Composition comprising an ATP degrading enzyme and uses thereof

Abstract

Provided herein, inter alia, are compositions and methods for treating and / or preventing conditions associated with skin and / or oral inflammation via use of exogenously administered ATP degrading enzymes such as NTPDase enzymes of the GDA1_CD39 superfamily, or phosphatase enzymes that are capable of functioning along a physiologically relevant pH range.

Description

COMPOSITION COMPRISING AN ATP DEGRADING ENZYME AND USES THEREOF

[0001] This application claims priority to Chinese International Patent Application No.PCT / CN2023 / 119945 filed September 20, 2023, the disclosures of which is incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing submitted in an XML file, in accordance with 37 C.F.R. §§2412 is incorporated herein by reference. The xml file name is “NB42172_WO_PCT_Seq_List.xml”, the date of creation of the xml file is September 12, 2024, and the size of the xml file in bytes is 149,120.FIELD OF THE INVENTION

[0003] Provided herein, inter alia, are compositions and methods for treating and / or preventing conditions associated with skin and / or oral inflammation via use of exogenously administered ATP degrading enzymes (NTPDase enzymes, nucleotidases such as apyrases and members of the GDA1 CD39 superfamily, and phosphatases).BACKGROUND

[0004] The skin and oral mucosa offer the first line of defense against external stimuli through their physical and immunological barrier functions, with commensal microorganisms playing crucial roles in maintenance, homeostasis and immunity. Dysregulation of the barrier 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 tri-phosphates (NTPs) such as extracellular ATP (eATP), and cytokines such as TNF-alpha, IL-ip, 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] Treatment and / or prophylaxis for skin and oral health has historically been driven by the etiology of the dysregulation, with possible negative consequences on maintenance of thehomeostatic balance with the microbiome. Therefore, there is a need for improved methods and compositions that function across a broad spectrum of pathologies by addressing a shared downstream inflammatory process, which could result not only in increased efficiency of product design, but the retention and health of commensal microbiota.

[0006] The subject matter disclosed herein addresses these needs and provides additional benefits as well.SUMMARY

[0007] Provided herein is a composition encompassing 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 from about pH 3.5 to pH 9. In some embodiments, the enzyme is an apyrase. In some embodiments, the enzyme is a member of the GDA1 CD39 superfamily and is not potato apyrase. In some embodiments, the enzyme is not a mammalian NTPDase. In some embodiments, the enzyme is a phosphatase.

[0008] In other embodiments, 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% compared to a reference. In some embodiments, the one or more inflammatory mediators are TNF-alpha, IL-6, IL17A, TFNy, IL-8 or a combination thereof. In some embodiments, the enzyme is capable of reducing the level cell death 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 a reference. 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 any one of 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:93.

[0009] In some of any embodiments, the enzyme is a polypeptide with one or more modifications. In some embodiments, the modification is a truncation. In some embodiments, the enzyme is a polypeptide with one or more modifications within the amino acid sequenceof SEQ ID NO: 19 or SEQ ID NO:34 (CRC22110). In some embodiments, the enzyme is a polypeptide containing a truncation in 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%, or100% identical to the amino acid sequence of SEQ ID NO:51. In some embodiments, the the enzyme is a polypeptide with one or more modifications within the amino acid sequence of SEQ ID NO:93 or SEQ ID NO:45.

[0010] In some of any embodiments, the composition contains an oral care component that is a toothpaste, a prophylactic paste, a tooth powder, a tooth polish, a tooth gel, a chewing gum, a lozenge, a mouthwash, mouth rinse, a whitening strip, patch, suspension, emulsion, hydrogel, paste, multiphase solution, a paint-on gel, varnish, veneer, and tube, syringe or dental tray comprising a gel or paste, or a gel or paste coated on an application support such as dental floss or a toothbrush. In some of any embodiments, the composition contains an skin care component that is an ointment, a serum, hydrogel, solution, dressing, moisturizer, exfoliator, body lotion, eye cream, sunscreen, toner, cleanser, skin wash, artificial skin, probiotic skin care component or spray. In some of any embodiments, the composition contains a skin and / or oral care component that is a wound care component.

[0011] Provided herein is a nucleic acid encoding any of the enzymes described herein.

[0012] Provided herein is a vector containing the nucleic acid of any of the embodiments described herein.

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

[0014] Provided herein is a method of treating and / or preventing an inflammatory response in a subject, wherein the method includes administering to the subject an effective amount of any of the compositions described herein. In some embodiments, the effective amount is a therapeutically effective amount of any of the compositions provided herein. In someembodiments, the composition is applied to the surface of the subject’s oral cavity selected from lips, the lining inside the cheeks and lips (buccal mucosa), the tongue, the upper and lower gums, the floor of the mouth under the tongue, the roof of the mouth (soft palate and hard palate), uvula, tonsils, and the area behind the wisdom teeth (retromolar trigone). 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 canker sores, cold sores, cuts, bite wounds, bums, surgical incisions, and tissue trauma. In some embodiments, the inflammatory response includes the release of inflammatory mediators. In some embodiments, the inflammatory mediators are TNF-alpha, IL-6, IFNy, IL17A or a combination thereof.

[0015] In some of any embodiments, the composition is applied to the skin of the subject. In some embodiments, the inflammatory response is associated with a skin condition. In some of any embodiments, the skin condition is selected from UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, an autoimmune disease, cuts, bite wounds, bums, surgical incisions, and tissue trauma. In some embodiments, the inflammatory response encompasses the release of inflammatory mediators. In some embodiments, the inflammatory mediators are IL-6, IL- 17 A, IFNy, IL-8 or a combination thereof.

[0016] Provided herein is a method of reducing the production of inflammatory mediators in a subject, wherein the method includes administering to the subject an effective amount of any of the compositions described herein. In some embodiments, the effective amount is a therapeutically effective amount of any of the compositions provided herein. In some embodiments, the inflammatory mediators are 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 comprises an open sore and / or wound selected from canker sores, cold sores, cuts, bite wounds, burns, surgical incisions, and tissue trauma. In some of any embodiments, the inflammatory mediators are TNF-alpha, IL-6, IFNy, IL17A or a combination thereof. In some embodiments, the inflammatory mediators are associated with a skin condition. In other embodiments, the skin condition is associated with UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopicdermatitis, autoimmune disease, cuts, bite wounds, bums, surgical incisions, and tissue trauma. In some embodiments, the inflammatory mediators are IL6, IL- 17 A, IFNy, IL-8 or a combination thereof. In some of any embodiments, the inflammatory mediators are produced by a macrophage, dendritic cell, neutrophil or epithelial cell.

[0017] In some embodiments, level of inflammatory mediators 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% in comparison to the level of inflammatory mediators in the subject before administration of the composition. In some embodiments, the composition is capable of reducing the level cell death 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 comparison to the level of inflammatory mediators in the subject before administration of the composition. In some embodiments, the subject is human. In some embodiments, the subject is an animal.

[0018] Provided herein is a method for enhancing and / or maintaining a subject's oral health, which encompasses administering to a subject an effective amount of any of the compositions described herein. In some embodiments, the effective amount is a therapeutically effective amount of any of the compositions provided herein.

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

[0020] Provided herein is a method of culturing artificial skin, comprising culturing epithelial cells in the presence of any one the compositions described herein. In some embodiments, the epithelial cells are from a mammal. In some embodiments, the mammal is a human.

[0021] Provided herein is a method of promoting wound healing in a subject, comprising administering to the subject a therapeutically effective amount of any one the compositions described herein. Also provided herein is the use of any of the compositions provided herein to promote wound healing in a subject.

[0022] Provided herein is a method of restoring a protective skin barrier, moisturizing skin, and / or reducing skin redness comprising applying any one the compositions described herein. to the skin of a human. Provided herein is the use of any one the compositionsdescribed herein for restoring a protective skin barrier, moisturizing skin, and / or reducing skin redness.

[0023] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0024] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 depicts the viability of 3D gingival tissues after addition of ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0026] FIG. 2 depicts the levels of TNF-a in the supernatant of 3D gingival tissues treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0027] FIG. 3 depicts the levels of IL-6 in the supernatant of 3D gingival tissues treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0028] FIG. 4 depicts the levels of ZFN-y in the supernatant of 3D gingival tissues treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0029] FIG. 5 depicts the levels of IL-17A in the supernatant of 3D gingival tissues treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0030] FIG. 6 depicts the levels of eATP in the supernatant of 3D gingival tissues treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0031] FIG. 7A and FIG. 7B depict the viability of a 3D psoriasis tissue model after addition of ATP or LPS at 6 hours (FIG. 7A) or 24 hours (FIG. 7B) in the presence or absence of NTPDase CRC22110-V1.

[0032] FIG. 8 depicts the levels of IL-6 in the supernatant of a 3D psoriasis tissue model treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0033] FIG. 9 depicts the levels of IL-17A in the supernatant of a 3D psoriasis tissue model treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0034] FIG. 10 depicts the levels of IFN-y in the supernatant of a 3D psoriasis tissue model treated with ATP or LPS in the presence or absence of NTPDase CRC22110-V1.

[0035] FIGs. 11A-11C depict levels of eATP in the supernatant of a 3D psoriasis tissue model after addition of ATP or LPS at 2 hours (FIG. 11 A), 6 hours (FIG. 11B) or 24 hours (FIG. 11C) in the presence or absence of NTPDase CRC22110-V1.

[0036] FIG. 12 depicts the levels of IL-8 in the supernatant of HaCaT cells treated LPS in the presence or absence of NTPDases CRC22110, CRC22110-V1, or potato apyrase.

[0037] FIGs. 13A-13B depict the levels of IL-8 in the supernatant of HaCaT cells treated with UV in the presence or absence of potato apyrase at 4hours (FIG. 13A) and 8 hours (FIG. 13B)

[0038] FIGs. 14A-14F depict the viability of a 3D psoriasis tissue model after addition of ATP or LPS in the presence of exemplary ATP degrading enzymes (FIG. 14A), and the levels of IL-6 (FIG. 14B), MCP-1 (FIG. 14C), IL- 17 (FIG. 14D), IL-ip (FIG. 14E), and IL- 33 (FIG. 14F), in the supernatant of a 3D psoriasis tissue model treated with ATP or LPS in the presence of exemplary enzymes.

[0039] FIGs. 15A-15F depict the viability of a 3D epigingival tissue model after addition of ATP or LPS in the presence of exemplary ATP degrading enzymes (FIG. 15A), and the levels of IL-8 (FIG. 15B), IL-18 (FIG. 15C), IFN-y (FIG. 15D), IL-33 (FIG. 15E), and MCP-1 (FIG. 15F), in the supernatant of a 3D epigingival tissue model treated with ATP or LPS in the presence of exemplary enzymes.DETAILED DESCRIPTION

[0040] The inflammatory process is a complex response mediated, in part, by the interplay 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. Vol. 9-2018; Dosch et al. Int. J. Mol Set. 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. The eATP released from damaged or dying cells can then activate the P2X7 receptor on immune cells, such as macrophages and dendritic cells, leading to the production and release of pro-inflammatory cytokines, such as interleukin- ip (IL-ip), tumor necrosis factor-alpha (TNF-a), and interleukin-6 (IL-6) (Zuccarini et al. Int. J. Mol Sci. 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 the health cells and induces cell death and accelerates the process of tissue damage and hinders wound healing.

[0041] In view of the above, targeting eATP presents a potential strategy for treating disorders associated with skin and oral inflammation. Enzymatic removal of extracellular nucleotides (e.g. eATP) may provide a means for reducing inflammatory cytokine secretion and overall downregulation of the inflammatory process in the skin and oral cavity. However, enzyme functionality is often regulated by the pH of the environment, with different tissues having varying pH ranges. Accordingly, the present disclosure relates, at least in part, to compositions and methods for decreasing and / or treating inflammation in the skin and / or oral cavity, through the administration of an ATP degrading enzyme such as NTPDase enzyme of the GDA1 CD39 superfamily, or phosphatase enzyme that is capable of functioning along a physiologically relevant pH range.I. Definitions

[0042] The term “ATP degrading enzyme” is used to mean an enzyme capable of degrading ATP substrate. ATP degrading enzymes can be found in multiple enzyme families, included but not limited to the apyrases (EC 3.6.1.5), NTPDases (EC 3.6.1.5, acid phosphatases (EC 3.1.3.2) and alkaline phosphatases (EC 3.1.3.1).

[0043] The term "acid phosphatase" (EC 3.1.3.2) (also known as acid phosphomonoesterase, phosphomonoesterase, glycerophosphatase, acid monophosphatase, acid phosphohydrolase, acid phosphomonoester hydrolase, uteroferrin, acid nucleoside diphosphate phosphatase, acid phosphatase (class A) or orthophosphoric-monoester phosphohydrolase (acid optimum)) is used to mean an enzyme having a pH optimum for mediating hydrolysis of a phosphate ester bond in a substrate at a pH less than about pH 6.5, such as less than about pH 4.0. Acidphosphatases free attached phosphoryl groups from other molecules during digestion. It can be further classified as a phosphomonoesterase. Acid phosphatase is stored in lysosomes and functions when these fuse with endosomes, which are acidified while they function; therefore, it has an acid pH optimum. 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 enzyme type is present in many microbial, animal and plant species. In some embodiments, the acid phosphatase used in the compositions and methods disclosed herein is not derived from Shigella. In some embodiments, the acid phosphatase used in the compositions and methods disclosed herein is not derived from a mammal.

[0044] As used herein “NTPDases” refer to enzymes also known as ectonucleotidases, Ecto- ATPases and ectonucleoside triphosphate diphosphohydrolase. NTPDases as used herein are capable of hydrolyzing extracellular nucleotides. NTPDases include four major families of ectonucleotidases: CD39 / NTPDases (ecto-nucleotide triphosphate diphosphohydrolases); ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs); alkaline phosphatases; and ecto-5'-nucleotidases / CD73 (See Robson et al. 2006 Purinergic Signalling 2:409-430). In some of any of the embodiments described herein, the NTDPase is an apyrase.

[0045] The term “apyrase,” as used herein, refers to one or more of a calcium-activated enzyme (i.e. proteins belonging to class EC. 3.6.1.5) which possesses ATP- diphosphohydrolase activity and catalyzes the hydrolysis of the gamma phosphate from ATP, and catalyzes the hydrolysis of the beta phosphate from ADP. As used herein, an apyrase can constitute an ATPase, an NTPase, or both. Apyrases are found in all eukaryotes and some prokaryotic organisms, indicating a preserved role for these enzymes across species. They possess a distinct phosphohydrolase activity, nucleotide substrate specificity, divalent cation requirement, and sensitivity to inhibitors. (See, Plesner, Int. Rev. Cytol., 158:141 (1995), and Handa & Guidotti, Biochem. Biophys. Res. Commun., 218(3):916 (1996)). In mammals, apyrase is believed to function primarily as an extracellular hydrolase specific for ATP and ADP, which function is important in the inactivation of synaptic ATP molecules following nerve stimulation. (See, Todorov et al., Nature, 387(6628):76 (1997)). Apyrase in mammals is also believed to be important in the inhibition of ADP-induced platelet aggregation. (See, Marcus et al., J. Clin. Invest., 99(6): 1351 (1997)). Recombinant potato apyrase is commercially available from Sigma-Aldrich. In some embodiments, the apyrase used in the compositions and methods disclosed herein is not derived from potato. In other embodiments,the apyrase used in the compositions and methods disclosed herein is not derived from a mammal.

[0046] As used herein, the “GDA1 CD39 superfamily refers to enzymes comprised of nucleoside triphosphate diphosphohydolases with common motifs in their protein sequences. The family is named after two proteins: the yeast GDPase (GDA1) and a lymphoid cell activation antigen, CD39. These proteins, in some embodiments, are cell-surface enzymes that hydrolyze a range of NTPs, including extracellular ATP. Non-limiting examples include ecto-ATPases, apyrases, CD39s, and ecto-ATP / Dases (Knowles, 2011, Purinergic Signalling volume 7, pages 21-45,' Robson et al. 2006 Purinergic Signalling 2:409-430,' incorporated by reference herein).

[0047] “Mucosa” is a mucus-secreting membrane lining all body cavities or passages that communicate with the exterior. Mucosa is a moist tissue that lines many organs (such as the mouth and intestines) and body cavities (such as nose, mouth, lungs, vagina, bile duct, esophagus) and secretes mucous (a thick fluid). The mucosa, or mucous membrane, is a type of tissue protects body cavities from environmental conditions, pathogens and toxic substances and are usually moist tissues that are bathed by secretions (such as secretions in the bowel, lung, nose, mouth and vagina).

[0048] As used herein, "oral cavity" refers to all interior and exterior components of the mouth, including but not limited to the lips, the lining inside the cheeks and lips (buccal mucosa), the tongue, the upper and lower gums, the floor of the mouth under the tongue, the roof of the mouth (soft palate and hard palate), uvula, tonsils, and the area behind the wisdom teeth (retromolar trigone).

[0049] As used herein, "microorganism" or “microbe” refers to a bacterium, a fungus, a virus, a protozoan, and other microbes or microscopic organisms.

[0050] The term “sequence identity” or “sequence similarity” as used herein, means that two polynucleotide sequences, a candidate sequence and a reference sequence, are identical (i.e. 100% sequence identity) or similar i.e. on a nucleotide-by-nucleotide basis) over the length of the candidate sequence. In comparing a candidate sequence to a reference sequence, the candidate sequence may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for determining sequence identity may beconducted using the any number of publicly available local alignment algorithms known in the art such as ALIGN or Megalign (DNASTAR), or by inspection.

[0051] The term “percent (%) sequence identity” or “percent (%) sequence similarity,” as used herein with respect to a reference sequence is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the residues in the reference polynucleotide sequence after optimal alignment of the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0052] As used herein with regard to amino acid residue positions, “corresponding to” or “corresponds to” or “correspond to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.

[0053] As used herein, “prevent,” “preventing,” “prevention” and grammatical variations thereof refers to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or condition and / or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing an subject's risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.

[0054] As used herein, the term “reducing” “or “decreasing” in relation to a particular trait, characteristic, feature, biological process, or phenomena refers to a decrease in the particular trait, characteristic, feature, biological process, or phenomena. The trait, characteristic, feature, biological process, or phenomena can be decreased 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%.

[0055] As used herein, the term “subject” or “patient” is meant a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition including, but not limited to, an oral health disease selected from periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and oral mucositis; an open sore and / or wound selected from canker sores, cold sores, cuts, bite wounds, burns, surgical incisions, and tissue trauma; or a skin condition is selected from UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, an autoimmune disease, cuts, bite wounds, bums, surgical incisions, and tissue trauma. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays oneor more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0056] As used herein “administer” or “administering” is meant the action of introducing one or more microbial strain, an exogenous feed enzyme and / or a strain and an exogenous feed enzyme to a subject, such as by feeding or by gavage.

[0057] As used herein, “effective amount” or “therapeutically effective amount” means a quantity of exogenous enzyme(s)s to improve one or more metrics in a subject. Improvement in one or more metrics of a subject (such as, without limitation, oral health status, skin health status, improved barrier integrity, reduced mortality, or reduced pathogen infection) can be measured as described herein or by other methods known in the art. The exogenous enzymes can also be administered in one or more doses.

[0058] As used herein, 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, the lining inside the cheeks and lips (buccal mucosa), the tongue, the upper and lower gums, the floor of the mouth under the tongue, the roof of the mouth (soft palate and hard palate), uvula, tonsils, and the area behind the wisdom teeth (retromolar trigone).

[0059] As used herein, the term “oral health condition” encompasses human and animal conditions, disorders, or diseases affecting the mouth, lips and / or oral cavity. Such oral conditions include “dental conditions,” which are oral conditions affecting the teeth and gums in particular. Non-limiting examples of oral conditions include infections, biofilms and / or plaques; mouth sores (including ulcers, cold sores and canker sores); stained teeth; as well as conditions that these oral health conditions may cause, e.g., oral cancers, tooth sensitivity, tooth decay and / or loss, cracked teeth, thrush, gum disease, gingivitis, periodontal disease, caries, halitosis, and / or inflammatory conditions, such as, e.g., tonsillitis, pharyngitis, laryngitis, glossitis, and stomatitis.

[0060] As used herein, “skin” refers to the organ of the body that covers the entire external surface. The term skin as used herein encompasses three layers, including the epidermis, the dermis and the hypodermis.

[0061] As used herein, the term “skin condition” encompasses human and animal conditions, disorders or diseases affecting the skin. Such skin conditions include UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, an autoimmune disease, cuts, bite wounds, burns, surgical incisions, and tissue trauma.

[0062] The term “commensal bacteria” refers to microflora (normal microflora, indigenous microbiota) consisting of those micro-organisms which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). These bacteria have been shown to impact the regulation of immunophysiological functions, including but not limited to, metabolism, ontogeny, and pathogen defense. Commensal bacteria have also been shown to promote resistance to skin and oral cavity pathogens that is mutually beneficial to the host and the commensal microbiota (Byrd et al. 2018. Nature Reviews Microbiology, 16:143-155; Kilian et al. 2016, British Dental Journal 221:657-666, incorporated by reference herein).

[0063] The term “probiotic” refers to live or viable microorganisms which, when administered in effective amounts to a subject, confer a health benefit on the subject. For example, the probiotics included in the probiotic formulations described herein, which are suitable for oral or topical administration to subjects, may improve the subjects' skin and / or oral health. The term “probiotic,” as used herein, encompasses live microorganisms as well as viable microorganisms in dormant states, including frozen microorganisms, desiccated or dried microorganisms, spores, cysts or microorganisms in various states of reduced metabolic activity, which can be reconstituted upon exposure to suitable conditions. In some embodiments, the microorganisms of the probiotic comprise or consist of one or more commensal bacteria. In other embodiments, probiotic the microorganisms of the probiotic are derived from a fecal microbial source (such as fecal microbial source obtained from an otherwise healthy subject). Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. Administration of non- viable bacterial compositions is also contemplated in certain embodiments of the methods disclosed herein.

[0064] As used herein, the term “engraftmenf ’ refers to the colonization of one or more bacterial species (such as commensal bacterial species provided, for example, as a probiotic) within the skin and / or oral cavity or to the adherence of one or more bacterial species (such as commensal bacterial species provided, for example, as a probiotic) onto the skin and / or oral cavity epithelial cells in a subject.

[0065] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of - 10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0066] As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0067] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0068] It is also noted that the term “consisting essentially of,” as used herein refers to a composition wherein the component(s) after the term is in the presence of other known component(s) in a total amount that is less than 30% by weight of the total composition and do not contribute to or interferes with the actions or activities of the component(s).

[0069] It is further noted that the term "comprising,” as used herein, means including, but not limited to, the component s) after the term “comprising.” The component(s) after the term “comprising” are required or mandatory, but the composition comprising the component(s) can further include other non-mandatory or optional component(s).

[0070] It is also noted that the term “consisting of,” as used herein, means including, and limited to, the component(s) after the term "consisting of.” The component(s) after the term “consisting of’ are therefore required or mandatory, and no other component(s) are present in the composition.

[0071] The terms “protein” and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and may be used interchangeably. A “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues. The single and 3-letter code foramino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. The single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Amino acid positions in a given polypeptide sequence can be named by the one letter code for the amino acid, followed by a position number. For example, a glycine (G) at position 87 is represented as “G087” or “G87.”

[0072] As used herein, where “amino acid sequence” is recited it refers to an 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 “polypeptide” or “protein” are not meant to limit the amino acid sequence to the deduced amino acid sequence but can include posttranslational modifications of the deduced amino acid sequences, such as amino acid deletions, additions, and modifications such as glycosylations and addition of lipid moieties. Also, 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”, unless indicated otherwise.

[0073] The terms “signal sequence” and “signal peptide” refer to a sequence of amino acid residues that may participate in the secretion or direct transport of the mature or precursor form of a protein. The signal sequence is typically located N-terminal to the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. A signal sequence is normally absent from the mature protein. A signal sequence is typically cleaved from the protein by a signal peptidase after the protein is secreted.

[0074] The term “mature” form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or enzyme without the signal peptide sequence and or a propeptide sequence.

[0075] The term “wild-type” in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term “naturally-occurring” refers to anything (e.g, proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term “non-naturally occurring” refers to anything that is not found in nature (e.g,recombinant / engineered nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

[0076] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0077] Unless defined otherwise 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.

[0078] Other definitions of terms may appear throughout the specification.II. CompositionsA. Enzymes

[0079] Provided herein are compositions comprising ATP degrading enzymes such as NTPDase enzymes and phosphatase enzymes. In some embodiments, the phosphatase enzymes are acid phosphatases or alkaline phosphatases. NTPDases, also known as ectonucleotidases, Ecto-ATPases and ectonucleoside triphosphate diphosphohydrolase, are ectoenzymes that hydrolyze extracellular nucleotides. There are four major families of ectonucleotidases: CD39 / NTPDases (ecto-nucleotide triphosphate diphosphohydrolases); ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs); alkaline phosphatases; and ecto-5'-nucleotidases / CD73.

[0080] In some embodiments, any of the compositions provided herein are capable of removing phosphatases from ATP. In some embodiments, any of the compositions provided herein are capable of removing phosphatases form NTP. In some embodiments, the composition is an apyrase and is capable of removing phosphates from ATP and / or NTP. In some embodiments, the composition is an NTPDase and is capable of removing phosphates from ATP and / or NTP. In some embodiments, the composition is an acid phosphatase and iscapable of removing phosphates from ATP and / or NTP. In some embodiments, the composition is an alkaline phosphatase and is capable of removing phosphates from ATP and / or NTP.

[0081] In some embodiments, any of the compositions provided herein are capable of breaking the bond between a sugar and a purine in ATP. In some embodiments, the composition is a purine nucleoside phosphorylase (PNP) and is capable of breaking the bond between a sugar and a purine in ATP.

[0082] NTPDases are constitutively expressed in many tissues and can be differentiated on the basis of cellular location. In some embodiments, the NTPDases provided herein comprise NTPDases 1, 2, 3 and 8, located on the cell surface; In some embodiments, the NTPDases provided herein comprise 5 and 6 and are localized within the cell and undergo secretion after heterologous expression. In some embodiments, the NTPDases provided herein comprise 4 and 7 and are entirely intracellularly located and face the lumen of cytoplasmic organelles. NTPDases located at the cell surface require Ca2+ or Mg2+ ions for activity. These subtypes contribute to the recycling of nucleosides from salvage pathways and extracellular nucleoside phosphates.

[0083] Due to their role in nucleotide metabolism, NTPDases help control the availability of extracellular nucleotide agonists at P2 receptors and modulate P2 receptor function as a result. The distribution of P2 receptors throughout the body, and the wide-spread nature of purinergic signaling, suggests an important role for NTPDases in a range of cellular processes.

[0084] Location, substrate preference and hydrolysis rates vary between subtypes; however, all NTPDases contain five highly conserved sequence domains, known as apyrase conserved regions (APCR1 to APCR5). In some of any of the embodiments provided herein, the enzyme has phosphatase activity. A phosphatase is an enzyme that dephosphorylates its substrates. Enzymes exhibiting phosphatase activity include: apyrases (also known as NTPDases, ATP-diphosphohydrolases (EC 3.6.1.5)), inorganic pyrophosphatases (EC 3.6.1.1,) acid phosphatase (EC 3.1.3.2), alkaline phosphatases (EC 3.1.3.1), trimetaphosphatases (EC 3.6.1.2) and exopolyphosphatases (EC 3.6.1.11). This phosphatase activity action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their substrates by using energetic molecules like ATP. In some of anyembodiments, any of the ATP degrading enzymes are capable of hydrolyzing ATP, ADP, CTP, CDP, UTP, UDP, GTP, and / or GDP.

[0085] The enzymes for use in the compositions and methods disclosed herein are active at least from about pH 3 to pH 9 (such as any of about 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 from pH 3.5 to 5.5 or from pH 3.5 to 7 or from pH 5 to 7. The enzyme can be a member of the GDA1 CD39 superfamily. In some embodiments, the enzyme can be a member of the GDA1 CD39 superfamily (excluding apyrases derived from potato). In some embodiments, the enzyme can be a member of the phosphatase enzyme class. In some embodiments, the enzyme has activity from pH 3 to pH 9. In other embodiments, the enzyme can be an acid phosphatase. In some embodiments, the enzyme comprises an amino acid sequence having at least 35% or at least 60% sequence identity (such as any 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) with the amino acid sequence ofSEQ ID NO: 19 or SEQ ID NO:34 (CRC22110). In other embodiments, the enzyme comprises an amino acid sequence having at least 50% or at least 66% sequence identity (such as any 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) with any of the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO:53.

[0086] Other enzymes for use in the compositions and methods disclosed herein are active at least from about pH 3 to pH 9 (such as any of about 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 from pH 3.5 to 5.5 or from pH 3.5 to 7 or from pH 5 to 7 and are a member of the GDA1 CD39 superfamily (excluding apyrases derived from potato) or the phosphatase enzyme class. In some embodiments, the enzyme comprises an aminoacid sequence having at least 35% or at least 60% sequence identity (such as any 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) with the predicted mature amino acid sequence of one or more 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, or SEQ ID NO: 45. In some of any embodiments, any of the sequences provided herein further comprises a signal sequence.

[0087] In some embodiments, the enzyme comprises a nucleic acid sequence having at least 35% or at least 60% sequence identity (such as any 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) with any of SEQ ID NOs: l-15 and 64-76. In some of any embodiments, any of the sequences provided herein further comprises a signal sequence.

[0088] In further embodiments, the enzyme comprises an amino acid sequence having a signal sequence and having at least 35% or at least 60% sequence identity (such as any 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) with the amino acid sequence of one or more of SEQ ID Nos: 16-30.

[0089] In further embodiments, the enzyme comprises an amino acid sequence having a signal sequence and having at least 35% or at least 60% sequence identity (such as any 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) with the full length amino acid sequence of one or more of SEQ ID Nos:77-102.

[0090] Provided herein are compositions comprising ATP degrading enzymes containing one or more modifications. In some embodiments, the modification comprises a deletion, insertion or substitution. In some embodiments, the modification comprises a truncation. In some embodiments, the truncation is of the N-terminus. In some embodiments, the truncation is of the C-terminus. In some embodiments, the modification comprises an insertion. In some embodiments, the insertion is on the N-terminus. In some embodiments, the insertion is on the C-terminus.

[0091] In some of any embodiments, any of the enzymes provided herein comprise a truncation in the C-terminus of an amino acid having the amino acid sequence of one or more 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 NOs: 90-102. In some of any embodiments, any of the enzymes provided herein comprise a truncation in the C-terminus of a polypeptide having the full-length amino acid sequence of one or more of SEQ ID NOs: 16-30 or SEQ ID NO:77-89.

[0092] In some embodiments, any of the enzymes provided herein comprises an amino acid sequence having at least 35% or at least 60% sequence identity (such as any 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) with the amino acid sequence of one or more of SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.B. Vectors

[0093] The terms “nucleic acid,” “ polynucleotide”, and “nucleic acid fragment” are used interchangeably herein and refer to a polymer of RNA or DNA that is single- or doublestranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolatednucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0094] A DNA construct comprising a nucleic acid encoding a phosphatase polypeptide disclosed herein (such as a nucleic acid comprising any of SEQ ID NOs:l-15) can be constructed such that it is suitable to be expressed in a host cell. Because of the known degeneracy in the genetic code, different polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also known that, depending on the desired host cells, codon optimization may be required prior to attempting expression.

[0095] A polynucleotide encoding a phosphatase polypeptide of the present disclosure can be incorporated into a vector. Vectors can be transferred to a host cell using known transformation techniques, such as those disclosed below.

[0096] A suitable vector may be one that can be transformed into and / or replicated within a host cell. For example, a vector comprising a nucleic acid encoding an 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 may also be suitably transformed into an expression host, such that the encoding polynucleotide is expressed as a functional phosphatase enzyme.

[0097] A non-limiting representative useful vector is pTrex3gM (see, Published US Patent Application 20130323798), pTTT (see, Published US Patent Application 20110020899), and p2JM103BBI (see Vogtentanz, Protein Expr Purif 55:40-52, 2007), which can be inserted into genome of host. The vectors pTrex3gM, pTTT, and p2JM103BBI can be modified with routine skill such that they comprise and express a polynucleotide encoding a phosphatase polypeptide of the invention.

[0098] An expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the phosphatase polypeptide to a host cell organelle such as a peroxisome, or to a particular host cell compartment. For expression under the direction of control sequences, the nucleic acid sequence of the phosphatase polypeptide is operably linked to the control sequences in proper manner with respect to expression.

[0099] For expression and production of a protein of interest (e.g., an ATP degrading enzyme described herein) in a cell, one or more expression vector or expression cassette comprising one or more copy of a polynucleotide encoding one or more enzymes described herein, and in some instances comprising multiple copies, is transformed into the cell under conditions suitable for expression of the variant. In some embodiments, a polynucleotide sequence encoding one or more ATP degrading enzyme described herein (as well as other sequences included in the vector) is integrated into the genome of the host cell, while in other embodiments, a plasmid vector comprising a polynucleotide sequence encoding one or more ATP degrading enzymes described herein remains as autonomous extra-chromosomal element within the cell. Some embodiments provide both extrachromosomal nucleic acid elements as well as incoming nucleotide sequences that are integrated into the host cell genome. The vectors described herein are useful for production of the one or more ATP degrading enzyme described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variant described herein is present on an integrating vector that enables the integration and optionally the amplification of the polynucleotide encoding the variant into the host chromosome. Examples of sites for integration are well known to those skilled in the art. In some embodiments, transcription of a polynucleotide encoding one or more ATP degrading enzymes described herein is effectuated by a promoter that is the wild-type promoter for the parent ATP degrading enzyme. In some other embodiments, the promoter is heterologous to the one or more ATP degrading enzyme described herein, but is functional in the host cell. Exemplary promoters for use in bacterial host cells include, but are not limited to the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpall promoters; the promoter of the B. stearothermophilus maltogenic amylase gene; the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene; the B. pumilis xylosidase gene; the B. thuringiensis crylllA; and the B. licheniformis alpha-amylase gene. Additional promoters include, but are not limited to the A4 promoter, as well as phage Lambda PR or PL promoters and the E. coli lac, trp or tac promoters, and the Bacillus rrn promoters such as rrnl, rrnB, rrnL and rrnE ribosomal RNA promoters and variants thereof.

[0100] A polynucleotide encoding a phosphatase polypeptide disclosed herein can be operably linked to a promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.Examples of promoters for directing the transcription of the DNA sequence encoding an phosphatase, for example in a bacterial, fungal, or mammalian host, include the aprE promoter (SEQ ID NO: 46), the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or cel A promoters, the promoters of the Bacillus licheniformis amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens amylase (amyQ), the promoters of the Bacillus subtilis xyl A and xylB genes, and the like.

[0101] For transcription in a fungal host, examples of useful promoters include those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, Aspergillus niger acid stable a-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase and the like. When a gene encoding a phosphatase polypeptide is expressed in a bacterial species such as an E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Along these lines, examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris A0X1 or A0X2 promoters. Expression in filamentous fungal host cells often involves cbhl, which is an endogenous, inducible promoter from T. reesei. See Liu et al.(2008) Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0102] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be a DNA sequence naturally associated with the phosphatase polypeptide gene of interest to be expressed or may be from a different genus or species from which a portion of the phosphatase is derived. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence may be the Trichoderma reesei cbhl signal sequence, which is operably linked to a cbhl promoter.

[0103] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a phosphatase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0104] The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xx.sf’, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., Published International PCT Application WO 91 / 17243.

[0105] Synthetic genes coding for the protein sequences corresponding to SEQ ID NOs. 1-15 can be generated using molecular biology methods known in the art. These genes can further be cloned into a suitable expression vector resulting in expression plasmids containing one or more of: a promoter (for example, the aprE promoter (SEQ ID NO: 46)); a signal sequence (for example, the aprE signal sequence (SEQ ID NO. 47) encoding a peptide of SEQ ID NO: 48); an oligonucleotide to facilitate the secretion of the target protein (for example, an oligonucleotide encoding the peptide Ala-Gly-Lys); a synthetic nucleotide sequence encoding the mature region of the gene of interest (for example, a synthetic oligonucleotide having at least 50% or at least 66% sequence identity (such as any 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) with the synthetic oligonucleotide of any of SEQ ID NOs: 1-15 or SEQ ID NOs: 64-76); and / or a terminator (such as the lat terminator (SEQ ID NO. 49)). In some embodiments, any of the oligonucleotides or encoded proteins provided herein can contain a C-terminal His tag. The expression plasmid can further be transformed into a suitable expression host cell (such as a mammalian or bacterial or fungal expression host cell).C. Host Cells

[0106] An isolated cell, either comprising a DNA construct (such as any of the DNA constructs disclosed herein) or an expression vector (such as any of the expression vectors disclosed herein, for example, an expression vector comprising any of the polynucleotides of SEQ ID NOs: 1-15 or SEQ ID NOs: 64-76 that encodes a polypeptide of any one of SEQ ID NOs: 16-45, SEQ ID NOs: 51-53 or SEQ ID NOs: 77-102), is advantageously used as a host cell in the recombinant production of a phosphatase polypeptide. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating theDNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector in connection with the different types of host cells.

[0107] Examples of suitable bacterial host cells are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus') stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis: Streptomyces species such as Streptomyces murinus: lactic acid bacterial species including Lactococcus sp. such as Lactococcus laclis Lactobacillus sp. including Lactobacillus reuteri,' Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.

[0108] A suitable yeast host cell can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species.

[0109] Suitable host cells among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces avA Mucor species. In addition, Trichoderma sp. such as Trichoderma ressei can be used as a host. A phosphatase polypeptide expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type phosphatase. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties.

[0110] Suitable mammalian host cells include, without limitation, Chinese hamster ovary (CHO) cells or human embryonic kidney HEK) cells. Additional host cells can include insect sells, such as S2 cells.

[0111] It is advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2, egll, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0112] General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding a phosphatase is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.

[0113] A method of producing any of the enzymes disclosed herein may comprise cultivating a host cell under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0114] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell and obtaining expression of a polypeptide. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0115] Any of the fermentation methods well known in the art can suitably be used to ferment the transformed or the derivative fungal strain as described above. In some embodiments, fungal cells are grown under batch or continuous fermentation conditions.

[0116] Separation and concentration techniques are known in the art and conventional methods can be used to prepare a concentrated solution or broth comprising a polypeptide of the invention.

[0117] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain a solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.

[0118] It may at times be desirable to concentrate a solution or broth containing the polypeptide to optimize recovery. Use of un-concentrated solutions or broth would typically increase incubation time in order to collect the enriched or purified enzyme precipitate.D. Enzyme Compositions

[0119] Any of the enzymes for use in the methods disclosed herein can be formulated in compositions (for example, pharmaceutical or nutritional compositions). As already mentioned, any of the enzyme compositions described herein can be useful in treating and / or preventing diseases associated with inflammation on the skin and the oral cavity. In one embodiment, the composition is preferably a pharmaceutical composition, comprising an enzyme according to the disclosure. Said pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the composition is not a pharmaceutical composition, and is sold to reduce the risk of disease.

[0120] In some embodiments, the composition includes between InM to 100 nM of any of the enzymes described herein, or any ranges therebetween. In some embodiments, the composition includes between 1 pM to InM of any of the enzymes described herein or any ranges therebetween. In some embodiments, the composition includes between lOOnM to 1 pM of any of the enzymes described herein or any ranges therebetween. In some embodiments, any of the compositions described herein contain about InM, 5nM, lOnM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM or lOOnM of any of the enzymes described herein or any ranges therebetween.

[0121] In some embodiments, the composition is in a form such as liquids, solids, semisolids or a combination thereof. The composition can be a complete oral nutritional supplement. The composition can also be in a form such as lozenges, lollipops, sachets, dissolvable films or a combination thereof.

[0122] In some embodiments, the composition is in a form such as a food, a beverage andcombinations thereof. The composition can include an ingredient such as a thickener. In an alternative embodiment, the composition is a topical compound that can be applied to the surface of the oral cavity. In an alternative embodiment, the composition is a topical compound that can be applied to the surface of the skin.

[0123] In some embodiments, the composition may include at least a portion of or be an oral care composition. Illustrative oral care compositions may include, but are not limited to, a toothpaste (dentifrice), a prophylactic paste, a tooth powder, a tooth polish, a tooth gel (e.g., whitening gel), a chewing gum, a lozenge, a mouthwash, mouth rinse, a whitening strip, patch, suspension, emulsion, hydrogel, paste, multiphase solution, a paint-on gel, varnish, veneer, and tube, syringe or dental tray comprising a gel or paste, liquid, powder, or a gel or paste coated on an application support such as dental floss or a toothbrush (e.g., a manual, electric, sound, a combination thereof or ultrasound toothbrush). In some embodiments, the oral care composition comprises a combination of one or more oral care compositions.

[0124] In some embodiments, the oral care composition may include humectants, surface active agents, gelling agents, and the like, and combinations thereof. Illustrative humectants may be or include, but are not limited to, glycerin, propylene glycol, and the like, and combinations thereof. In some embodiments, the humectant is present in an amount of from about 20 wt% to about 60 wt% based on a total weight of the oral care product. In at least one embodiment, the oral care product and / or the oral care whitening composition thereof is free or substantially free of polyol humectants. For example, the oral care composition does not contain any polyols as a humectant. In another implementation, the propylene glycol is present in an amount of from about 10 wt% to about 20 wt% based on a total weight of the oral care composition. In another embodiment, the glycerin is present in an amount of from about 25 wt% to about 40 wt% based on a total weight of the oral care composition.

[0125] Alternatively, the oral care composition may be may be a liquid, gel, paste or the like which may be applied as a coating to a non-consumable product, such as a toy for an animal. The oral care composition may be may be incorporated within the product. When the animal chews the toy, the composition comes into contact with some or all of the oral cavity of the animal and improves or maintains the oral health of the animal.

[0126] In some of any embodiments, the oral care composition may be a gel composition with the following ingredients: thickeners, moisteners, such as sorbitol, glycerin, polyethylene glycol and / or mixtures thereof, binders such as carboxymethyl cellulose,hydroxyethyl cellulose, xanthan gum, silica, thickener and / or mixtures thereof, conservatives (for example, methylparaben, propylparaben or mixtures thereof), acidifiers (typically phosphoric acid, citric acid, tartaric acid, maleic acid), essence (mint, etc.), flavoring agents (sodium saccharine or others).

[0127] 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, but are not limited to, colloidal silica, fumed silica, a cross-linked polyvinylpyrrolidone (PVP) polymer, crosslinked polyvinylpyrrolidone (PVP), and the like, and mixtures or combinations thereof. In a typical implementation, the thickening system includes a cross-linked polyvinylpyrrolidone (PVP) polymer. In a more typical implementation, the thickening system may be or include, but is not limited to, POLYPLASDONE™ XL- 10, which is commercially available from Ashland Inc. of Covington, KY.

[0128] The composition can be presented in any form, for example as a tablet, as an injectable fluid or as an infusion fluid etc. Moreover, the composition, protein, nucleotide and / or vector according to the invention can be administered via different routes, for example bronchially, topically, or orally.

[0129] In some embodiments, the skin care component may contain an ointment, a serum, hydrogel, solution, dressing, moisturizer, exfoliator, body lotion, eye cream, sunscreen, toner, cleanser, skin wash, artificial skin, probiotic skin care component or spray.

[0130] 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 undue toxicity, incompatibility, instability, allergic response, and the like.

[0131] In some embodiments, any of the skin care components or compositions herein are configured for topical application to keratinous tissue. In some embodiments, any of the skin care components or compositions may include dimethicone and a particulate material as well as a water-soluble UV-blocking active at specific amounts, and such compositions may provide the desired feels including non-greasy, non-sticky (indicated by Mean Break Time), non-shiny, refreshing and easy to spread (indicated by dynamic viscosity). In some embodiments, any of the skin care components or compositions provided herein are UV- blocking compositions or sunscreen compositions.

[0132] In some embodiments, any of the skin care components or compositions herein may comprise 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 from about 3.5% to about 40%, more preferably from about 4% to about 30%, yet more preferably from about 4.5% to about 20%, and most preferably from about 4.5% to about 10%, for example 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 ranges therebetween, by weight of the composition, of a particulate material (also called cosmetic powder or dry powder). Non-limiting examples of suitable particulate materials include inorganic powders, organic powders, composite powders, optical brightener particles, and mixtures of any of the foregoing. These particulates can, for instance, be platelet shaped, spherical, elongated or 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 oxides, titanium dioxides, zinc oxides and any mixtures thereof.

[0133] The present composition may include a variety of optional ingredients that are known for use in personal care composition, as long as the optional ingredient(s) do not unduly alter product stability, aesthetics, or performance. The optional ingredients, when incorporated into the composition, should be suitable for contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like within the scope of sound judgment. The compositions herein may comprise from about 0.0001% to about 50%; from about 0.001% to about 20%; or, alternately, from about 0.01% to about 10%, by weight of the composition, of optional ingredients. Some non-limiting examples of optional ingredients include abrasives, absorbents, opacifying agents, colorings / colorants (e.g., pigments, dyes, and lakes), particles, essential oils, anti-caking agents, foaming agents, anti-foaming agents, oil control agents, binders, biological additives, vitamins, minerals, peptides, sugar amines, flavonoid compounds, anti-oxidants, preservatives, plant extract, phytosterols, protease inhibitors, tyrosinase inhibitors, exfoliating agents, skin lightening agents, sunless tanning agents, anti-acne actives, anti-cellulite actives, anti-wrinkle actives, phytosterols and / or plant hormones, N-acyl amino acid compounds, antimicrobials, antifungals, moisturizers, emollients, humectants, lubricating agents, fragrances, anti-dandruff agents, buffering agents, bulking agents, chelating agents, biocides, denaturants, astringents, external analgesics, antiinflammatory agents, sunscreen agents, film formers and / or polymers for aiding film-formingproperties and substantivity of the composition, propellants, reducing agents, sequestrants, conditioning agents, and combinations of these.

[0134] In some embodiments, any of the compositions provided herein can optionally comprise pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeators, propellants, disinfectants, diluents and preservatives. Suitable excipients are commonly known in the art of pharmaceutical formulation and may be readily found and applied by the skilled artisan, references for instance Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia Pa., 17th ed. 1985.

[0135] For oral administration, the protein can, for example, be administered in solid dosage forms, such as capsules, tablets (e.g., with an enteric coating), and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. A In some embodiments, any of the enzymes provided herein (e.g. apyrases) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulphate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0136] Enteric coatings arrest the release of the active compound from orally ingestible dosage forms. Depending upon the composition and / or thickness, the enteric coatings are resistant to stomach acid for required periods of time before they begin to disintegrate and permit slow release of ATP degrading enzymes in the lower stomach, small intestines, or large intestine. Examples of some enteric coatings are disclosed in U.S. Pat. No. 5,225,202 (incorporated by reference). Examples of enteric coatings comprise beeswax and glyceryl monostearate; beeswax, shellac and cellulose, optionally with neutral copolymer of polymethacrylicacid esters; copolymers of methacrylic acid and methacrylic acid methylesters or neutral copolymer of polymethacrylic acid esters containing metallic stearates(for references enteric coatings see: U.S. Pat. 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 coating polymers begin to become soluble at pH 5.5 and above, with a maximum solubility rates at pH above 6.5. Enteric coatings may also comprise subcoating and outer coating steps, for instance for pharmaceutical compositions intended for specific delivery in the lower GI tract, i.e. in the colon (pH 6.4 to 7.0, ileum pH 6.6), as opposed to a pH in the upper intestines, in the duodenum of the small intestines the pH ranges 7.7-8 (after pancreatic juices and bile addition). The pH differences in the intestines may be exploited to target the enteric-coated ATP degrading enzyme composition to a specific area in the gut. It also allows the selection of a specific ATP degrading enzyme that is most active at a particular pH in the intestine.

[0137] In addition, the pH differences on the skin and oral cavity may be exploited to target the ATP degrading enzyme composition to a specific area on the skin and / or oral cavity. It also allows the selection of a specific ATP degrading enzyme that is most active at a particular pH on the skin and / or oral cavity. For example, the mean value of the pH on the skin lies in the range of 4-7, with an average of 4.7 (Lambers et al. 2006. Int J Cosmet Sci. 28(5): 359-70), although the parameter "skin pH" depends mainly on the area of skin, but it also depends to a lesser extent on sex, race and the time of day when values are determined. In addition, the mean pH value saliva has a pH normal range of 6.2-7.6 with 6.7 being the average pH. Resting pH of mouth does not fall below 6.3. In the oral cavity, the pH is maintained near neutrality (6.7-7.3) by saliva (Baliga et al. 2013. J Indian Soc Periodontol;17(4):461-465).

[0138] Besides the fact that a protein according to the invention can be incorporated in a pharmaceutical composition such an ATP degrading enzyme can also be part of a nutritional composition or a nutraceutical.

[0139] A protein according to the invention can be added to a nutrient (such as milk) but can also be produced within said nutrient (for instance by molecular engineering). Moreover, tablets and / or capsules can be prepared which are subsequently added to a nutrient or which can be taken directly by a human being.

[0140] In yet another aspect, the invention features beverage and food products comprising an ATP degrading enzyme effective to treat or prevent skin and / or oral inflammation in a subject in need thereof. The beverage products can contain from 1 to 10,000, e.g., 1 to 200, 200 to 500, 500 to 1,000, 1,000 to 5,000, or 5,000 to 10,000, units per milliliter. The foodproducts can contain from 1 to 10,000, e.g., 1 to 200, 200 to 500, 500 to 1,000, 1,000 to 5,000, or 5,000 to 10,000, units per gram.E. Inflammatory MediatorsOral Cavity

[0141] In oral inflammation, eATP can be released from damaged or dying cells, such as epithelial cells, immune cells, and nerve cells. eATP can then activate the P2X7 receptor on immune cells, such as macrophages and dendritic cells, leading to the production and release of inflammatory mediators, such as cytokines including interleukin- ip (IL-ip), tumor necrosis factor-alpha (TNF-a), and interleukin-6 (IL-6). 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 the health cells and induces cell death and accelerates the process of tissue damage and hinders wound healing (Zuccarini et al. Int. J. Mol. Sci. 2022, 23, 7790; Biderman et al. Arch. Oral Biol. 2017, 81, 131 -135; Yilmaz et al. Cell. Microbiol. 2008, 10, 863-875; Chan et. al. Front Immunol. 2019 Mar 4;10:364).

[0142] Tumor Necrosis Factor alpha (TNF-alpha) 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 the oral tissues, including macrophages, monocytes, and dendritic cells, in response to infection or injury. TNF-alpha 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 actions lead to the recruitment of immune cells to the site of inflammation and the destruction of infected or damaged tissue. In oral inflammation, TNF-alpha has been implicated in the pathogenesis of various conditions, such as periodontitis, gingivitis, and oral mucositis. In periodontitis, TNF-alpha plays a key role in the destruction of periodontal tissues by promoting osteoclastogenesis and bone resorption. In gingivitis, TNF-alpha is involved in the inflammation and destruction of the gingival tissue. In oral mucositis, TNF-alpha contributes to the ulceration and inflammation of the oral mucosa, which is a common side effect of chemotherapy and radiation therapy.

[0143] Interleukin-6 (IL-6) is a pro-inflammatory cytokine that plays an important role in the development of oral inflammation. It is produced by various cells in the oral tissues, including epithelial cells, fibroblasts, and immune cells, in response to infection, injury, orother stimuli. Interleukin-8 is a potential diagnostic salivary biomarker in periodontal diseases (Chand et. al. ,A Systematic Review (March 31, 2020), increased of IL- 18 levels are associated with periodontitis (Alarcon-Sanchez et. al. Oral Health 24, 981 (2024), and MCP- 1 has been shown to be associated with regulation and involvement in disease (Singh et. al. International Immunopharmacology. 2021 Dec; 101).

[0144] 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 actions 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 has been implicated 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 tissues by promoting osteoclastogenesis and bone resorption. In gingivitis, IL-6 is involved in the inflammation and destruction of the gingival tissue. In oral cancer, IL-6 contributes to tumor growth and metastasis by promoting angiogenesis and suppressing immune responses.

[0145] Interferon gamma (IFN-gamma) 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-gamma 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 actions lead to the recruitment of immune cells to the site of inflammation and the destruction of infected or damaged tissue. In oral inflammation, IFN-gamma has been implicated in the pathogenesis of various conditions, such as periodontitis and oral lichen planus. In periodontitis, IFN-gamma is involved in the destruction of periodontal tissues by promoting the activation of osteoclasts and the production of matrix metalloproteinases. In oral lichen planus, IFN-gamma is involved in the inflammation and damage of the oral mucosa.

[0146] IL-17A (Interleukin- 17 A) 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 known as Thl7 cells, which are involved in the regulation of immune response and tissue inflammation. In the context of oral inflammation, IL-17A activates and recruits neutrophils,a type of white blood cells, to the site of inflammation. This leads to increased production of pro-inflammatory cytokines and chemokines, resulting in tissue damage and destruction. Studies have shown that the levels of IL-17A are increased in the gingival tissue of patients with periodontitis, a common oral inflammatory disease characterized by the destruction of the periodontium and loss of teeth. Additionally, IL-17A has been found to be elevated in the saliva and serum of patients with oral lichen planus and recurrent aphthous stomatitis, two other common oral inflammatory conditions.

[0147] In some of any embodiments, the enzymes provided herein are capable of reducing an inflammatory response associated with an oral condition. In some embodiments, the enzymes are capable of reducing the release of inflammatory mediators. In some of any of the embodiments, the enzyme provided herein 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% compared to a reference. In some of any embodiments, the one or more inflammatory mediators is TNF-alpha, IL-6, IL17A, IFNy, IL- 8 or a combination thereof.

[0148] In some of any embodiments, any of the enzymes provided herein is capable of reducing the level cell death 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 a reference.

[0149] In some of any embodiments, a reference can be an untreated reference. In some embodiments, a reference can refer to a reference in which any ATP degrading enzyme was added. In some embodiments, a reference can refer to a reference in which an enzyme that was not an ATP degrading was added. In some of any embodiments, a reference can refer to a reference in which any of the enzymes described herein was previously added, and then stopped.Skin

[0150] In skin inflammation, eATP can be released from damaged or dying cells, such as epithelial cells, immune cells, and nerve cells. eATP can then activate the P2X7 receptor on immune cells, such as macrophages and dendritic cells, leading to the production and release of inflammatory mediators, such as cytokines including IL6, IL-17A, IFNy, and IL-8 These cytokines can further promote the recruitment and activation of immune cells, leading to a vicious cycle of inflammation and tissue damage on the skin. eATP interacts with the health cells and induces cell death and accelerates the process of tissue damage and hinders woundhealing Manica et al. J Cell Biochem. 2018 May; 119(5):3980-3988; Burnstock et al. J Invest Dermatol. 2012 Mar; 132(3 Pt l):526-46; Spari et al. Host. Int. J. Mol. Set. 2020, 21, 5590; Holzer et al. J Cutan Med Surg. 2004 Mar-Apr ;8(2):90-6; Trier et. al. J. Immunol. 2019; 202 (10): 2829-2835).

[0151] The IL-1 family is a large group of cytokines, all of which are expressed in human epidermis, and have well established roles in skin immunopathology. (Macleod et.al. Front Immunol. 2021 Dec 23; 12:808012.)

[0152] Interleukin-6 (IL-6) is a pro-inflammatory cytokine that plays an important 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 actions 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 has been implicated in the pathogenesis of various conditions, such as psoriasis, atopic dermatitis, and skin cancer. In psoriasis, IL-6 plays a key role in the thickening of the epidermis and the formation of psoriatic plaques by promoting the proliferation of keratinocytes and inducing the differentiation of Thl7 cells. In atopic dermatitis, IL-6 is involved in the inflammation and barrier dysfunction of the skin. In skin cancer, IL-6 contributes to tumor growth and metastasis by promoting angiogenesis and suppressing immune responses.

[0153] IL-17A (Interleukin- 17 A) is a cytokine that plays a significant role in the pathogenesis of skin inflammation. It is produced by a specific subset of T-helper cells known as Thl7 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 the site of inflammation. It also stimulates the production of other pro-inflammatory cytokines, including IL-ip, TNF-a, and IL-6, by keratinocytes, fibroblasts, and other immune cells. These cytokines contribute to the amplification and maintenance of the inflammatory response, leading to tissue damage and destruction. IL-17A has been implicated in the pathogenesis of several skin inflammatory disorders, including psoriasis, atopic dermatitis, and acne. In psoriasis, for example, elevated levels of IL-17A and its associated cytokines have been found in lesional skin, and targeting IL-17A has been shown to be an effective therapeutic approach for the treatment of 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 contribute to the development of inflammatory acne lesions.

[0154] In some of any embodiments, the enzymes provided herein are capable of reducing an inflammatory response associated with a skin condition. In some embodiments, the enzymes are capable of reducing the release of inflammatory mediators. In some of any of the embodiments, the enzyme provided herein is 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% compared to a reference. In some of any embodiments, the one or more inflammatory mediators is IL6, IL- 17 A, IFNy, IL-8 or a combination thereof.

[0155] In some of any embodiments, any of the enzymes provided herein is capable of reducing the level cell death 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.

[0156] In some of any embodiments, a reference can be an untreated reference. In some embodiments, a reference can refer to a reference in which any ATP degrading enzyme was added. In some embodiments, a reference can refer to a reference in which an enzyme that was not an ATP degrading was added. In some of any embodiments, a reference can refer to a reference in which any of the enzymes described herein was previously added, and then stopped.III. MethodsA. Methods for treatins a disorder characterized by inflammation of the skin and / or oral cavity

[0157] Provided herein are methods and compositions for decreasing or preventing a disorder characterized by inflammation of the skin and / or oral cavity in a subject in need thereof. In some embodiments, the methods and compositions provided herein are for decreasing or preventing a disorder characterized by inflammation on the skin in a subject in need thereof.In some embodiments, the methods and compositions provided herein are for decreasing or preventing a disorder characterized by inflammation in the oral cavity in a subject in need thereof. Without being bound to theory, it is believed that administration of ATP degrading enzyme, such as apyrases, including apyrases of the GDA1 CD39 superfamily, including acid phosphatates, including 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 to the amino acid sequence of any 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, or SEQ ID NO: 53; or 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 the amino acid sequence of SEQ ID Nos: 16-30 or 77-102) can alleviate inflammation by removing skin and / or oral cavity generated sources of inflammation by, for example, removal of eATP, CpG DNA, and / or flagellin. In some embodiments, the removal is of eATP.

[0158] One non-limiting aim of removal of eATP in the body is to prevent or reduce local inflammatory responses. Furthermore, in some embodiments, the detoxified eATP is no longer available for passage through mucosal layers and thus cannot enter the circulation where it will exert its toxic effects and / or cause a further local and / or systemic inflammatory response.

[0159] A source of ATP degrading enzyme can be any ATP degrading enzyme, or any composition comprising the ATP degrading enzyme and any means which is capable of producing a functional ATP degrading enzyme in the context of the current invention, such as DNA or RNA nucleic acids encoding an ATP degrading enzyme. The nucleic acid encoding the ATP degrading enzyme may be embedded in suitable vectors such as plasmids, phagemids, phages, (retro)viruses, transposons, gene therapy vectors and other vectors capable of inducing or conferring production of ATP degrading enzyme. Also, native orrecombinant microorganisms, such as bacteria, fungi, protozoa and yeast may be applied as a source of ATP degrading enzyme in the context of the current invention.

[0160] In one embodiment, the invention provides a method for the prevention or reduction of 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 comprises an open sore and / or wound selected from canker sores, cold sores, cuts, bite wounds, burns, surgical incisions, and tissue trauma. In some embodiments, the method includes the step of applying the ATP degrading enzyme to the surface of the subject’s oral cavity selected from lips, the lining inside the cheeks and lips (buccal mucosa), the tongue, the upper and lower gums, the floor of the mouth under the tongue, the roof of the mouth (soft palate and hard palate), uvula, tonsils, and the area behind the wisdom teeth (retromolar trigone). In some of any embodiments, the method includes the step of administering a source of an ATP degrading enzyme (such as an apyrase 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 to the amino acid sequence of any 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, or SEQ ID NO: 53; or 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 the amino acid sequence of SEQ ID Nos: 16-30 or SEQ ID NOs:77-102) to the the surface of the subject’s oral cavity selected from lips, the lining inside the cheeks and lips (buccal mucosa), the tongue, the upper and lower gums, the floor of the mouth under the tongue, the roof of the mouth (soft palate and hard palate), uvula, tonsils, and the area behind the wisdom teeth (retromolar trigone). In some embodiments, provided herein is a method of treating a disorder associated withinflammation of the oral cavity, wherein the treating consists of administering any of the enzymes or compositions provided herein. For those jurisdictions where methods of treatment are unpatentable by law, the invention likewise pertains to the use of ATP degrading enzyme as defined above, or the use of a composition containing a source of apyrase as defined above. The source of ATP degrading enzyme is used for the manufacture of a medicament for delivery of ATP degrading enzyme at the oral cavity for the prevention or reduction of inflammation of oral cavity tissues.

[0161] In one embodiment, the invention provides a method for the prevention or reduction of inflammation on the skin. In some of any embodiments, the skin includes the epidermis and / or the 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, an autoimmune disease, cuts, bite wounds, bums, surgical incisions, and tissue trauma. In some embodiments, the method includes the step of applying the ATP degrading enzyme to the surface of the subject’s skin or keratinous tissue. In some of any embodiments, the method includes the step of administering a source of ATP degrading enzyme (such as an apyrase, or an acid phosphatase 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 to the amino acid sequence of any 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, or SEQ ID NO: 53; or 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 the amino acid sequence of SEQ ID Nos: 16-30 or SEQ ID NOs: 77-102) to the skin or epidermal tissue of a subject. For those jurisdictions where methods of treatment are unpatentable by law, the invention likewise pertains to the use of ATP degrading enzyme as defined above, or the use of a composition containing a source of ATP degrading enzyme asdefined above. The source of ATP degrading enzyme is used for the manufacture of a medicament for delivery of ATP degrading enzyme at the skin for the prevention or reduction of inflammation of skin tissues.III. In particular the above-mentioned method of administering a source of ATP degrading enzyme at the skin and / or oral cavity is suited for the treatment or prophylaxis of inflammation and associated diseases, although the method may also be advantageously used for healthy subjects as a prophylactic treatment aimed at the prevention of inflammation and associated diseases. In some embodiments, the associated disease does not include irritant contact dermatitis. The beneficial effects of ATP degrading enzyme administration to reduce inflammation of the skin and / or oral cavity according to the current invention will generate a general health promoting effect regardless of the medical condition of the subject treated. The health promoting effect may be further augmented by the consequent decrease in LPS, eATP, and / or CpG DNA influx through mucosal layers.

[0162] An LPS, eATP, or CpG DNA mediated or induced disease may be any disease, symptom or group of symptoms caused by LPS, eATP, or CpG DNA toxicity. An LPS, eATP, or CpG DNA exacerbated disease may be any disease or symptom that is not directly caused by LPS, eATP, or CpG DNA but a disease which symptoms and clinical features may be aggravated by LPS, eATP, or CpG DNA and the clinical state of the subject suffering from such a disease is worsened by the presence LPS, eATP, or CpG DNA in the skin and / or oral caviy.

[0163] Preferably the method is aimed at the treatment of an LPS, eATP, and / or CpG DNA mediated or exacerbated diseases such as inflammatory diseases associated with the oral cavity, in particular in patients diagnosed with periodontitis, gingivitis, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis, and / or oral mucositis. In periodontitis, eATP is released from damaged or dying cells in the inflamed periodontium and acts as a chemoattractant for immune cells, such as neutrophils and macrophages, to the site of infection. Once recruited, these immune cells inflammatory mediators including cytokines and chemokines, leading to the destruction of the periodontal tissues and bone loss characteristic of periodontitis. In oral mucositis, eATP is released from damaged or dying cells in the oral mucosa as a result of 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-ip and IL- 18. These cytokines can further amplify the inflammatory response and contribute to the development of oral mucositis.

[0164] Preferably the method is aimed at the treatment of an LPS, eATP, and / or CpG DNA mediated or exacerbated diseases such as inflammatory diseases associated with the skin, in particular in patients diagnosed with UV radiation, bacterial infection, psoriasis, allergic contact dermatitis, atopic dermatitis, autoimmune disease, cuts, bite wounds, burns, surgical incisions, and / or tissue trauma. In psoriasis, eATP is released from keratinocytes and immune cells in the psoriatic plaques and acts as a pro-inflammatory mediator. eATP can activate the P2X7 receptor on immune cells, leading to the production and release of pro-inflammatory cytokines, such as IL-ip and IL- 18, which further promote the development and maintenance of psoriatic lesions. In atopic dermatitis, eATP is released from damaged or dying cells in the inflamed skin, and contributes to the activation and recruitment of immune cells to the site of inflammation. eATP can activate the P2X7 receptor on immune cells, leading to the production and release of pro-inflammatory cytokines and chemokines, such as IL-8, which contribute to the development and chronicity 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 the P2X7 receptor on immune cells, leading to the production and release of pro- inflammatory cytokines and chemokines, such as IL-ip and IL-6, which further promote the development of skin inflammation.

[0165] Tissues to be treated according to the present disclosure include lips, the lining inside the cheeks and lips (buccal mucosa), the tongue, the upper and lower gums, the floor of the mouth under the tongue, the roof of the mouth (soft palate and hard palate), uvula, tonsils, and the area behind the wisdom teeth (retromolar trigone).

[0166] Other tissues to be treated according to the present disclosure include the skin, which includes the epidermis, dermis and hypodermis.

[0167] In some embodiments, the compositions according to the disclosure provided herein can be used to promote wound healing in a subject. In some embodiments, provided herein is a method of promoting wound healing in a subject, comprising administering to the subject a therapeutically effective amount of any one the compositions described herein. Also provided herein is the use of any of the compositions provided herein to promote wound healing in a subject.

[0168] In some embodiments, provided herein is a method of culturing artificial skin, comprising culturing epithelial cells in the presence of any one the compositions described herein. In some embodiments, the epithelial cells are from a mammal. In some embodiments, the mammal is a human. In some embodiments, the compositions according to the disclosure provided herein can be used to culture artificial skin. In some embodiments, the artificial skin is made of 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 engraftment. In some embodiments, the epithelial cells are from a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human.

[0169] In some embodiments, provided herein is a method of restoring a protective skin barrier, moisturizing skin, and / or reducing skin redness comprising applying any of the compositions described herein to the skin of a human. In some embodiments, the composition is applied topically and reduces skin redness in a subject compared to a subject who has not used the composition. In some embodiments, the composition is applied topically and increases moisture in the skin of a subject compared to a subject who has not used the composition. In some embodiments, the composition is applied topically and restores the protective skin barrier of a subject compared to a subject who has not used the composition.

[0170] Also provided herein is the use of any of the compositions described herein for restoring a protective skin barrier, moisturizing skin, and / or reducing skin redness.

[0171] The compositions comprising a source of ATP degrading enzyme according to the current disclosure are particularly suited for oral and / or topical administration to prevent treat, reduce, treat or alleviate inflammatory diseases of the skin and / or oral cavity. Inflammatory diseases of the skin and / or oral cavity tract may be induced and / or exacerbated significantly by the influx of bacterially derived substances such as LPS, eATP, and / orCpG DNA, by exposure to environmental or chemical factors, including UV radiation, or trauma to the tissue or surrounding tissue.

[0172] The current disclosure also provides compositions comprising a source of ATP degrading enzyme, amongst which are pharmaceutical and nutraceutical compositions comprising a source of ATP degrading enzyme. The compositions may optionally comprise pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeators, propellants, disinfectants, diluents and preservatives. Suitable excipients are commonly known in the art of pharmaceutical formulation and may be readily found and applied by theskilled artisan, references for instance Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia Pa., 17th ed. 1985. In a preferred embodiment the compositions comprising a source of ATP degrading enzyme are suitable for administration with one or more oral care components including a toothpaste, a prophylactic paste, a tooth powder, a tooth polish, a tooth gel, a chewing gum, a lozenge, a mouthwash, mouth rinse, a whitening strip, patch, suspension, emulsion, hydrogel, paste, multiphase solution, a paint-on gel, varnish, veneer, and tube, syringe or dental tray comprising a gel or paste, or a gel or paste coated on an application support such as dental floss or a toothbrush. In a preferred embodiment the compositions comprising a source of ATP degrading enzyme are suitable for administration with one or more skin care components including ointment, a serum, hydrogel, solution, dressing, moisturizer, exfoliator, body lotion, eye cream, sunscreen, toner, cleanser, skin wash, artificial skin, probiotic skin care component or spray. In a preferred embodiment the compositions comprising a source of ATP degrading enzyme are suitable for administration with one or more wound care components.

[0173] In another embodiment, the compositions comprising a source of ATP degrading enzyme are suitable for oral administration and comprise an enteric coating to protect the ATP degrading enzyme from the adverse effects of gastric juices and low pH. Enteric coating and controlled release formulations are well known in the art (references as described above). Enteric coating compositions in the art may comprise of a solution of a water-soluble enteric coating polymer mixed with the active ingredient(s) such as ATP degrading enzyme and other excipients, which are dispersed in an aqueous solution and which may subsequently be dried and / or pelleted. The enteric coating formed offers resistance to attack of ATP degrading enzyme by atmospheric moisture and oxygen during storage and by gastric fluids and low pH after ingestion, while being readily broken down under the alkaline conditions which exist in the lower intestinal tract.

[0174] In some embodiments, any of the ATP degrading enzyme containing compositions for the delivery of ATP degrading enzyme to the skin and / or oral cavity for treatment and prevention inflammation according to the current disclosure can comprise a microbially- derived (i.e. 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.B. Methods for promoting engraftment of commensal bacteria on the skin and / or oral cavity

[0175] Also provided herein is a method for promoting the engraftment of one or more commensal bacteria onto the skin and / or oral cavity of a subject. The method requires at least the administering to the subject any of the ATP degrading enzyme compositions disclosed herein (such as an apyrase or an acid phosphatase 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 to the amino acid sequence of any 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, or SEQ ID NO: 53; or 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 the amino acid sequence of SEQ ID NOs: 16-30 or SEQ ID NOs: 77-102) and a source of one or more commensal bacteria. In some embodiments, the method promotes engraftment of the one or more commensal bacteria onto the skin and / or oral cavity of a subject to a greater extent (such as about a 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, inclusive of percentages falling in between these values, greater extent) compared to when the source of one or more commensal bacteria is administered in the absence of the ATP degrading enzyme compositions disclosed herein. Non-limiting examples of commensal bacteria include organisms from the genera Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphromonas, Prevotella, Treponema, Nisseria, Haemophilis, Eubacteria, Lactobacterium, Capnocytophaga, Eikenella, Leptotrichia, Peptostreptococcus, Staphylococcus, and Propionibacterium, Corynebacterium, Propionibacterium, Dermabacter, and Brevibacterium. The source of one or more commensal bacteria can be administered as a probiotic (for example, orally, topically or rectally) or as a fecal microbial transplant (forexample, rectally or orally in a capsule).

[0176] Generally, the source of one or more commensal bacteria (such as probiotic compositions) comprise bacteria, such as one or more bacterial strains. In some embodiments of the disclosure, the source of one or more commensal bacteria is formulated in freeze-dried or lyophilized form. For example, the source of one or more commensal bacteria can comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain disclosed herein.

[0177] In some embodiments, the source of one or more commensal bacteria comprise lyophilized bacteria. Lyophilization of bacteria is a well-established procedure in the art. Alternatively, the microbial-containing compositions can comprise a live, active bacterial culture.

[0178] In some embodiments, the source of one or more commensal bacteria is formulated to enable delivery of the bacterial strain to the skin and / or oral cavity. In some embodiments, the formulation is suitable for administration with one or more oral care components including a toothpaste, a prophylactic paste, a tooth powder, a tooth polish, a tooth gel, a chewing gum, a lozenge, a mouthwash, mouth rinse, a whitening strip, patch, suspension, emulsion, hydrogel, paste, multiphase solution, a paint-on gel, varnish, veneer, and tube, syringe or dental tray comprising a gel or paste, or a gel or paste coated on an application support such as dental floss or a toothbrush. In a preferred embodiment the formulation is suitable for administration with one or more skin care components including ointment, a serum, hydrogel, solution, dressing, moisturizer, exfoliator, body lotion, eye cream, sunscreen, toner, cleanser, skin wash, artificial skin, probiotic skin care component or spray. In a preferred embodiment the compositions comprising a source of ATP degrading enzyme are suitable for administration with one or more wound care components.

[0179] In some embodiments, the source of one or more commensal bacteria is encapsulated to enable delivery of the bacterial strain to the skin, oral cavity or intestine. Encapsulation protects the composition from degradation until delivery at the target location through, for example, rapturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporousmembrane or a microcapsule.

[0180] The source of one or more commensal bacteria can be administered orally and may be in the form of a tablet, capsule or powder. Other ingredients (such as vitamin C or minerals, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonization and / or engraftment and / or survival in vivo. Alternatively, the source of one or more commensal bacteria (such as probiotic compositions) can be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.

[0181] The source of one or more commensal bacteria can be formulated as a probiotic. Alternatively, the source of one or more commensal bacteria can be formulated as a non- viable bacterial compositions, such as a pasteurized or heat-treated bacterial composition.

[0182] A source of one or more commensal bacteria includes a therapeutically effective amount of a bacterial strain disclosed herein (for example, without limitation, one or more from the genera Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphromonas, Prevotella, Treponema, Nisseria, Haemophilis, Eubacteria, Lactobacterium, Capnocytophaga, Eikenella, Leptotrichia, Peptostreptococcus, Staphylococcus, and Propionibacterium, Corynebacterium, Propionibacterium, Dermabacter, and Brevibacterium). A therapeutically effective amount of a bacterial strain is sufficient to exert a beneficial effect upon a patient. A therapeutically effective amount of a source of one or more commensal bacteria may be sufficient to result in delivery to and / or partial or total engraftment and / or colonization of the subject’s skin and / or oral cavity.

[0183] A suitable daily dose of the commensal bacteria, for example for an adult human, may be from about 1 x 103to about 1 X 1011colony forming units (CPU); for example, from about 1 x 107to about 1 x 1010CPU; in another example from about 1 x 106to about 1 x 1010GPU; in another example from about 1 x 107to about 1 x 1011CPU; in another example from about 1 x 108to about 1 x 1010CPU; in another example from about 1 x 108to about 1 x 1011CPU. In certain embodiments, the dose of the bacteria is at least 109cells per day, such as at least 1010, at least 1011or at least 1012cells per day.

[0184] In certain embodiments, the source of one or more commensal bacteria contains the bacterial strain in an amount of from about 1 x 106to about 1 x 1011CFU / g, respect to the weight of the composition; for example, from about 1 x 108to about 1 x 1010CFU / g. The dose may be, for example, 1 g, 3g, 5g, and 10 g. In certain embodiments, the amount of thebacterial strain is from about 1 x 103to about 1 x 1011colony forming units per gram with respect to a weight of the composition.

[0185] In certain embodiments, any of the sources of one or more commensal bacteria is administered at a dose of between 500mg and lOOOmg, between 600mg and 900mg, between 700mg and 800mg, between 500mg and 750mg or between 750mg and lOOOmg. In certain embodiments, the lyophilized bacteria in any of the microbial-containing compositions disclosed herein is administered at a dose of between 500mg and lOOmg, between 600mg and 900mg, between 700mg and 800mg, between 500mg and 750mg or between 750mg and lOOOmg.

[0186] Typically, a probiotic, is optionally combined with at least one suitable prebiotic compound. A prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract Known prebiotics include commercial products such as inulin and transgalacto- oligosaccharides.

[0187] In certain embodiments, a probiotic composition is formulated to include a prebiotic compound in an amount of from about 1 to about 30% by weight respect to the total weight composition, (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo- oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomaltoseoligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosanoligosaccharides (or COS), human milk oligosaccharides, beta- glucans, gum arabic modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers.In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded. In another embodiment, the prebiotic can include one or more polyphenols (such as a plant polyphenol). In some embodiments, any of the probiotics disclosed herein can be formulated with additional probiotics derived from the genera Lactobacillus and Bifidobacterium (such as B. lactis B420).

[0188] The source of one or more commensal bacteria can further comprise pharmaceutically acceptable excipients or carriers. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art. Examples of suitable carriers include, without limitation, lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.Examples of suitable diluents include, without limitation, ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binders, lubricants, suspending agents, coating agents (such as a gastric-resistant enteric coating agent that does not dissolve or degrade until reaching the small or large intestine), or solubilizing agents. Examples of suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include, without limitation, sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

[0189] The source of one or more commensal bacteria can be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a pharmaceutical composition. In certain embodiments, the microbial-containing composition is formulated as a milk-based product. The term "milk-based product," as used herein, means any liquid or semi-solid milk- or whey- based product having a varying fat content. The milk- based product can be, e.g., cow's milk, goa s milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavored milks, ice cream; milk-containing food such as sweets.

[0190] In certain embodiments, the source of one or more commensal bacteria contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free fromother species of organism. In certain 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 certain embodiments, the compositions consist of from 1 to 10, such as from 1 to 5 bacterial strains or species.

[0191] The source of one or more commensal bacteria for use in accordance with the methods disclosed herein may or may not require marketing approval.

[0192] In certain embodiments, the source of one or more commensal bacteria is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere having 50% relative humidity, at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the bacterial strain as measured in colony forming units, remains after 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.

[0193] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.EXAMPLESEXAMPLE 1. CLONING, EXPRESSION AND PROTEIN PREPARATION OF ENZYME CANDIDATES

[0194] Phylogenetically diverse sequences from enzyme families known to have ATP hydrolysis activity were identified from the NCBI (worldwwideweb.ncbi.nlm.nih.gov / protein) or internal databases and annotated as members of the enzyme families GDA1 / CD39 or acid phosphatase . Gene sequences of interest were codon-optimized based on codon preference of Bacillus subtilis and synthetic genes encoding the sequences for various enzymes were generated (by Generay Biotech (Shanghai) Co., Ltd) using techniques known in the art. The sequences of 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 on Table 1. Genes were cloned into expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif. 55:40-52, 2007). The expression of the codon-optimized nucleotide sequence encodes the predicted mature polypeptide driven by aprE promoter (SEQ ID NO: 46, using the nucleotide sequence SEQ ID NO: 47 encoding the 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 genes of interest (to facilitate the secretion of the target protein in B. subtilis), and is stopped by to terminator (SEQ ID NO: 49). Competent B. subtilis cells were transformedwith each corresponding expression vector and plated on Luria Agar plates supplemented with 5 ppm chloramphenicol. The colony was inoculated in a flask containing LB medium and grew at 37°C with 200 rpm shaking for 5-6 h. Seed culture was inoculated into a production medium containing minerals (e.g., potassium sulfate, magnesium sulfate, ferrous sulfate, calcium chloride, citric acid, etc.), one or more carbon sources (e.g., glucose), a complex nitrogen source (e.g. hydrolyzed soy peptone, yeast extract) and a buffering component (e.g. MOPS, Tris, etc.). Cultures were grown at 32°C with 250 rpm shaking for 24-40 h.

[0195] General purification procedure was as follows. Culture broth was collected by centrifugation, concentrated and supplemented with ammonium sulfate to a final concentration of IM, then applied to a hydrophobic interaction column (e.g. phenyl FF, butyl FF) which was pre-equilibrated with 50 mM Tris pH 7.5 containing IM ammonium sulfate. The column was eluted with a stepwise mode (0.75, 0.5, 0.25, 0.02 M ammonium sulfate, H2O, 20% ethanol) and fractions containing active enzymes of interest were pooled and buffer-exchanged to 20 mM Tris pH7.5. Pooled sample was loaded on an anion-exchange chromatography (e.g. Q FF) and eluted with a gradient mode (0-0.5 M NaCl). Fractions containing purified enzymes were identified based on SDS-PAGE analysis and activity tests, and they were pooled. Purified samples were stored at -20°C in buffer containing 20 mM Tris pH7.5, 150 mM NaCl, and 40% glycerol until use.EXAMPLE 2. BIOCHEMICAL CHARACTERIZATION OF ENZYME CANDIDATES

[0196] Samples of the phylogenetically diverse enzymes described in Example 1 were assayed to determine their ability to hydrolyze ATP. Reference enzymes were purchased from Sigma Aldrich, including Potato apyrase (catalog # A6535), recombinant human CD39 His-tag (catalog # SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog #P0114).

[0197] Enzyme activity towards 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 detection kit (Sigma Aldrich cat no. MAK307). The enzyme reactions were carried out in 96-well plates with a total volume of 150 pL and contained 250 pM of a substrate, 50 mM of a buffer, 5 mM CaCh, and a variable concentration of enzyme. The substrate was ATP or ADP. The buffer was glycine (pH 3), acetate (pH 4, 4.5, 5), bis-Tris (pH 5.5, 6, 6.5), or Tris (pH 7, 8, 9). The concentration ofenzyme varied from about 1 pg / mL to 1 ng / mL and was adjusted based on the activity of the enzyme so that the amount of phosphate released during the assay was with the dynamic range of the assay (5 - 100 pM phosphate). The enzyme concentration was determined by the absorbance at 280 nm and using the predicted molecular weight and molar extinction coefficient to calculate the concentration. The enzyme reaction was initiated by the addition of 15 pL enzyme with 135 pL of the other reaction components such that the final reaction composition was as previously described. The reaction was stopped after 5 min by mixing 80 pL of the reaction mixture with 20 pL of the malachite green working reagent. The malachite green working reagent was prepared according to the manufacturer’s protocol by mixing 100 parts reagent A with 1 part reagent B. Phosphate standard solutions (0 - 80 pM) and a negative control reaction without enzyme were also mixed with the malachite green working reagent. All samples were tested in duplicate. The malachite green reaction mixtures were allowed to rest at room temperature for 45-60 min and the absorbance at 620 nm was measured. Lastly, the absorbance of the phosphate standards were used to convert the absorbance readings of each sample into phosphate concentrations and the negative control was subtracted from each reaction. The amount of phosphate in micromoles released per milligram of protein per minute of reaction time (pmol / mg / min) with ATP or ADP as a substrate is displayed in Table 2 and Table 3.

[0198] As shown in Table 2 and Table 3, CRC22110 was the most active enzyme, demonstrating activity along a wide pH range including from pH 3 to pH 9. Potato apyrase, CRC22110, CRC22105, CRC22112, CRC22383, 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. The acid phosphatase enzymes have an acidic pH optimum around pH 4.5 whereas the enzymes from the GDA1 CD39 superfamily had pH optima near neutral pH (pH 6-8) and have a broad activity range between pH 5 - 7, with some maintaining activity even to pH 9. CRC21320 has a narrower pH range with an optimum at pH 7.

[0199] Enzyme substrate specificity: The activity of the enzymes on different nucleotide substrates was tested using the malachite green assay. The assay was set up as described in above with the following modifications. The substrates tested were ATP, ADP, CTP, CDP, UTP, UDP, GTP, and GDP. CRC21323 and CRC21322 were tested with a buffer of acetate pH 4.5. CD39 was tested with a buffer of bis-Tris pH 7. All other enzymes were tested with a buffer of bis-Tris pH 6.5. After converting the absorbance to phosphate concentration, the activity of each enzyme was set relative to the activity on ATP. Test results are shown on Table 4.

[0200] As shown in Table 4, the nucleotide triphosphate is a better substrate than the diphosphate. Most enzymes have similar activity on UTP as ATP, except for CRC22105 which has noticeably reduced activity on UTP / UDP and CTP / CDP. However, each enzyme has slightly difference preferences.

[0201] Enzyme acid resistance: Candidate enzymes were tested for their ability to resist inactivation at low pH by incubating the enzymes in an “acid buffer” of 50 mM glycine and 5 mM CaCh at pH 2 for 15 or 60 minutes and comparing their activity to enzymes in a “control buffer” at their optimum pH. The optimum buffer for CRC21323 and CRC21322 was 50 mM acetate pH 4.5 with 5 mM CaCh. The optimum buffer for all other enzymes was 50 mM bisTris pH 6.5 with 5 mM CaCh. After incubating in the acidic glycine buffer, the enzyme mixtures were neutralized by mixing with an equal volume of neutralization buffer. The neutralization buffer for CRC21323 and CRC21322 was 100 mM acetate and 10 mM CaCh at pH 4.5. The neutralization buffer for the other enzymes was 100 mM bis-Tris and 10 mM CaCh at pH 6.5. Next, the acid-stressed and control enzyme were tested for their ability to release phosphate from ATP using the reaction conditions and malachite green assay as described in with the following changes. The only substrate tested was ATP. CRC21323 and CRC21322 were tested with a buffer of acetate pH 4.5, but all other enzymes were tested in a buffer of bis-Tris pH 6.5. After determining the concentration of phosphate in each reaction, the residual activity of each enzyme after 15 or 60 min incubation in the “acid buffer” was calculated by dividing the acid-stressed by the control enzyme phosphate concentrations for each enzyme. Test results are shown on Table 5 below.

[0202] As shown in Table 5, the acid resistance experiment indicates that CRC21328 and CRC22110 had the greatest resistance to the “acidic buffer” as they maintained the greatest activity after a 60 min incubation. Potato apyrase was stable after 15 min but appeared to lose most of its activity after 60 min incubation. Although CRC21323 and CRC21322 are members of the acid phosphatase family, they were the least stable under this assay condition.

[0203] Effect of pH on ATPase activity: The ATPase activity was measured at 0.05 ppm concentration in 50 mM MES buffer pH 6 and pH 5 with 0.1 mM ATP as substrate as described in Example 1. Commercial potato apyrase was used as reference._The absorbance at 620 nm was measured and the absorbance of a blank sample (water) was subtracted from each sample. Results are shown on Table 6.EXAMPLE 3. EXPRESSION OF TRUNCATED FORMS OF ENZYMES OF INTERESTThe CRC22110 G. xiamenensis apyrase molecule, expressed in B subtilis with N-terminal AGK peptide was selected for further evaluation, including truncations of the predicted sequence (SEQ ID NO: 50). Various C-terminal truncations of the CRC22110 apyrase were designed to remove residues C-terminal to the final Cysteine which is involved in 1 of the 7 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 methods of expression of truncation forms of CRC22110 are described below, wherein certain instances an additional 3 residues, AGK, are included at the predicted N-terminus of the mature polypeptide sequence. A first DNA fragment comprising a (5’) skfA gene flanking region (5’ skfA gene FR, SEQ ID NO:54) was operably linked to a polynucleotide construct (eg. expression cassette) comprising an upstream (5’) B. subtilis rrnl-p2 promoter region DNA sequence (SEQ ID NO:55) operably linked to DNA sequence of the B. subtilis aprE 5’ untranslated region (5’ UTR) (SEQ ID NO:56) operably linked to DNA encoding the B. subtilis aprE signal sequence (SEQ ID NO:47) operably linked to a DNA sequence encoding the desired C-terminal truncated G. xiamenensis apyrases with the addition of the nucleotide sequences encoding the tripeptide AGK at the N-termini (polypeptide sequences SEQ ID NO: 57, 58 or 59), operably linked to the Bacillus amyloliquefaciens BPN terminator (SEQ ID NO: 60) operably linked to a (3’) skfA gene flanking region (3’ skfA gene FR) (SEQ ID NO:61). A second DNA fragment comprising a (5’) amyE gene flanking region (5’ amyE gene FR) (SEQ ID NO:62) was operably linked to a polynucleotide construct (eg. expression cassette) comprising an upstream (5’) B. subtilis rrnl-p2 ( SEQ ID NO:55) promoter region DNA sequence operably linked to DNA sequence of the B. subtilis aprE 5’ untranslated region (5’ UTR) (SEQ ID NO:56) operably linked to DNA encoding the B. subtilis aprE signal sequence (SEQ ID NO:47) operably linked to a DNA sequence encoding the desired C-terminal truncated G. xiamenensisapyrases with the addition of the nucleotide sequences encoding the tripeptide AGK at the N- termini (polypeptide sequences SEQ ID NO: 57, 58 or 59), and operably linked to Bacillus amyloliquefaciens BPN terminator (SEQ ID NO: 60) operably linked to a (3’) amyE gene flanking region (3’ amyE gene FR) (SEQ ID NO:63). More particularly, these DNA fragments were assembled using standard molecular biology techniques and were used as templates to develop linear DNA expression cassettes for generation of a 2 copy strain. A suitable Bacillus subtilis strain, comprising nine protease deletion was used to integrate the first and second linear DNA expression cassette fragment described above in the genome using standard molecule biology techniques.EXAMPLE 4. BIOCHEMICAL EVALUATION OF TRUNCATED FORMS OF CRC22110 APYRASEThe relative enzyme activity on ATP substrate was determined for truncated forms of the CRC22110 apyrase 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.Enzyme Isolation: Culture supernatants from B subtilis fermentations were obtained by filtration and ammonium sulfate and IM Tris pH8 was added to a final concentration of IM ammonium sulfate and 20mM Tris pH8. The sample was centrifuged and filtered. The filtrate was loaded onto 300ml phenyl Sepharose column on equilibrated in 20mM Tris and IM ammonium sulfate pH8. A linear gradient from 0% to 100% 20mM Tris pH8 was run at lOml / min over 300 minutes. Elution fractions were collected and ATPase activity was monitored using ATPase activity assay. The active fractions were pooled, concentrated and buffer exchanged into 20mM Tris pH8. This pool was loaded onto 25ml Q Sepharose column that was equilibrated in 20mM Tris pH8. They were then eluted with step gradient from 50mM NaCl in 20mM Tris, followed by lOOmM NaCl, 200mM NaCl, 400mM and 500mM NaCl in 20mM Tris pH8. The active and pure fractions (>95% by inspection on SDS-PAGE) were pool as purified protein, they were then quantitated.Protein quantitation by UPLC: Protein quantitation methods: Protein concentration was determined by UPLC (Ultra Performance Liquid Chromatography) and by OD280 densitometry). For UPLC determination, the purified enzyme was diluted in 20mM Tris pH8 and concentration was determined by separation of protein components using a Zorbax 300 SB-C3 column (Agilent) and running a linear gradient of 0.1% Trifluoroacetic acid in water (Buffer A) and 0.1% Trifluoroacetic acid in Acetonitrile (Buffer B) with detection at 220nmcolumn on UHPLC. A lOul sample was loaded onto the column and the peak area of the diluted sample was determined. The enzyme concentration of the samples was calculated using a standard curve of the purified reference enzyme for example full length CRC22110. The protein concentration was also determined by OD280 measurement. The purified enzyme was diluted in 20mM Tris pH8, their OD280 was measured in a quartz cuvette. The protein concentration was calculated based on their respective extinction coefficients. The final concentration was calculated based on the average of UPLC method and OD280 determination.ATPase activity comparison: ATPase activity assay was performed using samples diluted to 0.003ppm or 0.0015ppm in assay buffer (50mM Tris pH8, 5mM CaC12, 0.01% Tween 80) using the ATPase assay described previously. Samples of lOul of diluted enzyme were added into 0.25mM ATP in assay buffer to start the reaction. The reaction mix was incubated at 25°C for lOmins, then 50ul of reaction mix was added into lOOul of QuantiChrom™ Malachite Green reagent (VWR cat # 75878) and reaction mixtures were then incubated at 25°C for 20mins, then the OD620 was measured. ATPase activity was compared after subtracting the blank (no enzyme control). The relative ATPase activity was calculated based on the background subtracted OD relative to full length CRC22110. The results are shown on Table 7 below.As shown on Table 7, the relative ATPase activity of CRC22110 and the C-terminal truncation is highly comparable, thus confirming the ability to truncate this region of the protein sequence without affecting enzyme function.EXAMPLE 5. USE OF EXEMPLARY ENZYMES IN AN EPIGINGIVAL TISSUE INFLAMMATORY MODELIn 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 exemplary enzymes in a 3D Epigingival tissue model oral inflammation.Briefly, a 3D Epigingival tissue model (Cat No GIN 100) was obtained from MatTek Corporation, MA, USA. Tissues were maintained and used in the experiment according to the manufactures protocol.To determine the role of ATP in oral inflammation, tissues were incubated with 10 mM ATP or LPS from Porphyromonas gingivalis (1 ug / mL) (Cat No SMB00610) or 10 mM GTP, or 100 nM AP429 or 10 mM ATP + LPS from Porphyromonas gingivalis (1 ug / mL) + 100 nM CRC22110-V1 for 2 and 6 hrs at 37C. Tissues were collected at 2 and 6 hrs and subjected to MTT assay to evaluate the viability of the tissues under different conditions according to the manufactures protocol. Briefly, tissues were incubated in the MTT solution for 3 hrs and then extraction was done overnight without shaking at room temperature in the dark. The optical density of the extracted solution was measured at 570 nm using 200 ul of sample.As shown in FIG. 1, ATP and LPS are capable of inducing cell death in 3D gingival tissues, while addition of exemplary NTPase CRC22110-V1 was able to negate the viability inhibiting impact of ATP and LPS.To determine the role of exemplary enzymes on the expression of cytokines including TNF-a, IL-6, IFN-y and IL-17A induced by ATP and LPS in 3D gingival tissues, supernatants from 6 hrs time points were measured using LEGENDplex Multi-Analyte Flow Assay (Cat No 740808). Cytokine levels were represented as Mean Fluorescent Units (MFI).As shown in FIG. 2, exposure of ATP or LPS resulted in a marked increase in TNF-a levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of TNF-a when tissues were treated with ATP and LPS.As shown in FIG. 3, exposure of ATP or LPS resulted in a measurable increase in IL-6 levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of IL-6 when tissues were treated with ATP and LPS.As shown in FIG. 4, exposure of ATP or LPS resulted in a marked increase in IFN-y levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of IFN-y when tissues were treated with ATP and LPS.As shown in FIG. 5, exposure of ATP or LPS resulted in a measurable increase in IL-17A levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of IL-17A when tissues were treated with ATP and LPS.To determine the levels of extracellular ATP (eATP) upon exposure to ATP and LPS, supernatants were collected from tissues incubated as described above, and ATP was measured using an ATP Determination Kit (Cat No A22066, Invitrogen) according to manufacturer’s protocol. As shown in FIG. 6, exposure of ATP or LPS resulted in an increase in eATP levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of eATP when tissues were treated with ATP and LPS.EXAMPLE 6. USE OF EXEMPLARY ATP DEGRADING ENZYMES IN A TISSUE MODEL OF SKIN INFLAMMATIONIn 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 exemplary enzymes in a 3D Psoriasis tissue model.Briefly, a 3D Psoriasis tissue model (Cat No SOR 300 FT) was obtained from MatTek Corporation, MA, USA. Tissues were maintained and used in the experiment according to the manufactures protocol.To determine the role of ATP in skin inflammation, tissues were incubated with 10 mM ATP or LPS from Porphyromonas gingivalis (1 ug / mL) or 10 mM GTP or 100 nM CRC22110-V1 or 10 mM ATP + LPS from Porphyromonas gingivalis (1 ug / mL) + 100 nM CRC22110-V1 for 2 and 6 hrs at 37C. Tissues were collected at 2 and 24 hrs and subjected to MTT assay to evaluate the viability of the tissues under different conditions according to the manufactures protocol. Briefly, tissues were incubated in the MTT solution for 3 hrs and then extraction was done overnight without shaking at room temperature in the dark. The optical density of the extracted solution was measured at 570 nm using 200 ul of sample.As shown in FIG. 7 ATP and LPS are capable of inducing cell death in 3D psoriasis tissues at 6 hours (FIG. 7A) and 24 hours (FIG. 7B), while the addition of exemplary NTPase CRC22110-V1 was able to negate the viability inhibiting impact of ATP and LPS.To determine the role of exemplary enzymes on the expression of cytokines including IL-6, IFN-y and IL-17A induced by ATP and LPS in 3D psoriasis tissues, supernatants from 6 hrs time points were measured using LEGENDplex Multi-Analyte Flow Assay (Cat No 740808). Cytokine levels were represented as Mean Fluorescent Units (MFI).As shown in FIG. 8, exposure of ATP or LPS resulted in a marked increase in IL-6 levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of IL-6 when tissues were treated with ATP and LPS.As shown in FIG. 9, exposure of ATP or LPS resulted in a measurable increase in IL-17A levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of IL-17A when tissues were treated with ATP and LPS.As shown in FIG. 10, exposure of ATP or LPS resulted in a marked increase in IFN-y levels in the supernatant, while addition of NTPase CRC22110-V1 was able to negate the accumulation of IFN-y when tissues were treated with ATP and LPS.To determine the levels of extracellular ATP (eATP) upon exposure to ATP and LPS, supernatants were collected from tissues incubated as described above, and ATP was measured at 2, 6 and 24 hrs using an ATP Determination Kit (Cat No A22066, Invitrogen™) according to manufacturer’s protocol. As shown in FIG. 11A, FIG. 11B and FIG. 11C, exposure of ATP or LPS resulted in an increase in eATP levels in the supernatant at 2, 6 and 24 hours, respectively, while addition of NTPase CRC22110-V1 was able to negate the accumulation of eATP when tissues were treated with ATP and LPS.Example 7. USE OF EXEMPLARY ATP DEGRADTING ENZYMES IN A SKINCELL DERIVED INFLAMMATION MODELThis Example describes the ability of exemplary enzymes to reverse damage caused by LPS and UV radiation in the human keratinocyte HaCaT skin cell line.Briefly, HaCaT cells were obtained from Acellerate and cultured in DMEM + GlutaMAX medium (4.5g / L D-glucose; lOOmg / mL Sodium Pyruvate) supplemented with 10% certified FBS (complete media). Cells were cultured in a T75 flask in a 37°C incubator containing 5% CO2 and humidity until cells reached 80-90% confluency. Cells were sub-cultured by detaching cells using 0.05% trypsin-EDTA, centrifuged and resuspended in lOmL of complete media. New flasks were seeded 1 : 10 and incubated under the same conditions above or cells were seeded into a 96 well plate for experiments.For an LPS induced HaCaT cell damage model, 2 x 104HaCaT cells were seeded in each well of a 96 well plate, and incubated for two days in a 37°C incubator containing 5% CO2 and humidity. Media was replaced and lOpg / mL LPS from E. coli O55:B8 was added separately or in combination with InM apyrase (Sigma-Cat No A6410), CRC22110 (WT) or AP429 to respective wells and cells were incubated overnight in a 37°C incubator containing 5% CO2 and humidity. Following incubation, supernatants were collected and stored in - 20°C until further use.For a UVB induced HaCaT cell damage model, 2 x 104HaCaT cells were seeded in each well of a 96 well, and incubated for two days in a 37°C incubator containing 5% CO2 andhumidity. Before exposure, media was removed and replaced with 50pL DPBS (w / o calcium). Cells were exposed to 25mJ / cm2and fresh media with or without IpM apyrase was added to the cells. Cells were incubated in a 37°C incubator containing 5% CO2 and humidity for 4 and 8 hours following UVB exposure. Supernatants were collected at each time-point and stored in -20°C until further use.Frozen supernatants from the LPS and UVB induced HaCaT cell damage models were thawed, and the concentration of IL-8 measured using BD OptEIA Human ELISA kit II according to manufacturer’s protocol. Briefly, supernatant was incubated in pre-coated wells at room temperature for 2hrs. Wells were washed 5x with wash buffer and subsequently incubated with a mixture of enzyme concentrate and detection antibody for Ihr at room temperature. Wells were washed 7x with wash buffer and then developed with the addition of TMB. The plate was incubated at room temperature in the dark until full development of standards and then the reaction was stopped by addition of stop solution (IM phosphoric acid). Using a spectramax M5 plate reader, the absorbance at 450nm was measured.As shown in FIG. 12, exposure to LPS induces IL-8 release from HaCat cells, while addition of potato apyrase, the CRC22110 apyrase or the truncated CRC22110-vl negates the IL-8 accumulation in the supernatant. Note that CRC22110 and CRC22110-V1 both showed higher improvement in IL-8 levels as compared to potato apyrase.As shown in FIG. 13A and FIG. 13B, exposure to UVB induces IL-8 release from HaCat cells at 4 and 8 hours, respectively, while addition of apyrase negates the IL-8 accumulation in the supernatant.EXAMPLE 8. BIOCHEMICAL CHARACTERIZATION OF ADDITIONAL ATP DEGRADING ENZYME CANDIDATES

[0204] Additional samples of the phylogenetically diverse enzymes described in Example 1 were assayed to determine their ability to hydrolyze ATP. Reference enzymes were purchased from Sigma Aldrich, including Potato apyrase (catalog # A6535), recombinant human CD39 His-tag (catalog # SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog #P0114).

[0205] The ability of candidate enzymes to degrade ATP was measured similar to as described in Example 2.

[0206] The amount of phosphate in micromoles released per milligram of protein per minute of reaction time (pmol / mg / min) with ATP as a substrate is displayed in Table 8 and Table 9.

[0207] As shown in Table 8 and Table 9, CRC34822 was the most active enzyme at pH 5 of the enzymes tested in this experiment.EXAMPLE 9. USE OF ADDITIONAL EXEMPLARY ATP DEGRADING ENZYMES IN A TISSUE MODEL OF SKIN INFLAMMATION

[0208] This Example describes the use of additional exemplary enzymes in a 3D Psoriasis model generally as described in Example 5 using a 3D Psoriasis tissue model (MatTek Life Sciences (Ashland, MA) Cat No SOR 300 FT).

[0209] Briefly, tissues were incubated with 10 mM ATP and LPS from Porphyromonas gingivalis (1 ug / mL) or 10 mM ATP + LPS from Porphyromonas gingivalis (1 ug / mL) + 1 uM CRC25100 or CRC34822 acid phosphatases for 2 and 4 hrs at 37C. Tissues were collected at 4 hrs and subjected to 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the viability of the tissues under different conditions according to the manufacture’s protocol. In summary tissues were incubated in the MTT solution for 3 hrs and then extraction was done overnight without shaking at room temperature in the dark. The optical density of the extracted solution was measured at 570 nm using 200 ul of sample.

[0210] Culture supernatants from 2 hrs time points were used to measure secreted cytokines by using the LEGENDplex Multi-Analyte Flow Assay (Cat No 740808). Cytokine levels were reported as Mean Fluorescent Units (MFI).

[0211] ATP was measured in the culture supernatants by using the ATP Determination Kit (ThermoFisher, Cat No A22066)

[0212] As shown in FIG. 14A, exposure of ATP and LPS resulted in significant decrease in cell viability. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the impact of ATP and LPS. As shown in FIG. 14B, exposure of ATP or LPS resulted in significant increase in IL-6 levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IL-6 when tissues were treated with ATP and LPS

[0213] As shown in FIG. 14C, exposure of ATP or LPS resulted in significant increase in MCP-1 levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IL-17A when tissues were treated with ATP and LPS. As shown in FIG. 14D, exposure of ATP or LPS resulted in significant increase in IL-8 levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IL-8 when tissues were treated with ATP and LPS.

[0214] As shown in FIG. 14E, exposure of ATP or LPS resulted in significant increase in IL-ip levels in the supernatant. Addition of all acid phosphatases tested was able to negate the accumulation of IL-ip when tissues were treated with ATP and LPS. As shown in FIG. 14F, exposure of ATP and LPS resulted in significant increase in IL-33 levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IL-33 when tissues were treated with ATP and LPS.EXAMPLE 10. USE OF ADDITIONAL EXEMPLARY ENZYMES IN AN EPIGINGIVAL TISSUE INFLAMMATORY MODEL

[0215] This Example describes the use of additional exemplary enzymes in a 3D Epigingival tissue model of oral inflammation. Briefly, a 3D Epigingival tissue model (Cat No GIN 100) was obtained from MatTek Corporation, MA, USA. Tissues were maintained and used in the experiment according to the manufacture’s protocol.

[0216] Tissues were incubated with 10 mM ATP and LPS from Porphyromonas gingivalis (1 ug / mL) Cat No SMB00610 or 10 mM ATP + LPS from Porphyromonas gingivalis (1 ug / mL) + 1 uM CRC25100 or CRC34822 enzyme sample and culture supernatants were collected at 2 and 4 hrs.

[0217] Tissues were collected at 4 hrs and subjected to MTT assay to evaluate the viability of the tissues under different conditions according to the manufacture’s protocol. In summary tissues were incubated in the MTT solution for 3 hrs and then extraction was done overnight without shaking at room temperature in the dark. The optical density of the extracted solution was measured at 570 nm using 200 ul of sample.

[0218] Supernatants from 2 hrs time points were used to measure cytokines by using the LEGENDplex Multi-Analyte Flow Assay (Cat No 740808). Cytokine levels were reported as Mean Fluorescent Units (MFI).

[0219] ATP was measured in the supernatants by using ATP Determination Kit (Cat No A22066) https: / / www.thermofisher.com / order / catalog / product / A22066.

[0220] As shown in FIG. 15A, exposure of ATP and LPS resulted in significant decrease in the cell viability. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the viability inhibiting impact of ATP and LPS. As shown in FIG. 15B, exposure of ATP or LPS resulted in significant increase in IL-8 levels in the culture supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IL-8 when tissues were treated with ATP and LPS.

[0221] As shown in FIG. 15C, exposure of ATP or LPS resulted in significant increase in IL- 18 levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IL-18 when tissues were treated with ATP and LPS. As shown in FIG. 15D, exposure of ATP and LPS resulted in significant increase in IFN-y levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IFN-y when tissues were treated with ATP and LPS.

[0222] As shown in FIG. 15E, exposure of ATP or LPS resulted in significant increase in IL-33 levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of IL-33 when tissues were treated with ATP and LPS. As shown in FIG. 15F, exposure of ATP and LPS resulted in significant increase in MCP-1 levels in the supernatant. Addition of acid phosphatase CRC25100 or CRC34822 was able to negate the accumulation of MCP-1 when tissues were treated with ATP and LPS.SEQUENCE TABLE

Claims

CLAIMSWe claim:

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 said enzyme is active at least from about pH 3.5 to pH 9.

3. The composition of claim 1 or claim 2, wherein the enzyme comprises an apyrase.

4. The composition of claim 1 or claim 2, wherein the enzyme comprises an 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 potato apyrase.

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

7. The composition of any one of claims 1-6, wherein 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% compared to a reference.

8. The composition of claim 7, wherein the one or more inflammatory mediators comprise TNF-alpha, IL-6, IL17A, IFNy, IL-8 or a combination thereof.

9. The composition of any one of claims 1-8, wherein the enzyme is capable of reducing the level cell death 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 a polypeptide that 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 the amino acid sequence of any one of 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:93.

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

12. The composition of any one of claims 1-11, wherein the enzyme comprises a polypeptide with 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 polypeptide containing a truncation in the C-terminal 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 with 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 a toothpaste, a prophylactic paste, a tooth powder, a tooth polish, a tooth gel, a chewing gum, a lozenge, a mouthwash, mouth rinse, a whitening strip, patch, suspension, emulsion, hydrogel, paste, multiphase solution, a paint-on gel, varnish, veneer, and tube, syringe or dental tray comprising a gel or paste, or a gel or paste coated on an application support such as dental floss or a toothbrush.

16. The composition of any one of claims 1-15, wherein the skin care component comprises an ointment, a serum, hydrogel, solution, dressing, moisturizer, exfoliator, body lotion, eye cream, sunscreen, toner, cleanser, skin wash, 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 the enzyme of any one of claims 1-17.

19. A vector comprising the nucleic acid of claim 18.

20. A recombinant host cell comprising the enzyme of any one of claims 1-17, the nucleic acid of claim 18, and / or the vector of 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 E. coli cell, a Yarrowia cell, an Aspergillus cell, Pichia cell or a Trichoderma reesei cell.

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

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

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

26. The method of claim 25, wherein the oral health condition comprises an oral health disease 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 comprises an open sore and / or wound selected from canker sores, cold sores, cuts, bite wounds, 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 mediators comprise TNF-alpha, IL-6, IFNy, 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 associated with 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, an autoimmune disease, cuts, bite wounds, burns, surgical incisions, and tissue trauma.

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

34. The method of claim 33, wherein the inflammatory mediators comprise IL6, IL-17A, IFNy, IL-8 or a combination thereof.

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

36. The method of claim 35, wherein the inflammatory mediators are associated with an oral health condition.

37. The method of claim 36, wherein the oral health condition comprises an oral health disease 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 comprises an open sore and / or wound selected from canker sores, cold sores, cuts, bite wounds, burns, surgical incisions, and tissue trauma.

39. The method of any one of claims 35-38, wherein the inflammatory mediators comprise TNF-alpha, IL-6, IFNy, IL17A or a combination thereof.

40. The method of claim 35, wherein the inflammatory mediators are 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 disease, cuts, bite wounds, burns, surgical incisions, and tissue trauma.

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

43. The method of any one of claims 35-42, wherein the inflammatory mediators are produced by a macrophage, dendritic cell, neutrophil or epithelial cell.

44. The method of any one of claims 35-41, wherein the level of inflammatory mediators are 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% in comparison to the level of inflammatory mediators in the subject before administration of the composition.

45. The method of any one of claims 23-44, wherein the composition is capable of reducing the level cell death 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% in comparison to the level of inflammatory mediators in the subject before administration of the composition.

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

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

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

49. A method of increasing commensal bacteria of the skin and / or oral cavity, comprising administering to the subject an effective amount of the composition of any one of claims 1- 17.

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

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

52. The method of claim 51, wherein the epithelial cells are from a mammal.

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

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

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

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

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

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