse microbial community thrives on the skin, and one square centimeter can contain up to a billion microorganisms. 39 These diverse communities of bacteria, fungi, mites and viruses provide protection from disease and form a dynamic but unique microhabitat on the skin. 40 There is growing evidence that 39,41 , especially AD 42,43 The skin microbiome is associated with changes in microbial communities or dysbiosis in the skin. Engineered probiotics are a novel approach based on the use of the skin microbiome for treatment. It is worth noting that engineered probiotics have important advantages over other drug delivery methods because they will establish residence on the patient's skin and continuously and stably deliver therapeutic proteins in situ. In addition, Staphylococcus epidermidis ( Staphylococcus epidermidis , SE) have demonstrated important beneficial immunomodulatory and antipathogenic effects on the skin that are associated with atopic dermatitis disease phenotype and severity. In addition, delivery of filaggrin, a structural protein derived from profilaggrin, further enhances the treatment regimen due to its role in the skin barrier and ability to reduce transepidermal water loss and improve skin hydration. The present invention has the following surprising advantages: methods and compositions (e.g., AZT-01) are provided for treating skin diseases (e.g., atopic dermatitis) using genetically engineered recombinant strains of Staphylococcus epidermidis as skin drug delivery systems that secrete human filaggrin to address the pathophysiology of atopic dermatitis. Once applied to the skin, stable colonization of the skin and subsequent in situ secretion of filaggrin can address the disease. Benefits of the present invention include its safety as a nonsteroidal treatment option, its efficacy due to the combination of the benefits of filaggrin secretion of the present invention with the benefits of topical application of Staphylococcus epidermidis, and its ability to be therapeutically effective even at low application frequencies (no more than once a day).

[0009] Thus, the present invention addresses the long-sought need for effective treatments for inflammatory skin diseases, particularly IV. The present invention is also one of the first reported demonstrations of commensal skin bacteria that can secrete therapeutic proteins to treat skin diseases. SUMMARY OF THE INVENTION

[0011] The present disclosure features a novel treatment modality for skin diseases, such as ichthyosis vulgaris (IV), which directly addresses the pathophysiology of ichthyosis vulgaris and consists of a live biotherapeutic product (LBP) comprising Staphylococcus epidermidis that secretes human filaggrin. The purpose of the present disclosure is to supplement the skin with long-term stable delivery of filaggrin by a chassis system based on beneficial microorganisms. Although the present invention utilizes and optimizes the potential probiotic properties of bacteria, disease correction is based on the rational targeted expression of related proteins that directly address the underlying pathophysiology. In addition, because the response to different microbial species and strains varies between individuals, a modular design that controls therapeutic delivery may provide significantly improved pharmacokinetics and disease regression compared to current methods that utilize naturally occurring strains. Importantly, because some strains can secrete antimicrobial peptides, the introduction of Staphylococcus epidermidis can help reestablish skin homeostasis by targeting pathogen-driven dysbiosis.

[0012] Thus, the present invention is particularly directed to methods and compositions for treating skin diseases (eg, IV) comprising engineered microorganisms capable of expressing a therapeutically relevant recombinant fusion polypeptide (ie, protein, peptide, or amino acid).

[0013] In the first aspect, the present invention features a recombinant microorganism capable of secreting a polypeptide, wherein the recombinant microorganism comprises an expression vector comprising a first coding sequence and a second coding sequence, wherein the first coding sequence comprises a gene capable of expressing the polypeptide, and the second coding sequence comprises a gene capable of expressing a cell penetrating peptide. In a related embodiment, the recombinant microorganism further comprises a third coding sequence comprising a gene capable of expressing an output signal. In another embodiment, the expression of the first coding sequence, the second coding sequence, and the third coding sequence is under the control of a promoter. In other embodiments, the arrangement of the first coding sequence, the second coding sequence, and the third coding sequence is in frame. In another related embodiment, the first coding sequence, the second coding sequence, and the third coding sequence are operably connected to a promoter. In one embodiment, the recombinant microorganism is a bacterium or a combination of bacteria. In another embodiment, the polypeptide is a silk polymer protein or a variant thereof. In other embodiments, the microorganism is selected from Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc or Oenococcus or a combination thereof. In other embodiments, the recombinant microorganism is Staphylococcus epidermidis. In some embodiments, the microorganism secretes a filaggrin fusion protein. In further embodiments, the filaggrin fusion protein comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1. In one embodiment, the filaggrin fusion protein consists of SEQ ID NO: 1.

[0014] In another aspect, the present invention features a method for producing a live biotherapeutic composition, the method comprising: (a) transfecting a cell with the following sequences: (i) a first coding sequence comprising a nucleic acid sequence capable of expressing a therapeutic polypeptide, and (ii) a second coding sequence comprising a nucleic acid sequence capable of expressing a cell penetrating peptide; and (b) causing the transfected cell to produce a therapeutic polypeptide fusion protein; and (c) obtaining the live biotherapeutic composition. In a related embodiment, the method further comprises: (iii) transfecting the cell with a third coding sequence, the third coding sequence comprising a nucleic acid sequence capable of expressing an output signal. In another embodiment, the first coding sequence, the second coding sequence, and the third coding sequence are arranged in a single plasmid. In another embodiment, the arrangement of the first coding sequence, the second coding sequence, and the third coding sequence is operably linked to a promoter. In other embodiments, the cell is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, or a combination thereof. In another embodiment, the cell is Staphylococcus epidermidis. In other embodiments, the therapeutic polypeptide fusion protein is a filaggrin fusion protein or a variant thereof. In further embodiments, the filaggrin fusion protein comprises an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 1. In one embodiment, the filaggrin fusion protein consists of SEQ ID NO: 1.

[0015] In another aspect, the disclosure features a composition comprising a filaggrin polypeptide or a variant thereof. In one embodiment, the filaggrin polypeptide is a fusion protein. In a further embodiment, the filaggrin fusion protein comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1. In one embodiment, the filaggrin fusion protein consists of SEQ ID NO: 1.

[0016] In another aspect, the invention features a composition obtained by any of the methods disclosed or described herein. In a related embodiment, the composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from an aqueous solution, an emulsion, a cream, a lotion, a gel or an ointment.

[0017] In another aspect, the present invention features a living biotherapeutic composition comprising a recombinant microorganism, wherein the recombinant microorganism comprises (i) a first coding sequence comprising a nucleic acid sequence capable of expressing a therapeutic polypeptide; (ii) a second coding sequence comprising a nucleic acid sequence capable of expressing a cell penetrating peptide; (iii) a third coding sequence comprising a nucleic acid sequence capable of expressing an export signal; and (iiv) a promoter operably linked to the first coding sequence, the second coding sequence, and the third coding sequence; wherein the first coding sequence, the second coding sequence, and the first coding sequence are capable of expressing a filaggrin fusion protein or a variant thereof. In a further embodiment, the filaggrin fusion protein comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1. In a related embodiment, the recombinant microorganism is Staphylococcus epidermidis. In another embodiment, the export signal exports the filaggrin fusion product or a variant thereof to the recombinant microorganism. In another embodiment, the cell penetrating peptide promotes the entry of the filaggrin fusion product or variant thereof into human keratinocytes.In another embodiment, the composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from an aqueous solution, an emulsion, a cream, a lotion, a gel or an ointment.

[0018] In another aspect, the invention features a kit comprising any of the compositions disclosed or described herein and instructions for use.

[0019] In another aspect, the invention features a method of treating a skin disease, the method comprising administering to a subject in need thereof a composition of any of the compositions disclosed or described herein. In one embodiment, the skin disease is IV. In another embodiment, the skin disease is atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a graph showing the characterization of strains grown in liquid culture. SE alone (solid black line, triangle markers) grows faster than SE-GFP alone or SE and SE-GFP together.

[0022] Figure 2A -C shows characterization of the growth of strains in RHE. (A) SE with empty vector (i.e., no protein production). (B) SE-GFP alone on RHE shows slower growth at 24 hours. (C) When mixed together 50:50 on RHE, SE empty vector outcompetes SE-GFP.

[0023] Figure 3A-C shows fluorescence and reflectance overlays. (A) Fluorescence SE on the surface and outlining individual cells in the stratum corneum (dashed lines show the outline of cornified cells as an example). (BC) Fluorescence and light wavelength overlays of RHE at 37°C for 3.5 hours after Dermaroller application followed by topical GFP application, showing that bacteria have localized into the crevices of the damaged stratum corneum, with no evidence of bacteria in the epidermis at this level. Depths were acquired at 50 µm (B) and 70 µm (C).

[0024] Figure 4A-C shows the results of GFP purification. Protein was purified from 225 mL SE culture (2.7-2.9 OD / mL) and lysed with CellB-Lyse (Sigma). 4 mL of eluate was generated and run on a Ni-NTA column in PBS pH 7.4 buffer and 0.4 mg was obtained. (A) InVision His-tag staining on SDS-PAGE gel with protein purified from SE. Lane (1) Lysate pellet; (2) Clarified supernatant; (3) Flowthrough; (4) Eluate; (5) Concentrate; (6) Marker; (7) Clarified supernatant; (8) Flowthrough; (9) Eluate; and (10) Concentrate. (C) GFP purified from SE.

[0025] Figure 5A-O shows characterization of proteins with and without RMR signal in RHE using 5 µg GFP as a reporter. (AD) Two-photon images of topically applied GFP with (C,D) or without (A,B) RMR signal at 30 min (A,C, E,G) or 60 min (B,D,F,H). Images were compiled as Z-stacks, which were projected onto a 2D plane. (EH) 3D surface analysis to examine the depth of protein penetration into RHE. (IN) Confocal images of GFP (K,N), GFP + RMR (J,M), or vehicle (I,L) using either optical (LN) or fluorescent (IK) wavelengths. (O) RHE punctured with a Dermaroller microneedle with 50 ug GFP-RMR. Images were acquired 30 min after application.

[0026] Fig. 6A-E shows a preliminary bioinformatics analysis of human filaggrin. (A) Homology of human filaggrin domains. (B) Sequence alignment of human filaggrin domains. (C) Hydrophobicity mapping of human filaggrin domains with corresponding illustration of the filaggrin sequence. (D) Example SAR training for human filaggrin monkey 9-10, using hydrophobicity mapping to remove the QSGEnSGRnSFLYQVSnHEQSES repeat at the N-terminus of hFLG 9-10, and (E) the resulting sequence of the protein with the RMR cell penetrating peptide added. (F) Overview of filaggrin structure. (Top) Profilaggrin and filaggrin gene structure. The majority of filaggrin is encoded by exon 3. (Bottom) Profilaggrin protein structure.

[0027] Fig. 7A -E shows assay development. (AC) Western blots of hFLG in culture media isolates from SE cultures producing filaggrin using various antibodies. (A) Sigma polyclonal anti-hFLG antibody. (B) SantaCruz polyclonal anti-hFLG antibody. (C) Polyclonal anti-hFLG antibody. (D) Proposed workflow for method development for MS-based filaggrin detection. (E) Identity of expressed human filaggrin from mass spectra for hFLG9-10. Colors indicate sequence confidence: dark blue (very high confidence), light blue (observed), and red (not observed).

[0028] Figure 8 Shown is a summary of Biacore surface plasmon resonance (SPR) for filaggrin activity.

[0029] Fig.9A -G shows experimental results for the generation of a standard curve for filaggrin decomposition products or natural moisturizing factor (NMF). Raman spectroscopy can detect individual NMF components (AD). Mass spectrometry (EG) is also very sensitive, with a limit of quantitation (LOQ) in the nanogram range in the figure.

[0030] Fig.10 Displays an overview of the workflow.

[0031] Fig.11 An experimental outline showing 16S sequencing. DETAILED DESCRIPTION OF THE INVENTION

[0033] Unless otherwise defined, all technical and scientific terms used in this article have the meanings commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many terms used in the present invention to technicians: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, the following terms used in this article have the meanings assigned to them below:

[0034] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0035] The term "including" is used herein to mean, and is interchangeable with, the phrase "including, but not limited to".

[0036] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0037] The term "such as" is used herein to mean, and is interchangeable with, the phrase "such as, but not limited to."

[0038] As used herein, the term "about" when used in reference to a specific described value means that the value may vary by no more than 1% from the described value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0039] The term "carrier", "carrier system" or "vehicle" as used herein refers to a compatible substance suitable for delivering, containing or "carrying" a pharmaceutically active ingredient or other substance for administration to a patient or subject in a topical composition. The carrier useful in this article should be pharmaceutically acceptable. The carriers and vehicles useful in this article include any such substances known in the art that are non-toxic and do not interact in a harmful manner with other components of the preparation containing it. The term "aqueous" refers to a preparation that contains water or becomes aqueous after being applied to the skin or mucosal tissue. Other examples of "carriers" include water, lower alcohols, higher alcohols, polyols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based mixtures and emulsion-based mixtures of such carriers.

[0040] As used herein, the term "engineered bacterial strain" or "recombinant bacterial strain" refers to a bacterial strain that has been "genetically modified" or "engineered" by introducing DNA prepared outside the organism into the bacterial strain. For example, the introduction of a plasmid containing new genes or other nucleic acid sequences into a bacterium will allow the bacterium to express those genes or other nucleic acid sequences. Alternatively, a plasmid containing new genes or other nucleic acid sequences can be introduced into a bacterium and then integrated into the genome of the bacterium, wherein the bacterium will express those genes or other nucleic acid sequences.

[0041] As used herein, the term "host cell" means a cell that has been transformed or transfected, or is capable of being transformed or transfected, by an exogenous polynucleotide sequence.

[0042] For the purposes of this disclosure, the term "isolated" refers to biological material (cells, nucleic acids or proteins) that has been removed from its original environment (the environment in which it naturally occurs). For example, a polynucleotide that exists in a plant or animal in its natural state is not isolated, but the same polynucleotide separated from its naturally occurring adjacent nucleic acids is considered "isolated."

[0043] An "isolated nucleic acid molecule" (e.g., an isolated promoter) is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules separated from the chromosome to which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid molecule does not contain sequences that naturally flank the nucleic acid molecule in the genomic DNA of the organism from which the nucleic acid molecule is derived.

[0044] As used herein, the term "genetic element" means a polynucleotide comprising a region encoding a polypeptide or a region of a polynucleotide regulating replication, transcription or translation or other processes important for expressing a polypeptide in a host cell, or a polynucleotide comprising both a region encoding a polypeptide and a region regulating expression operably linked thereto. Genetic elements may be contained in vectors that replicate as episomal elements (that is, as molecules that are physically independent of the host cell genome). They may be contained in plasmids. Genetic elements may also be contained in the host cell genome; not in their native state, but in purified DNA form or in a vector, etc., after manipulations such as isolation, cloning, and introduction into a host cell.

[0045] As used herein, the term "live biotherapeutic product" (or LBP) refers to a product candidate containing bacteria, yeast and / or other microorganisms.

[0046] As used herein, the term "patient" or "subject" refers to a human or an animal (in the case of an animal, more typically a mammal, such as a domesticated mammal, or an animal, such as poultry and fish and other seafood or freshwater organisms) who is to receive the treatments and compositions of the present invention.

[0047] As used herein, the expression "a subject in need thereof" refers to a human or non-human animal that exhibits one or more symptoms or signs of atopic dermatitis, and / or has been diagnosed with IV.

[0048] As used herein, the phrase "pharmaceutically acceptable" refers to active compounds, materials, engineered bacterial strain(s), compositions, carriers, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0049] As used herein, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. Therefore, for example, the polynucleotide used herein refers especially to single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions or single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, comprising DNA and RNA hybrid molecules (can be single-stranded or more generally double-stranded or triple-stranded, or a mixture of single-stranded and double-stranded regions). In addition, the polynucleotide used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chain in such a region can come from the same molecule or from different molecules. The region can include all of one or more molecules, but more generally only includes the region of some molecules. One of the molecules in the triple helix region is typically an oligonucleotide. As used herein, the term polynucleotide includes DNA or RNA as described above, which includes one or more modified bases. Therefore, DNA or RNA with a skeleton modified for stability or for other reasons is "polynucleotide" as the term means herein. In addition, only two examples are pointed out, DNA or RNA containing unusual bases (such as inosine) or modified bases (such as tritylated bases) are polynucleotides, as the term is used in this article. It should be understood that various modifications have been carried out to DNA and RNA, which are used for many useful purposes known to those skilled in the art. As used herein, the term polynucleotide includes such chemical, enzymatic or metabolic modified forms of polynucleotides, and chemical forms of DNA and RNA that are particularly unique to viruses and cells (including simple and complex cells). The term polynucleotide also includes short polynucleotides, which are commonly referred to as oligonucleotides. "Polynucleotide" and "nucleic acid" are usually used interchangeably in this article.

[0050] As used herein, the term "polypeptide" or "protein" refers to a biomolecule or macromolecule consisting of amino acid residues bonded together in a chain. The definition of polypeptide used herein is intended to encompass proteins (generally higher molecular weight) consisting of long chains of one or more amino acid residues and small peptides (generally lower molecular weight) of several amino acids. In other embodiments, single amino acids, although not technically polypeptides, are also considered within the scope of the present invention.

[0051] As used herein, the term "prevention" refers to completely or nearly completely preventing an abnormal skin disorder (e.g., IV) from occurring, such as when a patient or subject is susceptible to or at risk of contracting an abnormal skin disorder. Prevention may also include inhibition, i.e., preventing the development of an abnormal skin disorder.

[0052] As used herein, "promoter" refers to a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, adjacent to the transcription start site of the structural gene. If the promoter is an inducible promoter, the transcription rate increases in response to an inducing agent. For example, a promoter can be regulated in a tissue-specific manner so that it is active only in a specific tissue type when transcribing the associated coding region.

[0053] As used herein, the term "reducing the risk of" refers to reducing the likelihood or probability of an abnormal skin condition (eg, IV) occurring, such as when the patient or subject is susceptible to or at risk of contracting an abnormal skin condition.

[0054] As used herein, the term "therapeutically effective amount" refers to the amount of a pharmaceutically active compound or combination of compounds, or the amount of a pharmaceutically active compound delivered by one or more engineered bacterial strains (e.g., one or more skin therapeutic agents), when administered alone or in combination, to treat, prevent, or reduce the risk of a disease state or condition, such as an abnormal skin disorder (e.g., IV). The term also refers to the amount of a pharmaceutical composition containing an active compound or combination of compounds or one or more engineered bacterial strains that deliver pharmaceutically active compounds. For example, an effective amount refers to the amount of a compound present in a preparation administered to a patient or subject, or the amount of a compound delivered by one or more engineered bacterial strains present in a preparation administered to a patient or subject, which is sufficient to induce biological activity, such as activity for treating or preventing abnormal skin disorders.

[0055] As used herein, the term "treating" refers to providing therapeutic intervention to cure or improve an abnormal skin condition (eg, IV).

[0056] Composition

[0057] The present invention provides skin-resident microorganisms (e.g., bacteria) for treating or preventing skin diseases, which are genetically altered to express recombinant therapeutic polypeptides (Figure 2). The use of genetically engineered protein-producing microorganisms (e.g., bacteria) has multiple advantages over prior art methods for treating skin diseases. The therapeutic protein is able to treat the root cause of the defect that leads to the skin condition. In addition, the microorganism (e.g., bacteria) is able to replicate itself while retaining the inserted nucleic acid (e.g., gene) to continuously produce the therapeutic protein.

[0058] The present invention provides skin-colonizing microorganisms (e.g., bacteria, e.g., Staphylococcus epidermidis) that are genetically altered to express a therapeutic protein (e.g., human filaggrin). The use of genetically engineered filaggrin-producing microorganisms (e.g., bacteria) has several advantages over the use of filaggrin supplementation. First, the microorganism (e.g., bacteria) is able to replicate itself while retaining the inserted filaggrin nucleic acid sequence (e.g., gene). Second, Staphylococcus epidermidis is commonly present on the skin and has been shown to inhibit the growth of Staphylococcus aureus, a bacterial species of the same genus that dominates the skin flora in atopic dermatitis outbreaks.

[0059] Bacterial strains

[0060] The present invention provides genetically altered microorganisms, such as bacteria, capable of expressing recombinant therapeutic proteins. A wide range of microorganisms are suitable for use in the present invention. Examples include, but are not limited to, non-pathogenic and symbiotic bacteria. Bacteria suitable for use in the present invention include, but are not limited to, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus (e.g., Staphylococcus epidermidis), Lactobacillus (e.g., Lactobacillus acidophilus), Pediococcus, Leuconostoc or Oenococcus. In certain embodiments of the present invention, the bacteria is Staphylococcus epidermidis. In a preferred embodiment of the present invention, the Staphylococcus epidermidis strain to be used cannot produce biofilm. One such example of a Staphylococcus epidermidis strain that cannot produce biofilm is Staphylococcus epidermidis strain ATCC12228. However, in yet other embodiments of the present invention, other related or similar species found on the skin can be used.

[0061] Therapeutic proteins

[0062] The present invention provides genetically altered microorganisms, such as bacteria, capable of expressing recombinant therapeutic proteins. In a preferred embodiment of the present invention, the therapeutic protein comprises human filaggrin. Human filaggrin is encoded by a human gene ( FLG ) is expressed. Filaggrin is a protein produced by differentiated keratinocytes and has the function of aggregating keratin filaments into a cytoskeleton that, in combination with other components, constitutes the cornified cell envelope. FLGis a large gene located on chromosome lq21 that produces profilin, an insoluble multimeric protein that is proteolytically cleaved to release functional filaggrin monomers (Armengot-Carbo et al. 2014). The therapeutic protein of the present invention (and the gene that expresses the protein) can be from any mammal. Non-limiting examples include, but are not limited to, mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate or human gene sequences. In a preferred embodiment of the present invention, the therapeutic protein comprises a recombinant fusion protein comprising filaggrin operably linked to a cell penetrating protein (CPP). In one embodiment, the cell penetrating protein is RMRRMRRMRR. In other embodiments of the present invention, the therapeutic protein comprises a recombinant fusion protein comprising filaggrin operably linked to an export or secretion signal that allows the recombinant filaggrin to be exported from a microorganism (e.g., bacteria). In another embodiment, the therapeutic protein comprises a recombinant fusion protein comprising filaggrin operably linked to a cell penetrating protein (CPP) and an export or secretion signal.

[0063] In one embodiment, a fusion protein comprising filaggrin operably linked to a cell penetrating protein is shown in SEQ ID NO. 1.

[0064] SEQ ID NO. 1

[0065]

[0066] In one embodiment, the filaggrin fusion protein comprises an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 1. In one embodiment, the filaggrin fusion protein consists of SEQ ID NO: 1.

[0067] In some aspects, the therapeutic protein is not expressed by a microorganism (e.g., bacteria). In some aspects, the therapeutic protein comprises a silk polymer protein polypeptide or a variant thereof. In one embodiment, the silk polymer protein polypeptide is a fusion protein. In a further embodiment, the silk polymer protein polypeptide comprises an amino acid sequence with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1. In one embodiment, the silk polymer protein fusion protein consists of SEQ ID NO: 1.

[0068] secretion signal

[0069] A secretion signal or export signal is a peptide sequence on a protein that promotes the export of the protein through the secretory pathway, which ultimately results in the protein being secreted from the cell. In the present invention, any secretion signal that promotes the export of a protein (such as a protein comprising filaggrin) from a microorganism (e.g., a bacterial cell) is contemplated as a secretion signal.

[0070] Cell Penetrating Peptides

[0071] Cell penetrating peptides are peptide sequences that facilitate or mediate the delivery of biomolecules (e.g., proteins) in vivo without the use of any receptors and without causing any significant membrane damage. Cell penetrating peptides that facilitate entry into skin keratinocytes are contemplated as cell penetrating peptides of the present invention.

[0072] In one embodiment, the cell penetrating protein is RMRRMRRMRR (SEQ ID NO. 2).

[0073] Genetic constructs

[0074] The present invention utilizes standard molecular biology techniques, such as those described in (Sambrook et al. 2001). An example of a genetic construct for use in the present invention is pAZT, which is based on pJB38 (an allelic exchange E. coli-Staphylococcus shuttle vector) and further includes other design features on the plasmid to improve functionality (Bose, JL et al. .Applied and environmental microbiology. 2013;79(7):2218-2224). Using standard molecular biology techniques, a plasmid is constructed by inserting the cDNA of a gene encoding a therapeutic protein into a restriction site (Figure 2). The insert further comprises a coding sequence driven by a promoter. Such promoters can be constitutive or inducible. Examples of inducible promoters include those activated by compounds such as alcohols, sugars, metals, or tetracyclines or by physical factors such as light or high temperatures.

[0075] The mRNA sequence of human FLG has Genebank accession number NM_002016. FLG A portion of the cDNA was inserted into the restriction site of pJB38 to construct plasmid pAZT. The insert contained a nucleic acid coding sequence driven by a promoter. The construct further contained a nucleic acid sequence encoding a secretion signal and a cell penetrating peptide, thereby producing a recombinant filaggrin fusion protein.

[0076] Applications of recombinant bacterial strains

[0077] It should be understood that the skin disease to be treated can be any disease or disorder related to the skin. In one embodiment, the disorder is selected from atopic dermatitis, psoriasis, acne, allergic contact dermatitis, epidermolytic hyperkeratosis, seborrheic dermatitis, eczema, dry skin, allergies, rashes, UV-irritated skin, detergent-irritated skin (including irritation caused by enzymes and molecules used in detergents and sodium lauryl sulfate), thinning of the skin (e.g., skin of the elderly and children), bullous pemphigoid, pemphigus vulgaris, impetigo, vitiligo, alopecia, and hirsutism. In a specific embodiment, the skin disease is ichthyosis vulgaris (IV).

[0078] Examples of proteins that can be administered according to the present invention are preferably eukaryotic proteins. These proteins include, but are not limited to, single amino acids, small peptides, and large proteins. More specifically, genes encoding proteins that can be used as recombinant therapeutic proteins in the present invention include, but are not limited to, the following: members of the interleukin gene family, including, but not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15 and genes encoding their receptor antagonists. Genes encoding hematopoietic growth factors are also contemplated in the present invention, including, but not limited to, erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, stem cell factor, leukemia inhibitory factor, and thrombopoietin. Genes encoding neurotrophic factors are also contemplated, including, but not limited to, nerve growth factor, brain-derived neurotrophic factor, and ciliary neurotrophic factor. In addition, genes encoding interferons are included, including, but not limited to, IFN-α, IFN-β, and IFN-γ. Genes encoding chemokines (such as the CC family and CXC family of cytokines), genes encoding hormones (such as proinsulin and growth hormone), and genes encoding thrombolytic enzymes (including tissue plasminogen activator, streptokinase, urokinase, or other enzymes such as trypsin inhibitors) are further encompassed in the present invention. The present invention also includes genes encoding tissue repair factors, growth and regulatory factors, including, but not limited to, oncostatin M, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, bone morphogenic protein, insulin-like growth factor, calcitonin, and transforming growth factor α and β. Further encompassed genes include genes encoding structural proteins, including filament polymerase, actin, collagen, microfibrillin, elastin, or hard protein.

[0079] In a specific embodiment, the gene is a gene encoding filaggrin.

[0080] Antibody

[0081] The present disclosure also provides antibodies against an epitope of hFLG that is common in all subdomain pairs.In certain embodiments, the antibody is chicken IgY.

[0082] preparation

[0083] The formulations used according to the invention can contain any pharmaceutically effective amount of a genetically engineered microorganism, e.g., a bacterium, to produce a therapeutically effective amount of a desired polypeptide, e.g., at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 21.0%, about 22.0%, about 23.0%, about 24.0%, about 25.0%, about 26.0%, about 27.0%, about 28.0%, about 29.0%, about 30.0%, about 31.0%, about 32.0%, about 33.0%, about 34.0%, about 35.0%, about 36.0%, about 37.0%, about 38.0%, about 39.0%, about 40.0%, about 41.0%, about 42.0%, about 43.0%, about 44.0%, about 45.0%, about 46.0%, about 47.0%, about 48.0%, about 49.0%, about 50.0%, about 51.0%, about 52.0%, about 53.0%, about 54.0%, about 55.0%, about 56.0 .0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% or more of a genetically engineered microorganism, e.g., bacteria, with an upper limit of about 90.0% by weight of a genetically engineered microorganism, e.g., bacteria.

[0084] In an alternative embodiment, a formulation for use in accordance with the present invention can include, for example, at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5%, or more by weight of a genetically engineered microorganism, e.g., bacteria.

[0085] In some aspects, the formulation used according to the present invention may comprise any pharmaceutically effective amount of a filaggrin polypeptide or a variant thereof. In one embodiment, the filaggrin polypeptide is a fusion protein. In a further embodiment, the filaggrin polypeptide comprises an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 1.

[0086] In some embodiments, the preparation is a topical preparation. The topical preparation for use in the present invention can be in any form suitable for application to the body surface, such as creams, lotions, sprays, solutions, gels, ointments, pastes, plasters, paints, bioadhesives, suspensions, emulsions, etc., and / or can be prepared to contain liposomes, micelles and / or microspheres. Such preparations can be used in combination with a closed covering layer so that the moisture evaporated from the body surface is maintained in the preparation when applied to the body surface and thereafter. The preparation can include a live biological therapeutic composition, and can include at least one genetically engineered microorganism, for example, an engineered bacterial strain that produces a recombinant polypeptide. The engineered live biological therapeutic composition can deliver the polypeptide directly to the skin for treatment or prevention of abnormal skin conditions and / or skin diseases (e.g., inflammatory skin diseases).

[0087] Topical formulations include those in which any other active ingredients are dissolved or dispersed in a dermatological vehicle known in the art, such as an aqueous or non-aqueous gel, ointment, water-in-oil or oil-in-water emulsion. The components of such a vehicle may include water, an aqueous buffer solution, a non-aqueous solvent (such as ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycogen or glycerol), an oil (e.g., a mineral oil such as liquid paraffin, a natural or synthetic triglyceride such as Miglyol™, or a silicone oil such as polydimethylsiloxane). Depending on the nature of the formulation and its intended use and site of administration, the dermatological vehicle used may contain one or more components selected from the following (e.g., components other than water when the formulation is an aqueous gel): a solubilizer or solvent (e.g., a β-cyclodextrin, such as hydroxypropyl β-cyclodextrin, or an alcohol or polyol such as ethanol, propylene glycol or glycerol); a thickener (e.g., hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose or carbomer); a gelling agent (e.g., a polyoxyethylene-polyoxypropylene copolymer); a preservative (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorobutanol, a benzoate, potassium sorbate or EDTA or a salt thereof); and a pH buffer (e.g., a mixture of dihydrogen phosphate and hydrogen phosphate, or a mixture of citric acid and hydrogen phosphate).

[0088] Pharmaceutically acceptable carriers can also be incorporated into the preparations of the present invention, and can be any carrier conventionally used in the art. Examples thereof include water, lower alcohols, higher alcohols, polyols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based mixtures and emulsion-based mixtures of such carriers. The term "pharmaceutically acceptable" or "pharmaceutically acceptable carrier" is used herein to represent such compounds or compositions: it can be incorporated into pharmaceutical preparations without causing undesirable biological effects or undesirable interactions with other components of the preparations. "Carrier" or "vehicle" used herein means a carrier material suitable for incorporation into topical compositions. Useful carriers and vehicles herein include any such material known in the art, which is nontoxic and does not interact with other components of the preparation containing it in a harmful manner. The term "aqueous" means a preparation containing water or becoming water-containing after being applied to the skin or mucosal tissue.

[0089] Cream bases are washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase is also referred to as the "internal" phase and is typically composed of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. Although not necessarily, the aqueous phase often exceeds the oil phase in volume and typically contains a humectant. The emulsifier in the cream formulation is typically a nonionic, anionic, cationic, or amphoteric surfactant.

[0090] Lotions are products to be applied to the surface of the skin without friction, and are generally liquid or semi-liquid preparations in which particles (including active agents) are present in a water or alcohol matrix. Lotions are often suspensions of solids, preferably containing liquid oily emulsions of the water-in-oil type. Lotions are preferred preparations for treating large body areas herein because more fluid compositions are easy to apply. Insoluble substances in lotions must usually be finely pulverized. Lotions generally contain suspending agents (to produce better dispersions) and compounds that can be used to position and maintain active agents in contact with the skin, such as methylcellulose, sodium carboxymethyl-cellulose, etc. A solution is a homogeneous mixture prepared as follows: one or more chemical substances (solutes) are dissolved in a liquid so that the molecules of the dissolved substances are dispersed in the molecules of the solvent. The solution may contain other pharmaceutically or cosmetically acceptable chemicals to buffer, stabilize or preserve the solute. Common examples of solvents for preparing solutions are ethanol, water, propylene glycol, or any other acceptable vehicle. Of course, it is well known that gels are semisolid suspension systems. Single-phase gels contain organic macromolecules that are substantially uniformly distributed in a carrier liquid (which is typically aqueous, but preferably also contains alcohol and optionally oil). Preferred organic macromolecules, i.e., gelling agents, are cross-linked acrylic acid polymers such as polymers of the "carbomer" family, for example, carboxypolyolefins commercially available under the Carbopol trademark. Also preferred are hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, a dispersant such as alcohol or glycerol may be added, or the gelling agent may be dispersed by grinding, mechanical mixing or stirring, or a combination thereof. As is also well known in the art, ointments are semisolid preparations that are generally based on petrolatum or other petroleum derivatives. Those skilled in the art will appreciate that the specific ointment base to be used is an ointment base that will provide many desirable features (e.g., emollients, etc.). As other carriers or vehicles, ointment bases should be inert, stable, non-irritating and insensitive. As explained in Remington: The Science and Practice of Pharmacy, the 19th edition (Easton, PA: Mack Publishing Co., 1995) at 1399-1404 pages, ointment bases can be grouped into 4 categories: oily bases, emulsifiable bases, emulsion bases and water-soluble bases. Oily ointment bases include, for example, vegetable oils, fats obtained from animals and semisolid hydrocarbons obtained from petroleum.

[0091] Emulsifiable ointment bases, also known as absorbable ointment bases, contain little or no water and include, for example, hydroxytristearin sulfate, anhydrous lanolin and hydrophilic petrolatum.

[0092] Emulsion ointment bases are water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weights; see Remington: The Science and Practice of Pharmacy for further information.

[0093] Pastes are semisolid dosage forms in which the active agent is suspended in a suitable matrix. Depending on the nature of the matrix, pastes are classified as fatty pastes or those made from single-phase aqueous gels. The matrix in fatty pastes is usually petrolatum or hydrophilic petrolatum, etc. Pastes made from single-phase aqueous gels usually incorporate carboxymethylcellulose, etc. as a matrix.

[0094] Enhancers are lipophilic co-enhancers, commonly referred to as "plasticizing" enhancers, i.e., enhancers having a molecular weight in the range of about 150-1000, less than about 1 wt%, preferably less than about 0.5 wt%, and most preferably less than about 0.2 wt% water solubility. The Hildebrand solubility parameter δ of the plasticizing enhancer is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilic enhancers are fatty acid esters, fatty alcohols, and fatty ethers. Specific and most preferred examples of fatty acid esters include methyl laurate, ethyl oleate, propylene glycol monolaurate, propylene glycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyl dodecyl myristate. Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol, while fatty ethers include compounds in which a diol or triol, preferably a C2-C4 alkane diol or triol, is substituted with one or two fatty ether substituents. Additional penetration enhancers are known to those of ordinary skill in the art of topical drug delivery, and / or are described in the relevant texts and literature. See, e.g., Percutaneous Penetration Enhancers, Smith et al., ed. (CRC Press, 1995) (incorporated herein by reference in its entirety).

[0095] 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 types of materials whose presence may be pharmaceutically or otherwise desirable. Typical examples of optional additives for inclusion in the formulations of the present invention are as follows: preservatives such as sorbates; solvents such as isopropyl alcohol and propylene glycol; astringents such as menthol and ethanol; emollients such as polyalkylene methyl glucoside; humectants such as glycerol; emulsifiers such as glyceryl stearate, PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol and other polyhydroxy alcohols such as polyethylene glycol; sunscreens such as octyl methoxycinnamate (commercially available as Parsol MCX) and butyl methoxybenzoylmethane (available under the trade name Parsol 1789); antioxidants such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζι ... Λ - tocopherol, n-tocopherol and retinol (vitamin A); essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g., soybean oil, palm oil, liquid fractions of shea butter, sunflower oil), animal oils (e.g., perhydrosqualene), synthetic oils, silicone oils or waxes (e.g., cyclomethicone and dimethicone), fluorinated oils (usually perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol) and waxes (e.g., beeswax, carnauba wax and paraffin); skin feel modifiers; and thickeners and structurants such as swelling clays and cross-linked carboxy polyolefins commercially available under the Carbopol trademark. Other additives include benefit agents such as those that condition the skin (particularly, the upper layer of the skin in the stratum corneum) and keep it soft and / or protect the skin by delaying the reduction of its water content. Such conditioning agents and moisturizers include, for example, pyrrolidine carboxylic acids and amino acids; organic antimicrobial agents such as 2,4,4'-trichloro-2-hydroxydiphenyl ether (triclosan) and benzoic acid; anti-inflammatory agents such as acetylsalicylic acid and glycyrrhetinic acid; anti-seborrheic agents such as retinoic acid; vasodilators such as niacin; melanin production inhibitors such as kojic acid; and mixtures thereof. Further other active agents include, for example, alpha hydroxy acids, alpha keto acids, polymeric hydroxy acids, moisturizers, collagen, marine extracts and antioxidants such as ascorbic acid (vitamin C), alpha-tocopherol (vitamin E), beta-tocopherol, gamma-tocopherol, 6-tocopherol, epsilon-tocopherol, zeta-tocopherol, zeta-2-tocopherol, eta-tocopherol and retinol (vitamin A) and / or pharmaceutically acceptable salts, esters, amides or other derivatives thereof. A preferred tocopherol compound is alpha-tocopherol. Additional agents include those capable of improving oxygen supply in skin tissue, such as, for example, in Gross et al. ,WO 94 / 00098 and Gross et al. , WO 94 / 00109 (both assigned to Lancaster Group AG, incorporated herein by reference in their entirety). 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, menthol anthranilate, octocrylene, octyl methoxycinnamate, octylsalicylate, oxybenzone, padimamate O, phenylbenzimidazole sulfonic acid, sulisophenone, titanium dioxide, triethanolamine salicylate, zinc oxide, ensolizole, meradiraate, octinoxate, octocrylene. See Title 21, Chapter 1, Subsection D, Section 352, "Sunscreen drug products for over-the-counterhuman use", which is incorporated herein in its entirety. Other embodiments may include a variety of non-carcinogenic, non-irritating healing materials that promote treatment with the formulations of the present invention. Such healing materials may include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, glandular or animal extracts or safe therapeutic agents that may be added to the formulation to promote healing of the skin condition.

[0096] The present invention contemplates these various additives in amounts equivalent to those conventionally used in the cosmetic art and ranging, for example, from about 0.01% to about 20% of the total weight of the topical formulation.

[0097] The formulations of the present invention may also include conventional additives such as opacifiers, fragrances, colorants, stabilizers, surfactants, etc. In certain embodiments, other agents such as antimicrobial agents may also be added to prevent deterioration during storage, i.e., inhibit the growth of microorganisms such as yeast and mold.

[0098] Suitable antimicrobial agents for the present invention include, but are not limited to, those selected from: methyl and propyl esters of p-hydroxybenzoic acid (i.e. methyl parahydroxybenzoate and propyl parahydroxybenzoate), sodium benzoate, sorbic acid, imidurea and their combinations. In other embodiments, other reagents may also be added, such as repressors and inducers, i.e., suppressing (i.e. glucose) or inducing (i.e. xylose) the generation of the target polypeptide. As long as it is compatible with the function of the preparation and does not interfere with the function of the preparation, such additives may be used.

[0099] The formulations may also contain irritation reducing additives to minimize or eliminate the possibility of skin irritation or skin damage caused by the chemical entity to be administered or other components of the composition.

[0100] Suitable additives to reduce irritation include, for example: alpha-tocopherol; monoamine oxidase inhibitors, especially phenyl alcohols such as 2-phenyl-1-ethanol; glycerol; salicylates; ascorbic acid; ionophores such as monensin; amphiphilic amines; ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine. If present, additives to reduce irritation can be incorporated into the composition at a concentration effective to reduce irritation or skin damage, usually accounting for no more than about 20% by weight of the formulation, more usually no more than about 5% by weight. Other suitable pharmacologically active agents that can be incorporated into the present formulation and thus topically applied with the active agent in certain embodiments include, but are not limited to, the following: agents that improve or eradicate pigmented or non-pigmented age spots, keratosis, and wrinkles; antimicrobial agents; antibacterial agents; antipruritic and anti-drying agents; anti-inflammatory agents; local anesthetics and analgesics; corticosteroids; retinoids; vitamins; hormones; and anti-metabolites. Some examples of topical pharmacologically active agents include acyclovir, amphotericin, chlorhexidine, clotrimazole, ketoconazole, econazole, miconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin, phenytoin, para-aminobenzoic acid esters, octyl methoxycinnamate, octyl salicylate, oxybenzone, dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide, zinc pyrithione, diphenhydramine, pramoxine, lidocaine, procaine, erythromycin, tetracycline, clindamycin, crotamiton, Hydroquinone and its monomethyl and benzyl ethers, naproxen, ibuprofen, cromoglycic acid, retinol, retinyl palmitate, retinyl acetate, coal tar, griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetate, hydrocortisone 17-valerate, hydrocortisone 17-butyrate, progesterone, betamethasone valerate, betamethasone dipropionate, triamcinolone acetonide, fluocinolone acetonide, clobetasol propionate, minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide and 5-fluorouracil. Creams, lotions, gels, ointments, pastes, etc. can be spread on the affected surface and gently rubbed. Solutions can be applied in the same manner, but more usually will be applied with a dropper, swab, etc. and carefully applied to the affected area.

[0101] The administration schedule will depend on a number of readily determinable factors, such as the severity of the condition and its responsiveness to initial treatment, but will generally not involve more than one administration per day. One of ordinary skill can readily determine the optimum amount of the formulation to be administered, the method of administration, and the repetition rate. In general, it is contemplated that the formulations of the invention will be administered within the range of once or twice per week up to once per day.

[0102] method

[0103] Treatment

[0104] The present invention provides methods for treating skin diseases, wherein the methods include administering to a subject in need of such treatment a genetically engineered microorganism, e.g., a genetically engineered bacterium, which is capable of expressing a recombinant therapeutic fusion protein of the present invention, thereby treating the subject. In some aspects, the present invention provides methods for treating skin diseases, wherein the methods include administering to a subject in need of such treatment a pharmaceutical composition comprising a silk polymer protein polypeptide as described herein. In some embodiments, the silk polymer protein polypeptide is a fusion protein. In further embodiments, the silk polymer protein polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 1.

[0105] In one embodiment, the disease is atopic dermatitis.

[0106] In one embodiment, the disease is ichthyosis vulgaris (IV). The clinical features of IV usually appear at about 2 months of age and include dryness of the whole body and fine, white to gray scales, mainly on the abdomen, chest and extensor surfaces of the limbs. IV is generally caused by a genetic mutation inherited from one or both parents. The scales of ichthyosis vulgaris (sometimes referred to as ichthyosis or ichthyosis) may be present at birth, but usually appear for the first time during early childhood. In one embodiment, the subject to be treated is a child. Sometimes other skin diseases, such as the allergic skin disorder eczema are associated with ichthyosis vulgaris.

[0107] In another preferred embodiment, the recombinant therapeutic fusion protein comprises filaggrin. In other embodiments, the recombinant therapeutic fusion protein comprises filaggrin operably linked to a cell penetrating peptide. In one embodiment, the filaggrin fusion protein comprises an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 1.

[0108] In additional embodiments, the recombinant therapeutic fusion protein is operably linked to an output signal.

[0109] Reagent test kit

[0110] The present invention also provides a kit. In one aspect, the kit of the present invention comprises (a) a composition of the present invention and (b) instructions for use thereof. In another aspect, the kit of the present invention comprises (a) any one of the live biological therapeutic compositions of the present invention and (b) instructions for use thereof. The composition of the present invention is as described above. In certain embodiments, the composition of the present invention is an engineered microorganism capable of expressing a therapeutically relevant recombinant fusion polypeptide as described above. In a preferred embodiment, the composition comprises an engineered bacterium (e.g., Staphylococcus epidermidis) capable of expressing a recombinant fusion polypeptide comprising filament polymerase.

[0111] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of all figures and all references, patents and published patent applications cited in this application, as well as the drawings, are expressly incorporated herein by reference in their entirety. Example

[0112] The following examples further describe and demonstrate embodiments within the scope of the present invention. The examples are given for the purpose of illustration only and are not to be construed as limitations of the present invention as many variations thereof are possible without departing from the spirit and scope of the present invention.

[0113] Example 1. Quantification and comparison of growth characteristics of transformed bacteria in liquid culture medium

[0114] It is important to obtain a basic understanding of the ability of the transformed Staphylococcus epidermidis (SE) to compete with wild-type SE. In order to understand the growth characteristics of the transformed bacteria and the growth dynamics of the protein-producing bacteria of the recombinant, a standard growth method in liquid culture medium was used. In order to determine the growth differences between SE-sGFP, SE-chl (empty vector control, resistance to chloramphenicol (chl)) and wild-type SE, each strain was grown for 6 hours in 100 mL culture. Every 45 minutes (approximately the doubling time of SE), 1 mL samples were obtained and measured at 600 nm to obtain the measurement results of the total bacterial concentration. Optical density was compared in all samples to understand the growth characteristics.

[0115] The results are shown in Figure 1 .like Figure 1 As shown, protein production only slightly reduced the competitive growth of SE-GFP relative to SE, as determined by CFU measurements. The reduced growth is expected to be due to increased metabolic demands in protein-producing SE.

[0116] Example 2. On reconstructed human epidermis (RHE) SE-GFP Growth quantification of the control strains

[0117] To characterize the feasibility of applying bacteria to the skin, the growth kinetics of externally applied bacteria on an in vitro skin model were determined. This model provides a preliminary estimate of the ecological competition these bacteria will encounter on human skin. RHE cultures were established and maintained in medium without antibiotics and antifungals (supplemented with chloramphenicol as needed). SE-sGFP Use a pipette to apply to the center of the RHE 3mm diameter. SE-chl and SE-WT A control RHE of bacteria was also applied side by side with the experimental groups. At predetermined time points, tissue inserts were removed, homogenized and passed through a 40 µm filter to allow collection of bacterial flow-through. The bacterial suspension was spun down, resuspended in culture medium, serially diluted and plated to determine the CFU of bacteria. All measurements were normalized to the maximum recovery of bacteria determined by the CFU present 15 minutes after application.

[0118] The results are shown in Figure 2. As shown in Figure 2, when applied to RHE, SE-GFP Compared with empty vector SE Growth was slower. SE The growth reached a stable level after 24 hours, but SE-GFP Bacteria reached a steady state by 48 hours, and final counts were low ~ ½ log. SE Faster growth and slightly better protein production SE , with a CFU difference of at least one log (Figure 2). These results combined with those above lead to the conclusion that protein production SE This may be at a slight disadvantage when colonizing the skin in a clinical setting, potentially requiring additional genetic modifications to promote growth and competition. However, as shown in Example 3, these bacteria are fully capable of colonizing a skin model.

[0119] Example 3. SE-GFP Qualitative characterization of the growth of the 20 and 25 control strains on RHE

[0120] The purpose of this experiment is to use RHE and Vivascope to add SE-GFP The spatial and temporal information of settlement. SE-GFPThe samples were imaged in both reflection and fluorescence modes in three standardized areas of 2 mm x 2 mm width and 100 µm depth using a 4.75 µm step size and linear increase in laser power applied to the RHE for 3.5 hours. Ultrasound gel (Parker Laboratories) was used to maintain the refractive index between the objective lens and the glass sample plate. Importantly, to mimic the ultrastructure of damaged skin in AD patients, the RHE was intentionally punctured with a Derma Microneedle device to determine the localization of bacteria in the presence of damaged skin. Results showed that the 10 8 Bacteria were placed in the perforations (arrows, Figure 3B and Figure 3C ). This suggests that topically applied bacteria are able to home to areas of damaged skin.

[0121] Results from these experiments showed that the modified bacteria localized to the surface and deep grooves of the stratum corneum and remained constantly present during the experiments. These experiments also showed that SE This is attributed to a skin barrier defect and suggests that our bacteria will colonize deeper into the damaged skin, as we would expect in AD patients ( Figure 3A -C).

[0122] Example 4. Characterization of bacterially secreted sGFP delivery to skin using an in vitro model system

[0123] Isolation of sGFP produced by SE

[0124] From 225 mL SE The culture (2.7-2.9 OD / mL) was purified for protein and lysed with CellB-Lyse (Sigma). 4 mL of eluate was generated and run from a Ni-NTA column with 500 mM imidazole in BPS buffer pH 7.4 to give 0.4 mg / 225 mL SE. An SDS-PAGE gel of purified sGFP is shown in FIG4 .

[0125] Characterization of bulk purified sGFP and sGFP+RMR delivery to RHE and characterization of the SE-GFP reporter and control strains 3. Cellular Compartment and Depth of Penetration of sGFP Protein

[0126] The localization of purified sGFP and sGFP+RMR was examined to answer three important questions: (1) does sGFP+RMR penetrate the stratum corneum; (2) if so, at what depth can it be detected; and (3) what are the penetration kinetics.

[0127] 5.0 μg of GFP + / - RMR was applied to the RHE. Two-photon microscopy and confocal imaging were used to examine the effects at 30 and 60 minutes to examine penetration and kinetics. Importantly, the intracellular localization of GFP due to the role of RMR in penetrating human cells, as well as the transdermal localization of GFP due to the role of RMR in promoting skin penetration, were examined.

[0128] The results of these experiments showed that GFP without RMR did not effectively penetrate the RHE (Figure 5A, B, E, F). However, compared with GFP without RMR signal ( Figure 5E , F, K) compared to the same amount of GFP-RMR delivered deeper into the tissue ( Figure 5G , H, J), supporting the efficiency and necessity of RMR for transdermal penetration. In addition, evidence of accumulation of GFP with RMR in keratinocytes (Fig. 5C, D) was found compared to GFP without this tag (Fig. 5 A, B), supporting intracellular penetration. Together, these results suggest that RMR facilitates transdermal delivery of our proteins and is important for the production of filaggrin by SE This is highly relevant as treatments deliver filaggrin deeper into the skin of IV patients. The depth and localization of GFP after disruption of the stratum corneum with a Dermaroller was examined and it was found that GFP preferentially localized and penetrated into the area of ​​the break ( Fig.5O ).

[0129] Example 5. Development and optimization of analytical methods for the production of human filaggrin from Staphylococcus epidermidis

[0130] Analytical methods for filaggrin quantification were developed, as well as structure-activity approaches for the rational selection of optimal filaggrin oligomers and sequences to move forward into preclinical and subsequent clinical development.

[0131] Rational design of filaggrin sequences

[0132] As a starting point, a so-called domain-containing filaggrin molecule based on proposed linker regions flanking both ends was used. hFLG contains 12 repeating units and is treated by intracellular proteases to release the individual units. An overview of the filaggrin structure is given in Fig. 6E For bioinformatic analysis investigating homology and sequence identity of human filaggrin domains, see Fig. 6A, B. The sequences within these units are highly homologous. It has been proposed that each unit is cleaved in a "joint region". The segments between these joint regions are the individual units, each containing a pair of subdomains. The spatial orientation or topography of these units has not yet been resolved because the molecule is not highly structurally organized. Based on the Kyte-Doolittle hydrophilicity score, the joint region is defined as the most hydrophilic amino acid segment in the hFLG protein (Figure 6C, D).

[0133] In an attempt to investigate the SAR of hFLG, the entire unit containing subdomains 9 and 10 flanked by their linker regions was recombinantly produced. In order to identify the minimal protein segment responsible for keratin binding activity, proteins were generated with systematic deletions of regions at the N-terminus. The role of the linker region was also examined. To address this question, proteins were prepared in which the linker region was centrally located within the sequence. The RMR peptide sequence is shown in Fig. 6E , Fig. 6E Based on the previous characterization of GFP delivery via RMR (Figure 5A-N), a structure-activity relationship (SAR) based approach was used to select the best filaggrin sequence from the possible sequences that had been identified, as described below.

[0134] Example 6. Identification and quantification of filaggrin secreted by SE

[0135] For the secretion of filaggrin SE Subsequent preclinical and clinical development of strains is important to develop identification and quantification of SE Methods for secreting filaggrin.

[0136] Antibody development and immunoreactivity testing of hFLG

[0137] An antibody was developed against an epitope of hFLG (RL-012-001B, chicken IgY) that is common in all subdomain pairs. This was necessary because commercially available filaggrin antibodies showed significant non-specific binding. SE The IgY antibody showed a clear band on Western blot ( Fig.7D ). IgY was able to recognize several different pairs of hFLG subunits produced (Figure 7 BD).

[0138] Mass spectrometry (MS)

[0139] Preliminary LC-DDA- (Data Dependent Acquisition) MS / MS analysis of 20 ng of human filaggrin fragment 9-10 is shown in Fig. 7E This identified peptide sequences that can be followed when monitoring filaggrin production, secretion, and delivery.

[0140] The protein samples were resuspended in 50 mM ammonium bicarbonate buffer and digested with trypsin by incubation overnight at 37 ° C. The samples were then acidified to stop trypsin activity and then desalted using a C18 spin column, the eluate of which was then dried under vacuum centrifugation and resuspended in 0.1% formic acid. The resulting tryptic digest was analyzed by reversed-phase LC-DDA-MS / MS using a nanoACQUITY UPLC system connected to an Orbitrap-FusionTribrid mass spectrometer equipped with a nano-electrospray source. After injection, the peptides were loaded into a capture column and then separated using a C18 column.

[0141] LC-DDA-MS / MS analyses were performed with the mass spectrometer operating in Top Speed ​​data-dependent mode with a maximum total cycle time of 3 s. During the MS1 full scan, precursor ions were separated in the quadrupole mass analyzer with a maximum injection time of 60 ms and an automatic gain control (AGC) target of 4 × 10 5 and Orbitrap with a resolution of 120,000 or more m / z 200 records. Full scans were acquired in the range 300–1500 m / z, followed by MS2 of the most intense ions. Ions were repeatedly isolated using a 1.6 Th window, a maximum injection time of 110 ms, and an AGC target of 1 × 10 5 The chromatograms were then fragmented using a higher energy collision dissociation (HCD) at 28 Å. The MS / MS spectra were then analyzed at a resolution of 60,000. m / z 200 records. Ions with singly charged or unassigned charge states were excluded from fragmentation.

[0142] The reliability and robustness of this assay are important for SE The biochemistry, manufacturing, and control of any final LBP are particularly important. The qualification of the MS analytical method uses the following metrics: (1) specificity; (2) sensitivity (limit of detection (LOD) and limit of quantification (LOQ)); and (3) linearity. This is achieved using a reference standard of purified hFLG and running multiple samples over a range of concentrations. Fig. 7E As shown, specific peptides are detectable, and if desired, several of these can be prepared synthetically as reference standards.

[0143] Example 7. Keratin Binding Assay to Measuring Filaggrin Activity

[0144] The keratin binding assay is used to measure filaggrin activity. This will be assessed by a surface plasmon resonance (SPR) method (Biacore GE Healthcare) to rank different filaggrin segments based on their cytokeratin interactions. This will measure binding affinity, specificity, concentration, and kinetics to examine filaggrin-cytokeratin binding dynamics. Expected metrics generated by this method include K on , K off and K d Different human cytokeratins will be evaluated starting with cytokeratins 1 and 10 which are known to interact with hFLG. The assay was developed with our current filaggrin 9-10 constructs and used to measure additional filaggrin sequences to guide SAR analysis. The assay workflow is shown in Figure 8 .

[0145] Example 8. Optimization of human filaggrin by structure-activity relationship (SAR)

[0146] Based on the methods described above and shown in Figure 6, the remaining 11 filaggrin domains were tested and a matrix of identity and activity was generated as outlined in the methods described above (i.e., Western blot, MS, and surface plasmon resonance (SPR) protein-protein interaction assays). The correlation of protein sequence and in vitro activity will guide the selection of proteins to be generated as potential therapeutic agents.

[0147] Example 9. Development and optimization of analytical methods for the delivery of human filaggrin from Staphylococcus epidermidis to human skin model

[0148] Important for subsequent pharmacokinetic (PK) characterization is the development of assays for the detection and distribution of filaggrin. This includes the use of MS to detect intact and degraded products (as described above), as well as immunofluorescence and immunohistochemistry to characterize the distribution of filaggrin from the cells that produce it. SE Distribution of filaggrin in a human skin model (reconstructed human epidermis) as well as in mouse skin. Measurement of filaggrin breakdown products will also complement the characterization of filaggrin activity, i.e. internalization and processing of filaggrin. Additionally, assays were developed to detect filaggrin breakdown products, including Raman spectroscopy.

[0149] Flg- / - reconstructed human epidermis (RHE)

[0150] Filaggrin KO reconstructs human epidermis, trademarked as EpiDerm, purchased from MatTek (Ashland, MA). The epidermal model is constructed from normal human epidermal keratinocytes (NHEK) from neonatal foreskin or adult breast skin of a single donor. The model is provided as a 9mm diameter tissue sample, which has been grown on Millicell CM (Millipore) exposed to an air-liquid interface. The epidermis includes a total of 8-12 layers of basal, spinous and granular differentiation and 10-15 layers of the stratum corneum. In terms of histology, metabolism and genetics, the epidermis is particularly consistent with human skin. The model maintains morphology for three weeks. The knockdown of filaggrin in keratinocytes is achieved by knockdown based on shRNA lentivirus of filaggrin. For this purpose, it is estimated that 96 RHE samples are needed.

[0151] Mouse Model

[0152] Using wild-type BALB / c and flg- / - BALB / c mice were purchased from Jackson Laboratory, Bar Harbor, ME, and flg- / - Obtained via Azitra from Riken Laboratory (Nagaski, Japan). For this purpose, an estimated 22 BALB / c and 22 flg- / - .

[0153] Immunofluorescence and immunohistochemistry

[0154] In order to understand and characterize the SE Uptake and biodistribution of secreted filaggrin Immunofluorescence (IF) microscopy and immunohistochemistry (IHC) were used to visualize the localization and distribution of filaggrin from the filaggrin-producing SEs. flg- / - Reconstructed human epidermis (RHE). 6 -10 8 The range of colony forming units (CFU) of filaggrin SE Applied to RHE and mouse skin and incubated for 24 hours in the absence of antibiotics. Samples were fixed in triplicate for IF and IHC analysis at 24, 48 and 72 hours, and for these analyses, untreated RHE and mouse samples were used as negative controls.

[0155] For IF, samples were fixed with 70% ethanol, 50 mM glycine for 1 hour. Immunofluorescence staining was performed with our developed anti-filaggrin primary antibody (RL-012-001B) at 1:200 for 2 hours, followed by incubation with rat anti-goat rhodamine secondary antibody (Jackson Laboratory) at 1:200 dilution in the presence of Hoechst StainSolution (Sigma). Slides were mounted with coverslips in Gel / Mount (Biomed).

[0156] For IHC, samples are fixed and paraffin embedded in 10% formalin. Paraffin sections are dewaxed and washed with 95% ethanol, followed by methanol peroxide washing. The sections are then processed with heat-induced epitope repair (HIER) procedures using rodent Decloaker solution (BiocareMedical, RD913) and Biocare decloaking chamber. After washing in TrispH 7.4, sections are incubated in the presence of rat serum and FcBlock (24G2), followed by incubation of rabbit anti-epidermidis diluted in blocking solution. Samples are washed in Tris, then incubated with goat anti-rabbit IgG-Texas Red antibody (Invitrogen, T2767). Tissues are then counterstained with HOECSHT, and imaged using Leica DM IRBE fluorescence microscope.

[0157] Raman spectroscopy

[0158] Raman spectroscopy is a known method for identifying NMF decomposition products of filaggrin 62 During terminal differentiation, profilaggrin is rapidly dephosphorylated and cleaved to yield functional filaggrin monomers, which bind and assemble keratin intermediate filaments. 63,64 Cleavage of the deiminated filaggrin monomers is catalyzed by several proteases, including caspase 14 65 , calpain 1 and bleomycin hydrolase 66 Filaggrin is then broken down into natural moisturizing factors (NMF), specifically urocanic acid (UA), pyroglutamic acid (PGA) and citrulline (CIT) 62 .

[0159] Raman spectroscopy is used to identify flg- / -Filaggrin decomposition products in the RHE model and mouse skin. The Raman effect consists of a shift in the energy of a photon due to inelastic collisions of the photon with a molecule. Raman spectroscopy displays the scattered intensity as a function of the frequency difference between the incident and scattered light; this difference is called the "Raman shift". Initial Raman spectroscopy methods have been developed to identify NMF, the decomposition products of filaggrin. Skin can be directly observed (RHE) without sample preparation for tissue destruction. At this time, commercially available standard samples have been used to test the method, and the results are provided in Fig.9A -D.

[0160] When optimized, Raman spectroscopy is capable of detecting micromolar concentrations. Nanomolar detection levels are possible using surface-enhanced Raman spectroscopy, where nanoparticles of a suitable material (gold or silver) can be placed on the skin sample. These particles undergo surface plasmon resonance, thus significantly increasing the sensitivity of the Raman analysis. This is used to identify the production of filaggrin from topical application of RHE. SE Decomposition of filaggrin. 8 CFU of filaggrin SE The NMF was applied to RHE and incubated for 24 h without antibiotics. Samples were collected at various time points and analyzed using Raman spectroscopy to identify NMF decomposition products.

[0161] Mass spectrometry

[0162] To evaluate and characterize the metabolism of filaggrin, mass spectrometry was used. Detection of commercially available standards of the NMF molecules urocanic acid (UA), pyroglutamic acid (PGA), and citrulline (CIT) was performed using an Agilent HP1200 mass spectrometer coupled to an AB Sciex API4000 QTrap. Standards of UA, PGA, and CIT were purchased from Sigma-Aldrich and separated using an Agilent Eclipse XDBC18 column (3.5u, 2.1x100mm), injected into the mass spectrometer and detected by MRM (multiple reaction monitor) and secondary ion mass spectrometry (SIMS). The sensitivity of SIMS was shown to be approximately 10 times that of MRM. The limits of quantification (LOQ) for UA, PCA, and CIT were 0.5, 1, and 0.25 ng / mL, respectively, and the standard curves showed a correlation coefficient of 0.99. When testing the production of NMF from FLG in RHE experiments, HE tissue samples were homogenized and extracted with perchloric acid / acetonitrile, and the resulting solutions were injected. The standard curves for these results are provided in Fig.9E -G. These preliminary data suggest that CIT may be detected using only MS and not Raman spectroscopy.

[0163] These parameters were used to measure filaggrin breakdown from filaggrin-producing SE topically applied to RHE.8 CFU production of filaggrin SE was applied to RHE and incubated for 24 h in the absence of antibiotics. Samples were collected at various time points and analyzed using MS to detect NMF breakdown products. The assay was also qualified using the above methods using (1) specificity and sensitivity; (2) accuracy and precision; and (3) assay limits. The results report the initial metabolism and distribution of filaggrin in both models.

[0164] Example 10. Evaluation of the colonization kinetics and activity of candidate filaggrin-secreting SEs in mice

[0165] Using a genetic IV mouse model ( Flg- / - ) and wild-type mice to evaluate the production of FLG in vivo SE The dynamics of settlement.

[0166] Mouse Model

[0167] To investigate the applicability of this approach, a genetic model mouse system was used. The filaggrin knockout mouse ( Flg - / -). Flg - / - mice are deficient in filaggrin and display dry, scaly skin 19 Although the levels of natural moisturizing factor (a degradation product of filaggrin) were significantly decreased, stratum corneum (SC) hydration and TEWL were Flg - / - mice are normal. Antigens penetrate more efficiently Flg - / - SC, resulting in enhanced responses in hapten-induced contact hypersensitivity reactions and higher anti-ovalbumin (OVA) IgG(1) and IgE serum levels. Flg- / - Mice were obtained from the RIKEN BioResource Research Center (RIKEN BRC, Tsukuba, Ibaraki, Japan) through a material transfer agreement. Wild-type mice (BALB / c) will also be used in this experiment.

[0168] Study Design

[0169] From Fig. 6E The designed filaggrin sequence is SE Used in Flg- / - and BALB / c mice. This study will be conducted for 4 weeks using five groups of each mouse type. Mice will be assigned to the following treatment groups: local vehicle control (50% glycerol, 50% sterile BHI medium), local wild type SE (1.0x10 8 CFU / cm 2in 50% glycerol) and three doses of SE constructs secreting filaggrin ( SE FLG ) (10 6 , 10 7 and 10 8 CFU / cm 2 In 50% glycerol). Each solution was applied to the same ear and tail of each mouse daily for 7 days, and the mice were evaluated for microbiome analysis at days 7, 14, 30, and 60 to assess colonization dynamics, and microscopy and histology at days 7 and 14 to assess localization and macroscopic changes in the skin (e.g., any signs of adverse events such as inflammation), etc. 12 mice per group were used per mouse type. An overview of the study design is shown in Fig.10 .

[0170] Example 11. Administration of secretory filaggrin SE Settlement dynamics and persistence

[0171] To understand the addition SE Effects on microbial load, microbial diversity, and stability of the skin microbiome, 16S rRNA sequencing was used to measure changes in the microbial community. Skin microbiome samples were collected from the tail skin of mice using flocculent swabs at baseline (day 0), 7, 14, 30, and 60. DNA was extracted using a skin-specific custom protocol developed by Oh Laboratory at The Jackson Laboratory for Genomic Medicine. To measure microbial load, qPCR using 16S rRNA V1-V3 primers was utilized as previously described. To examine microbial community dynamics, V1-V3 was amplified and sequenced using 2x300bp read chemistry on the Illumina MiSeq platform. Sequence analysis to the genus level was as previously described 40 , and operational taxonomic units (OTUs) were defined at 97% similarity. A custom computational tool to distinguish Staphylococcus species was used to track the applied SE Relative abundance of [Oh et al., Genome Research 2013] 67 (Figure 6).

[0172] Dysbiosis was measured using ecological metrics and community structure analysis. First, dysbiosis was assessed as a function of diversity using the Shannon diversity index, an ecological metric for the microbial community under consideration, and compared before and after application. Additionally, the community structure of the local microbiome was compared before and after treatment. Dysbiosis was measured as the % overall deviation from (1) the baseline microbiome, and (2) the deviation from the average community structure in our controls. Statistics such as the Yue-Clayton index for comparing community structure were used. Finally, microbiome trends were analyzed at a per-species level.

[0173] Because S. epidermidis was not expected to cause depletion of the endogenous bacterial population, the longitudinal dynamics of each species across treatments were tracked to identify whether species were lost from the community at a targeted sequencing depth of 50,000 reads / sample.

[0174] The experimental overview of 16S sequencing is shown in Fig.11 .

[0175] Histological and Safety Analysis

[0176] Histology for examination SE Localization and exploration of safety and monitoring of any signs of inflammation were performed. The excised ears of each group were fixed in 4% paraformaldehyde for 16 h and embedded in paraffin. Then, 6 μm sections were stained with hematoxylin (SigmaAldrich, St Louis, MO, USA) and eosin (Sigma Aldrich, St Louis, MO, USA) (H&E). Lymphocytic infiltration in the dermis and edema of the spinous cell layer were observed by microscopy (100X, 200X).

[0177] Statistical analysis

[0178] If the data were normally distributed, between-group differences in the primary outcome macroscopic clinical disease score were assessed using a two-sided student t-test. If not (e.g., microbiome data), nonparametric equivalent tests such as the Wilcoxon-rank sum test were used. Differences across groups were assessed using ANOVA or nonparametric equivalent tests (Kruskal-Wallis). The same technique was used to assess TEWL and epidermal thickening. Finally, differences in ordinal variables were assessed using the chi-square test. All variables were corrected for false discovery rate, if necessary. P Values ​​for multiple comparisons.

[0179] For microbiome analysis, community variation between samples was calculated using a quantitative taxonomy-based Yue-Clayton distance. A beta diversity metric (e.g., the Yue-Clayton theta (θ) index), a measure of similarity between two samples that accounts for shared species and their abundance, was used. Pairwise comparisons between samples within a time series will produce a baseline for longitudinal variation within the mouse. For each group of sequential samples, a theta index was calculated. The ratio between sequential θ and baseline θ is then used as a flux score to replace instability over time. This allows stability to be scored based on a sliding window rather than a differential cluster comparison over time (which can be a confounding factor in longitudinal data). Dynamical system models for time series data, such as the generalized Lotka-Volterra nonlinear differential equation, are studied. 63,64 , time-dependent general additive model 65 or non-parametric methods such as Gaussian Process Dynamical Models 66 For each species, a model such as the Augmented Dickey-Fuller test was used. 67 To test the homeostasis of species. This determines which species contribute to variation over time, because the selection of microbes accounts for most of the time variation is possible.

[0180] Statistical analysis

[0181] Unless otherwise specified, the experiment was performed in triplicate and the mean and standard deviation were reported. For comparisons between groups, a two-sided t-test or analysis of variance was used. If the data were not normally distributed, these were replaced with nonparametric equivalent tests (Wilcoxon-rank sum test and Kruskal-Wallis test).

[0182] Security and the Kill Switch

[0183] A key requirement for almost all recombinant microorganisms for clinical applications is the ability to prevent unwanted introduction into other individuals or environments. To ensure the safety of engineered strains, in one embodiment, the present invention uses auxotrophic strains that require supplementation of key amino acids (D-ala) or certain metabolic genes ( AlaR) to survive, and at the same time replaces the need to select for antibiotic resistant strains, which are not commercially viable. In another embodiment, the present invention integrates a "kill switch" based on CRISPR / Cas9 self-cleavage after induction of a dual-xylose-riboswitch promoter. In another embodiment, the present invention provides a cell counter that recombines the AZT locus after a defined number of divisions, although this method requires re-application of the vector. In order to ensure the safety of the engineered S. epidermidis of the present invention, a CRISPR / Cas9-based kill switch is used, which is xylose-inducible and dually regulated by theophylline riboswitch. The basis of this approach is that Cas9 is extremely efficient in chromosome cleavage given a targeted guide, and since Staphylococci lack a canonical non-homologous end joining repair pathway, genome cleavage leads to death in the absence of a homologous recombination template. The use of a CRISPR-based system also confers great specificity, as comparative genomics can be used to design instructions that are unique to the engineered S. epidermidis strains of the present invention, such that the construct is inactive if it is spread to other microorganisms by horizontal gene transfer. Finally, in one embodiment, the invention provides a construct designed to express multiple CRISPR spacers to simultaneously target multiple genomic regions to ensure cleavage and minimize survival through reversion.

[0184] Equivalent solution

[0185] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

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Claims

1. A recombinant microorganism capable of secreting a filaggrin fusion protein, wherein the recombinant microorganism is Staphylococcus epidermidis ( Staphylococcus epidermidis ), wherein the recombinant microorganism comprises an expression vector containing a first coding sequence and a second coding sequence, the first coding sequence comprises a gene capable of expressing a filaggrin polypeptide, the second coding sequence comprises a gene capable of expressing a cell-penetrating peptide, wherein the filaggrin polypeptide consists of SEQ ID NO:

1.

2. The recombinant microorganism according to claim 1, further comprising a third coding sequence comprising a gene capable of expressing an output signal.

3. The recombinant microorganism of claim 2, wherein expression of the first coding sequence, the second coding sequence, and the third coding sequence is under the control of a promoter.

4. The recombinant microorganism of claim 3, wherein the first coding sequence, the second coding sequence, and the third coding sequence are arranged in frame.

5. The recombinant microorganism of claim 2, wherein the first coding sequence, the second coding sequence, and the third coding sequence are operably linked to a promoter.

6. A method for producing a live biotherapeutic composition, the method comprising: (a) transfecting Staphylococcus epidermidis cells with an expression vector comprising the following sequences: (i) a first coding sequence comprising a gene capable of expressing a therapeutic filaggrin polypeptide, wherein the filaggrin polypeptide consists of SEQ ID NO: 1, and (ii) a second coding sequence comprising a gene capable of expressing a cell penetrating peptide; and (b) allowing the transfected cells to produce a protein comprising the filaggrin polypeptide and the cell-penetrating peptide; and (c) obtaining the live biotherapeutic composition.

7. The method of claim 6, further comprising (iii) transfecting the cell with a third coding sequence comprising a nucleic acid sequence capable of expressing an export signal.

8. The method of claim 7, wherein the first coding sequence, the second coding sequence and the third coding sequence are arranged in a single plasmid.

9. The method of claim 7, wherein the first coding sequence, the second coding sequence, and the third coding sequence are operably linked to a promoter.

10. A composition obtainable by the method according to any one of claims 6 to 9.

11. The composition according to claim 10, comprising a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, an emulsion, a cream, a lotion, a gel and an ointment.

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