Modified catalytically inactive forms of chymotrypsin like elastase 2a (cela2a) and uses thereof

Abstract

The present application provides modified Chymotrypsin-like ELAstase 2A (CELA2A) proteins such as catalytically inactivate and protease resistant CELA2A proteins. Also provided herein are the uses of the CELA2A proteins for preserving pancreatic islet size, number as well as enhancing insulin secreting and insulin signaling and for the treatment of diabetes, pancreatitis and other related diseases.

Description

MODIFIED CATALYTICALLY INACTIVE FORMS OF CHYMOTRYPSIN LIKE ELASTASE 2A (CELA2A) AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No. 63 / 718,183, filed November 08, 2024, the contents of which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under HL135767 and DK134329 awarded by National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 3, 2025, is named 251609_000151_SL.xml and is 85,191 bytes in size.FIELD OF THE INVENTION

[0004] The present invention relates to modified Chymotrypsin-like ELAstase 2A (CELA2A) proteins, and their uses for the treatment of diabetes, pancreatitis and other related diseases.BACKGROUND

[0005] Metabolic syndrome is a complex set of traits that includes obesity, insulin resistance, hyperlipidemia and hypertension and is the fastest-growing risk factor for type 2 diabetes and atherosclerosis. However, the underlying mechanisms that link these diverse traits are not well understood.SUMMARY OF INVENTION

[0006] In one aspect, the present disclosure provides an isolated recombinant Chymotrypsin-like ELAstase 2A (rCELA2A) protein, comprising a substitution of the arginine residue at position 28 with another basic amino acid residue, wherein the residue position numbering iswith respect to a wild-type CELA2A preproenzyme, and wherein the rCELA2A protein is a proenzyme or preproenzyme, or a functional fragment or derivative thereof.

[0007] In some embodiments of the rCELA2A protein described herein, the wild-type CELA2A preproenzyme comprises the amino acid sequence of SEQ ID NO: 1.

[0008] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein has at least 80% amino acid sequence identity to SEQ ID NO: 1.

[0009] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein is resistant to proteolytic cleavage by trypsin.

[0010] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein exhibits reduced or no elastase activity as compared to a wild-type CELA2A enzyme.

[0011] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein does not comprise a signal peptide. In some embodiments of the rCELA2A protein described herein, the signal peptide comprises the amino acid sequence of SEQ ID NO: 7.

[0012] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein comprises an activation peptide. In some embodiments of the rCELA2A protein described herein, the activation peptide comprises the amino acid sequence of SEQ ID NO: 8.

[0013] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein comprises a substitution of the arginine residue at position 28 with a histidine residue (R28H). In some embodiments of the rCELA2A protein described herein, the rCELA2A protein comprising a R28H substitution comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments of the rCELA2A protein described herein, the rCELA2A protein comprising a R28H substitution consists of the amino acid sequence of SEQ ID NO: 9.

[0014] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein comprises a substitution of the arginine residue at position 28 with a lysine residue (R28K). In some embodiments of the rCELA2A protein described herein, the rCELA2A protein comprising a R28K substitution comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments of the rCELA2A protein described herein, the rCELA2A protein comprising a R28K substitution consists of the amino acid sequence of SEQ ID NO: 11.

[0015] In some embodiments of the rCELA2A protein described herein, the rCELA2A protein is produced in a eukaryotic host cell. In some embodiments, the eukaryotic host cell is THP-1 cell or a human embryonic kidney (HEK) cell. In some embodiments, the rCELA2A protein is produced in a prokaryotic host cell.

[0016] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding the rCELA2A protein described herein.

[0017] In one aspect, the present disclosure provides a vector comprising the polynucleotide described herein.

[0018] In one aspect, the present disclosure provides a host cell comprising the polynucleotide described herein, or the vector described herein. In some embodiments, the host cell is a THP- 1 cell or a human embryonic kidney (HEK) cell.

[0019] In one aspect, the present disclosure provides a pharmaceutical composition comprising the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, and a pharmaceutically acceptable excipient or carrier.

[0020] In one aspect, the present disclosure provides a method of treating a metabolic syndrome in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein. In some embodiments, the metabolic syndrome is diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis.

[0021] In one aspect, the present disclosure provides a method for preserving pancreatic beta cell structure and / or function in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein.

[0022] In one aspect, the present disclosure provides a method of preserving pancreatic islet size and / or number in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein of described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein.

[0023] In one aspect, the present disclosure provides a method of augmenting insulin secretion and / or enhancing insulin signaling in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein.

[0024] In various embodiments of the methods described herein, the subject has or is at risk of developing diabetes.

[0025] In various embodiments of the methods described herein, the diabetes is a type 1 diabetes or type 2 diabetes.

[0026] In one aspect, the present disclosure provides a method of treating pancreatitis in a subject in need thereof, comprising administering to the subject an effective amount of aCELA2A protein, or a polynucleotide or a vector comprising a nucleotide sequence encoding the CELA2A protein, or a pharmaceutical composition comprising said protein or polynucleotide or vector.

[0027] In some embodiments of the method of treating pancreatitis, the CELA2A protein is a wild-type CELA2A enzyme, proenzyme, or preproenzyme, or a functional fragment or derivative thereof. In some embodiments, the wild-type CELA2A protein comprises the amino acid sequence of SEQ ID NO: 1, 3 or 5. In some embodiments, the CELA2A protein is a mutant protein resistant to proteolytic cleavage by trypsin.

[0028] In some embodiments of the method of treating pancreatitis, the CEL, A2A protein is a mutant protein which exhibits reduced, or no elastase activity as compared to a wild-type CELA2A enzyme. In some embodiments, the mutant CELA2A protein is a proenzyme, or preproenzyme, or a functional fragment or derivative thereof.

[0029] In some embodiments of the method of treating pancreatitis, the method comprises administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein.

[0030] In some embodiments of the method of treating pancreatitis, the pancreatitis is chronic pancreatitis.

[0031] In some embodiments of the method of treating pancreatitis, administration of said CELA2A protein, polynucleotide, vector, or pharmaceutical composition reduces pancreatic inflammation in pancreatic islets.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 depicts amino acid sequences of the CELA2 A region flanking Asp 121, Leu85, and Thr70 showing their conservation in a variety of vertebrate species (top panel), and a schematic representation of CELA2A protein primary structure depicting the locations of amino acid substitutions and the splice site mutation in relation to different protein domains (bottom panel).

[0033] FIGS. 2A-2L depict mouse and human Cela2a expression. Cela2a mRNA (FIG.2A) and protein levels (FIG. 2B) in different mouse tissues. Immunohistochemical (IH) staining of CEL A2A in the human pancreas and small and large intestine (FIG.2C; for other tissues please see Esteghamat & Mani, 2019

[0034] ). Total plasma CELA2A levels (FIG. 2D) and elastase activity (FIG.2E) in p. D121N carriers vs. noncarriers measured by validated ELISA. Baseline and postprandial plasma CELA2A (FIG.2F), insulin (FIG.2G) and C -peptide (FIG.2H) andthe correlation of baseline and postprandial plasma insulin (FIG. 21) and C-peptide (FIG. 2 J) with plasma CELA2A in random healthy subjects. Plasma glucose and CELA2A levels during hyperglycemic clamps (FIG. 2K) and oral glucose tolerance test (OGTT) (FIG. 2L) in obese subjects. Unpaired t-test 2-tailed, Welch-corrected. ** denotes p <0.01.

[0034] FIGS. 3A-3E depict Cela2a induction of insulin secretion in vivo and rat islets. Plasma glucose (FIG. 3A), C-Peptide (FIG. 3B) and insulin to glucose ratios (FIG. 3C) (mean + s.e.m.) in hyperlipidemic and hyperglycemic Ldlr- / -mice injected with rhCela2a or vehicle. Insulin response of rat islets to rCela2a (FIG. 3D). Area under the curve (AUC) of Ca2+transient upon treatment of INS-1 cells with rCela2a (FIG. 3E). Unpaired t-test 2-tailed, Welch-corrected. ** denotes p <0.01.

[0035] FIGS. 4A-4P depict Cela2a KO mouse construct (FIG. 4A), gross appearance and white adipose tissues (WAT) (FIG. 4B), body weight (FIG. 4C), free fatty acids (FFA) (FIG.4D), cholesterol (FIG.4E) and triglycerides (TG) (FIG.4F) compared to wild-type (WT) mice. Intraperitoneal insulin tolerance test (ipITT) (FIG. 4J) and glucose and insulin during intraperitoneal glucose tolerance test ipGTT (FIGS. 4K, 4L) pancreatic islet quantity and size (FIG. 4M), apoptosis (FIG. 4N), senescence (FIG. 40) and pyroptosis (FIG. 4P) in Cela2a KO mice compared to WT mice are shown, n > 5 mice each, 1-way analysis of variance (ANOVA), Tukey’s post hoc test. * and ** denote p<0.05, and p<0.005, respectively.

[0036] FIGS. 5A-5L depict pancreatic islet matrix proteins (FIGS.5A-5B), volcano plot of the proteomics (FIG. 5C), pathway analysis of the proteomics data (FIG. 5D), western blot analysis of the matrix metalloproteinases (MMPs) and integrins (FIG. 5E), integrin-linked kinase (IL K)- protein kinase B (PKB / AKT) signaling, and immunofluorescence staining for selected MMPs, integrins, ILK and Collagen A1 (FIGS.5G-5L) for Cela2a KO mice compared to WT mice are shown. Unpaired t-test 2-tailed, Welch-corrected. * and ** denote p<0.()5, and p<0.005, respectively. Scale bars: 20 pm.

[0037] FIGS. 6A-6E depict increased immune cell infiltration and reduced vascularization in the islets of Cela2a KO mice. Increased number of F4 / 80 and CD11C-positive cells (FIG.6A), CD68-positive T cells (FIG. 6B), CD3-positive T cells (FIG. 6C), and CD45-positive leukocytes (FIG. 6D) in the islets of Cela2a KO vs. WT mice. Reduced islet vascularization in Cela2a KO mice vs. WT mice, as indicated by diminished CD31 staining (FIG. 6E). The quantitative analysis represents mean ± SEM from n = 10 mice per group. Statistical significance was determined using [statistical test]; *p < 0.05, **p < 0.01, ***p < 0.001 in the islet of Cela2a KO mice vs. wild-type littermates. Unpaired t-test 2-tailed, Welch-corrected. * and ** denote p<0.05, and p<0.005, respectively. Scale bars: 20 pm.

[0038] FIGS. 7A-7L show total blood cell count (FIG. 7A), IL-2 (FIG. 7B), IL- 16 (FIG.7C), IL-20 (FIG. 7D), the C-C motif chernokine 17 (CCL17) (FIG. 7E), Macrophage-derived chemokine (MDC) (FIG. 7F), Eotaxin (FIG. 7G), IFNb-1 (FIG. 7H), and IL-10 (FIG. 71) in Cela2a KO vs. WT mice. CELA2A protein in mice and human macrophages (FIGS. 7J-7K). The rise of CELA2A mRNA levels in human macrophages in response to lipopolysaccharide (LPS) and combined LPS and phorbol 12-myristate 13-acetate (PMA) stimulation is shown (FIG. 7L). Unpaired t-test 2-tailed, Welch-corrected. *, ** and *** denote p <0.05, p<0.005 and p<0.001, respectively.

[0039] FIGS.8A-8I show induction of CELA2A under different conditions and its PAR Independent effects on insulin secretion. CELA2A expression in THP-1 cells upon stimulation with PMA to transform to macrophages (100 ng / mL) (FIG. 8A). CELA2A levels rise in response to higher doses of LPS (to induce inflammation) (FIG. 8B). CELA2A levels rose in the plasma after the meal (FIG. 8C). The parallel rise of C -peptide before and after total pancreatectomy and intrahepatic autotransplantation (TPIAT) (FIG. 8D). Nonsignificant increase in insulin secretion from INS - 1 cells treated with the not purified supernatant of THP-1 cells (FIG. 8E). Insulin secretion from INS-1 cells in response to treatment with the purified wild-type but not mutant rCELA2A proenzymes with and without treatment with PMA (same figure) (FIG. 8F). The effect of wild-type rCELA2A proenzyme in the induction of insulin secretion faded after the treatment of INS-1 cells with PAR1 and combined PAR1 and PAR2 inhibitors (FIG.8G) and after treatment with trypsin (FIG.8H). ERK activation in INS-1 cells after treatment with wild-type rCELA2A vs. D121N CELA2A, assayed by immunofluorescence staining using antibodies against pERK (FIG. 8I). Unpaired t-test 2-tailed, Welch-corrected. *, ** and *** denote p<0.05, p<0.005 and p<0.001. Scale bars: 20 pm. CTL denotes control, WTAV: wild-type CELA2A; D: D121N; L: L85M; T: T70M.

[0040] FIGS. 9A-9D depict ERK and NF-kB in THP-1 cells in response to mutant vs. WT CELA2As with and without treatment with PMA and LPS stimulation before (FIGS. 9A, 9B) and after (FIGS. 9C, 9D) PAR2 inhibition Unpaired t-test, 2-tailed, Welch-corrected. * Denotes p<0.05. CTL denotes control, WT / W: wild-type CELA2A, D: D121N, L: L85M, T: T70M.

[0041] FIGS. 10A-10F depict the expression levels of CELA2A (FIG. 10A), TNF-a (FIG.10B), CXCL8 (FIG. 10C), CCL5 (FIG. 10D), IL- 10 (FIG. 10E), ABCA1 (FIG. 10F) in THP-1 cells expressing wild-type CELA2A and different mutant CELA2A. Unpaired t-test, 2-tailed, Welch-corrected. *, ** and *** denote p<0.05, p<0.005 and p<0.001, respectively. Scale bars: 20 pm. CTL denotes control, WTAV: wild-type CELA2A; D: D121N; L: L85M; T: T70M.

[0042] FIGS. 11A-11F show effects of Cela2a-deficient bone marrow-derived cell (BMDC) transplantation on insulin resistance, pancreatic islet function, and inflammation in wild-type (WT) mice. Intraperitoneal insulin tolerance test (ipITT) conducted in 12-week-old mice transplanted with Cela2a-deficient BMDCs (FIG. HA). Intraperitoneal glucose tolerance test (ipGTT) (FIG. 11B, FIG. 11C) and histological analysis of pancreatic islets (FIG. HD) in 18 weeks old mice transplanted with Cela2a-deficient BMDCs. Reduced extracellular matrix (ECM) (FIG. HE, FIG. HF), increased expression of MMP2 (FIG. 11G), MMP12 (FIG.11H), MMP9 (FIG. HI), and MMP7 (FIG. HJ), elevated infiltration of CD3-positive (FIG. HI) and CD45-positive cells (FIG. HJ), and reduced levels of integrins ITGB4 (FIG. HL) and ITGA5 (FIG. HK) in the islets of mice transplanted with Cela2a-deficient BMDCs compared to WT controls. Unpaired t-test 2-tailed, Welch-corrected. *, and ** denote p<0.05, and p<0.005.

[0043] FIGS. 12A-12B show induction of insulin secretion in INS-1 cells by wild-type, and modified rhCELA2A proenzymes R28H, R28K, and the PAR2 agonist 2-Furoyl-LIGRL-Amide vs. control (PBS) with and without PAR2 and PAR1+ PAR2 antagonists (FIG. 12A). The elastase activities of the wild-type and modified CELA2A proenzyme before and after trypsin-digestion (FIG. 12B).

[0044] FIGS. 13A-13M show protective effects of modified CEL A2A proenzymes on pancreatic islet structure, cilia maintenance, and inflammation. (FIG. 13A) Loss of beta cell cilia in the pancreatic islets of Cela2a knockout (KO) mice and (FIG. 13B) in the islets from mice transplanted with Cela2aKO bone marrow-derived cells (BMDCs). (FIGS. 13C-13D) Ciliary structure in INS-1 cells under baseline conditions, treated with (FIGS. 13E-13F) wildtype CELA2A, (FIGS. 13G-13H) the pathogenic CELA2A variant D121, (FIGS. 13I-13J) modified CELA2A variant R28H, and (FIGS. 13K-13L) modified CELA2A variant R28K, each one before and after digestion with trypsin. Only modified CELA2A variants R28K and R28H maintain the ciliary structure after treatment with trypsin. (FIG. 13M) Decreased expression of CD11C and F4 / 80, markers of Ml macrophages, in human islets treated with the modified R28H CELA2A variant compared to increased expression in islets treated with the pathogenic D121N CEL. A2A variant. Quantitative data represent mean + SEM from n = 3 experiments. Statistical significance was determined using [statistical test]: *p < 0.05, ***p < 0.001, ***p < 0.0001.

[0045] FIG. 14 depicts the functions of the wild-type CELA2A and the CELA2A variants.DETAILED DESCRIPTIONDefinitions

[0046] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present application. For purposes of the present application, the following terms are defined.

[0047] It is understood that embodiments of the application described terms of “comprising” herein include “consisting of” and / or “consisting essentially of’ embodiments.

[0048] The terms “polypeptide” and “protein” used interchangeably herein encompass native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide or protein may be monomeric or polymeric.

[0049] The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation has one to four of the following: (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide or protein that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

[0050] The term “functional fragment” as used herein refers to a fragment of the polypeptide or protein, or a polynucleotide encoding the polypeptide or protein, that retains at least one function of the full-length reference polypeptide or protein. A functional fragment may comprise an amino acid sequence of at least 5 contiguous amino acid residues, at least 6 contiguous amino acid residues, at least 7 contiguous amino acid residues, at least 8 contiguous amino acid residues, at least 9 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 11 contiguous amino acid residues, at least 12 contiguous amino acid residues, at least 13 contiguous amino acid residues, at least 14 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 aminoacid residues, at least contiguous 100 amino acid residues, at least contiguous 12.5 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues of the amino acid sequence of the full-length polypeptide or protein. The functional fragment of a polypeptide or protein may retain one, two, three, four, five, or more functions of the full-length protein or polypeptide. For example, a functional fragment of a recombinant Chymotrypsin-like ELAstase 2A (rCELA2A) protein may retain the ability to be resistant to the proteolytic effects of trypsin or other proteases, and / or to induce insulin secretion, and / or to protect pancreatic structure and function by guarding human pancreatic islets against inflammation and preserving the ciliary structure of beta cells.

[0051] The term “derivative” as used herein refers to a polypeptide, or polynucleotide, or a variant or analog thereof, comprising one or more mutations and / or chemical modifications as compared to a reference polypeptide or polynucleotide. Mutations and / or chemical modifications are further detailed below and can include, for example, insertions, substitutions, deletions, transversions, and / or inversions at one or more locations in the amino acid or nucleotide sequence.

[0052] In certain embodiments, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the derivative polypeptide molecule. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N. Y. (1991)); and Thornton et al., Nature 354:105 (1991), which are each incorporated herein by reference. As used herein, the twenty naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology — A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0053] The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.

[0054] The term “isolated polynucleotide” as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin or source of derivation, die “isolated polynucleotide” has one to three of the following: (1) is not associated with all or a portion of a polynucleotides with which the “isolated polynucleotide”is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0055] The terms “percent (%) sequence identity” or “homology” with respect to the polypeptide and nucleotide sequences described herein is defined as the percentage of amino acid or nucleic acid residues in a candidate sequence that are identical with the amino acid or nucleic acid residues in the reference sequence being compared after aligning the sequences. In some cases, conservative substitutions are considered as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R. C., Nucleic Acids Research 32(5): 1792- 1797, 2004; Edgar, R. C., BMC Bioinformatics 5(1): 113, 2004, each of which are incorporated herein by reference in their entirety for all purposes).

[0056] The term “vector”, as used herein, means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).

[0057] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease or disorder, diminishing the extent of the disease or disorder, stabilizing the disease or disorder (e.g., preventing or delaying the worsening of tiredisease or disorder), preventing or delaying the spread of the disease or disorder, preventing or delaying the recurrence of the disease or disorder, delay or slowing the progression of the disease or disorder, ameliorating the disease or disorder state, providing a remission (partial or total) of the disease or disorder, decreasing the dose of one or more other medications required to treat the disease or disorder, increasing the quality of life, and / or prolonging survival. The methods of the application contemplate any one or more of these aspects of treatment. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0058] The term “effective amount” used herein refers to an amount of an agent or composition sufficient to treat a specified state, disorder, condition, or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms (e.g., clinical or sub-clinical symptoms). For therapeutic use, beneficial or desired results include, e.g., decreasing one or more symptoms resulting from the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and / or prolonging survival of patients. An effective amount can be administered in one or more administrations. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0059] The term “simultaneous administration,” as used herein, means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition comprising both a first and second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition).

[0060] As used herein, the term “sequential administration” means that the first therapy and second therapy in a combination therapy are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either tlie first therapy or the second therapy may be administered first. The first and second therapiesare contained in separate compositions, which may be contained in the same or different packages or kits.

[0061] As used herein, the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.

[0062] As used herein, by “pharmaceutically acceptable” or “pharmacologi cally compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U. S. Food and Drug administration or other state / federal government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0063] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin, incorporated by reference in its entirety for all purposes.

[0064] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the subject is a human. In a preferred embodiment, the subject is a human.

[0065] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In certain embodiments, a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variationencompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0066] The term “about X-Y” used herein has the same meaning as “about X to about Y.”

[0067] As used herein and in the appended claims, the singular forms “a,” “an,” “or” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure. As is apparent to one skilled in the art, a subject assessed, selected for, and / or receiving treatment is a subject in need of such activities.

[0068] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rded. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ: Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan etal. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U. S. Patent No. 7,912,698 and U. S. Patent Appl. Pub. Nos. 2011 / 0202322 and 2011 / 0307437, each of which is incorporated by reference in their entirety for all purposes.

[0069] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.Recombinant Chymotrypsin-like ELAstase 2A (rCELA2A) proteins

[0070] The present application describes, among other things, modified, catalytically inactive and protease resistance recombinant Chymotrypsin-like ELAstase 2A (rCELA2A) proteins as a treatment to preserve the pancreatic beta cell structure and function. Non-limiting examples of such proteins include, e.g., CELA2A with R28K and R28H modification,

[0071] CELA2 A is a pancreatic elastase that was initially recognized for its role as a digestive enzyme. It is secreted into the pancreatic duct as a catalytically inactive proenzyme that is activated in the gut by proteases such as trypsin. Rare nonconservative loss of function mutations in the CELA2A gene have been identified as the underlying cause of early-onset atherosclerosis, type 2 diabetes (T2D) and metabolic traits such as obesity, hypertriglyceridemia, and hypertension (Esteghamat & Mani, Nature Genetics. 2019).

[0072] CELA2A is a circulating protein, with plasma levels that rise in response to meals, in parallel to insulin levels. The data presented herein demonstrate that rCELA2A stimulates insulin secretion in hyperglycemic mice but not in normoglycemic ones. Tills effect was confirmed to be direct by its ability to enhance insulin secretion in isolated rat islets.Additionally, CELA2A was found to improve insulin sensitivity in peripheral tissues.

[0073] By generating mice deficient for Cela2a, the role of CELA2A in preserving islet size and number was discovered herein. Mice deficient for Cela2a are hypoinsulinemic, have diminutive and reduced number of pancreatic islets that have altered structure due to the loss of matrix proteins. The pancreatic tissues in Cela2aKO mice are infiltrated with inflammatory cells. The present disclosure shows that CELA2A is expressed in monocytes, neutrophils and macrophages. Transplantation of bone marrow-derived cells from Cela2a-deficient mice into wild-type mice resulted in the same phenotype, suggesting that pancreatic inflammation is a cell-autonomous effect. Conversely, transplantation of wild-type bone marrow-derived cells rescued the phenotype in Cela2a knockout mice.

[0074] CELA2A monomer is a 25kDa molecule that is rapidly filtered and excreted by the kidney. Its biological half-life time is increased by binding to protein such as albumin and lipoproteins as a 75kDa oligomer. The secreted proenzyme is activated by digestive proteases in the gut and potentially by circulating proteases when it enters the blood circulation.

[0075] The present disclosure is based, in pail, on the discovery that the catalytic activity of CELA2A is not necessary for its insulinotropic effect. This significant discovery highlights catalytically inactive CELA2A as a promising therapeutic candidate, offering potential safetyadvantages as elastases can be destructive to the artery.

[0076] The CELA2A proenzyme is activated through catalytic cleavage at arginine 28. By mutating arginine 28 to lysine and histidine— amino acids in the same group that cause only minor structural changes - two CELA2A proenzymes were generated that are resistant toproteases but retain two key functions: (1) inducing insulin secretion, and (2.) protecting pancreatic structure and function by guarding human pancreatic islets against inflammation and preserving the ciliary structure of beta cells.

[0077] Accordingly, in some embodiments, the present disclosure provides a modified CELA2A protein (e.g., a recombinant human CELA2A proenzyme) that is catalytically inactive. The modified CELA2A protein may be resistant to the proteolytic effects of trypsin or other proteases. The modified CELA2A proenzyme can have significantly more potent insulinotropic effects compared to the wild-type proenzyme. The detailed results described in the Examples section below' support the role of modified CEL, A2A as a potential therapy for the treatment of diabetes and pancreatitis.

[0078] In one aspect, the present disclosure provides an isolated recombinant Chymotrypsin-like ELAstase 2A (rCEL, A2A) protein, comprising a substitution of the arginine residue at position 28 with another basic amino acid residue, wherein the residue position numbering is with respect to a wild-type CELA2A preproenzyme.

[0079] In some embodiments, the rCELA2A protein is a proenzyme or preproenzyme, or a functional fragment or derivative thereof.

[0080] In some embodiments, the rCELA2A protein is a proenzyme.

[0081] In some embodiments, the rCELA2 A protein is a preproenzyme.

[0082] Amino acid sequences of exemplary wild-type CEL, A2A preproenzyme, proenzyme, and active enzyme are listed below.

[0083] Wild-type CELA2A preproenzyme amino acid sequence WRIZ, Z, L. S,77AAGAL. S,CGDPTYPPYVTRVVGGEEARPNSWPWOVSLOYSSNGKWYHT CGGSLIANSWVLTAAHCISSSRTYRVGLGRHNLYVAESGSLAVSVSKIVVHKDWNS NQISKGNDIALLKLANPVSLTDKIQLACLPPAGTILPNNYPCYVTGWGRLQTNGAVP DVLQQGRLLVVDYATCSSSAWWGSSVKTSMICAGGDGVISSCNGDSGGPLNCQASD GRWQVHGIVSFGSRLGCNYYHKPSVFTRVSNYIDWINSVIANN (SEQ ID NO: 1) (signal sequence is in italics; activation peptide is underlined)

[0084] Wild-type CEL, A2A preproenzyme nucleotide sequence ATGATAAGGACGCTGCTGCTGTCCACTTTGGTGGCTGGAGCCCTCAGTiGGGGGGACCCC Z\CTTZ\CCCACCTTATGTGZ\CTAGGGTGGTTGGCGGTGZAG-AZ\GCGZ\GGCCCZACAGCTGG CCCTGGCAGGTCTCCCTGCAGTACAGCTCCAATGGCAAGTGGTACCACACCTGCGGAGGG TCCCTGATAGCCZVACAGCTGGGTCCTGACGGCTGCCCACTGCATCAGCTCCTCCAGGACC TZcCCGCGTGGGGCTGGGCCGGCACZ CTCTZcCGTTGCGGAGTCCGGCTCGCTGGCAGTC AGTGTCTCTAAGATTGTGGTGCACAAGGACTGGAACTCCAACCAAATCTCCAAAGGGAAC GACATTGCCCTGCTCAZVACTGGCTxAACCCCGTCTCCCTCACCGACAAGATCCAGCTGGCCTGCCTCCCTCCTGCCGGCZ\CCATTCTZ\CCCZZ\CZZ\CTZ\CCCCTGCTACGTCZ\CGGGCTGGGGAAGGCTGCAGACCAACGGGGCTGTTCCTGATGTCCTGCAGCAGGGCCGGTTGCTGGTTGTGGAC ±A±GCCACCTGCTCCAGCTCTGCCTGGTGGGGCAGCAGCG±GAAAACCAG±ATG ATCTGTGCTGGGGGTGATGGCGTGATCTCCAGCTGCAACGGAGACTCTGGCGGGCCACTG AACTGTCAGGCGTCTGACGGCCGGTGGCAGGTGCACGGCATCGTCAGCTTCGGGTCTCGC CTCGGCTGCZVACTACTACCACZkAGCCCTCCGTCTTCACGCGGGTCTCCZkATTACATCGAC TGGATC. AATTCGGTGATTGCA. AATAACTAA (SEQ ID NO: 2 )(signal sequence is in italics; activation peptide is underlined)

[0085] Wild-type CELA2A proenzyme amino acid sequence CGDPTYPPYVTRVVGGEEARPNSWPWOVSLOYSSNGKWYHTCGGSLIANSWVLTA AHCISSSRTYRVGLGRHNLYVAESGSLAVSVSKIVVHKDWNSNQISKGNDIALLKLA NPVSLTDKIQLACLPPAGTILPNNYPCYVTGWGRLQTNGAVPDVLQQGRLLVVDYA TCSSSAWWGSSVKTSMICAGGDGVISSCNGDSGGPLNCQASDGRWQVHGIVSFGSR LGCNYYHKPSVFI’RVSNYIDWINSVIANN (SEQ ID NO: 3)(activation peptide is underlined)

[0086] Wild-type CELA2A proenzyme nucleotide sequence TGTGGGGACCCCACTTACCCACCTTATGTGACTAGGGTGGTTGGCGGTGAAGAAGCGAGG CCCAACAGCTGGCCCTGGCAGGTCTCCCTGCAGTACAGCTCCAATGGCAAGTGGTACCAC ACCTGCGGAGGGTCCCTGATAGCCZVACAGCTGGGTCCTGACGGCTGCCCACTGCATCAGC TCCTCCAGGACC TACCGCGTGGGGCTGGGCCGGCACZVACCTCTACGTTGCGGAGTCCGG CTCGCTGGCAGTCAGTGTCTCTAAGATTGTGGTGCACAAGGACTGGAACTCCAACCAAAT CTCCZ\AAGGGZ\ACGACATTGCCCTGCTCAZ\ACTGGCTZ\ACCCCGTCTCCCTCACCGACAA GATCCAGCTGGCCTGCCTCCCTCCTGCCGGCACCATTCTACCCZ\ACAACTACCCCTGCTA CGTCACGGGCTGGGGAAGGCTGCAGACCAACGGGGCTGTTCCTGATGTCCTGCAGCAGGG CCGGTTGCTGGTTGTGGACTATGCCACCTGCTCCAGCTCTGCCTGGTGGGGCAGCAGCGT GZIZWICCZIGTZITGATCTGTGCTGGGGGTGATGGCGTGZITCTCCZIGCTGCZIACGGAGACTC TGGCGGGCCACTGAACTGTCAGGCGTCTGACGGCCGGTGGCAGGTGCACGGCATCGTCAG CTTCGGGTCTCGCCTCGGCTGCZkACTACTACCACAAGCCCTCCGTCTTCACGCGGGTCTC CAATTACATCGACTGGATCAATTCGGTGATTGCAAATAACTAA (SEQ ID NO: 4)

[0087] Wild-type CELA2A active enzyme amino acid sequence VVGGEEARPNSWPWQVSLQYSSNGKWYHTCGGSLIANSWVLTAAHCLSSSRTYRVG LGRHNLYVAESGSLAVSVSKIVVHKDWNSNQISKGNDIALLKLANPVSLTDKIQLAC LPPAGTILPNNYPCYVTGWGRLQTNGAVPDVLQQGRLLVVDYATCSSSAWWGSSV KTSMICAGGDGVISSCNGDSGGPLNCQASDGRWQVHGIVSFGSRLGCNYYHKPSVF TRVSNYIDWINSVIANN (SEQ ID NO: 5)

[0088] Wild-type CELA2A active enzyme nucleotide sequence GTGGTTGGCGGTGAAGAAGCGAGGCCCAACAGCTGGCCCTGGCAGGTCTCCCTGCAGTAC AGCTCCAATGGCAAGTGGTACCACACCTGCGGAGGGTCCCTGATAGCCAACAGCTGGGTC CTGACGGCTGCCCACTGCATCAGCTCCTCCAGGACCTACCGCGTGGGGCTGGGCCGGCAC AACCTCTACGTTGCGGAGTCCGGCTCGCTGGCAGTCAGTGTCTCTAAGATTGTGGTGCACAAGGACTGGAACTCCAACCAAATCTCCAAAGGGAACGACATTGCCCTGCTCAAACTGGCT AACCCCGTCTCCCTCACCGACAAGATCCAGCTGGCCTGCC TCCCTCCTGCCGGCACCAT T CTACCCAACAACTACCCCTGCTACGTCACGGGCTGGGGAAGGCTGCAGACCAACGGGGCT GTTCCTGATGTCCTGCAGCAGGGCCGGTTGCTGGTTGTGGACTATGCCACCTGCTCCAGC TCTGCCTGGTGGGGCAGCAGCGTGAAAACCAGTATGATCTGTGCTGGGGGTGATGGCGTG ATCTCCAGCTGCAACGGAGACTCTGGCGGGCCACTGAACTGTCAGGCGTCTGACGGCCGG TGGCAGGTGCACGGCATCGTCAGCTTCGGGTCTCGCCTCGGCTGCAACTACTACCACAAG CCCTCCGTCTTCACGCGGGTCTCCAATTACATCGACTGGATCAATTCGGTGATTGCAAAT AACIAA (SEQ ID NO: 6)

[0089] The human gene sequences encoding for CELA2A are provided below. > ENST00000359621. 5 CELA2A-201 cdna: protein_coding ACAGAACTCCCACGGACACACCATGATAAGGACGCTGCTGCTGTCCACTTTGGTGGCTGG AGCCCTCAGTTGTGGGGACCCCACTTACCCACCTTATGTGACTAGGGTGGTTGGCGGTGA AGAAGCGAGGCCCAACAGCTGGCCCTGGCAGGTCTCCCTGCAGTACAGCTCCAATGGCAA GTGGTACCACACCTGCGGAGGGTCCCTGATAGCCAACAGCTGGGTCCTGACGGCTGCCCA CTGCATCAGCTCCTCCAGGACCTACCGCGTGGGGCTGGGCCGGCACAACCTCTACGTTGC GGAGTCCGGCTCGCTGGCAGTCAGTGTCTCTAAGATTGTGGTGCACAAGGACTGGAACTC CAACCAAATCTCCAAAGGGAACGACATTGCCCTGCTCAAACTGGCTAACCCCGTCTCCCT CACCGACAAGATCCAGCTGGCCTGCCTCCCTCCTGCCGGCACCATTCTACCCAACAACTA CCCCTGCTACGTCACGGGCTGGGGAAGGCTGCAGACCAACGGGGCTGTTCCTGATGTCCT GCAGCAGGGCCGG GC GG G GGAC GCC CC GC CCAGC C GCC GG GGGG CAGCAGCGTGAAAACCAGTATGATCTGTGCTGGGGGTGATGGCGTGATCTCCAGCTGCAA CGGAGACTCTGGCGGGCCACTGAACTGTCAGGCGTCTGACGGCCGGTGGCAGGTGCACGG CATCG CAGC CGGGTC CGCCTCGGC GCAAC C CCACAAGCCCTCCG C C C GCGGGTCTCCAATTACATCGACTGGATCAATTCGGTGATTGCAAATAACTAACCZIAAAGA AGTCCCTGGGACTGTTTCAGACTTGGAAAGGTCACAGAAGGAAAATAATATAATAAAGTG ACAACT TGCAAATCA (SEQ ID NO: 13)> CELA2A-201 ENSE00001693671 exon: protein__c.oding ACAGAACTCCCACGGACACACCATGATAAGGACGCTGCTGCTGTCCACTTTGGTGGCTGG AG (SEQ ID MO: 14)> CELA2A~201 ENSE00003612435 exon: protein_coding CCCICAGTIGTGGGGACCCCACITACCCACCTTATGTGACIAGGGTGGITGGCGGTGAAG AAGCGAGGCCCAACAGCTGGCCCTGGCAG (SEQ ID NO: 15)> CELA2A-201 ENSE00003549273 exon: protein_coding GTCICCCTGCAGTACAGCTCCAATGGCAAGTGGTACCACACCIGCGGAGGGTCCCTGATA GCCAACAGCTGGGTCCTGACGGCTGCCCACTGCATCAG (SEQ ID NO: 16) > CELA2A-201 ENSE00002685587 exon: protei n__c.odi ng CTCCTCCAGGACCTACCGCGTGGGGCTGGGCCGGCACAACCTCTACGTTGCGGAGTCCGG CTCGCTGGCAGTCAGTGTCTCTAAGATTGTGGTGCA. CAAGGACTGGAACTCCAACCAAAT CTCCAAAGG (SEQ ID NO: 17 )> CELA2A-201 ENSE00001595685 exon: protein_coding GAA. CGACATTGCCCTGCTCAAA. CTGGCTAA. CCCCGTCTCCCTCACCGACAAGATCCA. GCT GGCCTGCCTCCCTCCTGCCGGCACC TTCT CCCAACAACTACCCCTGCTACGTCACGGG CTGGGGAAGGCTGCAGA (SEQ ID NO: 18)> CELA2A-201 ENSE00001637775 exon: protein_coding CC ACGGGGCTGTTCCTGATGTCCTGC GCAGGGCCGGTTGCTGGTTGTGGACT TGCC CCTGCTCCAGCTCTGCCTGGTGGGGCAGCAGCGTGAAHACCAGTATGATCTGTGCTGGGG GTGATGGCGTGATC TCCAGCTGCAAC (SEQ ID NO: 19)> CELA2A-201 ENSE00003532875 exon: protei n__c.odi ngGGAGAC iCi GGCGGGC CAO I GAAC I G I CAGGCG I C i GAO G GO OGG I GGCAGG I GCACGGC ATCGTCAGCTTCGGGTCTCGCCTCGGCTGCAACTACTACCACAAGCCCTCCGTCTTCACG CGGGTCTCCAATTACATCGACTGGATCAATTCG (SEQ ID NO: 20 )> CELA2A-201 ENSE00001885503 exon: protein_coding GTGATTGCAAATAACTZIACCAZIAAGAAGTCCCTGGGACTGTTTCAGACTTGGAAAGGTCA CAGAAGGAAAATAATATAATAAAGTGACAACTATGCAAATCA (SEQ ID NO: 21)CEDA2A-201 intron 1: prote in_ cod ingGTAAGTCC TGTTACCCAGAGGCACTGGTT TCCCATGCCCTGGTGGGGC TGGAAATGGGAT CTTCCTGTCCTCCCCTCTCGCCCCCACCCAACCCCTACTGCATTCAGACCTATAATCATA AGAACATTGGAATGGAGTTTCAAAGAATTGGAGCAAAGAGCAGGATTCTATGACCTCTTG GGATCCTTCTAGAACAAGGGTTTTCTATTTGGGGGGTCAGAATGCGCAGCAAGTGTAGTT TACATTGTGTGGGTCGCTGCTTCCTGACTCAAGACCCTTTCTCTTTTCACAG ( SEQ ID NO:> CELA2A-201 intron 2: prote in_ cod ing GTGAGTTGACCACACTGTACTTCTCCCCGTCCCTGCCCCACTCCCTTTACATCTCCCCTT TGCCCTTCCACCATGGCGTCTATTGTGCTGGCAAAGTGAGTATTGATGGGACTCAGAGAG CAGCACAGGCAACTTTAGCAATAAGATATATTTCCGGCTGGGCGCAGTGGCTCATGCCTG TTATCCCAGCACTGTGGGAGGCTGTGGTGGGCAGATCACCTGAAGTCAGGAGTTTGAGAC CAGCCTGGCCAACATGGTGAAACCTGCCTCTACTAATAATACACAAGTAGCCAGGTGTGG TGGCACACGCCTGTAATCCCAGCAACTGGGGAGGCTTAGGCAGGAGGATCGCTTGAACCC AGGAGGCAGAGGTTGCAGTGAGCTGAAATTGCACCACTGTACTCCAGCCTGGGCAACAAA GCAAGACTCTGTCTCAAAAAAAAAAAAAAAGATACATTTCTGTGGCCTTGGCCAACCTTG GGAGTGGAGGGAGAGGTGCCTCCTCCAGCTGCAGTAAGGAGCTGTCCCCTCCCCACTGCC CATAGGCAGC AAAT AT AGTAT TAG TC T AT TAACCAATCAGAGGC T T GT TTAC AAAT GTAC CATCAGTCTAGGAACCATTCAAGACTACCTATGCAAACATTCCTTGCTTTAAGGAACCAA TCAGTGCTATTTGCGCAGATTAATCTTTAACCACAGGCAAATCAATGTTACCAATGAAAA AAATGTTTTCTTAAGTTGATATAATCATAGTGGTATCAAGACTAAACTGCCAAGTGGGGC AC AGGT GT AAAT TACT CAGAC GTGT TAAT GGTGGGGAT T T TAAGAGAAATGTGT AAAGT T TACAT T GACATA CAAATAT AGTGAAAT CTCAT T TATC T GATGTAAT TGGGAC CGAGT A ATTGAAAAGTTGGTTATAAAGAAATTATTTTAAATCATATATATATAGTAAAGATTTTAT CTTAGACTATGTAAACAATTTTTTGAGTTCTTAACTGTCTTTTTCATATAAGCCATGCCC CATATCTTATCTACAAACCCGTTTCTTTAAAGCAGAGCAGTAACTTTGAGACGTTCACAA AGCAATCTGAGTTATGAGCCCATCCAATCTTTCCATTTTCTCAC GACACT CTTCAAGA GCAATTTTTTAAGAGTCATGGTGATCCAGTCTCTTCCAAGCATTTAACTTTGTTTCCATA TAAATAACGACTCTCTTTCTGAACTCATATTTCATTGGTTACATGTTTAATCAAATTTCA TTATT CAAGC T ATTGATATAAACTGCTTTGGGAGCAAACCCAGCTGAATCAGGATGA GT T TC T AC C T T AAG TAGGAGGAGC AAC TAGC AGC C CAC T AGC C T T GAGCAT T C AGAGT GA CCCATGCAGCAGTCACTGTGTTTAGAGAGGCCAGCGTTCATTCATCTGCTCCTTGATTAA GAGACTGTCAATGATAGGCCAGGCGTGGTGGCTCATGCCTGTAATCCCAGC CTTTGGCA GGCCGAGGCAGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTG AAACTCTGCCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGCGGGCACCTGTAGT CCCAGCTACTTGGGAGGCTGAGGC C AGAATCGCTTGAACCTGGGAGGCGC GGTTGCG GTGAGCCAAGATCACACCTCTGCACTCCAGCCTGGGTGACAGAGTGAGATCCATAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGACTGTCAACGATAGAA TGATGGCAGAT TTTCAC TTTTTTTCTTTTTTTGAGACAGAGTCTTGCTCTGTCACCCA GGCTGGAGTGCAGTGGCACAATCATAGCTCACTGCAGCCTCCAACTCCTGGGCTCTGGCC ATCTACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGCACCCACCACTGTGCTCACC CATAGTAATTTTTTTTTTTTTTTGAGACAGTGTCACATTCTGTCACCCAGGCTGGAGT C AGTGGCTGGTGAAACATGGCTCACAGCGGCCTCGAACTCCTAGGCTCAAGCGATCCTCCC ACCTCAGCCTCCCTATCAGCTGAGACCACAGGTGCATGCCCCCACACCCGGTTAAGTTTC TTTTTTTTTTTTTAGAGATGGGTCTC CTATGTTGCCCAGGCTGGTCTTGAACTTI ITGGG C I CAAG i GAI CC I C C I GCG I C GGCC I C CC AAAG I GC I GGGAI CACAGGCG I GAGCC i C I C AACCCAGCCTCACAATTTATATACTTGGCTTTGTGTGCTTTTGCCAGTTCTTTTGCTTTT CTCACAGTCCC C GTATTTTTTCCCTGAGAATTTGGAAGAAGGGCCACAGTCCCTCTGC TACAGGGCCCCATAACACCGGGCGCATTTGTGCTAAGAACTAATCATTGCTGCCTACAAA AATACTTCCTCACCCTCTTGCACAAAAGTATTCTTGCCTTGAGAACCAGTGAATGTTAAT CCATGAAGAGTCCCTATGCTTTGTGCTTCTGTTTCCCTGCGGAAATAAGGCAGATCTAGC TGGGTGGGGACTCAGATGAGGCAAGTAAGACATTCACTCTGGAGGCAAAATGTAAGGGGG TAGCACAAAACTCAGCAACCAGCCAGGAACAGTGGCACACACCTTTAATCCCAGCTATTC AAGGGGCTGAGGCAGGAGGATTGCTTGAGCCCAAGAGTTTGAGACCAGCATGGGCAACAT AGATAGCCATCTCAAAAAAAAAAAAAAAATACAAACAAACAAAAAACCTCACTAACGAAG ATGAATGATATTTTAATGCAATTTTGTTTCTTCTCCAGGAGACAGAGTCTCATTCTGTCA CCCAGGCTTAATTACAGTATGAGTCACCCTGAGAGCCCTGCACAGTGGCTTATGTTCCCC ATTTCCAGAGAAGGCAACTGGGACTTGGAAAGGTTACAGAATTCCCCAAAGGCACTGGGT TAGCCCGGGGGGCGAATGAGGATTCAAATCTGGTTCAGTGCTACCTCTTTAGCTACACTTTGCTGGCCACACATAATACGTATTATGTGGGCTAAATTTCACATCTGTACGATGGTTTCA GCACATTTTTAAATTGGGGGGGGGGTACATCCTTTTAAGTATCTTTTTTCACTTTTTCTA AT T T T AAAC T C T T T T T T AG CC T C T TAAT GT C CAC AAC GAGAA TGC C AAAT GGC T AAT TAAAAACAACAAC GT TCAGAGAAAAGCAGGCATC TGC CAACAAAC AGGTCATC GT T C CCCACAGTTATAGGGAGCCCGATCAGAGCAGTCCATGCCCTACTGTCACCTCCCTCCCCT CTGCTTGGCCGTGGCAGGGAATTGAAAGACAGGGGAGGAGGAATGAAGGAACAGGAAGGG CATGGAACAGCTTCATTAGAGACTAAAACAGTGTCAAATGTCACCACCTTAAAGTTCTTT TTTTTTTTTTTTAAAGACCTGGTCTCACTCTGTTAATTTTATTTTATTTTAAAATTTTTT TGGTAGGGTCAAGTTCTCACTATGGTGCCCAGGCTGGTTCACAAACTCCTGGGTTCAAGC AATACTCCCACCTCAGCCTTCTGAGTAACCGGGACTATAGGCATGCACCGCCGCACCCAG CGGCTCATTTTGGGCTATTTTTGTTGTAGAGACAATGTATTTAGTCCTGCCTCACACGGC T AATAAAGAC AT AT C T GAGAC T GGGT AAT T T AT AAAGGAAAGAGGT T T AAC T GAC T CAT A GCTCAGCGTGGCTGGGGAGGCCTCAGGAAACTTATAATCATGGCAGAAGGGAAAGGAAAC ACATCCTTGTTCACGCAGCGGTAGCAGGGAGAAGTGCCGAGCAAAAGTGGGGGAAAGACC CTTATAAAACCATCAGATCTTGTGAGAACTTACTCACTATCATGAAAACAGCATGGAGGT GACCACCCTCATGATTCAATTACCTCCCACCAGGTCCCTCGCACGACAAATGGGGATTAT AAGAACTATAATTCAAGATGAGATTTGGGTAGGGACACAGCCAAACCTTATCAGATGGGG TTTCACCATGTTGCCCAGGCTGGTCTCAAACTCCTGGGCTCAAGTGATCCTCCTTCCTTG GCCTCCCGAAGTGTTGGGATTACAAGCATGAGCCACCATGCCTGGCCTGATTGTTTTTCA TGTAGATTGCATTAACACGTTTTTTATCTTGATGGCTGAGGTTTTGGGTGCTGCCTTAAG TTGTGCACATGAGGCGACTGCCTCACTCCCCCTTACCCTTGTCCCAGCCCCGTTCTTGGG AAACTGGAGTGCAGGGAGGCCCTGCTCTTTCAAACCTAGCCACAGAGCTCCTTTGCTTCT CCCCAG ( SEQ ID NO: 23 )> CELA2A-201 intron 3: protein_coding GTAACTGCCTTTCCCTGGGCGCTTGGCCTGCTCACCAGCTGGTGCTCATTTCTGAGCTGG GGGCTCAAATGGCCTGAACCATGCT CATAAAGCAGCCTTGCAAATAACC CT TACTGG CTGAGACACAAGCTGTAGTCAATCAATGGTTCAGTGTGTTGGCCCATCAATGTCAGTACA TGGCATGGATGGAGTGTCTGTGCCAGGCAGGCACTGAGGGAGGATGAAGAGGAGGGGAAG GCCCAATCTCTGCCCTCTTGGGGAAACT C TGTGGCCTCTGGATCTGGAATTGGGTTCC CTTGAACAACTTAACTGACTTCTCAAAGCTTCAATACCATCACCTGCAGAAAAATGGAGT AACAGAATTTAGTGTGAATTCAATGAGTTGATTTACCAAAGGGCCTGGCACTTAGTAAGT GCTCAGTGAAAGCTAATTCTGAATT TAGATGAATTAT AAGGCCCACAAGGTTCTGCAA CGTTGTTCACTTATCCTCTTCTTTGTCCGTGACCTCTGCTCATCACAGCTGGAACTCACC GGAGTTTCTGCCGGGTCGGCGTGACTCCCTACCAGGAGTGATCCCTTCTCCCATCCGATT TATAATAAGCTCTAGGAGGTATTGGGGAAAAT ATAGAGGCCTACAGTCAGGAGTCCTGG CI 1GIAGI 1CIGGI 1 ICI CAC 1 AACGG 1 GGGAC I C 1 GGGC CAGG 11 AC GCC I C I C I GGGC CTCAGTTTCCCCATCTGCAAATGAAAGGTGGGAAAAATGTCCTTGAGGGCAAATGACTCC TGTTTTCTCTGTGTTCATCTCATTCCCTCCATAAGAACTATAGAGTTTGTGTT TTAT A CT TTTATT TTATAT TGTAGTT TATAGT TAGTATAGTAACAAT TATTATATGATCATAGT T CTTAGGTTATATACTTTAGGCCCTATTATAACTAACTGATTGTCCCAGGGGAGGAAAGAT CCCCAAGTCCCATCTAGTGGCTCACGCAGCAGCCAGTT CTTGGGTCTATCCCCAGC TT A I CCAAAGCCAGGC C I C I GGAGG I GAC CC 1CICCC 1GGGCCCCCI 1 1 C 1 CCCAGGGGGGICICA CAGAGGCAAGGGTCTCAACCACACCACACCCCCTCTGCTTCTTCTACAGGGAGGGGGAAG GAGCTCTGGCCTCTTAGATGTGGAACCAGCTCCAAAGATGTCTGGGGTGGGGATGTGACC TGGGGAGGGTGAAGCAAAGACTGCCAGAGCGACCTGGGTTTGCTGCCAGTTACACCTGCA GGGCCCCTGTCCGCTGAGCATGTATCTACTTCATGCCAGGTCCTGGGGACACAGGGACAG TGCTGTCAACCCTGTGACTGTCCCTGGAGTAGGACACAATCTCTCCTGCCTTCCCCCTTT CAACAAGGCCCTCTGCATTTTCAAACATGCCCTGGGGCCCTGCCAGTCAGAGCAACCTGG GGTAACAGGGTACAGGGAAGATGACAAGGTCTCCAAGCCCTCCAAAGCCCACAGGGCAGG AAAAGTCAACCCGGTCCTCATGCTTCGCCTCCACACTCACCCAG ( SEQ ID NO: 25 ) > CELA2A-201 intron 5: prote in_ cod ing GTAAGTGGGAGCCAGGAGCCCCCAGGCCTGGGAGGGAAGGGAGGTGATTCACGTCACCCC TGTCTGGCCGGGGCCTCTCACCTGTCATCCCAGGGTGTGTGGCTGCCTTGGAGAGACGGG ATGGCATAGGCTGACGCCTGCCTGGGATCAAATGTCAGCTCTCCCACTTAATGACTGTGA CACCTTGGACACATTACTAGGTCTCTCCATGCAGCACTTTTTTCATCTGTAAAATGCAAATATTTGGCTGGGCGCAGTGGCTCATACCTGTAATCCCAGCACTTTGGGAGGCCCATCCCG CGGATCAC TTGAGGTCAGGAGACCAGCCTGACCAACACGGAGAAAC TC TGTCTC TACTAA ZVtATATTZW WtZWGAATAGCZtGGATGTGGCAGTGTGCACCTGTAGTCCCAGCTAC TCAGGAGGCTGAGGCACGAGAATCACTTGAACCTGGGAGATGGAGGTTGCAGTGAGCCAA GATTTCGCTACTGCACTCCAGCCTGGGGGACAGAGCGAGACTCCGCCTC L ACAJA AG GCATZ< ATZ\ATZ\GCACCTZ\CATCACZ\GAGTTGTCZ\CGZ\GGATTZ kGGAGATZ< ATCCATAG GAAGCCCAGAATGGGGTAGGTGCTGTGTTAGTTGTTGTCACTGTCCCTATGATGGAGAGA ZVAGTTACAGAGACCATGTGACATCTTCTGTGTGGCCCZVAGGCTAGAGTTCAGZVACAGCAT TTTCCTCTGTGACCTGAGACCCCTGGAGCCCTCATCAGACCCTCCATGCCCCATAGC iAA AGCTGTCCCCTGTGCCATTAGGTCTTGCTGCTCAGAACCTGAATGAGGGCGGGTCTCCTT T GACAT AGAC T C C AAGGGT C A GAGA T C CAAT GAG AGT AGC CACCZVAGAGGZVAC AGAGT T C CCGCCCTTCCCTTGTGA iATCACCAGAGGTTTGGAAGATTCTAGGAAGGGATGAGTAATA ATGA. CCTTA. TGACCA. TAATGAGA. TTATGAGTGTTAA. TCACA. TTATGA. CA. TTTTTTCCTAA. TGACATZkAACCCATZkAGTATAATGACATGTTTTTCCAGGGGTACATCCACATACGTACCC TCAAATAATTCCCATTGCCCTCTTGACTGGCTTATCTGTAAACACAAGGACACATCAAAT CCTGCGGTGAGCGGTGGTGGAA. GAGGCA. TTTCATGCTTATTAA. GATA. CAAGCACAACCTC CTGTTTGTTTCCCCAGAC VACCCTGGGGCAGGCGCATTTCATCTGAGACAGTGAGAGTGG GGAGGCCTCC iATGCTTGGCTTTTCGGCCCAGCTCTGTCAGTCACGGTG i iACCTTGGG CAA. GCCA TTCCGAA CTCAGTTTCCTCATCTGTAAZ\ATGGAAACACTGTTGGTGAA. GAC TAZ ^GAGGTGZAAAGGTGZAAAATGCATCATACATTGCZGAAATGTTATGCZVAGTGTTZkATT Z\ATTACTTATTAGTGGTCTTAAGCCAGACACTTZ\ACTTCCCTTTTTTTTTTTTTTTTTTT TTTTTTTGAGACAGAATCTTACTCTGTTACCCTGGCTGGAATGCAGTGGTGCAATCTCGG CTCACTGCZkACCTCTGCCTCCCGAGCTCAAGCGATTCTCCTGCCTCAGCCTCCCZkAGTAG CTGGCATTATAGGCGAGTGCCACCATGCCCGGGTAATTTTTGTATTTTAGTAGAGGTGGG GTTTCACCA. TGTTGGCCAGGCTGGTCTCAAACTCTTGACCTCAAGTGATCTGCCCACCTT GGCCTCCCTAAGCGCTGGGATTAGAGGGTGAGC CCGCGCCCGACCGAC CTTAACTTCC CTAGGCTTCZ\ATCGGAAGTGAAGZ\ATTGTGTTZ\ATCTCATCCTTGCCACTCATAGCTGTG GTA CAGTAATGGAGGTGATGGTGGCTGTGTCATTGGGGGCCA. TTATGAZ\AGCCCTCCCT TCACC CACC GC TATTTTTCCCAAGGAGGGGGCTCTAACAGCCAGGAGGCTCACTTTC TTATTTCAGATZ\AGTGACAGAAAGCCATGGAGCTAGCTCAGCAZ Z\AAAAGGGZ\AATTGTT ATAAGGA. TTTZ\Z\Z\AGTTTTATCCCZ\AGGGCAGAGATACAGCCA. GGCA. TCAGGZ\ACAA. GCT AGAATCAGGGGCTGGAAACCTGTAAGAAACACAGGAAACCCTCCCTCAGAATTTCTGTTC TTTGTGGGTCTACTTCATTCTCCACATAAAACA \AATGTCAGTCATTGCCAACAGCTTCC CAGAATTATGTCCTCTGZ\Z\ACCGGZ\ATCTCATAGACCCCATCTCAGAATCCCAGGGGAGG G GCTCATTGGCTTAGCTTG GTC GGTGTTC CTGCTG ACC TC ACCGTGAGG CA CAGTATAGACAAACGTGGCTGTTCGCATGTTGCAATGGATGGAAGACAGGAACAGGGGAA ACCTZ\AGACGGGTCCATCACTTCCTTTTTCTCAGGA. GTCCCTGCATCCCTZ\ATGGCTTCT CTCTGATCTCATTCAG (SEQ ID NO: 26)> CELA2A-201 intron 6: protein_coding GTGA. GTAGCZ\JAAATCAGGGGCTCCGCTCCA. TGACAAZ\ATGTGGCTGGGGATZ\AGGCTATA. GAGGTCCATCCCTCCAT GGTCCATCCTCCCACCTCCTGGCAGAATTACCCCGAAACATG TTCCAGATATATCTTTGGCCAGGCACGGTGGCTCACACCTGTAATCCCAGCACTTTGAGA GGCCAGGCGGGCAGACCACTTGAGGTCAGCAGTTCZAAGACCAGCCTGGCCZkACATGGTGA AACCTTGTGTCTACTAAAACTAAAAAAATTAGCCGGGCATGGTGGCAGGCGCCTGTACTC ACAGCTACTCGGGAGGCTGAGGCAGGAGAATCACTTGAACCCATGAGGCGGAGGTTGCAG TGAGCTGAGATCACACCGCTGCZAATCCAGCCTGGGTGACAGAGCGAZAACTCZGAAZAAZAAAA AACAAAAAAACTTGATCTTGGTTCTTTTTTGTTCTTGTTTTGAGATGTTTTGAGATTCTC CTTGTAGTATCCACCCATCTCACTGCTGGGAAGTCCTTCCCTGGGTCTCATCAAGGTCTC TCGTGACTGTCACTATGTATCC TTTCC TC TATGCTGGZkAGGGZ\ACCZ\AAGGZGAAGTATTC CCTGGCTTAGGGCCTTTCTZtCGATGZtCTTCTGGTTTTTCTGTTGZtATTAGTCTCATCZtAT GGCGCAGCGTGTCCCCTCCAGGTAGCTGCCACGGAAACCTTCCTGGAGACAGTGTCTTAA CCACCAGGZAACTGC TC TGTGT TGGACT ±AGCAGTGZ\AGTGGGGATGGAGTAGGAZ\AGAG± TCCACZ\CGCCCTTCAGATCZ\CACTGTGCCCTZ\CZ\CACTCTGCCCZ\CATAGZ\CCZ\CCCGCG GGAAGACGGAACCAGTGGGGAAGAGCCTGGCAGCTGAAGTCTGGGATGTGGCCTCAGCTC CTTTACZ\AACTGCCTGTGCZ\ACCTTGGGCZ\AGTCCC*AACCTCTCZLACACTCCAGTTTCTC CTCTTCCGTGGCCTGGGGZ\TGCTZ\CGZ\ATATTTZ\CCTCZ\TAGGZ\TTTTGTGZ\GGATTZ< AT GAGATGAGGCATAGZXACATGCTTAGCTCAGCTCTGGCCACACAGGAAGZXATZ\AGTGTTGG CTCTTGTTGGZkATTATGGTACCACCTTGGGCTATGACCACZAAGGGTCAGCTTCCGAGGACAGTGACCTGCAGCAGAACAATAGAAATGCATTGAGAACAATGGTTCCAATGGGCAGCCCC TTCCTCTCCCTTTACCTGCCTATAACTCTGGCCTTCCTCAG (SEQ ID NO: 27 ) > CELA2A-201 intron 7: protein..coding GTAAGAACCGGACCAGCCCTGAGCCCCAAGGCACTACCCTGCTCACCTGGCCTCGGGAGT GCCATGCCCACCTGGTGACTGAGAATCCCCTCCTTCCTCTTGAGAGCTAGATGGGAACCC CTTGGAGGAGGCTGCAGACCTGAGTAACTGCTGGGCCTGCCATGGGTCCCCCAAATTTCT GTGTGGATAAAGCTGAGTGAAAAGGAACATAGAGGGTGGCCTTGTCCAAAGAGGTTGGAC ACTCCTCAGGCATATGAAGAGTGAGTTCCGCTGGGCGCCGTGGCTCATGCCTGTAATCCC AGCTCTTTGGGAGGCCAAGGCGGGCAGATCACGAGGTCAGAAGTTCAAGACCAGCCTGAC CAACC T GGCAAAAC CC CATGT C TAG TAAAAAAATC CAAAAAAAAT T AGCCAGGT GT GGT A GCGCACTCCTGTAATCCCAGCTACTCAGGAGACTGAGGCAGGAGACTCCCTTGAACCTGG GAGATGGAGGTTGCAGTGAGCCAAAATTGCACCATTGCACTCCAGCCTGGACAAGAAGAG TGAAACTCCATCTAAAAAAAAAAAAAAAAAAAAAAAGAGTGAGTGCCATAGAAATGGTGA TTTTATTTTTGTTTATCTGTGTGTAGGCCCAGACTCCACCATCCAGTGCTATAAACAGGT ATATTTATCTGCAAAGCCCAAAACCTGATATCCCCATAGCATTAATTATTGGAAATTAGT CCACCTCAGGGGTCCTCCAGCTATTCTGTAGGGTGACCAACCATCATGGTTTGCCCAGGA CTGAGAGGTTTTCCAGGATGTGGGACTTCCTGTTTTACACTGGGACCATCCCAGGCAAAT AGAGCTGAGTTGGTCCCCCTGTCTTGTAAAAGTAAACAACTCAGAGAGGTGTCTCCTTTC AGCTTCCACAATAACTCAATTTGTTTTTAACAATGAACACATTTGTTTATAACAATGAGC ATTAAAATTATTTATTAAAAATAATAGGCCAGGTGCCATGGCTCATGCCTGTAATCGCAG CACTTTGGGAGGCCAAGGAGGGAGGATTGCTTGAGCCCAGGAGTTCAAGAGCATCCTGGG CAACATAGCAAGACTCTGTCTTTACAAAAAAATTTTTTTTAATTACTCAGGCACGGTGGT ACATGCCTGTAGTCTCAGCTACTTGGGAGGCTTGGGTGGAAGGATCACTTGGGCCCGGGA GGCCAACGCTGCAGTGAGCACTTAAGCCTGGGCGACATAGCAAGACCCTGTCTCCAAAAA TAATGATAATAATACCTGGCATCAATATTAAGGAGCAGCCATGGATACACGCAGCAGTAG GTGAAAGCAGCCAGAACAGGAAACCCCGTCACAAGAAAGGGATGCCTGGTGGCTCACGCC TGTAATCCAGGCACTTTGGGAGGTCAAGATGGGCAGATCACCAGAGGTCAGGAGTTCGAG ACCAGCCTGGTCAACACGGGGAAACCCCGTCTCTACTAAAAATACAAAAATAAGGTGGGT GTGGTGATACGTGCTTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGGGAATCACTTGA ACCTGGGAGGTGGAGGTTGCAGTAACCCGAGTTCGCACCACTGCACTCCAGCCTGGGCGA C AGAGC GAGAC T C C AT C T C AAAAGAAAAAAAAAC ACAC AAAAAAGAAAGAGT T GCC CAT G AGAGGGCAGAGGAGCCACC TGCCTCGTACTGGATGCTAGAAGGAAAGAGTCGGACACTC TGTGCCAATGAGGGACCAGCTCAGTTTGCTCTTCTGTAAAATGGGACTTGAACTAGACCA GGACTCCTCCACAATTTAGGCGCTAGAGCAAATGGGAGTTATGGTATTCGTGGCAGAGCC CCTAAAGAAGGGGAAAGCTGGCATCTGACTTC CTAAGTTTAATTTGCAGAGAAGTGTTA AAT TGAAT TAAAC T GACAC T T ATAAAACC AAATAT GAAAC TGCAGC CATGAGCACGTGGT CACAGGAGGAAAACTAAACTGTCAAAATAAGCAGGTGGCTGCTTCCAAGTGTTCCCACTG ATTCTTGCCAGATCCTTGTTCATTTGTCTGTCTGCTGGCATCCTTTTGCCAACAAGTGGT C AC AAGCAGAGAT T CC AAAGAC GT T T G T T GGAC AAAAAT GAAAAAT CAAAAAGACAGT AG CCATTAATCATTAATGGTGCTTTTTTATATCTAAATTGGGAAAAACCCATCTGAGTCCAC CTTTGGAGGCGCCACCCACATGTCAAAGGAGCCTGGCCTGAGACTGACTGGACGAGAGGC TGCTTAGGGGCCTTTCTCAGCCAAATCCTAAGACCGTTTTCCAGCCCCAGCTGCTGTGGG AGTCACCATGCAGCTGGTCCTGGCAGCAACCTGGGGACCTGAAGCAGGGGCAGGGGCAAT TGTCCCAATTTTTTTTTCCCAGCATCAGCTGTTCCCAGCCACAGACACAAGC CAACAGA ACAGGCCAGATCCCGAAGCATGGCCAGATGGCAGCAGGGATCCACGGGGCAGCACGCCTC GCAAGGCAGCTGGGCAGAGGCCCTGTGTTCATGGGAGAGCAGTCGGGACAGGCACAGATG CCCGGTAACGGGTTTCAGAAGGGGGAGAGCCAAGCAACCAGCTGTCCTTGGGTGGGGGCT GCCCCTGGCAGCAGCGGAGGAGCTAGTGAAACCTTGCAGTAGCCCCAACCCAGGGACACA GTGTGTGCAGCCTGGAGTCACATACCACTAGAGCCCTGGGGGTGCCTGAGAGCACCTGCC TGGATAAACCCAGACAGGTGATGAGCCAAGGTCAGGAAATACAAGCAAAAGAGCAGAGAA GCAGAGGAGACAAGTGGCCTGGGGCATTCATCCTTTCAACAAATGTCCATTGAATGACAG TCACATGTGAACCTTCAGCAATCCTTAGTTTTTATTCTTTTTCCGGAAGGTAATACATAC TCTTTGTAAAAATATTAAAAGACTATAAGAGTATCAGGTAAAAAAGTAAGAAGTCCCCCC TCATTTTACATCTCCAATTCCACTCTCCAGAGGTAATCATTGGTTTTGTTTTTGTTTTTT AAGACAGAGTCTCGCTCTGTCACCCAGGCTGAAATGCAGTGGCTTGATCGCAGCTCAATG CAACCTCCACCTCCCGGGTTCAAGCGATTCTTCTGCCTCAGCCTCCTGAGTAGCTGGGAT TACAGGCACACACCACCATGCCCAACTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCA CCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGGTGATCTGCCCGCCTTGGCCTCCAAAAGTGCTGGGATTACAGGCATGAGCCACTGTGCCTGGCCAAGTTGTCATTGTTAACA GC T TGTGTGTGGTC TCAACTT TATGTGCATATATAGCTATATATTTACGTAGATAGACAC TGTCTTCTATGCAAAAACTAAGATCTGAGTCTTCATACTGTTCTGCAGCTGTTTTTCACT TAACACCTTATCACATTTTACATGTTAGTACATACATGTCATTCTTTTGAACAGCTAAAT AGTATTCCATAGTTTGAGTGTAATGAATGCAGCTTTCATGTTTCCTATTTTTCACTCTTA CATTGTGCAATGAACATCCTTGGACATAATTCTTGCACACTGGTCTGAGTATTTGTGCTG TAGCAGCTGGATCTCCTGCAGTTTACAGCACTGGATCATGAAGTTGGCATATCAAAATGT TTAACAGGCTGGGCGCGGTGGCTCACGCCTGTAATCTGAGCACTTTGGGAAGCTGAGGCG AGTAGATCACTTGAGGTCAGCAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCTCATC TCTACTAAAAATGCAAAAAATTAGCCAGGCATGGTGGTGCATGACTATAATCTCAGCTAC CCGGGAGGCTGATGCATGAGAATCACTTAAACCTGCATAGTGGAGGTTGCAGTGAGCCCA GATCGCCCTACTGCACTCCAGCCTGGGCAACAGAGTGAGACTGTCTCAAAAAGAAAACAA ACAAACAAATAAAAAAAAAAACGTTTAACAGACACTGATCAACAGCCTCTGGAAACAGTT ATAGCCCCAACCACAGTGCATAACTGCCTTGCAAACAGCAGTTTTGATTTAAGAAACAAA TGAAATCTAAAAATATGCCATCCACACATTTGGAATATAATTTTTAGGGGTTCGAAGGCC CCCTACCCTGAAGCCCACACAGGACTCCTGGCTAAGACCCCCTTTACAGGAAGACCCTAA CAGGTCAATGAAAAGCTGTGATCACATCTTTTTTTCCGAAACGTCATCAGAACTCCTCAG GCAGGAGC lACTGTAGiGTGGGCTGCC iGiAACTCACATGAGiAGC 1TAGCCCAGGAGGA CAGAGACAGGAAACTGCCATGCACAGCTCTGCGGTTAGGTGAACCTGACGATTATCTTGT GTGTCCTGCAG (SEQ ID NO: 28)> CELA2A-201 peptide: ENSP00000352639 pep: protein.. coding MIRTLLLSTLVAGALSCGDPTYPPYVTRVVGGEEARPNSWPWQVSLQYSSNGKWYHTCGG SL IAN S WVLTAAHC I S SSRT Y RVGLGRHN LYVAE SGSLAVSVSKIVVHKDWN SN QI SKGN DIALLKLANPVSLTDKIQLACLPPAGTILPNNYPCYVTGWGRLQTNGAVPDVLQQGRLLV VDYATCSSSAWWGSSVKTSMICAGGDGVISSCNGDSGGPLNCQASDGRWQVHGIVSFGSR LGCNYYHKPSVFTRVSNYIDWINSVIANN (SEQ ID NO: 29)> CELA2A-201 utr3: protein_coding CCAAAAGAAGTCCCTGGGACTGTTTCAGACTTGGAAAGGTCACAGAAGGAAAATAATATA ATAAAGTGACAACTATGCAAATCA (SEQ ID NO: 30)> CELA2A-201 utr5: protein_codingACAGAACTCCCACGGACACACC (SEQ ID NO: 31)>1 dna: chromosome: GRC 38: 1: 15456732: 15472091: 1 ACAGAACTCCCACGGACACACCATGATAAGGACGCTGCTGCTGTCCACTTTGGTGGCTGG AGGTAAGTCCTGTTACCCAGAGGCACTGGTTTCCCATGCCCTGGTGGGGCTGGAAATGGG ATCTTCCTGTCCTCCCCTCTCGCCCCCACCCAACCCCT CTGC TTCAGACCT T ATCA TAAGAACATTGGAATGGAGTTTCAAAGAATTGGAGCAAAGAGCAGGATTCTATGACCTCT TGGGATCCTTCTAGAACAAGGGTTTTCTATTTGGGGGGTCAGAATGCGCAGCAAGTGTAG TTTAC TTGTGTGGGTCGCTGCTTCCTGACTCAAGACCCTTTCTCTT TC C GCCCTCA GTTGTGGGGACCCCACTTACCCACCTTATGTGACTAGGGTGGTTGGCGGTGAAGAAGCGA GGCCCAACAGCTGGCCCTGGCAGGTGAGTTGACCACACTGTACTTCTCCCCGTCCCTGCC CCACTCCCTTT C TCTCCCCTTTGCCCTTCC CCATGGCGTCT TTGTGCTGGCAAAGT GAGTATTGATGGGACTCAGAGAGCAGCACAGGCAACTTTAGCAATAAGATATATTTCCGG CTGGGCGCAGTGGC TCATGCC TGTTATCCCAGCAC TGTGGGAGGCTGTGGTGGGCAGATC ACCTGAAGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCTGCCTCTACTAAT AATACACAAGTAGCCAGGTGTGGTGGCACACGCCTGTAATCCCAGCAACTGGGGAGGCTT AGGCAGGAGGATCGCT TGAACCCAGGAGGCAGAGGTTGCAGTGAGC TGAAATTGCACCAC TGTACTCCAGCCTGGGCAACAAAGCAAGACTCTGTCTCAAAAAAAAAAAAAAAGATAC T TTCTGTGGCCTTGGCCAACCTTGGGAGTGGAGGGAGAGGTGCCTCCTCCAGCTGCAGTAA GGAGCTGTCCCCTCCCCACTGCCCATAGGCAGCAAATATAGTATTACTCTATTAACCAAT CAGAGGCTTGTTTACAAATGTACCATCAGTCTAGGAACCATTCAAGACTACCTATGCAAA CATTCCTTGCTTTAAGGAACCAATCAGTGCTATTTGCGCAGATTAATCTTTAACCACAGG CAAATCAATGTTACCAATGAAAAAAATGTTTTCTTAAGTTGATATAATCATAGTGGTATC AAGACTAAACTGCCAAGTGGGGCACAGGTGTAAATTACTCAGACGTGTTAATGGTGGGGA TTTTAAGAGAAATGTGTAAAGTTTACATTGACATAAACAAATATAGTGAAATCTCATTTA TCTGATGTAATTGGGACCGAGTAATTGAAAAGTTGGTTATAAAGAAATTATTTTAAATCA TATATATATAGT AAAGAT T T T ATC T TAGACTATGT AAACAAT TT T T TGAGT TC T TAAC T G TCTTTTTCATATAAGCCATGCCCCATATCTTATCTACAAACCCGTTTCTTTAAAGCAGAG CAGTAACTTTGAGACGTTCACAAAGCAATCTGAGTTATGAGCCCATCCAATCTTTCCATTTTCTCACAGACACTACTTCAAGAGCAATTTTTTAAGAGTCATGGTGATCCAGTCTCTTCC AAGCATTTAACTTTGTTTCCATATAAATAACGACTCTCTTTCTGAACTCATATTTCATTG GT TACATGTT T AZ T C A T T T CAT TAT TACAAGCATZiAT T GATATAAACTGC T T TGGGAG CAAACCCAGCTGAATCAGGATGAGTTTCTACCTTAAGTAGGAGGAGCAACTAGCAGCCCA CTAGCCTTGAGCATTCAGAGTGACCCATGCAGCAGTCACTGTGTTTAGAGAGGCCAGCGT TCATTCATCTGCTCCTTGATTA GAGACTGTCZATGATAGGCCAGGCGTGGTGGCTCATG CCTGTAATCCCAGCACTTTGGCAGGCCGAGGCAGGTGGATCACCTGAGGTCAGGAGTTTG AGACCAGCCTGGCCAACATGGTGAAACTCTGCCTCTACTAAAAATACAAAAATTAGCCAG GCATGGTGGCGGGCACCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCACAAGAATCGCTT GAACCTGGGAGGCGCAGGTTGCGGTGAGCCAAGATCACACCTCTGCACTCCAGCCTGGGT GACAGAGTGAGATCCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAGACTGTCAACGATAGAATGATGGCAGATATTTCACATTTTTTTCTTTTTTTGAG ACAGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGCACAATCATAGCTCACTGCAG CCTCCAACTCCTGGGCTCTGGCCATCTACCTGCCTCAGCCTCCCAAAGTGCTGGGATTAC AGGCACCCACCACTGTGCTCACCCATAGTAATTTTTTTTTTTTTTTGAGACAGTGTCACA TTCTGTCACCCAGGCTGGAGTACAGTGGCTGGTGAAACATGGCTCACAGCGGCCTCGAAC TCCTAGGCTCAAGCGATCCTCCCACCTCAGCCTCCCTATCAGCTGAGACCACAGGTGCAT GCCCCCACACCCGGTTAAGTTTCTTTTTTTTTTTTTAGAGATGGGTCTCACTATGTTGCC CAGGCTGGTCTTGAACTTCTGGGCTCAAGTGATCCTCCTGCGTCGGCCTCCCAAAGTGCT GGGATCACAGGCGTGAGCCTCTCZVACCCAGCCTCACZVATTTATATACTTGGCTTTGTGTG CTTTTGCCAGTTCTTTTGCTTTTCTCACAGTCCCACAGTATTTTTTCCCTGAGAATTTGG AAGAAGGGCCACAGTCCCTCTGCTACAGGGCCCCATAACACCGGGCGCATTTGTGCTAAG AACTAATCATTGCTGCCTACAAAAATACTTCCTCACCCTCTTGCACAAAAGTATTCTTGC CTTGAGAACCAGTGAATGTTAATCCATGAAGAGTCCCTATGCTTTGTGCTTCTGTTTCCC TGCGGAAATAAGGCAGATCTAGCTGGGTGGGGACTCAGATGAGGCAAGTAAGACATTCAC TCTGGAGGCAAAATGTAAGGGGGTAGCACAAAACTCAGCAACCAGCCAGGAACAGTGGCA CACACCTTTAATCCCAGCTATTCAAGGGGCTGAGGCAGGAGGATTGCTTGAGCCCAAGAG T T TGAGAC CAGCAT GGGCAAC ATAGAT AGCCATC T CAAAAAAAAAAAAAAAATACAAAC A AAC AAAAAAC C T C AC T A C G AG T G AT GAT AT T T T AAT GC AAT T T T GT T T C T TC TC C A GGAGACAGAGTCTCATTCTGTCACCCAGGCTTAATTACAGTATGAGTCACCCTGAGAGCC CTGCACAGTGGCTTATGTTCCCCATTTCCAGAGAAGGCAACTGGGACTTGGAAAGGTTAC AGAATTCCCCAAAGGCACTGGGTTAGCCCGGGGGGCGAATGAGGATTCAAATCTGGTTCA GTGCTACC CTTTAGC ACAC TTGCTGGCCACACATAATACGTA ATGTGGGCTAAAT TTCACATCTGTACGATGGTTTCAGCACATTTTTAAATTGGGGGGGGGGTACATCCTTTTA AGTATCTTTTTTC CTTTTTCTAATTTTAAACATCTTTTTTAAGACCTCTTAATGTCACA C AAAC GAG AAA GC CAAAT GGC AA AAAAAC AACAAC G CAGAGAAAAGC AGGCA C TGCCAACAAACAGGTCATCGTTCCCCACAGTTATAGGGAGCCCGATCAGAGCAGTCCATG CCCT CTGTC CCTCCCTCCCCTCTGCTTGGCCGTGGC GGGAATTGAAAGACAGGGGAG GAGGAATGAAGGAACAGGAAGGGCATGGAACAGCTTCATTAGAGACTAAAACAGTGTCAA ATGTCACCACCTTAAAGTTCTTTTTTTTTTTTTTTAAAGACCTGGTCTCACTCTGTTAAT TTTATTTTATTTTAAAATTTTTTTGGTAGGGTCAAGTTCTCACTATGGTGCCCAGGCTGG TTCACAAACTCCTGGGTTCAAGCAATACTCCCACCTCAGCCTTCTGAGTAACCGGGACTA TAGGCATGCACCGCCGCACCCAGCGGCTCATTTTGGGCTATTTTTGTTGTAGAGACAATG TATTTAGTCCTGCCTCAC CGGCT ATAAAGAC TATCTGAGACTGGGTAATTTATAAAG GAAAGAGGTTTAAC GACTCATAGCTCAGCGTGGC GGGGAGGCCTCAGGAAAC TATAA TCATGGCAGAAGGGAAAGGAAACACATCCTTGTTCACGCAGCGGTAGCAGGGAGAAGTGC CGAGCAAAAGTGGGGGAAAGACCCTTATAAAACCATCAGATCTTGTGAGAACTTACTC C TATCATGAAAACAGCATGGAGGTGACCACCCTCATGATTCAATTACCTCCCACCAGGTCC C T CGCACGACAAAT GGGGAT T ATAAGAAC TATAAT TCAAGAT GAGATT TGGGTAGGGAC A CAGCCA-AACCTTATCAGATGGGGTTTCACCATGTTGCCCAGGCTGGTCTCZ ACTCCTGG GCTCAAGTGATCCTCCTTCCTTGGCCTCCCGAAGTGTTGGGATTACAAGCATGAGCCACC ATGCCTGGCCTGATTGTTTTTCATGTAGATTGCATTAACACGTTTTTTATCTTGATGGCT GAGGTTTTGGGTGCTGCCTTAAGTTGTGCACATGAGGCGACTGCCTCACTCCCCCTTACC CT GTCCCAGCCCCGT CTTGGGAAAC GGAGTGCAGGGAGGCCCTGC CTTTCAAACC AGCCACAGAGCTCCTTTGCTTCTCCCCAGGTCTCCCTGCAGTACAGCTCCAATGGCAAGT GGTACCACACCTGCGGAGGGTCCCTGATAGCCZ< ACAGCTGGGTCCTGACGGCTGCCCACT GCA i CAGG i AAC IGCC 11 ICCC i GGGC GC 11 GGCC i GC I C AC CAGC i GG I GC ICAI 11 C i GAGCTGGGGGCTCAAATGGCCTGAACCATGCTACATAAAGCAGCCTTGCAAATAACCACTATACTGGCTGAGACACAAGCTGTAGTCAATCAATGGTTCAGTGTGTTGGCCCATCAATGT CAGTACATGGCATGGATGGAGTGTCTGTGCCAGGCAGGCACTGAGGGAGGATGAAGAGGA GGGGAAGGCCCAATCTCTGCCCTCTTGGGGAiAACTACATGTGGCCTCTGGATCTGGAATT GGGTTCCCTTGAACAACTTAACTGACTTCTCAAAGCTTCAATACCATCACCTGCAGAAAA ATGGAGTAACAGAATTTAGTGTGAATTCAATGAGTTGATTTACCAAAGGGCCTGGCACTT AG T AG TGC T C GT GAAAGC T AT T C T GAAT T T GAT GAAT TAT AAGGC C C C AGG T TCTGCAACGTTGTTCACTTATCCTCTTCTTTGTCCGTGACCTCTGCTCATCACAGCTGGA ACTCACCGGAGTTTCTGCCGGGTCGGCGTGACTCCCTACCAGGAGTGATCCCTTCTCCCA TCCGATTTATAATAAGCTCTAGGAGGTATTGGGGAAAATAATAGAGGCCTACAGTCAGGA GTCCTGGCTTGTAGTTCTGGTTTCTCACTAACGGTGGGACTCTGGGCCAGGTTACGCCTC TCTGGGCCTCAGTTTCCCCATCTGCAAATGAAAGGTGGGAAAAATGTCCTTGAGGGCAAA TGACTCCTGTTTTCTCTGTGTTCATCTCATTCCCTCCATAAGAACTATAGAGTTTGTGTT AT ATAAC TTTAT T ATAT GTAGT TATAGTTAGTATAG AACAATTATTATATGAT CATAGTTCTTAGGTTATATACTTTAGGCCCTATTATAACTAACTGATTGTCCCAGGGGAG GAAAGATCCCCAAGTCCCATCTAGTGGCTCACGCAGCAGCCAGTTACTTGGGTCTATCCC CAGCATTATCCAAAGCCAGGCCTCTGGAGGTGACCCTCTCCCTGGGCCCCCTTTCTCCCA GCTCCTCCAGGACCTACCGCGTGGGGCTGGGCCGGCACAACCTCTACGTTGCGGAGTCCG GCTCGCTGGCAGTCAGTGTCTCTAAGATTGTGGTGCACAAGGACTGGAACTCCAACCAAA TCTCCAAAGGGTTCGTTTCTGTCTGGGTGCACTTGGGGGTGAGGTTGTCAGGGAACAGAT GGGGGTCTCACAGAGGCAAGGGTCTCAACCACACCACACCCCCTCTGCTTCTTCTACAGG GAGGGGGAAGGAGCTCTGGCCTCTTAGATGTGGAACCAGCTCCAAAGATGTCTGGGGTGG GGATGTGACCTGGGGAGGGTGAAGCAAAGACTGCCAGAGCGACCTGGGTTTGCTGCCAGT TACACCTGCAGGGCCCCTGTCCGCTGAGCATGTATCTACTTCATGCCAGGTCCTGGGGAC ACAGGGACAGTGCTGTCAACCCTGTGACTGTCCCTGGAGTAGGACACAATCTCTCCTGCC TTCCCCCTTTCAACAAGGCCCTCTGCATTTTCAAACATGCCCTGGGGCCCTGCCAGTCAG AGCAACCTGGGGTAACAGGGTACAGGGAAGATGACAAGGTCTCCAAGCCCTCCAAAGCCC ACAGGGCAGGAAAAGTCAACCCGGTCCTCATGCTTCGCCTCCACACTCACCCAGGAACGA CATTGCCCTGCTCAAACTGGCTAACCCCGTCTCCCTCACCGACAAGATCCAGCTGGCCTG CCTCCCTCCTGCCGGCACC TTCT CCCAACAACTACCCCTGCT CGTCACGGGCTGGGG AAGGCTGCAGAGTAAGTGGGAGCCAGGAGCCCCCAGGCCTGGGAGGGAAGGGAGGTGATT CACGTCACCCCTGTCTGGCCGGGGCCTCTCACCTGTCATCCCAGGGTGTGTGGCTGCCTT GGAGAGACGGGATGGCAT GGCTGACGCCTGCCTGGGATCAAATGTCAGCTCTCCCACTT AATGACTGTGACACCTTGGACACATTACTAGGTCTCTCCATGCAGCACTTTTTTCATCTG TAAAATGCAAATATTTGGCTGGGCGCAGTGGCTCATACCTGTAATCCCAGCACTTTGGGA GGCCCATCCCGCGGATC CTTGAGGTCAGGAGACCAGCCTGACCAACACGGAGAAACTCT GTCTCTACTAAAAATATTAAAAAAAAAAAAAATAGCAGGATGTGGCAGTGTGCACCTGTA GTCCCAGCTACTCAGGAGGCTGAGGCACGAGAATCACTTGAACCTGGGAGATGGAGGTTG CAGTGAGCCAAGATTTCGCT CTGC CTCCAGCCTGGGGGACAGAGCGAGACTCCGCCTC AAAAACAAAAGGCATAATAATAGCACCTACATCACAGAGTTGTCACGAGGATTAAAGGAG ATAATCCATAGGAAGCCCAGAATGGGGTAGGTGCTGTGTTAGTTGTTGTCACTGTCCCTA TGATGGAGAGAAAGTTACAGAGACCATGTGACATCTTCTGTGTGGCCCAAGGCTAGAGTT CAGAACAGCATTTTCCTCTGTGACCTGAGACCCCTGGAGCCCTCATCAGACCCTCCATGC CCCATAGCAAAAGCTGTCCCCTGTGCCATTAGGTCTTGCTGCTCAGAACCTGAATGAGGG CGGGTCTCCTTTGACATAGACTCCAAGGGTCAGAGATCCAATGAC GTAGCCACCAAGAG GAACAGAGTTCCCGCCCTTCCCTTGTGAAATCACCAGAGGTTTGGAAGATTCTAGGAAGG GATGAGTAATAATGACCTTATGACCATAATGAGATTATGAGTGTTAATCACATTATGACA TTTTTTCCTAATGACATAAACCCATAAGTAT ATGACATGTTTTTCCAGGGGTACATCCA CATACGTACCCTCAAATAATTCCCATTGCCCTCTTGACTGGCTTATCTGTAAAC ACAAGG ACACATCAAATCCTGCGGTGAGCGGTGGTGGAAGAGGCATTTCATGCTTATTAAGATACA AGCACAACCTCCTGTTTGTTTCCCCAGACAACCCTGGGGCAGGCGCATTTCATCTGAGAC AGTGAGAGTGGGGAGGCCTCCAAATGCTTGGCTTTTCGGCCCAGCTCTGTCAGTCACGGT GAAACCTTGGGCAAGCCACTTCCGAACCTCAGTTTCCTCATCTGTAAAATGGAAACACTG TTGGTGAAGACTZiAAAGAGGTGAAAGGTGAAAATGCATCATACATTGCAAAATGTTATGC AAGTGTTAATTAATTACTTATTAGTGGTCTTAAGCCAGACACTTAACTTCCCTTTTTTTTTT TTTT TT TTTTTT TT TTGAGACAGAATC TTACTC TGTTACCCTGGCTGGAATGCAGTGGTGCAATCTCGGCTCACTGCAACCTCTGCCTCCCGAGCTCAAGCGATTCTCCTGCCTCAGC CTCCCAAGTAGCTGGCA ATAGGCGAGTGCCACCATGCCCGGGTAATTTTTGTATTTTA GTAGAGGTGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCTTGACCTCAAGTGATCTGCCCACCTTGGCCTCCCTAAGCGCTGGGATTAGAGGGTGAGCACCGCGCCCGACCGAC ACTTAACTTCCCTAGGCTTCAATCGGAAGTGAAGAATTGTGTTAATCTCATCCTTGCCAC TCATAGCTGTGGTAACAGT2AATGGAGGTGATGGTGGCTGTGTCATTGGGGGCCATTATGA AAGCCCTCCCTTCACCACACCAGCATATTTTTCCCAAGGAGGGGGCTCTAACAGCCAGGA GGCTCACTTTCTTATTTCAGATAAGTGACAGAAAGCCATGGAGCTAGCTCAGCAAAAAAA GGGAAATTGTTATAAGGATTTAAAAGTTTTATCCCAAGGGCAGAGATACAGCCAGGCATC AGGAACAAGCTAGAATCAGGGGCTGGAAACCTGTAAGAAACACAGGAAACCCTCCCTCAG AATTTCTGTTCTTTGTGGGTCTACTTCATTCTCCACATAAAACAAAATGTCAGTCATTGC CAACAGCTTCCCAGAATTATGTCCTCTGAAACCGGAATCTCATAGACCCCATCTCAGAAT CCCAGGGGAGGGAGCTCATTGGCTTAGCTTGAGTCAGGTGTTCACTGCTGAACCAATCAA CCGTGAGGACACAGTATAGACAAACGTGGCTGTTCGCATGTTGCAATGGATGGAAGACAG GAACAGGGGAAACCTAAGACGGGTCCATCACTTCCTTTTTCTCAGGAGTCCCTGCATCCC TAATGGCTTCTCTCTGATCTCATTCAGCCAACGGGGCTGTTCCTGATGTCCTGCAGCAGG GCCGGTTGCTGGTTGTGGACTATGCCACCTGCTCCAGCTCTGCCTGGTGGGGCAGCAGCG TGAAAACCAGTATGATCTGTGCTGGGGGTGATGGCGTGATCTCCAGCTGCAACGTGAGTA CCAAAATCAGGGGCTCCGCTCCATGACAAAATGTGGCTGGGGATAAGGCTATAGAGGTCC ATCCCTCCATAGGTCCATCCTCCCACCTCCTGGCAGAATTACCCCGAAACATGTTCCAGA TATATCTTTGGCCAGGCACGGTGGCTCACACCTGTAATCCCAGCACTTTGAGAGGCCAGG CGGGCAGACCACTTGAGGTCAGCAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCTTG TGTCTACTAAAACTAAAAAAATTAGCCGGGCATGGTGGCAGGCGCCTGTACTCACAGCTA CTCGGGAGGCTGAGGCAGGAGAATCACTTGAACCCATGAGGCGGAGGTTGCAGTGAGCTG AGATCACACCGCTGCAATCCAGCCTGGGTGACAGAGCGAAACTCAAAAAAAAAAACAAAA AAACTTGATCTTGGTTCTTTTTTGTTCTTGTTTTGAGATGTTTTGAGATTCTCCTTGTAG TATCCACCCATCTCACTGCTGGGAAGTCCTTCCCTGGGTCTCATCAAGGTCTCTCGTGAC TGTCACTATGTATCCTTTCCTCTATGCTGGAAGGGAACCAAAGGAAAGTATTCCCTGGCT TAGGGCCTTTCTACGATGACTTCTGGTTTTTCTGTTGAATTAGTCTCATCAATGGCGC G CGTGTCCCCTCCAGGTAGCTGCCACGGAAACCTTCCTGGAGACAGTGTCTTAACCACCAG GAACTGCTCTGTGTTGGACTTAGCAGTGAAGTGGGGATGGAGTAGGAAAGAGTTCCACAC GCCCTTCAGATC CACTGTGCCCT C CACTCTGCCC C TAGACCACCCGCGGGAAGAC GGAACCAGTGGGGAAGAGCCTGGCAGCTGAAGTCTGGGATGTGGCCTCAGCTCCTTTACA AACTGCCTGTGCAACCTTGGGCAAGTCCCAACCTCTCAACACTCCAGTTTCTCCTCTTCC GTGGCCTGGGGATGCTACGAATATTTACCTC T GGATTTTGTGAGGATTAATGAGATGA GGCATAGAACATGCTTAGCTCAGCTCTGGCCACACAGGAAGAATAAGTGTTGGCTCTTGT TGGAATTATGGTACCACCTTGGGCTATGACCACAAGGGTCAGCTTCCGAGGACAGTGACC TGCAGCAGAACAATAGAAATGCATTGAGAACAATGGTTCCAATGGGCAGCCCCTTCCTCT CCCTTTACCTGCCTATAACTCTGGCCTTCCTCAGGGAGACTCTGGCGGGCCACTGAACTG TCAGGCGTCTGACGGCCGGTGGCAGGTGCACGGCATCGTCAGCTTCGGGTCTCGCCTCGG CTGCAACTACT CCACAAGCCCTCCGTCTTCACGCGGGTCTCCAATTACATCGACTGGAT CAATTCGGTAAGAACCGGACCAGCCCTGAGCCCCAAGGCACTACCCTGCTCACCTGGCCT CGGGAGTGCCATGCCCACCTGGTGACTGAGAATCCCCTCCTTCCTCTTGAGAGCTAGATG GGAACCCCTTGGAGGAGGCTGCAGACCTGAGTAACTGCTGGGCCTGCCATGGGTCCCCCA AATTTCTGTGTGGATAAAGCTGAGTGAAAAGGAACATAGAGGGTGGCCTTGTCCAAAGAG GT TGGACACTCCTCAGGCATATGAAGAGTGAGTTCCGCTGGGCGCCGTGGCTCATGCCTG TAATCCCAGCTCTTTGGGAGGCCAAGGCGGGCAGATCACGAGGTCAGAAGTTCAAGACCA GCCTGACCAACCTGGCAAAACCCCATGTCTACTAAAAAAATCCAAAAAAAATTAGCCAGG TGTGGTAGCGCACTCC GTAATCCCAGCTACTCAGGAGAC GAGGCAGGAGACTCCCTTG AACCTGGGAGATGGAGGTTGCAGTGAGCCAAAATTGCACCATTGC CTCCAGCCTGGACA AGAAGAGTGAAACTCCATCTAAAAAAAAAAAAAAAAAAAAAAAGAGTGAGTGCCATAGAA ATGGTGATTTTATTTTTGTTTATCTGTGTGTAGGCCCAGACTCCACCATCCAGTGC ATA2AACAGGTATATTTATCTGCAAAGCCCAAAACCTGATATCCCCATAGCATT2AATTATTGGA AATTAGTCCACCTCAGGGGTCCTCCAGCTATTCTGTAGGGTGACCAACCATCATGGTTTG CCCAGGACTGAGAGGTTTTCCAGGATGTGGGACTTCCTGTTTTACACTGGGACCATCCCA GGCAAATAGAGCTGAGTTGGTCCCCCTGTCTTGTAAAAGTAAACAACTCAGAGAGGTGTC TCCTTTCAGCTTCCACAATAACTCAATTTGTTTTTAA AATGAA ACATTTGTTTATAAC AATGAGCATTAAAATTATTTATTAAAAATAATAGGCCAGGTGCCATGGCTCATGCCTGTA ATCGCAGCACTTTGGGAGGCCAAGGAGGGAGGATTGCTTGAGCCCAGGAGTTCAAGAGCA TCCTGGGCAACATAGCAAGACTCTGTCTTTACAAAAAAATTTTTTTTAATTACTCAGGCA CGGTGGTACATGCC GTAGTC CAGCTAC TGGGAGGCTTGGGTGGAAGGATCACT GGGC C C GGGAGGC C AAC GC TGC AG T GAGC AC T TAAGC C TGGGC GACAT AGC AAGAC C C T GT C T CCAAAAATAATGATAATAATACCTGGCATCAATATTAAGGAGCAGCCATGGATACACGCA GCAGTAGGTGZiAAGCAGCCAGAACAGGAAACCCCGTCACAAGAAAGGGATGCCTGGTGGC TCACGCCTGTAATCCAGGCACTTTGGGAGGTCAAGATGGGCAGATCACCAGAGGTCAGGA GTTCGAGACCAGCCTGGTCAACACGGGGAAACCCCGTCTCTACTAAAAATACAAAAATAA GGTGGGTGTGGTGATACGTGCTTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGGGAAT CACTTGAACCTGGGAGGTGGAGGTTGCAGTAACCCGAGTTCGCACCACTGCACTCCAGCC TGGGCGACAGAGCGAGACTCCATCTCAAAAGAAAAAAAAACACACAAAAAAGAAAGAGTT GCCCATGAGAGGGCAGAGGAGCCACCATGCCTCGTACTGGATGCTAGAAGGAAAGAGTCG GACACTCTGTGCCAATGAGGGACCAGCTCAGTTTGCTCTTCTGTAAAATGGGACTTGAAC TAGACCAGGACTCCTCCACAATTTAGGCGCTAGAGCAAATGGGAGTTATGGTATTCGTGG CAGAGCCCCTAAAGAAGGGGAAAGCTGGCATCTGACTTCACTAAGTTTAATTTGCAGAGA AGTGTTAAATTGAATTAAACTGACACTTATAAAACCAAATATGAAACTGCAGCCATGAGC ACGTGGTCACAGGAGGAAAACTAAACTGTCAAAATAAGCAGGTGGCTGCTTCCAAGTGTT CCCACTGATTCTTGCCAGATCCTTGTTCATTTGTCTGTCTGCTGGCATCCTTTTGCCAAC AAGTGGTC ACAAGC AGAGAT T CCAAAGAC GT T TGT TGGAC AAAAAT GAAAAATC AAAAAG ACAGTAGCCATTAATCATTAATGGTGCTTTTTTATATCTAAATTGGGAAAAACCCATCTG AGTCCACCTTTGGAGGCGCCACCCACATGTCAAAGGAGCCTGGCCTGAGACTGACTGGAC GAGAGGCTGCTTAGGGGCCTTTCTCAGCCAAATCCTAAGACCGTTTTCCAGCCCCAGCTG CTGTGGGAGTCACCATGCAGCTGGTCCTGGCAGCAACCTGGGGACCTGAAGCAGGGGCAG GGGCAATTGTCCCAATTTTTTTTTCCCAGCATCAGCTGTTCCCAGCCACAGACACAAGCA CAACAGAACAGGCCAGATCCCGAAGCATGGCCAGATGGCAGCAGGGATCCACGGGGCAGC ACGCCTCGCAAGGCAGCTGGGCAGAGGCCCTGTGTTCATGGGAGAGCAGTCGGGACAGGC ACAGATGCCCGGTAACGGGTTTCAGAAGGGGGAGAGCCAAGCAACCAGCTGTCCTTGGGT GGGGGCTGCCCCTGGCAGCAGCGGAGGAGCTAGTGAAACCTTGCAGTAGCCCCAACCCAG GGAC CAGTGTGTGCAGCCTGGAGTC C T CC CTAGAGCCCTGGGGGTGCCTGAGAGC AC C TGC CT GGATAAAC CCAGACAGGTGAT GAGCCAAGGTC AGGAAATACAAGCAAAAGAG CAGAGAAGCAGAGGAGACAAGTGGCCTGGGGCATTCATCCTTTCAACAAATGTCCATTGA ATGACAGTCACATGTGAACCTTCAGCAATCCTTAGTTTTTATTCTTTTTCCGGAAGGTAA TACATACTCTTTGTAAAAATATTAAAAGACTATAAGAGTATCAGGTAAAAAAGTAAGAAG TCCCCCCTCATTTTACATCTCCAATTCCACTCTCCAGAGGTAATCATTGGTTTTGTTTTT GTTTTTTAAGAC GAGTCTCGCTCTGTCACCCAGGCTGAAATGCAGTGGCTTGATCGCAG CTCAATGCAACCTCCACCTCCCGGGTTCAAGCGATTCTTCTGCCTCAGCCTCCTGAGTAG CTGGGATTACAGGCACACACCACCATGCCCAACTAATTTTTGTATTTTTAGTAGAGATGG GGTTTCACC TGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGGTGATCTGCCCGCCT TGGCCTCCAAAAGTGCTGGGATTACAGGCATGAGCCACTGTGCCTGGCCAAGTTGTCATT GTTAACAGCTTGTGTGTGGTCTCAACTTTATGTGCATATATAGCTATATATTTACGTAGA TAGACACTGTCTTCTATGCAAAAACTAAGATCTGAGTCTTCATACTGTTCTGC GCTGTT TTTCACTTAACACCTTATCACATTTTACATGTTAGTACATACATGTCATTCTTTTGAACA GCTAAATAGTATTCCATAGTTTGAGTGTAATGAATGCAGCTTTCATGTTTCCTATTTTTC ACTCTTACATTGTGCAATGAACATCCTTGGACATAATTCTTGCACACTGGTCTGAGTATT TGTGCTGTAGCAGCTGGATCTCCTGCAGTTTACAGCACTGGATCATGAAGTTGGCATATC AAAATGTTTAACAGGCTGGGCGCGGTGGCTCACGCCTGTAATCTGAGCACTTTGGGAAGC TGAGGCGAGTAGATCACTTGAGGTCAGCAGTTTGAGACCAGCCTGGCCAACATGGTGAAA CCTCATCTCTACTAAAAATGCAAAAAATTAGCCAGGCATGGTGGTGCATGACTATAATCT CAGCTACCCGGGAGGCTGATGCATGAGAATCACTTAAACCTGCATAGTGGAGGTTGCAGT G GCC C AG AT C GC C C T AC T GC AC T C C AGC C T GGGC A C AG AG TG G AC TGT C T C AAAAAG AAAACAAACAAACAAATAAAAAAAAAAAC GT T TAACAGAC AC TGAT CAACAGCC TC TGGA AACAGTTATAGCCCCAACCACAGTGCATAACTGCCTTGCAAACAGCAGTTTTGATTTAAG AAACAAATGA2AATCTAA2A2AATATGCCATCCACACATTTGGAATATAATTTTTAGGGGTTC GAAGGCCCCCTACCCTGAAGCCCACACAGGACTCCTGGCTAAGACCCCCTTTACAGGAAG ACCCTAACAGGTCAATGAAAAGCTGTGATCACATCTTTTTTTCCGAAACGTCATCAGAAC TCCTCAGGCAGGAGCTACTGTAGTGTGGGCTGCCTGT2AACTCACATGAGTAGCTTAGCCC AGGAGGACAGAGACAGGAAACTGCCATGCACAGCTCTGCGGTTAGGTGAACCTGACGATT ATCTTGTGTGTCCTGCAGGTGATTGCAAATAACTAACCAAAAGAAGTCCCTGGGACTGTT TCAGACTTGGA2AAGGTCACAGAAGGAAAAT2AATATAAT2AAAGTGACAACTATGCAAATCA (SEQ ID NO: 32 )

[0090] In some embodiments, the wild-type CEL A2A preproenzyme comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the wild-type CELA2A preproenzyme consists of the amino acid sequence of SEQ ID NO: 1.

[0091] In some embodiments, the rCELA2A protein has at least 80% amino acid sequence identity to SEQ ID NO: 1. In some embodiments, the rCELA2A protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1.

[0092] In some embodiments, the rCELA2A protein has at least 80% amino acid sequence identity to SEQ ID NO: 3. In some embodiments, the rCELA2A protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3.

[0093] In some embodiments, the rCELA2A protein is resistant to proteolytic cleavage by trypsin.

[0094] In some embodiments, the rCELA2A protein exhibits reduced or no elastase activity as compared to a wild-type CEL A2A enzyme.

[0095] In some embodiments, the rCELA2A protein does not comprise a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of MIRTLLLSTLVAGALS (SEQ ID NO: 7). In some embodiments, the signal peptide consists of the amino acid sequence of MIRTLLLSTLVAGALS (SEQ ID NO: 7).

[0096] In some embodiments, the rCELA2A protein comprises an activation peptide, with a substitution of the arginine residue at position 28 with another basic amino acid residue. In some embodiments, the activation peptide comprises the amino acid sequence of CGDPTYPPYVTR (SEQ ID NO: 8). In some embodiments, the activation peptide consists of the amino acid sequence of CGDPTYPPYVTR (SEQ ID NO: 8).

[0097] In some embodiments, the rCELA2A protein comprises a substitution of the arginine residue at position 28 with a histidine residue (R28H). In some embodiments, the rCELA2A protein comprising a R28H substitution comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the rCELA2A protein comprising aR28H substitution consists of the amino acid sequence of SEQ ID NO: 9.

[0098] CELA2A R28H amino acid sequence CGDPTYPPYVTHVVGGEEARPNSWPWQVSLQYSSNGKWYHTCGGSLIANSWVLTA AHC1SSSRTYRVGLGRHNLYVAESGSLAVSVSKIVVHKDWNSNQISKGNDIALLKLA NPVSLTDKIQLACLPPAGTILPNNYPCYVTGWGRLQTNGAVPDVLQQGRLLVVDYATCSSSAWWGSSVKTSMICAGGDGV1SSCNGDSGGPLNCQASDGRWQVHG1VSFGSR LGCNYYHKPSVFl’RVSNYIDWINSVIANN (SEQ ID NO: 9)(R28H substitution is bolded)

[0099] CELA2A R28H nucleotide sequence TGTGGGGACCCCACTTACCCACCTTATGTGACTCACGTGGTTGGCGGTGAAGAAGCGAGG CCCAACAGCTGGCCCTGGCAGGTCTCCCTGCAGTACAGCTCCAATGGCAAGTGGTACCAC ACCTGCGGAGGGTCCCTGATAGCCAACAGCTGGGTCCTGACGGCTGCCCACTGCATCAGC TCCTCCAGGACCTACCGCGTGGGGCTGGGCCGGCACAACCTCTACGTTGCGGAGTCCGG CTCGCTGGCAGTCAGTGTCTCTAAGATTGTGGTGCACAAGGACTGGAACTCCAACCAAAT CTCCAAAGGGAACGACATTGCCCTGCTCAAACTGGCTAACCCCGTCTCCCTCACCGACAA GATCCAGCTGGCCTGCCTCCCTCCTGCCGGCACCATTCTACCCAACAACTACCCCTGCTA CGTCACGGGCTGGGGAAGGCTGCAGACCAACGGGGCTGTTCCTGATGTCCTGCAGCAGGG CCGGTTGCTGGTTGTGGACTATGCCACCTGCTCCAGCTCTGCCTGGTGGGGCAGCAGCGT GAAAACCAGTATGATCTGTGCTGGGGGTGATGGCGTGATCTCCAGCTGCAACGGAGACTC TGGCGGGCCACTGAACTGTCAGGCGTCTGACGGCCGGTGGCAGGTGCACGGCATCGTCAG CTTCGGGTCTCGCCTCGGCTGCAACTACTACCACAAGCCCTCCGTCTTCACGCGGGTCTC CAATTACATCGACTGGATCAATTCGGTGATTGCAAATAACTAA (SEQ ID NO: 10) (R28H substitution is bolded)

[0100] In some embodiments, the rCELA2A protein comprising a substitution of the arginine residue at position 28 with a lysine residue (R28K). In some embodiments, the rCELA2A protein comprising a R28K substitution comprises the amino acid sequence of SEQ ID NO: 11, In some embodiments, the rCELA2A protein comprising a R28K substitution consists of the amino acid sequence of SEQ ID NO: 11.

[0101] CELA2A R28K amino acid sequence CGDPrYPPYVTKVVGGEFARPNSWPWQVSLQYSSNGKWYHTCGGSLIANSWVLTA AHCISSSRTYRVGLGRHNLYVAESGSLAVSVSKIVVHKDWNSNQISKGNDIALLKLA NPVSLTDKIQLACLPPAGTILPNNYPCYVTGWGRLQTNGAVPDVLQQGRLLVVDYA TCSSSAWWGSSVKTSMICAGGDGVISSCNGDSGGPLNCQASDGRWQVHGIVSFGSR LGCNYYHKPSVFTRVSNYIDWINSVIANN (SEQ ID NO: 11)(R28K substitution is bolded)

[0102] CELA2A R28K nucleotide sequence TGTGGGGACCCCACTTACCCACCTTATGTGACTAAGGTGGTTGGCGGTGAAGAAGCGAGG CCCAACAGCTGGCCCTGGCAGGTCTCCCTGCAGTACAGCTCCAATGGCAAGTGGTACCAC ACCTGCGGAGGGTCCCTGATAGCCAACAGCTGGGTCCTGACGGCTGCCCACTGCATCAGC TCCTCCAGGACCTACCGCGTGGGGCTGGGCCGGCACAACCTCTACGTTGCGGAGTCCGG CTCGCTGGCAGTCAGTGTCTCTAAGATTGTGGTGCACAAGGACTGGAACTCCAACCAAATCTCCAAAGGGAACGACATTGCCCTGCTCAAACTGGCTAACCCCGTCTCCCTCACCGACAA GATCCAGCTGGCCTGCCTCCCTCCTGCCGGCACCATTCTACCCAACAACTACCCCTGCTA CGTCACGGGCTGGGGAAGGCTGCAGACCAACGGGGCTGTTCCTGATGTCCTGCAGCAGGG CCGGTTGCTGGTTGTGGACTATGCCACCTGCTCCAGCTCTGCCTGGTGGGGCAGCAGCGT GAAAACCAGTATGATCTGTGCTGGGGGTGATGGCGTGATCTCCAGCTGCAACGGAGACTC TGGCGGGCCACTGAACTGTCAGGCGTCTGACGGCCGGTGGCAGGTGCACGGCATCGTCAG CTTCGGGTCTCGCCTCGGCTGCAACTACTACCACAAGCCCTCCGTCTTCACGCGGGTCTC CAATTACATCGACTGGATCAATTCGGTGATTGCAAATAACTAA (SEQ ID NO: 12) (R28K substitution is bolded)

[0103] The rCELA2A proteins described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., insul inotropic effect) of the described rCELA2A protein.

[0104] These variants may include: (i) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (ii) variants in which one or more amino acids are added to or deleted from the polypeptide, (iii) variants in which one or more amino acids include a substituent group, and (iv) variants in which the described rCELA2A protein is fused or conjugated with another peptide or polypeptide (e.g., a fusion partner, a protein tag) or other chemical moiety, that may confer useful properties to the rCELA2A protein, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. The rCELA2A proteins described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0105] Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar- chemical properties such as size, charge and polarity: Group I (Ala, Ser, Thr, Pro, Gly); Group II (Asp, Asn, Glu, Gin); Group III (His, Arg, Lys); Group IV (Met, Leu, He, Vai, Cys); Group V (Phe, Thy, Trp).

[0106] In some embodiments, a rCELA2A protein described herein is fused or conjugated with a moiety that specifically binds albumin. In some embodiments, the albumin binding moiety is a peptide, an antibody, or an antibody fragment (e.g., a Fab, scFv, VHH, scFab and dAb). Non-limiting examples of albumin binding peptides are described in U. S. patentpublication No. US20050287153, the disclosure of which is incorporated by reference in its entirety. In some embodiments, the albumin is rat albumin, rabbit albumin, or human albumin. In some embodiments, the moiety that specifically binds albumin is genetically fused or chemically conjugated to the N-terminus of the rCELA2A protein, or a fragment or a variant thereof. In some embodiments, the moiety that specifically binds albumin is genetically fused or chemically conjugated to the C-terminus of the rCELA2A protein, or a fragment or a variant thereof. In some embodiments, the moiety that specifically binds albumin is genetically fused or chemically conjugated to the rCELA2A protein, or a fragment or a variant thereof via a linker (e.g., peptidyl linker or nonpeptidyl linker),

[0107] Accordingly, embodiments of the rCELA2A proteins can include variants having about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the described rCELA2A proteins (e.g., SEQ ID NO: 9 or 11).

[0108] In some embodiments, a rCELA2A protein of lire present disclosure has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9.

[0109] In some embodiments, a rCELA2A protein of the present disclosure has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 11.

[0110] In some embodiments, the rCELA2A protein is produced in a eukaryotic host cell.

[0111] In some embodiments, the eukaryotic host cell is THP-1 cell or a human embryonic kidney (HEK) cell.

[0112] In some embodiments, the rCELA2A protein is produced in a prokaryotic host cell.

[0113] In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding the rCELA2A protein as described herein.

[0114] In some embodiments, a polynucleotide of the present disclosure comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, a polynucleotide of the present disclosure has at least 70%, 75%>, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.

[0115] In some embodiments, a polynucleotide of the present disclosure comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, a polynucleotide of the present disclosure has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12.

[0116] In some embodiments, a polynucleotide of the present disclosure comprises the nucleotide sequence of SEQ ID NO: 2, 4 or 6. In some embodiments, a polynucleotide of the present disclosure has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, 4, or 6.

[0117] In another aspect, the present disclosure provides a vector comprising the polynucleotide as described herein.

[0118] In another aspect, the present disclosure provides a host cell comprising the polynucleotide or the vector described herein.

[0119] In some embodiments of the recombinant expression vectors described herein, a nucleotide sequence encoding a rCELA2A protein may be operably linked to a regulatory sequence that control the expression of the rCELA2A protein in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) tliat control the transcription or translation of the nucleotide sequence encoding a rCELA2A protein. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the ail that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Vims 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma.Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRD promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia vims type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0120] In addition to the nucleotide sequences encoding a rCELA2A protein and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U. S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confersresistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0121] For expression of the rCELA2A proteins, the expression vector(s) encoding the rCELA2A protein may be transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the rCELA2A proteins of the disclosure in either prokaryotic or eukaryotic host cells, expression of rCELA2A proteins in eukaryotic cells, and most preferably mammalian host ceils, is the most preferred because such eukaryotic cells, and in particular' mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded protein.

[0122] Suitable mammalian host cells for expressing the recombinant proteins of the disclosure include THP-1 cells, human embryonic kidney (HEK) cells, Chinese Hamster Ovary (CHO) cells (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad, Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Moi. Biol. 759:601-621), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding rCELA2A genes are introduced into mammalian host cells, the rCELA2A proteins are produced by culturing the host ceils for a period of time sufficient to allow for expression of the rCELA2A proteins in the host cells or, more preferably, secretion of the rCELA2A proteins into the culture medium in which the host cells are grown. Recombinant proteins can be recovered from the culture medium using standard protein purification methods.

[0123] In some embodiments, the eukaryotic host cell is THP-1 ceil or a human embryonic kidney (HEK) cell.

[0124] Once expressed, rCELA2A proteins can be purified according to standard procedures known in the art. Such procedures include, but are not limited to, ammonium sulfate precipitation, the use of affinity columns, routine column chromatography, gel electrophoresis, and the like (see, generally, R. Scopes, “Protein Purification”, Springer-Verlag, N. Y. (1982)). Once purified, the rCELA2A proteins may then be used to practice the method of the disclosure, or to prepare a pharmaceutical composition useful in practicing the method of the disclosure.Pharmaceutical Compositions

[0125] In another aspect, the present disclosure provides a pharmaceutical composition comprising the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, and a pharmaceutically acceptable excipient or carrier.

[0126] There are a wide variety of suitable formulations of the composition comprising a rCELA2A protein disclosed herein. The following formulations and methods are merely exemplary and are in no way limiting. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0127] Examples of suitable carriers, excipients, and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. In some embodiments, the composition comprising the described rCELA2A protein with a carrier as discussed herein is present in a dry formulation (such as lyophilized composition). The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.

[0128] In some embodiments, the compositions are formulated to be administered by any route which results in a therapeutically effective outcome. These include but are not limited to administered intravenously, intraarterially, intraperitoneally, intravesicularly,subcutaneously, intrathecally, intrapulmonarily, intramuscularly, intratracheally, intraocularly, transdennally, orally, or by inhalation.

[0129] Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Injectable formulations are preferred.

[0130] In some embodiments, the composition is formulated to have a pH range of about 4.5 to about 9.0, including for example pH ranges of about any of 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. In some embodiments, the pH of the composition is formulated to no less than about 6, including for example no less than about any of 6.5, 7, or 8 (such as about 8). The composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.

[0131] In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.

[0132] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is advisable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.

[0133] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effectiveamount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder contemplated in the disclosure.

[0134] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for any suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired, with other active agents, e.g., analgesic agents.

[0135] Suitable compositions and dosage forms include, for example, dispersions, suspensions, solutions, syrups, granules, beads, powders, pellets, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, and the like. Powdered and granular formulations of a pharmaceutical preparation of the disclosure may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form a material that is suitable to administration to a subject. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0136] Pharmaceutical compositions of the disclosure may also be formulated to provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface-active agent, or a preservative such as methylhydroxybenzoate. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0137] In certain embodiments, pharmaceutical compositions of the disclosure may comprise the described 1CELA2A proteins in combination with at least one additional therapeutic agent useful for treating or preventing a metabolic syndrome (e.g., diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis) or pancreatitis. This additional therapeutic agent may comprise therapeutic agents identifiedherein or another therapeutic agent, e.g., commercially available therapeutic agent, known to treat, prevent or reduce the symptoms of a metabolic syndrome (e.g., diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis) or pancreatitis.Methods of the Invention

[0138] The modified CELA2A protein described herein can be used, for example, to improve the postprandial insulin secretion, to enhance the survival and function of pancreatic islets, developing a cure for a metabolic syndrome, such as type 2 diabetes, dyslipidemia, and atherosclerosis.

[0139] In one aspect, the present disclosure provides a method of treating a metabolic syndrome in a subject in need thereof, comprising administering to the subject an effective amount of a rCELA2A protein, or a polynucleotide or a vector comprising a nucleotide sequence encoding the rCELA2A protein, or a pharmaceutical composition comprising said protein or polynucleotide or vector, wherein the rCELA2A protein exhibits reduced or no elastase activity as compared to the wild-type CE, A2A protein and / or is resistant to proteolytic cleavage by trypsin or other proteases. In some embodiments, the metabolic syndrome is diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis.

[0140] In another aspect, the present disclosure provides a method for preserving pancreatic beta cell structure and / or function in a subject in need thereof, comprising administering to the subject an effective amount of a rCELA2A protein, or a polynucleotide or a vector comprising a nucleotide sequence encoding the rCELA2A protein, or a pharmaceutical composition comprising said protein or polynucleotide or vector, wherein the rCELA2A protein exhibits reduced or no elastase activity as compared to the wild-type CELA2A protein and / or is resistant to proteolytic cleavage by trypsin or oilier proteases.

[0141] In another aspect, the present disclosure provides a method of augmenting insulin secretion and / or enhancing insulin signaling in a subject in need thereof, comprising administering to the subject an effective amount of a rCELA2A protein, or a polynucleotide or a vector comprising a nucleotide sequence encoding the rCELA2A protein, or a pharmaceutical composition comprising said protein or polynucleotide or vector, wherein the rCELA2A protein exhibits reduced or no elastase activity as compared to the wild- type CELA2A protein and / or is resistant to proteolytic cleavage by trypsin or other proteases.

[0142] In various embodiments, the rCELA2A protein is resistant to proteolytic cleavage by trypsin and oilier proteases. In some embodiments, the rCELA2A protein exhibits reduced orno elastase activity as compared to the wild-type CELA2A protein. In some embodiments, the rCEEA2A protein does not comprise a signal peptide. In some embodiments, the rCEEA2A protein comprises an activation peptide.

[0143] In another aspect, the present disclosure provides a method of treating a metabolic syndrome in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein. In some embodiments, the metabolic syndrome is diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis.

[0144] In another aspect, the present disclosure provides a method for preserving pancreatic beta cell structure and / or function in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein.

[0145] In another aspect, the present disclosure provides a method of preserving pancreatic islet size and / or number in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein.

[0146] In another aspect, the present disclosure provides a method of augmenting insulin secretion and / or enhancing insulin signaling in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or die pharmaceutical composition o described herein.

[0147] In some embodiments, the subject has or is at risk of developing diabetes. In some embodiments, the diabetes is a type 1 diabetes or type 2 diabetes.

[0148] In another aspect, the present disclosure provides a method of treating pancreatitis in a subject in need thereof, comprising administering to the subject an effective amount of a CELA2A protein, or a polynucleotide or a vector comprising a nucleotide sequence encoding the CELA2A protein, or a pharmaceutical composition comprising said protein or polynucleotide or vector.

[0149] In some embodiments, the CELA2A protein is a wild-type CELA2A enzyme, proenzyme, or preproenzyme, or a functional fragment or derivative thereof.

[0150] In some embodiments, the wild-type CELA2. A protein comprises the amino acid sequence of SEQ ID NO: 1, 3, or 5.

[0151] In some embodiments, the CELA2A protein is a mutant protein resistant to proteolytic cleavage by trypsin.

[0152] In some embodiments, the CELA2A protein is a mutant protein which exhibits reduced, or no elastase activity as compared to a wild-type CELA2A enzyme.

[0153] In some embodiments, the mutant CELA2A protein is a proenzyme, or preproenzyme, or a functional fragment or derivative thereof.

[0154] In some embodiments, the method comprises administering to the subject an effective amount of the modified rCELA2A protein described herein, or the polynucleotide described herein, or the vector described herein, or the pharmaceutical composition described herein.

[0155] In some embodiments, the pancreatitis is chronic pancreatitis.

[0156] In some embodiments, administration of the CELA2A protein, polynucleotide, vector, or pharmaceutical composition described herein reduces pancreatic inflammation in pancreatic islets.

[0157] In certain embodiments, the described rCELA2A proteins of the disclosure are useful in the methods of the disclosure in combination with at least one additional therapeutic agent useful for treating or preventing a metabolic syndrome (e.g., diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis) or pancreatitis. This additional therapeutic agent may comprise therapeutic agents identified herein or another therapeutic agent, e.g., commercially available therapeutic agent, known to treat, prevent or reduce the symptoms of a metabolic syndrome (e.g., diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis) or pancreatitis.

[0158] A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 19981, Clin. Pharmacokinet.6: 429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.Kits

[0159] Kits provided herein include one or more containers comprising the described rCELA2A protein or a pharmaceutical composition comprising the described rCELA2A protein and / or other agent(s), and in some embodiments, further comprise instructions for use in accordance with any of the methods described herein. The kit may further comprise a description of selection of subject suitable for treatment. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0160] In some embodiments, the kit comprises a) a composition comprising a rCELA2A protein described herein, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the described rCEEA2A protein for treatment of a disease or disorder.

[0161] The kits of the disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0162] In some embodiments, the kits comprise one or more components that facilitate delivery of the described rCELA2A protein, or a composition comprising the agent, and / or additional therapeutic agents to the subject. In some embodiments, the kit comprises, e.g., syringes and needles suitable for delivery of cells to the subject, and the like. In such embodiments, the described rCELA2A protein, or a composition comprising the agent may be contained in the kit in a bag, or in one or more vials. In some embodiments, the kit comprises components that facilitate intravenous or intra-arterial delivery of the described rCEXA2A protein, or a composition comprising the agent to the subject. In some embodiments, the described rCELA2A protein, or a composition comprising the agent may be contained, e.g., within a bottle or bag (for example, a blood bag or similar bag able to contain up to about 1.5 L solution comprising the cells), and the kit further comprises tubing and needles suitable for the delivery of the described rCELA2A protein, or a composition comprising the agent to the subject.

[0163] The instructions relating to the use of the compositions generally include information as to dosage, dosing schedule, and route of administration for the intendedtreatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. For example, kits may be provided that contain sufficient dosages of the described rCELA2A protein as disclosed herein to provide effective treatment of a subject for an extended period, such as any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of the pharmaceutical compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.EXAMPLES

[0164] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.Example 1. Whole exome sequencing (WES) identifies mutations in CELA2A underlying CAD and all metabolic syndrome traits.

[0165] Through WES of multiplex and nuclear kindreds with early-onset myocardial infarction, and metabolic syndrome traits including obesity, hypertension, dyslipidemia, insulin resistance and / or T2DM, and fatty liver disease at age 30 or younger multiple independent, novel, and deleterious non-conservative missense mutations were identified in the CELA2A gene, which perfectly segregate with all metabolic traits (FIG. 1). In the largest kindred, a perfectly segregating CELA2A mutation (LOD= 6.9) was found, that causes loss of elastase activity due to the substitution of an evolutionarily conserved aspartic acid for asparagine in the catalytic domain in codon 121 (p. D121N). Of significance, common CELA2A variants rs 1042010 and rs3820068 have been associated with elevated systolic blood pressure by independent genome-wide association studies [1-3] (p<3x10-13and p< 1 x 1012, respectively ). The rsl042010(G) allele is in disequilibrium with the eSNP rs3753326(A) (D’:0.8674, R2: 0.7134) that is in an open chromatin region and is associated with reduced CELA2A transcripts in the adrenal gland (GTEX database, p=2.4 xlO-9). Several other variants in CELA2A locus are associated with obesity and high cholesterol in GWAS (Common Metabolic Disease Knowledge Portal). Strikingly, the variant rs!44878061 in the CELA2A gene, which is in ED with rs 1042010, is associated with chronic pancreatitis [4], suggesting links between chronic pancreatitis, metabolic traits and T2DM,Example 2. CELA2A is a widely expressed protein.

[0166] Initially known only as an exocrine pancreatic digestive enzyme, the highest CELA2A mRNA and protein levels after exocrine pancreas were found in white (WAT) and brown adipose tissues (BAT), intestine, spleen, heart, and liver in mice (FIGS. 2A, 2B). Using immunohistochemistry analysis in human surgical specimens, the most intense CELA2A immunoreactivity was found in the exocrine pancreas, followed by the adrenal cortex, and in the gut-associated lymphatic tissue (GALT) (FIG. 2C). In addition, high levels of CELA2A mRNA and protein were found in human BMDCs (shown in later sections), as well as in the adipose tissues, pancreatic islets, and the liver. Based on Tabula Muris, Cela2a expression in the pancreas was primarily observed in the acinar, ductal cells, and leukocytes, with relatively lower levels detected in the islets.Example 3. CELA2A is a circulating protein, its levels rise postprandially and in parallel with plasma insulin levels in humans.

[0167] In p. D 12 IN-carriers, it was found that the total plasma levels of CELA2A was increased but the elastase activity was reduced by almost half (FIGS. 2D, 2E), indicative of a substantial reduction of total circulating elastase activity. In healthy controls (mean age 35 y) the plasma levels of CELA2A rose postprandially (FIG. 2F, 2G, 2H), perfectly parallel with insulin and C- peptide (R“ coefficient 0.9 and 0.88, respectively) (FIGS. 21, 2J). Similarly, a hyperglycemic clamp, aimed at plasma glucose levels of 200 mg / dl resulted in both higher plasma insulin and CELA2A levels in obese subjects (FIG. 2K). The oral glucose tolerance test (OGTT) of the same subjects showed a parallel rise of plasma CELA2A and insulin (FIG. 2L). These findings indicate a physiological role of CELA2A.Example 4. CELA2A promotes glucose-induced and calcium ion-dependent insulin secretion.

[0168] The administration of the endotoxin-free recombinant Cela2a (<0. lEU / ml) into 3 m-old male diabetic Ldlr- / -mice resulted in a significant drop in plasma glucose levels compared to saline (FIG. 3A). There was a parallel rise in circulating levels of C -peptide (FIGS. 3B, 3C). No effect was seen in normoglycemic mice. To investigate the direct effect of rCela2a on insulin secretion, size-matched primary rat islets were treated with purified rCela2a (MyBioSource, 2.8 ug / ml) or with vehicles at 2.5 mM and 9 mM glucose concentrations and assessed insulin secretion by measuring insulin levels. It was found that insulin secretion was augmented by rCela2a at 9 mM glucose, compared to vehicles alone (FIG. 3D). The intracellular Ca2+ transient after treatment with rCela2a vs. vehicle was dramatically increased in the presence of 9mM glucose (FIG. 3E). The MyBioSourcerCela2a is generated in E coli and is catalytically inactive. Remarkably, subsequent studies have revealed that the catalytically inactive form of Cela2a elicits the highest levels of insulin secretion in INS-1 cells.Example 5. Reduced pancreatic islet size and number and reduced glucose-stimulated insulin secretion in Cela2a KO mice on a chow diet.

[0169] The disease-associated human CELA2A mutations either disrupt the catalytic domain or are splice variants, resulting in the loss of the catalytic domain. This prompted the creation of a global Cela2a KO mouse model to examine disease pathways. Cela2a KO mice on a C57bl / 6 background were generated using a targeting plasmid developed by KOMP. The plasmid contained originally a poly-A stop site, a beta gal gene trap cassette distal to exon2 flanked by FRTs and followed by a floxed exon 3-4 of Cela2a (FIG. 4A). Unexpectedly, the poly-A stop site was noted to be leaky, randomly resulting in incomplete disruption of the mouse Cela2a gene. To bypass this problem, flip recombination and crossbreeding was employed with a strain that expresses Cre recombinase under alpha-actin promoter, and mice that are globally deficient for Cela2a by greater than 90% were generated. Cela2a KO mice exhibited higher body size and white adipose tissue despite similar food intake (FIGS. 4B), higher total body, lean, and fat weight, and higher percent fat weight (FIGS. 4C-4F) compared to littermates. Accordingly, plasma FFA (FIG. 4G), cholesterol (FIG. 4H) and triglycerides (TG) (FIG. 41) levels were higher in Cela2a KO compared to WT mice. The 12-week-old male Cela2a KO mice and age, gender, and weight-matched wild-type underwent intraperitoneal glucose and insulin tolerance tests. While Cela2a KO mice exhibited normal insulin sensitivity by intraperitoneal insulin tolerance test (ipITT) (FIG. 4J) they displayed higher glucose levels (FIG. 4K) and a dramatic reduction in glucose-stimulated insulin secretion (GSIS ) (FIG. 4L) during intraperitoneal glucose tolerance test (ipGTT) while fed a chow diet. The intraperitoneal insulin tolerance test did not reveal any difference in insulin sensitivity between Cela2a KO vs. WT mice (FIG.4H). As expected, the plasma Cela2a levels were lower in KO mice compared to WT mice during ipGTT (FIG. 41). The examination of the pancreas revealed dramatically reduced pancreatic islet quantity and size (FIGS. 5A-5B) compared to WT mice.Example 6. Reduced pancreatic isle size and number in Cela2a KO mice is associated with loss of matrix proteins.

[0170] The examination of the pancreas in 12-week-old (homozygous) Cela2a KO mice fed a chow diet revealed a dramatic reduction in both the quantity and size of pancreatic islets compared to WT mice (FIG. 4M). Interestingly, heterozygous mice exhibited a mixture ofenlarged and diminutive islets relative to WT mice. Consistent with these findings there was increased sign of apoptosis, pyroptosis, and senescence, assayed by poly (ADP-ribose) polymerase- I (PARP) (FIG. 4N), Caspasel (FIG. 4P), and nuclear localization of p27 (FIG.40) in the beta cells of Cela2a KO vs. WT mice.

[0171] The survival, growth, differentiation, and proliferation of beta cells is highly dependent on the composition of the extracellular' matrix (ECM), made of collagens, fibronectin, and integrins [3-6]. Strikingly, the trichrome (FIG. 5A) and Sirius red stainings (FIG. 5B) revealed loss of matrix proteins / collagen in Cela2a KO mice possibly due to degradation.

[0172] To gain an unbiased understanding of the effect of the altered composition of tire ECM on the signaling pathway, a proteomic analysis was performed. The proteomic analysis of the Cela2a KO mice pancreas revealed a dramatic reduction in the expression levels of the integrin protein alpha 6 (ITGA6), integrin-linked kinase (ILK), as well as their downstream effector mTOR (FIG. SC). The Reactome pathway analysis identified inflammation as one of the major pathways altered in the proteome of the mutant vs. wild-type mice (FIG. 5D). The Integrins are the major ECM adhesion molecules which are involved in signaling events, determining the cell fate, polarity, migration, differentiation, proliferation, and survival [7]. Their interaction with the ECM plays a critical role in the differentiation of endocrine progenitors into insulin- and glucagon-positive cells. Therefore, the levels of most abundant integrins in the pancreas, ITGA6 ITGA5, ITGB3 and ITGB4 were measured in the mice. Surprisingly, there was a drastic reduction in the levels of all 4 integrins in the islets and surrounding tissues of Cela2a KO vs. WT mice by western blot analysis (FIG. 5E). The matrix metalloproteinases (MMPs) are the main group of enzymes responsible for the degradation of the extracellular matrix (ECM) secreted by the infiltrating immune cells like macrophages and T cells. The beta cells in the islets may also produce MMPs and this is in response to cytokines during inflammation. MMP2, 7, 9, and 12 are the key MMPs in the pancreatic islets [8]. Accordingly, there were increased levels of these proteins in the Cela2a KO vs. WT mice (FIG. 5E).

[0173] ECM imparts spatial context for signaling events by adhesion molecules.Accordingly, there were lower levels of pAKT, pGSK, pFOXO, pERK, and pAMPK in Cela2a KO vs. WT mice pancreatic tissues (FIG. 5F), suggesting impaired Integrin-ILK-AKT pathway caused by altered ECM.

[0174] The reduced levels of ITGA6, ITGA5, ITGB3, and ITGB4 in pancreatic tissues of Cela2a KO mice compared to WT mice were further validated by immunostaining (FIGS.5G-5J), which also revealed decreased levels of Collagenlal (FIG. 5K) and ILK1 (FIG. 5L) in Cela2a KO mice versus WT mice. Growing evidence suggests that intact integrin-ILK-AKT signaling plays a crucial role in preserving pancreatic islets [9, 10] by limiting apoptosis and senescence of islet cells

[0011] . Taken together, these findings suggest that inflammation and MMPs contribute to the loss of matrix proteins and integrins, thereby impairing AKT signaling and leading to the deterioration of pancreatic islets.Example 7. The massive infiltration of hematopoietic cells in Cela2a KO mice islets.

[0175] MMPs in the pancreatic islets are excreted by a variety of cells, including macrophages, neutrophils, lymphocytes, and beta cells under inflammatoryconditions. Inflammation has long been considered a hallmark of Type 1 diabetes. However, the emerging evidence implicates its role in the pathogenesis of type 2 diabetes

[0012] .Additionally, pancreatitis, a common inflammatory disease of the pancreas, has been associated with type 3C diabetes

[0012] . The presence of inflammatory cells in the islets and the exocrine pancreatic tissues of the Cela2a KO mice was investigated. Unexpectedly, a significantly larger number of F4 / 80 and CD11C-positive cells was observed, the latter being a specific marker of Ml macrophages, in the islets of Cela2a KO mice compared to their wild-type littermates (FIG. 6A). Consistent with these finding, there was an increased number of CD68 cells (FIG. 6B) in the islets of Cela2a KO mice compared to their wild-type littermates. Furthermore, the islets of Cela2a KO mice exhibited an increased number of CD3 (FIG. 6C), and CD45-positive cells (FIG. 6D) compared to their wild-type littermates. These findings were accompanied by reduced islet vascularization, as indicated by CD31 staining (FIG. 6E). These unexpected results led to investigating the potential causal role of inflammation in the impaired development of pancreatic islets. To address this, pancreatic size and inflammation during the early postnatal period when the pancreas is still developing were examined. Analysis of Cela2a KO mice at postnatal day 6 (P6) revealed smaller islets compared to wild-type mice. Additionally, there was an increased presence of CD68-, CD11C-, and F4 / 80-positive cells in Cela2a KO mice compared to WT mice. These findings suggest that inflammation is an early event in Cela2a KO mice and likely plays a causal role in impairing pancreatic islet structure and function.Example 8. The activation of systemic inflammation in mice deficient for Cela2a.

[0176] Cela2a KO mice had an increased number of lymphocytes, and a reduced number of neutrophils compared to the wild-type littermates (FIG. 7A). The quantified multiplex cytokine array revealed increased plasma levels of inflammatory cytokines and reduced levels of anti-inflammatory cytokines. Specifically, the levels of IL-2 (FIG. 7B), IL- 16 (FIG.7C), IL-20 (FIG. 7D), the C-C motif chemokine 17 (CCL17) (FIG. 7E), and Macrophage-derived chemokine (MDC) (FIG. 7F), which are both chemoattractants for Th2 lymphocytes were increased in Cela2a KO vs. WT mice. Recruitment of T-helper 2 (Th2) lymphocytes and macrophages in fetal islets has been shown to result in localized inflammation and a permanent reduction in islet vascularity and impaired insulin secretion [13 J. In addition, the plasma levels of Eotaxin (FIG. 7G), an eosinophil chemotactic protein, and IFNb-1 (FIG. 7H), known to induce beta cell death [14, 15] were increased and those of immunosuppressant IL-10 (FIG. 7I) were reduced in Cela2a KO vs. WT mice.Example 9. The expression of CELA2A in the primary monocytes, neutrophils, and THP1 cells and the induction of insulin secretion.

[0177] Increased plasma cytokine levels in Cela2a KO mice raised the question of the cell-autonomous effect of Cela2a and prompted the examination of its expression in mouse and human macrophages isolated from peripheral blood by magnetic beads. Both 25kDa and 75kDa (oligomer) were abundantly present in mice and human macrophages (FIGS. 7J-7K). The CELA2A mRNA levels of human macrophages increased with LPS and combined LPS and PMA (FIG. 7L).

[0178] CELA2A was also expressed and secreted from THP1 monocytes (FIG. 8A). The CELA2A expression in THP-1 cells is similarly enhanced upon stimulation with LPS (FIGS.8A-8B). These findings prompted an investigation into the contribution of monocytes to circulating levels of CELA2A in plasma. We first measured the circulating levels of CELA2A in patients before and after total pancreatectomy and intrahepatic autotransplantation (TPIAT) during mixed meal tolerance tests. As expected, the CELA2A levels rose in the plasma after the meal (FIG. 8C). There was a substantial drop in CELA2A plasma levels in patients after TPIAT indicating that the exocrine pancreas is a major contributor to plasma CELA2A. Strikingly, however, high levels of circulating CELA2A were detectable after TPIAT during a mixed meal tolerance test. There was a parallel rise of C-peptide before and to a much lower degree after TPIAT (FIG.8D). These findings suggested an extrapancreatic source of CELA2A, which potentially includes monocytes and neutrophils. THP-1 cells were then transfected with plasmids containing HA-tagged wildtype and mutant CELA2As, which substituted aspartic acid for asparagine 121, leucine 85 for methionine, and threonine 70 for methionine discovered in humans with metabolic syndrome. INS-1 cells were treated with the supernatant, which showed a slight nonsignificant increase in insulin secretion (FIG. 8E). Next, CELA2A was purified by affinity chromatography. Treatment of INS-1 cells with wild-type but not mutant rCELA2A proenzymes led to insulinsecretion from INS-1 cells (FIG. 8F). The insulin secretion from INS-1 cells was reduced when THP-1 cells were treated with PMA (FIG. 8F). The effect of wild-type rCELA2A proenzymes in the induction of insulin secretion faded after treating INS-1 cells with PAR1 and combined PAR1 and PAR2. inhibitors (FIG. 8G). PAR1 was previously recognized as the receptor for CELA2A in the gut

[0016] and PAR2 as the receptor for pancreatic elastase 1 in mice

[0017] . Interestingly, trypsin activation of rCELA2A proenzymes completely abolished its insulinotropic effect (FIG. 8H), indicating the CELA2A proenzymes but not its catalytically active form induces insulin secretion. It has been shown that ERK activation is critical for insulin secretion as well as islet growth. Treatment of INS-1 cells with wild-type rCELA2A led to higher ERK activation compared to D121N CELA2A, assayed by immunofluorescence staining using antibodies against pERK (FIG. 8I). In contrast ERK activation in inflammatory cells has been linked to NF-kB activation and macrophage polarization in response to LPS or inflammatory signals [18, 19]. There was higher activation of ERK and NF-kB in THP-1 cells expressing mutant vs. WT CELA2As in response to LPS, suggesting gain of function effects (FIGS. 9A, 9B). Interestingly, these activations were neutralized by a PAR2 antagonist, identifying PAR2 as the main receptor for CELA2A in macrophages and the mutant proteins as its biased agonist (FIGS. 9C, 9D).Example 10. Wild-type CELA2A shifts macrophages toward the M2 phenotype, and its loss causes insulin resistance and hypoinsulinemia.

[0179] The expression levels of TNFa and CXCL8 mRNAs were decreased in THP-1 cells expressing wild-type CELA2A and increased in THP- 1 cells expressing mutant CELA2As (FIGS. 10A, 10B). The CXCL8 levels have been shown to be increased in diabetic patients

[0020] and act as a critical determinant for islet survival after transplantation and its blockade has been shown to improve intrahepatic islet engraftment and reduce intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion

[0021] , Furthermore, the expression levels of CCL5 were increased and those of IL-10 and ABCA1 were decreased in THP-1 cells expressing mutant CELA2As (FIGS. 10C-10E). CCL5 has been shown to impair glucose-induced insulin secretion in mice

[0022] . In contrast, IL-10 has been shown to exert cytoprotective effects in beta-cells

[0023] and prevents the onset of diabetes in the nonobese diabetic mouse

[0024] . ABCA1 can regulate insulin secretion in the pancreatic betacells

[0025] , Carriers of loss-of-function mutations in ABCA1 display pancreatic beta-cell dysfunction [26, 2.7]. These findings indicate, the cell-autonomous effect of CELA2a in the induction of systemic inflammation that likely contributes to hypoinsulinemia and islet destruction in Cela2a KO mice.Example 11. Transplantation of Cela2a deficient bone marrow-derived cells (BMDCs) into WT mice induces hypoinsulinemia and insulin resistance, accompanied by increased inflammation in the pancreatic islets.

[0180] Given the cell-autonomous role of pancreatic inflammatory cells in systemic inflammation and the loss of Cela2a's insulinotropic effects due to mutations, it was decided to transplant BMDCs into 4-week-old C57BL / 6 WT mice to compare their effects on insulin secretion and sensitivity with wild-type BMDCs. In 12-week-old mice transplanted with Cela2a deficient BMDCs hyperglycemia was observed during an intraperitoneal insulin tolerance test (ipITT), indicating insulin resistance (FIG. 11A). An intraperitoneal glucose tolerance test (ipGTT) at 18 weeks revealed hypoinsulinemia in mice transplanted with Cela2a-deficient BMDCs compared to those with wild-type BMDCs, suggesting a decline in beta cell function with aging (FIGS. 11B, 11C). Examination of pancreatic islets showed smaller islet sizes (FIG. 11D), reduced extracellular matrix (ECM) (FIGS. 11E, 11F), and increased levels of MMP2 (FIG. 11G), MMP12 (FIG. 11H), MMP9 (FIG. 11I), and MMP7 (FIG. 11J). There were also elevated CD3 (FIG. 11K) and CD45-positive cells (FIG. 11L) in mice transplanted with Cela2a deficient BMDCs compared to wild-type controls.Additionally, levels of ITGB4 (FIG. 11K) and ITGA5 (FIG. 11L) were reduced in the pancreatic islets of mice receiving Cela2a-deficient BMDCs compared to those transplanted with wild-type BMDCs.Example 12. Genetically modified rCELA2A proenzymes are stronger insulin inducers and resistant to proteases

[0181] CELA2A was engineered to resist to proteolytic cleavage by mutating the cleavage site at arginine 28 to lysine or histidine (R28K or R28H) amino acids that cause only minor structural changes. Treatment of INS-1 cells with the purified modified recombinant proenzymes resulted in increased insulin secretion. Notably, the R28H variant demonstrated a stronger insulinotropic effect compared to the wild-type protein (FIG. 12A). Both R28H and R28K variants were resistant to protease activation (FIG. 12B) and exhibited reduced elastase activity relative to wild-type CELA2A. Given the abundance of proteases in plasma, these findings suggest a significant advantage of the modified proenzymes over the wild-type CELA2A.Example 13. Modified rCELA2A proenzymes have anti-inflammatory effects and preserve pancreatic islet structure.

[0182] Significant progress has been made in the treatment of diabetes, with new antidiabetic drugs like Semaglutide, demonstrating, for the first time, proven efficacy inreducing cardiovascular mortality. However, a cure for diabetes remains elusive, and cardiovascular mortality continues to be a major concern. The present studies have shown the importance of CELA2A in maintaining islet size and function. Beta cell cilia play an important role in pancreatic beta cell development and function, partly by the regulation of insulin cell signaling. Disruption of ciliary function has been associated with impaired insulin secretion and the development of diabetes

[0028] , The study of the pancreatic islets in Cela2a KO mice showed almost complete disappearance of the beta cell cilia (FIG. 13A). In addition, mice transplanted with Cela2a KO BMDCs also showed loss of cilia in the pancreatic islets (FIG. 13B). The ciliary structure was maintained in INS-1 cells treated with wild-type (FIGS. 13E-13F) and modified CELA2As (R28H and R28K, FIGS. 13I-13J and 13K-13L) vs. baseline (FIGS. 13C-13D), while it was fully lost when these cells were treated with the pathological variant D121N (FIGS. 13G-13H). However, these functions were preserved only in the modified CELA2As and were lost in the wild-type CELA2A after the treatment with trypsin.

[0183] The effect of WT vs. modified R28H CELA2A on pancreatic inflammation in human islets was examined. CD11C and F4 / 80 expression, the markers of Ml macrophages were increased in the human islets treated with the human pathogenic D121N CELA2A variant and were diminished in islets treated with R28H CELA2A variant (FIG. 13M).Discussion

[0184] In this study, the potential therapeutic applications of a modified, enzymatically inactive form of CELA2A proenzyme in preserving beta cell structure and function was explored.

[0185] The present investigation into CELA2A’s physiological role revealed that it is a circulating protein whose levels increase postprandially and parallel insulin levels.Functionally, it was demonstrated that the modified CELA2A proenzyme stimulates insulin secretion and enhances insulin signaling, even in the absence of insulin, indicating its potential as an insulin secretagogue. Notably, the present research revealed that the catalytically inactive form of purified CELA2A is more potent in inducing insulin secretion, positioning it as a promising and safe candidate for therapeutic use.

[0186] Furthermore, the present study delved into the effects of CELA2A mutations, showcasing their impact on pancreatic islet cells. Cela2a knockout mice exhibited reduced islet size and number, leading to impaired glucose-stimulated insulin secretion. These mice also displayed altered expression levels of matrix proteins and integrin activation pathways, suggesting a role for CELA2A in maintaining islet structure and growth. Additionally, thepresence of inflammatory markers and immune cell infiltration in Cela2a knockout mice's islets indicated a potential link between CELA2A mutations, inflammation, and pancreatic dysfunction.

[0187] The present findings also shed light on the association between CELA2A mutations and chronic pancreatitis, with affected individuals showing signs of pancreatitis preceding the onset of diabetes. Purified CELA2A was found to play a role in suppressing inflammation, and its loss or mutation exacerbated inflammation in both mice and humans exocrine and endocrine pancreas. These results position CELA2A as a therapeutic target not only for diabetes but also for pancreatitis.

[0188] Moreover, the present study explored the molecular mechanisms underlying CELA2A’s insulinotropic effects. CELA2A was engineered to be resistant to proteolytic cleavage, resulting in a modified proenzyme with significantly enhanced insulin secretion capabilities. This modified CELA2A variant exhibited potent insulinotropic effects in INS-1 cells, outperforming the wild-type protein. Importantly, these effects were observed in a proenzyme state, indicating the potential of this modified form as an effective insulin secretagogue.

[0189] In conclusion, the present disclosure highlights the multifaceted roles of CELA2A in regulating insulin secretion, preserving pancreatic islet structure, and suppressing inflammation. The enzymatically inactive, modified CELA2A proenzyme emerged as a promising therapeutic candidate for diabetes and pancreatitis. Its potent insulinotropic effects, coupled with its ability to mitigate inflammation and maintain pancreatic integrity, position it as a valuable target for future therapeutic interventions. Further studies are warranted to explore the clinical applications of this modified CELA2A proenzyme and its potential to revolutionize the treatment of diabetes and related pancreatic disorders.

[0190] Although CELA2A is known for its function as a digestive enzyme, it was discovered herein that it is also a circulating protein that is widely expressed, with the highest levels found in bone marrow-derived cells. Its plasma levels increase after each meal in parallel to plasma insulin levels in healthy individuals and is reduced but still present after pancreatectomy. It was found herein that CELA2A triggers insulin secretion and sensitivity, while catalytically inactive mutant CELA2A proteins increase platelet aggregation and reduce insulin sensitivity. Mechanistically, wild-type CELA2A induces PAR2-dependent activation of AMPK in THP-1 human monocytes, while mutant CELA2As trigger different PAR2 -depen dent pathways, resulting in increased ERK and reduced AMPK activation. Interestingly, mutation carriers had lower levels of the anti-inflammatory cytokine IL- 10compared to noncarriers. These findings suggest that CELA2A is a novel risk factor and a potential therapeutic target for T2DM and prompted the generation of a global Cela2a KO mouse model. Cela2a KO mice on a chow diet are initially insulin resistant but exhibit fewer and considerably smaller pancreatic islets associated with reduced glucose-stimulated insulin secretion starting at age 12 weeks of age.

[0191] The present groundbreaking therapy holds significant economic advantages due to the absence of existing treatments for beta cell survival. Beta cells play a vital role in insulin production and are crucial for managing diabetes. With no existing therapies specifically designed to ensure the survival of these cells, the present innovation fills a critical gap in the field.

[0192] By preserving and protecting beta cell function, the modified rCELA2A proenzyme described herein has the potential to revolutionize diabetes management. This not only leads to enhanced patient outcomes, reducing the long-term healthcare costs associated with diabetes-related complications, but also contributes to a healthier, more productive workforce. Individuals with diabetes, especially those with type 1 diabetes, often face substantial medical expenses, including hospitalizations and medications. The present therapy has the economic advantage of potentially reducing these costs significantly over the long term.

[0193] Moreover, the economic benefits extend beyond individual patients. Healthcare systems and insurance providers could potentially save substantial amounts in diabetes-related treatments, reducing the burden on public health resources. Additionally, the overall productivity and quality of life for people living with diabetes may improve, leading to a more economically active population.References1 Warren, H. R. et al. Genome- wide association analysis identifies novel blood pressure loci and offers biological insights into cardiovascular risk. Nat Genet 49, 403-415, doi:10.1038 / ng.3768 (2017).2 Wain, L. V. et al. Novel Blood Pressure Locus and Gene Discovery Using Genome-Wide Association Study and Expression Data Sets from Blood and the Kidney. Hypertension, doi: 10.1161 / HYPERTENSIONAHA.l 17.09438 (2017).3 Hoffmann, T. J. et al. Genome-wide association analyses using electronic health records identify new loci influencing blood pressure variation. Nat Genet 49, 54-64, doi:10.1038 / ng.3715 (2017).

Claims

4 Rosendahl, J. et al. Genome-wide association study identifies inversion in the CTRB1-CTRB2 locus to modify risk for alcoholic and non-alcoholic chronic pancreatitis. Gut 61, 1855-1863, doi:10.1136 / gutjnl-2017-314454 (2018).5 Legate, K. R., Montanez, E., Kudlacek, O. & Fassler, R. ILK, PINCH and parvin: the tIPP of integrin signalling. Nat Rev Mol Cell Biol 7, 20-31, doi:10.1038 / nrm1789 (2006).6 Weber, L. M., Hayda, K. N. & Anseth, K. S. Cell-matrix interactions improve beta-cell survival and insulin secretion in three-dimensional culture. Tissue Eng Part A 14, 1959-1968, doi: 10.1089 / ten.tea.2007.0238 (2008).7 Townsend, S. E. & Gannon, M. Extracellular Matrix-Associated Factors Play Critical Roles in Regulating Pancreatic beta-Cell Proliferation and Survival. Endocrinology 160, 1885-1894, doi:10.1210 / en.2019-00206 (2019).8 Llacua, L. A., Faas, M. M. & de Vos, P. Extracellular matrix molecules and their potential contribution to the function of transplanted pancreatic islets. Diabetologia 61, 1261-1272, doi:10.1007 / s00125-017-4524-8 (2018).9 Fornaro, M. et al. p27(kipl) acts as a downstream effector of and is coexpressed with the betalC integrin in prostatic adenocarcinoma. J Clin Invest 103, 321-329, doi: 10.1172 / JCI4585 (1999).10 Diaferia, G. R. et al. betal integrin is a crucial regulator of pancreatic beta-cell expansion. Development 140, 3360-3372, doi:10.1242 / dev.098533 (2013).11 Georgia, S. & Bhushan, A. p27 Regulates the transition of beta-cells from quiescence to proliferation. Diabetes 55, 2950-2956, doi:10.2337 / db06-0249 (2006).12 Hart, P. A. et al. Type 3c (pancreatogenic) diabetes mellitus secondary to chronic pancreatitis and pancreatic cancer. Lancet Gastroenterol Hepatol 1, 226-237,doi: 10.1016 / S2468- 1253( 16)30106-6 (2016).13 Jaeckle Santos, L. J. et al. Neutralizing Th2 inflammation in neonatal islets prevents betacell failure in adult IUGR rats. Diabetes 63, 1672-1684, doi:10.2337 / db13-1226 (2014). 14 Thomas, II. E. et al. Interferon signalling in pancreatic beta cells. Front Biosci (Landmark Ed) 14, 644-656, doi: 10.2741 / 3270 (2009).15 Hessner, M. J. et al. Involvement of eotaxin, eosinophils, and pancreatic predisposition in development of type 1 diabetes mellitus in the BioBreeding rat. J Immunol 173, 6993-7002, doi: 10.4049 / jimmunol.173.11.6993 (2004).16 Motta, J. P. et al. Epithelial production of elastase is increased in inflammatory bowel disease and causes mucosal inflammation. Mucosal Immunol 14, 667-678,doi: 10.1038 / s41385-021-00375-w (2021).17 Basile, G. et al. Excess pancreatic elastase alters acinar-beta cell communication by impairing the mechano-signaling and the PAR2 pathways. Cell Metab 35, 1242-1260 el249, doi: 10.1016 / j.cmet.2023.05.007 (2023).18 Chen, L. et al. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget 9, 7204-7218, doi:10.18632 / oncotarget.23208 (2018).19 Zhang, X. & Mosser, D. M. Macrophage activation by endogenous danger signals. J Pathol 214, 161-178, doi:10.1002 / path.2284 (2008).20 Cimini, F. A. et al. Circulating IL-8 levels are increased in patients with type 2 diabetes and associated with worse inflammatory and cardiometabolic profile. Acta Diabetol 54, 961-967, doi:10.1007 / s00592-017-1039-1 (2017).21 Citro, A. et al. CXCR1 / 2 inhibition enhances pancreatic islet survival after transplantation. J Clin Invest 122, 3647-3651, doi:10.1172 / JCI63089 (2012).22 Pais, R., Zietek, T., Hauner, H.. Daniel, H. & Skurk, T. RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice. Am J Physiol Gastrointest Liver Physiol 307, G330-337, doi: 10.1152 / ajpgi.00329.2013 (2014).23 Russell, M. A. & Morgan, N. G. The impact of anti-inflammatory cytokines on the pancreatic beta-cell. Islets 6, e950547, doi:10.4161 / 19382014.2014.950547 (2014).24 Pennline, K. J., Roque-Gaffney, E. & Monahan, M, Recombinant human IL-10 prevents the onset of diabetes in the nonobese diabetic mouse. Clin Immunol Immunopathol 71, 169-175, doi: 10.1006 / clin.1994.1068 (1994).25 Kang, M. H. et al. Regulation of ABC Al protein expression and function in hepatic and pancreatic islet cells by miR-145. Arteriosclerosis, thrombosis, and. vascular biology 33, 2724-2732, doi: 10.1161 / ATVBAHA.113.302004 (2013).26 Vergeer, M. et al. Carriers of loss-of-function mutations in ABCA1 display pancreatic beta-cell dysfunction. Diabetes Care 33, 869-874, doi:10.2337 / dc09-1562 (2010).27 Koseki, M. et al. Impaired insulin secretion in four Tangier disease patients with ABCA1 mutations. J Atheroscler Thromb 16, 292-296, doi: 10.5551 / jat. e599 (2009).28 Cowley, S. C. MAIT cells and pathogen defense. Cell Mol Life Sci 71, 4831-4840, doi: 10.1007 / s00018-014-1708-y (2014).29 Riva, A. et al. Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease. Gut 67, 918-930, doi: 10.1136 / gutjnl-2017-314458 (2018).30 Nhu, Q. M., Shirey, K. A., Pennini, M. E., Stiltz, J. & Vogel, S. N. Proteinase-activated receptor 2 activation promotes an anti-inflammatory and alternatively activated phenotype in LPS-stimulated murine macrophages. Innate Immun 18, 193-203,doi: 10.1177 / 1753425910395044 (2012).31 Gillis, E. E., Musall, J. B., Baban, B. & Sullivan, J. C. IL-10 treatment decreases blood pressure in male, but not female, spontaneously hypertensive rats. Am J Physiol Renal Physiol 319, F359-F365, doi:10.1152 / ajprenal.00206.2020 (2020).32 Hong, E. G. et al. Interleukin- 10 prevents diet-induced insulin resistance by attenuating macrophage and cytokine response in skeletal muscle. Diabetes 58, 2525-2535,doi: 10.2337 / db08-1261 (2009).33 Rongione, A. J. et al. Interleukin 10 reduces the severity of acute pancreatitis in rats. Gastroenterology 112, 960-967, doi:10.1053 / gast.1997.v112.pm9041259 (1997).34 Esteghamat, F. et al. CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation. Nat Genet 51, 1233-1243, doi:10.1038 / s41588-019-0470-3 (2019).* * *[00194] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all values are approximate and are provided for description.[00195] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.Claims1. An isolated recombinant Chymotrypsin-like ELAstase 2A (rCELA2A) protein, comprising a substitution of an arginine residue at position 28 with another basic amino acid residue, wherein residue position numbering is with respect to a wild-type CELA2A preproenzyme, and wherein the rCELA2A protein is a proenzyme or preproenzyme, or a functional fragment or derivative thereof.

2. The rCELA2A protein of claim 1, wherein the wild-type CELA2A preproenzyme comprises the amino acid sequence of SEQ ID NO: 1.

3. The rCELA2A protein of claim 1, wherein the rCELA2A protein has at least 80% amino acid sequence identity to SEQ ID NO: 1.

4. The rCELA2A protein of any one of claims 1-3, wherein the rCELA2A protein is resistant to proteolytic cleavage by trypsin.

5. The rCELA2A protein of any one of claims 1-4, wherein the rCELA2A protein exhibits reduced or no elastase activity as compared to a wild-type CELA2A enzyme.

6. The rCELA2A protein of any one of claims 1-5, wherein the rCELA2A protein does not comprise a signal peptide.

7. The rCELA2A protein of claim 6, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 7.

8. The rCELA2A protein of any one of claims 1-7, wherein the rCELA2A protein comprises an activation peptide.

9. The rCELA2A protein of claim 8, wherein the activation peptide comprises the amino acid sequence of SEQ ID NO: 8.

10. The rCELA2A protein of any one of claims 1-9, wherein the rCELA2A protein comprises a substitution of the arginine residue at position 28 with a histidine residue (R28H).

11. The rCELA2A protein of claims 10, wherein the rCELA2A protein comprising a R28H substitution, comprises the amino acid sequence of SEQ ID NO: 9.

12. The rCELA2A protein of claims 10 or 11, wherein the rCELA2A protein comprising a R28H substitution, consists of the amino acid sequence of SEQ ID NO: 9.

13. The rCELA2A protein of any one of claims 1-9, wherein the rCELA2A protein comprises a substitution of the arginine residue at position 28 with a lysine residue (R28K).

14. The rCELA2A protein of claims 13, wherein the rCELA2A protein comprising a R28K substitution, comprises the amino acid sequence of SEQ ID NO: 11.

15. The rCELA2A protein of claims 14, wherein the rCELA2A protein comprising a R28K substitution, consists of the amino acid sequence of SEQ ID NO: 11.

16. The rCELA2A protein of any one of claims 1-15, wherein the rCELA2A protein is produced in a eukaryotic host cell.

17. The rCELA2A protein of claim 16, wherein the eukaryotic host cell is THP-1 cell or a human embryonic kidney (HEK) cell.

18. The rCELA2A protein of any one of claims 1-15, wherein the rCELA2A protein is produced in a prokaryotic host cell.

19. A polynucleotide comprising a nucleotide sequence encoding the rCELA2A protein of anyone of claims 1-18.

20. A vector comprising the polynucleotide of claim 19.

21. A host cell comprising the polynucleotide of claim 19 or the vector of claim 20.

22. The host cell of claim 21, wherein the host cell is a THP-1 cell or a human embryonic kidney (HEK) cell.

23. A pharmaceutical composition comprising the rCELA2A protein of any one of claims 1- 18, or the polynucleotide of claim 19, or the vector of claim 20, and a pharmaceutically acceptable excipient or carrier.

24. A method of treating a metabolic syndrome in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein of any one of claims 1-18, or the polynucleotide of claim 19, or the vector of claim 20, or the pharmaceutical composition of claim 23.

25. The method of claim 24, wherein the metabolic syndrome is diabetes, dyslipidemia, hypertriglyceridemia, obesity, hypertension, and / or atherosclerosis.

26. A method for preserving pancreatic beta cell structure and / or function in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein of any one of claims 1-18, or the polynucleotide of claim 19, or the vector of claim 20, or the pharmaceutical composition of claim 23.

27. A method of preserving pancreatic islet size and / or number in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein of any one of claims 1-18, or the polynucleotide of claim 19, or the vector of claim 20, or the pharmaceutical composition of claim 23.

28. A method of augmenting insulin secretion and / or enhancing insulin signaling in a subject in need thereof, comprising administering to the subject an effective amount of the rCELA2A protein of any one of claims 1-18, or the polynucleotide of claim 19, or the vector of claim 20, or the pharmaceutical composition of claim 23.

29. The method of any one of claims 26-28, wherein the subject has or is at risk of developing diabetes.

30. The method of claim 25 or 29, wherein the diabetes is a type 1 diabetes or type 2 diabetes.

31. A method of treating pancreatitis in a subject in need thereof, comprising administering to the subject an effective amount of a CELA2A protein, or a polynucleotide or a vectorcomprising a nucleotide sequence encoding the CELA2A protein, or a pharmaceutical composition comprising said protein or polynucleotide or vector.

32. The method of claim 31, wherein the CELA2A protein is a wild-type CELA2A enzyme, proenzyme, or preproenzyme, or a functional fragment or derivative thereof.

33. The method of claim 32, wherein the wild-type CELA2A protein comprises the amino acid sequence of SEQ ID NO: 1, 3 or 5.

34. The method of claim 31, wherein the CELA2A protein is a mutant protein resistant to proteolytic cleavage by trypsin.

35. The method of claim 31 or claim 34, wherein the CELA2A protein is a mutant protein which exhibits reduced, or no elastase activity as compared to a wild-type CELA2A enzyme.

36. The method of any one of claims 31, 34 and 35, wherein the mutant CELA2A protein is a proenzyme, or preproenzyme, or a functional fragment or derivative thereof.

37. The method of any one of claims 31 and 34-36, wherein the method comprises administering to the subject an effective amount of the rCELA2A protein of any one of claims 1-18, or the polynucleotide of claim 19, or the vector of claim 20, or the pharmaceutical composition of claim 23.

38. The method of any one of claims 31-37, wherein the pancreatitis is chronic pancreatitis.

39. The method of any one of claims 31-38, wherein administration of the CEL A2A protein, polynucleotide, vector, or pharmaceutical composition reduces pancreatic inflammation in pancreatic islets.

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