Compositions Comprising Antibody-Cleaving Enzymes and Methods of Using the Same

Abstract

Disclosed herein are compositions for cleaving antibodies and in vivo methods of using these compositions. The compositions comprise recombinant enzymes having human and non-human primate IgM-specific protease activity and human and non-human primate IgG-specific protease activity, providing a means of directly addressing pathogenic IgM and IgG antibodies, which are key players in the initiation of immune dysfunction. The enzymes may comprise a sequence having at least 50% identity to the sequence set forth in SEQ ID NO:17 or SEQ ID NO:18.

Description

[Technical Field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 421,996, filed November 2, 2022, U.S. Provisional Application No. 63 / 492,985, filed March 29, 2023, and U.S. Provisional Application No. 63 / 502,920, filed May 17, 2023, each of which is incorporated herein in its entirety.

[0002] II. Federal Subsidy Statement This invention was made with government support under Federal Grant Numbers R01 HL089221, R01 Al166969, and U19 AI131471 awarded by the National Institutes of Health. The federal government has certain rights in this invention.

[0003] III. REFERENCE TO SEQUENCE LISTING The Sequence Listing, created on October 31, 2023, and submitted on November 1, 2023 as an XML file entitled "22-2060-WO-Sequence_Listing," having a size of 289 kilobytes, is hereby incorporated by reference herein pursuant to Rule 1.52(e)(5) of the U.S. Patent Act. [Background technology]

[0004] IV. Background Monomeric IgM is the first antibody to appear as a B cell antigen receptor (BCR) on the surface of B cells, accompanied by the secretion of pentameric IgM from plasma cells. Class switching during B cell activation generates different isotypes or subtypes of IgG, which constitute the most abundant secreted class of antibodies (Engels N, et al. (2018) Immunol Rev. 283:150-160). Due to the highly diverse BCR repertoire, recognition of self-antigens can sometimes lead to the development of harmful IgG and IgM autoantibodies, resulting in autoimmune disease (Suurmond J, et al. (2015) J Clin Invest. 125:2194-2202). Similarly, antibodies against human leukocyte antigens (HLA) expressed on the surface of cells in transplanted organs can lead to rejection (Choi AY, et al. (2021) Front Immunol. 12:694-763). Furthermore, antibodies against viral vectors such as recombinant adeno-associated viruses (AAVs) are beneficial in the context of immunization against pathogens such as viruses, but may preclude the administration of gene therapy in future patients (Earley J, et al. (2023) Trends Biotechnol. 41:836-845). Another pathophysiological manifestation of autoantibodies in the context of autoimmune disease, organ transplantation, or gene therapy is the activation of the complement cascade and its lytic components, which can result in significant tissue damage (Goldberg BS, et al. (2020) Immunol Cell Biol. 98:305-317; Grafals M, et al. (2019) Front Immunol. 10:2380; West C, et al. (2023) Hum Gene Ther. 34:554-566; Smith CJ, et al. (2022) Front Immunol. 13:999-1021). Therefore, understanding and manipulating IgG and IgM levels can have profound implications for the development of therapeutic modalities with broad clinical impact. To this end, a wide range of approaches, including plasmapheresis, B cell targeting agents, complement inhibitors, neonatal Fc receptor (FcRn) blockers, and IgG-degrading enzymes, are being explored to modulate humoral immunity in autoimmune diseases, organ transplantation, and gene therapy applications (Choi AY, et al. (2021) Front Immunol. 12:694-763; Lee DSW, et al. (2020) Nature Reviews Drug Discov. 20:179-199; Zelek WM, et al. (2019) Mol Immunol. 114:341-352; Corti M, et al. (2014) Mol Ther Methods Clin Dev. 1:14033; Elmore ZC, et al. (2020) JCI Insight. 5(19):e139881; Leborgne C, et al. (2020) Nature Med. 26:1096-1101). Despite this spectrum of agents, strategies that directly address pathogenic IgM and IgG antibodies, which are key players in the initiation of immune dysfunction, are lacking. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Engels N, et al. (2018) Immunol Rev. 283:150-160 [Non-patent document 2] Suurmond J, et al. (2015) J Clin Invest. 125:2194-2202 [Non-patent document 3] Choi AY, et al. (2021) Front Immunol. 12:694-763 [Non-patent document 4] Earley J, et al. (2023) Trends Biotechnol. 41:836-845 [Non-Patent Document 5] Goldberg BS, et al. (2020) Immunol Cell Biol. 98:305-317 [Non-patent document 6] Grafals M, et al. (2019) Front Immunol. 10:2380 [Non-Patent Document 7] West C, et al. (2023) Hum Gene Ther. 34:554-566 [Non-patent document 8] Smith CJ, et al. (2022) Front Immunol. 13:999-1021 [Non-Patent Document 9] Choi AY, et al. (2021) Front Immunol. 12:694-763 [Non-Patent Document 10] Lee DSW, et al. (2020) Nature Reviews Drug Discov. 20:179-199 [Non-Patent Document 11] Zelek WM, et al. (2019) Mol Immunol. 114:341-352 [Non-Patent Document 12] Corti M, et al. (2014) Mol Ther Methods Clin Dev. 1:14033 [Non-Patent Document 13] Elmore ZC, et al. (2020) JCI Insight. 5(19):e139881 [Non-Patent Document 14] Leborgne C, et al. (2020) Nature Med. 26:1096-1101 Summary of the Invention

[0006] Therefore, there remains an urgent need to generate and characterize antibody-cleaving enzymes and to develop therapies based on these antibody-cleaving enzymes. V. Brief description of the drawings [Brief explanation of the drawings]

[0007] [Figure 1-1]Figures 1A-1H show structural models and in vitro characterization of human IgM cleavage enzymes. Figure 1A shows a representative structural model of the core domain of a candidate cysteine ​​protease containing the active site in the endothelial cells. Figure 1B confirms the protein expression system and IgM cleavage assay. We used a known porcine IgM protease (IdeSsuis) from Streptococcus suis, which is known to cleave porcine IgM but not human IgM. Immunoblot analysis shows the banding pattern of IgM from human serum after treatment with multiple candidate proteins. Cleavage bands were observed for protein candidates 2 and 6, hereafter referred to as IceM and IceM2, respectively. Figure 1C is a schematic diagram illustrating the predicted cleavage site motif and the molecular weight of the IgM cleavage product (CP). Figure 1D shows a structural docking analysis of IceM in complex with the human IgM heavy chain (HC) containing the active site in the endothelial cells. Figure 1E is an SDS-PAGE gel showing recombinant IceM (41.6 kDa) expression and purification with a BSA loading control. Figure 1F shows an SDS-PAGE gel of purified human IgM, IceM, or IgM treated with 10 μg mL of IceM at 37°C for 1 hour. Figure 1G shows a concentration curve for IceM enzymatic activity in human serum. Figures 1I and 1J show immunoblot analyses showing the banding patterns of IgM, IgA, IgD, IgE, or IgG from human serum (Figure 1H) and IgM from serum across species (Figure 1I) treated with PBS or 10 μg mL of IceM at 37°C for 1 hour. * indicates full-length HC (IgM, approx. 73 kDa; IgG, approx. 53 kDa; IgA, approx. 55 kDa IgE, approx. 73 kDa; IgD, approx. 63 kDa), and arrows indicate cleavage products. Figure 1J shows the alignment of IgM sequences from multiple species with the predicted human and NHP cleavage site residues (SEQ ID NO: 110), shown in red boxes. Error bars represent the mean ± standard deviation. All experiments were completed with at least three biological replicates. Analysis was performed using GraphPad Prism version 9.5, and a P value of less than 0.05 was significant. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above.

[0008] [Figure 2] Figures 2A-2C show structural modeling and analysis of IceM contact residues. Figure 2A shows a structural model of IdeS, with residues that contact human IgG shown in red. Figures 2B-2C show structural models of the IgM-cleaving enzymes IceM (Figure 2B) and IceM2 (Figure 2C), with residues that contact human IgM shown in red.

[0009] [Figure 3-1] Figures 3A-3C show the optimization of linkers for recombinant IceM fusion proteins. Figure 3A (top) shows structural modeling of IceM fusion proteins with three different linkers (rigid linker, flexible linker 1, and flexible linker 2, with the corresponding linker amino acid sequences directly below the structure). The models are colored by pLDDT. Figure 3A (bottom) shows immunoblot analysis of His-tagged fusion protein expression. The fusion protein with a rigid linker, hereafter referred to as IceMG, showed the best expression and was selected for further evaluation. Figure 3B shows a structural model of the dual-cleavage enzyme IceMG. Figure 3C shows a structural docking analysis of IceMG in complex with human IgM (gray) and human IgG (green) heavy chains, with the active site in red. [Figure 3-2] Same as above.

[0010] [Figure 4-1]Figures 4A-4H show in vitro characterization of the human IgM and IgG cleavage enzyme IceMG. Figure 4A shows an SDS-PAGE gel demonstrating recombinant IceMG (70.4 kDa) expression and purification with a BSA loading control. Figure 4B shows an SDS-PAGE gel of purified human IgM, IceMG, or IgM treated with 10 μg mL of IceMG for 1 hour at 37°C. Figure 4C shows a schematic illustrating the predicted IgG cleavage products (CPs). Figure 4D shows an SDS-PAGE gel of purified human IgG, IceMG, or IgG treated with 10 μg mL of IceMG for 1 hour at 37°C. Figures 4E and 4F show concentration curves for IceMG enzyme activity against IgM (Figure 4E) or IgG (Figure 4F) in human serum. Figure 4G shows immunoblot analysis showing the banding patterns of IgM, IgG, IgA, IgE, or IgD from human serum treated with PBS or 10 μg mL of IceMG at 37°C for 1 hour, and IgM or IgG from serum across species. Figure 4H shows the IgG cleavage site residues for each IgG subtype: IgG1 (SEQ ID NO: 115), IgG2 (SEQ ID NO: 116), IgG3 (SEQ ID NO: 117), and IgG4 (SEQ ID NO: 118). * indicates full-length HC, and arrows indicate cleavage products. Error bars represent the mean ± standard deviation. All experiments were completed with at least three biological replicates. Analysis was performed using GraphPad Prism version 9.5, and a P value of less than 0.05 was significant. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above.

[0011] [Figure 5-1]Figures 5A-5G show in vitro evaluation of recombinant IceM and IceMG on B cells. Figure 5A shows immunoblot analysis of Daudi cells treated with 10 μg mL of recombinant IceM for 1 hour at 37°C. Figure 5B shows immunofluorescence staining of surface-bound IgM on Daudi cells. Figure 5C shows representative plots of flow cytometry analysis of IgM surface expression on Daudi cells after 1 hour of treatment with PBS or IceM. Data were gated to analyze the IgM+ (FITC+) cohort. Figure 5D shows quantification of the percent IgM+ Daudi cells. Figure 5E shows representative plots of flow cytometry analysis of IgM surface expression on CD19+ B cells (BCR) after treatment with PBS, IceM, or IceMG at 1 hour, 12 hours, and 24 hours after treatment. Data were gated on live cells, followed by CD19+ (APC+) to analyze the IgM+ (FITC+) cohort. Figure 5F shows quantification of the percent IgM+ B cells from (Figure 5A). Figure 5G shows a dose curve for IceM enzyme activity in B cells treated for 1 hour at 37°C. Error bars represent the mean ± standard deviation. All experiments were completed with at least three biological replicates. Statistical significance was determined using an unpaired, two-tailed Student's t-test (Figure 5D) or a two-way ANOVA with Sidak correction for multiple comparisons (Figure 5F). Analysis was performed using GraphPad Prism version 9.5, and a P value of less than 0.05 was considered significant. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0012] [Figure 6]Figures 6A-6C show in vitro evaluation of recombinant IceM and IceMG complement activation. Figure 6A shows a diagram of the AAV9- or platelet factor-based complement activation assay. Figures 6B and 6C show ELISA-based quantification of C3a levels in human serum treated with PBS or 10 μg mL of IceM for 1 hour at 37°C to cleave IgM, followed by incubation with AAV9 (Figure 6B) or PF4 / heparin complex (Figure 6C). Error bars represent the mean ± standard deviation. All experiments were completed with at least three biological replicates. Statistical significance was determined using one-way analysis of variance. Analysis was performed using GraphPad Prism version 9.5, and a P value of less than 0.05 was considered significant.

[0013] [Figure 7-1] Figures 7A-7N show in vivo evaluation of recombinant IceM and IceMG in passively immunized mice. Figure 7A shows a schematic of the experimental design for the mouse study. Figures 7B-7E show immunoblots for circulating human IgM in mice treated with the indicated doses of PBS or IceM. Figures 7F-7I show immunoblots for circulating human IgM (upper panels) or IgG (lower panels) in mice treated with the indicated doses of PBS or IceM. Five mice (numbered 1-5) were tested per cohort, with human serum (Hu) included as a positive control. Figure 7J shows a schematic of the experimental design for the AAV mouse study. Luciferase expression was assessed in heart and liver tissue 21 days after injection for AAV8 (Figures 7K and 7L) and AAV9 (Figures 7M and 7N). Luciferase expression levels were normalized to total tissue protein concentration and expressed as log relative luminescence units per gram of tissue (log RLU / g tissue). [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0014] [Figure 8-1]Figures 8A-8H show in vivo evaluation of recombinant IceM in rhesus macaques. Figure 8A outlines the experimental design for non-human primate studies of IceM. Figures 8B and 8C show immunoblot analysis of His-tagged IceM (41.6 kDa) from NHP N303 (Figure 8B) and NHP M741 (Figure 8C) at various time intervals after IceM administration. Figures 8D and 8E show immunoblots for circulating IgM in animals treated with IceM. Days of treatment are indicated in red. Figure 8F shows quantification of uncleaved IgM and cleavage product bands from the immunoblots in Figures 8G and 8H. * indicates full-length HC, and arrows indicate cleavage products. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0015] [Figure 9-1] Figures 9A-9H show in vivo evaluation of recombinant IceMG on circulating IgM in rhesus macaques. Figure 9A shows an outline of the experimental design for non-human primate study 1 of IceMG. Figures 9B-9D (top) show immunoblot analysis of His-tagged IceMG (70.4 kDa) from animals 49873 (Figure 9B), 49813 (Figure 9C), and 499682 (Figure 9D) at various time intervals after IceMG administration. Figures 9B-9D (bottom) show immunoblots for circulating IgM in animals treated with the indicated doses of IceMG. Figures 9E-9G show quantification of IgM immunoblots from Figures 9B-9D. Figure 9H shows ELISA-based quantification of C3a levels in serum from the NHPs in Figures 9B-9D after treatment with AAV9 to induce complement activation. * indicates full-length HC, and arrows indicate cleavage products. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above.

[0016] [Figure 10-1]Figures 10A-10G show in vivo evaluation of recombinant IceMG on circulating IgM in rhesus monkeys. Figure 10A shows an outline of the experimental design for IceMG non-human primate study 2 using animals 36725, 37662, 35788, 38131, 38718, and 38721. Figures 10B-10D show immunoblot analysis for circulating IgM (Figures 10B-10D) in animals treated with the indicated doses of IceMG. Figures 10E-10G show quantification of IgM immunoblots from Figures 10B-10D. * indicates full-length HC, and arrows indicate cleavage products. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0017] [Figure 11-1] Figures 11A-11F show in vivo evaluation of recombinant IceMG on circulating IgG in rhesus monkeys. Figures 11A-11F show immunoblots for circulating IgG in animals treated with IceMG at the doses indicated for non-human primate study 1 (Figures 11A-11C) or non-human primate study 2 (Figures 11D-11F). * indicates full-length HC, and arrows indicate cleavage products. [Figure 11-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0018] VI. Brief Overview Disclosed herein is a recombinant enzyme having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgG-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an enzyme comprising any one of the sequences of SEQ ID NOs: 01 to 46 and having IgM-specific protease activity and / or IgG-specific protease activity.

[0019] Disclosed herein are recombinant enzymes capable of binding to and cleaving at one or more of the sequences set forth in SEQ ID NO: 111 to SEQ ID NO: 118 in IgG molecules, and the sequence set forth in SEQ ID NO: 110 in IgM molecules.

[0020] Disclosed herein are enzymes comprising any one of the sequences set forth in SEQ ID NO: 01 through SEQ ID NO: 46. Disclosed herein are isolated nucleic acid molecules encoding any of the disclosed recombinant enzymes. Disclosed herein are codon-optimized isolated nucleic acid molecules encoding any of the disclosed recombinant enzymes.

[0021] Disclosed herein is an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NOs: 47 to 92. Disclosed herein is an isolated nucleic acid molecule encoding a recombinant enzyme having the sequence of any one of SEQ ID NOs: 01 to 46. Disclosed herein is a vector comprising a nucleic acid sequence encoding a recombinant enzyme comprising the sequence of any one of SEQ ID NOs: 01 to 46. Disclosed herein is a vector comprising a nucleic acid sequence comprising the sequence of any one of SEQ ID NOs: 47 to 92, or a fragment thereof.

[0022] Disclosed herein is a method for cleaving immunoglobulin M (IgM) and / or IgM and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence of any one of SEQ ID NOs: 01 to 46.

[0023] Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising any one of the sequences of SEQ ID NO: 01 to SEQ ID NO: 46.

[0024] Disclosed herein is a method for reducing the level of circulating intact IgM and / or IgG and IgM in the serum of a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence of any one of SEQ ID NO: 01 to SEQ ID NO: 46.

[0025] Disclosed herein is a method for inhibiting complement activation in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence of any one of SEQ ID NOs: 01 to 46. Disclosed herein is a method for reducing and / or minimizing an immune response in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence of any one of SEQ ID NOs: 01 to 46.

[0026] Disclosed herein are methods for reducing the levels of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to a subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG-specific protease activity, and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM-specific protease activity.

[0027] Disclosed herein is a method for improving and / or enhancing the efficacy and / or expression of a transgene in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an enzyme comprising any one of the sequences set forth in SEQ ID NO: 01 to SEQ ID NO: 46. VII. Detailed Description

[0028] The present disclosure describes formulations, combination compositions, kits, capsules, containers, and / or methods thereof. It should be understood that the present invention is not limited to specific synthetic methods or, unless otherwise specified, to specific reagents, which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein.

[0029] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. A.Definition

[0030] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods, unless otherwise specified, or to particular reagents, unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described.

[0031] This disclosure describes the inventive concepts with reference to specific examples. However, it is intended to cover all modifications, equivalents, and alternatives to the inventive concepts consistent with this disclosure.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0033] The phrase "consisting essentially of" limits the scope of a claim to the recited components in a composition or recited steps in a method and to those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase "consisting of" excludes any component, step, or element not recited in the claim. The phrase "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended. "Comprising" does not exclude additional, unrecited components or steps.

[0034] As used herein, when referring to any numerical value, the term "about" means a value within a range that is ±10% of the stated value.

[0035] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, a further embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that the particular value forms a further embodiment by use of the antecedent "about." It will be further understood that each of the endpoints of a range is significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] References in this specification and the concluding claims to parts by weight of a particular element or component in a composition represent the weight relationship between the element or component and any other element or component in the composition or article to which the parts by weight are expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are included in the compound.

[0037] As used herein, the term "as needed" or "as needed" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur. In certain aspects, the disclosed methods can include one or more additional steps, such as, for example, repeating or modifying the administering step, as needed.

[0038] As used herein, "isolated" refers to a nucleic acid molecule or sequence that has been substantially separated from, produced away from, or purified away from other biological components in the cells or tissues of an organism in which it resides, such as other cellular, chromosomal and extrachromosomal DNA and RNA, and proteins. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in host cells and chemically synthesized nucleic acids and proteins. Isolated IceM, IceM2, IceM Full, IceM2 Full, IceM Mid, IceM Read, IceM2 Read, IceMG, IceMG2, IceMG Optilinker1, IceMG2 Optilinker1, IceMG-Optimlinker2, and IceMG-Optinlinker3 can be at least 50% pure, e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.

[0039] As used herein, the term "subject" refers to the target of administration, e.g., a human. The term "subject" also includes domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). Thus, the subject of the methods disclosed herein can be a vertebrate, e.g., a mammal, fish, bird, reptile, or amphibian. Alternatively, the subject of the methods disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus is intended to encompass adult and pediatric subjects, as well as fetuses, regardless of male or female. In some embodiments, the subject can be a human patient. In certain embodiments, the subject may have a disease or disorder, may be suspected of having a disease or disorder, or may be at risk for developing a disease or disorder (e.g., a genetic disease or disorder). In certain embodiments, the subject may have AD or AIF disease and / or disorder. In certain embodiments, the subject may be in need of one or more transplanted organs. In certain embodiments, the subject may have received one or more transplanted organs. In certain embodiments, the subject may be in need of, be able to receive, or have received gene therapy.

[0040] As used herein, the term "diagnosed" means to have been subjected to testing by a person skilled in the art, e.g., a physician, and to have been found to have a condition that can be diagnosed or treated by a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical preparation, or any combination thereof, or by one or more of the disclosed methods. For example, "diagnosed with a disease or disorder" means to have been subjected to testing by a person skilled in the art, e.g., a physician, and to have been found to have a condition (e.g., a genetic disease or disorder) that can be treated by one or more of the disclosed enzymes, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical preparation, or any combination thereof, or by one or more of the disclosed methods. For example, "suspected of having a disease or disorder" can mean having been subjected to testing by a person of ordinary skill in the art, e.g., a physician, and found to have a condition (e.g., a genetic disease or disorder) that can possibly be treated by one or more of the disclosed enzymes, disclosed recombinant enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or any combination thereof, or by one or more of the disclosed methods. In certain embodiments, the testing can be physical and can include various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzyme assays), or a combination thereof.

[0041] "Patient" refers to a subject suffering from a disease or disorder (e.g., a genetic disease or disorder). In certain embodiments, a patient may refer to a subject who has been diagnosed with or is suspected of having a disease or disorder. In certain embodiments, a patient may refer to a subject who has been diagnosed with or is suspected of having a disease or disorder and who is seeking or undergoing treatment for the disease or disorder. In certain embodiments, a patient may have AD or AIF disease and / or disorder. In certain embodiments, a patient may need one or more transplanted organs. In certain embodiments, a patient may have received one or more transplanted organs. In certain embodiments, a patient may have received one or more transplanted organs. In certain embodiments, a patient may need gene therapy, be able to receive gene therapy, or have received gene therapy.

[0042] As used herein, phrases such as "identified as needing treatment for disease or disorder" refer to the selection of a subject based on the need for treatment for disease or disorder.For example, a subject can be identified as having a need for treatment for disease or disorder (e.g., genetic disease or disorder, AD, AIF disease, transplanted organ) based on earlier diagnosis by a person skilled in the art, and then be subjected to treatment for genetic disease or disorder.In some embodiments, the identification can be performed by a person different from the person who makes the diagnosis.In some embodiments, the administration can be performed by the person who makes the diagnosis.

[0043] As discussed herein, humoral immunity is mediated by circulating proteins called antibodies, which are produced by B lymphocytes. Antibodies exist either as membrane-bound antibodies on the surface of B lymphocytes, which function as receptors for antigens, or as secreted antibodies present in the circulation, tissues, and mucosal sites to neutralize toxins, prevent the invasion and spread of pathogens, and eliminate microorganisms. B lymphocyte activation is initiated by specific recognition of antigens by their surface Ig receptors. Activation, in cooperation with Th cells, leads to the proliferation of antigen-specific cells and their differentiation, giving rise to memory B lymphocytes and antibody-secreting plasma cells. The type and quantity of antibodies produced vary depending on the antigen driving the immune response, the involvement of T lymphocytes, previous antigen exposure, and the site where activation occurs. Antibody responses to protein antigens require the internalization of specifically recognized antigens by B lymphocytes and the presentation of their peptide fragments to CD4+ helper T lymphocytes, which subsequently activate these B lymphocytes. Because of the involvement of helper T lymphocytes, this type of response is called a "T-dependent antibody response" (TDAR). In contrast, antibody responses to polyvalent nonprotein antigens with repeating determinants, such as polysaccharides, some lipids, and nucleic acids, do not involve antigen-specific helper T lymphocytes and are called "T-independent antibody responses." In TDAR, plasma cells or their precursors migrate from germinal centers in peripheral lymphoid organs where they are produced to the bone marrow, where they live for many years and ensure specific protection against microorganisms for an extended period of time.

[0044] As used herein, "immune response" refers to the body's response to foreign substances and infectious organisms and includes (i) the body's innate or nonspecific immune system, and (ii) the body's adaptive or specific immune system (including B cells, cytotoxic T cells, and helper T cells).

[0045] As used herein, "primary and secondary antibody responses" to protein antigens are qualitatively and quantitatively distinct. While primary responses result from the activation of naive B lymphocytes not previously stimulated by the antigen, secondary immune responses are elicited when the same antigen stimulates antigen-specific memory B lymphocytes. In secondary responses, antibody production is greater, peak antibody levels are reached more quickly (approximately 2-3 days vs. 7 days), the predominant isotype is IgG instead of IgM, and antibody affinity is much higher. Once released, various forms of antibodies (Ig) possess several effector functions for engaging antigens. Microorganisms, tumor cells, or foreign proteins can express several types of antigens and multiple copies of these antigens. When the microbial antigen targeted by the antibody associates with the toxic portion of the molecule, the antibody can neutralize the toxin. The production of neutralizing antibodies is frequently a problem in the therapeutic application of recombinant proteins derived from biotechnology, as they can be a rate-limiting step during preclinical trials because animals can perceive the recombinant protein as non-self, especially when humanized.

[0046] Although most effector functions of humoral immunity are mediated by antibody activation processes, they are also important players in innate immunity. For example, the classical activation pathway of the complement cascade is triggered by antigen-antibody complexes and is specifically mediated by the constant region of Ig molecules. Both IgM and IgG can activate the complement system, resulting in previously identified biologically active components, including lytic units, chemotactic factors, and complement peptides, that opsonize microorganisms to facilitate phagocytosis. IgM and IgG can function to opsonize some microorganisms in an antigen-dependent manner and independently of complement activation because macrophages and neutrophils have receptors on their surface that recognize the constant region of Ig (Fc receptors, FcR). FcRs also play a key role in IgG's ability to participate in a process known as "antibody-dependent cellular cytotoxicity," whereby antigen-specific antibodies attach to certain types of cells, including NK cells, via FcR, enabling these cells to closely adhere to target cells and induce cell death. Finally, FcRs are also the primary effector mechanism for IgE, which is the primary immune defense against certain types of parasitic infections (most notably helminths) and is primarily produced by the external immune system along secretory surfaces. IgE binds to FcRs on the surface of mast cells and basophils. Once bound to FcRs, IgE can act as an antigen-specific receptor and trigger the release of proinflammatory factors, including vasoactive amines (e.g., histamine) and products of the arachidonic acid cascade (e.g., leukotrienes and prostaglandins). Systemic immunity is mediated by IgM and IgG, the latter being the predominant form of Ig found in the blood. Local immunity is primarily mediated by IgA and IgE.

[0047] As known to those skilled in the art, autoimmune diseases (AD) originate in the adaptive immune system. AD ​​occurs when adaptive immune cells lose their ability to maintain self-tolerance of human cells. As a result, the immune system launches an attack on healthy tissues as if they were infectious pathogens. Some ADs are organ-specific, while others can affect multiple parts of the body. Recurrent fever and chronic inflammation can result from autoimmune diseases. However, many possible symptoms are unique to AD, including hair loss, dry mouth, temperature sensitivity, muscle weakness, reproductive problems, and more. Various conditions, including the environment, hormones, injury, infection, and genes, can all play a role in initiating autoimmunity. AD is thought to be triggered by a combination of multiple factors, not limited to genetics. While the exact cause remains unknown, researchers believe that various conditions, including the environment, hormones, injury, infection, and genes, can all play a role in initiating autoimmunity. Autoimmune conditions are very common. Current data estimate that AD affects at least 14 million people in the United States alone. There are 80-100 known autoimmune and autoimmune-related conditions, including Hashimoto's thyroiditis, type 1 diabetes, Sjogren's, and celiac disease. The gender prevalence in AD is well documented; in fact, approximately 80% of those diagnosed are women. AD can affect anyone at any age, but the majority tend to emerge during adulthood.

[0048] Autoinflammatory (AIF) diseases involve only the innate immune system. AIF occurs when innate immune cells are activated without an existing infection or injury. This unfortunate event directs the release of cytokines and other immune responses, causing fever and inflammation. Recurrent episodes of high fever are the primary symptom of AIF. Similar to some autoimmune diseases, autoinflammatory conditions can spread and affect the joints, gastrointestinal tract, skin, eyes, and internal organs.

[0049] As used herein, "regulatory element" can refer to promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, e.g., polyadenylation signals and poly U sequences). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0050] As used herein, "inhibit," "inhibiting," and "inhibition" mean to decrease or reduce an activity, level, response, condition, severity, disease, or other biological parameter (e.g., levels of circulating IgG and / or circulating IgM, or levels of surface-bound IgM on B cells). This can include, but is not limited to, a complete abolition of an activity, level, response, condition, severity, disease, or other biological parameter (e.g., levels of circulating IgG and / or IgM, or levels of surface-bound IgM on B cells). This also includes, for example, a 10% inhibition or reduction of an activity, level, response, condition, severity, disease, or other biological parameter compared to native or control levels (e.g., a subject without a disease or disorder, such as a genetic disease or disorder). Thus, in certain embodiments, inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, reduction compared to native or control levels. In some embodiments, inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% compared to native or control levels. In some embodiments, inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75-100% compared to native or control levels. In some embodiments, native or control levels can be pre-disease or pre-disorder levels.

[0051] The term "treat" or "treating" or "treatment" includes palliative treatment, i.e., alleviating symptoms but not curing the disease, condition, or disorder; preventative treatment, i.e., treatment aimed at minimizing or partially or completely preventing the occurrence of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy toward ameliorating the associated disease, condition, or disorder. In certain embodiments, the term encompasses any treatment of a subject, including a mammal (e.g., a human), and includes (i) preventing an undesirable physiological change, disease, condition, or disorder from occurring in a subject who has not yet been diagnosed as having it but may be susceptible to the disease; (ii) inhibiting a physiological change, disease, condition, or disorder, i.e., halting its development; or (iii) relieving a physiological change, disease, condition, or disorder, i.e., causing regression of the disease. For example, in certain embodiments, treating a disease or disorder can reduce the severity of an established disease or disorder in a subject by 1% to 100% compared to a control (e.g., an individual without a genetic disease or disorder). In certain embodiments, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (e.g., a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of the disease or disorder in a subject by 1% to 100% compared to a control (e.g., an individual without a genetic disease or disorder). In some embodiments, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in one or more symptoms of an established disease or disorder. It is understood that treating does not necessarily refer to a cure or complete disappearance or eradication of a disease or disorder. However, in some embodiments, treating can refer to a cure or complete disappearance or eradication of a disease or disorder.

[0052] As used herein, the term "prevent" or "preventing" or "prevention" refers to preventing, preventing, making unnecessary, forestalling, terminating, or impeding something from occurring, especially by a progressive effect. When reduce, inhibit, or prevent are used herein, it is understood that the use of the other two words is also expressly disclosed unless specifically indicated otherwise. In certain embodiments, it is intended to prevent a disease or disorder having chromatin deregulation and / or chromatin dysregulation. The terms "prevent," "preventing," and "prevention" also refer to prophylactic or preventative measures to protect or hinder a subject (e.g., an individual) who does not have a given disease or disorder (e.g., a genetic disease or disorder) or related complications from progressing to the complications.

[0053] As used herein, the terms "administering" and "administration" refer to any method of providing a subject with one or more of the disclosed enzymes, disclosed recombinant enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or any combination thereof. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous administration, intraCSF administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can also include administration into the hepatic artery or through the hepatic portal vein (HPV). Administration of the disclosed enzymes, disclosed recombinant enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical compositions, disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed small molecules, disclosed endonucleases, disclosed oligonucleotides, and / or disclosed RNA therapeutics can include direct administration to the CNS or PNS. Administration can be continuous or intermittent. Administration can include a combination of one or more routes.

[0054] In certain aspects, one of skill in the art can determine an effective dose, an effective schedule, and an effective route of administration for a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof, to treat or prevent a disease or disorder (e.g., a genetic disease or disorder). In certain aspects, one of skill in the art can also change, vary, or modify aspects of the administering step to improve the efficacy of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof.

[0055] "Determining the amount" refers to both the absolute quantification of a particular analyte (e.g., the level of IgG and / or IgM) or the determination of the relative abundance of a particular analyte (e.g., the level of IgG and / or IgM compared to a control / reference level). This phrase includes both direct or indirect measurement of abundance, or both.

[0056] As used herein, IdeZ is a cysteine ​​protease that inactivates IgG antibodies by cleaving them at the lower hinge region of the heavy chain, generating one F(ab')2 and one homodimeric Fc fragment.

[0057] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. In some embodiments, the pharmaceutical carrier used can be solid, liquid, or gaseous. In some embodiments, examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In some embodiments, examples of liquid carriers include sugar syrup, peanut oil, olive oil, and water. In some embodiments, examples of gaseous carriers include carbon dioxide and nitrogen. Any convenient pharmaceutical medium can be used when preparing the disclosed compositions for oral dosage form. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. can be used to form oral liquid preparations, such as suspensions, elixirs, and solutions, while carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. can be used to form oral solid preparations, such as powders, capsules, and tablets. Due to their ease of administration, tablets and capsules are the preferred oral dosage forms, and solid pharmaceutical carriers are used. If necessary, tablets can be coated by standard aqueous or nonaqueous techniques. Suitable fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions can also contain auxiliary agents, such as preservatives, wetting agents, emulsifying agents, and dispersing agents.Prevention of microbial activity can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). The rate of drug release can be controlled depending on the ratio of drug to polymer and the characteristics of the particular polymer used. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Suitable inert carriers may include sugars such as lactose. Optionally, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0058] As used herein, the term "excipient" refers to an inert substance commonly used as a diluent, vehicle, preservative, binder, or stabilizer, including, but not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkylsulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). Also, for reference, see Remington's Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0059] As used herein, "concurrently" means (1) simultaneously in time or (2) at different times during the course of a common treatment schedule.

[0060] The term "contacting," as used herein, refers to bringing one or more of the disclosed enzymes, disclosed recombinant enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or any combination thereof, together with a target region or intended target region (e.g., a cleavage site on IgG (SEQ ID NOs:111-118) and / or IgM (SEQ ID NO:110)) in a manner such that one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof have an effect, either directly or indirectly, on the intended target or targeted region. The target region can comprise one or more cells, and in certain embodiments, the one or more cells can be in a subject. The target region or intended target region can be one or more of the subject's organs (e.g., lung, heart, liver, kidney, brain, etc.). In certain embodiments, the target region or intended target region can be any organ, tissue, or cell affected by a disease or disorder (e.g., a genetic disease or disorder).

[0061] As used herein, " determining " can refer to measuring or confirming the existence and severity of disease or disorder, such as genetic disease or disorder.The methods and techniques used to determine the existence and / or severity of disease or disorder are typically known in the medical field.For example, the technical field is familiar with the methods for identifying and / or diagnosing the existence, severity, or both of disease or disorder (such as genetic disease or disorder).

[0062] As used herein, "effective amount" and "effective amount" can refer to an amount that is sufficient to achieve a desired result, such as, for example, treatment and / or prevention of a disease or disorder (e.g., a genetic disease or disorder) or a suspected disease or disorder. As used herein, the terms "effective amount" and "effective amount" can refer to an amount that is sufficient to achieve a desired effect on an undesired condition (e.g., a disease or disorder). For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve a desired therapeutic result, or that is generally insufficient to cause adverse side effects, but has an effect on undesired symptoms. In certain aspects, a "therapeutically effective amount" refers to an amount of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation that (i) treats a particular disease, condition, or disorder (e.g., a genetic disease or disorder), (ii) attenuates, ameliorate, or eliminates one or more symptoms of a particular disease, condition, or disorder (e.g., a genetic disease or disorder), or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder (e.g., a genetic disease or disorder) described herein. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the disclosed enzyme, disclosed recombinant enzyme, disclosed isolated nucleic acid molecule, disclosed vector, or disclosed pharmaceutical preparation used; the disclosed method used; the patient's age, weight, overall health, sex, and diet; the number of administrations; the route of administration; the rate of excretion of the disclosed isolated nucleic acid molecule, disclosed vector, or disclosed pharmaceutical preparation used; the duration of treatment; drugs used in conjunction with or concurrently with the disclosed isolated nucleic acid molecule, disclosed vector, or disclosed pharmaceutical preparation used, as well as other similar factors well known in the medical field. For example, it is within the skill of the art to start with a dose of the disclosed enzyme, disclosed recombinant enzyme, disclosed isolated nucleic acid molecule, disclosed vector, or disclosed pharmaceutical preparation lower than the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.If desired, the effective daily dose can then be divided into multiple doses for administration purposes. Consequently, a single dose of the disclosed isolated nucleic acid molecule, the disclosed vector, or the disclosed pharmaceutical formulation can contain such amounts or submultiples that make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. The dosage can vary and can be administered daily for one or several days, in one or multiple doses. Guidance can be found in the literature regarding appropriate dosages for a given class of pharmaceutical product. Furthermore, in various embodiments, the preparation can be administered in a "prophylactically effective amount," i.e., an amount effective for preventing a disease or condition, such as a disease or disorder caused by a missing, defective, and / or mutant protein or enzyme.

[0063] As used herein, "RNA therapeutics" may refer to the use of oligonucleotides that target RNA. RNA therapeutics may offer greater specificity, improved efficacy, and reduced toxicity, along with the promise of specifically targeting the exact nucleic acid involved in a particular disease. This may be particularly potent for genetic diseases, where it is most advantageous to target RNA, as opposed to proteins. In certain embodiments, therapeutic RNAs may comprise one or more expression sequences. As known in the art, expression sequences may include RNAi, shRNA, mRNA, non-coding RNA (ncRNA), antisense, e.g., antisense RNA, miRNA, morpholino oligonucleotides, peptide-nucleic acids (PNAs), or ssDNA (natural and modified nucleotides, including, but not limited to, LNA, BNA, 2'-O-Me-RNA, 2'-MEO-RNA, 2'-F-RNA), or analogs or conjugates thereof. In certain embodiments, the disclosed therapeutic RNAs can include one or more long non-coding RNAs (lncRNAs), such as long intergenic non-coding RNAs (lincRNAs), pre-transcripts, pre-miRNAs, pre-mRNAs, competitive endogenous RNAs (ceRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), pseudogenes, rRNAs, or tRNAs. In certain embodiments, the ncRNAs can be piwi-interacting RNAs (piRNAs), primary miRNAs (pri-miRNAs), or premature miRNAs (pre-miRNAs). In certain embodiments, the disclosed therapeutic RNAs or RNA therapeutics can include antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function through the RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form structures recognized by RNase H, resulting in cleavage of the mRNA target. In certain embodiments, RNA therapeutics can include RNAi and ASOs that inhibit mRNA translation.Generally speaking, as is known in the art, RNAi operates in a sequence-specific and post-transcriptional manner by activating ribonucleases, which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cleaved into smaller pieces, while antisense oligonucleotides bind to their target nucleic acids through Watson-Crick base pairing and inhibit or alter gene expression through steric hindrance, alteration of splicing, initiation of target degradation, or other events.

[0064] As used herein, "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids, and there is no limit on the maximum number of amino acids, which may include a protein sequence. The term "peptide" may refer to a short chain of amino acids, including, for example, a natural peptide, a recombinant peptide, a synthetic peptide, or any combination thereof. Proteins and peptides may include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0065] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. A reference to a single strand may also define the sequence of the complementary strand. Thus, a nucleic acid may encompass the complementary strand of a referenced single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid may encompass a substantially identical nucleic acid and its complement. A single strand can provide a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid may encompass a probe that hybridizes under stringent hybridization conditions. A nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, where the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods. Also, as used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "nucleotide sequence," and "polynucleotide" can refer to RNA or DNA, linear or branched, single-stranded or double-stranded, or a hybrid thereof. This term can encompass RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc., can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind to RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modifications to the phosphodiester backbone or the 2'-hydroxy of the ribose sugar group of RNA, can also be made.A "synthetic" nucleic acid or polynucleotide, as used herein, refers to a nucleic acid or polynucleotide that is not found in nature but is constructed by the hand of man and, therefore, is not a product of nature.

[0066] A "polynucleotide" is a sequence of nucleotide bases, which may be RNA, DNA, or a DNA-RNA hybrid sequence (containing both naturally occurring and non-naturally occurring nucleotides).

[0067] A "fragment" or "portion" of a nucleotide sequence may be understood to mean a nucleotide sequence that is reduced in length (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) relative to a reference nucleic acid or nucleotide sequence and that comprises, consists essentially of, or consists of a nucleotide sequence of contiguous nucleotides that is identical or nearly identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments or portions according to the present disclosure may, where appropriate, be included in a larger constituent polynucleotide. In certain aspects, a fragment or portion of a nucleotide sequence or nucleic acid sequence may include a sequence encoding an exon having one or more mutations.

[0068] A "fragment" or "portion" of an amino acid sequence is reduced in length relative to the reference amino acid sequence (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more amino acids) and is identical or nearly identical (e.g., reduced by 70 or more amino acids) to the reference amino acid sequence. "A fragment or portion of an amino acid sequence that is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to or greater than ...

[0069] "Complement" or "complementary," as used herein, means that a nucleic acid can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between the nucleotides or nucleotide analogs of a nucleic acid molecule. "Complementarity" refers to the property shared between two nucleic acid sequences such that when they are aligned antiparallel to each other, the nucleotide bases at each position are complementary.

[0070] As used herein, "operably linked" means that the expression of a gene or transgene is under the control of the promoter to which it is spatially connected. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0071] As used herein, the terms "promoter" or "promoters" are known in the art. A variety of promoter elements can be used depending on the desired level and tissue-specific expression. Promoters can be tissue-specific or ubiquitous, constitutive or inducible, depending on the desired pattern of gene expression. Promoters can be native (endogenous) or foreign (exogenous) and can be natural or synthetic sequences. By foreign or exogenous, it is intended that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced.

[0072] "Tissue-specific promoters" are known in the art and include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and cardiac-specific promoters.

[0073] "Liver-specific promoters" are known in the art and include, but are not limited to, the thyroxine-binding globulin (TBG) promoter, the alpha 1-microglobulin / bikunin enhancer / thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone-binding globulin promoter, the alpha 1-antitrypsin promoter, the bovine albumin (bAlb) promoter, the mouse albumin (mAlb) promoter, the human alpha 1-antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC172 promoter comprising the hAAT promoter and the alpha 1-microglobulin enhancer, the DC190 promoter comprising the human albumin promoter and the prothrombin enhancer, or any other natural or synthetic liver-specific promoter. In one embodiment, the liver-specific promoter comprises approximately 845 bp and may include the thyroid hormone-binding globulin promoter sequence (2382-13) described in Ill CR, et al. (1997), two copies of the α1-microglobulin / bikunin enhancer sequence (22,804 to 22,704), and a 71 bp leader sequence.

[0074] Ubiquitous / constitutive promoters are known in the art and include, but are not limited to, CMV major immediate early enhancer / chicken beta-actin promoter, cytomegalovirus (CMV) major immediate early promoter, elongation factor 1-alpha (EF1-alpha) promoter, simian vacuolating virus 40 (SV40) promoter, AmpR promoter, PγK promoter, human ubiquitin C gene (Ubc) promoter, MFG promoter, human beta-actin promoter, CAG promoter, EGR1 promoter, FerH promoter, FerL promoter, GRP78 promoter, GRP94 promoter, HSP70 promoter, β-kin promoter, mouse phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, ROSA promoter, human ubiquitin B promoter, Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous / constitutive promoter.

[0075] As used herein, an "inducible promoter" refers to a promoter that can be regulated by a positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemicals (e.g., metallothionein promoters or hormone-inducible promoters), temperature, and light.

[0076] As used herein, the term "serotype" refers to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological uniqueness can be determined by the lack of cross-reactivity between antibodies and one AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0077] As used herein, "tropism" refers to the specificity of the AAV capsid protein present in the AAV viral particle to affect a particular type of cell or tissue. The tropism of an AAV capsid for a particular cell or tissue type may be determined by measuring the ability of an AAV vector particle containing a hybrid AAV capsid protein to infect or transduce a particular cell or tissue type using standard assays well known in the art, such as those disclosed in the Examples of the present application. As used herein, the term "hepatotropism" or "hepatotropism" refers to tropism for the liver or liver tissues and cells, including hepatocytes.

[0078] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences can then be referred to as "substantially identical" or "essentially similar" when they are optimally aligned. For example, sequence similarity or identity can be determined by searching databases, such as FASTA, BLAST, etc., but hits must be searched and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences, can have "substantial sequence identity" if the sequence identity percentage is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher, preferably 90%, 95%, 98%, 99% or higher. Such sequences are also referred to herein as "variants," e.g., truncated, deleted, and / or other variants of mutant proteins or enzymes. It should be understood that sequences that have substantial sequence identity do not necessarily have the same length, and may vary in length. For example, sequences that have the same nucleotide sequence but have additional nucleotides 3' and / or 5' thereto are 100% identical.

[0079] A "heterologous" or "recombinant" nucleotide or amino acid sequence, as used interchangeably herein, can refer to a nucleotide or amino acid sequence that is not naturally associated with the host cell into which it is introduced, and includes non-naturally occurring multiple copies of a naturally occurring nucleotide or amino acid sequence.

[0080] Different nucleic acids or proteins that share homology can be referred to as "homologs." The term homolog includes homologous sequences from the same and other species, and orthologous sequences from the same and other species. "Homology" refers to the level of similarity between two or more nucleic acid and / or amino acid sequences in terms of percent positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Thus, the disclosed compositions and disclosed methods can include homologs to the disclosed nucleotide sequences and / or disclosed polypeptide sequences.

[0081] "Ortholog," as used herein, can refer to homologous nucleotide and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. Homologs of the disclosed nucleotide sequences or disclosed polypeptides can have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100%) to the disclosed nucleotide sequences or disclosed polypeptides.

[0082] As used herein, "codon optimization" can refer to the process of modifying a nucleic acid sequence for enhanced expression in a target host cell by replacing one or more codons or multiple native sequences with codons that are more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. Different species exhibit specific biases for certain codons of specific amino acids. As contemplated herein, genes can be adjusted for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database." Many methods and software tools for codon optimization have been previously reported (see, for example, genomes.urv.es / OPTIMIZER / ).

[0083] For example, a codon is a sequence of three nucleotides that encodes a specific amino acid in protein synthesis. Although there are only 20 amino acids, there are 64 distinct codons, resulting in the degeneracy of the genetic code. For example, the amino acid leucine can be coded for by six synonymous codons: CUU, CUC, CUA, CUG, UUA, and UUG, while cysteine ​​can be coded for by two codons: UGU and UGC. Overall, two of the 20 amino acids can be coded for by one codon, nine by two codons, one by three codons, five by four codons, and three by six codons, resulting in 61 essential codons. The remaining three codons are stop codons, terminating protein formation. Codon degeneracy results in many possible ways to code proteins; for example, a typical 375-amino acid protein in humans can potentially be coded for by 10,207 different codon sequences. All possible coding and resulting sequences are likely not identically observed in nature; however, some synonymous codons are used more frequently than others to encode certain amino acids in certain organisms. This phenomenon is called "codon usage bias" or "codon bias." For example, leucine is encoded by the codon CUG in 39.5% of cases in Homo sapiens, while the same codon is used to code for the same amino acid in 11.1% of cases in Saccharomyces cerevisiae (Nakamura et al., 2000).

[0084] As used herein, "immune tolerance," "immunological tolerance," and "immunotolerance" refer to a state of unresponsiveness or reduced response of the immune system to a substance (e.g., protein replacement therapy, enzyme replacement therapy, recombinant product, transplanted organ, vector protein, etc.) capable of eliciting an immune response in a subject. Immune tolerance is induced by prior exposure to a specific antigen. Immune tolerance can be determined in a subject by measuring antibodies to a particular antigen (e.g., HLA for a transplanted organ, an enzyme or therapeutic protein as part of a replacement therapy, a viral vector, an AAV capsid protein, etc.). Low or absent antibody titers over time are indicative of immune tolerance or efficacy of the disclosed enzymes or disclosed recombinant enzymes. For example, immune tolerance can be established by having an IgG antibody titer of less than or equal to about 12,000, 11,500, 11,000, 10,500, 10,000, 9,500, 9,000, 8,500, 8,000, 7,500, 7,000, 6,500, or 6,000 after gene therapy (e.g., administration of a transgene encoding a missing, defective, and / or mutant protein or enzyme), protein replacement therapy, enzyme replacement therapy, or recombinant product.

[0085] As is known in the art, antibodies (Abs) can attenuate AAV infection through multiple mechanisms, including by binding to the AAV capsid and blocking key steps of transduction, such as cell surface attachment and uptake, endosomal escape, productive transport to the nucleus, or uncoating, as well as by promoting AAV opsonization by phagocytes, thereby mediating their rapid clearance from the circulation. For example, in humans, serological studies reveal a high prevalence of NAbs in populations worldwide, with approximately 67% of individuals having antibodies to AAV1, 72% having antibodies to AAV2, and approximately 40% having antibodies to AAV serotypes 5 to 9. Vector immunogenicity represents a major challenge in AAV vector re-administration.

[0086] Also disclosed herein in certain embodiments are partially self-complementary parvovirus (e.g., the disclosed AAV) genomes, plasmid vectors encoding the parvovirus genomes, and parvovirus (e.g., the disclosed AAV) particles comprising such genomes. In certain embodiments, provided herein are plasmid vectors comprising a nucleotide sequence encoding a disclosed parvovirus genome, such as a disclosed AAV. In certain embodiments, provided herein are partially self-complementary parvovirus genomes comprising a payload construct, parvovirus ITRs flanking the payload construct, and a self-complementary region flanking one of the ITRs. The self-complementary region can comprise a nucleotide sequence complementary to the payload construct. The disclosed self-complementary region can have a length shorter than the entire length of the payload construct.

[0087] In some embodiments, the disclosed self-complementary regions of the disclosed parvovirus genomes can comprise a minimum length, but can also have a length that is less than the entire length of the payload construct. In some embodiments, the disclosed self-complementary regions can comprise a length of at least 50 bases, at least 100 bases, at least 200 bases, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, or at least 1,000 bases.

[0088] In certain embodiments, a "self-complementary parvovirus genome" can be a single-stranded polynucleotide having, from 5' to 3', a first parvovirus ITR sequence, a heterologous sequence (e.g., comprising a desired gene, e.g., a payload construct), a second parvovirus ITR sequence, a second heterologous sequence that is complementary to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a "partially self-complementary genome" does not include three parvovirus ITRs, and the second heterologous sequence that is complementary to the first heterologous sequence has a length that is shorter than the entire length of the first heterologous sequence (e.g., a payload construct). Thus, a partially self-complementary genome is a single-stranded polynucleotide having, in the 5' to 3' or 3' to 5' direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., a payload construct), a second parvovirus ITR sequence, and a self-complementary region that is complementary to a portion of the heterologous sequence, and has a length that is less than the entire length of the heterologous sequence.

[0089] As used herein, "immunomodulating" refers to the ability of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system. The immune system functions to protect organisms from infection and foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells, which regulate each other through interactions between multiple cells and by constituting soluble factors, including lymphokines and antibodies, which have autocrine, paracrine, and endocrine effects on immune cells.

[0090] As used herein, "immunomodulator" refers to an agent that can modulate a given immune response to a desired level (e.g., as an immunopotentiator, immunosuppressant, or inducer of immunological tolerance). Examples of immune modulators include, but are not limited to, aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxyspergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen, IL6 inhibitors (e.g., sarilumab), indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofetil, and the like. Disclosed immunomodulators may include rituximab, naproxen, prednisolone, prednisone, prednisolone-indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-rapamycin or ImmTOR), thalidomide, tocilizumab, tolmetin, triamcinolone acetonide, anti-CD3 antibody, anti-CD4 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD40 antibody, anti-FcRN antibody, anti-IL6 antibody, anti-IGF1R antibody, IL2 mutein, BTK inhibitor, or a combination thereof. In certain embodiments, the disclosed immunomodulators may include one or more Treg (regulatory T cell) infusions (e.g., antigen-specific Treg cells and AAV). In certain embodiments, the disclosed immunomodulators may be bortezomib or SVP-rapamycin. In certain embodiments, the immune modulators can be administered by any suitable route of administration, including, but not limited to, intrauterine, intraCSF, intrathecal, intravenous, subcutaneous, transdermal, intradermal, intramuscular, oral, transdermal, intraperitoneal (IP), or intravaginal. In certain embodiments, the disclosed immune modulators can be administered using a combination of routes. Administration can also include administration into the hepatic artery or through the hepatic portal vein (HPV). Administration of the immune modulator can be continuous or intermittent, and administration can include a combination of one or more routes.

[0091] As used herein, the term "immunotolerant" refers to non-responsiveness to an antigen (e.g., a vector, a therapeutic protein, a transgene product, etc.). An immune-tolerant promoter can reduce, ameliorate, or prevent a transgene-induced immune response that may be associated with gene therapy. Whether one or more promoters can confer immune tolerance can be determined using assays known in the art for measuring immune responses, such as immunohistochemical detection of cytotoxic T cell responses.

[0092] As used herein, the term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products, which contain information about the indications, usage, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic products.

[0093] As used herein, the term "in combination with" includes the use of two or more therapies (e.g., drug therapies) in the context of the administration of other therapies (e.g., other agents). Administration "in combination with" one or more additional therapeutic agents includes simultaneous (e.g., concurrent) and sequential administration in any order. The use of the term "in combination with" does not limit the order in which therapies are administered to a subject. As a non-limiting example, a first therapy (e.g., a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof) can be administered (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours ...50 minutes, 60 minutes, 75 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 200 minutes, 24 hours, 30 minutes, 32 minutes, 36 minutes, 38 minutes, 40 minutes, 42 minutes, 44 hours, 46 hours, 48 ​​hours, 50 minutes, 52 hours, 54 hours, 56 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours The administration may be performed within 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), simultaneously therewith, or thereafter (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer).

[0094] Disclosed are the disclosed enzymes, disclosed recombinant enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical preparations, and components used to prepare the disclosed enzymes, disclosed recombinant enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical preparations used in the methods disclosed herein. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific references to the various individual and collective combinations of each, and permutations of these compounds, are not expressly disclosed, but each is specifically contemplated and described herein. For example, when a particular compound is disclosed and discussed, and several modifications that can be made to several molecules comprising the compound are discussed, any and all combinations and permutations of possible compounds and modifications are specifically contemplated unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C are disclosed, as well as classes of molecules D, E, and F, and examples of combinations of molecules A-D, each is considered to be disclosed, individually and collectively, even if not individually listed, and each of the combinations they refer to is contemplated, and A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F. Likewise, any subset or combination of these is also disclosed. Thus, for example, the subgroups A-E, B-F, and C-E are considered to be disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention. B. Compositions for Transcriptome Manipulation 1. Enzymes

[0095] Disclosed herein are enzymes having IgM-specific protease activity. Disclosed herein are enzymes having IgG-specific protease activity. Disclosed herein are enzymes having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein are recombinant enzymes having IgM-specific protease activity. Disclosed herein are recombinant enzymes having IgG-specific protease activity. Disclosed herein are recombinant enzymes having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein are fusion proteins having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein are recombinant enzymes capable of binding to and cleaving at one or more of the sequences set forth in SEQ ID NOs: 111 to 118, and the sequence set forth in SEQ ID NO: 110.

[0096] Disclosed herein is an enzyme comprising any one of the sequences set forth in SEQ ID NOs: 01 to 46. Disclosed herein is an enzyme comprising any one of the sequences set forth in SEQ ID NOs: 01 to 46 and having IgM-specific protease activity and / or IgG-specific protease activity.

[0097] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 01. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 04.

[0098] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 07. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 10. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 13.

[0099] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 16. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 19.

[0100] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 02 or SEQ ID NO: 03, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 02 or SEQ ID NO: 03, or a fragment thereof.

[0101] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 05 or SEQ ID NO: 06, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 05 or SEQ ID NO: 06, or a fragment thereof.

[0102] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 08 or SEQ ID NO: 09, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 08 or SEQ ID NO: 09, or a fragment thereof.

[0103] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or a fragment thereof.

[0104] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof.

[0105] Disclosed herein are recombinant fusion proteins comprising a proteolytic domain derived from a disclosed IgG-specific protease linked to a proteolytic domain derived from a disclosed IgM-specific protease. In certain embodiments of the disclosed recombinant fusion proteins, the linker can comprise a flexible linker or a rigid linker. In certain embodiments, the disclosed linker can comprise the sequence of any one of SEQ ID NOs: 94 to 104. In certain embodiments, the disclosed linker can comprise the sequence of SEQ ID NO: 96. In certain embodiments, the disclosed linker can be a rigid linker.

[0106] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme comprising a sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof, and having IgM-specific protease activity and IgG-specific activity.

[0107] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, or a fragment thereof.

[0108] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, or a fragment thereof.

[0109] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 24. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 27. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 30. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 33. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 36.

[0110] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 39. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 42.

[0111] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26, or a fragment thereof.

[0112] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29, or a fragment thereof.

[0113] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32, or a fragment thereof.

[0114] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 35, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 35, or a fragment thereof.

[0115] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38, and having IgM-specific protease activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising the sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 38, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 38, or a fragment thereof.

[0116] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof.

[0117] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44, or a fragment thereof.

[0118] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46, or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46, and having IgM-specific protease activity and IgG-specific activity. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46, or a fragment thereof. Disclosed herein is a recombinant enzyme having IgM-specific protease activity and IgG-specific activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46, or a fragment thereof.

[0119] In certain embodiments, the disclosed recombinases can have IgM-specific protease activity. In certain embodiments, the disclosed recombinases can cleave IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3. In certain embodiments, the disclosed recombinases can cleave human IgM. In certain embodiments, the disclosed recombinases can cleave non-human primate IgM. In certain embodiments, the disclosed recombinases do not cleave non-human IgM. For example, in certain embodiments, the disclosed recombinases do not cleave canine IgM, porcine IgM, and mouse IgM. In certain embodiments, the disclosed IgM-specific recombinases do not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0120] In certain embodiments, the disclosed recombinases can have a binding domain specific for IgM. In certain embodiments, the disclosed recombinases can cleave IgM between positions 350 and 365 of SEQ ID NO: 105. In certain embodiments, SEQ ID NO: 105 can represent the linear sequence of IgM (e.g., a substrate). In certain embodiments, the disclosed recombinases can have a binding domain capable of specifically binding to one or more amino acid residues 355 to 360 of IgM. In certain embodiments, the disclosed recombinases can have affinity for human IgM. In certain embodiments, the disclosed IgM can be cleaved at SEQ ID NO: 110. In certain embodiments, the disclosed IgM can be cleaved at a sequence including 355D, 356T, 357A, 358I, 359R, and 360V. In certain embodiments, the disclosed IgM can be cleaved at positions 355 to 360 of SEQ ID NO: 105.

[0121] In certain embodiments, the disclosed recombinases can have IgG- and IgM-specific protease activity. In certain embodiments, the disclosed recombinases can cleave IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3, and can cleave IgG below the hinge region. In certain embodiments, the disclosed recombinases can cleave IgG below the hinge region, thereby generating F(ab')2 and Fc fragments. In certain embodiments, the disclosed recombinases can cleave IgG and separate the Cμ2 and Cμ3 domains from the Cμ1 domain.

[0122] In certain embodiments, the disclosed recombinant enzymes are capable of cleaving human IgG and human IgM. In certain embodiments, the disclosed recombinant enzymes are capable of cleaving non-human primate IgG and non-human primate IgM. In certain embodiments, the disclosed recombinant enzymes with IgG and IgM protease activity do not cleave IgA, IgD, IgE, or any combination thereof. In certain embodiments, the disclosed recombinant enzymes with IgG and IgM protease activity do not cleave non-human IgM or non-human IgG. For example, in certain embodiments, the disclosed recombinant enzymes with IgG and IgM protease activity do not cleave canine IgM, porcine IgM, and mouse IgM, or canine IgG, porcine IgG, or mouse IgG.

[0123] In certain embodiments, the disclosed recombinant enzymes can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or any combination thereof. In certain embodiments, the disclosed recombinant enzymes can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, or any combination thereof. In certain embodiments, the disclosed recombinant enzymes can cleave IgG at amino acid residues 109 to 124 of the IgG of SEQ ID NO:106, residues 106 to 120 of the IgG of SEQ ID NO:107, residues 156 to 171 of the IgG of SEQ ID NO:108, residues 106 to 121 of the IgG of SEQ ID NO:109, or any combination thereof. In certain embodiments, the disclosed recombinant enzymes are capable of cleaving IgG at amino acid residues 116 to 122 of the IgG of SEQ ID NO: 106, residues 112 to 118 of the IgG of SEQ ID NO: 107, residues 163 to 169 of the IgG of SEQ ID NO: 108, residues 113 to 119 of the IgG of SEQ ID NO: 109, or any combination thereof. In certain embodiments, the disclosed recombinant enzymes are capable of cleaving one or more IgG molecules at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0124] In certain embodiments, the disclosed recombinases can be specific for IgM and IgG and can cleave IgM between positions 355 and 360 of SEQ ID NO: 105, and can cleave at residues 116 to 122 of the IgG of SEQ ID NO: 106, residues 112 to 118 of the IgG of SEQ ID NO: 107, residues 163 to 169 of the IgG of SEQ ID NO: 108, and residues 113 to 119 of the IgG of SEQ ID NO: 109. In certain embodiments, the disclosed recombinases can be specific for IgM and IgG and can recognize and cleave SEQ ID NO: 110 in IgM and can cleave SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118 in IgG.

[0125] In certain embodiments, the disclosed recombinases are capable of clearing and / or removing IgM and / or IgM and IgG from the circulation in a subject. In certain embodiments, the disclosed recombinases are capable of clearing and / or removing IgM and / or IgM and IgG from the circulation in a subject's serum. In certain embodiments, the disclosed recombinases are capable of cleaving and / or degrading circulating IgM and / or IgM and IgG in a subject. In certain embodiments, the disclosed recombinases are capable of transiently clearing and / or removing IgM and / or IgM and IgG from the circulation in a subject's serum, or transiently cleaving and / or degrading circulating IgM and / or IgM and IgG in a subject. In certain embodiments, the disclosed recombinases are capable of reducing and / or lowering circulating IgM and / or IgG and IgM levels in a subject, or transiently reducing and / or lowering circulating IgM and / or IgG and IgM levels in a subject. In embodiments, the disclosed transient reduction and / or decrease can include about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0126] In certain embodiments, the disclosed recombinant enzymes are capable of blocking and / or minimizing activation of the complement cascade. In certain embodiments, the disclosed recombinant enzymes are capable of blocking and / or minimizing activation of C1 complex and / or C3 complex formation. In certain embodiments, the disclosed recombinant enzymes are capable of blocking and / or minimizing activation of the classical complement pathway. In certain embodiments, the disclosed recombinant enzymes are capable of transiently blocking and / or minimizing activation of the complement cascade, transiently blocking and / or minimizing activation of C1 complex and / or C3 complex formation, transiently blocking and / or minimizing activation of the classical complement pathway, or any combination thereof.

[0127] In certain embodiments, the disclosed recombinases are capable of removing and / or cleaving surface-bound IgM from B cells and / or removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed removal and / or cleavage of IgM from the surface of B cells can be reversible and / or transient. In certain embodiments, the disclosed recombinases are capable of transiently removing and / or cleaving surface-bound IgM from B cells and / or transiently removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed recombinases are capable of removing and / or cleaving surface-bound IgM from B cells and / or removing and / or cleaving IgM from the surface of B cells without killing and / or damaging the B cells.

[0128] In certain embodiments, the disclosed recombinases can reduce and / or minimize vector-mediated immunotoxicity in a subject. In certain embodiments, the disclosed recombinases can enable and / or tolerate re-administration of a vector in a subject. In certain embodiments, the disclosed recombinases can reduce and / or minimize the immunogenicity of a transgene in a subject.

[0129] In certain embodiments, the disclosed recombinases can improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, the disclosed recombinases can improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, improving and / or enhancing the efficacy and / or expression of a transgene in a subject can include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of improvement and / or enhancement when compared to an existing level of transgene efficacy and / or expression, such as, for example, a pre-treatment level (before administering one or more disclosed recombinases). In certain embodiments, the amount of improvement and / or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than existing levels, such as pre-treatment levels (before administering one or more disclosed recombinant enzymes). In certain embodiments, the improvement and / or enhancement can be measured relative to a control level or reference level (e.g., determined using one or more subjects who have not received or been administered one or more disclosed recombinant enzymes). In certain embodiments, the improvement and / or enhancement can be partial or incomplete restoration. In certain embodiments, the improvement and / or enhancement can be complete or near-complete, such that levels of neutralizing antibodies or neutralizing IgG and / or IgM molecules are similar to those of wild-type or control levels.

[0130] In certain embodiments, the disclosed recombinases can reduce and / or minimize the severity of a rejection event of one or more transplanted organs. In certain embodiments, the disclosed recombinases can prevent rejection of one or more transplanted organs. In certain embodiments, the disclosed rejection events can be classified as hyperacute rejection events, acute rejection events, or chronic rejection events. In certain embodiments, after administration and / or delivery of the disclosed recombinases, one or more transplanted organs are not rejected by the subject. In certain embodiments, after administration and / or delivery of the disclosed recombinases, the viability of one or more transplanted organs can be improved and / or increased. In certain embodiments, after administration and / or delivery of the disclosed recombinases, the functionality of one or more transplanted organs can be improved and / or increased. In certain embodiments, after administration and / or delivery of the disclosed recombinases, the functionality of one or more transplanted organs is not reduced and / or impaired. In certain embodiments, the disclosed recombinases can degrade one or more anti-HLA antibodies against one or more transplanted organs.

[0131] In certain aspects, the disclosed enzymes can be used to modulate humoral immunity in a subject, hi certain aspects, modulating humoral immunity in a subject can be an increase in the functionality of the subject's humoral immunity or a decrease in the functionality of the subject's humoral immunity.

[0132] In certain embodiments, the disclosed enzymes can comprise one or more labels and / or tags. In certain embodiments, the disclosed labels and / or tags can comprise fluorescent labels and / or tags. In certain embodiments, the disclosed labels and / or tags can comprise a carboxy-terminal label and / or tag or an amino-terminal label and / or tag. In certain embodiments, the disclosed labels and / or tags can comprise a carboxy-terminal fluorescent label and / or tag or an amino-terminal fluorescent label and / or tag. In certain embodiments, the disclosed labels and / or tags include ALFA tags, AviTag, C tags, calmodulin tags, iCapTag (intein capture tags), polyglutamate tags, polyarginine tags, E tags, FLAG tags, HA tags, His tags, Myc tags, NE tags, Rho1D4 tags, S tags, SBP tags, Softag 1, Softag 3, Spot-tag, Strep-tag, T7-tag, TC-tag, Ty-tag, V5-tag, VSV-tag, Xpress-tag, Isopeptag, Spy-tag, SnoopTag, SnoopTagJr, DogTag, SdyTag, BCCP (biotin carboxy carrier protein), glutathione-S-transferase tag, green fluorescent protein tag, Halo-tag, SNAP-tag, CLIP-tag, HUH-tag, maltose binding protein tag, Nus-tag, thioredoxin-tag, Fc-tag, designed intrinsically disordered tag containing disorder-promoting amino acids, carbohydrate recognition domain or CRDSAT-tag, HiBiT-tag, or any combination thereof.

[0133] In certain embodiments, the disclosed labels and / or tags can include fluorescent labels or fluorescent tags. In certain embodiments, the disclosed fluorescent labels or disclosed fluorophores can include enhanced green fluorescent protein (EGFP), mEmerald, enhanced yellow fluorescent protein (EYFP), mApple, TdTomato, mCherry, miRFP670, any known fluorescent label or tag, or any combination thereof. In certain embodiments, the disclosed fluorescent labels or disclosed fluorophores can include any fluorescent label or fluorophore that is amenable to analysis via flow cytometry. In certain embodiments, the disclosed fluorescent label or disclosed fluorophore pair can include any pair of fluorescent labels or fluorophores that are amenable to analysis via flow cytometry and have excitation and emission spectra that can be isolated or separated from each other, thereby enabling interrogation of docking interactions. Fluorophores and fluorescent labels are known in the art.

[0134] In certain embodiments, the disclosed enzymes or disclosed recombinant enzymes can be therapeutically effective when the dose comprises about 0.01 mg / kg to about 100 mg / kg body weight, hi certain embodiments, the disclosed enzymes or disclosed recombinant enzymes can be therapeutically effective when the dose comprises about 0.01 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg body weight.

[0135] Disclosed herein is a recombinant enzyme comprising the sequence of SEQ ID NO: 119. Disclosed herein is a recombinant enzyme encoded by the codon-optimized nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a recombinant enzyme encoded by a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the codon-optimized sequence of SEQ ID NO: 160 or SEQ ID NO: 161.

[0136] Disclosed herein is a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a recombinant enzyme comprising the sequence of any one of SEQ ID NOs: 122 to 159. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NOs: 122 to 159.

[0137] Disclosed herein is a recombinant enzyme comprising the sequence of SEQ ID NO: 119 and having human and non-human primate IgG-specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121 and having human and non-human primate IgG-specific protease activity. Disclosed herein is a recombinant enzyme comprising any one of the sequences of SEQ ID NOs: 122 to 159 and having human and non-human primate IgG-specific protease activity. Disclosed herein is a recombinant enzyme having human and non-human primate IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. Disclosed herein is a recombinant enzyme having human and non-human primate IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a recombinant enzyme having human and non-human primate IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NOs: 122 to 159.

[0138] Disclosed herein are recombinant enzymes having human and non-human primate IgG-specific protease activity that are encoded by the codon-optimized nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein are recombinant enzymes having human and non-human primate IgG-specific protease activity that are encoded by a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the codon-optimized nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. 2. Nucleic acid molecules

[0139] Disclosed herein are isolated nucleic acid molecules encoding any of the disclosed enzymes. Disclosed herein are isolated nucleic acid molecules encoding any of the disclosed recombinant enzymes. Disclosed herein are isolated nucleic acid molecules comprising the sequence of any one of SEQ ID NOs: 47 to 92.

[0140] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 01. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 24. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 47. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 70.

[0141] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 04. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 27. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 50. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 73.

[0142] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 07. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 30. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 53. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 76.

[0143] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 10. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 33. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 56. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 79.

[0144] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 13. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 36. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 59. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 82.

[0145] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 16. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 39. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 62. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 85.

[0146] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 19. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 42. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 65. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 88.

[0147] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:48 or SEQ ID NO:49. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:71 or SEQ ID NO:72.

[0148] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:51 or SEQ ID NO:52. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:74 or SEQ ID NO:75.

[0149] Disclosed herein are isolated nucleic acid molecules encoding enzymes, including recombinant enzymes, comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein are isolated nucleic acid molecules encoding enzymes comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein are isolated nucleic acid molecules encoding enzymes comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, or a fragment thereof. Disclosed herein are isolated nucleic acid molecules encoding enzymes comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, or a fragment thereof. Disclosed herein are isolated nucleic acid molecules encoding enzymes, including recombinant enzymes, comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:54 or SEQ ID NO:55. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:77 or SEQ ID NO:78.

[0150] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:57 or SEQ ID NO:58. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:80 or SEQ ID NO:81.

[0151] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 38. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 61. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 83 or SEQ ID NO: 84.

[0152] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, and having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 64.Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:86 or SEQ ID NO:87.

[0153] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, and having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:66 or SEQ ID NO:67.Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:89 or SEQ ID NO:90.

[0154] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, and having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity, comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:68 or SEQ ID NO:69.Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:91 or SEQ ID NO:92.

[0155] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having the sequence set forth in SEQ ID NO: 24. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having the sequence set forth in SEQ ID NO: 27. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having the sequence set forth in SEQ ID NO: 30. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having the sequence set forth in SEQ ID NO: 33. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having the sequence set forth in SEQ ID NO: 36.

[0156] Disclosed herein is an isolated nucleic acid molecule that encodes an enzyme having the sequence set forth in SEQ ID NO: 39. Disclosed herein is an isolated nucleic acid molecule that encodes an enzyme having the sequence set forth in SEQ ID NO: 42.

[0157] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:25 or SEQ ID NO:26, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26, which encodes an enzyme having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26, or a fragment thereof ...

[0158] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:28 or SEQ ID NO:29, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:28 or SEQ ID NO:29, and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29, and encoding an enzyme having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29, or a fragment thereof ...

[0159] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:31 or SEQ ID NO:32, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32, and encoding an enzyme having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32, or a fragment thereof ...

[0160] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 35, and encoding an enzyme having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 35, or a fragment thereof ...

[0161] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:37 or SEQ ID NO:38, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:37 or SEQ ID NO:38, and having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 38, which encodes an enzyme having IgM-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 37 or SEQ ID NO: 38, or a fragment thereof ...

[0162] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, and having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity.

[0163] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:43 or SEQ ID NO:44, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:43 or SEQ ID NO:44, and having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44, or a fragment thereof, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44, or a fragment thereof, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity.

[0164] Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:45 or SEQ ID NO:46, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46, or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding an enzyme having a sequence set forth in SEQ ID NO:45 or SEQ ID NO:46, and having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46, or a fragment thereof, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46, or a fragment thereof, and encoding an enzyme having IgM-specific protease activity and IgG-specific protease activity.

[0165] In certain embodiments, the disclosed isolated nucleic acid molecules can comprise nucleic acid sequences for one or more labels and / or tags. In certain embodiments, the disclosed labels and / or tags can comprise fluorescent labels and / or tags. In certain embodiments, the disclosed labels and / or tags can comprise carboxy-terminal labels and / or tags or amino-terminal labels and / or tags. In certain embodiments, the disclosed labels and / or tags can comprise carboxy-terminal fluorescent labels and / or tags or amino-terminal fluorescent labels and / or tags.

[0166] In certain embodiments, the disclosed labels and / or tags include ALFA tag, AviTag, C tag, calmodulin tag, iCapTag (intein capture tag), polyglutamate tag, polyarginine tag, E tag, FLAG tag, HA tag, His tag, Myc tag, NE tag, Rho1D4 tag, S tag, SBP tag, Softag 1, Softag 3, Spot-tag, Strep-tag, T7-tag, TC-tag, Ty-tag, V5-tag, VSV-tag, Xpress-tag, Isopeptag, Spy-tag, SnoopTag, SnoopTagJr, DogTag, SdyTag, BCCP (biotin carboxy carrier protein), glutathione-S-transferase tag, green fluorescent protein tag, Halo-tag, SNAP-tag, CLIP-tag, HUH-tag, maltose binding protein tag, Nus-tag, thioredoxin-tag, Fc-tag, designed intrinsically disordered tag containing disorder-promoting amino acids, carbohydrate recognition domain or CRDSAT-tag, HiBiT-tag, or any combination thereof.

[0167] In certain embodiments, the disclosed labels and / or tags can include fluorescent labels or fluorescent tags. In certain embodiments, the disclosed fluorescent labels or disclosed fluorophores can include enhanced green fluorescent protein (EGFP), mEmerald, enhanced yellow fluorescent protein (EYFP), mApple, TdTomato, mCherry, miRFP670, any known fluorescent label or tag, or any combination thereof. In certain embodiments, the disclosed fluorescent labels or disclosed fluorophores can include any fluorescent label or fluorophore that is amenable to analysis via flow cytometry. In certain embodiments, the disclosed fluorescent label or disclosed fluorophore pair can include any pair of fluorescent labels or fluorophores that are amenable to analysis via flow cytometry and have excitation and emission spectra that can be isolated or separated from each other, thereby allowing for interrogation of interactions. Fluorophores and fluorescent labels are known in the art.

[0168] In certain embodiments of the disclosed nucleic acid molecules, the disclosed encoded recombinase can have IgM-specific protease activity. In certain embodiments, the disclosed encoded recombinase can cleave IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3. In certain embodiments, the disclosed encoded recombinase can cleave human IgM. In certain embodiments, the disclosed encoded enzyme can cleave non-human primate IgM. In certain embodiments, the disclosed encoded recombinase does not cleave non-human IgM. For example, in certain embodiments, the disclosed encoded recombinase does not cleave canine IgM, porcine IgM, and mouse IgM. In certain embodiments, the disclosed encoded IgM-specific recombinase does not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0169] In certain embodiments, the disclosed encoded recombinases can have a binding domain specific for IgM. In certain embodiments, the disclosed encoded recombinases can cleave IgM between positions 350 and 365 of SEQ ID NO: 105. In certain embodiments, SEQ ID NO: 105 can represent the linear sequence of IgM (e.g., a substrate). In certain embodiments, the disclosed encoded recombinases can have a binding domain capable of specifically binding to one or more amino acid residues 355 to 360 of IgM. In certain embodiments, the disclosed encoded recombinases can have affinity for human IgM. In certain embodiments, the disclosed IgM can be cleaved at SEQ ID NO: 110. In certain embodiments, the disclosed IgM can be cleaved at a sequence including 355D, 356T, 357A, 358I, 359R, and 360V. In certain embodiments, the disclosed IgM can be cleaved at positions 355 to 360 of SEQ ID NO: 105.

[0170] In certain embodiments, the disclosed encoded recombinases can have IgG- and IgM-specific protease activity. In certain embodiments, the disclosed encoded recombinases can cleave IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3, and can cleave IgG below the hinge region. In certain embodiments, the disclosed encoded recombinases can cleave IgG below the hinge region, thereby generating F(ab')2 and Fc fragments. In certain embodiments, the disclosed encoded recombinases can cleave IgG and separate the Cμ2 and Cμ3 domains from the Cμ1 domain.

[0171] In certain embodiments, the disclosed encoded recombinant enzymes are capable of cleaving human IgG and human IgM. In certain embodiments, the disclosed encoded recombinant enzymes are capable of cleaving non-human primate IgG and non-human primate IgM. In certain embodiments, the disclosed encoded recombinant enzymes with IgG and IgM protease activity do not cleave IgA, IgD, IgE, or any combination thereof. In certain embodiments, the disclosed encoded recombinant enzymes with IgG and IgM protease activity do not cleave non-human IgM or non-human IgG. For example, in certain embodiments, the disclosed encoded recombinant enzymes with IgG and IgM protease activity do not cleave canine IgM, porcine IgM, and mouse IgM, or canine IgG, porcine IgG, or mouse IgG.

[0172] In certain embodiments, the disclosed encoded recombinant enzymes can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or any combination thereof. In certain embodiments, the disclosed encoded recombinant enzymes can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, or any combination thereof. In certain embodiments, the disclosed encoded recombinant enzymes can cleave IgG at amino acid residues 109 to 124 of the IgG of SEQ ID NO:106, residues 106 to 120 of the IgG of SEQ ID NO:107, residues 156 to 171 of the IgG of SEQ ID NO:108, residues 106 to 121 of the IgG of SEQ ID NO:109, or any combination thereof. In certain embodiments, the disclosed encoded recombinant enzymes are capable of cleaving IgG at amino acid residues 116 to 122 of the IgG of SEQ ID NO: 106, residues 112 to 118 of the IgG of SEQ ID NO: 107, residues 163 to 169 of the IgG of SEQ ID NO: 108, residues 113 to 119 of the IgG of SEQ ID NO: 109, or any combination thereof. In certain embodiments, the disclosed encoded recombinant enzymes are capable of cleaving one or more IgG molecules at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0173] In certain embodiments, the disclosed encoded recombinase may be specific for IgM and IgG and can cleave IgM between positions 355 and 360 of SEQ ID NO: 105, and can cleave at residues 116 to 122 of the IgG of SEQ ID NO: 106, residues 112 to 118 of the IgG of SEQ ID NO: 107, residues 163 to 169 of the IgG of SEQ ID NO: 108, and residues 113 to 119 of the IgG of SEQ ID NO: 109. In certain embodiments, the disclosed encoded recombinase may be specific for IgM and IgG and can recognize and cleave SEQ ID NO: 110 in IgM and can cleave SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118 in IgG.

[0174] In certain embodiments, the disclosed encoded recombinases are capable of clearing and / or removing IgM and / or IgM and IgG from the circulation in a subject. In certain embodiments, the disclosed recombinases are capable of clearing and / or removing IgM and / or IgM and IgG from the circulation in the serum of a subject. In certain embodiments, the disclosed recombinases are capable of cleaving and / or degrading circulating IgM and / or IgM and IgG in a subject. In certain embodiments, the disclosed recombinases are capable of transiently clearing and / or removing IgM and / or IgM and IgG from the circulation in the serum of a subject, or transiently cleaving and / or degrading circulating IgM and / or IgM and IgG in a subject. In certain embodiments, the disclosed recombinases are capable of reducing and / or decreasing circulating IgM levels and / or IgM and IgG levels in a subject, or transiently reducing and / or decreasing circulating IgM levels and / or IgM and IgG levels in a subject. In embodiments, the disclosed transient reduction and / or decrease can include about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0175] In certain embodiments of the disclosed nucleic acid molecules, the disclosed encoded enzymes are capable of blocking and / or minimizing activation of the complement cascade. In certain embodiments, the disclosed encoded enzymes are capable of blocking and / or minimizing activation of C1 complex and / or C3 complex formation. In certain embodiments, the disclosed encoded enzymes are capable of blocking and / or minimizing activation of the classical complement pathway. In certain embodiments, the disclosed encoded enzymes are capable of transiently blocking and / or minimizing activation of the complement cascade, transiently blocking and / or minimizing activation of C1 complex and / or C3 complex formation, transiently blocking and / or minimizing activation of the classical complement pathway, or any combination thereof.

[0176] In certain embodiments of the disclosed nucleic acid molecules, the disclosed encoded enzymes are capable of removing and / or cleaving surface-bound IgM from B cells and / or are capable of removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed removal and / or cleavage of IgM from the surface of B cells can be reversible and / or transient. In certain embodiments, the disclosed encoded enzymes are capable of transiently removing and / or cleaving surface-bound IgM from B cells and / or are capable of transiently removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed encoded enzymes are capable of removing and / or cleaving surface-bound IgM from B cells and / or are capable of removing and / or cleaving IgM from the surface of B cells without killing and / or damaging the B cells.

[0177] In certain embodiments of the disclosed nucleic acid molecules, the disclosed encoded enzymes can reduce and / or minimize vector-mediated immunotoxicity in a subject. In certain embodiments of the disclosed nucleic acid molecules, the disclosed encoded enzymes can enable and / or tolerate vector re-administration in a subject. In certain embodiments of the disclosed nucleic acid molecules, the disclosed encoded enzymes can reduce and / or minimize immunogenicity of a transgene in a subject. In certain embodiments of the disclosed nucleic acid molecules, the disclosed encoded enzymes can improve and / or enhance the efficacy and / or expression of a transgene in a subject.

[0178] In certain embodiments, the disclosed nucleic acid molecules can improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, the disclosed nucleic acid molecules can improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, improving and / or enhancing the efficacy and / or expression of a transgene in a subject can include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of improvement and / or enhancement when compared to an existing level of transgene efficacy and / or expression, such as, for example, a pre-treatment level (before administering one or more disclosed nucleic acid molecules). In certain embodiments, the amount of improvement and / or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than existing levels, such as pre-treatment levels (before administering one or more disclosed nucleic acid molecules). In certain embodiments, the improvement and / or enhancement can be measured relative to a control level or reference level (e.g., determined using one or more subjects who have not received or been administered one or more disclosed nucleic acid molecules). In certain embodiments, the improvement and / or enhancement can be partial or incomplete restoration. In certain embodiments, the improvement and / or enhancement can be complete or near-complete, such that levels of neutralizing antibodies or neutralizing IgG and / or IgM molecules are similar to those of wild-type or control levels.

[0179] In some embodiments, the disclosed nucleic acid molecules and / or disclosed nucleic acid sequences can be CpG-depleted and codon-optimized for expression in human cells. In some embodiments, "CpG-free" can mean completely free of CpGs or partially free of CpGs. In some embodiments, "CpG-free" can mean "CpG-depleted." In some embodiments, "CpG-depleted" can mean "free of CpGs." In some embodiments, "CpG-depleted" can mean completely depleted of CpGs or partially depleted of CpGs. In some embodiments, "CpG-free" can mean "CpG-optimized" for desired and / or ideal expression levels. CpG depletion and / or optimization are known to those skilled in the art.

[0180] In certain embodiments, a "codon-optimized" gene can be a gene whose codon usage is designed to mimic the preferred codon usage of a host cell (i.e., a human cell or a human subject). In certain embodiments, the disclosed nucleic acid molecules and / or disclosed nucleic acid sequences can be codon-optimized in whole or in part. Because any single amino acid (except methionine and tryptophan) is coded for by several codons, the sequence of a nucleic acid molecule can be altered without changing the coded amino acid. In certain embodiments, the disclosed nucleic acid sequences can be codon-optimized for expression in a human cell or organ, or in one or more human cells or organs. In certain embodiments, the disclosed nucleic acid sequences can be codon-optimized for expression in a human subject.

[0181] In certain aspects, the disclosed codon-optimized nucleic acid sequences can include SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, or any combination thereof.

[0182] In some embodiments, the disclosed nucleic acid sequences are encoded by the following genes: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18 , ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC 88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, CO L27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREB BP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1,DOCK2、DOCK3、DOCK6、DOCK7、DOCK8、DSCAM、DSP、DST、DUOX2、DYNC1H1、DYNC 2H1、DYSF、EIF2AK4、EP300、EPG5、ERCC6、ERCC6L2、EXPH5、EYS、F5、F8、FANC A、FANCD2、FANCM、FAT1、FAT4、FBN1、FBN2、FLG、FLG2、FLNA、FLNB、FLNC、FLT 4、FMN2、FN1、FRAS1、FREM1、FREM2、FSIP2、FYCO1、GLI2、GLI3、GPR179、GREB1 L、GRIN2A、GRIN2B、GRIN2D、HCFC1、HECW2、HERC1、HERC2、HFM1、HIVEP1、HIV EP2、HMCN1、HSPG2、HTT、HUWE1、HYDIN、IFT140、IFT172、IGF1R、IGF2R、IGSF1 INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1 549、KIDINS220、KIF14、KIF1A、KIF1B、KIF21A、KIF26B、KIF7、KMT2A、KMT2B 、KMT2C、KMT2D、KMT2E、KNL1、LAMA1、LAMA2、LAMA3、LAMA4、LAMA5、LAMB1、LAM B2, LAMC3, LCT, LOXHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8 IP3、MAPKBP1、MAST1、MBD5、MCM3AP、MED12、MED12L、MED13、MED13L、MED23、 MEGF8、MET、MLH3、MPDZ、MSH6、MTOR、MYH10、MYH11、MYH14、MYH2、MYH3、MYH6、 MYH7、MYH7B、MYH8、MYH9、MYLK、MYO15A、MYO18B、MYO3A、MYO5A、MYO5B、MYO7 A、MYO9A、NALCN、NBAS、NBEA、NBEAL2、NCAPD2、NCAPD3、NEB、NEXMIF、NEXMIF、NF1、NFASC、NHS、NIN、NIPBL、NLRP1、NOTCH1、NOTCH2、NOTCH3、NPHP4、NRXN1 、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、OBSCN、OBSL1、OTOF、OTOG、OT OGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI4KA、PIEZO1、PIEZO2、PIK3 C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC、PLEKHG2、PNPLA6、POGZ、PO LA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PRKDC、PRPF8、PRR12、PRX、PTCH1 、PTPN23、PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、RAB3GAP2、RAI1、RALGAPA1、R ANBP2、RB1CC1、RELN、RERE、REV3L、RIC1、RIMS1、RIMS2、RNF213、ROBO1、ROB O2、ROBO3、ROS1、RP1、RP1L1、RTTN、RUSC2、RYR1、RYR2、SACS、SAMD9、SAMD9L、 SBF2、SCAPER、SCN10A、SCN11A、SCN1A、SCN2A、SCN3A、SCN4A、SCN5A、SCN8A、 SCN9A、SETBP1、SETD1A、SETD1B、SETD2、SETD5、SETX、SHANK2、SHANK3、SHRO OM4、SI、SIPA1L3、SLIT2、SLX4、SMARCA2、SMARCA4、SMCHD1、SNRNP200、SON、 SPEF2、SPEG、SPG11、SPTA1、SPTAN1、SPTB、SPTBN2、SPTBN4、SRCAP、STRC、SVI L, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TN R、TNRC6B、TNXB、TOGARAM1、TONSL、TRIO、TRIOBP、TRIP11、TRIP12、TRPM1、T RPM6、TRPM7、TRRAP、TSC2、TTC37、TTN、TUBGCP6、UBR1、UNC80、USH2A、USP9X、It can encode a protein or a portion thereof related to VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, ZNF469, or a portion thereof (e.g., exon 1 or exon 4, etc.).

[0183] In certain embodiments, the disclosed nucleic acid sequences can encode genes or portions thereof (e.g., specific exons, e.g., exons with mutations) as well as gene products directly or indirectly associated with one or more genetic disorders. Such genes include, but are not limited to, dystrophin (DMD), including mini- and micro-dystrophin; titin (TTN); titin cap (TCAP), α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD); alpha-1-antitrypsin (A1-AT); myosin heavy chain 6 (MYH6); myosin heavy chain 7 (MYH7); myosin heavy chain 11 (MYH11); myosin light chain 2 (ML2); myosin light chain 3 (ML3); myosin light chain kinase 2 (MYLK2); myosin-binding protein C (MYBPC3); desmin (DES); dynamin 2 (DNM2); laminin alpha 2 (LAMA2); lamin A / C (LMNA); lamin B (LMNB); lamin B receptor (LBR); dysferlin (DYSF); emerin (EMD); insulin; blood clotting factors, including, but not limited to, factor VIII and factor IX; erythropoietin (E PO); lipoprotein lipase (LPL); sarcoplasmic reticulum Ca2++-ATPase (SERCA2A), S100 calcium-binding protein A1 (S100A1); myotubularin (MTM); DM1 protein kinase (DMPK); glycogen phosphorylase L (PYGL); glycogen phosphorylase, muscle-associated (PYGM); glycogen synthase 1 (GYS1); glycogen synthase 2 (GYS2); α-galactosidase A (GLA); α-N-acetylgalactosidase Lactosaminidase (NAGA); acid α-glucosidase (GAA), sphingomyelinase phosphodiesterase 1 (SMPD1); lysosomal acid lipase (LIPA); collagen type I α1 chain (COL1A1); collagen type I α2 chain (COL1A2); collagen type III α1 chain (COL3A1); collagen type V α1 chain (COL5A1); collagen type V α2 chain (COL5A2); collagen type VI α1 chain (COL6A1); collagen type VI α2 chain (COL6A2);Collagen type VI α3 chain (COL6A3); procollagen lysine 2-oxoglutarate 5-dioxygenase (PLOD1); lysosomal acid lipase (LIPA); frataxin (FXN); myostatin (MSTN); β-N-acetylhexosaminidase A (HEXA); β-N-acetylhexosaminidase B (HEXB); β-glucocerebrosidase (GBA); adenosine monophosphate deaminase 1 (AMPD1); β-globin (HBB); iduronidase (IDUA); iduronate 2-sulfate (IDS); troponin 1 (TNNI3); Troponin T2 (TNNT2); troponin C (TNNC1); tropomyosin 1 (TPM1); tropomyosin 3 (TPM3); N-acetyl-α-glucosaminidase (NAGLU); N-sulfoglucosamine sulfohydrolase (SGSH); heparan-α-glucosaminide N-acetyltransferase (HGSNAT); integrin a7 (IGTA7); integrin a9 (IGTA9); glucosamine (N-acetyl)-6-sulfatase (GNS); galactosamine (N-acetyl)-6-sulfatase (GALNS); β-galactosamine Glucosidase (GLB1); β-glucuronidase (GUSB); hyaluronoglucosaminidase 1 (HYAL1); acid ceramidase (ASAHI); galactosylcermidase (GALC); cathepsin A (CTSA); cathepsin D (CTSA); cathepsin K (CTSK); GM2 ganglioside activator (GM2A); arylsulfatase A (ARSA); arylsulfatase B (ARSB); formylglycine synthase (SUMFI); neuraminidase 1 (NEU1); N-acetylglucosidase glucosamine-1-phosphate transferase a (GNPTA); N-acetylglucosamine-1-phosphate transferase beta (GNPTB); N-acetylglucosamine-1-phosphate transferase gamma (GNPTG); mucolipin-1 (MCOLN1); NPC intracellular transporter 1 (NPC1); NPC intracellular transporter 2 (NPC2); ceroid lipofuscinosis 5 (CLN5); ceroid lipofuscinosis 6 (CLN6); ceroid lipofuscinosis 8 (CLN8); palmitoyl protein thioesterase 1 (PPT1);Tripeptidyl peptidase 1 (TPP1); battenin (CLN3); DNAJ heat shock protein family 40 member C5 (DNAJC5); major facilitator superfamily domain-containing 8 (MFSD8); mannosidase alpha class 2B member 1 (MAN2B1); mannosidase R (MANBA); aspartylglucosaminidase (AGA); alpha-L-fucosidase (FUCA1); cystinosin, lysosomal cysteine ​​transporter (CTNS); sialin; soluble carrier family 2 member 10 (SLC2A10) ); soluble carrier family 17 member 5 (SLC17A5); soluble carrier family 6 member 19 (SLC6A19); soluble carrier family 22 member 5 (SLC22A5); soluble carrier family 37 member 4 (SLC37A4); lysosomal-associated membrane protein 2 (LAMP2); sodium voltage-gated channel alpha subunit 4 (SCN4A); sodium voltage-gated channel beta subunit 4 (SCN4B); sodium voltage-gated channel alpha subunit 5 (SCN5A); sodium voltage-gated channel alpha subunit subunit 4 (SCN4A); calcium voltage-gated channel subunit α1c (CACNA1C); calcium voltage-gated channel subunit α1s (CACNA1S); phosphoglycerate kinase 1 (PGK1); phosphoglycerate mutase 2 (PGAM2); amylo-α-1,6-glucosidase, 4-α-glucanotransferase (AGL); potassium voltage-gated channel ISK-related subfamily member 1 (KCNE1); potassium voltage-gated channel ISK-related subfamily member 2 (KCNE2); potassium voltage-gated Mouth-type channel subfamily J member 2 (KCNJ2); potassium voltage-gated channel subfamily J member 5 (KCNJ5); potassium voltage-gated channel subfamily H member 2 (KCNH2); potassium voltage-gated channel KQT-like subfamily member 1 (KCNQ1); hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4); chloride voltage-gated channel 1 (CLCN1); carnitine palmitoyltransferase 1A (CPT1A); ryanodine receptor 1 (RYR1); ryanodine receptor 2 (RYR2);Crosslinking integrator 1 (BIN1); Large xylosyl and glucuronyltransferase 1 (LARGE1); Docking protein 7 (DOK7); Fukutin (FKTN); Fukutin-related protein (FKRP); Selenoprotein N (SELENON); Protein O-mannosyltransferase 1 (POMT1); Protein O-mannosyltransferase 2 (POMT2); Protein O-linked mannose N-acetylglucosaminyltransferase 1 (POMGNT1); Protein O-linked mannose N-acetylglucosaminyltransferase 2 (POMGNT2); Protein-O-mannose kinase (POMK); Isoprenoid synthase domain-containing (ISPD); Plectin (PLEC); Cholinergic receptor nicotinic epsilon subunit (CHRNE); Choline O-acetyltransferase (CHAT); Choline kinase beta (CHKB); Collagen-like tail subunit of asymmetric acetylcholinesterase (COLQ); Receptor-associated protein at synapse (RAPSN); Four and a half LIM domain 1 (FHL1); β-1,4-glucuronyltransferase 1 (B4GAT1); β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2); dystroglycan 1 (DAGI); transmembrane protein 5 (TMEM5); transmembrane protein 43 (TMEM43); SECIS-binding protein 2 (SECISBP2); glucosamine (UDP-N-acetyl)-2-epimerase / N-acetylmannosamine kinase (GNE); anoctamin 5 (ANO5); structural maintenance of chromosome flexible hinge domain containing 1 (SMCH) D1); lactate dehydrogenase A (LDHA); lactate dehydrogenase B (LHDB); calpain 3 (CAPN3); caveolin 3 (CAV3); tripartite motif-containing 32 (TRIM32); CCHC-type zinc finger nucleic acid-binding protein (CNBP); nebulin (NEB); actin, α1, skeletal muscle (ACTA1); actin, α1, cardiac muscle (ACTC1); actinin α2 (ACTN2); poly(A)-binding protein nuclear 1 (PABPN1); LEM domain-containing protein 3 (LEMD3); zinc metalloproteinase STE24 (ZMPSTE24);Microsomal triglyceride transfer protein (MTTP); cholinergic receptor nicotinic α1 subunit (CHRNA1); cholinergic receptor nicotinic α2 subunit (CHRNA2); cholinergic receptor nicotinic α3 subunit (CHRNA3); cholinergic receptor nicotinic α4 subunit (CHRNA4); cholinergic receptor nicotinic α5 subunit (CHRNA5); cholinergic receptor nicotinic α6 subunit (CHRNA6); cholinergic receptor nicotinic α7 subunit (CHRNA7); cholinergic receptor nicotinic α8 subunit (CHRNA8); cholinergic receptor nicotinic α9 subunit (CHRNA9); cholinergic receptor nicotinic α10 subunit (CHRNA10); cholinergic receptor nicotinic β1 subunit (CHRNB1); cholinergic receptor nicotinic β2 subunit (CHRNB2); cholinergic receptor nicotinic β3 subunit unit (CHRNB3); cholinergic receptor nicotinic beta 4 subunit (CHRNB4); cholinergic receptor nicotinic gamma subunit (CHRNG1); cholinergic receptor nicotinic a subunit (CHRND); cholinergic receptor nicotinic E subunit (CHRNE1); ATP-binding cassette subfamily A member 1 (ABCA1); ATP-binding cassette subfamily C member 6 (ABCC6); ATP-binding cassette subfamily C member 9 (ABCC9); ATP-binding cassette subfamily D member 1 (ABCD1); ATPase sarcoplasmic / endoplasmic reticulum Ca2+ transporter 1 (ATP2A1); ATM serine / threonine kinase (ATM); tocopherol transferase protein (TTPA); kinesin family member 21A (KIF21A); paired-like homeobox 2a (PHOX2A); heparan sulfate proteoglycan 2 (HSPG2); stromal interaction molecule 1 (STIM1); notch 1 (NOTCHI); notch 3 (NOTCH3); dystrobrevin alpha (DTNA); protein kinase AMP-activated, non-catalytic gamma 2 (PRKAG2); cysteine- and glycine-rich protein 3 (CSRP3); vinculin (VCL); myozenin 2 (MyoZ2);myopalladin (MYPN); junctophilin 2 (JPH2); phospholamban (PLN); calreticulin 3 (CALR3); nexilin F-actin-binding protein (NEXN); LIM domain binding 3 (LDB3); eyes absent 4 (EYA4); huntingtin (HTT); androgen receptor (AR); protein tyrosine phosphate non-receptor type 11 (PTPN11); JA; Joint plakoglobin (JUP); desmoplakin (DSP); plakophilin 2 (PKP2); desmoglein 2 (DSG2); desmocollin 2 (DSC2); catenin alpha 3 (CTNNA3); NK2 homeobox 5 (NKX2-5); A-kinase anchor protein 9 (AKAP9); A-kinase anchor protein 10 (AKAP10); guanine nucleotide-binding protein alpha-inhibitory activity polypeptide 2 (GNAI2); ankyrin 2 (ANK2); syntrophin alpha-1 (SNTAT); calmodulin 1 (CALM 1); calmodulin 2 (CALM2); HTRA serine peptidase 1 (HTRA1); fibrillin 1 (FBN1); fibrillin 2 (FBN2); xylosyltransferase 1 (XYLT1); xylosyltransferase 2 (XYLT2); tafazzin (TAZ); homogentisate 1,2-dioxygenase (HGD); glucose-6-phosphatase catalytic subunit (G6PC); 1,4-alpha-glucan enzyme 1 (GBE1); phosphofructokinase, muscle (PFKM); phosphorylase kinase regulatory subunit phosphorylase kinase regulatory subunit alpha 1 (PHKA1); phosphorylase kinase regulatory subunit alpha 2 (PHKA2); phosphorylase kinase regulatory subunit beta (PHKB); phosphorylase kinase catalytic subunit gamma 2 (PHKG2); phosphoglycerate mutase 2 (PGAM2); cystathionine beta-synthase (CBS); methylenetetrahydrofolate reductase (MTHFR); 5-methyltetrahydrofolate-homocysteine ​​methyltransferase (MTR); 5-methyltetrahydrofolate-homocysteine ​​methyltransferase methylmalonic aciduria and homocystinuria, cblD type (MMADHC); mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1); mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 5 (MT-ND5); mitochondrially encoded tRNA glutamate (MT-TE); mitochondrially encoded tRNA histadine (MT-TH);Mitochondrial DNA, including mitochondrial-encoded tRNA leucine 1 (MT-TL1); mitochondrial-encoded tRNA lysine (MT-TK); mitochondrial-encoded tRNA serine 1 (MT-TS1); mitochondrial-encoded tRNA valine (MT-TV); mitogen-activated protein kinase 1 (MAP2K1); B-Raf proto-oncogene, serine / threonine kinase (BRAF); raf-1 proto-oncogene, serine / threonine kinase (RAF1); including, but not limited to, insulin growth factor 1 (IGF-1); trans Growth factors include transforming growth factor beta 3 (TGFβ3); transforming growth factor beta receptor, type I (TGFβR1); transforming growth factor beta receptor, type II (TGFβR2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 4 (FGF4), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B); vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2), or any combination thereof;

[0184] In some embodiments, the disclosed nucleic acid sequences can encode RNA. In some embodiments, the disclosed encoded RNA can include ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof. In some embodiments, the disclosed encoded RNA can include lncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof.

[0185] In certain embodiments, the disclosed encoded RNAs can include functional non-coding RNA elements. In certain embodiments, the disclosed nucleic acid molecules can include sequences for one or more regulatory elements. In certain embodiments, the disclosed regulatory elements can be promoters, enhancers, internal ribosome entry sites (IRES), or other expression control elements (e.g., transcription termination signals, e.g., polyadenylation signals and poly-U sequences). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). In certain embodiments, the disclosed promoters can be tissue-specific or ubiquitous, constitutive or inducible, depending on the desired pattern of gene expression. The promoters can be native or foreign, and can be natural or synthetic sequences. By foreign, it is meant that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced. In certain embodiments, the disclosed promoters can be promoter / enhancers. In certain embodiments, the disclosed promoters can be endogenous promoters. In some embodiments, the disclosed endogenous promoter can be an endogenous promoter / enhancer. In some embodiments, the disclosed endogenous promoter or the disclosed endogenous promoter / enhancer can generally be obtained from the non-coding region upstream of the transcription start site of the gene of interest. In some embodiments, the disclosed endogenous promoter or the disclosed endogenous promoter / enhancer can be used for constitutive and efficient expression of the disclosed gene. In some embodiments, the disclosed promoter can be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known in the art. In some embodiments, the disclosed promoter can be any eukaryotic RNA polymerase II promoter.

[0186] In certain embodiments, the disclosed nucleic acid molecules can be in an expression cassette. Expression cassettes are known in the art and can include a promoter sequence, an open reading frame (for a transgene of interest), and a 3' UTR (which typically contains polyA in eukaryotic cells).

[0187] In certain embodiments, the disclosed isolated nucleic acid molecules are capable of restoring one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, hi certain embodiments, the disclosed isolated nucleic acid molecules are capable of restoring functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0188] In certain embodiments, upon expression, the disclosed isolated nucleic acid molecules can reduce and / or minimize the severity of rejection events of one or more transplanted organs. In certain embodiments, upon expression, the disclosed isolated nucleic acid molecules can prevent rejection of one or more transplanted organs. In certain embodiments, the disclosed rejection events can be classified as hyperacute rejection events, acute rejection events, or chronic rejection events. In certain embodiments, upon expression of the disclosed isolated nucleic acid molecules, one or more transplanted organs are not rejected by the subject. In certain embodiments, upon expression of the disclosed isolated nucleic acid molecules, the viability of one or more transplanted organs can be improved and / or increased. In certain embodiments, upon expression of the disclosed isolated nucleic acid molecules, the functionality of one or more transplanted organs can be improved and / or increased. In certain embodiments, upon expression of the disclosed isolated nucleic acid molecules, the functionality of one or more transplanted organs is not reduced and / or impaired. In certain embodiments, the disclosed encoded recombinase can degrade one or more anti-HLA antibodies against one or more transplanted organs.

[0189] Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 119. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NOs: 120 to 121. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NOs: 122 to 159. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 119, which has IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NOs: 120 to 121, which has IgG-specific protease activity. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NOs: 122 to 159, which has IgG-specific protease activity.

[0190] Disclosed herein is an isolated nucleic acid molecule encoding a recombinant enzyme comprising the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a nucleic acid sequence encoding a recombinant enzyme and having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a nucleic acid sequence encoding a recombinant enzyme having human and non-human primate IgG-specific protease activity and having the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a recombinant enzyme having human and non-human primate IgG-specific protease activity encoded by a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the codon-optimized nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161.

[0191] Disclosed herein are expression cassettes comprising the disclosed nucleic acid sequences. Disclosed herein are expression cassettes comprising isolated nucleic acid molecules encoding any of the disclosed enzymes. Disclosed herein are expression cassettes comprising isolated nucleic acid molecules encoding any of the disclosed recombinant enzymes. Disclosed herein are expression cassettes comprising isolated nucleic acid molecules comprising the sequence of any one of SEQ ID NOs: 47 to 92. Disclosed herein are expression cassettes comprising isolated nucleic acid molecules comprising the sequence of SEQ ID NO: 160 or SEQ ID NO: 161. 3. Vector

[0192] Disclosed herein are vectors comprising the disclosed isolated nucleic acid molecules. Disclosed herein are vectors comprising one or more of the disclosed isolated nucleic acid molecules. Disclosed herein are vectors comprising the disclosed isolated nucleic acid molecules having IgM-specific protease activity. Disclosed herein are vectors comprising the disclosed isolated nucleic acid molecules having IgM- and IgG-specific protease activity. Disclosed herein are vectors comprising the disclosed isolated nucleic acid molecules having IgM-specific protease activity. Disclosed herein are vectors comprising the disclosed isolated nucleic acid molecules having IgM- and IgG-specific protease activity. Disclosed herein are vectors comprising a nucleic acid sequence encoding any one of the enzymes set forth in SEQ ID NO: 01 to SEQ ID NO: 46, or a fragment thereof. Disclosed herein are vectors comprising a nucleic acid sequence encoding any one of the recombinant enzymes set forth in SEQ ID NO:02, SEQ ID NO:03, SEQ ID NO:05, SEQ ID NO:06, SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46. Disclosed herein are vectors comprising a nucleic acid sequence encoding any one of the enzymes set forth in SEQ ID NO:01, SEQ ID NO:04, SEQ ID NO:07, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:39, and SEQ ID NO:42. Disclosed herein are vectors comprising the disclosed expression cassettes.

[0193] Disclosed herein are vectors containing a nucleic acid sequence comprising any one of the sequences of SEQ ID NOs: 47 to 92, or a fragment thereof. Disclosed herein are vectors containing any one of the nucleic acid sequences of SEQ ID NOs: 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66, 67, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81, 83, 84, 86, and 87. Disclosed herein are vectors containing any one of the nucleic acid sequences of SEQ ID NOs: 01, 47, 50, 53, 56, 59, 62, 65, 70, 73, 76, 79, and 82.

[0194] Disclosed herein are vectors containing a nucleic acid sequence comprising any one of the sequences of SEQ ID NOs: 47 to 92, or a fragment thereof. Disclosed herein are vectors containing any one of the nucleic acid sequences of SEQ ID NOs: 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 66, 67, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81, 83, 84, 86, and 87. Disclosed herein are vectors containing any one of the nucleic acid sequences of SEQ ID NOs: 01, 47, 50, 53, 56, 59, 62, 65, 70, 73, 76, 79, and 82.

[0195] Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinase of SEQ ID NO: 119. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinase of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinase of any one of SEQ ID NOs: 122 to 159. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinase of SEQ ID NO: 119, or a fragment thereof. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinase of SEQ ID NO: 120 or SEQ ID NO: 121, or a fragment thereof. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinase of any one of SEQ ID NOs: 122 to 159, or a fragment thereof.

[0196] Disclosed herein is a vector comprising the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a vector comprising the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161, or a fragment thereof.

[0197] In certain embodiments, the disclosed vectors can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, hi certain embodiments, the disclosed vectors can restore functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0198] In one embodiment, a therapeutically effective amount of the disclosed vectors can be delivered via intravenous (IV) administration and is about 1 x 10 10 vg / kg ~ approx. 2×10 14 In one embodiment, for example, the disclosed vectors can be administered in a range of about 1 x 10 vg / kg. 11 ~Approx. 8×10 13 vg / kg or approximately 1 × 10 12 ~Approx. 8×10 13 In one embodiment, the disclosed vectors can be administered at a dose of about 1 x 10 vg / kg. 13 ~Approx. 6×10 13In certain embodiments, the disclosed vectors can be administered at a dose of at least about 1 x 10 vg / kg. 10 , at least about 5 × 10 10 , at least about 1 x 10 11 , at least about 5 × 10 11 , at least about 1 x 10 12 , at least about 5 × 10 12 , at least about 1 x 10 13 , at least about 5 × 10 13 , or at least about 1 × 10 14 In certain embodiments, the disclosed vectors can be administered at a dose of about 1 x 10 vg / kg or less. 10 Below, about 5×10 10 Below, approximately 1×10 11 Below, about 5×10 11 Below, approximately 1×10 12 Below, about 5×10 12 Below, approximately 1×10 13 Below, about 5×10 13 or less, or about 1 x 10 14 In one embodiment, the disclosed vectors can be administered at a dose of about 1 x 10 vg / kg or less. 12 In one embodiment, the disclosed vectors can be administered at a dose of about 1 x 10 vg / kg. 11 In one embodiment, a therapeutically effective amount of the disclosed vectors can be delivered via intravenous (IV) administration, for a total of about 1 x 10 vg / kg per subject. 12 vg ~ Total per subject is about 1 × 10 17 In certain embodiments, a therapeutically effective amount of the disclosed vectors can be delivered via intravenous (IV) administration, totaling about 1 x 10 per subject. 12 vg, approximately 1 × 10 per subject in total 13 vg, approximately 1 × 10 per subject in total 14 vg, approximately 1 × 10 per subject in total 15 vg, approximately 1 × 10 per subject in total 16 vg, or approximately 1 × 10 per subject in total 17In some embodiments, the disclosed vectors can be administered in a single dose, or in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) if needed for the desired therapeutic result. In some embodiments, the therapeutically effective amount of the disclosed vectors can include a range determined by one of skill in the art. In some embodiments, the therapeutically effective amount of the disclosed vectors can be delivered via intravenous (IV) administration, totaling about 1 x 10 per subject. 12 vg ~ Total per subject is about 1 × 10 17 In certain embodiments, a therapeutically effective amount of the disclosed vectors can be delivered via intravenous (IV) administration, totaling about 1 x 10 per subject. 12 vg, approximately 1 × 10 per subject in total 13 vg, approximately 1 × 10 per subject in total 14 vg, approximately 1 × 10 per subject in total 15 vg, approximately 1 × 10 per subject in total 16 vg, or approximately 1 × 10 per subject in total 17 vg range.

[0199] In certain embodiments, the disclosed nucleic acid sequences can have coding sequences that are less than about 4.5 kilobases.

[0200] In some embodiments, the disclosed vector can be a viral vector or a non-viral vector.In some embodiments, the disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector.In some embodiments, the disclosed vector can comprise exosomes, extracellular vesicles, and virus-like particles.In some embodiments, the disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease virus vector, a poxvirus vector, or a picornavirus vector.

[0201] In some embodiments, the disclosed viral vectors can be adeno-associated viral (AAV) vectors. In some embodiments, the disclosed AAV vectors can include naturally isolated serotypes, including but not limited to AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7, and bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified as AAV by the International Committee on Taxonomy of Viruses (ICTV). In certain embodiments, the AAV capsid may be chimeric, created either by capsid evolution or by rational capsid engineering from naturally isolated AAV variants to capture desired serotype traits, such as enhanced or specific tissue tropism and / or host immune response escape. Naturally isolated AAV variants include, but are not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In certain embodiments, the disclosed AAV vectors can be AAV-Rh74 or related variants (e.g., capsid variant-like RHM4-1). In some embodiments, the disclosed AAV vectors can be AAV8. In some embodiments, the disclosed AAV vectors can be AAVhum.8. In some embodiments, the disclosed AAV vectors can be self-complementary AAVs disclosed herein.

[0202] In some embodiments, the disclosed vectors can include one or more promoters operably linked to the disclosed transgenes and / or disclosed nucleic acid sequences. In some embodiments, the disclosed promoters can be located 5' (upstream) or 3' (downstream) of the disclosed transgenes and / or disclosed nucleic acid sequences under their control. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0203] In certain embodiments, the disclosed vectors can further comprise one or more regulatory elements. In certain embodiments, the disclosed "regulatory elements" can refer to promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements are discussed below and can include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0204] In certain embodiments, the disclosed promoters for one or more of the disclosed isolated nucleic acid molecules can be tissue-specific or ubiquitous, constitutive or inducible, depending on the desired pattern of gene expression. The promoters can be native or foreign, and can be natural or synthetic sequences. By foreign, it is meant that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced. In certain embodiments, the disclosed promoters can be promoter / enhancers. In certain embodiments, the disclosed promoters for one or more of the disclosed isolated nucleic acid molecules can be endogenous promoters. In certain embodiments, the disclosed endogenous promoters or disclosed endogenous promoters / enhancers can generally be obtained from the non-coding region upstream of the transcription start site of the gene of interest. In certain embodiments, the disclosed endogenous promoters or disclosed endogenous promoters / enhancers can be used for constitutive and efficient expression of the disclosed genes. In some embodiments, the disclosed promoter for one or more of the disclosed isolated nucleic acid molecules can be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoter / enhancers are well known in the art. In some embodiments, the disclosed promoter can be any eukaryotic RNA polymerase II promoter.

[0205] In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of disclosed recombinases having IgM-specific protease activity. In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of disclosed recombinases capable of cleaving IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3. In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of disclosed recombinases capable of cleaving human IgM. In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of disclosed recombinases capable of cleaving non-human primate IgM. In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of disclosed recombinases that do not cleave non-human IgM. For example, in certain embodiments, the disclosed vectors can comprise nucleic acid sequences of disclosed recombinases that do not cleave canine IgM, porcine IgM, and mouse IgM. In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of disclosed IgM-specific recombinases that do not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0206] In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of the disclosed recombinases that can have a binding domain specific for IgM. In certain embodiments, the disclosed recombinases can cleave IgM between positions 350 and 365 of SEQ ID NO: 105. In certain embodiments, SEQ ID NO: 105 can represent the linear sequence of IgM (e.g., a substrate). In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of the disclosed recombinases that can have a binding domain that can specifically bind to one or more amino acid residues 355 to 360 of IgM. In certain embodiments, the disclosed vectors can comprise nucleic acid sequences of the disclosed recombinases that can have affinity for human IgM. In certain embodiments, the disclosed IgM can be cleaved at SEQ ID NO: 110. In certain embodiments, the disclosed IgM can be cleaved at a sequence including 355D, 356T, 357A, 358I, 359R, and 360V. In some embodiments, the disclosed IgM can be truncated at positions 355 to 360 of SEQ ID NO:105.

[0207] In some embodiments, the disclosed vectors can comprise nucleic acid sequences of the disclosed recombinases that can have IgG- and IgM-specific protease activity. In some embodiments, the disclosed vectors can comprise nucleic acid sequences of the disclosed recombinases that can cleave IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3 and cleave IgG below the hinge region. In some embodiments, the disclosed recombinases can cleave IgG below the hinge region, thereby generating F(ab')2 and Fc fragments. In some embodiments, the disclosed recombinases can cleave IgG and separate the Cμ2 and Cμ3 domains from the Cμ1 domain.

[0208] In certain embodiments, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase capable of cleaving human IgG and human IgM. In certain embodiments, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase capable of cleaving non-human primate IgG and non-human primate IgM. In certain embodiments, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase having IgG and IgM protease activity that does not cleave IgA, IgD, IgE, or any combination thereof. In certain embodiments, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase having IgG and IgM protease activity that does not cleave non-human IgM or non-human IgG. For example, in certain embodiments, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase having IgG and IgM protease activity that does not cleave canine IgM, porcine IgM, and mouse IgM, or canine IgG, porcine IgG, or mouse IgG.

[0209] In certain aspects, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase, which can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or any combination thereof. In certain aspects, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase, which can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, or any combination thereof. In certain aspects, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase capable of cleaving IgG at amino acid residues 109 to 124 of the IgG of SEQ ID NO:106, residues 106 to 120 of the IgG of SEQ ID NO:107, residues 156 to 171 of the IgG of SEQ ID NO:108, residues 106 to 121 of the IgG of SEQ ID NO:109, or any combination thereof. In certain aspects, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase capable of cleaving IgG at amino acid residues 116 to 122 of the IgG of SEQ ID NO: 106, residues 112 to 118 of the IgG of SEQ ID NO: 107, residues 163 to 169 of the IgG of SEQ ID NO: 108, residues 113 to 119 of the IgG of SEQ ID NO: 109, or any combination thereof. In certain aspects, the disclosed vectors can comprise the nucleic acid sequence of a disclosed recombinase capable of cleaving one or more IgG molecules at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0210] In certain embodiments, the disclosed encoded recombinases are capable of clearing and / or removing IgM and / or IgM and IgG from the circulation in a subject. In certain embodiments, the disclosed recombinases are capable of clearing and / or removing IgM and / or IgM and IgG from the circulation in the serum of a subject. In certain embodiments, the disclosed recombinases are capable of cleaving and / or degrading circulating IgM and / or IgM and IgG in a subject. In certain embodiments, the disclosed recombinases are capable of transiently clearing and / or removing IgM and / or IgM and IgG from the circulation in the serum of a subject, or transiently cleaving and / or degrading circulating IgM and / or IgM and IgG in a subject. In certain embodiments, the disclosed recombinases are capable of reducing and / or decreasing circulating IgM levels and / or IgM and IgG levels in a subject, or transiently reducing and / or decreasing circulating IgM levels and / or IgM and IgG levels in a subject. In embodiments, the disclosed transient reduction and / or decrease can include about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0211] In certain embodiments, the disclosed vectors are capable of blocking and / or minimizing activation of the complement cascade. In certain embodiments, the disclosed vectors are capable of blocking and / or minimizing activation of C1 complex and / or C3 complex formation. In certain embodiments, the disclosed vectors are capable of blocking and / or minimizing activation of the classical complement pathway. In certain embodiments, the disclosed vectors are capable of transiently blocking and / or minimizing activation of the complement cascade, transiently blocking and / or minimizing activation of C1 complex and / or C3 complex formation, transiently blocking and / or minimizing activation of the classical complement pathway, or any combination thereof.

[0212] In certain embodiments, the disclosed vectors are capable of removing and / or cleaving surface-bound IgM from B cells and / or removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed removal and / or cleavage of IgM from the surface of B cells can be reversible and / or transient. In certain embodiments, the disclosed vectors are capable of transiently removing and / or cleaving surface-bound IgM from B cells and / or transiently removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed vectors are capable of removing and / or cleaving surface-bound IgM from B cells and / or removing and / or cleaving IgM from the surface of B cells without killing and / or damaging the B cells.

[0213] In certain embodiments, the disclosed vectors can reduce and / or minimize the severity of a rejection event of one or more transplanted organs. In certain embodiments, the disclosed vectors can prevent rejection of one or more transplanted organs. In certain embodiments, the disclosed rejection events can be classified as hyperacute rejection events, acute rejection events, or chronic rejection events. In certain embodiments, after administration and / or delivery of the disclosed vectors, the one or more transplanted organs are not rejected by the subject. In certain embodiments, after administration and / or delivery of the disclosed vectors, the viability of one or more transplanted organs can be improved and / or increased. In certain embodiments, after administration and / or delivery of the disclosed vectors, the functionality of one or more transplanted organs can be improved and / or increased. In certain embodiments, after administration and / or delivery of the disclosed vectors, the functionality of one or more transplanted organs is not reduced and / or impaired. In certain embodiments, the disclosed recombinase enzymes can degrade one or more anti-HLA antibodies against one or more transplanted organs.

[0214] In certain embodiments, the disclosed vectors can reduce and / or minimize vector-mediated immunotoxicity in a subject. In certain embodiments, the disclosed vectors can reduce and / or minimize immunogenicity of a transgene in a subject.

[0215] In certain aspects, the disclosed vectors can be used to modulate humoral immunity in a subject, hi certain aspects, modulating humoral immunity in a subject can be an increase in the functionality of the subject's humoral immunity or a decrease in the functionality of the subject's humoral immunity.

[0216] In certain embodiments, the disclosed vectors may enable and / or tolerate re-dosing of the vector in a subject. In certain embodiments, the disclosed vectors of the disclosed embodiments may improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, the disclosed vectors may improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, the disclosed vectors may improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, improving and / or enhancing the efficacy and / or expression of a transgene in a subject may include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of improvement and / or enhancement when compared to an existing level of transgene efficacy and / or expression, such as, for example, a pre-treatment level (before administering one or more disclosed vectors). In certain embodiments, the amount of improvement and / or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than existing levels, such as pre-treatment levels (before administering one or more disclosed vectors). In certain embodiments, the improvement and / or enhancement can be measured relative to a control level or reference level (e.g., determined using one or more subjects who have not received or been administered one or more disclosed vectors). In certain embodiments, the improvement and / or enhancement can be partial or incomplete recovery. In certain embodiments, the improvement and / or enhancement can be complete or near-complete, such that levels of neutralizing antibodies or neutralizing IgG and / or IgM molecules are similar to those of wild-type or control levels. 4. Pharmaceutical preparations

[0217] Disclosed herein are pharmaceutical formulations comprising the disclosed enzymes. Disclosed herein are pharmaceutical formulations comprising the disclosed enzymes and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein are pharmaceutical formulations comprising the disclosed recombinant enzymes. Disclosed herein are pharmaceutical formulations comprising the disclosed recombinant enzymes and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein are pharmaceutical formulations comprising the disclosed vectors. Disclosed herein are pharmaceutical formulations comprising the disclosed vectors and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein are pharmaceutical formulations comprising enzymes having IgM-specific protease activity. Disclosed herein are pharmaceutical formulations comprising enzymes having IgG-specific protease activity. Disclosed herein are pharmaceutical formulations comprising enzymes having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein are pharmaceutical formulations comprising recombinant enzymes having IgM-specific protease activity. Disclosed herein are pharmaceutical formulations comprising recombinant enzymes having IgG-specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a fusion protein having IgM-specific protease activity and IgG-specific protease activity. Disclosed herein is a pharmaceutical formulation comprising an enzyme comprising any one of the sequences set forth in SEQ ID NOs: 01 to 46. Disclosed herein is a pharmaceutical formulation comprising an enzyme comprising any one of the sequences set forth in SEQ ID NOs: 01 to 46 and having IgM-specific protease activity and / or IgG-specific protease activity.

[0218] Disclosed herein are pharmaceutical preparations comprising the disclosed recombinant enzymes having human or non-human primate IgM-specific protease activity, and the disclosed recombinant enzymes having human or non-human primate IgG-specific protease activity. Disclosed herein are pharmaceutical preparations comprising the recombinant enzymes comprising any one of the sequences set forth in SEQ ID NOs: 01 to 46, and the recombinant enzymes comprising the sequence of SEQ ID NO: 119. Disclosed herein are pharmaceutical preparations comprising the recombinant enzymes comprising any one of the sequences set forth in SEQ ID NOs: 01 to 46, and the recombinant enzymes comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein are pharmaceutical preparations comprising the recombinant enzymes comprising any one of the sequences set forth in SEQ ID NOs: 01 to 46, and the recombinant enzymes comprising the sequence of SEQ ID NOs: 122 to 159.

[0219] Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of SEQ ID NO: 119. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of any one of SEQ ID NOs: 122 to 159. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NOs: 122 to 159. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of SEQ ID NO: 119 and having human and non-human primate IgG-specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121 and having human and non-human primate IgG-specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising any one of the sequences of SEQ ID NOs: 122 to 159 and having human and non-human primate IgG-specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119 and having human and non-human primate IgG-specific protease activity. Disclosed herein are pharmaceutical formulations comprising a recombinant enzyme having human and non-human primate IgG-specific protease activity, the recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121.Disclosed herein are pharmaceutical formulations comprising a recombinant enzyme having human and non-human primate IgG-specific protease activity, the recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to any one of SEQ ID NOs: 122 to 159.

[0220] Disclosed herein are pharmaceutical formulations comprising the disclosed isolated nucleic acid molecules. Disclosed herein are pharmaceutical formulations comprising the disclosed isolated nucleic acid molecules and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein are pharmaceutical formulations comprising an isolated nucleic acid molecule comprising any one of SEQ ID NOs: 47 to 92. Disclosed herein are pharmaceutical formulations comprising an isolated nucleic acid molecule comprising any one of SEQ ID NOs: 47 to 92 and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein are pharmaceutical formulations comprising an isolated nucleic acid molecule comprising any one of SEQ ID NOs: 160 or 161. Disclosed herein are pharmaceutical formulations comprising an isolated nucleic acid molecule comprising the sequence of SEQ ID NO: 160 or 161 and one or more pharmaceutically acceptable carriers and / or excipients.

[0221] In certain embodiments, the disclosed pharmaceutical formulations can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In certain embodiments, the disclosed pharmaceutical formulations can restore functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0222] In certain embodiments, the disclosed pharmaceutical preparations can have IgM-specific protease activity. In certain embodiments, the disclosed pharmaceutical preparations can cleave IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3. In certain embodiments, the disclosed recombinant enzymes can cleave human IgM. In certain embodiments, the disclosed pharmaceutical preparations can cleave non-human primate IgM. In certain embodiments, the disclosed pharmaceutical preparations do not cleave non-human IgM. For example, in certain embodiments, the disclosed pharmaceutical preparations do not cleave canine IgM, porcine IgM, and mouse IgM. In certain embodiments, the disclosed IgM-specific pharmaceutical preparations do not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0223] In certain embodiments, the disclosed pharmaceutical preparations can have a binding domain specific for IgM. In certain embodiments, the disclosed pharmaceutical preparations can cleave IgM between positions 350 and 365 of SEQ ID NO: 105. In certain embodiments, SEQ ID NO: 105 can represent the linear sequence of IgM (e.g., a substrate). In certain embodiments, the disclosed pharmaceutical preparations can have a binding domain capable of specifically binding to one or more amino acid residues 355 to 360 of IgM. In certain embodiments, the disclosed pharmaceutical preparations can have affinity for human IgM. In certain embodiments, the disclosed IgM can be cleaved at SEQ ID NO: 110. In certain embodiments, the disclosed IgM can be cleaved at a sequence including 355D, 356T, 357A, 358I, 359R, and 360V. In certain embodiments, the disclosed IgM can be cleaved at positions 355 to 360 of SEQ ID NO: 105.

[0224] In certain embodiments, the disclosed pharmaceutical preparations can have IgG- and IgM-specific protease activity. In certain embodiments, the disclosed pharmaceutical preparations can cleave IgM heavy chains at the interdomain region between the constant domains Cμ2 and Cμ3, and can cleave IgG below the hinge region. In certain embodiments, the disclosed pharmaceutical preparations can cleave IgG below the hinge region, thereby producing F(ab')2 and Fc fragments. In certain embodiments, the disclosed pharmaceutical preparations can cleave IgG to separate the Cμ2 and Cμ3 domains from the Cμ1 domain.

[0225] In certain embodiments, the disclosed pharmaceutical preparations are capable of cleaving human IgG and human IgM. In certain embodiments, the disclosed pharmaceutical preparations are capable of cleaving non-human primate IgG and non-human primate IgM. In certain embodiments, the disclosed pharmaceutical preparations with IgG and IgM protease activity do not cleave IgA, IgD, IgE, or any combination thereof. In certain embodiments, the disclosed pharmaceutical preparations with IgG and IgM protease activity do not cleave non-human IgM or non-human IgG. For example, in certain embodiments, the disclosed pharmaceutical preparations with IgG and IgM protease activity do not cleave canine IgM, porcine IgM, and mouse IgM, or canine IgG, porcine IgG, or mouse IgG.

[0226] In certain embodiments, the disclosed pharmaceutical formulations can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or any combination thereof. In certain embodiments, the disclosed pharmaceutical formulations can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, or any combination thereof. In certain embodiments, the disclosed pharmaceutical formulations can cleave IgG at amino acid residues 109 to 124 of the IgG of SEQ ID NO:106, residues 106 to 120 of the IgG of SEQ ID NO:107, residues 156 to 171 of the IgG of SEQ ID NO:108, residues 106 to 121 of the IgG of SEQ ID NO:109, or any combination thereof. In certain embodiments, the disclosed pharmaceutical formulations are capable of cleaving IgG at amino acid residues 116 to 122 of the IgG of SEQ ID NO: 106, residues 112 to 119 of the IgG of SEQ ID NO: 107, residues 163 to 169 of the IgG of SEQ ID NO: 108, residues 113 to 119 of the IgG of SEQ ID NO: 109, or any combination thereof. In certain embodiments, the disclosed pharmaceutical formulations are capable of cleaving one or more IgG molecules at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0227] In certain embodiments, the disclosed pharmaceutical formulations are capable of clearing and / or removing IgM and / or IgG and IgM from the circulation in a subject. In certain embodiments, the disclosed recombinant enzymes are capable of cleaving and / or degrading circulating IgM and / or IgG and IgM in a subject. In certain embodiments, the disclosed pharmaceutical formulations are capable of transiently clearing and / or removing IgM and / or IgG and IgM from the circulation in a subject, or transiently cleaving and / or degrading circulating IgM and / or IgG and IgM in a subject. In certain embodiments, the disclosed pharmaceutical formulations are capable of reducing and / or lowering circulating IgM and / or IgG and IgM levels in a subject, or transiently reducing and / or lowering circulating IgM and / or IgG and IgM levels in a subject. In embodiments, the disclosed transient reduction and / or decrease can include about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0228] In certain embodiments, the disclosed pharmaceutical formulations are capable of blocking and / or minimizing activation of the complement cascade. In certain embodiments, the disclosed pharmaceutical formulations are capable of blocking and / or minimizing activation of the C1 complex and / or C3 complex formation. In certain embodiments, the disclosed pharmaceutical formulations are capable of blocking and / or minimizing activation of the classical complement pathway. In certain embodiments, the disclosed pharmaceutical formulations are capable of transiently blocking and / or minimizing activation of the complement cascade, transiently blocking and / or minimizing activation of the C1 complex and / or C3 complex formation, transiently blocking and / or minimizing activation of the classical complement pathway, or any combination thereof.

[0229] In certain embodiments, the disclosed pharmaceutical formulations are capable of removing and / or cleaving surface-bound IgM from B cells and / or removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed removal and / or cleavage of IgM from the surface of B cells can be reversible and / or transient. In certain embodiments, the disclosed pharmaceutical formulations are capable of transiently removing and / or cleaving surface-bound IgM from B cells and / or transiently removing and / or cleaving IgM from the surface of B cells. In certain embodiments, the disclosed pharmaceutical formulations are capable of removing and / or cleaving surface-bound IgM from B cells and / or removing and / or cleaving IgM from the surface of B cells without killing and / or damaging the B cells.

[0230] In certain embodiments, the disclosed pharmaceutical formulations can reduce and / or minimize vector-mediated immunotoxicity in a subject. In certain embodiments, the disclosed pharmaceutical formulations can enable and / or tolerate re-dosing of the vector in a subject.

[0231] In certain embodiments, the disclosed pharmaceutical formulations can reduce and / or minimize the immunogenicity of a transgene in a subject. In certain embodiments, the disclosed pharmaceutical formulations can improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, improving and / or enhancing the efficacy and / or expression of a transgene in a subject can include an improvement and / or enhancement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of improvement and / or enhancement when compared to an existing level of transgene efficacy and / or expression, such as, for example, a pre-treatment level (before administering one or more disclosed pharmaceutical formulations). In certain embodiments, the amount of improvement and / or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than existing levels, such as pre-treatment levels (before administering one or more disclosed pharmaceutical formulations). In certain embodiments, the improvement and / or enhancement can be measured relative to a control level or reference level (e.g., determined using one or more subjects who have not received or been administered one or more disclosed pharmaceutical formulations). In certain embodiments, the improvement and / or enhancement can be partial or incomplete recovery. In certain embodiments, the improvement and / or enhancement can be complete or near-complete, such that levels of neutralizing antibodies or neutralizing IgG and / or IgM molecules are similar to those of wild-type or control levels.

[0232] In certain embodiments, the disclosed pharmaceutical formulations can include (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In certain embodiments, the disclosed compositions can include one or more immune modulators. In certain embodiments, the disclosed compositions can include one or more proteasome inhibitors. In certain embodiments, the disclosed compositions can include one or more immunosuppressants or immunosuppressants. In certain embodiments, the immunosuppressant can be antithymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof. In certain embodiments, the disclosed formulations can include an anaplerotic agent (e.g., a C7 compound such as triheptanoin or MCT).

[0233] In certain embodiments, the pharmaceutical formulations can reduce and / or minimize the severity of rejection events of one or more transplanted organs. In certain embodiments, the pharmaceutical formulations can prevent rejection of one or more transplanted organs. In certain embodiments, the disclosed rejection events can be classified as hyperacute rejection events, acute rejection events, or chronic rejection events. In certain embodiments, after administration of the disclosed pharmaceutical formulations, the disclosed isolated nucleic acid molecules, one or more transplanted organs are not rejected by the subject. In certain embodiments, after administration of the disclosed pharmaceutical formulations, the viability of one or more transplanted organs can be improved and / or increased. In certain embodiments, after administration of the disclosed pharmaceutical formulations, the functionality of one or more transplanted organs can be improved and / or increased. In certain embodiments, after administration of the disclosed pharmaceutical formulations, the functionality of one or more transplanted organs does not decrease and / or decline. In certain embodiments, the disclosed pharmaceutical formulations can degrade one or more anti-HLA antibodies against one or more transplanted organs. In certain embodiments, any of the disclosed pharmaceutical formulations can include one or more excipients and / or pharmaceutically acceptable carriers. Excipients and / or pharmaceutically acceptable carriers are known in the art and are discussed above.

[0234] In certain embodiments of the disclosed pharmaceutical formulations, the disclosed enzymes or disclosed recombinant enzymes can be therapeutically effective when the dosage comprises about 0.01 mg / kg to about 100 mg / kg of body weight. In certain embodiments, the disclosed enzymes or disclosed recombinant enzymes can be therapeutically effective when the dosage comprises about 0.01 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg of body weight. 5. Plasmids

[0235] The present specification discloses a plasmid that comprises one or more of the disclosed isolated nucleic acid molecules.The present specification discloses a plasmid that comprises one or more of the disclosed vectors.For example, the present specification discloses a plasmid that is used in the method for producing the disclosed composition, such as the disclosed enzyme, the disclosed recombinant enzyme, the disclosed isolated nucleic acid molecule, the disclosed vector, or the disclosed pharmaceutical preparation.Plasmids and the use of plasmids are known in the art.

[0236] Disclosed herein are plasmids comprising the sequence set forth in any one of SEQ ID NO: 93, or a fragment thereof. Disclosed herein are plasmids comprising a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 93, or a fragment thereof. Disclosed herein are plasmids comprising a sequence having at least 40% to 60%, at least 60% to 80%, at least 80% to 90%, or at least 90% to 100% identity to the sequence set forth in SEQ ID NO: 93, or a fragment thereof.

[0237] Disclosed herein is a plasmid comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 47 to 92. Disclosed herein is a plasmid comprising a nucleic acid molecule that, upon expression, can produce an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 01 to 46. Disclosed herein is a plasmid comprising a nucleic acid sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a plasmid comprising a nucleic acid molecule that, upon expression, can produce an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 119 to 121. 6.Cells

[0238] Disclosed herein are cells comprising the disclosed isolated nucleic acid molecules, the disclosed vectors, and / or the disclosed plasmids. Disclosed herein are cells transduced with one or more of the disclosed viral vectors. Disclosed herein are cells transduced with one or more of the disclosed recombinant viral vectors. Disclosed herein are cells transfected with one or more of the disclosed isolated nucleic acid molecules. In some embodiments, the disclosed cells are transfected, or the disclosed cells are transfected with one or more nucleic acid sequences having the sequence set forth in SEQ ID NO:93.

[0239] Disclosed herein are cells transfected with a nucleic acid molecule comprising a sequence set forth in any one of SEQ ID NOs: 47 to 92. Disclosed herein are cells that produce the disclosed enzymes or the disclosed recombinant enzymes for harvesting and purification. Disclosed herein are cells that produce an enzyme comprising a sequence of any one of SEQ ID NOs: 01 to 46 for harvesting and purification. Disclosed herein are cells that, upon expression of a nucleic acid molecule comprising a sequence of any one of SEQ ID NOs: 47 to 92, can produce an enzyme comprising a sequence of any one of SEQ ID NOs: 01 to 46 for harvesting and purification.

[0240] Disclosed herein are cells transfected with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein are cells that produce the disclosed enzymes or the disclosed recombinant enzymes for harvesting and purification. Disclosed herein are cells that produce an enzyme comprising any one of the sequences of SEQ ID NO: 119-121 for harvesting and purification. Disclosed herein are cells that produce an enzyme comprising any one of the sequences of SEQ ID NO: 122-159 for harvesting and purification. Disclosed herein are cells that, upon expression of a nucleic acid molecule comprising the sequence of SEQ ID NO: 160 or SEQ ID NO: 161, can produce an enzyme comprising the sequence of SEQ ID NO: 119 for harvesting and purification.

[0241] In some embodiments, the disclosed cell or cells can be humanized. Techniques for achieving transfection and transduction are known in the art, and the use of transfected or transduced cells is known in the art. In some embodiments, disclosed herein are human immortalized cell lines transfected with one or more disclosed viral vectors or transfected with one or more disclosed isolated nucleic acids or disclosed plasmids. In some embodiments, disclosed herein are human immortalized cell lines contacted with one or more disclosed pharmaceutical preparations. Disclosed herein are cells obtained for subjects treated with one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed plasmids, or one or more disclosed pharmaceutical preparations. 7.Animals

[0242] Disclosed herein are animals treated with one or more of the disclosed enzymes, one or more of the disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more of the disclosed vectors, one or more of the disclosed pharmaceutical formulations, and / or one or more of the disclosed plasmids. Transgenic animals are known in the art, as are techniques for producing transgenic animals. C. Methods for Cleaving IgM and / or IgG

[0243] Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules. Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed vectors. Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed pharmaceutical formulations.

[0244] Disclosed herein is a method for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 01 to 15. Disclosed herein is a method for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 24 to 38. Disclosed herein is a method for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 02 or SEQ ID NO: 03. Disclosed herein is a method for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 05 or SEQ ID NO: 06. Disclosed herein is a method for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 08 or SEQ ID NO: 09. Disclosed herein is a method for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a method for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0245] Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in any one of SEQ ID NOs: 47 to 61. Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in any one of SEQ ID NOs: 70 to 84. Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. Disclosed herein are methods for cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52. Disclosed herein is a method of cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO: 54 or SEQ ID NO: 55. Disclosed herein is a method of cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 58. Disclosed herein is a method of cleaving immunoglobulin M (IgM), comprising contacting one or more IgM molecules with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 61.

[0246] Disclosed herein are methods for cleaving immunoglobulin G (IgG), comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 160-161. Disclosed herein are methods for cleaving immunoglobulin G (IgG), comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein are methods for cleaving immunoglobulin G (IgG), comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein are methods for cleaving immunoglobulin G (IgG), comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein are methods for cleaving immunoglobulin G (IgG), comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein are methods for cleaving immunoglobulin G (IgG), comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising a sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), comprising contacting one or more IgG molecules with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), comprising contacting one or more IgM molecules with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161.

[0247] Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with one or more disclosed nucleic acid molecules. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with one or more disclosed vectors. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with one or more disclosed pharmaceutical formulations. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 16 to 23. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 39 to 46. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21.Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with an enzyme comprising a sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 39 to 46. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 119 to 121.

[0248] Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising a sequence set forth in any one of SEQ ID NOs: 62 to 69. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising a sequence set forth in any one of SEQ ID NOs: 85 to 92. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising a sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 64. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising a sequence set forth in SEQ ID NO: 66 or SEQ ID NO: 67. Disclosed herein is a method for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 69. Disclosed herein is a method for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 86 or SEQ ID NO: 87. Disclosed herein is a method for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 89 or SEQ ID NO: 90. Disclosed herein is a method for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 91 or SEQ ID NO: 92.

[0249] In certain embodiments, the cleaved IgM and / or IgG molecules can be in the serum of a subject. In certain embodiments, the cleaved IgM and / or IgG molecules can circulate in a subject. In certain embodiments, the disclosed IgM and / or IgG molecules can be neutralized against exogenously administered proteins. In certain embodiments, the disclosed IgM and / or IgG molecules can be neutralized against enzyme replacement therapy or protein replacement therapy or recombinant products. In certain embodiments, the disclosed IgM and / or IgG molecules can be neutralized against non-viral or viral vectors. In certain embodiments, the disclosed IgM and / or IgG molecules can be neutralized against proteins associated with non-viral or viral vectors. For example, in certain embodiments, the disclosed IgM and / or IgG molecules can be neutralized against viral vector capsid proteins (e.g., AAV capsid proteins).

[0250] In certain aspects, the disclosed methods can further include diagnosing the subject as in need of one or more of the disclosed enzymes, one or more of the disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more of the disclosed vectors, one or more of the disclosed pharmaceutical formulations, or any combination thereof.

[0251] In certain embodiments of the disclosed methods, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in levels of circulating intact IgM and / or intact IgG.

[0252] In certain embodiments, the disclosed IgM and / or IgG molecules can be neutralized against one or more transplanted organs. In certain embodiments, the disclosed methods can reduce and / or minimize the severity of rejection events of one or more transplanted organs. In certain embodiments, the disclosed methods can prevent rejection of one or more transplanted organs. In certain embodiments, the disclosed rejection events can be classified as hyperacute rejection events, acute rejection events, or chronic rejection events. In certain embodiments, the disclosed methods can reduce the risk of rejection of one or more transplanted organs. In certain embodiments, the disclosed methods can reduce or lower a subject's need for long-term immunosuppression. In certain embodiments, the disclosed methods can improve and / or increase the viability of one or more transplanted organs. In certain embodiments, the disclosed methods can improve and / or increase the functionality of one or more transplanted organs. In certain embodiments, the disclosed methods can reduce and / or not reduce the functionality of one or more transplanted organs. In certain embodiments, the disclosed methods can degrade one or more anti-HLA antibodies against one or more transplanted organs.

[0253] In certain embodiments, the subject can have high persistent antibody titers (HSAT—defined as a titer of ≧12,800). In certain embodiments, the subject can have high persistent antibody titers (HSAT) to one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral or viral vectors, one or more proteins associated with non-viral vectors, one or more proteins associated with viral vectors, or any combination thereof. In certain embodiments, after the contacting step, the disclosed HSAT can be reduced and / or eliminated. In certain embodiments, after the reduction and / or elimination of one or more HSATs, the subject can be re-medicated. In certain embodiments, after reduction and / or elimination of one or more HSATs, the subject may be re-medicated with one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0254] In certain aspects, the disclosed methods can include modifying one or more of the disclosed steps. For example, modifying one or more steps of the disclosed methods can include modifying or changing one or more features or aspects of one or more steps of the disclosed methods. For example, in certain aspects, the methods can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof, administered to the subject; by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to the subject; or by changing the period of time over which one or more disclosed enzymes or recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to the subject.

[0255] In certain aspects, the disclosed methods can further include measuring the levels of IgM and / or IgG in the subject (e.g., in the subject's serum) one or more times.

[0256] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can clear and / or remove IgM and / or IgG from the circulation in a subject. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can leave and / or degrade circulating IgM and / or IgG in a subject. In certain embodiments, the disclosed methods can transiently clear and / or remove IgM and / or IgG from the circulation in a subject, or transiently cleave and / or degrade circulating IgM and / or IgG in a subject. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can reduce and / or decrease circulating IgM and / or IgG levels in a subject, or transiently reduce and / or decrease circulating IgM and / or IgG levels in a subject. In embodiments, the disclosed transient reduction and / or decrease can include about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0257] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can block and / or minimize activation of the complement cascade. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can block and / or minimize activation of C1 complex and / or C3 complex formation. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can block and / or minimize activation of the classical complement pathway. In certain embodiments, the disclosed methods can transiently block and / or minimize activation of the complement cascade, transiently block and / or minimize activation of C1 complex and / or C3 complex formation, transiently block and / or minimize activation of the classical complement pathway, or any combination thereof.

[0258] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can remove and / or cleave surface-bound IgM from B cells and / or can remove and / or cleave IgM from the surface of B cells.

[0259] In certain embodiments, the disclosed removal and / or cleavage of IgM from the surface of B cells can be reversible and / or transient. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can transiently remove and / or cleave surface-bound IgM from B cells and / or can transiently remove and / or cleave IgM from the surface of B cells. In certain embodiments, the disclosed methods can remove and / or cleave surface-bound IgM from B cells and / or can remove and / or cleave IgM from the surface of B cells without killing and / or damaging the B cells.

[0260] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can reduce and / or minimize vector-mediated immunotoxicity in a subject, hi certain embodiments, the disclosed methods of cleaving IgM and / or IgG can enable and / or tolerate re-dosing of the vector in a subject.

[0261] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can modulate humoral immunity in a subject, hi certain embodiments, modulating humoral immunity in a subject can be an increase in the functionality of the subject's humoral immunity or a decrease in the functionality of the subject's humoral immunity.

[0262] In certain embodiments of the disclosed methods, intact IgM and / or intact IgG can reduce and / or decrease the efficacy and / or potency of an administered gene therapy. In certain embodiments of the disclosed methods, intact IgM and / or intact IgG can reduce and / or decrease the efficacy and / or potency of an administered therapeutic protein or therapeutic recombinant protein. In certain embodiments of the disclosed methods, intact IgM and / or intact IgG can reduce and / or decrease the efficacy and / or potency of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0263] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can reduce and / or minimize the immunogenicity of a transgene in a subject. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, improving and / or enhancing the efficacy and / or expression of a transgene in a subject can include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of improvement and / or enhancement when compared to the existing level of transgene efficacy and / or expression, such as, for example, a pre-treatment level (before administering one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In certain embodiments, the amount of improvement and / or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than an existing level, such as a pre-treatment level (before administering one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In certain embodiments, the improvement and / or enhancement can be measured relative to a control level or reference level (e.g., determined using one or more subjects who have not received or been administered one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In certain embodiments, the improvement and / or enhancement can be a partial or incomplete recovery. In certain embodiments, the improvement and / or enhancement can be complete or near complete, such that the levels of neutralizing antibodies or neutralizing IgG and / or IgM molecules are similar to that of wild-type or control levels.

[0264] In certain aspects of the disclosed methods, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof, can be delivered and / or administered prior to, concurrently with, or after the delivery and / or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.

[0265] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can further include reducing and / or minimizing the vector-mediated immunotoxicity and / or immunogenicity of the transgene (e.g., ability to induce specific immunity). In certain embodiments, the vector-mediated immunotoxicity and / or immunogenicity of the transgene can reduce and / or decrease the efficacy of a recombinant product encoded by the transgene. In certain embodiments, the vector-mediated immunotoxicity and / or immunogenicity of the transgene can reduce and / or decrease the ability and / or likelihood of re-medicating a subject with one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In certain embodiments, the vector-mediated immunotoxicity and / or immunogenicity of the transgene can reduce and / or decrease the ability and / or likelihood of re-medicating a subject with gene therapy, enzyme replacement therapy, protein replacement, a recombinant product, or any combination thereof.

[0266] In certain embodiments of the disclosed methods of cleaving IgM and / or IgG, IgM can be cleaved in the heavy chain at the interdomain region between constant domains Cμ2 and Cμ3. In certain embodiments, IgM can be cleaved between positions 350 and 365 of SEQ ID NO: 105. In certain embodiments, SEQ ID NO: 105 can represent the linear sequence of IgM (e.g., a substrate). In certain embodiments, IgM can be cleaved between positions 355 and 360 of IgM. In certain embodiments, IgM can be cleaved at SEQ ID NO: 110. In certain embodiments, IgM can be cleaved at a sequence including 355D, 356T, 357A, 358I, 359R, and 360V. In certain embodiments, IgM can be cleaved at positions 355 to 360 of IgM of SEQ ID NO: 105. In certain embodiments, IgG can be cleaved below the hinge region, thereby providing F(ab')2 and Fc fragments. In certain embodiments, IgG can be cleaved to separate the Cμ2 and Cμ3 domains from the Cμ1 domain. In certain embodiments, IgG can be cleaved at SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or any combination thereof. In certain embodiments, IgG can be cleaved at SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, or any combination thereof. In certain embodiments, IgG can be cleaved at positions 109 to 124 of the IgG of SEQ ID NO:106, positions 106 to 120 of the IgG of SEQ ID NO:107, positions 156 to 171 of the IgG of SEQ ID NO:108, positions 106 to 121 of the IgG of SEQ ID NO:109, or any combination thereof. In certain embodiments, IgG may be cleaved at positions 116 to 122 of the IgG of SEQ ID NO: 106, positions 112 to 118 of the IgG of SEQ ID NO: 107, positions 163 to 169 of the IgG of SEQ ID NO: 108, positions 113 to 119 of the IgG of SEQ ID NO: 109, or any combination thereof. In certain embodiments, IgG may be cleaved at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0267] Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the methods comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes having IgM-specific protease activity and one or more disclosed recombinant enzymes having IgG-specific protease activity.

[0268] Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes comprising a sequence of any one of SEQ ID NOs: 01-46, and one or more disclosed recombinant enzymes comprising a sequence of any one of SEQ ID NOs: 119-159.

[0269] Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more disclosed recombinases comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12, and one or more disclosed recombinases comprising the sequence of any one of SEQ ID NOs: 119 through 159. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more disclosed recombinases comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12, and one or more disclosed recombinases comprising the sequence of SEQ ID NO: 119. Disclosed herein are methods for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more disclosed recombinases comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15, and one or more disclosed recombinases comprising the sequence of any one of SEQ ID NOs: 119 through 159. Disclosed herein is a method for cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinases comprising the sequence of SEQ ID NO:14 or SEQ ID NO:15, and one or more disclosed recombinases comprising the sequence of SEQ ID NO:119.

[0270] In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can further comprise contacting one or more IgM and IgG molecules with a recombinase comprising the sequence of SEQ ID NO: 119. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can further comprise contacting one or more IgM and IgG molecules with a recombinase comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can further comprise contacting one or more IgM and IgG molecules with a recombinase comprising the sequence of any one of SEQ ID NOs: 122 through 159. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can further comprise contacting one or more IgM and IgG molecules with a recombinase comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can further comprise contacting one or more IgM and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In certain embodiments, the disclosed methods of cleaving IgM and / or IgG can further comprise contacting one or more IgM and IgG molecules with a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NOs: 122 through 159.

[0271] In certain aspects, the disclosed methods can further include administering one or more doses of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0272] In certain embodiments, the disclosed methods can further include measuring and / or determining the subject's pre-treatment levels of circulating IgG and / or IgM and / or the subject's pre-treatment levels of surface-bound IgM. In certain embodiments, the disclosed methods can further include measuring and / or determining the subject's levels of circulating IgG and / or IgM and / or the subject's levels of surface-bound IgM one or more times (e.g., before, during, and after the disclosed administering step).

[0273] In some embodiments, the disclosed methods can be performed using a single disclosed enzyme that has both IgG- and IgM-specific protease activity, hi some embodiments, the disclosed methods can be performed using a disclosed enzyme that has IgG-specific protease activity and a disclosed enzyme that has IgM-specific protease activity. D. Methods for Cleaving Surface-Bound IgM

[0274] Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid molecules. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed vectors. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed pharmaceutical formulations. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 01 to 15. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 24 to 38. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in SEQ ID NO: 02 or SEQ ID NO: 03. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in SEQ ID NO: 05 or SEQ ID NO: 06. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising the sequence set forth in SEQ ID NO: 08 or SEQ ID NO: 09.Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0275] Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with one or more disclosed nucleic acid molecules comprising a sequence set forth in any one of SEQ ID NOs: 47 through 61. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with one or more disclosed nucleic acid molecules comprising a sequence set forth in any one of SEQ ID NOs: 70 through 84.

[0276] Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO: 54 or SEQ ID NO: 55. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 58. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with one or more disclosed nucleic acids comprising the sequence set forth in SEQ ID NO:60 or SEQ ID NO:61.

[0277] Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 16 to 23. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 39 to 46. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with an enzyme comprising a sequence set forth in any one of SEQ ID NOs: 39 to 46.

[0278] Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NOs: 62 to 69. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NOs: 85 to 92. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 64. Disclosed herein are methods for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 66 or SEQ ID NO: 67. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 68 or SEQ ID NO: 69. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 86 or SEQ ID NO: 87. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 89 or SEQ ID NO: 90. Disclosed herein is a method for cleaving bound immunoglobulin M (IgM), comprising contacting one or more IgM molecules bound to the surface of a B cell with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 91 or SEQ ID NO: 92.

[0279] In some embodiments, the disclosed B cells can be in the serum of a subject. In some embodiments, the disclosed B cells can be circulating in a subject.

[0280] In certain embodiments, the disclosed surface-bound IgM molecules can be neutralized against exogenously administered proteins. In certain embodiments, the disclosed surface-bound IgM molecules can be neutralized against enzyme replacement therapy or protein replacement therapy or recombinant products. In certain embodiments, the disclosed surface-bound IgM molecules can be neutralized against non-viral vectors or viral vectors. In certain embodiments, the disclosed surface-bound IgM molecules can be neutralized against proteins associated with non-viral vectors or viral vectors. For example, in certain embodiments, the disclosed surface-bound IgM molecules can be neutralized against viral vector capsid proteins (e.g., AAV capsid proteins).

[0281] In certain aspects, the disclosed methods can further include diagnosing the subject as in need of one or more of the disclosed enzymes, one or more of the disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more of the disclosed vectors, one or more of the disclosed pharmaceutical formulations, or any combination thereof.

[0282] In certain embodiments of the disclosed methods, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in levels of circulating intact IgM and / or intact IgG.

[0283] In certain embodiments, the disclosed surface-bound IgM molecules can be neutralized against one or more transplant organs. In certain embodiments, the disclosed methods can degrade one or more anti-HLA antibodies against one or more transplant organs.

[0284] In certain embodiments, the subject can have high persistent antibody titers (HSAT—defined as a titer of ≧12,800). In certain embodiments, the subject can have high persistent antibody titers (HSAT) to one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral or viral vectors, one or more proteins associated with non-viral vectors, one or more proteins associated with viral vectors, or any combination thereof. In certain embodiments, after the contacting step, the disclosed HSAT can be reduced and / or eliminated. In certain embodiments, after the reduction and / or elimination of one or more HSATs, the subject can be re-medicated. In certain embodiments, after reduction and / or elimination of one or more HSATs, the subject may be re-medicated with one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0285] In certain aspects, the disclosed methods can further include measuring the levels of IgM and / or IgG in the subject (e.g., in the subject's serum) one or more times.

[0286] In certain embodiments, the disclosed methods of cleaving surface-bound IgM are capable of clearing and / or removing IgM from the circulation in a subject. In certain embodiments, the disclosed methods of cleaving surface-bound IgM are capable of cleaving and / or degrading circulating IgM in a subject. In certain embodiments, the disclosed methods of cleaving surface-bound IgM are capable of transiently clearing and / or removing IgM from the circulation in a subject, or transiently cleaving and / or degrading circulating IgM in a subject. In certain embodiments, the disclosed methods of cleaving surface-bound IgM are capable of reducing and / or decreasing the level of circulating IgM levels in a subject, or transiently reducing and / or decreasing the level of circulating IgM levels in a subject. In certain embodiments, the disclosed methods are capable of removing and / or cleaving surface-bound IgM from B cells and / or removing and / or cleaving IgM from the surface of B cells without killing and / or damaging the B cells.

[0287] In embodiments, the disclosed transient reduction and / or decrease can include about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0288] In certain embodiments, the disclosed methods of cleaving surface-bound IgM can block and / or minimize activation of the complement cascade. In certain embodiments, the disclosed methods of cleaving surface-bound IgM can block and / or minimize activation of the C1 complex and / or C3 complex formation. In certain embodiments, the disclosed methods of cleaving surface-bound IgM can block and / or minimize activation of the classical complement pathway.

[0289] In certain embodiments, the disclosed methods of cleaving surface-bound IgM can transiently block and / or minimize activation of the complement cascade, transiently block and / or minimize activation of C1 complex and / or C3 complex formation, transiently block and / or minimize activation of the classical complement pathway, or any combination thereof.

[0290] In certain embodiments, the disclosed methods of cleaving surface-bound IgM can reduce and / or minimize vector-mediated immunotoxicity in a subject. In certain embodiments, the disclosed methods of cleaving surface-bound IgM can enable and / or allow for re-administration of a vector in a subject. In certain embodiments, the disclosed methods of cleaving surface-bound IgM can enable and / or allow for re-administration of one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof in a subject.

[0291] In certain embodiments, the disclosed methods of cleaving surface-bound IgM can reduce and / or minimize the immunogenicity of a transgene in a subject. In certain embodiments, the disclosed methods of cleaving surface-bound IgM can improve and / or enhance the efficacy and / or expression of a transgene in a subject. In certain embodiments, improving and / or enhancing the efficacy and / or expression of a transgene in a subject can include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of improvement and / or enhancement when compared to the existing level of transgene efficacy and / or expression, such as, for example, a pre-treatment level (prior to administering one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In certain embodiments, the amount of improvement and / or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than an existing level, such as a pre-treatment level (before administering one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In certain embodiments, the improvement and / or enhancement can be measured relative to a control level or reference level (e.g., determined using one or more subjects who have not received or been administered one or more of the disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In certain embodiments, the improvement and / or enhancement can be a partial or incomplete recovery. In certain embodiments, the improvement and / or enhancement can be complete or near complete, such that the levels of neutralizing antibodies or neutralizing IgG and / or IgM molecules are similar to that of wild-type or control levels.

[0292] In certain embodiments, the disclosed methods of cleaving surface-bound IgM can modulate humoral immunity in a subject, hi certain embodiments, modulating humoral immunity in a subject can be an increase in the functionality of the subject's humoral immunity or a decrease in the functionality of the subject's humoral immunity.

[0293] In certain embodiments of the disclosed methods, intact IgM and / or intact IgG can reduce and / or decrease the efficacy and / or potency of an administered gene therapy. In certain embodiments of the disclosed methods, intact IgM and / or intact IgG can reduce and / or decrease the efficacy and / or potency of an administered therapeutic protein or therapeutic recombinant protein. In certain embodiments of the disclosed methods, intact IgM and / or intact IgG can reduce and / or decrease the efficacy and / or potency of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0294] In certain aspects, the disclosed methods can include administering to a subject in need thereof a theoretically effective amount of one or more of the disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more of the disclosed vectors, one or more of the disclosed pharmaceutical formulations, or any combination thereof.

[0295] In some embodiments, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzyme can comprise from about 0.01 mg / kg to about 100 mg / kg body weight. In some embodiments, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg / kg, ab...

Claims

1. A recombinant enzyme having human and non-human primate IgM-specific protease activity and human and non-human primate IgG-specific protease activity.

2. 2. The recombinant enzyme of claim 1, comprising a sequence having at least 50% identity to the sequence shown in SEQ ID NO: 17 or SEQ ID NO:

18.

3. 2. The recombinant enzyme of claim 1, comprising a sequence having at least 50% identity to the sequence shown in SEQ ID NO:20 or SEQ ID NO:

21.

4. The recombinase according to any one of claims 1 to 3, wherein the recombinase cleaves human IgM in the heavy chain in the interdomain region between the constant domains Cμ2 and Cμ3.

5. The recombinant enzyme according to any one of claims 1 to 4, wherein the IgM-specific protease activity occurs between positions 350 and 365 of IgM of SEQ ID NO:

105.

6. 6. The recombinase of claim 1, wherein the recombinase cleaves human or non-human primate IgG below the hinge region, thereby separating the Cμ2 and Cμ3 domains from the Cμ1 domain.

7. 7. The recombinant enzyme of any one of claims 1 to 6, wherein the IgG-specific protease activity occurs between positions 116 to 122 of the IgG of SEQ ID NO: 106, positions 112 to 118 of the IgG of SEQ ID NO: 107, positions 163 to 169 of the IgG of SEQ ID NO: 108, positions 113 to 119 of the IgG of SEQ ID NO: 109, or any combination thereof.

8. 7. The recombinant enzyme of any one of claims 1 to 6, wherein said IgM-specific protease activity occurs with the sequence of SEQ ID NO: 110 of an IgM molecule and said IgG-specific protease activity occurs with the sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 of a human IgG molecule, or any combination thereof.

9. An isolated nucleic acid molecule comprising a codon-optimized nucleic acid sequence encoding the recombinant enzyme according to any one of claims 1 to 8.

10. 10. The isolated nucleic acid molecule of claim 9, comprising the sequence set forth in SEQ ID NO: 63 or SEQ ID NO:

64.

11. 10. The isolated nucleic acid molecule of claim 9, comprising the sequence set forth in SEQ ID NO: 66 or SEQ ID NO:

67.

12. Recombinant enzyme with human and non-human primate IgM-specific protease activity.

13. 13. The recombinant enzyme of claim 12, comprising a sequence having at least 50% identity to the sequence shown in SEQ ID NO: 11 or SEQ ID NO:

12.

14. 13. The recombinant enzyme of claim 12, comprising a sequence having at least 50% identity to the sequence shown in SEQ ID NO: 14 or SEQ ID NO:

15.

15. The recombinase according to any one of claims 12 to 14, wherein the recombinase cleaves the heavy chain in the interdomain region between the constant domains Cμ2 and Cμ3 of human or non-human primate IgM.

16. The recombinant enzyme according to any one of claims 12 to 15, wherein the IgM-specific protease activity occurs between positions 350 and 365 of IgM of SEQ ID NO:

105.

17. The recombinant enzyme according to any one of claims 12 to 15, wherein said IgM-specific protease activity occurs at the sequence of SEQ ID NO: 110 of an IgM molecule.

18. An isolated nucleic acid molecule comprising a codon-optimized nucleic acid sequence encoding the recombinant enzyme according to any one of claims 12 to 17.

19. 19. The isolated nucleic acid molecule of claim 18, comprising the sequence set forth in SEQ ID NO: 57 or SEQ ID NO:

58.

20. 19. The isolated nucleic acid molecule of claim 18, comprising the sequence set forth in SEQ ID NO: 60 or SEQ ID NO:

61.

21. A pharmaceutical preparation comprising the recombinant enzyme of any one of claims 1 to 8 or claims 12 to 17.

22. 18. A method for reducing the level of circulating IgM or the levels of circulating IgM and IgG in a subject, comprising the step of administering a therapeutically effective amount of a recombinant enzyme according to any one of claims 1 to 8 or claims 12 to 17 to said subject in need of a reduction in the level of circulating IgM or the levels of circulating IgM and IgG, wherein after said administering step, the level of circulating IgM or the levels of circulating IgM and IgG are reduced when compared to the level before the administering step.

23. 23. The method of claim 22, wherein the reduction in levels of circulating IgM or levels of circulating IgM and IgG is transient.

24. 24. The method of claim 23, wherein the transient reduction in levels of circulating IgM or levels of circulating IgM and IgG lasts for about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, or more than 2 weeks.

25. 25. The method of claim 24, wherein the transient reduction in circulating levels of IgM or circulating IgM and IgG levels comprises a reduction of at least 80% when compared to levels prior to the administering step.

26. 26. The method of any one of claims 22 to 25, wherein the subject has received, is receiving or will receive a recombinant product.

27. 26. The method of any one of claims 22 to 25, wherein the subject has been, is currently undergoing, or will be undergoing enzyme or protein replacement therapy.

28. 26. The method of any one of claims 22 to 25, wherein the subject has received, is receiving, or will receive one or more transplanted organs.

29. 30. The method of claim 28, wherein after the administering step, the severity of rejection events of the one or more transplanted organs is reduced and / or minimized.

30. The method of any one of claims 22 to 25, wherein the subject has an autoimmune disease or disorder.

31. The method of any one of claims 22 to 25, wherein the subject has been, is undergoing, or will undergo gene therapy.

32. 18. A method for inhibiting activation of the classical complement pathway in a subject, comprising administering to said subject in need of inhibition of activation of the classical complement pathway a therapeutically effective amount of a recombinant enzyme according to any one of claims 1 to 8 or claims 12 to 17, wherein after said administering step, formation of the C1 and / or C3 complex is reduced and / or prevented.

33. 18. A method for reducing and / or minimizing an immune response, comprising administering a therapeutically effective amount of a recombinant enzyme according to any one of claims 1 to 8 or claims 12 to 17 to a subject in need of reducing and / or minimizing an immune response, wherein after said administering step, the level of circulating IgM or the levels of circulating IgM and IgG targeting the recombinant product is reduced when compared to the level before the administering step.

34. 19. A method for reducing and / or minimizing an immune response, comprising administering to a subject in need thereof (i) a therapeutically effective amount of a recombinant enzyme according to any one of claims 1 to 8 or claims 12 to 17, and (ii) a therapeutically effective amount of a recombinant enzyme having a sequence as set forth in any of SEQ ID NOs: 119 to 159, wherein after said administering step, the levels of circulating IgM and IgG targeting the recombinant product are reduced when compared to the levels before the administering step.

35. 35. The method of claim 33 or claim 34, further comprising the step of re-administering the recombinant product to the subject.

36. 35. The method of claim 33 or claim 34, wherein the recombinant product comprises a vector or a portion thereof.

37. 35. The method of claim 33 or claim 34, wherein the recombinant product comprises an encoded transgene product.

38. 35. The method of claim 33 or claim 34, wherein the recombinant product comprises a therapeutic protein or enzyme.

39. 39. The method of any one of claims 33 to 38, further comprising the step of reducing and / or minimizing immunogenicity to said recombinant product.

40. 38. The method of claim 37, further comprising improving and / or enhancing the efficacy and / or expression of the transgene in the subject.

41. 41. The method of claim 40, wherein the efficacy and / or expression of the transgene in the subject is improved and / or enhanced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% when compared to levels prior to the administering step.

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