Compositions comprising antibody cleaving enzymes and methods of use thereof

Abstract

Disclosed herein are compositions for cleaving antibodies and methods of using these compositions.

Description

COMPOSITIONS COMPRISING ANTIBODY CLEAVING ENZYMES AND METHODS OF USE THEREOFI. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional App. No. 63 / 651,163 filed 23 May 2024, which is incorporated herein in its entirety.II. REFERENCE TO THE SEQUENCE LISTING

[0002] The Sequence Listing submitted 23 May 2025 as an XML file named ‘'22-2060-W02- Sequence_Li sting”, created on 23 May 2025 and having a size of 65,536 bytes, is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).III. BACKGROUND

[0003] Monomeric IgM is the first antibody to emerge as the B cell antigen receptor (BCR) on the surface of B cells followed by secreted pentameric IgM from plasma cells. Class switching upon 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 generation of harmful IgG and IgM autoantibodies leading to autoimmune disease (Suurmond J, et al. (2015) J Clin Invest. 125:2194-2202). Similarly, antibodies directed against human leukocyte antigens (HLAs) expressed on the surface of cells in a transplanted organ can lead to rejection (Choi AY, et al. (2021) Front Immunol. 12:694-763). Further, while beneficial in the context of immunity against pathogens such as viruses, antibodies against viral vectors such as recombinant adeno-associated viruses (AAV) can preclude administration of gene therapies in prospective 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 therapies is the activation of the complement cascade and lytic components thereof, which can lead to 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).

[0004] Thus, understanding and manipulating IgG and IgM levels can have profound implications in 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) blocking agents and IgG degrading enzy mes are being explored to modulate humoral immunity in autoimmune disease, organ transplant 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):el39881;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 initiating immune dysfunction, are lacking.

[0005] Thus, there remains an urgent need to generate and characterize antibody-cleaving enzymes and to develop therapeutics based on those antibody-cleaving enzymes.IV. BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1 shows circulating IgG and IgM after repeated administration of IceM (SEQ ID NO:01) and IceMG (SEQ ID NO:06).V. BRIEF SUMMARY

[0007] Disclosed herein is an enzymatic polypeptide fragment of SEQ ID NO:01, comprising 350 or fewer consecutive amino acids of SEQ ID NO: 01, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:02. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 340 or fewer, 330 or fewer, or 320 or fewer consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 350 or fewer consecutive amino acids of SEQ ID NO: 01 and can comprise an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 350 or fewer consecutive amino acids of SEQ ID NO:01 and can comprise SEQ ID NO:02. In an aspect, a disclosed enzymatic polypeptide fragment can comprise at least 300 consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed enzymatic polypeptide fragment can consist of SEQ ID NO:02.

[0008] Disclosed herein is an enzymatic polypeptide fragment of SEQ ID NO: 03, comprising 320 or fewer consecutive amino acids of SEQ ID NO: 03, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:04. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 320 or fewer, 319 or fewer, 318 or fewer, 317 or fewer, or 316 or fewer consecutive amino acids of SEQ ID NO: 03. In an aspect, a disclosed enzymatic polypeptide can comprise 320 or fewer consecutive amino acids of SEQ ID NO:03 and can comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 320 or fewer consecutive amino acids of SEQ ID NO:03 and can comprise the amino acid sequence of SEQ ID NO:04. In an aspect, a disclosed enzy matic polypeptide fragment can comprise at least 300 consecutive amino acids of SEQ ID NO:03. In an aspect, a disclosed enzymatic polypeptide fragment can consist of SEQ ID NO:04.

[0009] Disclosed herein is an enzymatic polypeptide fragment of SEQ ID NO:07, comprising 295 or fewer consecutive amino acids of SEQ ID NO: 07, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:08. In an aspect, a disclosed enzymatic polypeptidefragment can comprise 290 or fewer consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed enzymatic polypeptide can comprise 295 or fewer consecutive amino acids of SEQ ID NO:07 and can comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 08. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 295 or fewer consecutive amino acids of SEQ ID NO:07 and can comprise the amino acid sequence of SEQ ID NO:08. In an aspect, a disclosed enzymatic polypeptide fragment can comprise at least 280 consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed enzymatic polypeptide fragment can consist of SEQ ID NO: 08.

[0010] Disclosed herein is a fusion enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant fusion enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant fusion protein comprising a proteolytic domain from a disclosed IgG specific protease linked to a proteolytic domain from a disclosed IgM specific protease. Disclosed herein is a fusion protein having IgM specific protease activity and IgG specific protease activity. In an aspect, a disclosed fusion protein can comprise the sequence of SEQ ID NO:05 or SEQ ID NO:06. In an aspect, a disclosed fusion protein can comprise a sequence having at least 90% identity to the sequence of SEQ ID NO:05 or SEQ ID NO:06. In an aspect, a disclosed fusion protein can comprise a sequence having at least 90% identity to the sequence of SEQ ID NO:05 or SEQ ID NO:06 and having one or more N terminus-truncations and one or more C terminus truncations.

[0011] Disclosed herein is a method of reducing circulating and / or surface-bound IgM in a subject, the method comprising administering to a subject a disclosed enzymatic polypeptide fragment or a disclosed fusion protein described herein. Disclosed herein is a method of reducing circulating and / or surface-bound IgM in a subject, the method comprising administering to a subject a pharmaceutical formulation comprising a disclosed enzymatic polypeptide fragment or a disclosed fusion protein described herein.VI. DETAILED DESCRIPTION

[0012] The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and / or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects 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, example methods and materials are now described.

[0013] All publications mentioned herein are incorporated herein 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 is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.A. Definitions

[0014] 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 may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects 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, example methods and materials are now described.

[0015] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0016] As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0017] The phrase “consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as 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 that is 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.

[0018] In an aspect, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value.

[0019] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of 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 are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0020] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0021] In an aspect, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0022] In an aspect, “isolated” refers to a nucleic acid or polypeptide that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA. and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

[0023] In an aspect, the term “subject” refers to the target of administration, e.g. a human being. The term “subject” also includes domesticated animals (e.g.. cats, dogs. etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g, mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods 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, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a disease or disorder, be suspected of having a disease or disorder, or be at risk of developing a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a subject can have an AD or AIF disease and / or disorder. In an aspect, a subject can be in need of one or more transplanted organs. In an aspect, a subject can have received one or more transplanted organs. In an aspect, a subject can have received one or moretransplanted organs. In an aspect, a subject can be in need of gene therapy, can be receiving gene therapy, or can have received gene therapy.

[0024] In an aspect, the term “diagnosed” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the disclosed enzymes, the disclosed recombinant enzymes, the disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof, or by one or more of the disclosed methods. For example, “diagnosed with a disease or disorder” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can be treated by one or more of the disclosed enzymes, the disclosed recombinant enzymes, the disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion enzymes the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, 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 an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can likely be treated by one or more of the disclosed enzymes, the disclosed recombinant enzymes, the disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof, or by one or more of the disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzymatic assay), or a combination thereof.

[0025] In an aspect, a “patient” refers to a subject afflicted with a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder. In an aspect, a patient can have an AD or AIF disease and / or disorder. In an aspect, a patient can be in need of one or more transplanted organs. In an aspect, a patient can have received one or more transplanted organs. In an aspect, a patient can have received one or more transplanted organs. In an aspect, a patient can be in need of gene therapy, can be receiving gene therapy, or can have received gene therapy.

[0026] In an aspect, the phrase “identified to be in need of treatment for a disease or disorder,” or the like, refers to selection of a subject based upon need for treatment of the disease or disorder. For example, a subject can be identified as having a need for treatment of a disease or disorder(e.g., a genetic disease or disorder, an AD, an AIF disease, transplanted organs) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the genetic disease or disorder. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0027] In an aspect, 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 that function as receptors for the antigen or as secreted antibodies that reside in the circulation, tissues, and mucosal sites to neutralize toxins, prevent the entry and spread of pathogens, and eliminate microbes. The activation of B lymphocytes is initiated by the specific recognition of antigens by the surface Ig receptors of these cells. Activation leads to proliferation of antigen-specific cells and their differentiation in cooperation with Th cells, generating memory’ B lymphocytes and antibody-secreting plasma cells. The ty pe and amount of antibodies produced vary according to the antigen dnving the immune response, the involvement of T lymphocytes, a prior history of antigen exposure, and the site where activation occurs. Antibody responses to protein antigens require that the specifically recognized antigen is internalized by B lymphocytes and that a peptide fragment of it is presented to CD4+ helper T lymphocytes that subsequently activate these B lymphocytes. Because of the involvement of helper T lymphocytes, this type of response is called "T-dependent antibody response’ (TDAR). In contrast, antibody responses to multivalent nonproteinic 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 TDARs, plasma cells or their precursors migrate from germinal centers in the penpheral lymphoid organs, where they are produced, to the bone marrow, where they live for many years and ensure specific protection against microbes for long periods of time.

[0028] In an aspect, "immune response’" refers to the body’s response to foreign agents and infections organisms and includes (i) the body’s innate or non-specific immune system and (ii) the body’s adaptive or specific immune system (including B cells, cytotoxic T cells, and helper T cells).

[0029] In an aspect, “primary’ and secondary antibody responses’" to protein antigens differ qualitatively and quantitatively. Primary responses result from the activation of naive B lymphocytes that have not been previously stimulated by the antigen, whereas a secondary’ immune response is elicited yvhen the same antigen stimulates antigen-specific memory B lymphocytes. In secondary responses as compared to primary responses, antibody production is greater and peak antibody levels attained more rapidly (approximately 2-3 vs. 7 days), the mainisotype is IgG instead of IgM, and antibody affinity is much greater. Once released, the various forms of antibodies (Ig) possess a number of effector functions to engage the antigen. Microbes, tumor cells, or foreign proteins can express several types of antigens and multiple copies of these antigens. If the microbial antigen to which an antibody is directed is associated with the toxic portion of a molecule, then the antibody can neutralize the toxin. The production of neutralizing antibodies is frequently a problem in the therapeutic application of biotechnology-derived recombinant proteins as neutralizing antibodies can become a rate-limiting step during preclinical testing because animals may perceive the recombinant protein, especially when humanized, as non-self.

[0030] Most of the effector functions of humoral immunity are mediated by antibody-activated processes, but they are also key 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 the Ig molecule. Both IgM and IgG can activate the complement system, which results in the biologically active components identified previously, including the lytic unit, the chemotactic factors, and the complement peptides, which opsonize the microbe to facilitate phagocytosis. IgM and IgG can function to opsonize some microbes independent of complement activation in an antigen-dependent fashion because macrophages and neutrophils have receptors on their surface, which recognize the constant region of Ig (Fc receptors, FcRs). FcRs also play a major role in the ability of IgG to participate in a process known as ‘antibody-dependent cellular cytotoxicity,’ whereby antigen-specific antibody attaches via FcRs to certain types of cells, including NK cells, enabling these cells to attach intimately to the target cell and trigger cell death. Finally, FcRs are also the primary’ effector mechanism for IgE, which is the principal immune defense against certain types of parasitic infections (most notably, helminths) and is produced primarily by the external immune system along secretory surfaces. IgE binds to FcR on the surface of mast cells and basophils. Once bound to FcR, IgE can serve as an antigen-specific receptor to trigger the release of proinflammatory factors, including vasoactive amines (e.g., histamine) and products of the arachidonic cascade (e.g., leucotrienes and prostaglandins). Systemic immunity is mediated by IgM and IgG, the latter being the major form of Ig found in the blood. Local immunity is mediated primarily by IgA and IgE.

[0031] In an aspect, a “regulatory element” can refer to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as 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 the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0032] In an aspect, “inhibit,’’ “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter (such as the level of circulating IgG and / or circulating IgM or the level of surface-bound IgM on B cells). This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter (such as the level of circulating IgG and / or IgM or the level of surface-bound IgM on B cells). This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameters as compared to the native or control level (e.g., a subject not having a disease or disorder such as a genetic disease or disorder). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction can be 0-25%, 25-50%, 50- 75%. or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level.

[0033] In an aspect, the words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, z.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity' of an established a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, 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 (such as a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1 %- 100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can referto 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder.

[0034] In an aspect, the term “prevent’’ or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and / or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a genetic disease or disorder) or related complication from progressing to that complication.

[0035] In an aspect, the terms “administering” and “administration” refer to any method of providing one or more disclosed enzy matic polypeptide fragments or fusion enzy mes or fusion products, 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 to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed enzymatic polypeptide fragment or fusion enzyme or fusion product, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, and / or a disclosed RNA therapeutic can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more route.

[0036] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more disclosed enzymatic polypeptidefragments or fusion enzymes or fusion products, 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 to treat or prevent a disease or disorder (such as genetic disease or disorder). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more disclosed enzymatic polypeptide fragments or fusion enzymes or fusion products, 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.

[0037] In an aspect, “determining the amount7' is meant both an absolute quantification of a particular analyte (e.g., level of IgG and / or IgM) or a determination of the relative abundance of a particular analyte (e.g., level of IgG and / or IgM compared to a control / reference level). The phrase includes both direct or indirect measurements of abundance or both.

[0038] In an aspect, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions: while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterialand antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, 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.

[0039] In an aspect, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is 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.. alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0040] In an aspect, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0041] The term “contacting” In an aspect refers to bringing one or more disclosed enzymatic polypeptide fragments or fusion enzymes or fusion products, 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 together with a target area or intended target area (e.g. a cleavage site on IgG and / or IgM in such a manner that the one or more disclosed enzymatic polypeptide fragments or fusion enzy mes or fusion products, 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 exert an effect on the intended target or targeted area either directly or indirectly. A target area can comprise one or more cells, and in an aspect, one or more cells can be in a subject. A target area or intended target area can be one or more of a subj ect’s organs (e.g., lungs, heart, liver, kidney, brain, etc ). In an aspect, a target area or intendedtarget area can be any organ, tissue, or cells that are affected by a disease or disorder (such as a genetic disease or disorder).

[0042] In an aspect, “determining’' can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as. for example, a genetic disease or disorder. Methods and techniques used to determine the presence and / or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and / or diagnose the presence, severity, or both of a disease or disorder (such as, for example, a genetic disease or disorder).

[0043] In an aspect, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and / or prevention of a disease or disorder (e.g., a genetic disease or disorder) or a suspected disease or disorder. In an aspect, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an 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 the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a genetic disease or disorder), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a genetic disease or disorder), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g.. a genetic disease or disorder). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed enzymatic polypeptide fragment, the disclosed fusion protein / fusion enzyme, the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations employed; the disclosed methods employed; the age. body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed, and other like factors well known in the medical arts. For example, it is within the skill of the art to start doses of the disclosed enzymatic polypeptide fragment, the disclosed fusion protein / fusion enzy me, the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations at levels lower thanthose required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and / or mutant protein or enzyme.

[0044] In an aspect, “RNA therapeutics” can refer to the use of oligonucleotides to target RNA. RNA therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA. morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2’-O-Me-RNA, 2’-MEO-RNA, 2’-F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non-coding RNA (IncRNA), such as, for example, along intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA. competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzy mes (ribozy mes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as known to the art, RNAi operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick basepairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0045] In an aspect, “peptide,"’ “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids and there is no limitation on the maximum number of amino acids that can comprise a protein’s sequence. The term “peptide” can refer to a short chain of amino acids including, for example, natural peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0046] In an aspect, “nucleic acid” or “oligonucleotide” or “polynucleotide” means at least two nucleotides covalently linked together. The depiction of a single strand can also define the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid can encompass substantially identical nucleic acids and complements thereof. A single strand can provide a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid can encompass a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded, or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxy ribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods. Also In an aspect, the terms “nucleic acid,"’ “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence”, and “polynucleotide” can refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term can encompass RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2’-hydroxy in the ribose sugar group of the RNA can also be made. In an aspect, a “synthetic” nucleic acid or polynucleotide 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.

[0047] In an aspect, a “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA, or DNA- RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides).

[0048] In an aspect, a “fragment” or “portion” of a nucleotide sequence can be understood to mean a nucleotide sequence of reduced length relative (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) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleotide sequence of contiguous nucleotides identical or almost 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 a nucleic acid fragment or portion according to the disclosure can be, where appropriate, included in a larger polynucleotide of which it is a constituent. In an aspect, a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations.

[0049] In an aspect, a “fragment” or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative (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) to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost 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 amino acid sequence. Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent.

[0050] In an aspect, “complement” or “complementary” means a nucleic acid can mean Watson- Crick ( e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary'.

[0051] In an aspect, “operably linked” means that expression of a gene or a transgene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance betw een that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0052] In an aspect, ‘'promoter” or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native (endogenous) or foreign (exogenous) and can be a natural or a synthetic sequence. By foreign or exogenous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0053] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may 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 against databases such as FASTA, BLAST, etc., but hits should be retrieved 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 percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and / or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and / or 5’-side are 100% identical.

[0054] A “heterologous” or a “recombinant” nucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide or amino acid sequence.

[0055] Different nucleic acids or proteins having homology' can be referred to as “homologues”. The term homologue 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’ betw een two or more nucleic acid and / or amino acid sequences in terms of percent of positional identity’ (i.e., sequence similarity’ or identity’). Homology’ also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the disclosed compositions and disclosed methods can comprise homologues to the disclosed nucleotide sequences and / or disclosed polypeptide sequences.

[0056] In an aspect, “orthologous” can refer to homologous nucleotide sequences and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a disclosed nucleotide sequence or a disclosed polypeptide can have substantialsequence 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 a disclosed nucleotide sequence or a disclosed polypeptide.

[0057] In an aspect, ‘"codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes.urv.es / OPTIMIZER / ). For example, a codon is a sequence of three nucleotides that encodes for a specific amino acid in the synthesis of a protein. There are 64 distinct codons, but only 20 amino acids, leading to the degeneracy of the genetic code. For instance, the amino acid Leucine can be encoded with six synonymous codons CUU, CUC, CUA, CUG, UUA and UUG whereas Cysteine can be encoded with two codons UGU and UGC. Overall, 2 of the 20 amino acids can be encoded with 1 codon, 9 with 2 codons, 1 with 3 codons. 5 with 4 codons and 3 with 6 codons leading to 61 essential codons. The remaining three codons are stop codons and are reserved for termination of protein formation. Codon degeneracy leads to many possible ways of encoding a protein, e.g. a typical 375-amino acid protein in humans can be potentially encoded by 10207 different codon sequences. All possible encodings and resulting sequences are not equally likely to be observed in nature; however, as some synonymous codons are more frequently used than others in encoding a particular amino acid in a particular organism. This phenomenon is called ‘codon usage bias’ or ‘codon bias’. As an example, Leucine is encoded 39.5% of the time with codon CUG in Homo sapiens, whereas the same codon is used 11.1% of the same amino acid’s encoding in Saccharomyces cerevisiae (Nakamura et al., 2000).

[0058] In an aspect, “immune tolerance,” “immunological tolerance,” and “immunotolerance” refers to a state of unresponsiveness or blunted response of the immune system to substances (e.g., protein replacement therapy, enzy me replacement therapy, recombinant product, a transplanted organ, a vector protein, etc.) that have the capacity' to elicit 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 against a particular antigen (such as, for example, to an HLA on a transplanted organ, an enzyme or therapeutic protein as part of replacement therapy, a viral vector, an AAV capsid protein). Low7or absent antibody titers over time is an indicator of immune tolerance or the efficacy of a disclosed enzymatic polypeptide fragment, adisclosed fusion protein / fusion enzyme. For example, immune tolerance can be established by having IgG antibody titers 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 following gene therapy (such as the administration of the transgene encoding, for example, a missing, deficient, and / or mutant protein or enzyme), protein replacement therapy, enzyme replacement therapy, or a recombinant product.

[0059] As known to the art, antibodies (Abs) can mitigate AAV infection through multiple mechanisms by binding to AAV capsids and blocking critical steps in transduction such as cell surface attachment and uptake, endosomal escape, productive trafficking to the nucleus, or uncoating as well as promoting AAV opsonization by phagocytic cells, thereby mediating their rapid clearance from the circulation. For example, in humans, serological studies reveal a high prevalence of NAbs in the worldwide population, with about 67% of people having antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV serotypes 5 through 9. Vector immunogenicity represents a major challenge in re-administration of AAV vectors.

[0060] In an aspect, "‘immune-modulating” refers to the ability of a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion 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 the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0061] In an aspect, “immune modulator” refers to an agent that is capable of adjusting a given immune response to a desired level (e.g., as in immunopotentiation, immunosuppression, or induction of immunologic tolerance). Examples of immune modulators include but are not limited to, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine. cyclophosphamide, cyclosporine, deoxyspergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen, IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, my cophenolate mofetil, 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 antibodies, anti-CD4 antibodies, anti-CD19 antibodies, anti- CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGFIR antibodies, an IL2 mutein. a BTK inhibitor, or a combination thereof. Inan aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, an immune modulator can be administered by any suitable route of administration including, but not limited to, in utero, intra-CSF, intrathecally. intravenously, subcutaneously, transdermally, intradermally, intramuscularly, orally, transcutaneously, intraperitoneally (IP), or intravaginally. In an aspect, a disclosed immune modulator can be administered using a combination of routes. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of an immune modulator can be continuous or intermittent, and administration can comprise a combination of one or more routes.

[0062] In an aspect, the term “immunotolerant” refers to unresponsiveness to an antigen (e.g., a vector, a therapeutic protein, a transgene product, etc.). An immunotolerant promoter can reduce, ameliorate, or prevent transgene-induced immune responses that can be associated with gene therapy. Assays known in the art to measure immune responses, such as immunohistochemical detection of cytotoxic T cell responses, can be used to determine whether one or more promoters can confer immunotolerant properties.

[0063] In an aspect, the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0064] In an aspect, the term “in combination” in the context of the administration of other therapies (e.g.. other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., one or more disclosed enzymatic polypeptide fragments or fusion enzymes or fusion products, 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) may be administered prior to (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, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (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) theadministration of a second therapy (e.g., gene therapy, enzyme replacement therapy, protein replacement therapy, recombinant product) to a subject having or diagnosed with a disease or disorder (such as a genetic disease or disorder).

[0065] Disclosed are the components to be used to prepare the disclosed enzymatic polypeptide fragments, disclosed fusion proteins / fusion enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed enzymatic polypeptide fragments, the disclosed fusion proteins / fusion enzymes, the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E. and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered 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, if there are a variety of 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. Compositions1. Enzymes

[0066] Disclosed herein is an enzy me having IgM specific protease activity. Disclosed herein is a recombinant enzyme having IgM specific protease activity. Disclosed herein is an enzy matic polypeptide fragment having IgM specific protease activity.

[0067] Disclosed herein is an enzymatic polypeptide fragment of SEQ ID NO:0L comprising 350 or fewer consecutive amino acids of SEQ ID NO: 01, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:02. In an aspect, a disclosed enzy matic polypeptide fragment can comprise 340 or fewer, 330 or fewer, or 320 or fewer consecutive amino acids ofSEQ ID NO:01. In an aspect a disclosed enzymatic polypeptide fragment can comprise 350 or fewer consecutive amino acids of SEQ ID NO: 01 and can comprise an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 350 or fewer consecutive amino acids of SEQ ID NO:01 and can comprise SEQ ID NO:02. In an aspect, a disclosed enzymatic polypeptide fragment can comprise at least 300 consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed enzy matic polypeptide fragment can consist of SEQ ID NO:02.

[0068] Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 350 or fewer consecutive amino acids of SEQ ID NO:01. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 340 or fewer consecutive amino acids of SEQ ID NO:01. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 330 or fewer consecutive amino acids of SEQ ID NO:01. Disclosed herein is an enzy matic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO:01. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 315 or fewer consecutive amino acids of SEQ ID NO:01.

[0069] Disclosed herein is an enzy matic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%. 96%. 97%, 98%, or 99% identical to SEQ ID NO: 02. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 340 or feyver consecutive amino acids of SEQ ID NO:01 and comprises an amino acid sequence that it at least 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO: 01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 330 or fewer consecutive amino acids of SEQ ID NO: 01 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. Disclosedherein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO:01 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 315 or fewer consecutive amino acids of SEQ ID NO:01 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02.

[0070] Disclosed herein is an enzymatic polypeptide fragment of SEQ ID NO: 03, comprising 320 or fewer consecutive amino acids of SEQ ID NO: 03, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:04. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 320 or fewer, 319 or fewer, 318 or fewer, 317 or fewer, or 316 or fewer consecutive amino acids of SEQ ID NO:03. In an aspect, a disclosed enzymatic polypeptide can comprise 320 or fewer consecutive amino acids of SEQ ID NO:03 and can comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 320 or fewer consecutive amino acids of SEQ ID NO:03 and can comprise the amino acid sequence of SEQ ID NO:04. In an aspect, a disclosed enzymatic polypeptide fragment can comprise at least 300 consecutive amino acids of SEQ ID NO:03. In an aspect, a disclosed enzy matic polypeptide fragment can consist of SEQ ID NO: 04.

[0071] Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO:03. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 319 or fewer consecutive amino acids of SEQ ID NO:03. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 318 or fewer consecutive amino acids of SEQ ID NO:03. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 317 or fewer consecutive amino acids of SEQ ID NO:03. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but forone or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 316 or fewer consecutive amino acids of SEQ ID NO:03.

[0072] Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO:03 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04.

[0073] Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 319 or fewer consecutive amino acids of SEQ ID NO:03 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 04. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 318 or fewer consecutive amino acids of SEQ ID NO:03 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO:04. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 317 or fewer consecutive amino acids of SEQ ID NO: 03 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO: 03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 316 or fewer consecutive amino acids of SEQ ID NO:03 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04.

[0074] In an aspect, a disclosed enzy matic polypeptide fragment can have IgM specific protease activity. In an aspect, a disclosed enzymatic polypeptide fragment can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, a disclosed enzymatic polypeptide fragment can cleave human IgM. In an aspect, a disclosed enzymatic polypeptide fragment can cleave non-human primate IgM. In an aspect, a disclosed enzy matic polypeptide fragment does not cleave non-human IgM. For example, in an aspect, a disclosed enzymatic polypeptide fragment does not cleave dog IgM, pig IgM, and mouse IgM. In an aspect, a disclosed enzymatic polypeptide fragment does not cleave human IgA, IgD, IgE, IgG, or any combination thereof. In an aspect, a disclosed enzymatic polypeptide fragment can have a bindingdomain specific for IgM. In an aspect, a disclosed enzymatic polypeptide fragment can have an affinity for human IgM.

[0075] In an aspect, a disclosed enzymatic polypeptide fragment having IgM specific protease activity can be isolated. In an aspect, a disclosed enzymatic polypeptide fragment having IgM specific protease activity can be isolated and purified. In an aspect, an isolated enzymatic polypeptide fragment having IgM specific protease activity' can be at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.

[0076] In an aspect, a disclosed enzymatic polypeptide fragment having IgM specific protease activity can be fused to or linked with a disclosed enzymatic polypeptide fragment having IgG specific protease activity.

[0077] Disclosed herein is an enzyme having IgG specific protease activity. Disclosed herein is a recombinant enzy me having IgG specific protease activity. Disclosed herein is an enzy matic polypeptide fragment having IgM specific protease activity.

[0078] Disclosed herein is an enzymatic polypeptide fragment of SEQ ID NO:07. comprising 295 or fewer consecutive amino acids of SEQ ID NO: 07, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:08. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 290 or fewer consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed enzymatic polypeptide can comprise 295 or fewer consecutive amino acids of SEQ ID NO:07 and can comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 08. In an aspect, a disclosed enzymatic polypeptide fragment can comprise 295 or fewer consecutive amino acids of SEQ ID NO:07 and can comprise the amino acid sequence of SEQ ID NO:08. In an aspect, a disclosed enzymatic polypeptide fragment can comprise at least 280 consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed enzymatic polypeptide fragment can consist of SEQ ID NO: 08.

[0079] Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzymatic polypeptide fragment comprises 295 or fewer consecutive amino acids of SEQ ID NO:07. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzymatic polypeptide fragment comprises 294 or fewer consecutive amino acids of SEQ ID NO:07. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzymatic polypeptide fragment comprises 293 or fewer consecutive amino acids of SEQ ID NO:07. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resultingenzymatic polypeptide fragment comprises 292 or fewer consecutive amino acids of SEQ ID NO:07. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzymatic polypeptide fragment comprises 291 or fewer consecutive amino acids of SEQ ID NO: 07.

[0080] Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzymatic polypeptide fragment comprises 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprises an amino acid sequence that it at least 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzy matic polypeptide fragment comprises 294 or fewer consecutive amino acids of SEQ ID NO: 07 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzy matic polypeptide fragment comprises 293 or fewer consecutive amino acids of SEQ ID NO: 07 and comprises an amino acid sequence that it at least 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO: 07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enzymatic polypeptide fragment comprises 292 or fewer consecutive amino acids of SEQ ID NO:07 and comprises an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99% identical to SEQ ID NO:08. Disclosed herein is an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the resulting enz matic polypeptide fragment comprises 291 or fewer consecutive amino acids of SEQ ID NO: 07 and comprises an amino acid sequence that it at least 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08.

[0081] In an aspect, a disclosed enzymatic polypeptide fragment having IgG specific protease activity7can be isolated. In an aspect, a disclosed enzymatic polypeptide fragment having IgG specific protease activity can be isolated and purified.

[0082] In an aspect, an isolated enzymatic polypeptide fragment having IgG specific protease activity can be at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure. In an aspect, a disclosed enzymatic polypeptide fragment having IgG specific protease activity' can be fused to or linked with a disclosed enzy matic polypeptide fragment having IgM specific protease activity.

[0083] Disclosed herein is a fusion enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant fusion enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant fusion protein comprising a proteolytic domain from a disclosed IgG specific protease linked to a proteolytic domain from a disclosed IgM specific protease. Disclosed herein is a fusion protein having IgM specific protease activity and IgG specific protease activity. In an aspect, a disclosed fusion protein can comprise the sequence of SEQ ID NO:05 or SEQ ID NO:06. In an aspect, a disclosed fusion protein can comprise a sequence having at least 90% identity to the sequence of SEQ ID NO:05 or SEQ ID NO:06. In an aspect, a disclosed fusion protein can comprise a sequence having at least 90% identity to the sequence of SEQ ID NO:05 or SEQ ID NO:06 and having one or more N terminus-truncations and one or more C terminus truncations.

[0084] In an aspect, a disclosed fusion protein can comprise a first domain and a second domain, wherein the first domain comprises a disclosed enzymatic polypeptide fragment. In an aspect, a disclosed fusion protein can comprise a first domain and a second domain, wherein the second domain comprises a disclosed enzymatic polypeptide fragment. In an aspect, a disclosed first domain and a disclosed second domain can be linked by a linker.

[0085] In an aspect, a first disclosed domain can comprise IgM protease activity or IgG protease activity. In an aspect, a second disclosed domain can comprise IgM protease activity or IgG protease activity. In an aspect, a first disclosed domain can comprise IgM protease activity’ and a second disclosed domain can comprise IgG protease activity. In an aspect, a first disclosed domain can comprise IgG protease activity and a second disclosed domain can comprise IgM protease activity. In an aspect, a first disclosed domain and a second disclosed domain can comprise differing specific protease activities.

[0086] In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment of SEQ ID NO:01, comprising 350 or fewer consecutive amino acids of SEQ ID NO:01, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 02. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising 340 or fewer, 330 or fewer, or 320 or fewer consecutive amino acids of SEQ ID NO:01 . In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO: 01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. or 99% identical to SEQ ID NO:02. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprising SEQ ID NO:02. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising atleast 300 consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment consisting of SEQ ID NO: 02.

[0087] In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 350 or fewer consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 340 or fewer consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 330 or fewer consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO:01. In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide comprising the sequence of SEQ ID NO: 01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 315 or fewer consecutive amino acids of SEQ ID NO:01.

[0088] In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide comprising the sequence of SEQ ID NO: 01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 340 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 02. In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 330 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide comprising the sequence of SEQ ID NO:01 but for one or more truncations at the N terminusand / or the C terminus such that the enzymatic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO: 01 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 315 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02.

[0089] In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment of SEQ ID NO:03, comprising 320 or fewer consecutive amino acids of SEQ ID NO:03, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 04. In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide fragment comprising 320 or fewer, 319 or fewer, 318 or fewer, 317 or fewer, or 316 or fewer consecutive amino acids of SEQ ID NO: 03. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising 320 or fewer consecutive amino acids of SEQ ID NO:03 and comprising an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising 320 or fewer consecutive amino acids of SEQ ID NO:03 and comprising the amino acid sequence of SEQ ID NO: 04. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising at least 300 consecutive amino acids of SEQ ID NO:03. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment consisting of SEQ ID NO: 04.

[0090] In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO:03. In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide comprising the sequence of SEQ ID NO: 03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 319 or fewer consecutive amino acids of SEQ ID NO:03. In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 318 or fewer consecutive amino acids of SEQ ID NO: 03. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 317 or fewer consecutive amino acids of SEQ IDNO:03. In an aspect a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 316 or fewer consecutive amino acids of SEQ ID NO:03.

[0091] In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 320 or fewer consecutive amino acids of SEQ ID NO: 03 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 319 or fewer consecutive amino acids of SEQ ID NO:03 and comprising an amino acid sequence that it at least 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 318 or fewer consecutive amino acids of SEQ ID NO:03 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 317 or fewer consecutive amino acids of SEQ ID NO: 03 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. or 99% identical to SEQ ID NO:04. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:03 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 316 or fewer consecutive amino acids of SEQ ID NO:03 and comprising an amino acid sequence that it at least 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04.

[0092] In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide fragment of SEQ ID NO:07, comprising 295 or fewer consecutive amino acids of SEQ ID NO:07, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 08. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising 290 or fewer consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprising an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 08. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising 295 or fewer consecutive amino acids of SEQ ID NO: 07 and comprising the amino acid sequence of SEQ ID NO: 08. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment comprising at least 280 consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide fragment consisting of SEQ ID NO: 08.

[0093] In an aspect, a disclosed fusion protein can comprise an enzy matic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 295 or fewer consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 294 or fewer consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 293 or fewer consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 292 or fewer consecutive amino acids of SEQ ID NO:07. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 291 or fewer consecutive amino acids of SEQ ID NO:07.

[0094] In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 295 or fewer consecutive amino acids of SEQ ID NO: 07 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzy matic polypeptide fragment comprises 294 or fewer consecutive amino acids of SEQ ID NO:07 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises293 or fewer consecutive amino acids of SEQ ID NO:07 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 292 or fewer consecutive amino acids of SEQ ID NO: 07 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08. In an aspect, a disclosed fusion protein can comprise an enzymatic polypeptide comprising the sequence of SEQ ID NO:07 but for one or more truncations at the N terminus and / or the C terminus such that the enzymatic polypeptide fragment comprises 291 or fewer consecutive amino acids of SEQ ID NO:07 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08.

[0095] In an aspect of a disclosed fusion protein, one or more components or one or more functional domains can be linked together, for example, using a "‘peptide linker’ or a “linker”. In an aspect, the terms “peptide linker” and “linker” are used interchangeably to refer to a short stretch of amino acids used to connect two functional domains together in a polypeptide chain.

[0096] In an aspect, the terms “peptide linker” and “linker” are used interchangeably to refer to a short stretch of amino acids used to connect two disclosed functional domains or two disclosed enzymatic polypeptides fragments together in a disclosed fusion protein.

[0097] Suitable peptide linkers are known in the art, and can include, for example, peptide linkers containing flexible amino acid residues (e.g., glycine, alanine, and serine). In an aspect, a disclosed linker can contain motifs, e.g., multiple or repeating motifs, of GA, GS. GG, GGA, GGS. GGG. GGGA (SEQ ID NO: 10), GGGS (SEQ ID NO: 11), GGGG (SEQ ID NO: 12), GGGGA (SEQ ID NO: 13), GGGGS (SEQ ID NO: 14), GGGGG (SEQ ID NO: 15), GGAG (SEQ ID NO: 16), GGSG (SEQ ID NO: 17), AGGG (SEQ ID NO: 18), or SGGG (SEQ ID NO: 19).

[0098] In an aspect, a disclosed linker can contain 2 to 12 amino acids including motifs of GA or GS, e.g., GA. GS, GAGA (SEQ ID NO:20), GSGS (SEQ ID NO:21), GAGAGA (SEQ ID NO:22), GSGSGS (SEQ ID NO:23), GAGAGAGA (SEQ ID NO:24), GSGSGSGS (SEQ ID NO 25), GAGAGAGAGA (SEQ ID NO:26), GSGSGSGSGS (SEQ ID NO:27), GAGAGAGAGAGA (SEQ ID NO:28), and GSGSGSGSGSGS (SEQ ID NO:29).

[0099] In an aspect, a disclosed linker can contain 3 to 12 amino acids including motifs of GGA or GGS including, for example, GGA, GGS, GGAGGA (SEQ ID NO:30), GGSGGS (SEQ ID NO:31), GGAGGAGGA (SEQ ID NO:32), GGSGGSGGS (SEQ ID NO:33), GGAGGAGGAGGA (SEQ ID NO:34), and GGSGGSGGSGGS (SEQ ID NO:35).

[0100] In an aspect, a disclosed linker can contain 4 to 12 amino acids including motifs of GGAG (SEQ ID NO:36), GGSG (SEQ ID NO:37), GGAGGGAG (SEQ ID NO:38), GGSGGGSG (SEQ ID NO:39), GGAGGGAGGGAG (SEQ ID NO:40), and GGSGGGSGGGSG (SEQ ID NO:41).

[0101] In an aspect, a disclosed linker can contain motifs of GGGGA (SEQ ID NO:42) or GGGGS (SEQ ID NO:43) including, for example. GGGGAGGGGAGGGGA (SEQ ID NO:44) and GGGGSGGGGSGGGGS (SEQ ID NO: 45).

[0102] In an aspect, a disclosed amino acid linker between an ActRIIB-ECD variant and a heterologous domain (e.g., an Fc domain monomer (e g., a wild-ty pe Fc domain monomer, an Fc domain monomer with one or more amino acid substitutions), an albumin-binding peptide, a fibronectin domain, or a human serum albumin domain) can be GGG, GGGA (SEQ ID NO: 10), GGGG (SEQ ID NO: 12), GGGAG (SEQ ID NO:47), GGGAGG (SEQ ID NO:48), or GGGAGGG (SEQ ID NO 49).

[0103] In an aspect, a disclosed linker can also contain amino acids other than glycine, alanine, and senne. e g., AAAL (SEQ ID NO:50), AAAK (SEQ ID NO:51), AAAR (SEQ ID NO:52), EGKSSGSGSESKST (SEQ ID NO:53), GSAGSAAGSGEF (SEQ ID NO 54), AEAAAKEAAAKA (SEQ ID NO:55), KESGSVSSEQLAQFRSLD (SEQ ID NO:56), GENLYFQSGG (SEQ ID NO:57), SACYCELS (SEQ ID NO:58), RSIAT (SEQ ID NO 59), RPACKIPNDLKQKVMNH (SEQ ID NO: 60),GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO 61), AAANSSIDLISVPVDSR (SEQ ID NO:62), orGGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:63).

[0104] In an aspect, a disclosed linker can contain motifs, e.g., multiple or repeating motifs, of EAAAK (SEQ ID NO:64) or GGGGS (SEQ ID NO:66). In an aspect, a disclosed linker can contain motifs including, for example, multiple or repeating motifs of praline-rich sequences such as (XP)n, in which X may be any amino acid (e.g., A, K, or E) and n is from 1-5, and PAPAP (SEQ ID NO:65).

[0105] In an aspect, a disclosed linker can be KDIWQKDKAERIKDTL (SEQ ID NO:09).

[0106] For example, in an aspect, a disclosed fusion protein can comprise a disclosed enzymatic polypeptide fragment having the sequence of SEQ ID NO: 02 linked to a disclosed enzymatic polypeptide fragment having the sequence of SEQ ID NO:08. For example, in an aspect, a disclosed fusion protein can comprise a disclosed enzymatic polypeptide fragment having the sequence of SEQ ID NO:04 linked to a disclosed enzymatic polypeptide fragment having the sequence of SEQ ID NO: 08. For example, in an aspect, a disclosed fusion protein can comprise a disclosed enzy matic polypeptide fragment having the sequence of SEQ ID NO:08 linked to a disclosed enzymatic poly peptide fragment having the sequence of SEQ ID NO:02. For example,in an aspect, a disclosed fusion protein can comprise a disclosed enzymatic polypeptide fragment having the sequence of SEQ ID NO:08 linked to a disclosed enzymatic polypeptide fragment having the sequence of SEQ ID NO: 04. In an aspect, any one of these disclosed fusions can comprise the linker of SEQ ID NO: 09.

[0107] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can be isolated. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can be isolated and purified. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can be at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%. at least 98%, or at least 100% pure.

[0108] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can have IgG and IgM specific protease activity. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3 and can cleave the IgG below the hinge region. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can cleave the IgG below the hinge region, thereby yielding F(ab’)2 and Fc fragments. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can cleave the IgG separating the Cp2 and Cp3 domains from the Cpl domain. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can cleave human IgG and human IgM. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can cleave non-human primate IgG and non-human primate IgM. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein having IgG and IgM protease activity does not cleave IgA, IgD, IgE, or any combination thereof. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein having IgG and IgM protease activity does not cleave non-human IgM or non-human IgG. For example, in an aspect, a disclosed fusion enzyme or a disclosed fusion protein having IgG and IgM protease activity does not cleave dog IgM, pig IgM, and mouse IgM, or dog IgG, pig IgG. or mouse IgG.

[0109] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can clear and / or remove IgM and / or IgM and IgG from circulation in a subject. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can clear and / or remove IgM and / or IgM and IgG from circulation in the sera of a subject. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can cleave and / or degrade circulating IgM and / or IgM and IgG in a subject. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can transiently clear and / or remove IgM and / or IgM and IgG from circulation in the sera of a subject, or can transiently cleave and / or degrade circulating IgM and / or IgM and IgG in a subject. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can reduce and / or diminish the level of circulating IgM and / or IgG and IgM levels in a subject, or can transiently reduce and / or diminish the level of circulating IgM and / or IgG and IgM levels in a subject. In aspect, a disclosed transient reduction and / or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about2 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.

[0110] In an aspect, a disclosed fusion enzy me or a disclosed fusion protein can block and / or minimize activation of the complement cascade. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can block and / or minimize activation of the formation of the Cl complex and / or C3 complex. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can block and / or minimize activation of the classical complement pathway. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can transiently block and / or minimize activation of the complement cascade, can transiently block and / or minimize activation of the formation of the Cl complex and / or C3 complex, can transiently block and / or minimize activation of the classical complement pathway, or any combination thereof.

[0111] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein 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. In an aspect, the disclosed removal and / or cleavage of IgM from the surface of B cells can be reversible and / or transient. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein 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 an aspect, a disclosed fusion enzyme or a disclosed fusion protein 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 injuring the B cells.

[0112] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can reduce and / or minimize vector mediated immunotoxicity in a subject. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can enable and / or allow vector re-dosing in a subject. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can reduce and / or minimize transgene immunogenicity in a subject.

[0113] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can improve and / or enhance transgene efficacy and / or expression in a subject. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can improve and / or enhance transgene efficacy and / or expression in a subject. In an aspect, improving and / or enhancing transgene efficacy and / or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and / or enhancement when compared to a pre-existing level of transgene efficacy and / or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion enzymes). In an aspect, 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% more than a pre-existing levelsuch as a pre-treatment level (prior to administering one or more disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion enzymes). In an aspect, an improvement and / or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzymatic polypeptide fragments or disclosed fusion proteins I fusion enzymes). In an aspect, an improvement and / or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and / or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and / or IgM molecules is similar to that of a wild-type or control level.

[0114] In an aspect, a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzy me can be administered to the subject one time. In an aspect, a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzy me can be administered to the subj ect one or more times, two or more times, three or more times, four or more times, five or more times, or more than five times. In an aspect, a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzyme can be administered to the subject at least twice (e.g., 2, 3, 4, 5, 6, or more times).

[0115] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can decrease and / or minimize the severity of a rejection event of the one or more transplanted organs. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can prevent the rejection of the one or more transplanted organs. In an aspect, a disclosed rejection event can be classified a hyperacute rejection event, an acute rejection event, or a chronic rejection event. In an aspect, following the administration and / or delivery of a disclosed fusion enzyme or a disclosed fusion protein, the one or more transplanted organs are not rejected by the subject. In an aspect, following the administration and / or delivery of a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzy me, the viability7of one or more transplanted organs can be improved and / or increased. In an aspect, following the administration and / or delivery of a disclosed fusion enzyme or a disclosed fusion protein, the functionality of one or more transplanted organs can be improved and / or increased. In an aspect, following the administration and / or delivery of a disclosed fusion enzyme or a disclosed fusion protein, the functionality7of one or more transplanted organs is not decreased and / or diminished. In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0116] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can be used to modulate humoral immunity7in a subject. In an aspect, modulating humoral immunity7in a subject can be an increase in the functionality7of the subject’s humoral immunity7, or can be a decrease in the functional of the subject's humoral immunity.

[0117] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can comprise one or more labels and / or tags. In an aspect, a disclosed label and / or a tag can comprise a fluorescent label and / or a tag. In an aspect, a disclosed label and / or a tag can comprise a carboxy -terminal label and / or tag or an amino-terminal label and / or tag. In an aspect, a disclosed label and / or a tag can comprise a carboxy-terminal fluorescent label and / or tag or an amino-terminal fluorescent label and / or tag. In an aspect, a disclosed label and / or tag can comprise an ALFA tag, an AviTag, a C tag, a Calmodulin-tag, an iCapTag (intein Capture Tag), a poly glutamate tag, a poly arginine tag. an E tag, a FLAG tag, a HA tag, a His tag. a Myc tag, a NE tag, a RholD4 tag, a S tag, a SBP tag. a Softag 1. a Softag 3. a Spot tag, a Strep tag. a T7 tag, a TC tag. a Ty tag. a V5 tag. a VSV- tag, an Xpress tag, an Isopeptag, a Spy tag, a SnoopTag, a SnoopTagJr, a DogTag, a SdyTag, a BCCP (Biotin Carboxyl Carrier Protein), a glutathi one- S -transferase tag, a green fluorescent protein tag, a Halo tag, a SNAP -tag, a CLIP tag, a HUH tag, a maltose binding protein tag, aNus tag. a thioredoxin tag. a Fc tag, a Designed Intrinsically Disordered tags containing disorder promoting amino acids, a Carbohydrate Recognition Domain or CRDSAT tag, a HiBiT-tag. or any combination thereof.

[0118] In an aspect, a disclosed label and / or a tag can comprise a fluorescent label or a fluorescent tag. In an aspect, a disclosed fluorescent label or disclosed fluorophore can comprise 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 an aspect, a disclosed fluorescent label or disclosed fluorophore can comprise any fluorescent label or fluorophore that is amendable to analysis via flow cytometry. In an aspect, a pair of disclosed fluorescent labels or disclosed fluorophores can comprise any pair of fluorescent labels or fluorophores that is amendable to analysis via flow cytometry and have excitation and emission spectra that can be isolated or separated from each other, thereby enabling the interrogation of the docking interactions. Fluorophores and fluorescent labels are known to those skilled in the art.

[0119] In an aspect, a disclosed amino acid sequence can comprise a signal peptide. In an aspect, a skilled person in the art can appreciate that the signal peptide is cleaved from a polypeptide and that a mature form of the polypeptide does not comprise a signal peptide. For example, in an aspect, a disclosed polypeptide can comprise a signal peptide that is cleaved during posttranslation processing. In an aspect, signal peptides are identified in Table 1.2. Nucleic Acid Molecules

[0120] Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding any disclosed enzy me or any disclosed recombinant enzy me. Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding any disclosed enzymatic polypeptidefragment. Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding any disclosed enzymatic polypeptide fragment having IgM specific protease activity. Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding any disclosed enzymatic polypeptide fragment having IgG specific protease activity. Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding any disclosed enzymatic polypeptide fragment having IgM specific protease activity and IgG specific protease activity.

[0121] Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding an enzymatic polypeptide fragment of SEQ ID NO:01, comprising 350 or fewer consecutive amino acids of SEQ ID NO:01. and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:02. In an aspect, a disclosed nucleic acid molecule can encode (i) an enzymatic polypeptide fragment comprising 340 or fewer, 330 or fewer, or 320 or fewer consecutive amino acids of SEQ ID NO:01; (ii) an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%. 92%. 93%, 94%, 95%. 96%. 97%, 98%, or 99% identical to SEQ ID NO: 02; (m) an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprising SEQ ID NO:02; (iv) an enzy matic polypeptide fragment comprising at least 300 consecutive amino acids of SEQ ID NO: 01; or (v) an enzymatic polypeptide fragment consisting of SEQ ID NO: 02.

[0122] Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding an enzymatic polypeptide fragment of SEQ ID NO:03, comprising 320 or fewer consecutive amino acids of SEQ ID NO: 03, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:04. In an aspect, a disclosed nucleic acid molecule can encode (i) an enzymatic polypeptide fragment comprising 320 or fewer. 319 or fewer. 318 or fewer, 317 or fewer, or 316 or fewer consecutive amino acids of SEQ ID NO:03; (ii) an enzymatic polypeptide comprising 320 or fewer consecutive amino acids of SEQ ID NO:03 and comprising an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04; (iii) an enzymatic polypeptide fragment comprising 320 or fewer consecutive amino acids of SEQ ID NO:03 and comprising the amino acid sequence of SEQ ID NO:04; (iv) an enzymatic polypeptide fragment can comprise at least 300 consecutive amino acids of SEQ ID NO: 03; or (v) an enzy matic polypeptide fragment consisting of SEQ ID NO: 04.

[0123] Disclosed herein is a nucleic acid molecule comprising a nucleotide sequence encoding an enzymatic polypeptide fragment of SEQ ID NO:07, comprising 295 or fewer consecutive amino acids of SEQ ID NO: 07, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:08. In an aspect, a disclosed nucleic acid molecule can encode (i) an enzymatic polypeptide fragment comprising 290 or fewer consecutive amino acids of SEQ ID NO: 07; (ii) anenzymatic polypeptide comprising 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprising an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:08; (iii) an enzymatic polypeptide fragment comprising 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprising the amino acid sequence of SEQ ID NO:08; (iv) an enzymatic polypeptide fragment comprising at least 280 consecutive amino acids of SEQ ID NO:07; or (v) an enzymatic polypeptide fragment consisting of SEQ ID NO:08.

[0124] In an aspect, a disclosed nucleic acid molecule can comprise the nucleic acid sequence for one or more labels and / or tags. In an aspect, a disclosed label and / or a tag can comprise a fluorescent label and / or a tag. In an aspect, a disclosed label and / or a tag can comprise a carboxyterminal label and / or tag or an amino-terminal label and / or tag. In an aspect, a disclosed label and / or a tag can comprise a carboxy-terminal fluorescent label and / or tag or an amino-terminal fluorescent label and / or tag. Labels and tags including fluorescent labels and fluorescent tags are discussed supra.

[0125] In an aspect, a disclosed nucleic acid molecule can be incorporated in whole or in part into a viral vector or a non-viral vector. In an aspect, a 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 an aspect, a disclosed vector can comprise exosomes, extracellular vesicles, and virus like particles. In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a Flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picomavirus vector.

[0126] In an aspect, a disclosed vector can further comprise one or more regulator)’ elements. In an aspect, a disclosed “regulatory element7’ can refer to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as 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 the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).3. Pharmaceutical Formulations

[0127] Disclosed herein is a pharmaceutical formulation comprising a disclosed enzy me. Disclosed herein is a pharmaceutical formulation comprising a disclosed enzyme and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein is a pharmaceutical formulation comprising a disclosed enzyme having IgM specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant enzy me having IgM specific protease activity.

[0128] Disclosed herein is a pharmaceutical formulation comprising a disclosed enzymatic polypeptide fragment of SEQ ID NO:01, comprising 350 or fewer consecutive amino acids of SEQ ID NO:01, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:02. In an aspect, a disclosed pharmaceutical formulation can comprise (i) an enzymatic polypeptide fragment comprising 340 or fewer, 330 or fewer, or 320 or fewer consecutive amino acids of SEQ ID NO:01; (ii) an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99% identical to SEQ ID NO:02; (lii) an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO:01 and can comprise SEQ ID NO:02; (iv) an enzymatic polypeptide fragment comprising at least 300 consecutive amino acids of SEQ ID NO:01; (vi) an enzymatic polypeptide fragment consisting of SEQ ID NO: 02.

[0129] Disclosed herein is a pharmaceutical formulation comprising a disclosed enzymatic polypeptide fragment of SEQ ID NO: 03. comprising 320 or fewer consecutive amino acids of SEQ ID NO:03, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:04. In an aspect, a disclosed pharmaceutical formulation can comprise (i) an enzymatic polypeptide fragment comprising 320 or fewer, 319 or fewer, 318 or fewer, 317 or fewer, or 316 or fewer consecutive amino acids of SEQ ID NO:03; (ii) an enzymatic polypeptide comprising 320 or fewer consecutive amino acids of SEQ ID NO:03 and comprising an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04; (iii) an enzy matic polypeptide fragment comprising 320 or fewer consecutive amino acids of SEQ ID NO: 03 and comprising the amino acid sequence of SEQ ID NO: 04; (iv) an enzymatic polypeptide fragment comprising at least 300 consecutive amino acids of SEQ ID NO:03; or (v) an enzymatic polypeptide fragment consisting of SEQ ID NO: 04.

[0130] Disclosed herein is a pharmaceutical formulation comprising a disclosed enzyme having IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant enzyme having IgG specific protease activity’. Disclosed herein is a pharmaceutical formulation comprising a disclosed enzymatic polypeptide fragment.

[0131] Disclosed herein is a pharmaceutical formulation comprising a disclosed enzymatic polypeptide fragment of SEQ ID NO: 07, comprising 295 or fewer consecutive amino acids of SEQ ID NO:07, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:08. In an aspect, a disclosed pharmaceutical formulation can comprise (i) an enzymatic polypeptide fragment comprising 290 or few er consecutive amino acids of SEQ ID NO: 07; (ii) an enzymatic polypeptide comprising 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprising an amino acid sequence that is at least 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical to SEQ ID NO:08; (iii) an enzymatic polypeptide fragment comprising 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprising the amino acid sequence of SEQ ID NO:08; (iv) an enzymatic polypeptide fragment comprising at least 280 consecutive amino acids of SEQ ID NO:07; or (v) an enzymatic polypeptide fragment consisting of SEQ ID NO:08.

[0132] Disclosed herein is a pharmaceutical formulation comprising a disclosed fusion enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant fusion enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant fusion protein comprising a proteolytic domain from a disclosed IgG specific protease linked to a proteolytic domain from a disclosed IgM specific protease. Disclosed herein is a pharmaceutical formulation comprising a disclosed a fusion protein having IgM specific protease activity and IgG specific protease activity.

[0133] In an aspect, a disclosed pharmaceutical formulation can comprise (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 an aspect, a disclosed composition can comprise one or more immune modulators. In an aspect, a disclosed composition can comprise one or more proteasome inhibitors. In an aspect, a disclosed composition can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).4. Kits

[0134] Disclosed herein is a kit comprising a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof. In an aspect, a kit can comprise a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and one or more agents. 'Agents" and “Therapeutic Agents'’ are known to the art and are described supra. In an aspect, the one or more agents can treat, prevent, inhibit, and / or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and / or ameliorate a disclosed antibody-related complication or a complication related to circulating and / or surface-bound IgM.

[0135] In an aspect a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having circulating and / or surface-bound IgM).

[0136] Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory' device or downloaded from an internet website, or as recorded presentation.

[0137] In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed polypeptide, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and a label or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0138] The label or package insert can indicate that a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof can be used for treating, preventing, inhibiting, and / or ameliorating a disclosed antibody-related complication or a complication related to circulating and / or surface-bound IgM. A kit can comprise additional components necessary' for administration such as. for example, other buffers, diluents, filters, needles, and syringes.C. Methods

[0139] Disclosed herein is a method of reducing circulating and / or surface-bound IgM in a subject, the method comprising administering to a subject a disclosed enzymatic polypeptide fragment or a disclosed fusion protein described herein. Disclosed herein is a method of reducing circulating and / or surface-bound IgM in a subject, the method comprising administering to a subject a pharmaceutical formulation comprising a disclosed enzymatic polypeptide fragment or a disclosed fusion protein described herein.

[0140] In an aspect, a disclosed method can comprise administering to the subject (i) an enzy matic polypeptide fragment of SEQ ID NO:01, comprising 350 or fewer consecutive amino acids ofSEQ ID NO:01, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:02; (ii) an enzymatic polypeptide fragment comprising 340 or fewer, 330 or fewer, or 320 or fewer consecutive amino acids of SEQ ID NO:01; (iii) an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprising an amino acid sequence that it at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:02; (iv) an enzymatic polypeptide fragment comprising 350 or fewer consecutive amino acids of SEQ ID NO:01 and comprising SEQ ID NO:02; (v) an enzymatic polypeptide fragment comprising at least 300 consecutive amino acids of SEQ ID NO:01; or (vi) an enzy matic polypeptide fragment consisting of SEQ ID NO: 02.

[0141] In an aspect, a disclosed method can comprise administering to the subject (i) an enzymatic polypeptide fragment of SEQ ID NO: 03, comprising 320 or fewer consecutive amino acids of SEQ ID NO:03, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:04; (ii) an enzymatic polypeptide fragment comprising 320 or fewer, 319 or fewer, 318 or fewer, 317 or fewer, or 316 or fewer consecutive amino acids of SEQ ID NO: 03; (iii) an enzymatic polypeptide comprising 320 or fewer consecutive amino acids of SEQ ID NO:03 and comprising an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:04; (iv) enzy matic polypeptide fragment comprising 320 or fewer consecutive amino acids of SEQ ID NO:03 and can comprise the amino acid sequence of SEQ ID NO:04. In an aspect, a disclosed enzymatic polypeptide fragment can comprise at least 300 consecutive amino acids of SEQ ID NO:03. In an aspect, a disclosed enzymatic polypeptide fragment can consist of SEQ ID NO: 04.

[0142] Disclosed herein is a method of reducing circulating and / or surface-bound IgGin a subject, the method comprising administering to a subject a disclosed enzymatic polypeptide fragment or a disclosed fusion protein described herein. Disclosed herein is a method of reducing circulating and / or surface-bound IgG in a subject, the method comprising administering to a subject a pharmaceutical formulation comprising a disclosed enzymatic polypeptide fragment or a disclosed fusion protein described herein.

[0143] Disclosed herein is a method of reducing circulating and / or surface-bound IgG in a subj ect, the method comprising administering to a subject an enzy matic polypeptide fragment of SEQ ID NO:07, comprising 295 or fewer consecutive amino acids of SEQ ID NO:07, and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:08. In an aspect, a disclosed method of reducing circulating and / or surface-bound IgG in a subject can comprise administering (i) an enzymatic polypeptide fragment comprising 290 or fewer consecutive amino acids of SEQ ID NO:07; (ii) an enzymatic polypeptide comprising 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprising an amino acid sequence that is at least 91%, 92%. 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 08; (iii) an enzymatic polypeptide fragment comprising 295 or fewer consecutive amino acids of SEQ ID NO:07 and comprising the amino acid sequence of SEQ ID NO:08; (iv) an enzymatic polypeptide fragment comprising at least 280 consecutive amino acids of SEQ ID NO:07; and (v) an enzymatic polypeptide fragment consisting of SEQ ID NO: 08.

[0144] Disclosed herein is a method of reducing circulating and / or surface-bound IgG and / or IgM in a subject, the method comprising administering to a subject a disclosed fusion enzy me or a disclosed fusion protein. Disclosed herein is a method of reducing circulating and / or surfacebound IgG and / or IgM in a subject, the method comprising administering to a subject a pharmaceutical formulation comprising a disclosed fusion enzyme or a disclosed fusion protein.

[0145] In an aspect, a disclosed fusion enzyme or a disclosed fusion protein can comprise (i) a disclosed fusion protein comprising the sequence of SEQ ID NO:05 or SEQ ID NO:06; (ii) a disclosed fusion protein can comprise a sequence having at least 90% identity to the sequence of SEQ ID NO:05 or SEQ ID NO:06. or (iii) a disclosed fusion protein can comprise a sequence having at least 90% identity to the sequence of SEQ ID NO:05 or SEQ ID NO:06 and having one or more N terminus-truncations and one or more C terminus truncations. In an aspect, a disclosed fusion enzy me or a disclosed fusion protein can comprise (i) a first domain and a second domain, wherein the first domain comprises a disclosed enzymatic polypeptide fragment, or (ii) a disclosed fusion protein can comprise a first domain and a second domain, wherein the second domain comprises a disclosed enzy matic polypeptide fragment. In an aspect, a disclosed first domain and a disclosed second domain can be linked by a linker. In an aspect, a disclosed first domain can comprise a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzyme having IgG-specific protease activity. In an aspect, a disclosed second domain can comprise a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzyme having IgM-specific protease activity7. In an aspect, a disclosed second domain can comprise a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzyme having IgG-specific protease activity. In an aspect, a disclosed first domain can comprise a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzyme having IgM-specific protease activity7.

[0146] In an aspect, a first disclosed domain can comprise IgM protease activity or IgG protease activity. In an aspect, a second disclosed domain can comprise IgM protease activity or IgG protease activity. In an aspect, a first disclosed domain can comprise IgM protease activity and a second disclosed domain can comprise IgG protease activity. In an aspect, a first disclosed domain can comprise IgG protease activity7and a second disclosed domain can comprise IgM proteaseactivity. In an aspect, a first disclosed domain and a second disclosed domain can comprise differing specific protease activities.

[0147] In an aspect, a disclosed method can comprise repeating the administering of a disclosed enzymatic polypeptide fragment or a disclosed fusion protein one or more times, two or more times, three or more times, four or more times, five or more times, or more than five times. In an aspect, a disclosed method can comprise repeating the administering of a disclosed enzymatic polypeptide fragment or a disclosed fusion protein at one or more scheduled or pre-determined intervals. In an aspect, a disclosed method can comprise repeating the administering of a disclosed enzymatic polypeptide fragment or a disclosed fusion protein indefinitely. In an aspect, a disclosed method can comprise repeating the administering of a disclosed enzymatic polypeptide fragment or a disclosed fusion protein for so long as a subject receives enzyme replacement therapy or gene replacement therapy. In an aspect, a disclosed method can comprise repeating the administering of a disclosed enzymatic polypeptide fragment or a disclosed fusion protein for so long as a subject experiences a transplant rejection event.

[0148] In an aspect of a disclosed method, a subject can be any age or any gender. In an aspect, a subject can be treatment naive or can have received a disclosed treatment one or more time.

[0149] In an aspect, a subject can be in need of one or more organ transplants. In an aspect, a subj ect can have received one or more organ transplants. In an aspect, a disclosed organ transplant can comprise the heart, one or both kidneys, the liver, one or both lungs, the pancreas, part or all of the colon or the intestines, or any combination thereof. In an aspect, a subject can be in need of gene therapy, can have received gene therapy , or can be receiving gene therapy. In an aspect, a subject can be treated with gene therapy or can have a disease and / or disorder that can be treated with gene therapy.

[0150] In an aspect, a disclosed method can administer a therapeutically effect amount of a disclosed enzy matic polypeptide fragment or a disclosed fusion protein or a disclosed fusion enzyme. In an aspect, a disclosed enzymatic polypeptide fragment or a disclosed fusion protein or a disclosed fusion enzyme can be therapeutically effective when the dose comprises about 0.01 mg / kg body weight to about 100 mg / kg body weight. In an aspect, a disclosed enzymatic polypeptide fragment or a disclosed fusion protein or a disclosed fusion enzyme 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.

[0151] In an aspect the post-administration levels of circulating and / or surface-bound IgM can be reduced in the subject by at least 10% when compared to the pre-administration levels of circulating and / or surface-bound IgM. In an aspect, post-administration levels of circulating and / or surface-bound IgM can be reduced by at least 15%, at least 20%, at least 30%, at least 35%, at least 40%. at least 45%. at least 50%. at least 55%. at least 60%. at least 65%. at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% when compared to the pre-administration levels of circulating and / or surface-bound IgM. For example, FIG. 1 shows circulating IgG and IgM after repeated administration of IceM (SEQ ID NO:01) and IceMG (SEQ ID NO:05).

[0152] In an aspect, the circulating and / or surface-bound IgM antibodies can be neutralizing antibodies. For example, the IgM antibodies can be neutralizing against an exogenously administered agent, such as a recombinant protein or a viral vector (e.g., neutralizing against a viral vector capsid protein).

[0153] In an aspect, the circulating and / or surface-bound IgG antibodies can be neutralizing antibodies. For example, the IgG antibodies can be neutralizing against an exogenously administered agent, such as a recombinant protein or a viral vector (<?.g, neutralizing against a viral vector capsid protein).

[0154] In an aspect, a disclosed method can increase the efficacy of a viral vector by, for example, increasing transgene expression and / or reducing neutralization of the viral vector. In an aspect, a disclosed method can minimize vector-mediated immunotoxicity and / or transgene immunogenicity (e.g., the ability7to induce specific immunity). In an aspect, vector-mediated immunotoxicity and / or transgene immunogenicity can decrease and / or reduce the efficacy of the encoded transgene. In an aspect, vector-mediated immunotoxicity and / or transgene immunogenicity can decrease and / or reduce the ability7and / or likelihood of re-dosing a subject with one or more disclosed enzy matic polypeptide fragments or disclosed fusion enzymes or disclosed fusion proteins. In an aspect, vector-mediated immunotoxicity and / or transgene immunogenicity can decrease and / or reduce the ability and / or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, recombinant product, or any combination thereof.

[0155] In an aspect, the IgM antibodies can be neutralizing against one or more transplanted organs. In an aspect, the IgG antibodies can be neutralizing against one or more transplanted organs. In an aspect, a disclosed method can decrease and / or can minimize the severity of a rejection event of the one or more transplanted organs. In an aspect, a disclosed method can prevent the rejection of one or more transplanted organs. In an aspect, a disclosed rejection event can be classified as hyperacute rejection event, an acute rejection event, or a chronic rejectionevent. In an aspect, a disclosed method can reduce the risk of rejection of the one or more transplanted organs. In an aspect, a disclosed method can reduce or diminish the subject’s need for long-term immunosuppression. In an aspect, a disclosed method can improve and / or increase the viability of one or more transplanted organs. In an aspect, a disclosed method can improve and / or increase the functionality of one or more transplanted organs. In an aspect, a disclosed method does not decrease and / or diminish the functionality of one or more transplanted organs. In an aspect, a disclosed method can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0156] In an aspect, a subject can have or can be suspected of having one of more disclosed autoimmune diseases and / or disorders, one or more autoinflammatory diseases and / or disorders, one or more inflammatory diseases and / or disorders, or any combination thereof. In an aspect, a disclosed autoimmune disease and / or disorder can be celiac disease, diabetes mellitus ty pe 1, Graves’ disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, or any combination thereof. In an aspect, a disclosed autoinflammatory diseases and / or disorder can be Familial Mediterranean Fever (FMF), hyper IgD syndrome (HIDS), TNF receptor-associated periodic syndrome (TRAPS), cryopyrin associated periodic syndromes (CAPS), Blau syndrome, pyogenic sterile arthritis pyoderma gangrenosum and acne syndrome (PAP A), or any combination thereof. In an aspect, a disclosed inflammatory' disease and / or disorder can be Encephalitis, Myelitis, Meningitis, Arachnoiditis, Neuritis, Dacryoadenitis, Scleritis, Episcleritis, Keratitis, Retinitis, Chorioretinitis, Blepharitis, Conjunctivitis, Uveitis, Otitis externa, Otitis media, Labyrinthitis, Mastoiditis, Carditis, Endocarditis. Myocarditis, Pericarditis, Vasculitis, Arteritis, Phlebitis. Capillaritis, Sinusitis, Rhinitis, Phary ngitis, Laryngitis Tracheitis, Bronchitis. Bronchiolitis, Pneumonitis. Pleuritis, Mediastinitis, Stomatitis, Gingivitis, Gingivostomatitis, Glossitis, Tonsillitis, Sialadenitis / Parotitis, Cheilitis, Pulpitis, Gnathitis, Esophagitis, Gastritis, Gastroenteritis, Enteritis, Colitis, Enterocolitis, Duodenitis, Ileitis. Caecitis. Appendicitis, Proctitis, Hepatitis, Ascending cholangitis, Cholecystitis, Pancreatitis, Peritonitis, Dermatitis, Folliculitis, Cellulitis, Hidradenitis, Arthritis, Dermatomyositis, Myositis, Synovitis / Tenosynovitis, Bursitis, Enthesitis, Fasciitis, Capsulitis, Epicondylitis, Tendinitis, Panniculitis, Osteochondritis, Spondylitis, Periostitis, Chondritis, Nephritis, Glomerulonephritis, Pyelonephritis, Ureteritis, Cystitis, Urethritis, Oophoritis. Salpingitis, Endometritis, Parametritis, Cervicitis, Vaginitis, Vulvitis, Mastitis, Orchitis, Epididymitis, Prostatitis, Seminal vesiculitis. Balanitis, Posthitis, Balanoposthitis, Chorioamnionitis, Funisitis, Omphalitis, Insulitis, Hypophy sitis, Thyroiditis, Parathyroiditis, Adrenalitis, Lymphangitis, Lymphadenitis, or any combination thereof.

[0157] In an aspect a disclosed method of cleaving IgM can further comprise administering to the subject one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0158] In an aspect, a disclosed method of cleaving IgM can further comprise administering to the subject one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed enz matic polypeptide fragment, a disclosed fusion enzyme or fusion product, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof.

[0159] In an aspect, a disclosed method can comprise administering to the subject one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed enzymatic polypeptide fragment, a disclosed fusion enzyme or fusion product, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof.

[0160] In an aspect, a disclosed method can comprise administering to the subject one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed enzymatic polypeptide fragment, a disclosed fusion enzyme or fusion product, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method can further comprise administering to the subject one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering to the subject one or more proteasome inhibitors repeatedly over time.

[0161] In an aspect, a disclosed method of cleaving IgM can further comprise administering to the subject one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be. but is not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG). cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering to the subject one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering to the subject one or more one or more immunosuppressive agents repeatedly overtime. In an aspect a disclosed method can comprise administering to the subject a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0162] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering to the subject a compound that exerts a therapeutic effect against B cells and / or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV).

[0013] In an aspect, a disclosed method of cleaving IgM can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and / or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.

[0164] In an aspect, a subject can have a high sustained antibody titer (HSAT - defined as titers of > 12,800). In an aspect, a subject can have a high sustained antibody titer (HSAT) against 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 vectors 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. In an aspect of a disclosed method, following the administering step, a disclosed HSAT can be reduced and / or eliminated. In an aspect, after the reduction and / or elimination of one or more HSATs, a subject can be re-dosed. In an aspect, after the reduction and / or elimination of one or more HSATs. a subject can be re-dosed 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 vectors 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. In an aspect, a disclosed method can comprise determining the subject’s pre-treatment antibody level. In an aspect, a disclosed method can comprise determining the subject’s post-treatment antibody level one or more times.

[0165] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymatic polypeptide fragments or fusion enzymes or fusion products, one or more of thedisclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more one or more disclosed enzymatic polypeptide fragments or fusion enzymes or fusion products, 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 to a subject, or by changing the duration of time one or more disclosed enzymatic polypeptide fragments or fusion enzymes or fusion products, 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 are administered to a subject.

[0166] In an aspect, a disclosed method can transiently clear and / or remove IgM from circulation in a subject, or can transiently cleave and / or degrade circulating IgM in a subject. In an aspect, a disclosed method of cleaving IgM can reduce and / or diminish the level of circulating IgM levels in a subject, or can transiently reduce and / or diminish the level of circulating IgM levels in a subject. In aspect, a disclosed transient reduction and / or diminishment can comprise 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.

[0167] In an aspect, a disclosed method of cleaving IgM can block and / or minimize activation of the complement cascade. In an aspect, a disclosed method of cleaving IgM can block and / or minimize activation of the formation of the C l complex and / or C3 complex. In an aspect, a disclosed method of cleaving IgM can block and / or minimize activation of the classical complement pathway. In an aspect, a disclosed method can transiently block and / or minimize activation of the complement cascade, can transiently block and / or minimize activation of the formation of the Cl complex and / or C3 complex, can transiently block and / or minimize activation of the classical complement pathway, or any combination thereof.

[0168] In an aspect, a disclosed method of cleaving IgM can modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject’s humoral immunity, or can be a decrease in the functional of the subject’s humoral immunity.

[0169] In an aspect, a disclosed method can transiently clear and / or remove IgG from circulation in a subject, or can transiently cleave and / or degrade circulating IgG in a subject. In an aspect, a disclosed method of cleaving IgG can reduce and / or diminish the level of circulating IgG levels in a subject, or can transiently reduce and / or diminish the level of circulating IgG levels in a subject. In aspect, a disclosed transient reduction and / or diminishment can comprise 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.

[0170] In an aspect, a disclosed method of cleaving IgG can block and / or minimize activation of the complement cascade. In an aspect, a disclosed method of cleaving IgM can block and / or minimize activation of the formation of the Cl complex and / or C3 complex. In an aspect, a disclosed method of cleaving IgG can block and / or minimize activation of the classical complement pathway. In an aspect, a disclosed method can transiently block and / or minimize activation of the complement cascade, can transiently block and / or minimize activation of the formation of the Cl complex and / or C3 complex, can transiently block and / or minimize activation of the classical complement pathway, or any combination thereof.

[0171] In an aspect, a disclosed method of cleaving IgG can modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject's humoral immunity, or can be a decrease in the functional of the subject's humoral immunity.

[0172] In an aspect, a disclosed method of cleaving IgM and / or IgG can reduce and / or minimize transgene immunogenicity in a subject. In an aspect, a disclosed method of cleaving IgM and / or IgG can improve and / or enhance transgene efficacy and / or expression in a subject. In an aspect, improving and / or enhancing transgene efficacy and / or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and / or enhancement when compared to a pre-existing level of transgene efficacy and / or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzymes or disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion 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 an aspect, 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% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed enzymes or disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion 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 an aspect, an improvement and / or enhancement can be measured against a control level or a reference level (e.g.. determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzymes, one or more disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion 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 an aspect, animprovement and / or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and / or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG molecules is similar to that of a wild-type or control level.

[0173] In an aspect, a disclosed method can further comprise measuring and / or determining a subject’s pre-treatment level of circulating IgM and / or IgG and / or a subject’s pre-treatment level of surface bound IgM and / or IgG. In an aspect, a disclosed method can further comprise measuring and / or determining one or more times a subject's level of circulating IgM and / or IgG and / or a subject’s level of surface bound IgM (e.g., before, during, and after a disclosed administering step).

[0174] In an aspect of a disclosed method of cleaving surface-bound IgM and / or IgG, contacting a cell can comprising methods known to the art. For example, contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more disclosed enzymatic polypeptide fragments or disclosed fusion proteins / fusion enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0175] In an aspect, administering can comprise intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic. hepatic intraarterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed composition, a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and / or a disclosed vector can be administered via intra-CSF administration in combination with RNAi, antisense oligonucleotides. miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more immune modulators, and / or a gene editing system. In an aspect, a disclosed composition, a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and / or a disclosed vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and / or a disclosed vector can be concurrently and / or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed composition, a disclosed nucleic acid molecule, a disclosed enzymatic polypeptide fragment, a disclosed fusion protein / fusion enzy me, a disclosed vector, and / or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistem magna (ICM) administration. In an aspect, administering a disclosed composition a disclosedenzymatic polypeptide fragment, a disclosed fusion protein / fusion enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and / or a disclosed vector can comprise IV administration and intrathecal (ITH) administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and / or subsequent routes of administration.

[0176] In an aspect, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzy me or a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzyme can comprise about 0.01 mg / kg body weight to about 100 mg / kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzyme or a disclosed enzymatic polypeptide fragment or a disclosed fusion protein / fusion enzyme 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.

[0177] In an aspect, a disclosed method of cleaving surface-bound IgM and / or IgG can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0178] In an aspect, a disclosed method of cleaving surface-bound IgM and / or IgG can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria. Such methods are known to the skilled person.

[0179] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gammaglobulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg / kg body weight to about 0.6 mg / kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg / kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg / kg body weight for 3 to 5 or greater cycles, with up to three daysper cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg / kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect. In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.Table 1 - Exemplary Enzymatic Polypeptide Fragment Sequences

[0180] The disclosed enzymatic polypeptide fragments and disclosed fusion proteins / fusion enzymes clear IgM and IgG from circulation in vivo and inactivate the antibody driven classical complement pathway by clearing IgM and IgG antibody complexes deposited on tissues. Taken together, the disclosed enzymatic polypeptide fragments and disclosed fusion proteins / fusion enzymes are promising candidates for modulating humoral immunity in primates and therapeutic removal of IgM or both IgM and IgG in pathophysiological settings that may present in autoimmune disorders, organ transplantation, and AAV gene therapy.

Claims

VII. CLAIMSWhat is claimed is:

1. An enzymatic polypeptide fragment of SEQ ID NO:01, comprising 350 or fewer amino acids and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 02.

2. The enzymatic polypeptide fragment of Claim 1, comprising 340 or fewer, 330 or fewer, or 320 or fewer amino acids.

3. The enzymatic polypeptide fragment of Claim 1 or 2, comprising SEQ ID NO:02.

4. The enzymatic polypeptide fragment of any one of Claims 1 - 3, consisting of SEQ ID NO: 2.

5. An enzymatic polypeptide fragment of SEQ ID NO:03, comprising 320 or fewer amino acids and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 04.

6. The enzymatic polypeptide fragment of Claim 5, comprising SEQ ID NO: 04.

7. The enzymatic polypeptide fragment of Claim 5 or Claim 6, consisting of SEQ ID NO:04.

8. A fusion protein comprising a first domain comprising the enzymatic fragment of any one ofClaims 1 - 7.

9. The fusion protein of Claim 8, further comprising a second domain comprising an enzymatic polypeptide or an enzymatic polypeptide fragment.

10. The fusion protein of Claim 9, wherein the first and second domains are linked via a linker.

11. A polynucleotide sequence encoding the enzymatic polypeptide fragment of any one ofClaims 1 - 7 or the fusion protein of any one of Claims 8 - 10.

12. A composition comprising the enzymatic polypeptide fragment of any one of Claims 1 - 7 or the fusion protein of any one of Claims 8 - 10.

13. A method of reducing the level of circulating IgM in a subject, the method comprising: administering to a subject the enzymatic polypeptide fragment of any one of Claims 1 - 7 or the fusion protein of any one of Claims 8 - 10.

14. A method of reducing an immune response in a subject, the method comprising: administering to the subject the enzymatic polypeptide fragment of any one of Claims 1 - 7 or the fusion protein of any one of Claims 8 - 10.

15. The method of Claim 13 or 14, wherein the subject: has received, is receiving, or will receive a recombinant product; has received, is receiving, or will receive enzyme replacement therapy or protein replacement therapy; has received, is receiving, or will receive one or more transplanted organs; has an autoimmune disease or disorder; and / or has received, is receiving, or will receive gene therapy.

16. The method of any one of Claims 13 - 15, comprising repeating the administering step two or more times.

17. An enzymatic polypeptide fragment of SEQ ID NO:07, comprising 295 or fewer amino acids and comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 08.

18. The enzymatic polypeptide fragment of Claim 17. comprising 290 or fewer amino acids.

19. The enzymatic polypeptide fragment of Claim 17 or Claim 18, comprising SEQ ID NO: 08.

20. The enzy matic polypeptide fragment of any one of Claims 17 - 19, consisting of SEQ IDNO:08.

21. A fusion protein comprising a first domain comprising the enzymatic fragment of any one ofClaims 17 - 20.

22. The fusion protein of Claim 21, further comprising a second domain comprising an enzymatic polypeptide or an enzymatic polypeptide fragment.

23. The fusion protein of Claim 22, wherein the first and second domains are linked via a linker.

24. A polynucleotide sequence encoding the enzymatic polypeptide fragment of any one ofClaims 17 - 20 or the fusion protein of any one of Claims 21 - 23.

25. A composition comprising the enzymatic polypeptide fragment of any one of Claims 17 - 20 or the fusion protein of any one of Claims 21 - 23.

26. A method of reducing the level of circulating IgM in a subject, the method comprising: administering to the subject the enzymatic polypeptide fragment of any one of Claims 17- 20 or the fusion protein of any one of Claims 21 - 23.

27. A method of reducing an immune response in a subject, the method comprising: administering to the subject the enzymatic polypeptide fragment of any one of Claims 17- 20 or the fusion protein of any one of Claims 21 - 23.

28. The method of Claim 26 or Claim 27, wherein the subject: has received, is receiving, or will receive a recombinant product; has received, is receiving, or will receive enzyme replacement therapy or protein replacement therapy; has received, is receiving, or will receive one or more transplanted organs; has an autoimmune disease or disorder; has received, is receiving, or will receive gene therapy.

29. The method of Claim 27 or Claim 28, comprising repeating the administering step two or more times.

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