Compositions comprising atp degrading enzymes and uses thereof

Abstract

Provided herein, inter alia, are compositions and methods for the treatment and / or prevention of conditions associated with skin and / or oral inflammation via the use of an exogenously administered ATP degrading enzyme, such as an NTPD enzyme of the GDA1CD39 superfamily or a phosphatase that can function in a physiologically related pH range.

Description

[0001] This application claims priority to Chinese International Patent Application No. PCT / CN2023 / 119945, filed on September 20, 2023, the disclosure of which is incorporated herein by reference in its entirety. By invoking the inclusion in the sequence list

[0002] The sequence list submitted in XML file format according to 37 CFR §§2412 is incorporated herein by reference. The XML file is named "NB42172_WO_PCT_Seq_List.xml", created on September 12, 2024, and is 149,120 bytes in size. Technical Field

[0003] This article provides, in particular, compositions and methods for treating and / or preventing conditions associated with skin and / or oral inflammation by using exogenously applied ATP-degrading enzymes (NTPD enzymes, nucleotidases such as adenosine triphosphate bisphosphatase and members of the GDA1_CD39 superfamily, and phosphatases). Background Technology

[0004] The skin and oral mucosa provide the first line of defense against external stimuli through their physical and immune barrier functions, in which symbiotic microorganisms play a crucial role in maintenance, homeostasis, and immunity. Barrier dysregulation caused by physical or chemical damage, environmental exposure, toxins, and pathogenic microorganisms can lead to alterations in the microenvironment, activation of immune cells, and the release of pro-inflammatory molecules and mediators, including extracellular nucleotide triphosphates (NTPs) (such as extracellular ATP (eATP)) and cytokines (such as TNF-α, IL-1β, and IL-6). Regardless of the source of disruption, the inflammatory process can lead to further barrier alterations, varying degrees of tissue damage, and loss of function.

[0005] Treatments and / or prevention of skin and oral health have historically been driven by dysregulation of etiology, potentially negatively impacting the maintenance of homeostasis with the microbiome. Therefore, there is a need for improved methods and compositions that target common downstream inflammatory processes across a wide range of pathologies, leading not only to more efficient product design but also to the survival and health of the symbiotic microbiome.

[0006] The topics disclosed in this article address these needs and offer additional benefits. Summary of the Invention

[0007] This document provides a composition comprising an ATP-degrading enzyme and at least one oral care component and / or at least one skin care component. In some embodiments, the enzyme is active at least at a pH of about 3.5 to pH 9. In some embodiments, the enzyme is an adenosine triphosphate (ATP) diphosphatase. In some embodiments, the enzyme is a member of the GDA1_CD39 superfamily and is not a potato ATP diphosphatase. In some embodiments, the enzyme is not a mammalian NTPD enzyme. In some embodiments, the enzyme is a phosphatase.

[0008] In other embodiments, compared to a reference, the enzyme is capable of reducing the level of one or more inflammatory mediators by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the one or more inflammatory mediators are TNF-α, IL-6, IL17A, IFNγ, IL-8, or a combination thereof. In some embodiments, compared to a reference, the enzyme is capable of reducing cell death levels by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the enzyme is associated with SEQ ID NO: 19, SEQ ID NO: 34 (CRC22110), SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 30, SEQ ID NO: 45, and SEQ ID NO: The amino acid sequence of any one of 93 is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide.

[0009] In some of the embodiments, the enzyme is a polypeptide having one or more modifications. In some embodiments, the modification is truncated. In some embodiments, the enzyme is a polypeptide having one or more modifications within the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34 (CRC22110). In some embodiments, the enzyme contains a truncated polypeptide at the C-terminus of SEQ ID NO: 19 or SEQ ID NO: 34 (CRC22110). In some embodiments, the enzyme is a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the enzyme is a polypeptide having one or more modifications within the amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 45.

[0010] In some embodiments, the composition contains an oral care component, which is toothpaste, preventative paste, tooth powder, tooth polish, tooth gel, chewing gum, lozenges, mouthwash, oral rinse, whitening strips, patches, suspensions, lotions, hydrogels, pastes, multiphase solutions, paint gels, varnishes, finishes, and tubes, syringes, or dental trays containing gels or pastes, or gels or pastes applied to application supports such as dental floss or toothbrushes. In some embodiments, the composition contains a skin care component, which is an ointment, serum, hydrogel, solution, dressing, moisturizer, exfoliant, body wash, eye cream, sunscreen, skin cream, cleanser, skin lotion, artificial skin, probiotic skin care component, or spray. In some embodiments, the composition contains skin and / or oral care components as wound care components.

[0011] This article provides a nucleic acid that encodes any of the enzymes described herein.

[0012] This document provides a vector containing the nucleic acid of any of the embodiments described herein.

[0013] This document provides a recombinant host cell containing any of the enzymes, nucleic acids, or vectors described herein. In some embodiments, the cell is a plant cell, bacterial cell, fungal cell, or yeast cell. In some embodiments, the cell is a Bacillus cell, an Escherichia coli cell, a Yarrowia cell, an Aspergillus cell, a Pichia cell, or a Trichoderma reesei cell.

[0014] This document provides a method for treating and / or preventing an inflammatory response in a subject, wherein the method comprises administering to the subject an effective amount of any composition described herein. In some embodiments, the effective amount is a therapeutically effective amount of any composition provided herein. In some embodiments, the composition is applied to the oral surface of the subject, the oral surface being selected from the lips, cheeks and the lining of the inner lips (buccal mucosa), tongue, upper and lower gingiva, the floor of the mouth under the tongue, the roof of the mouth (soft and hard palate), the uvula, tonsils, and the area behind the wisdom teeth (posterior trigone of the molars). In some embodiments, the inflammatory response is associated with an oral health condition. In some embodiments, the oral health condition is an oral health disease selected from periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and oral mucositis. In some embodiments, the oral health condition is an open sore and / or wound selected from oral ulcers, cold sores, cuts, bites, burns, surgical incisions, and tissue trauma. In some embodiments, the inflammatory response includes the release of inflammatory mediators. In some embodiments, these inflammatory mediators are TNF-α, IL-6, IFNγ, IL17A, or combinations thereof.

[0015] In some of the embodiments, the composition is applied to the skin of a subject. In some embodiments, the inflammatory response is associated with a skin condition. In some of the embodiments, the skin condition is selected from UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and tissue trauma. In some embodiments, the inflammatory response encompasses the release of inflammatory mediators. In some embodiments, these inflammatory mediators are IL-6, IL-17A, IFNγ, IL-8, or combinations thereof.

[0016] This document provides a method for reducing the production of inflammatory mediators in a subject, wherein the method comprises administering to the subject an effective amount of any composition described herein. In some embodiments, the effective amount is a therapeutically effective amount of any composition provided herein. In some embodiments, these inflammatory mediators are associated with oral health conditions. In some embodiments, the oral health condition is an oral health disease selected from periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and oral mucositis. In some embodiments, the oral health condition includes open sores and / or wounds selected from oral ulcers, cold sores, cuts, bites, burns, surgical incisions, and tissue trauma. In some of any embodiments, these inflammatory mediators are TNF-α, IL-6, IFNγ, IL17A, or combinations thereof. In some embodiments, these inflammatory mediators are associated with skin conditions. In other embodiments, the skin condition is associated with UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and tissue trauma. In some embodiments, these inflammatory mediators are IL-6, IL-17A, IFNγ, IL-8, or combinations thereof. In some of any embodiments, these inflammatory mediators are produced by macrophages, dendritic cells, neutrophils, or epithelial cells.

[0017] In some embodiments, the inflammatory mediator levels are reduced by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the inflammatory mediator levels in the subject prior to administration of the composition. In some embodiments, the composition is capable of reducing cell death levels by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the inflammatory mediator levels in the subject prior to administration of the composition. In some embodiments, the subject is a human. In some embodiments, the subject is an animal.

[0018] This document provides a method for improving and / or maintaining the oral health of a subject, the method encompassing administering an effective amount of any of the compositions described herein to the subject. In some embodiments, the effective amount is a therapeutically effective amount of any of the compositions provided herein.

[0019] This document provides a method for increasing symbiotic bacteria on the skin and / or in the oral cavity, the method encompassing administering an effective amount of any of the compositions described herein to the subject. In some embodiments, the effective amount is a therapeutically effective amount of any of the compositions provided herein.

[0020] This document provides a method for culturing artificial skin, the method comprising culturing epithelial cells in the presence of any of the compositions described herein. In some embodiments, these epithelial cells are derived from mammals. In some embodiments, the mammal is a human.

[0021] This document provides a method for promoting wound healing in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the compositions described herein. This document also provides the use of any of the compositions provided herein for promoting wound healing in a subject.

[0022] This document provides a method for restoring the protective skin barrier, moisturizing the skin, and / or reducing skin redness, the method comprising applying any of the compositions described herein to human skin. This document also provides the use of any of the compositions described herein for restoring the protective skin barrier, moisturizing the skin, and / or reducing skin redness.

[0023] Each of the aspects and embodiments described herein may be used together unless explicitly or clearly excluded from the context of the embodiment or aspect.

[0024] Throughout this specification, references have been made to various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.). For all purposes, the disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated in their full text by reference. Attached Figure Description

[0025] Figure 1 The viability of 3D gingival tissue was depicted after the addition of ATP or LPS in the presence or absence of the NTPD enzyme CRC22110-V1.

[0026] Figure 2 The levels of TNF-α in the supernatant of 3D gingival tissue treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0027] Figure 3 The levels of IL-6 in the supernatant of 3D gingival tissue treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0028] Figure 4 The levels of IFN-γ in the supernatant of 3D gingival tissue treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0029] Figure 5The levels of IL-17A in the supernatant of 3D gingival tissue treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0030] Figure 6 The levels of eATP in the supernatant of 3D gingival tissue treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0031] Figure 7A and Figure 7B The study depicted the effects of ATP or LPS addition on the treatment of NTPD enzyme CRC22110-V1 in the presence or absence of NTPD enzyme 6 hours later. Figure 7A ) or 24 hours ( Figure 7B Viability of 3D psoriasis tissue model.

[0032] Figure 8 The levels of IL-6 in the supernatant of 3D psoriasis tissue models treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0033] Figure 9 The levels of IL-17A in the supernatant of 3D psoriasis tissue models treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0034] Figure 10 The levels of IFN-γ in the supernatant of 3D psoriasis tissue models treated with ATP or LPS were depicted in the presence or absence of the NTPD enzyme CRC22110-V1.

[0035] Figures 11A-11C The study depicted the effects of ATP or LPS addition on the treatment of NTPD enzyme CRC22110-V1 in the presence or absence of 2 hours. Figure 11A ), 6 hours ( Figure 11B ) or 24 hours ( Figure 11C The level of eATP in the supernatant of a 3D psoriasis tissue model.

[0036] Figure 12 The levels of IL-8 in the supernatant of HaCaT cells treated with LPS were depicted in the presence or absence of NTPD enzymes CRC22110, CRC22110-V1, or potato triphosphate diphosphatase.

[0037] Figures 13A-13B The study described the effects of potato adenosine triphosphate bisphosphatase on the growth of potatoes in the presence or absence of 4 hours ( ). Figure 13A ) and 8 hours ( Figure 13B The level of IL-8 in the supernatant of HaCaT cells treated with UV.

[0038] Figures 14A-14F The viability of 3D psoriasis tissue models after the addition of ATP or LPS in the presence of exemplary ATP-degrading enzymes is depicted. Figure 14A ) and IL-6 in the supernatant of a 3D psoriasis tissue model treated with ATP or LPS in the presence of exemplary enzymes. Figure 14B ), MCP-1 ( Figure 14C ), IL-17 Figure 14D ), IL-1β ( Figure 14E ) and IL-33 ( Figure 14F ) level.

[0039] Figures 15A-15F The viability of a 3D gingival epithelial tissue model after the addition of ATP or LPS in the presence of an exemplary ATP-degrading enzyme is depicted. Figure 15A ) and IL-8 in the supernatant of a 3D gingival epithelial tissue model treated with ATP or LPS in the presence of exemplary enzymes. Figure 15B ), IL-18 Figure 15C ), IFN-γ ( Figure 15D ), IL-33 ( Figure 15E ) and MCP-1 ( Figure 15F ) level. Detailed Implementation

[0040] Inflammation is a complex process mediated in part by the interaction between extracellular nucleotides (e.g., ATP, ADP, UTP, UDP) and inflammatory cytokines. Extracellular ATP (eATP) is a danger-associated molecular pattern (DAMP) that can activate the immune system and contribute to the development of inflammation (Vilches et al. Front. Immunol. [Immunology Frontiers] Vol. 9-2018; Dosch et al. Int. J. Mol Sci. [International Journal of Molecular Sciences] 2018.19(4):1222.). In skin inflammation, eATP has been shown to play an important role in the pathogenesis of various conditions such as psoriasis, atopic dermatitis, and contact dermatitis. In oral inflammation, eATP has been shown to play an important role in the pathogenesis of various conditions such as periodontitis and oral mucositis. eATP released from damaged or dead cells can then activate P2X7 receptors on immune cells such as macrophages and dendritic cells, leading to the production and release of pro-inflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) (Zuccarini et al., Int. J. Mol Sci. [International Journal of Molecular Sciences] 2022, 23(14):7790). These cytokines can further promote the recruitment and activation of immune cells, leading to a vicious cycle of inflammation and tissue damage in the oral cavity. eATP interacts with healthy cells and induces cell death, accelerating the process of tissue damage and hindering wound healing.

[0041] In light of the above, targeting eATP presents a potential strategy for treating barriers associated with skin and oral inflammation. Enzymatic removal of extracellular nucleotides (e.g., eATP) can provide a means of reducing the overall downregulation of inflammatory cytokine secretion and inflammatory processes in the skin and oral cavity. However, enzyme functionality is generally regulated by the pH of the environment, and different tissues have different pH ranges. Therefore, this disclosure relates at least in part to compositions and methods for reducing and / or treating inflammation in the skin and / or oral cavity by administering ATP-degrading enzymes such as NTPD enzymes of the GDA1_CD39 superfamily or phosphatases capable of functioning within a physiologically relevant pH range.

[0042] I. Definition

[0043] The term "ATP-degrading enzyme" is used to refer to enzymes that can degrade ATP substrates. ATP-degrading enzymes can be found in several enzyme families, including but not limited to adenosine triphosphate diphosphatase (EC 3.6.1.5), NTPD enzyme (EC 3.6.1.5), acid phosphatase (EC 3.1.3.2), and alkaline phosphatase (EC 3.1.3.1).

[0044] The term "acid phosphatase" (EC 3.1.3.2) (also known as acid phosphomonoesterase, phosphomonoesterase, glycerol phosphatase, acid monophosphatase, acid phosphohydrolase, acid phosphomonoester hydrolase, uterine transferrin, acid nucleoside diphosphate phosphatase, acid phosphatase (class A), or orthophosphomonoester phosphohydrolase (acid-optimal)) is used to refer to an enzyme that has an optimal pH for mediating the hydrolysis of phosphate ester bonds in a substrate at a pH less than about 6.5, such as less than about 4.0. Acid phosphatases release attached phosphoryl groups from other molecules during digestion. It can be further classified as a phosphomonoesterase. Acid phosphatases are stored in lysosomes and function when the lysosome is fused with an endosome, which is acidified during function; therefore, acid phosphatases have an optimal acidic pH. Acid phosphatases degrade ATP [Feder et al. (202) Plant Sci. 294: 1-11, Sharma et al. (2023) J. Enz. Inh. & Med. Chem. 38: 1-12]. This type of enzyme is found in many microbial, animal, and plant species. In some embodiments, the acid phosphatases used in the compositions and methods disclosed herein are not derived from the genus Shigella. In some embodiments, the acid phosphatases used in the compositions and methods disclosed herein are not derived from mammals.

[0045] As used herein, “NTPD enzyme” refers to an enzyme also known as an extracellular nucleotidase, extracellular ATPase, and extracellular nucleoside triphosphate diphosphate hydrolase. As used herein, NTPD enzymes hydrolyze extracellular nucleotides. NTPD enzymes comprise four major families of extracellular nucleotidases: CD39 / NTPD enzymes (extracellular nucleotidases); extracellular nucleotidase phosphodiesterase (E-NPP); alkaline phosphatases; and extracellular 5'-nucleotidases / CD73 (see Robson et al. 2006 Purinergic Signalling 2:409-430). In some of the embodiments described herein, the NTPD enzyme is an adenosine triphosphate diphosphate phosphatase.

[0046] As used herein, the term “adenosine triphosphate diphosphatase” refers to one or more of the calcium-activating enzymes (i.e., proteins belonging to class EC.3.6.1.5) that have ATP-bisphosphate hydrolase activity and catalyze the hydrolysis of γ-phosphate from ATP and β-phosphate from ADP. As used herein, adenosine triphosphate diphosphatases can constitute ATPases, NTPases, or both. Adenosine triphosphate diphosphatases have been found in all eukaryotes and some prokaryotes, indicating that these enzymes retain their function across different species. They possess unique phosphate-hydrolyzing activities, nucleotide substrate specificity, divalent cation requirements, and sensitivity to inhibitors. (See Plesner, Int. Rev. Cytol. [International Review of Cell Science], 158:141 (1995) and Handa and Guidotti, Biochem. Biophys. Res. Commun. [Biochemical and Biophysical Research Communications], 218(3):916 (1996)). In mammals, adenosine triphosphate (ATP) bisphosphatases are thought to function primarily as extracellular hydrolases specific for ATP and ADP, a function crucial for the inactivation of ATP molecules at the synapse after neural stimulation (see, Todorov et al., Nature, 387(6628):76 (1997)). ATP bisphosphatases in mammals are also thought to be important for inhibiting ADP-induced platelet aggregation (see, Marcus et al., J. Clin. Invest., 99(6):1351 (1997)). Recombinant potato ATP bisphosphatase is commercially available from Sigma-Aldrich. In some embodiments, the ATP bisphosphatase used in the compositions and methods disclosed herein is not derived from potatoes. In other embodiments, the ATP bisphosphatase used in the compositions and methods disclosed herein is not derived from mammals.

[0047] As used herein, the “GDA1_CD39 superfamily” refers to enzymes composed of nucleoside triphosphate diphosphate hydrolases that share a common motif in their protein sequences. This family is named after two proteins: yeast GDP enzyme (GDA1) and lymphocyte activation antigen CD39. In some embodiments, these proteins are cell surface enzymes that hydrolyze a range of NTPs, including extracellular ATP. Non-limiting examples include extracellular ATPases, adenosine triphosphate diphosphate phosphatases, CD39, and extracellular ATP diphosphate hydrolases (ecto-ATP / Dase) (Knowles, 2011, Purinergic Signalling, Vol. 7, pp. 21–45; Robson et al., 2006, Purinergic Signalling, 2:409–430; incorporated herein by reference).

[0048] A mucous membrane is a mucous-secreting membrane that covers the surface of all body cavities or passages that communicate with the outside world. Mucous membranes are moist tissues that cover the surfaces of many organs (such as the mouth and intestines) and body cavities (such as the nose, mouth, lungs, vagina, bile ducts, and esophagus) and secrete mucus (a viscous liquid). Mucous membranes are tissues that protect body cavities from environmental conditions, pathogens, and toxic substances, and are typically moist tissues permeated with secretions (such as those in the intestines, lungs, nose, mouth, and vagina).

[0049] As used in this article, "oral cavity" refers to all internal and external parts of the oral cavity, including but not limited to the lips, cheeks and the lining of the inner lips (buccal mucosa), tongue, upper and lower gums, floor of the mouth under the tongue, roof of the mouth (soft and hard palate), uvula, tonsils and the area behind the wisdom teeth (posterior trigone of the molars).

[0050] As used in this article, “microorganism (or microbe)” refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0051] As used herein, the terms “sequence identity” or “sequence similarity” mean that two polynucleotide sequences (candidate sequence and reference sequence) are identical in length (i.e., 100% sequence identity) or similar (i.e., on a nucleotide-by-nucleotide basis). When comparing a candidate sequence to a reference sequence, the candidate sequence may contain additions or deletions (i.e., vacancies) compared to the optimal alignment used for the two sequences (which does not contain additions or deletions). The optimal sequence alignment used to determine sequence identity can be performed using any number of publicly available local alignment algorithms known in the art (such as ALIGN or Megalign (DNASTAR)) or by inspection.

[0052] As used herein, the terms “percentage (%) sequence identity” or “percentage (%) sequence similarity” with respect to a reference sequence are defined as the percentage of nucleotide residues in a candidate sequence that are identical to residues in a reference polynucleotide sequence after optimal alignment of the sequences and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity.

[0053] As used herein, “corresponding to” or “corresponds to” or “corresponding to” refers to an amino acid residue at a position listed in a protein or peptide, or an amino acid residue that is similar to, homologous to, or equivalent to a residue listed in a protein or peptide.

[0054] As used in this article, “preventing” and its grammatical variations refer to methods that partially or completely delay or eliminate the occurrence or recurrence of one or more of a disorder or condition and / or its accompanying symptoms, or prevent a subject from acquiring or reacquiring a disorder or condition, or reduce the risk of a subject acquiring or reacquiring one or more of a disorder or condition or its accompanying symptoms.

[0055] As used herein, the term “reduction” or “lowering” in relation to a particular trait, characteristic, feature, biological process, or phenomenon means a reduction in that trait, characteristic, feature, biological process, or phenomenon. A trait, characteristic, feature, biological process, or phenomenon may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater than 100%.

[0056] As used herein, the terms “subject” or “patient” refer to a mammal (e.g., a human). In some embodiments, the subject has a relevant disease, disorder, or condition, including but not limited to oral health conditions selected from periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and oral mucositis; open sores and / or wounds selected from oral ulcers, cold sores, cuts, bites, burns, surgical incisions, and tissue trauma; or skin conditions selected from UV radiation, bacterial infections, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and tissue trauma. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics that are a predisposition to or risk of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has received and / or has received diagnostic and / or therapeutic administration.

[0057] As used herein, “administer or administering” means the act of introducing one or more strains of microorganisms, exogenous feed enzymes and / or strains and exogenous feed enzymes into a subject by feeding or tube feeding.

[0058] As used herein, "effective amount" or "therapeutic effective amount" means the amount of exogenous enzyme that improves one or more of a subject's indicators. Improvements in one or more of a subject's indicators (such as, but not limited to, oral health, skin health, improved barrier integrity, reduced mortality, or reduced pathogen infection) can be measured as described herein or by other methods known in the art. Exogenous enzymes can also be administered in one or more doses.

[0059] As used in this article, the term "oral health" refers to the health of any part of the internal and external anatomy of the mouth and oral cavity, including the lips, cheeks and the lining of the inner lips (buccal mucosa), tongue, upper and lower gums, floor of the mouth under the tongue, roof of the mouth (soft and hard palate), uvula, tonsils and the area behind the wisdom teeth (posterior trigone of the molars).

[0060] As used herein, the term "oral health condition" encompasses conditions, disorders, or diseases in humans and animals that affect the mouth, lips, and / or oral cavity. Such oral conditions include "dental conditions," which are oral conditions that specifically affect the teeth and gums. Non-limiting examples of oral conditions include infections, biofilms and / or plaque; aphthous ulcers (including ulcers, cold sores, and oral canker sores); tooth stains; and conditions that these oral health conditions may cause, such as oral cancer, tooth sensitivity, cavities and / or missing teeth, cracked teeth, thrush, gum disease, gingivitis, periodontitis, dental caries, halitosis, and / or inflammatory conditions such as tonsillitis, pharyngitis, laryngitis, glossitis, and stomatitis.

[0061] As used herein, “skin” refers to a body organ that covers the entire outer surface of the body. As used herein, the term skin encompasses three layers: the epidermis, the dermis, and the hypodermis.

[0062] As used in this article, the term "dermatitis" encompasses all human and animal conditions, disorders, or diseases that affect the skin. Such dermatitis includes UV radiation, bacterial infections, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and tissue trauma.

[0063] The term "symbiotic bacteria" refers to the microbial community (normal microbiome, indigenous microbiota) composed of microorganisms that exist on the body surface covered by epithelial cells and exposed to the external environment (gastrointestinal and respiratory tracts, vagina, skin, etc.). These bacteria have been shown to influence the regulation of immune physiological functions, including but not limited to metabolism, ontogenesis, and pathogen defense. Symbiotic bacteria have also been shown to promote resistance to skin and oral pathogens, which is mutually beneficial for both the host and the symbiotic microbiota (Byrd et al. 2018. Nature Reviews Microbiology, 16:143-155; Kilian et al. 2016. British Dental Journal, 221:657-666, incorporated herein by reference).

[0064] The term "probiotic" refers to a live or viable microorganism that, when administered to a subject in an effective amount, confers a health benefit to the subject. For example, probiotic formulations described herein, suitable for oral or topical administration to a subject, may contain probiotics that improve the subject's skin and / or oral health. As used herein, the term "probiotic" encompasses both live microorganisms and viable microorganisms in a dormant state, including frozen microorganisms, dehydrated or dried microorganisms, spores, cysts, or microorganisms in various states of reduced metabolic activity that can be reconstituted upon exposure to suitable conditions. In some embodiments, the probiotic microorganism comprises or consists of one or more symbiotic bacteria. In other embodiments, the probiotic microorganism is derived from a fecal microbial source (e.g., a microbial source obtained from the feces of another healthy subject). Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. In some embodiments of the methods disclosed herein, the administration of a non-viable bacterial composition is also contemplated.

[0065] As used herein, the term “implantation” refers to the colonization of one or more bacterial species (such as symbiotic bacterial species provided as, for example, probiotics) in the skin and / or oral cavity, or to the attachment of one or more bacterial species (such as symbiotic bacterial species provided as, for example, probiotics) to the skin and / or oral epithelial cells of a subject.

[0066] Certain ranges are presented herein with the term "approximately" preceding the numerical value. The term "approximately" is used herein to provide textual support for the exact number that follows it, as well as for numbers that are close to or approximate to the number that follows the term. In determining whether a number is close to or approximate to a particular stated number, the close to or approximate unstated number may be a number that is substantially equivalent to the number in the context in which the particular statement is presented. For example, with respect to numerical values, the term "approximately" refers to the range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0067] Unless the context clearly indicates otherwise, as used herein, the singular terms “a / an” and “the” include plural indicators.

[0068] It should be further noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for the use of exclusive terms such as “alone,” “only,” etc., or the use of “negative” to limit the description of the claim elements.

[0069] It should be noted that, as used herein, the term “consisting essentially of” refers to a composition in which the component following the term constitutes less than 30% by weight of the total composition in the presence of other known components, and does not affect or interfere with the function or activity of the components.

[0070] It should be further noted that, as used herein, the term “comprising” means, but is not limited to, the components following the term “comprising.” The components following the term “comprising” are essential or mandatory, but compositions comprising components may further include other non-mandatory or optional components.

[0071] It should also be noted that, as used herein, the term "composed of" means including but not limited to the components following the term "composed of". Therefore, the components following the term "composed of" are essential or mandatory, and no other components are present in the composition.

[0072] The terms "protein" and "peptide" refer to compounds containing amino acids linked by peptide bonds and are used interchangeably. A "protein" or "peptide" comprises a polymeric sequence of amino acid residues. Throughout this disclosure, single-letter and three-letter codes for amino acids are used in accordance with the definitions of the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC-IUB. The single-letter X refers to any one of the twenty amino acids. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. The position of an amino acid in a given polypeptide sequence can be named by the single-letter code of the amino acid followed by a position number. For example, glycine (G) at position 87 is represented as "G087" or "G87".

[0073] As used herein, when referring to an "amino acid sequence," it means the amino acid sequence of a protein or peptide molecule. An "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. However, terms such as "peptide" or "protein" are not intended to limit the amino acid sequence to the deduced sequence, but can include post-translational modifications of the deduced amino acid sequence, such as amino acid deletions, additions, and modifications (e.g., glycosylation and the addition of lipid moieties). Additionally, unless otherwise stated, the use of non-natural amino acids, such as D-amino acids, to improve stability or pharmacokinetic behavior falls within the scope of the term "amino acid sequence."

[0074] The terms "signal sequence" and "signal peptide" refer to amino acid residue sequences that can participate in the secretion or directed transport of proteins in their mature or precursor forms. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. Signal sequences are generally absent in mature proteins. Typically, after protein secretion, the signal sequence is cleaved from the protein by a signal peptidase.

[0075] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of a protein, polypeptide, or enzyme that does not have a signal peptide sequence and / or a precursor peptide sequence.

[0076] Regarding amino acid or nucleic acid sequences, the term "wild-type" indicates that the amino acid or nucleic acid sequence is natural or naturally occurring. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., protein, amino acid, or nucleic acid sequences). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant / engineered nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences).

[0077] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as such lower numerical limit is explicitly stated herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as such higher numerical limit is explicitly stated herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as such narrower numerical range is explicitly stated in its entirety herein.

[0078] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0079] Other definitions of terms may appear throughout this specification.

[0080] II. Composition

[0081] A. enzymes

[0082] This document provides compositions comprising ATP-degrading enzymes, such as NTPD enzymes and phosphatases. In some embodiments, the phosphatase is an acid phosphatase or an alkaline phosphatase. NTPD enzymes, also known as extracellular nucleotidases, extracellular ATPases, and extracellular nucleoside triphosphate diphosphate hydrolases, are extracellular enzymes that hydrolyze extracellular nucleotides. There are four main families of extracellular nucleotidases: CD39 / NTPD enzymes (extracellular nucleotidases); extracellular nucleotidase phosphodiesterase (E-NPP); alkaline phosphatases; and extracellular 5'-nucleotidases / CD73.

[0083] In some embodiments, any composition provided herein is capable of removing phosphatase from ATP. In some embodiments, any composition provided herein is capable of removing phosphatase from NTPs. In some embodiments, the composition is an adenosine triphosphate bisphosphatase and is capable of removing phosphate from ATP and / or NTPs. In some embodiments, the composition is an NTPD enzyme and is capable of removing phosphate from ATP and / or NTPs. In some embodiments, the composition is an acid phosphatase and is capable of removing phosphate from ATP and / or NTPs. In some embodiments, the composition is an alkaline phosphatase and is capable of removing phosphate from ATP and / or NTPs.

[0084] In some embodiments, any composition provided herein is capable of breaking the bond between sugars and purines in ATP. In some embodiments, the composition is a purine nucleoside phosphorylase (PNP) and is capable of breaking the bond between sugars and purines in ATP.

[0085] NTPD enzymes are constitutively expressed in many tissues and can differentiate based on cellular location. In some embodiments, the NTPD enzymes provided herein comprise NTPD enzymes 1, 2, 3, and 8 located on the cell surface; in some embodiments, the NTPD enzymes provided herein comprise 5 and 6, and are located intracellularly and undergo secretion following heterologous expression. In some embodiments, the NTPD enzymes provided herein comprise 4 and 7, and are entirely intracellular and face the lumen of cytoplasmic organelles. NTPD enzymes located on the cell surface require Ca2+ or Mg2+ ions to be active. These isoforms facilitate the recovery of nucleosides from salvage pathways and extracellular nucleoside phosphates.

[0086] Due to their role in nucleotide metabolism, NTPD enzymes help control the availability of extracellular nucleotide agonists at P2 receptors, and thus regulate P2 receptor function. The systemic distribution of P2 receptors and the widespread propagation of purinergic signaling suggest that NTPD enzymes play an important role in a range of cellular processes.

[0087] Location, substrate preference, and hydrolysis rate vary between subtypes; however, all NTPD enzymes contain five highly conserved sequence domains, known as the conserved regions of adenosine triphosphate diphosphatase (APCR1 to APCR5). In some of the examples provided herein, the enzyme exhibits phosphatase activity. A phosphatase is an enzyme that dephosphorylates its substrate. Enzymes exhibiting phosphatase activity include: adenosine triphosphate diphosphatase (also known as NTPD enzyme, ATP-bisphosphate hydrolase (EC 3.6.1.5)), inorganic pyrophosphatase (EC 3.6.1.1), acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase (EC 3.6.1.2), and exonuclease (EC 3.6.1.11). This phosphatase activity acts in the opposite way to phosphorylases and kinases, which attach phosphate groups to their substrates using high-energy molecules such as ATP. In some of any embodiments, any of the ATP-degrading enzymes is capable of hydrolyzing ATP, ADP, CTP, CDP, UTP, UDP, GTP, and / or GDP.

[0088] The enzymes used in the compositions and methods disclosed herein are at least at a pH of about 3 to 9 (e.g., about pH 9). 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or any one of 9) or pH 3.5 to 5.5, or pH It is active at pH 3.5 to 7, or pH 5 to 7. The enzyme may be a member of the GDA1_CD39 superfamily. In some embodiments, the enzyme may be a member of the GDA1_CD39 superfamily (excluding potato-derived adenosine triphosphate diphosphatases). In some embodiments, the enzyme may be a member of a phosphatase class. In some embodiments, the enzyme is active at pH 3 to pH 9. In other embodiments, the enzyme may be an acid phosphatase. In some embodiments, the enzyme comprises the same as SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 34 (CRC22110) has at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).In other embodiments, the enzyme comprises an amino acid sequence having at least 50% or at least 66% sequence identity with any one of the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 (such as any one of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity).

[0089] Other enzymes used in the compositions and methods disclosed herein are at least at pH 3 to pH 4. 9 (such as any of pH 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 9) or pH The enzyme is active at pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7, and is a member of the GDA1_CD39 superfamily (excluding potato-derived adenosine triphosphate bisphosphatases) or the acid phosphatase class. In some embodiments, the enzyme comprises the same as SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: The predicted mature amino acid sequences of one or more of the 45 have an amino acid sequence with at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some embodiments, any sequence provided herein further comprises a signal sequence.

[0090] In some embodiments, the enzyme comprises the same as SEQ ID NO: Any one of 1-15 and 64-76 has a nucleic acid sequence with at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some embodiments, any sequence provided herein further comprises a signal sequence.

[0091] In another embodiment, the enzyme comprises a signal sequence and is associated with SEQ ID No: One or more of the amino acid sequences in 16-30 have an amino acid sequence with at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0092] In another embodiment, the enzyme comprises a signal sequence and is associated with SEQ ID No: One or more of the full-length amino acid sequences in 77-102 have an amino acid sequence with at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0093] This document provides compositions comprising an ATP-degrading enzyme containing one or more modifications. In some embodiments, the modification comprises deletion, insertion, or substitution. In some embodiments, the modification comprises truncation. In some embodiments, the truncation is a truncation of the N-terminus. In some embodiments, the truncation is a truncation of the C-terminus. In some embodiments, the modification comprises insertion. In some embodiments, the insertion is located at the N-terminus. In some embodiments, the insertion is located at the C-terminus.

[0094] In some embodiments, any enzyme provided herein comprises, at its C-terminus, a truncated amino acid sequence having one or more of the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 90-102. In some embodiments, any enzyme provided herein comprises, at its C-terminus, a truncated polypeptide having one or more of the full-length amino acid sequences of SEQ ID NO: 16-30 or SEQ ID NO: 77-89.

[0095] In some embodiments, any enzyme provided herein comprises the same as SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: One or more of the amino acid sequences in 53 have an amino acid sequence with at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0096] B. carrier

[0097] The terms “nucleic acid,” “polynucleotide,” and “nucleic acid fragment” are used interchangeably herein and refer to a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or modified nucleotide bases. Isolated nucleic acid molecules in the form of DNA polymers may consist of one or more fragments of cDNA, genomic DNA, or synthetic DNA.

[0098] DNA constructs containing nucleic acids encoding the phosphatase polypeptides disclosed herein (such as nucleic acids containing any one of SEQ ID NO: 1-15) can be constructed to make them suitable for expression in host cells. Due to the known degeneracy in the genetic code, different polynucleotides encoding the same amino acid sequence can be designed and prepared using conventional techniques. It is also known that codon optimization may be required before attempting expression, depending on the desired host cell.

[0099] Polynucleotides encoding the disclosed phosphatase polypeptides can be bound to the vector. The vector can be transferred to host cells using known transformation techniques, such as those disclosed below.

[0100] Suitable vectors can be vectors that can be transformed into and / or replicated within host cells. For example, a vector containing a nucleic acid encoding a phosphatase polypeptide disclosed herein can be transformed and / or replicated in a bacterial host, fungal, or mammalian cell as a means of propagating and amplifying the vector. The vector can also be appropriately transformed into an expression host such that the encoding polynucleotide is expressed as a functional phosphatase.

[0101] Useful, non-limiting representative vectors are pTrex3gM (see, published U.S. Patent Application 20130323798), pTTT (see, published U.S. Patent Application 20110020899), and p2JM103BBI (see, Vogtentanz, Protein Expr Purif, 55:40-52, 2007), which can be inserted into the host genome. The vectors pTrex3gM, pTTT, and p2JM103BBI can be modified using conventional techniques to contain and express polynucleotides encoding the phosphatase polypeptide of the present invention.

[0102] Expression vectors typically contain control nucleotide sequences, such as promoters, operons, ribosome binding sites, translation initiation signals, and optionally repressor genes or one or more activator genes. Additionally, expression vectors may contain sequences encoding amino acids that enable the phosphatase polypeptide to target host cell organelles (such as peroxisomes) or specific host cell compartments. For expression guided by the control sequence, the nucleic acid sequence of the phosphatase polypeptide is operatively ligated to the control sequence in a manner appropriate for expression.

[0103] To express and produce a target protein (e.g., an ATP-degrading enzyme as described herein) in a cell, one or more expression vectors or expression cassettes containing one or more copies (and in some cases, multiple copies) of a polynucleotide encoding one or more enzymes as described herein are transformed into the cell under conditions suitable for variant expression. In some embodiments, a polynucleotide sequence encoding one or more ATP-degrading enzymes as described herein (and other sequences contained in the vector) is integrated into the genome of a host cell; however, in other embodiments, a plasmid vector containing a polynucleotide sequence encoding one or more ATP-degrading enzymes as described herein remains an autonomous extrachromosomal element within the cell. Some embodiments provide extrachromosomal nucleic acid elements and imported nucleotide sequences integrated into the host cell genome. The vectors described herein can be used to produce one or more ATP-degrading enzymes as described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variants as described herein is present on an integration vector capable of integrating the polynucleotide encoding the variant into the host chromosome and optionally amplifying it in the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, transcription of the polynucleotide encoding one or more ATP-degrading enzymes as described herein is achieved via a promoter that is a wild-type promoter of the parental ATP-degrading enzyme. In some other embodiments, the promoters are heterologous to one or more ATP-degrading enzymes described herein, but are functional in the host cell. Exemplary promoters for bacterial host cells include, but are not limited to, the promoters amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, and pHpaII; the promoter of the raw maltose amylase gene from *Bacillus stearothermophilus*; the amylase gene from *Bacillus amyloliquefaciens* (BAN); the alkaline protease gene from *Bacillus subtilis*; the alkaline protease gene from *Bacillus clausii*; the xylosidase gene from *Bacillus pumilis*; cryIIIA from *Bacillus thuringiensis*; and the α-amylase gene from *Bacillus licheniformis*. Other promoters include, but are not limited to, the A4 promoter, as well as the phage λ PR or PL promoter, and the Escherichia coli lac, trp or tac promoter, and the Bacillus rrn promoters (such as rrnI, rrnB, rrnL and rrnE ribosomal RNA promoters) and their variants.

[0104] The polynucleotide encoding the phosphatase polypeptide disclosed herein can be operatively linked to a promoter that allows transcription in a host cell. The promoter can be any DNA sequence exhibiting transcriptional activity in a selected host cell and can be derived from a gene encoding a protein homologous to or heterologous to the host cell. Examples of promoters used to direct transcription of DNA sequences encoding phosphatases, for example, in bacterial, fungal, or mammalian hosts, include the aprE promoter (SEQ ID NO:46), the promoter of the lac operon of *Escherichia coli*, the dagA or celA promoter of the *Streptomyces coelicolor* agarase gene, the promoter of the *Bacillus licheniformis* amylase gene (amyL), the promoter of the *Bacillus stearothermophilus* raw maltose amylase gene (amyM), the promoter of the *Bacillus amyloliquefaciens* amylase gene (amyQ), and the promoters of the *Bacillus subtilis* xylA and xylB genes, etc.

[0105] For transcription in fungal hosts, examples of useful promoters include those derived from genes encoding the following: *Aspergillus oryzae* TAKA amylase, *Rhizomucor miehei* aspartic protease, *Aspergillus niger* neutral α-amylase, *Aspergillus niger* acid-stable α-amylase, *Aspergillus niger* glucosylamylase, *Rhizomucor miehei* lipase, *Aspergillus oryzae* alkaline protease, *Aspergillus oryzae* triose phosphate isomerase, *Aspergillus nidulans* acetamase, etc. When genes encoding phosphatase polypeptides are expressed in bacterial species (such as *Escherichia coli*), suitable promoters can be selected from, for example, bacterial phage promoters, including the T7 promoter and the phage λ promoter. Following these lines of thought, examples of suitable promoters for expression in yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of *Saccharomyces cerevisiae* and the AOX1 or AOX2 promoters of *Pichia pastoris*. Expression in filamentous fungal host cells typically involves cbh1, an endogenously inducible promoter from *T. reesei*. See Liu et al. (2008) *Acta Biochim. Biophys. Sin (Shanghai)* 40(2): 158-65.

[0106] The coding sequence can be operatively linked to the signal sequence. The DNA encoding the signal sequence can be a DNA sequence naturally associated with the gene for the target phosphatase polypeptide to be expressed, or it can originate from a different genus or species from which a portion of the phosphatase is derived. The signal sequence and promoter sequence constituting the DNA construct or vector can be introduced into the fungal host cell and can be derived from the same source. For example, the signal sequence could be the *Trichoderma reesei* cbh1 signal sequence operatively linked to the cbh1 promoter.

[0107] Expression vectors may also contain a suitable transcription terminator, and in eukaryotes, a polyadenylated sequence that is operatively linked to a DNA sequence encoding a phosphatase. The terminator and polyadenylated sequence may be appropriately derived from the same source as the promoter.

[0108] The vector may also contain selective markers, such as genes whose products compensate for defects in isolated host cells, like the dal gene from Bacillus subtilis or Bacillus licheniformis, or genes conferring antibiotic resistance (e.g., resistance to ampicillin, kanamycin, chloramphenicol, or tetracycline). Furthermore, the vector may contain Aspergillus selective markers (such as amdS, argB, niaD, and xxsC), markers inducing hygromycin resistance, or selection that can be achieved through co-transformation (as is known in the art). See, for example, published international PCT application WO 91 / 17243.

[0109] Synthetic genes encoding protein sequences corresponding to SEQ ID NO: 1-15 can be generated using molecular biology methods known in the art. These genes can be further cloned into suitable expression vectors to obtain expression plasmids containing one or more of the following: a promoter (e.g., the aprE promoter (SEQ ID NO: 46)); a signal sequence (e.g., the aprE signal sequence encoding the peptide of SEQ ID NO: 48 (SEQ ID NO: 47)); oligonucleotides promoting the secretion of target proteins (e.g., oligonucleotides encoding the peptide Ala-Gly-Lys); and synthetic nucleotide sequences encoding the maturation region of the target gene (e.g., sequences corresponding to SEQ ID NO: 1-15 or SEQ ID NO: 48). The synthetic oligonucleotides of any one of 64-76 have at least 50% or at least 66% sequence identity (such as any one of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity); and / or a terminator (such as the lat terminator (SEQ ID NO. 49)). In some embodiments, any oligonucleotide or encoded protein provided herein may contain a C-terminal His tag. Expression plasmids can also be further transformed into suitable expression host cells (such as mammalian, bacterial, or fungal expression host cells).

[0110] C. host cells

[0111] Isolated cells containing a DNA construct (such as any DNA construct disclosed herein) or an expression vector (such as any expression vector disclosed herein, for example, an expression vector containing any polynucleotide of SEQ ID NO: 1-15 or SEQ ID NO: 64-76 encoding a polypeptide of any of SEQ ID NO: 16-45, SEQ ID NO: 51-53, or SEQ ID NO: 77-102) are advantageously used as host cells in the recombinant production of phosphatase polypeptides. Cells can be conveniently transformed with a DNA construct encoding the enzyme by integrating the DNA construct (in one or more copies) into the host chromosome. This integration is generally considered advantageous because the DNA sequence is more likely to be stably maintained in the cell. The DNA construct can be integrated into the host chromosome according to conventional methods, such as by homologous or heterologous recombination. Alternatively, cells can be transformed with an expression vector associated with a different type of host cell.

[0112] Examples of suitable bacterial host cells are Gram-positive bacterial species, such as those in the genus *Bacillaceae*, including *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus lentus*, *Bacillus brevis*, *Geobacillus stearothermophilus* (formerly *Geobacillus stearothermophilus*), *Bacillus alkalophilus*, *Bacillus amyloliquefaciens*, *Bacillus coagulans*, *Bacillus lautus*, *Bacillus megaterium*, and *Bacillus thuringiensis*; *Streptomyces* species, such as *Streptomyces murinus*; and lactic acid bacteria species, including those in the genus *Lactococcus*. *Lactococcus lactis* sp., including *Lactobacillus reuteri*, *Leuconostoc* sp., *Pediococcus* sp., and *Streptococcus* sp. Alternatively, strains of Gram-negative bacteria belonging to the family Enterobacteriaceae (including *Escherichia coli*) or the family Pseudomonadaceae can be selected as host organisms.

[0113] Suitable yeast host cells can be selected from biotechnology-related yeast species, such as, but not limited to, species of the genera *Pichia*, *Hansenula*, *Kluyveromyces*, *Yarrowinia*, *Schizosaccharomyces*, or *Saccharomyces* (including *Saccharomyces cerevisiae*), or species belonging to the genus *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*). The methyltrophic yeast strain *Pichiapastoris* can be used as a host organism. Alternatively, the host organism can be a species of the genus *Hansenula*.

[0114] Suitable host cells among filamentous fungi include species of the genus *Aspergillus*, such as *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus tubigensis*, *Aspergillus awamori*, or *Aspergillus nidus*. Alternatively, strains of the genus *Fusarium* (e.g., *Fusarium oxysporum*) or strains of the genus *Rhizomucor* (e.g., *Rhizomucor oryzae*) can serve as host organisms. Other suitable strains include species of the genera *Thermomyces* and *Mucor*. Additionally, species of the genus *Trichoderma* (e.g., *Trichoderma reesei*) can serve as hosts. Phosphatase polypeptides expressed by fungal host cells can be glycosylated, i.e., will include a glycosyl moiety. The glycosylation pattern can be the same as or different from that present in wild-type phosphatases. The type and / or degree of glycosylation may confer alterations to enzymatic and / or biochemical properties.

[0115] Suitable mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells. Other host cells may include insect cells, such as S2 cells.

[0116] It is advantageous to express a host-deleted gene, where the gene defect can be cured by the transformed expression vector. Known methods can be used to obtain fungal host cells with one or more inactivated genes. Any cloned gene from a Trichoderma species or other filamentous fungal host, such as cbh1, cbh2, egl1, and egl2 genes, can be deleted. Gene deletion can be accomplished by methods known in the art by inserting the desired gene, in its desired form, into a plasmid.

[0117] Universal transformation techniques are known in the art. See, for example, Sambrook et al. (2001), ibid. Expression of heterologous proteins in *Trichoderma* is described, for example, in U.S. Patent No. 6,022,725. For transformation of *Aspergillus* strains, also refer to Cao et al. (2000) Science 9:991-1001. Genetically stable transformants can be constructed using vector systems, thereby stably integrating nucleic acids encoding phosphatases into the host cell chromosome. The transformants are then selected and purified using known techniques.

[0118] Methods for producing any enzyme disclosed herein may include culturing host cells under conditions favorable to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0119] The culture medium used to culture cells can be any conventional medium suitable for the growth of host cells and the acquisition of peptide expression. Suitable culture media and media components can be obtained from commercial suppliers or can be prepared according to published formulations (e.g., as described in the catalog of the American Type Culture Collection).

[0120] Any fermentation method well known in the art can be suitably used to ferment the transformed or derived fungal strains as described above. In some embodiments, the fungal cells are grown under batch or continuous fermentation conditions.

[0121] Separation and concentration techniques are known in the art, and conventional methods can be used to prepare concentrated solutions or broths containing the peptides of the present invention.

[0122] After fermentation, a fermented broth is obtained. Microbial cells and various suspended solids (including residual crude fermentation material) are removed using conventional separation techniques to obtain a solution. Commonly used methods include filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, post-centrifugation ultrafiltration, extraction, or chromatography.

[0123] Sometimes it may be necessary to concentrate the solution or broth containing the peptides to optimize recovery. Using an unconcentrated solution or broth typically increases the incubation time in order to collect the enriched or purified enzyme precipitate.

[0124] D. Enzyme composition

[0125] Any enzyme used in the methods disclosed herein can be formulated into a composition (e.g., a pharmaceutical or nutritional composition). As already mentioned, any enzyme composition described herein can be used to treat and / or prevent diseases associated with inflammation of the skin and mouth. In one embodiment, the composition is preferably a pharmaceutical composition comprising an enzyme according to the disclosure. The pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition is not a pharmaceutical composition and is marketed to reduce the risk of disease.

[0126] In some embodiments, the composition comprises any enzyme described herein in any range between 1 nM and 100 nM or between. In some embodiments, the composition comprises any enzyme described herein in any range between 1 pM and 1 nM or between. In some embodiments, the composition comprises any enzyme described herein in any range between 100 nM and 1 µM or between. In some embodiments, any composition described herein contains any enzyme described herein in any range of about 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, or 100 nM or between.

[0127] In some embodiments, the composition is in the form of a liquid, solid, semi-solid, or a combination thereof. The composition may be a complete oral nutritional supplement. The composition may also be in the form of tablets, lollipops, sachets, soluble films, or combinations thereof.

[0128] In some embodiments, the composition is in the form of food, beverage, or combinations thereof. The composition may contain ingredients such as thickeners. In alternative embodiments, the composition is a topical compound that can be applied to the oral cavity. In alternative embodiments, the composition is a topical compound that can be applied to the skin.

[0129] In some embodiments, the composition may comprise at least a portion of an oral care composition or an oral care composition. Illustrative oral care compositions may include, but are not limited to, toothpaste (teeth cleaning agent), preventative paste, tooth powder, tooth polish, tooth gel (e.g., whitening gel), chewing gum, lozenges, mouthwash, oral rinses, whitening strips, patches, suspensions, emulsions, hydrogels, pastes, multiphase solutions, varnish gels, varnishes, finishes, and tubes, syringes, or dental trays comprising gels or pastes, liquids, powders, or gels or pastes applied to an application support such as dental floss or a toothbrush (e.g., a manual toothbrush, an electric toothbrush, a sonic toothbrush, a combination thereof, or an ultrasonic toothbrush). In some embodiments, the oral care composition comprises a combination of one or more oral care compositions.

[0130] In some embodiments, the oral care composition may include humectants, surfactants, gelling agents, and combinations thereof. Illustrative humectants may include, but are not limited to, glycerin, propylene glycol, and combinations thereof. In some embodiments, the humectant is present in an amount of about 20 wt% to about 60 wt% based on the total weight of the oral care product. In at least one embodiment, the oral care product and / or its oral care whitening composition is free of or substantially free of polyol humectants. For example, the oral care composition does not contain any polyols as humectants. In another embodiment, propylene glycol is present in an amount of about 10 wt% to about 20 wt% based on the total weight of the oral care composition. In another embodiment, glycerin is present in an amount of about 25 wt% to about 40 wt% based on the total weight of the oral care composition.

[0131] Alternatively, the oral care composition can be a liquid, gel, paste, etc., which can be applied as a coating to non-consumable products (such as animal toys). The oral care composition can be incorporated into the product. When the animal chews the toy, the composition comes into contact with some or all of the animal's mouth and improves or maintains the animal's oral health.

[0132] In some of any embodiments, the oral care composition may be a gel composition having the following components: thickener, wetting agent (such as sorbitol, glycerin, polyethylene glycol and / or mixtures thereof), binder (such as carboxymethyl cellulose, hydroxyethyl cellulose, xanthan gum, silica, thickener and / or mixtures thereof), conservatist (e.g., methylparaben, propylparaben or mixtures thereof), acidifier (typically phosphoric acid, citric acid, tartaric acid, maleic acid), flavor (mint, etc.), and flavoring agent (sodium saccharin or other flavoring agents).

[0133] In some embodiments, the oral care composition may include a thickening system having one or more thickeners. The one or more thickeners may be any orally acceptable thickener or thickening agent. Illustrative thickeners may be, or include, colloidal silica, fumed silica, cross-linked polyvinylpyrrolidone (PVP) polymers, cross-linked polyvinylpyrrolidone (PVP), and mixtures or combinations thereof. In a typical embodiment, the thickening system comprises a cross-linked polyvinylpyrrolidone (PVP) polymer. In a more typical embodiment, the thickening system may be, or includes, POLYPLASDONE. TM XL-10 is commercially available from Ashland Inc. of Covington, KY.

[0134] The composition can be presented in any form, such as as a tablet, as an injectable fluid, or as an infusion fluid. Furthermore, the compositions, proteins, nucleotides, and / or carriers according to the invention can be administered via various routes, such as bronchial, topical, or oral administration.

[0135] In some embodiments, the skin care component may contain ointment, serum, hydrogel, solution, dressing, moisturizer, exfoliant, body wash, eye cream, sunscreen, skin cream, cleanser, skin lotion, artificial skin, probiotic skin care component, or spray.

[0136] In some embodiments, the skin care component comprises any "dermatologically acceptable" composition. In some embodiments, the dermatologically acceptable composition is suitable for use in contact with human skin tissue without excessive toxicity, incompatibility, instability, allergic reactions, etc.

[0137] In some embodiments, any skincare component or composition herein is configured for topical application to keratinized tissue. In some embodiments, any skincare component or composition may comprise dimethicone and particulate material, as well as a specific amount of water-soluble UV-blocking active ingredient, and such compositions may provide a desired feel, including non-greasy, non-sticky (indicated by mean break-up time), non-shine, refreshing, and easily spreadable (indicated by dynamic viscosity). In some embodiments, any skincare component or composition provided herein is a UV-blocking composition or a sunscreen composition.

[0138] In some embodiments, any skincare component or composition herein may contain particulate material (also referred to as cosmetic powder or dry powder) in any range from about 0.25% to about 0.5%, 0.5% to about 1%, 1% to about 1.5%, 2% to about 2.5%, 2.5% to about 3%, 3% to about 45%, preferably about 3.5% to about 40%, more preferably about 4% to about 30%, even more preferably about 4.5% to about 20%, and most preferably about 4.5% to about 10% by weight of the composition, such as about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, or any range thereof. Non-limiting examples of suitable particulate materials include inorganic powders, organic powders, composite powders, fluorescent whitening agent particles, and mixtures of any of the foregoing substances. These particles can be, for example, flake-shaped, spherical, elongated, needle-shaped, or irregularly shaped; surface-coated or uncoated; porous or non-porous; charged or uncharged. In some embodiments, the particulate material is hydrophobically coated. Suitable inorganic particulate materials include, but are not limited to, talc, silicates, silica, iron oxide, titanium dioxide, zinc oxide, and any mixtures thereof.

[0139] The compositions of the present invention may contain a variety of optional ingredients known for use in personal care compositions, provided that one or more optional ingredients do not unduly alter the stability, aesthetics, or performance of the product. When incorporated into the composition, the optional ingredients should be suitable for contact with human keratinized tissue without undue toxicity, incompatibility, instability, allergic reactions, etc., within a reasonable judgment. The compositions herein may contain from about 0.0001% to about 50%; from about 0.001% to about 20%; or alternatively from about 0.01% to about 10% of optional ingredients by weight of the composition. Some non-limiting examples of optional ingredients include abrasives, absorbents, opacifiers, colorants (e.g., pigments, dyes, and lakes), particles, essential oils, anti-caking agents, foaming agents, defoamers, oil-controlling agents, binders, bio-additives, vitamins, minerals, peptides, glycosamines, flavonoids, antioxidants, preservatives, plant extracts, phytosterols, protease inhibitors, tyrosinase inhibitors, exfoliants, skin brighteners, non-tanning agents, anti-acne actives, anti-cellulite actives, anti-wrinkle actives, phytosterols and / or phytohormones, N-acyl amino acid compounds, antimicrobial agents, antifungal agents, moisturizers, emollients, humectants, lubricants, fragrances, anti-dandruff agents, buffers, swelling agents, chelating agents, biocides, denaturants, astringents, topical analgesics, anti-inflammatory agents, sunscreens, film-forming agents, and / or polymers, propellants, reducing agents, chelates, conditioning agents, and combinations thereof that contribute to the film-forming properties and directness of the composition.

[0140] In some embodiments, any composition provided herein may optionally contain pharmaceutically acceptable excipients, stabilizers, activators, carriers, penetrants, propellants, disinfectants, diluents, and preservatives. Suitable excipients are well known in the field of pharmaceutical formulation and can be readily identified and applied by a person skilled in the art, as referenced in, for example, Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pennsylvania, 17th edition, 1985.

[0141] For oral administration, proteins can be administered, for example, in solid dosage forms such as capsules, tablets (e.g., with enteric coating), and powders, or in liquid dosage forms such as elixirs, syrups, and suspensions. In some embodiments, any enzymes provided herein (e.g., adenosine triphosphate bisphosphatase) can be encapsulated in gelatin capsules along with inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc. Examples of additional inactive ingredients that can be added to provide desired color, taste, stability, buffering capacity, dispersibility, or other known desired characteristics are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be manufactured as sustained-release products for use in the sustained release of the drug over several hours. Compressed tablets may be coated with sugar or film to mask any unpleasant taste and protect the tablets from atmospheric effects, or with enteric coating for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colorings and flavorings to improve patient acceptability.

[0142] Enteric coatings prevent the release of active compounds from orally ingestible dosage forms. Depending on their composition and / or thickness, enteric coatings resist gastric acid for a required time before they begin to disintegrate and allow ATP-degrading enzymes to release slowly in the lower stomach, small intestine, or large intestine. Some examples of enteric coatings are disclosed in U.S. Patent No. 5,225,202 (incorporated by reference). Examples of enteric coatings include beeswax and glyceryl monostearate; beeswax, shellac, and cellulose, optionally comprising a neutral copolymer with polymethacrylate; copolymers of methacrylic acid and methyl methacrylate; or neutral copolymers of polymethacrylate containing metal stearates (for references on enteric coatings, see: U.S. Patent Nos. 4,728,512, 4,794,001, 3,835,221, 2,809,918, 5,225,202, 5,026,560, 4,524,060, 5,536,507). Most enteric-coated polymers become soluble at pH 5.5 and higher, with maximum solubility above pH 6.5. Enteric coatings can also include a base coat and an outer coat step, as in pharmaceutical compositions designed for specific delivery in the lower GI tract, i.e., in the colon (pH 6.4 to 7.0, ileum pH 6.6), in contrast to the upper intestine, where the small intestine and duodenum have a pH range of 7.7–8 (after the addition of pancreatic juice and bile). The pH differences in the intestine can be utilized to target enteric-coated ATP-degrading enzyme compositions to specific regions of the gut. It also allows for the selection of specific ATP-degrading enzymes that are most active at specific pH levels in the intestine.

[0143] Additionally, the pH difference between the skin and oral cavity can be utilized to target ATP-degrading enzyme compositions to specific areas on the skin and / or oral cavity. It also allows for the selection of specific ATP-degrading enzymes that exhibit the highest activity at a particular pH on the skin and / or oral cavity. For example, the average pH on the skin ranges from 4 to 7, with an average of 4.7 (Lambers et al. 2006. Int J Cosmet Sci. [International Journal of Cosmetic Science] 28(5):359-70). Although the parameter “skin pH” depends primarily on skin area, it also depends to a lesser extent on sex, ethnicity, and the time of day at which the value was determined. Furthermore, the average pH of saliva has a normal pH range of 6.2-7.6, with 6.7 being the average pH. The resting pH of the oral cavity is not lower than 6.3. In the oral cavity, the pH is maintained near neutral (6.7-7.3) by saliva (Baliga et al. 2013. J Indian Soc Periodontol [Journal of the Indian Society of Periodontology]; 17(4):461-465).

[0144] In addition to the fact that proteins according to the invention can be incorporated into pharmaceutical compositions, such ATP-degrading enzymes can also be part of nutritional compositions or nutritional products.

[0145] The proteins according to the invention can be added to nutrients (such as milk), but can also be produced within said nutrients (e.g., through molecular engineering). Furthermore, tablets and / or capsules can be prepared and subsequently added to nutrients or taken directly by humans.

[0146] In another aspect, the present invention is characterized by beverage and food products comprising an ATP-degrading enzyme that effectively treats or prevents skin and / or oral inflammation in subjects in need. Beverage products may contain 1 unit / mL to 10,000 units / mL, for example, 1 unit / mL to 200 units / mL, 200 units / mL to 500 units / mL, 500 units / mL to 1,000 units / mL, 1,000 units / mL to 5,000 units / mL, or 5,000 units / mL to 10,000 units / mL. Food products may contain 1 unit / g to 10,000 units / g, for example, 1 unit / g to 200 units / g, 200 units / g to 500 units / g, 500 units / g to 1,000 units / g, 1,000 units / g to 5,000 units / g, or 5,000 units / g to 10,000 units / g.

[0147] E. Inflammatory mediators

[0148] oral cavity

[0149] In oral inflammation, eATP can be released from damaged or dead cells, such as epithelial cells, immune cells, and nerve cells. eATP can then activate P2X7 receptors on immune cells, such as macrophages and dendritic cells, leading to the production and release of inflammatory mediators, including cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). These cytokines can further promote the recruitment and activation of immune cells, resulting in a vicious cycle of inflammation and tissue damage in the oral cavity. eATP interacts with healthy cells and induces cell death, accelerating tissue damage and hindering wound healing (Zuccarini et al. Int. J. Mol. Sci. [International Journal of Molecular Sciences] 2022, 23, 7790; Biderman et al. Arch. Oral Biol. [Archives of Oral Biology] 2017, 81, 131-135; Yilmaz et al. Cell. Microbiol. [Cell Microbiology] 2008, 10, 863-875; Chan et al. Front Immunol. [Frontiers in Immunology] 2019, March 4; 10:364).

[0150] Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine that plays a crucial role in the initiation and development of oral inflammation. It is produced by various cells in oral tissues, including macrophages, monocytes, and dendritic cells, in response to infection or injury. TNF-α promotes inflammation by stimulating the production of other cytokines and chemokines, activating immune cells such as neutrophils and macrophages, and increasing the expression of adhesion molecules on endothelial cells. These effects lead to the recruitment of immune cells to the site of inflammation and the destruction of infected or damaged tissue. In oral inflammation, TNF-α is involved in the pathogenesis of various conditions, such as periodontitis, gingivitis, and oral mucositis. In periodontitis, TNF-α plays a key role in the destruction of periodontal tissue by promoting osteoclastogenesis and bone resorption. In gingivitis, TNF-α is involved in the inflammation and destruction of gingival tissue. In oral mucositis, TNF-α contributes to ulceration and inflammation of the oral mucosa, a common side effect of chemotherapy and radiotherapy.

[0151] Interleukin-6 (IL-6) is a pro-inflammatory cytokine that plays a crucial role in the development of oral inflammation. It is produced by various cells in oral tissues, including epithelial cells, fibroblasts, and immune cells, in response to infection, injury, or other stimuli. Interleukin-8 is a potential diagnostic salivary biomarker for periodontal disease (Chand et al., A Systematic Review (March 31, 2020)), increased IL-18 levels are associated with periodontitis (Alarcón-Sánchez et al., Oral Health 24, 981 (2024)), and MCP-1 has been shown to be associated with disease regulation and involvement (Singh et al., International Immunopharmacology, Dec 2021; 101).

[0152] IL-6 promotes inflammation by stimulating the production of other cytokines, activating immune cells such as T cells and B cells, and increasing the expression of adhesion molecules on endothelial cells. These effects lead to the recruitment of immune cells to the site of inflammation and the destruction of infected or damaged tissue. In oral inflammation, IL-6 is involved in the pathogenesis of various conditions, such as periodontitis, gingivitis, and oral cancer. In periodontitis, IL-6 plays a key role in the destruction of periodontal tissue by promoting osteoclastogenesis and bone resorption. In gingivitis, IL-6 is involved in the inflammation and destruction of gingival tissue. In oral cancer, IL-6 promotes tumor growth and metastasis by promoting angiogenesis and suppressing the immune response.

[0153] Interferon-γ (IFN-γ) is a pro-inflammatory cytokine that plays a crucial role in the development of oral inflammation. It is produced by various immune cells, such as T cells and natural killer cells, in response to infection or other stimuli. IFN-γ promotes inflammation by stimulating the production of other cytokines and chemokines, activating immune cells, such as macrophages and dendritic cells, and increasing the expression of adhesion molecules on endothelial cells. These effects lead to the recruitment of immune cells to the site of inflammation and the destruction of infected or damaged tissue. In oral inflammation, IFN-γ is involved in the pathogenesis of various conditions, such as periodontitis and oral lichen planus. In periodontitis, IFN-γ participates in the destruction of periodontal tissues by promoting osteoclast activation and the production of matrix metalloproteinases. In oral lichen planus, IFN-γ is involved in inflammation and damage to the oral mucosa.

[0154] IL-17A (interleukin-17A) is a cytokine that plays a crucial role in inflammatory responses. In the context of oral inflammation, IL-17A has been found to contribute to the pathogenesis of several oral inflammatory diseases, including periodontitis, oral lichen planus, and recurrent aphthous stomatitis. IL-17A is produced by a specific group of T cells called Th17 cells, which are involved in the regulation of immune responses and tissue inflammation. In the context of oral inflammation, IL-17A activates and recruits neutrophils (a type of white blood cell) to sites of inflammation. This leads to increased production of pro-inflammatory cytokines and chemokines, resulting in tissue damage and destruction. Studies have shown increased levels of IL-17A in the gingival tissue of patients with periodontitis (a common oral inflammatory disease characterized by periodontal destruction and tooth loss). Additionally, elevated levels of IL-17A have been found in the saliva and serum of patients with oral lichen planus and recurrent aphthous stomatitis (two other common oral inflammatory conditions).

[0155] In some of the embodiments, the enzymes provided herein are capable of reducing inflammatory responses associated with oral conditions. In some embodiments, these enzymes are capable of reducing the release of inflammatory mediators. In some of the embodiments, compared to reference, the enzymes provided herein are capable of reducing the level of one or more inflammatory mediators by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some of the embodiments, the one or more inflammatory mediators are TNF-α, IL-6, IL17A, IFNγ, IL-8, or combinations thereof.

[0156] In some of the embodiments, any enzyme provided herein, compared with the reference, is capable of reducing cell death levels by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0157] In some embodiments, a reference may be an untreated reference. In some embodiments, a reference may refer to a reference in which any ATP-degrading enzyme has been added. In some embodiments, a reference may refer to a reference in which a non-ATP-degrading enzyme has been added. In some embodiments, a reference may refer to a reference in which any enzyme described herein has been previously added and then stopped.

[0158] skin

[0159] In skin inflammation, eATP can be released from damaged or dead cells, such as epithelial cells, immune cells, and nerve cells. eATP can then activate P2X7 receptors on immune cells, such as macrophages and dendritic cells, leading to the production and release of inflammatory mediators, including cytokines such as IL-6, IL-17A, IFNγ, and IL-8. These cytokines can further promote the recruitment and activation of immune cells, resulting in a vicious cycle of inflammation and tissue damage in the skin. eATP interacts with healthy cells and induces cell death, and accelerates the process of tissue damage and hinders wound healing (Manica et al. J Cell Biochem. May 2018; 119(5):3980-3988; Burnstock et al. J Invest Dermatol. March 2012; 132(3 Pt 1):526-46; Spari et al. Host. Int. J. Mol. Sci. 2020, 21, 5590; Holzer et al. J Cutan Med Surg. March-April 2004; 8(2):90-6; Trier et al. J. Immunol. 2019; 202(10): 2829-2835).

[0160] The IL-1 family is a large class of cytokines, all of which are expressed in the human epidermis and have recognized roles in skin immunopathology. (Macleod et al. Front Immunol. [Immunology Frontiers] 2021 Dec 23; 12:808012.)

[0161] Interleukin-6 (IL-6) is a pro-inflammatory cytokine that plays a crucial role in the development of skin inflammation. It is produced by various cells in the skin, including keratinocytes, fibroblasts, and immune cells, in response to infection, injury, or other stimuli. IL-6 promotes inflammation by stimulating the production of other cytokines, activating immune cells such as T cells and B cells, and increasing the expression of adhesion molecules on endothelial cells. These effects lead to the recruitment of immune cells to the site of inflammation and the destruction of infected or damaged tissue. In skin inflammation, IL-6 is involved in the pathogenesis of various conditions, such as psoriasis, atopic dermatitis, and skin cancer. In psoriasis, IL-6 plays a key role in epidermal thickening and psoriatic plaque formation by promoting keratinocyte proliferation and inducing Th17 cell differentiation. In atopic dermatitis, IL-6 is involved in skin inflammation and barrier dysfunction. In skin cancer, IL-6 promotes tumor growth and metastasis by promoting angiogenesis and suppressing the immune response.

[0162] IL-17A (interleukin-17A) is a cytokine that plays a crucial role in the pathogenesis of skin inflammation. It is produced by a specific subset of T helper cells called Th17 cells, which are involved in the regulation of immune responses and tissue inflammation. In the context of skin inflammation, IL-17A promotes the recruitment of immune cells, such as neutrophils and macrophages, to sites of inflammation. It also stimulates keratinocytes, fibroblasts, and other immune cells to produce other pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6. These cytokines contribute to the amplification and maintenance of the inflammatory response, leading to tissue damage and destruction. IL-17A is associated with the pathogenesis of several inflammatory skin disorders, including psoriasis, atopic dermatitis, and acne. For example, in psoriasis, elevated levels of IL-17A and its associated cytokines are found in damaged skin, and targeting IL-17A has proven to be an effective treatment for psoriasis. In atopic dermatitis, IL-17A has been shown to play a role in the amplification of the inflammatory response, contributing to the development of chronic and severe skin inflammation. Similarly, in acne, IL-17A has been found to be involved in the recruitment and activation of neutrophils, which contributes to the development of inflammatory acne lesions.

[0163] In some of the embodiments, the enzymes provided herein are capable of reducing inflammatory responses associated with skin conditions. In some embodiments, these enzymes are capable of reducing the release of inflammatory mediators. In some of the embodiments, compared to reference, the enzymes provided herein are capable of reducing the level of one or more inflammatory mediators by at least 1%, at least 5%, 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some of the embodiments, the one or more inflammatory mediators are IL-6, IL-17A, IFNγ, IL-8, or combinations thereof.

[0164] In some of the embodiments, any enzyme provided herein, compared with the reference, is capable of reducing cell death levels by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0165] In some embodiments, a reference may be an untreated reference. In some embodiments, a reference may refer to a reference in which any ATP-degrading enzyme has been added. In some embodiments, a reference may refer to a reference in which a non-ATP-degrading enzyme has been added. In some embodiments, a reference may refer to a reference in which any enzyme described herein has been previously added and then stopped.

[0166] III. Methods

[0167] A. Methods for treating disorders characterized by skin and / or oral inflammation

[0168] This document provides methods and compositions for reducing or preventing barriers characterized by skin and / or oral inflammation in subjects of need. In some embodiments, the methods and compositions provided herein are used to reduce or prevent barriers characterized by skin inflammation in subjects of need. In some embodiments, the methods and compositions provided herein are used to reduce or prevent barriers characterized by oral inflammation in subjects of need. Unbound by theory, it is believed that the application of ATP-degrading enzymes (such as adenosine triphosphate diphosphatases, including those of the GDA1_CD39 superfamily, including acid phosphatases, including those associated with SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: The amino acid sequence of any one of 53 is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical; or identical to SEQ ID No: An amino acid sequence of 16-30 or 77-102, comprising at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% (the same) can reduce inflammation by removing, for example, sources of inflammation generated in the skin and / or mouth. In some embodiments, this removal is the removal of eATP.

[0169] One non-limiting purpose of removing eATP from the body is to prevent or reduce local inflammatory responses. Furthermore, in some embodiments, the detoxified eATP is no longer able to cross the mucosal layer and therefore cannot enter the circulation, where it would exert its toxic effects and / or cause further local and / or systemic inflammatory responses.

[0170] The source of the ATP-degrading enzyme can be any ATP-degrading enzyme or any composition containing an ATP-degrading enzyme, and any tool capable of producing a functional ATP-degrading enzyme in the context of this invention, such as DNA or RNA nucleic acids encoding the ATP-degrading enzyme. The nucleic acid encoding the ATP-degrading enzyme can be embedded in a suitable vector (such as plasmids, phage particles, bacteriophages, (retro)viruses, transposons, gene therapy vectors, and other vectors capable of inducing or conferring the production of the ATP-degrading enzyme). Additionally, natural or recombinant microorganisms, such as bacteria, fungi, protozoa, and yeast, can be used as a source of ATP-degrading enzymes in the context of this invention.

[0171] In one embodiment, the present invention provides a method for preventing or reducing inflammation in the oral cavity. In some embodiments, the inflammation is associated with an oral health condition. In some embodiments, the oral health condition is an oral health disease selected from periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and oral mucositis. In some embodiments, the oral health condition includes open sores and / or wounds selected from oral ulcers, cold sores, cuts, bites, burns, surgical incisions, and tissue trauma. In some embodiments, the method includes the step of applying an ATP-degrading enzyme to the oral surface of a subject, the oral surface being selected from the lips, cheeks and the lining of the inner lips (buccal mucosa), tongue, upper and lower gingiva, the floor of the mouth under the tongue, the roof of the mouth (soft and hard palate), uvula, tonsils, and the area behind the wisdom teeth (retromolar triangle).In some of any embodiments, the method includes dissolving an ATP-degrading enzyme (such as SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 53). The amino acid sequence of any one of 53 is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical; or identical to SEQ ID No: 16-30 or SEQ ID NO: The step of applying a source of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the same adenosine triphosphate bisphosphatase to the oral surface of a subject, the oral surface being selected from the lips, cheeks and the lining of the inner lips (buccal mucosa), tongue, upper and lower gingiva, floor of the mouth under the tongue, roof of the mouth (soft and hard palate), uvula, tonsils, and the area behind the wisdom teeth (posterior trigone). In some embodiments, this document provides a method for treating a disorder associated with oral inflammation, wherein the treatment consists of the administration of any enzyme or composition provided herein. In jurisdictions where treatment methods cannot be patented under their laws, this invention also relates to the use of ATP-degrading enzymes as defined above, or the use of compositions containing a source of adenosine triphosphate diphosphatase as defined above. The source of the ATP-degrading enzyme is used to manufacture medicaments for oral delivery of the ATP-degrading enzyme to prevent or reduce inflammation of oral tissues.

[0172] In one embodiment, the present invention provides a method for preventing or reducing inflammation on the skin. In some embodiments, the skin includes the epidermis and / or dermis. In some embodiments, the inflammation is associated with a skin condition. In some embodiments, the skin condition is selected from UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and tissue trauma. In some embodiments, the method includes the step of applying an ATP-degrading enzyme to the surface of the subject's skin or keratinous tissue. In some of any embodiments, the method includes dissolving an ATP-degrading enzyme (such as SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: The amino acid sequence of any one of 53 is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical; or identical to SEQ ID No: 16-30 or SEQ ID NO: The procedure involves administering a source of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the same adenosine triphosphate bisphosphatase or acid phosphatase to the skin or epidermal tissue of a subject. For those jurisdictions where treatment methods are not patentable under certain laws, the invention also relates to the use of ATP-degrading enzymes as defined above, or the use of compositions containing a source of ATP-degrading enzymes as defined above. The source of the ATP-degrading enzyme is used to manufacture medicaments for the skin delivery of the ATP-degrading enzyme to prevent or reduce inflammation of skin tissue.

[0173] III. In particular, the methods described above for applying ATP-degrading enzymes to the skin and / or mouth are suitable for treating or preventing inflammation and related diseases, although the methods can also be advantageously used in healthy subjects as a preventative treatment aimed at preventing inflammation and related diseases. In some embodiments, related diseases do not include irritant contact dermatitis. The beneficial effects of applying ATP-degrading enzymes according to the invention to reduce skin and / or oral inflammation will produce an overall health-promoting effect, regardless of the medical condition of the treated subject. The subsequent reduction of LPS, eATP, and / or CpG DNA inflow through the mucosal layer can further enhance the health-promoting effect.

[0174] LPS, eATP, or CpG DNA-mediated or induced diseases can be any disease, symptom, or symptom group caused by LPS, eATP, or CpG DNA toxicity. LPS, eATP, or CpG DNA-aggravated diseases can be any disease or symptom not directly caused by LPS, eATP, or CpG DNA, but whose symptoms and clinical features may be exacerbated by LPS, eATP, or CpG DNA, and in which the clinical condition of the subject with such a disease is worsened by the presence of LPS, eATP, or CpG DNA in the skin and / or mouth.

[0175] Preferably, this method is intended to treat LPS, eATP, and / or CpG DNA-mediated or exacerbated conditions, such as oral cavity-related inflammatory diseases, particularly in patients diagnosed with periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and / or oral mucositis. In periodontitis, eATP is released from damaged or dead cells in the inflamed periodontium and acts as a chemical attractant for immune cells, such as neutrophils and macrophages, to the site of infection. Once recruited, these immune cells release inflammatory mediators, including cytokines and chemokines, leading to periodontal tissue destruction and bone loss characteristic of periodontitis. In oral mucositis, eATP is released from damaged or dead cells in the oral mucosa due to chemotherapy or radiation therapy and contributes to the development of inflammation and ulceration. eATP can activate the NLRP3 inflammasome in oral epithelial cells, leading to the production and release of inflammatory mediators, including cytokines and chemokines such as IL-1β and IL-18. These cytokines can further amplify the inflammatory response and contribute to the development of oral mucositis.

[0176] Preferably, this method is intended to treat diseases mediated or exacerbated by LPS, eATP, and / or CpG DNA, such as skin-related inflammatory diseases, particularly in patients diagnosed with UV radiation, bacterial infections, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and / or tissue trauma. In psoriasis, eATP is released from keratinocytes and immune cells in psoriatic plaques and acts as a pro-inflammatory mediator. eATP can activate P2X7 receptors on immune cells, leading to the production and release of pro-inflammatory cytokines such as IL-1β and IL-18, which further promotes the development and maintenance of psoriatic lesions. In atopic dermatitis, eATP is released from damaged or dead cells in inflamed skin and contributes to the activation and recruitment of immune cells to the site of inflammation. eATP can activate P2X7 receptors on immune cells, leading to the production and release of pro-inflammatory cytokines and chemokines such as IL-8, which contributes to the development and longevity of atopic dermatitis. In contact dermatitis, eATP is released from damaged skin cells in response to exposure to various irritants or allergens. eATP can activate P2X7 receptors on immune cells, leading to the production and release of pro-inflammatory cytokines and chemokines such as IL-1β and IL-6, which further promotes the development of skin inflammation.

[0177] According to this disclosure, the tissues to be treated include the lips, cheeks and the lining of the inner lips (buccal mucosa), tongue, upper and lower gums, floor of the mouth under the tongue, roof of the mouth (soft and hard palate), uvula, tonsils and the area behind the wisdom teeth (posterior triangle of the molars).

[0178] Other tissues to be treated according to this disclosure include the skin, which includes the epidermis, dermis and hypothalamus.

[0179] In some embodiments, the compositions provided herein according to this disclosure can be used to promote wound healing in a subject. In some embodiments, this document provides a method for promoting wound healing in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the compositions described herein. This document also provides the use of any of the compositions provided herein for promoting wound healing in a subject.

[0180] In some embodiments, this document provides a method for culturing artificial skin, the method comprising culturing epithelial cells in the presence of any of the compositions described herein. In some embodiments, these epithelial cells are derived from mammals. In some embodiments, the mammal is a human. In some embodiments, the compositions provided herein according to this disclosure can be used to culture artificial skin. In some embodiments, the artificial skin is made from epithelial cells. In some embodiments, the artificial skin can be used to promote wound healing. In some embodiments, the artificial skin can be used for implantation. In some embodiments, these epithelial cells are derived from mammals. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human mammal.

[0181] In some embodiments, this document provides a method for restoring the protective skin barrier, moisturizing the skin, and / or reducing skin redness, the method comprising applying any of the compositions described herein to human skin. In some embodiments, the composition is applied topically and reduces skin redness in the subject compared to a subject who has not used the composition. In some embodiments, the composition is applied topically and increases skin moisture in the subject compared to a subject who has not used the composition. In some embodiments, the composition is applied topically and restores the subject's protective skin barrier compared to a subject who has not used the composition.

[0182] This document also provides for the use of any of the compositions described herein for restoring the protective skin barrier, moisturizing the skin, and / or reducing skin redness.

[0183] Compositions containing ATP-degrading enzymes according to this disclosure are particularly suitable for oral and / or topical application to prevent, treat, reduce, cure, or alleviate inflammatory diseases of the skin and / or oral cavity. Inflammatory diseases of the skin and / or oral cavity can be significantly induced and / or aggravated by exposure to environmental or chemical factors (including UV radiation) or trauma to tissues or surrounding tissues, allowing bacterial derivatives such as LPS, eATP, and / or CpG DNA to enter.

[0184] This disclosure also provides compositions containing ATP-degrading enzyme sources, including pharmaceutical and nutritional compositions containing ATP-degrading enzyme sources. The compositions may optionally contain pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeabilizers, propellants, disinfectants, diluents, and preservatives. Suitable excipients are well known in the field of pharmaceutical formulation and can be readily identified and applied by those skilled in the art, as referenced in, for example, Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pennsylvania, 17th edition, 1985. In preferred embodiments, compositions containing ATP-degrading enzymes are suitable for application with one or more oral care components, including toothpaste, preventative pastes, tooth powder, tooth polish, tooth gel, chewing gum, lozenges, mouthwash, oral rinses, whitening strips, patches, suspensions, lotions, hydrogels, pastes, multiphase solutions, varnish gels, varnishes, finishes, and tubes, syringes, or dental trays containing gels or pastes, or gels or pastes applied to application supports such as dental floss or toothbrushes. In preferred embodiments, compositions containing ATP-degrading enzymes are suitable for application with one or more skin care components, including ointments, serums, hydrogels, solutions, dressings, moisturizers, exfoliants, body washes, eye creams, sunscreens, skin creams, cleansers, skin lotions, artificial skin, probiotic skin care components, or sprays. In preferred embodiments, compositions containing ATP-degrading enzymes are suitable for application with one or more wound care components.

[0185] In another embodiment, the composition containing an ATP-degrading enzyme source is suitable for oral administration and includes an enteric coating to protect the ATP-degrading enzyme from the adverse effects of gastric juice and low pH. Enteric coatings and controlled-release formulations are well known in the art (references are provided above). Enteric-coated compositions in the art may comprise a solution of a water-soluble enteric-coating polymer mixed with one or more active ingredients (such as ATP-degrading enzymes) and other excipients, dispersed in an aqueous solution and subsequently dried and / or granulated. The resulting enteric coating resists the attack of atmospheric moisture and oxygen during storage, as well as the attack of gastric juice and low pH on the ATP-degrading enzyme after ingestion, while readily decomposing under the alkaline conditions present in the lower intestine.

[0186] In some embodiments, any composition containing an ATP-degrading enzyme for delivering an ATP-degrading enzyme to the skin and / or mouth for the treatment and prevention of inflammation, according to this disclosure, may comprise a microbial-derived (i.e., derived from bacteria, archaea, fungi, or yeast) ATP-degrading enzyme or a eukaryotic ATP-degrading enzyme. In some embodiments, the ATP-degrading enzyme is not a mammalian ATP-degrading enzyme.

[0187] B. Methods for promoting the implantation of symbiotic bacteria on the skin and / or in the mouth

[0188] This document also provides a method for facilitating the implantation of one or more symbiotic bacteria onto the skin and / or oral cavity of a subject. This method requires, at least, administration to the subject of any ATP-degrading enzyme composition disclosed herein (such as that with SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: The amino acid sequence of any one of 53 is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical; or identical to SEQ ID NO: 16-30 or SEQ ID NO: The amino acid sequence of 77-102 is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to adenosine triphosphate bisphosphatase or acid phosphatase and one or more symbiotic bacteria. In some embodiments, compared with the application of a source of one or more symbiotic bacteria in the absence of the ATP-degrading enzyme composition disclosed herein, the method promotes the implantation of the one or more symbiotic bacteria onto the subject's skin and / or mouth to a greater extent (e.g., an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125% or more (including percentages falling between these values)).Non-limiting examples of symbiotic bacteria include organisms from the following genera: *Streptococcus*, *Actinomyces*, *Veillonella*, *Fusobacterium*, *Porphromonas*, *Prevotella*, *Treponema*, *Nisseria*, *Haemophilis*, *Eubacteria*, and *Lactobacillus*. The bacterial strains include *C. ctobacterium*, *Capnocytophaga*, *Eikenella*, *Leptotrichia*, *Peptostreptococcus*, *Staphylococcus*, *Propionibacterium*, *Corynebacterium*, *Dermabacter*, and *Brevibacterium*. The bacterial source of one or more of these symbiotic bacteria can be administered as a probiotic (e.g., orally, topically, or rectally) or as a fecal microbiota graft (e.g., rectally or orally in capsule form).

[0189] Generally, the source of the one or more symbiotic bacteria (such as a probiotic composition) includes bacteria, such as one or more bacterial strains. In some embodiments of this disclosure, the source of the one or more symbiotic bacteria is formulated in a freeze-dried or lyophilized form. For example, the source of the one or more symbiotic bacteria may include granules or gelatin capsules, such as hard gelatin capsules, containing the bacterial strains disclosed herein.

[0190] In some embodiments, the source of the one or more symbiotic bacteria comprises lyophilized bacteria. Lyophilization of bacteria is a well-established procedure in the art. Alternatively, the composition containing microorganisms may comprise live bacterial cultures.

[0191] In some embodiments, the bacterial source of the one or more symbiotic bacteria is formulated to enable the bacterial strain to be delivered to the skin and / or oral cavity. In some embodiments, the formulation is suitable for application with one or more oral care components, including toothpaste, preventative paste, tooth powder, tooth polish, tooth gel, chewing gum, lozenges, mouthwash, oral rinses, whitening strips, patches, suspensions, lotions, hydrogels, pastes, multiphase solutions, paint gels, varnishes, finishes, and tubes, syringes, or dental trays containing gels or pastes, or gels or pastes coated on application supports such as dental floss or toothbrushes. In a preferred embodiment, the formulation is suitable for application with one or more skin care components, including ointments, serums, hydrogels, solutions, dressings, moisturizers, exfoliants, body washes, eye creams, sunscreens, skin creams, cleansers, skin lotions, artificial skin, probiotic skin care components, or sprays. In a preferred embodiment, compositions containing an ATP-degrading enzyme source are suitable for application with one or more wound care components.

[0192] In some embodiments, the bacterial source of one or more symbiotic bacteria is encapsulated to enable delivery of the bacterial strain to the skin, mouth, or intestine. Encapsulation protects the composition from degradation before delivery to the target site, degradation pathways such as rupture due to chemical or physical stimuli (e.g., stress, enzyme activity, or physical disintegration, which may be triggered by pH changes). Any suitable encapsulation method can be used. Exemplary encapsulation techniques include retention within a porous matrix, attachment or adsorption on a solid carrier surface, self-aggregation by flocculation or with a crosslinking agent, and mechanical containment by a microporous membrane or microcapsule.

[0193] The bacterial source of the one or more symbiotic bacteria may be administered orally and may be in the form of tablets, capsules, or powder. Other ingredients (e.g., vitamin C or minerals) may be included as oxygen scavengers and prebiotic substrates to improve delivery and / or partial or complete colonization and / or implantation and / or survival in the body. Alternatively, the bacterial source of the one or more symbiotic bacteria (such as a probiotic composition) may be administered orally as a food or nutritional product (such as milk or whey-based fermented dairy products) or as a pharmaceutical product.

[0194] The bacterial source of the one or more symbiotic bacteria can be formulated as a probiotic. Alternatively, the bacterial source of the one or more symbiotic bacteria can be formulated as a non-viable bacterial composition, such as a pasteurized or heat-treated bacterial composition.

[0195] The source of one or more symbiotic bacteria includes therapeutically effective amounts of the bacterial strains disclosed herein (e.g., but not limited to, from one or more of the following genera: Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphyromonas, Prevotella, Treponema, Neisseria, Haemophilus, Eubea, Lactobacillus, Carbonylophilus, Eikebium, Ciliophora, Peptostreptococcus, Staphylococcus, Propionibacterium, Corynebacterium, Propionibacterium, Dermocactus, and Brevibacterium). The therapeutically effective amount of the bacterial strain is sufficient to provide a beneficial effect to the patient. The source of one or more symbiotic bacteria may be sufficient to result in delivery to the skin and / or oral cavity of the subject, and / or partial or complete implantation and / or colonization on the skin and / or oral cavity of the subject.

[0196] For example, for adults, a suitable daily dose of symbiotic bacteria could be approximately 1 x 10⁻⁶. 3 To approximately 1 x 10 11 One colony-forming unit (CPU); for example, approximately 1 x 10⁻⁶. 7 To approximately 1 x 10 10 CPU; in another instance, approximately 1 x 10 6 To approximately 1 x 10 10 GPU; in another instance, approximately 1 x 10 7 To approximately 1 x 10 11 CPU; in another instance, approximately 1 x 10 8 To approximately 1 x 10 10 CPU; in another instance, approximately 1 x 10 8 To approximately 1 x 10 11 CPU. In some embodiments, the bacterial dose is at least 10. 9 Cells / day, such as at least 10 10 At least 10 11 Or at least 10 12 Cells / day.

[0197] In some embodiments, the bacterial source of the one or more symbiotic bacteria contains about 1 x 10⁻⁶ bacteria relative to the weight of the composition. 6 To approximately 1 x 10 11 The bacterial strain at a CFU / g level; for example, approximately 1 x 10⁻⁶. 8 To approximately 1 x 10 10 CFU / g. This dosage can be, for example, 1 g, 3 g, 5 g, and 10 g. In some embodiments, the amount of bacterial strain relative to the weight of the composition is about 1 x 10⁻⁶. 3 To approximately 1 x 10 11 Colony forming units / gram.

[0198] In some embodiments, the source of any one or more of the symbiotic bacteria is administered at doses between 500 mg and 1000 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg, or between 750 mg and 1000 mg. In some embodiments, the lyophilized bacteria in any of the microbial-containing compositions disclosed herein are administered at doses between 500 mg and 100 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg, or between 750 mg and 1000 mg.

[0199] Typically, probiotics are optionally combined with at least one suitable prebiotic compound. Prebiotic compounds are usually indigestible carbohydrates, such as oligosaccharides or polysaccharides or sugar alcohols, which are not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and trans-galacto-oligosaccharides.

[0200] In some embodiments, the probiotic composition is formulated to include a prebiotic compound in an amount of about 1 to about 30% by weight (e.g., 5 to 20% by weight) relative to the total weight of the composition. The carbohydrate may be selected from the group consisting of: fructooligosaccharides (or FOS), short-chain fructooligosaccharides, inulin, isomaltooligosaccharides, pectin, xylooligosaccharides (or XOS), chitosan oligosaccharides (or COS), human milk oligosaccharides, β-glucan, gum arabic modified starch and resistant starch, polydextrose, D-tagatose, gum arabic fiber, carob, oat and citrus fiber. In one aspect, the prebiotic is a short-chain fructooligosaccharide (hereinafter referred to as FOSs-cc for simplicity); said FOSs-cc is not a digestible carbohydrate, is typically obtained by the conversion of beet sugar and comprises a sucrose molecule bound with three glucose molecules. In another embodiment, the prebiotic may include one or more polyphenols (such as plant polyphenols). In some embodiments, any probiotic disclosed herein may be formulated together with other probiotics derived from the genera *Lactobacillus* and *Bifidobacterium* (such as *Bifidobacterium lactis* B420).

[0201] The bacterial source of one or more symbiotic bacteria may further include pharmaceutically acceptable excipients or carriers. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field. Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include, but are not limited to, ethanol, glycerol, and water. The choice of drug carrier, excipient, or diluent may be based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain (or in addition to a carrier, excipient, or diluent) any suitable binder, lubricant, suspending agent, coating agent (such as a gastrointestinal-resistant coating agent that does not dissolve or degrade before reaching the small or large intestine), or solubilizer. Examples of suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include, but are not limited to, esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0202] The source of one or more symbiotic bacteria can be formulated as a food product. For example, in addition to the therapeutic effects of the present invention, the food product can also provide nutritional benefits, such as in nutritional supplements. Similarly, food products can be formulated to enhance the flavor of the compositions of the present invention, or to make the compositions more consumer-appealing by making them more similar to ordinary food rather than pharmaceutical compositions. In some embodiments, the microbial-containing composition is formulated as a milk-based product. As used herein, the term "milk-based product" means any liquid or semi-solid milk-based or whey-based product with a different fat content. Milk-based products can be, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, unprocessed milk reconstituted from milk powder and whey, or processed products such as yogurt, curdled milk, curd, acidic milk, acidic whole milk, buttermilk, and other acidic milk products. Another important group includes milk beverages such as whey beverages, fermented milk, condensed milk, infant or toddler milk; flavored milk, ice cream; and milk-containing foods such as confectionery.

[0203] In some embodiments, the source of the one or more symbiotic bacteria contains a single bacterial strain or species and contains no other bacterial strains or species. Such compositions may contain only trace amounts or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be cultures substantially free of other biological species. In some embodiments, the compositions of the invention consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 bacterial strains or species. In some embodiments, these compositions consist of 1 to 10 (e.g., 1 to 5) bacterial strains or species.

[0204] The source of the one or more symbiotic bacteria used according to the methods disclosed herein may or may not require market authorization.

[0205] In some embodiments, when the source of the one or more symbiotic bacteria is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere with 50% relative humidity, at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the bacterial strain, as measured in colony-forming units, persists for a period of at least about 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years.

[0206] The invention can be further understood by referring to the following examples, which are provided for illustration and not for limitation. Example

[0207] Example 1. Cloning, expression, and protein preparation of enzyme candidates

[0208] From NCBI ( worldwwideweb.ncbi.nlm.nih.gov / proteinPhylogenetically diverse sequences of enzyme families known to have ATP hydrolytic activity were identified from internal databases and annotated as members of enzyme families GDA1 / CD39 or acid phosphatases. Codon optimization of the target gene sequence was performed based on Bacillus subtilis codon preferences, and synthetic genes encoding sequences of various enzymes were generated using techniques known in the art (Generay Biotech (Shanghai) Co., Ltd). The sequences of the codon-optimized genes (SEQ ID NO: 1-15, 64-76), the native full-length polypeptide sequences (SEQ ID NO: 16-30, 77-89), and the predicted mature enzymes (SEQ ID NO: 31-45, 90-102) are listed in Table 1. The genes were cloned into the expression vector p2JM103BBI (Vogtentanz, ProteinExpr Purif. [Protein Expression and Purification] 55:40-52, 2007). Expression of codon-optimized nucleotide sequences was performed using the nucleotide sequence SEQ ID NO: 47, which encodes an aprE signal peptide (SEQ ID NO: 48) with an additional 3 amino acids (Ala-Gly-Lys) between the signal sequence and the predicted mature sequence of the target gene (to promote the secretion of the target protein in Bacillus subtilis). This sequence encodes the predicted mature polypeptide driven by the aprE promoter (SEQ ID NO: 46) and terminated by the lat terminator (SEQ ID NO: 49). Competent Bacillus subtilis cells were transformed with each corresponding expression vector and plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies were inoculated into flasks containing LB medium and grown at 37°C with shaking at 200 rpm for 5–6 h. Seed cultures were inoculated into production media containing minerals (e.g., potassium sulfate, magnesium sulfate, ferrous sulfate, calcium chloride, citric acid, etc.), one or more carbon sources (e.g., glucose), complex nitrogen sources (e.g., hydrolyzed soybean peptone, yeast extract), and buffer components (e.g., MOPS, Tris, etc.). Cultures were grown at 32°C and shaken at 250 rpm for 24–40 h.

[0209] The general purification procedure is as follows: Collect the culture broth by centrifugation, concentrate and replenish with ammonium sulfate to a final concentration of 1 M, then apply it to a hydrophobic interaction column (e.g., phenyl FF, butyl FF), pre-equilibrate with 50 mM Tris (pH 7.5) containing 1 M ammonium sulfate. Elute the column in stepwise mode (0.75 M, 0.5 M, 0.25 M, 0.02 M ammonium sulfate, H2O, 20% ethanol), and combine the fractions containing the target active enzyme, exchanging the buffer to 20 mM Tris pH (7.5). Load the combined sample onto anion exchange chromatography (e.g., QFF) and elute in gradient mode (0–0.5 M NaCl). Identify the fractions containing the purified enzyme based on SDS-PAGE analysis and activity assays, and combine them. Store the purified sample at -20°C in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 40% glycerol until use.

[0210]

[0211] Example 2. Biochemical characterization of enzyme candidates

[0212] Samples of the phylogeneticly diverse enzymes described in Example 1 were assayed to determine their ability to hydrolyze ATP. Reference enzymes were purchased from Sigma-Aldrich and included potato adenosine triphosphate bisphosphatase (catalog number A6535), recombinant human CD39His tag (catalog number SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog number P0114).

[0213] Enzymatic activity against ATP or ADP at pH 3 to 9: The ability of candidate enzymes to degrade ATP or ADP to AMP and free phosphate (Pi) was measured using a standard malachite green phosphate assay kit (Sigma-Aldrich, catalog number MAK307). Enzyme reactions were performed in 96-well plates with a total volume of 150 µL containing 250 µM substrate, 50 mM buffer, 5 mM CaCl2, and varying concentrations of enzyme. The substrate was either ATP or ADP. Buffers were glycine (pH 3), acetate (pH 4, 4.5, 5), bis-Tris (pH 5.5, 6, 6.5), or Tris (pH 7, 8, 9). Enzyme concentrations varied from approximately 1 µg / mL to 1 ng / mL and were adjusted based on enzyme activity to ensure that the amount of phosphate released during the assay conformed to the dynamic range of the assay (5–100 µM phosphate). The enzyme concentration was calculated by determining the absorbance at 280 nm and using the predicted molecular weight and molar extinction coefficient. The enzyme reaction was initiated by adding 15 µL of enzyme and 135 µL of other reaction components, resulting in the final reaction composition as previously described. After 5 min, the reaction was stopped by mixing 80 µL of the reaction mixture with 20 µL of the malachite green working reagent. The malachite green working reagent was prepared according to the manufacturer's protocol by mixing 100 parts of reagent A with 1 part of reagent B. A phosphate standard solution (0–80 µM) and a negative control reaction without the enzyme were also mixed with the malachite green working reagent. All samples were tested in duplicate. The malachite green reaction mixture was allowed to stand at room temperature for 45–60 min, and the absorbance at 620 nm was measured. Finally, the absorbance readings of each sample were converted to phosphate concentration using the absorbance of the phosphate standard, and the negative control was subtracted from each reaction. Using ATP or ADP as substrates, the number of micromoles of phosphate released per milligram of protein per minute of reaction time (µmol / mg / min) is shown in Tables 2 and 3.

[0214]

[0215]

[0216] As shown in Tables 2 and 3, CRC22110 is the most active enzyme, exhibiting activity over a wide pH range (including pH 3 to pH 9). Potato adenosine triphosphate bisphosphatase, CRC22110, CRC22105, CRC22112, CRC22383, and CRC21328 are members of the GDA1_CD39 superfamily. CRC21320 is a member of the purple acid phosphatase family. CRC21323 and CRC21322 are members of the acid phosphatase (class A) superfamily. Acid phosphatases have an acidic pH optimum of approximately pH 4.5, while enzymes from the GDA1_CD39 superfamily have a near-neutral pH optimum (pH 6–8) and a wide range of activity between pH 5 and 7, with some enzymes even maintaining activity at pH 9. CRC21320 has a narrower pH range, with an optimum at pH 7.

[0217] Enzyme substrate specificity: The enzyme activity for different nucleotide substrates was tested using the malachite green assay. The assay was set up as described above, with the following modifications. The substrates tested were ATP, ADP, CTP, UTP, UDP, GTP, and GDP. CRC21323 and CRC21322 were tested using acetate buffer at pH 4.5. CD39 was tested using bis-Tris buffer (pH 7). All other enzymes were tested using bis-Tris buffer (pH 6.5). The activity of each enzyme was set relative to its activity against ATP after converting absorbance to phosphate concentration. The test results are shown in Table 4.

[0218]

[0219] As shown in Table 4, nucleotide triphosphates are better substrates than diphosphates. Most enzymes exhibit similar activity to UTP as ATP, except for CRC22105, which shows significantly reduced activity to both UTP / UDP and CTP / CDP. However, each enzyme has a slightly different preference.

[0220] Enzyme acid resistance: The ability of candidate enzymes to resist inactivation at low pH was tested by incubating the enzymes in an “acidic buffer” of 50 mM glycine and 5 mM CaCl2 at pH 2 for 15 min or 60 min and comparing their activity with that of the enzyme in a “control buffer” at its optimal pH. The optimal buffer for CRC21323 and CRC21322 was 50 mM acetate containing 5 mM CaCl2 (pH 4.5). The optimal buffer for all other enzymes was 50 mM bis-Tris containing 5 mM CaCl2 (pH 6.5). After incubation in the acidic glycine buffer, the enzyme mixture was neutralized by mixing with an equal volume of neutralization buffer. The neutralization buffer for CRC21323 and CRC21322 was 100 mM acetate and 10 mM CaCl2 (pH 4.5). The neutralization buffer for other enzymes was 100 mM bis-Tris and 10 mM CaCl2 (pH 6.5). Next, the ability of acid-stressed enzymes and control enzymes to release phosphate from ATP was tested using the reaction conditions and malachite green assay described in… with the following modifications. The only substrate for the test was ATP. CRC21323 and CRC21322 were tested using acetate buffer at pH 4.5, but all other enzymes were tested in bis-Tris buffer at pH 6.5. After determining the phosphate concentration in each reaction, the residual activity of each enzyme after incubation in “acid buffer” for 15 min or 60 min was calculated by dividing the acid stress result of each enzyme by the phosphate concentration of the control enzyme. The test results are shown in Table 5 below.

[0221]

[0222] As shown in Table 5, the acid tolerance test indicated that CRC21328 and CRC22110 exhibited the greatest tolerance to the "acid buffer" because they maintained maximum activity after 60 min of incubation. Potato adenosine triphosphate bisphosphatase was stable after 15 min, but appeared to lose most of its activity after 60 min of incubation. Although CRC21323 and CRC21322 are members of the acid phosphatase family, they were the least stable under these assay conditions.

[0223] Effect of pH on ATPase activity: As described in Example 1, ATPase activity was measured at a concentration of 0.05 ppm in 50 mM MES buffer (pH 6 and pH 5) with 0.1 mM ATP as the substrate. Commercial potato adenosine triphosphate bisphosphatase was used as a reference. Absorbance was measured at 620 nm, and the absorbance of the blank sample (water) was subtracted from each sample. The results are shown in Table 6.

[0224]

[0225] Example 3. Expression of the truncated form of the target enzyme

[0226] The CRC22110 *Gallic Monomospora leucosus* molecule with an N-terminal AGK peptide, expressed in *Bacillus subtilis*, was selected for further evaluation, including truncation of the predicted sequence (SEQ ID NO: 50). Various C-terminal truncations of the CRC22110 ATP-bisphosphatase were designed to remove C-terminal residues to a final cysteine ​​residue involving one of the seven disulfide bonds in the mature enzyme: CRC22110-V1 (SEQ ID NO: 51), CRC22110-V2 (SEQ ID NO: 52), and CRC22110-V3 (SEQ ID NO: 53). The expression methods of the truncated forms of CRC22110 are described below, wherein in some cases, an additional three AGK residues are included at the predicted N-terminus of the mature polypeptide sequence. A first DNA fragment containing the flanking region of the (5') skfA gene (5' skfA gene FR, SEQ ID NO: 54) is operatively linked to a polynucleotide construct (e.g., an expression cassette) containing an upstream (5') Bacillus subtilis rrnI-p2 promoter region DNA sequence (SEQ ID NO: 55), which is operatively linked to a DNA sequence of the Bacillus subtilis aprE 5' untranslated region (5' UTR) (SEQ ID NO: 56), which is operatively linked to DNA encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO: 47), which is operatively linked to a DNA sequence encoding a desired C-terminal truncated Adenosine triphosphate diphosphatase of *Galium spp.* (polypeptide sequence SEQ ID NO: 57, 58, or 59) with an added nucleotide sequence encoding the N-terminal tripeptide AGK, which is operatively linked to the *Bacillus amyloliquefaciens* BPN terminator (SEQ ID NO: 58, 59). 60), the terminator is operatively linked to the (3') skfA gene flanking region (3' skfA gene FR) (SEQ ID NO: 61).A second DNA fragment containing the (5') amyE gene flanking region (5' amyE gene FR) (SEQ ID NO: 62) is operatively linked to a polynucleotide construct (e.g., an expression cassette) containing an upstream (5') Bacillus subtilis rrnI-p2 (SEQ ID NO: 55) promoter region DNA sequence, which is operatively linked to a DNA sequence of the Bacillus subtilis aprE 5' untranslated region (5' UTR) (SEQ ID NO: 56), which is operatively linked to DNA encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO: 47), which is operatively linked to a DNA sequence encoding the desired C-terminal truncated Adenosine triphosphate diphosphatase of *Galium spp.* (polypeptide sequence SEQ ID NO: 57, 58, or 59) with added nucleotide sequence encoding the N-terminal tripeptide AGK, and which is operatively linked to the *Bacillus amyloliquefaciens* BPN terminator (SEQ ID NO: 57, 58, or 59). 60), the terminator is operatively linked to the (3') amyE gene flanking region (3' amyE gene FR) (SEQ ID NO: 63). More specifically, these DNA fragments were assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes for producing two-copy strains. Using standard molecular biology techniques, suitable Bacillus subtilis strains containing nine protease deletions were used to integrate the above-described first and second linear DNA expression cassette fragments into the genome.

[0227] Example 4. Biochemical evaluation of the truncated form of CRC22110 adenosine triphosphate diphosphatase

[0228] The relative enzymatic activity of the truncated form of CRC22110 adenosine triphosphate diphosphatase against ATP substrates was determined as described below. Purified samples of CRC2210 (SEQ ID NO: 50), CRC22110-V1 (SEQ ID NO: 51), and CRC22110-V3 (SEQ ID NO: 53) were prepared and quantified as described below.

[0229] Enzyme separation: The culture supernatant from Bacillus subtilis fermentation was obtained by filtration, and ammonium sulfate and 1 M Tris (pH 8) were added to a final ammonium sulfate concentration of 1 M and a final Tris (pH 8) concentration of 20 mM. The sample was centrifuged and filtered. The filtrate was loaded onto a 300 mL phenyl agarose column equilibrated in 20 mM Tris and 1 M ammonium sulfate (pH 8). A linear gradient of 0% to 100% 20 mM Tris (pH 8) was run at 10 mL / min over 300 min. The elution fractions were collected, and ATPase activity was monitored using an ATPase activity assay. The active fractions were combined, concentrated, and buffer-exchanged to 20 mM Tris (pH 8). This fraction was loaded onto a 25 mL Q agarose column equilibrated in 20 mM Tris (pH 8). The protein was then eluted using a stepwise gradient: first with 50 mM NaCl in 20 mM Tris (pH 8), followed by elution with 100 mM NaCl, 200 mM NaCl, 400 mM NaCl, and 500 mM NaCl in 20 mM Tris (pH 8). The active fraction and the pure fraction (>95% as checked by SDS-PAGE) were combined to form the purified protein, which was then quantified.

[0230] Protein quantification by UPLC: Protein concentration was determined by UPLC (ultra-high performance liquid chromatography) and OD280 density determination. For UPLC determination, the purified enzyme was diluted in 20 mM Tris (pH 8), and the protein fraction was separated using a Zorbax 300 SB-C3 column (Agilent Technologies). A linear gradient was run between 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B), and detection was performed at 220 nm on UHPLC to determine the concentration. 10 μL of sample was loaded onto the column, and the peak area of ​​the diluted sample was determined. The enzyme concentration of the sample was calculated using a standard curve of a purified reference enzyme (e.g., full-length CRC22110). Protein concentration was also determined by OD280 measurement. The purified enzyme was diluted in 20 mM Tris (pH 8), and its OD280 was measured in a quartz cuvette. The protein concentration was calculated based on its respective extinction coefficient. The final concentration was calculated based on the average value determined by the UPLC method and OD280.

[0231] ATPase activity comparison: ATPase activity was measured using the previously described ATPase assay, with samples diluted to 0.003 ppm or 0.0015 ppm in assay buffer (50 mM Tris (pH 8), 5 mM CaCl2, 0.01% Tween 80). 10 μL of the diluted enzyme sample was added to 0.25 mM ATP in the assay buffer to initiate the reaction. The reaction mixture was incubated at 25°C for 10 min, then 50 μL of the reaction mixture was added to 100 μL of QuantiChrom™ Malachite Green reagent (VWR catalog number 75878), and the reaction mixture was incubated at 25°C for 20 min before measuring OD620. ATPase activity (without enzyme control) was compared after subtracting the blank. Relative ATPase activity was calculated relative to full-length CRC22110 based on OD after background subtraction. The results are shown in Table 7 below.

[0232]

[0233] As shown in Table 7, the relative ATPase activities of CRC22110 and C-terminal truncated ATPases are highly comparable, thus confirming that this region of the protein sequence can be truncated without affecting enzyme function.

[0234] Example 5. Use of an exemplary enzyme in a gingival epithelial tissue inflammation model

[0235] In oral inflammation, eATP has been shown to play an important role in the pathogenesis of various conditions, such as periodontitis and oral mucositis. This example describes the use of an exemplary enzyme in a 3D gingival epithelial tissue model of oral inflammation.

[0236] In short, the 3D gingival epithelial tissue model (catalog number GIN 100) was obtained from MatTek, Inc., Massachusetts, USA. The tissue was maintained and used in experiments according to the manufacturer's protocol.

[0237] To determine the role of ATP in oral inflammation, tissues were incubated with 10 mM ATP or LPS (1 μg / mL) from *Porphyromonas gingivalis* (catalog number SMB00610), or 10 mM GTP, 100 nM AP429, or 10 mM ATP + LPS (1 μg / mL) + 100 nM CRC22110-V1 at 37°C for 2 and 6 hours. Tissues were collected at 2 and 6 hours for MTT assays to evaluate tissue viability under different conditions according to the manufacturer's protocol. Briefly, tissues were incubated in MTT solution for 3 hours and then extracted overnight in the dark at room temperature without shaking. The optical density of the extraction solution was measured at 570 nm using 200 μL of sample.

[0238] like Figure 1 As shown, ATP and LPS can induce cell death in 3D gingival tissue, while the addition of the exemplary NTP enzyme CRC22110-V1 can counteract the inhibitory effect of ATP and LPS activity.

[0239] To determine the role of exemplary enzymes in the expression of ATP- and LPS-induced cytokines, including TNF-α, IL-6, IFN-γ, and IL-17A, in 3D gingival tissue, supernatant from 6-hour time points was measured using a LEGENDplex multianalyte flow assay (catalog number 740808). Cytokine levels are expressed as mean fluorescence units (MFI).

[0240] like Figure 2 As shown, exposure to ATP or LPS led to a significant increase in TNF-α levels in the supernatant, while the addition of NTP enzyme CRC22110-V1 could counteract the accumulation of TNF-α when tissues were treated with ATP and LPS.

[0241] like Figure 3 As shown, exposure to ATP or LPS resulted in a measurable increase in IL-6 levels in the supernatant, while the addition of the NTP enzyme CRC22110-V1 was able to counteract the accumulation of IL-6 when tissues were treated with ATP and LPS.

[0242] like Figure 4 As shown, exposure to ATP or LPS leads to a significant increase in IFN-γ levels in the supernatant, while the addition of NTP enzyme CRC22110-V1 can counteract the accumulation of IFN-γ when tissues are treated with ATP and LPS.

[0243] like Figure 5As shown, exposure to ATP or LPS resulted in a measurable increase in IL-17A levels in the supernatant, while the addition of the NTP enzyme CRC22110-V1 was able to counteract the accumulation of IL-17A when tissues were treated with ATP and LPS.

[0244] To determine the level of extracellular ATP (eATP) after exposure to ATP and LPS, supernatant was collected from tissues incubated as described above, and ATP was measured using the ATP determination kit (catalog number A22066, Invitrogen) according to the manufacturer's protocol. Figure 6 As shown, exposure to ATP or LPS leads to an increase in eATP levels in the supernatant, while the addition of the NTP enzyme CRC22110-V1 can counteract the accumulation of eATP when tissues are treated with ATP and LPS.

[0245] Example 6. Use of exemplary ATP-degrading enzymes in a tissue model of skin inflammation

[0246] In skin inflammation, eATP has been shown to play an important role in the pathogenesis of various conditions, such as psoriasis, atopic dermatitis, and contact dermatitis. This example describes the use of an exemplary enzyme in a 3D psoriasis tissue model.

[0247] In short, the 3D psoriasis tissue model (catalog number SOR 300 FT) was obtained from MatTek, Inc., Massachusetts, USA. The tissue was maintained and used in experiments according to the manufacturer's protocol.

[0248] To determine the role of ATP in skin inflammation, tissues were incubated with 10 mM ATP or LPS (1 μg / mL) from *Porphyromonas gingivalis*, or 10 mM GTP, or 100 nM CRC22110-V1, or 10 mM ATP + LPS (1 μg / mL) + 100 nM CRC22110-V1 at 37°C for 2 h and 6 h. Tissues were collected at 2 h and 24 h for MTT assay to evaluate tissue viability under different conditions according to the manufacturer's protocol. Briefly, tissues were incubated in MTT solution for 3 h and then extracted overnight in the dark at room temperature without shaking. The optical density of the extraction solution was measured at 570 nm using 200 μL of sample.

[0249] As shown in Figure 7, ATP and LPS can react in 6 hours ( Figure 7A ) and 24 hours ( Figure 7B It induces cell death in 3D psoriasis tissue, and the addition of the exemplary NTP enzyme CRC22110-V1 can counteract the inhibitory effects on ATP and LPS activity.

[0250] To determine the role of exemplary enzymes in the expression of ATP- and LPS-induced cytokines, including IL-6, IFN-γ, and IL-17A, in 3D psoriasis tissue, supernatants from 6-hour time points were measured using a LEGENDplex multianalyte flow assay (catalog number 740808). Cytokine levels are expressed as mean fluorescence units (MFI).

[0251] like Figure 8 As shown, exposure to ATP or LPS led to a significant increase in IL-6 levels in the supernatant, while the addition of the NTP enzyme CRC22110-V1 was able to counteract the accumulation of IL-6 when tissues were treated with ATP and LPS.

[0252] like Figure 9 As shown, exposure to ATP or LPS resulted in a measurable increase in IL-17A levels in the supernatant, while the addition of the NTP enzyme CRC22110-V1 was able to counteract the accumulation of IL-17A when tissues were treated with ATP and LPS.

[0253] like Figure 10 As shown, exposure to ATP or LPS leads to a significant increase in IFN-γ levels in the supernatant, while the addition of NTP enzyme CRC22110-V1 can counteract the accumulation of IFN-γ when tissues are treated with ATP and LPS.

[0254] To determine the level of extracellular ATP (eATP) after exposure to ATP and LPS, supernatant was collected from tissues incubated as described above, and ATP was measured at 2, 6, and 24 hours using the ATP determination kit (catalog number A22066, Invitrogen™) according to the manufacturer's protocol. Figure 11A , Figure 11B and Figure 11C As shown, exposure to ATP or LPS led to an increase in eATP levels in the supernatant at 2, 6, and 24 hours, respectively, while the addition of NTP enzyme CRC22110-V1 was able to counteract the accumulation of eATP when tissues were treated with ATP and LPS.

[0255] Example 7. Use of exemplary ATP-degrading enzymes in a skin cell-derived inflammation model

[0256] This example describes the ability of an exemplary enzyme to reverse damage caused by LPS and UV radiation in the human keratinocyte HaCaT skin cell line.

[0257] In summary, HaCaT cells were obtained from Acellerate and cultured in DMEM + GlutaMAX medium (4.5 g / L D-glucose; 100 mg / mL sodium pyruvate) supplemented with 10% premium FBS (complete medium). Cells were cultured in T75 flasks at 37°C in an incubator with 5% CO2 and humidity until 80%-90% confluence. Cells were subcultured using 0.05% trypsin-EDTA, centrifuged, and resuspended in 10 mL of complete medium. New flasks were seeded 1:10 and incubated under the same conditions as above, or cells were seeded into 96-well plates for experiments.

[0258] For the LPS-induced HaCaT cell injury model, 2 x 10 4 One HaCaT cell was seeded in each well of a 96-well plate and incubated for two days in a 37°C incubator with 5% CO2 and humidity. The medium was changed, and 10 μg / mL of LPS from *E. coli* O55:B8 was added to the corresponding well alone or in combination with 1 nM adenosine triphosphate bisphosphatase (Sigma-Aldrich - catalog number A6410), CRC22110 (WT), or AP429. The cells were then incubated overnight in a 37°C incubator with 5% CO2 and humidity. After incubation, the supernatant was collected and stored at -20°C until further use.

[0259] For the UVB-induced HaCaT cell damage model, 2 x 10 4 HaCaT cells were seeded in each of 96 wells and incubated for two days in a 37°C incubator with 5% CO2 and humidity. Prior to exposure, the culture medium was removed and replaced with 50 μL DPBS (calcium-free). Cells were then exposed to 25 mJ / cm². 2 Fresh culture medium with or without 1 μM adenosine triphosphate bisphosphatase was added to the cells. After UVB exposure, the cells were incubated for 4 and 8 hours in a 37°C incubator containing 5% CO2 and humidity. The supernatant was collected at each time point and stored at -20°C until further use.

[0260] Thaw the frozen supernatant from the LPS and UVB-induced HaCaT cell damage model and measure the IL-8 concentration using the BD OptEIA Human ELISA Kit II according to the manufacturer's protocol. Briefly, incubate the supernatant in pre-coated wells at room temperature for 2 hours. Wash the wells 5x with wash buffer and then incubate for 1 hour at room temperature with a mixture of enzyme concentrate and detection antibody. Wash the wells 7x with wash buffer and then develop by adding TMB. Incubate the plate in the dark at room temperature until the standards are fully developed, and then stop the reaction by adding stop solution (1M phosphate). Measure the absorbance at 450 nm using a Spectramax M5 plate reader.

[0261] like Figure 12 As shown, LPS exposure induced the release of IL-8 from HaCat cells, while the addition of potato ATP bisphosphatase, CRC22110 ATP bisphosphatase, or truncated CRC22110-v1 counteracted the accumulation of IL-8 in the supernatant. It should be noted that both CRC22110 and CRC22110-v1 showed significantly higher IL-8 levels compared to potato ATP bisphosphatase.

[0262] like Figure 13A and Figure 13B As shown, UVB exposure induced the release of IL-8 from HaCat cells at 4 and 8 hours, respectively, while the addition of adenosine triphosphate bisphosphatase counteracted the accumulation of IL-8 in the supernatant.

[0263] Example 8. Biochemical characterization of other ATP-degrading enzyme candidates

[0264] Additional samples of the phylogenetic diversification enzymes described in Example 1 were assayed to determine their ability to hydrolyze ATP. Reference enzymes were purchased from Sigma-Aldrich and included potato adenosine triphosphate bisphosphatase (catalog number A6535), recombinant human CD39 His tag (catalog number SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog number P0114).

[0265] Similar to measuring the ability of candidate enzymes to degrade ATP as described in Example 2.

[0266] Using ATP as a substrate, the number of micromoles of phosphate released per milligram of protein per minute of reaction time (µmol / mg / min) is shown in Tables 8 and 9.

[0267]

[0268]

[0269] As shown in Tables 8 and 9, CRC34822 is the most active enzyme at pH 5 among the enzymes tested in this experiment.

[0270] Example 9. Other exemplary uses of ATP-degrading enzymes in tissue models of skin inflammation

[0271] Using a 3D psoriasis tissue model (MatTek Life Sciences, Ashland, MA, catalog number SOR 300 FT), this example describes the use of additional exemplary enzymes in a 3D psoriasis model as generally described in Example 5.

[0272] In summary, tissues were incubated with 10 mM ATP and LPS (1 μg / mL) or 10 mM ATP + LPS (1 μg / mL) + 1 μM CRC25100 or CRC34822 acid phosphatase from *Porphyromonas gingivalis* at 37°C for 2 h and 4 h, respectively. At 4 h, tissues were collected and subjected to 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays to evaluate tissue viability under different conditions according to the manufacturer's protocol. In general, tissues were incubated in MTT solution for 3 h and then extracted overnight in the dark at room temperature without shaking. The optical density of the extraction solution was measured at 570 nm using 200 μL of sample.

[0273] Secreted cytokines were measured using culture supernatant from 2-hour time points using the LEGENDplex multianalyte flow assay (catalog number 740808). Cytokine levels were reported as mean fluorescence units (MFI).

[0274] ATP in culture supernatant was measured using an ATP determination kit (Thermo Fisher Scientific, catalog number A22066).

[0275] like Figure 14A As shown, exposure to ATP and LPS significantly reduced cell viability. The addition of acid phosphatases CRC25100 or CRC34822 could counteract the effects of ATP and LPS. Figure 14B As shown, exposure to ATP or LPS led to a significant increase in IL-6 levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 could counteract the accumulation of IL-6 when tissues were treated with ATP and LPS.

[0276] like Figure 14C As shown, exposure to ATP or LPS led to a significant increase in MCP-1 levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 could counteract the accumulation of IL-17A in tissues treated with ATP and LPS. Figure 14D As shown, exposure to ATP or LPS led to a significant increase in IL-8 levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 was able to counteract the accumulation of IL-8 when tissues were treated with ATP and LPS.

[0277] like Figure 14E As shown, exposure to ATP or LPS resulted in a significant increase in IL-1β levels in the supernatant. The addition of all the acid phosphatases tested was able to counteract the accumulation of IL-1β when tissues were treated with ATP and LPS. Figure 14F As shown, exposure to ATP and LPS led to a significant increase in IL-33 levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 was able to counteract the accumulation of IL-33 when tissues were treated with ATP and LPS.

[0278] Example 10. Use of other exemplary enzymes in a gingival epithelial tissue inflammation model

[0279] This example describes the use of another exemplary enzyme in a 3D gingival epithelial tissue model of oral inflammation. Briefly, the 3D gingival epithelial tissue model (catalog number GIN 100) was obtained from MatTek, Inc., Massachusetts, USA. The tissue was maintained and used in experiments according to the manufacturer's protocol.

[0280] Tissues were incubated with 10 mM ATP and LPS (1 ug / mL, catalog number SMB00610) from *Porphyromonas gingivalis* or 10 mM ATP + LPS (1 ug / mL) + 1 uM CRC25100 or CRC34822 enzyme samples from *Porphyromonas gingivalis*, and the culture supernatant was collected at 2 and 4 hours.

[0281] Tissues were collected after 4 hours and subjected to MTT assays to evaluate tissue viability under different conditions according to the manufacturer's protocol. In summary, tissues were incubated in MTT solution for 3 hours and then extracted overnight at room temperature in the dark without shaking. The optical density of the extraction solution was measured at 570 nm using 200 μL of sample.

[0282] Cytokines were measured using supernatant from 2-hour time points using the LEGENDplex multianalyte flow assay (catalog number 740808). Cytokine levels were reported as mean fluorescence units (MFI).

[0283] Using the ATP assay kit (catalog number A22066) https: / / www.thermofisher.com / order / catalog / product / A22066 )Measure the ATP in the supernatant.

[0284] like Figure 15A As shown, exposure to ATP and LPS significantly reduced cell viability. The addition of acid phosphatases CRC25100 or CRC34822 could counteract the inhibitory effects of ATP and LPS on cell viability. Figure 15B As shown, exposure to ATP or LPS led to a significant increase in IL-8 levels in the culture supernatant. The addition of acid phosphatases CRC25100 or CRC34822 was able to counteract the accumulation of IL-8 when tissues were treated with ATP and LPS.

[0285] like Figure 15C As shown, exposure to ATP or LPS led to a significant increase in IL-18 levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 could counteract the accumulation of IL-18 when tissues were treated with ATP and LPS. Figure 15D As shown, exposure to ATP and LPS led to a significant increase in IFN-γ levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 was able to counteract the accumulation of IFN-γ during tissue treatment with ATP and LPS.

[0286] like Figure 15E As shown, exposure to ATP or LPS led to a significant increase in IL-33 levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 could counteract the accumulation of IL-33 when tissues were treated with ATP and LPS. Figure 15F As shown, exposure to ATP and LPS led to a significant increase in MCP-1 levels in the supernatant. The addition of acid phosphatases CRC25100 or CRC34822 was able to counteract the accumulation of MCP-1 when tissues were treated with ATP and LPS. sequence list

[0287] 。

Claims

1. A composition comprising an ATP-degrading enzyme and at least one oral care component and / or at least one skin care component.

2. The composition of claim 1, wherein the enzyme is active at least at a pH of about 3.5 to pH 9.

3. The composition of claim 1 or claim 2, wherein the enzyme comprises adenosine triphosphate diphosphatase.

4. The composition of claim 1 or claim 2, wherein the enzyme comprises acid phosphatase.

5. The composition of any one of claims 1-3, wherein the enzyme comprises a member of the GDA1_CD39 superfamily and is not a potato ATP bisphosphatase.

6. The composition according to any one of claims 1-5, wherein the enzyme is not a mammalian NTPD enzyme.

7. The composition of any one of claims 1-6, wherein, compared with a reference, the enzyme is capable of reducing the level of one or more inflammatory mediators by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

8. The composition of claim 7, wherein the one or more inflammatory mediators include TNF-α, IL-6, IL17A, IFNγ, IL-8, or a combination thereof.

9. The composition of any one of claims 1-8, wherein the enzyme is capable of reducing cell death levels by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a reference.

10. The composition of any one of claims 1-9, wherein the enzyme comprises SEQ ID NO: 19, SEQ ID NO: 34 (CRC22110), SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 30, SEQ ID NO: 45 and SEQ ID NO: The amino acid sequence of any one of 93 is at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of the polypeptide.

11. The composition of any one of claims 1-10, wherein the enzyme comprises a polypeptide having one or more modifications.

12. The composition of any one of claims 1-11, wherein the enzyme comprises a polypeptide having one or more modifications within the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 34 (CRC22110).

13. The composition of any one of claims 1-11, wherein the enzyme comprises a truncated polypeptide at the C-terminus of SEQ ID NO: 19 or SEQ ID NO: 34 (CRC22110).

14. The composition of any one of claims 1-11, wherein the enzyme comprises a polypeptide having one or more modifications within the amino acid sequence of SEQ ID NO: 93 or SEQ ID NO:

45.

15. The composition of any one of claims 1-14, wherein the oral care component comprises toothpaste, preventative paste, tooth powder, tooth polish, tooth gel, chewing gum, lozenges, mouthwash, oral rinse, whitening strips, patches, suspensions, emulsions, hydrogels, pastes, multiphase solutions, varnish gels, varnishes, finishes, and tubes, syringes, or dental trays containing gels or pastes, or gels or pastes coated on application supports such as dental floss or toothbrushes.

16. The composition of any one of claims 1-15, wherein the skin care component comprises an ointment, serum, hydrogel, solution, dressing, moisturizer, exfoliant, body wash, eye cream, sunscreen, skin cream, cleanser, skin lotion, artificial skin, probiotic skin care component, or spray.

17. The composition of any one of claims 1-16, wherein the skin and / or oral care component comprises a wound care component.

18. A nucleic acid encoding an enzyme as described in any one of claims 1-17.

19. A vector comprising the nucleic acid as described in claim 18.

20. A recombinant host cell comprising the enzyme as described in any one of claims 1-17, the nucleic acid as described in claim 18, and / or the vector as described in claim 19.

21. The cell of claim 20, wherein the cell is a plant cell, a bacterial cell, a fungal cell, or a yeast cell.

22. The cell of claim 20 or claim 21, wherein the cell is a Bacillus cell, an Escherichia coli cell, a Yarrowia cell, an Aspergillus cell, a Pichia cell, or a Trichoderma reesei cell.

23. A method for treating and / or preventing an inflammatory response in a subject, the method comprising administering to the subject an effective amount of the composition as described in any one of claims 1-17.

24. The method of claim 23, wherein the composition is applied to the oral surface of the subject, the oral surface being selected from the lips, cheeks and the lining of the inner lips (buccal mucosa), tongue, upper and lower gingiva, the floor of the mouth under the tongue, the roof of the mouth (soft and hard palate), uvula, tonsils and the area behind the wisdom teeth (posterior trigone of the molars).

25. The method of claim 23 or claim 24, wherein the inflammatory response is related to an oral health condition.

26. The method of claim 25, wherein the oral health condition includes oral health diseases selected from periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and oral mucositis.

27. The method of any one of claims 23-26, wherein the oral health condition includes open sores and / or wounds selected from oral ulcers, cold sores, cuts, bites, burns, surgical incisions, and tissue trauma.

28. The method of any one of claims 23-27, wherein the inflammatory response comprises the release of inflammatory mediators.

29. The method of claim 28, wherein the inflammatory mediator comprises TNF-α, IL-6, IFNγ, IL17A, or a combination thereof.

30. The method of claim 23, wherein the composition is applied to the skin of the subject.

31. The method of claim 23 or claim 30, wherein the inflammatory response is related to a skin condition.

32. The method of any one of claims 23 or 30-31, wherein the skin condition is selected from UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and tissue trauma.

33. The method of any one of claims 23 or 30-31, wherein the inflammatory response includes the release of inflammatory mediators.

34. The method of claim 33, wherein the inflammatory mediator comprises IL6, IL-17A, IFNγ, IL-8, or a combination thereof.

35. A method for reducing the production of inflammatory mediators in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition as described in any one of claims 1-17.

36. The method of claim 35, wherein the inflammatory mediator is associated with oral health conditions.

37. The method of claim 36, wherein the oral health condition includes oral health diseases selected from periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and oral mucositis.

38. The method of claim 36 or claim 37, wherein the oral health condition includes open sores and / or wounds selected from oral ulcers, cold sores, cuts, bites, burns, surgical incisions, and tissue trauma.

39. The method of any one of claims 35-38, wherein the inflammatory mediator comprises TNF-α, IL-6, IFNγ, IL17A, or a combination thereof.

40. The method of claim 35, wherein the inflammatory mediator is associated with a skin condition.

41. The method of claim 40, wherein the skin condition is associated with UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune diseases, cuts, bites, burns, surgical incisions, and tissue trauma.

42. The method of any one of claims 35 or 40-41, wherein the inflammatory mediator comprises IL6, IL-17A, IFNγ, IL-8, or a combination thereof.

43. The method of any one of claims 35-42, wherein the inflammatory mediators are produced by macrophages, dendritic cells, neutrophils or epithelial cells.

44. The method of any one of claims 35-41, wherein the level of inflammatory mediators in the subject is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the level of inflammatory mediators in the subject prior to administration of the composition.

45. The method of any one of claims 23-44, wherein the composition is capable of reducing cell death levels by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the levels of inflammatory mediators in the subject prior to administration of the composition.

46. ​​The method of any one of claims 23-45, wherein the subject is a human being.

47. The method of any one of claims 23-45, wherein the subject is an animal.

48. A method for improving and / or maintaining the oral health of a subject, the method comprising administering to the subject an effective amount of the composition as described in any one of claims 1-17.

49. A method for increasing symbiotic bacteria on the skin and / or in the oral cavity, the method comprising administering to a subject an effective amount of the composition as described in any one of claims 1-17.

50. The method of any one of claims 23-49, wherein the effective amount of the composition comprises the therapeutically effective amount of the composition of any one of claims 1-17.

51. A method for culturing artificial skin, the method comprising culturing epithelial cells in the presence of the composition as described in any one of claims 1-17.

52. The method of claim 51, wherein the epithelial cells are derived from mammals.

53. The method of claim 52, wherein the mammal is a human.

54. A method for promoting wound healing in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition as described in any one of claims 1-17.

55. Use of the composition according to any one of claims 1-17 for promoting wound healing in a subject.

56. A method for restoring the protective skin barrier, moisturizing the skin, and / or reducing skin redness, the method comprising applying the composition of any one of claims 1-17 to human skin.

57. Use of the composition according to any one of claims 1-17 for restoring the protective skin barrier, moisturizing the skin, and / or reducing skin redness.

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