WO2025171163A1

WO2025171163A1 - Compositions for and methods of treating lysosomal storage diseases

Info

Publication number: WO2025171163A1
Application number: PCT/US2025/014837
Authority: WO
Inventors: Baodong Sun
Original assignee: Duke University
Current assignee: Duke University
Priority date: 2024-02-06
Filing date: 2025-02-06
Publication date: 2025-08-14
Anticipated expiration: 2026-08-06

Abstract

Disclosed herein are compositions for and methods of treating and/or preventing disease progression for one or more lysosomal storage diseases.

Description

COMPOSITIONS FOR AND METHODS OF TREATING LYSOSOMAL STORAGE DISEASES

I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claim priority to U.S. Provisional Application No. 63/550,215 filed 6 February 2024 and U.S. Provisional Application No. 63/562,380 filed 7 March 2024, both of which are incorporated herein in their entirety.

II. REFERENCE TO THE SEQUENCE LISTING

[0002] The Sequence Listing submitted 6 February 2025 as a text file named “25-4027-WO_SL”, created on 6 February 2025 and having a size of 180,224 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

III. BACKGROUND

[0003] Lysosomes are subcellular organelles responsible for the physiologic turnover of cell constituents. The deficiencies of lysosomal enzymes and lysosome-related proteins cause lysosomal storage diseases (LSDs), a group of more than 50 inherited metabolic disorders that affect between 1/5000 to 1/8000 people. LSDs are characterized by the over-accumulation of various undegraded substrates within the lysosomes, resulting in the damage and symptoms in a broad range of organs. The most common LSDs include Fabry disease, Mucopolysaccharidoses (MPSs), Gaucher disease, and Pompe disease.

[0004] Currently approved treatment for LSDs is mainly cation-independent mannose 6- phosphate receptor (CI-M6PR)-mediated enzyme replacement therapy (ERT), which is available for some of the LSDs such as Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease, Mucopolysaccharidoses (MPSs), Metachromatic leukodystrophy, Farber disease, and LALD disease. However, major limitations of current ERTs exist including the poor enzyme uptake efficiency in skeletal muscle and bone, the inability of the infused enzyme to cross the brain-blood barrier (BBB) to correct neuronal deficits in the CNS, the requirement for lifetime intravenous enzyme administration, high drug cost, and the development of antibody responses and allergic reactions from repeated infusions of high-dose enzyme that comprise the therapeutic efficacy and cause safety issues.

[0005] Thus, there remains an urgent need for a minimally invasive, definitive therapy to address the underlying cause of as well as the sequelae of symptoms associated with these various lysosomal diseases and disorders. Consequently, the present disclosure provides compositions for and methods of treating lysosomal disease and/or disorder, which can be used alone or in combination with other treatments. IV. BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1 is a schematic of the 3D structure of human IGF2 and its critical binding sites to the IR and IGF1R (Phe26, Tyr27, and Val43) and IGFBPs (Glu6).

[0007] FIG. 2 is a schematic diagram showing the components of IGF-hGAA fusion proteins.

[0008] FIG. 3A provides data showing enzyme uptake of regeneration IGF2-hGAA variants in rat L6 myoblast cells. FIG. 3B is a representative Western blot showing enzyme uptake of 1^st- generation IGF2-hGAA variants in rat L6 myoblast cells.

[0009] FIG. 4A is a representative Western blot showing binding of unmodified IGF2-GAA to HEK293 cells was enhanced by over-expression of IR or IGF1R. FIG. 4B provides data showing mutagenesis of the IGF2 tag suppressed the binding of regeneration IGF2-GAA variants to the IR over-expressed in the HEK293 cells. FIG. 4C provides data showing mutagenesis of the IGF2 tag suppressed the binding of regeneration IGF2-GAA variants to the IGF1R over-expressed in the HEK293 cells.

[0010] FIG. 5 is a schematic showing a representative AAV construct expressing an IGF2-hGAA fusion protein.

[0011] FIG. 6A provides data showing GAA activity in the liver of GAA-KO mice 4 weeks after administration of the AAV vectors expressing regeneration IGF2-GAA variants. FIG. 6B provides data showing GAA activity in the plasma of GAA-KO mice 4 weeks after administration of the AAV vectors expressing regeneration IGF2-GAA variants.

[0012] FIG. 7A provides data showing the detection of GAA activity in tissues of GAA-KO mice 4 weeks after administration of the AAV vectors expressing regeneration IGF2-GAA variants. FIG. 7B provides data showing the reduction of glycogen content in tissues of GAA-KO mice 4 weeks after administration of the AAV vectors expressing regeneration IGF2-GAA variants.

[0013] FIG. 8A provides data showing enzyme uptake of 2^nd-generation IGF2-hGAA variants in rat L6 myoblast cells. FIG. 8B is a representative Western blot showing enzyme uptake of 2^nd- generation IGF2-hGAA variants in rat L6 myoblast cells.

[0014] FIG. 9A provides data showing mutagenesis of the IGF2 tag suppressed the binding of 2^nd-generation IGF2-GAA variants to the IR over-expressed in the HEK293 cells in accordance with one embodiment of the present disclosure. FIG. 9B provides data showing mutagenesis of the IGF2 tag suppressed the binding of 2^nd-generation IGF2-GAA variants to the IGF1R overexpressed in the HEK293 cells.

[0015] FIG. 10A provides data showing the detection of GAA activity in tissues of GAA-KO mice 4 weeks after administration of AAV vectors expressing untagged hGAA, unmodified IGF2- hGAA, and a 2^nd-generation IGF2-GAA variant. FIG. 10B provides data showing the reduction of glycogen content in tissues of GAA-KO mice 4 weeks after administration of AAV vectors expressing untagged hGAA, unmodified IGF2-hGAA, and a 2nd-generation IGF2-GAA variant. [0016] FIG. 11 is a representative Western blot showing the detection GAA enzymes in the plasma of GAA-KO mice 4 weeks after administration of AAV vectors expressing untagged hGAA, unmodified IGF2-hGAA, and a 2nd-generation IGF2-GAA variant.

V. BRIEF SUMMARY

[0017] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0018] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0019] Disclosed herein is polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0020] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0021] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0022] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0023] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0024] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0025] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0026] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. [0027] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0028] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0029] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0030] Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0031] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, and increasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0032] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed mutant IGF2-hGAA polypeptide or a pharmaceutical formulation thereof, and increasing the expression level and/or activity level of GAA. [0033] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed mutant IGF2-hGAA polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of GAA is increased.

[0034] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule encoding a mutant IGF2-hGAA polypeptide or a disclosed vector comprising the disclosed nucleic acid, and increasing the expression level and/or activity level of GAA.

[0035] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule encoding a mutant IGF2-hGAA polypeptide or a disclosed vector comprising the disclosed nucleic acid, wherein the expression level and/or activity level of GAA is increased.

VI. DETAILED DESCRIPTION

[0036] 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.

[0037] 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

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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. [0045] 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.

[0046] 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 herein 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 an LSD, be suspected of having an LSD, or be at risk of developing an LSD.

[0047] 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 one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “diagnosed with an LSD” 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 treated by one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “suspected of having an LSD” can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can likely be treated by one or more of by one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a 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.

[0048] A “patient” refers to a subject afflicted with a glycogen storage disease. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a glycogen storage disease. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a lysosomal storage disease (LSD) and is seeking treatment or receiving treatment for an LSD. [0049] In an aspect, the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder. For example, a subject can be identified as having a need for treatment of a disorder (e.g., such an LSD) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder (e.g., such as an LSD). 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.

[0050] In an aspect, “glycogenosis” (plural is glycogenoses) refers to a metabolic disorder caused by a defective glycogen metabolism resulting in the extra glycogen storage in cells.

[0051] In an aspect, “inhibit,” “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not having an LSD). 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.

[0052] 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, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating an LSD (such as any in Table 1) can reduce the severity of an established LSD in a subject by 1%-100% as compared to a control (such as, for example, an individual not having a glycogen storage disease). 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 an LSD (such as any in Table 1). For example, treating an LSD can reduce one or more symptoms of the LSD in a subject by l%-100% as compared to a control (such as, for example, an individual not having an LSD). In an aspect, treating can refer to 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 LSD (such as any in Table 1). It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a LSD (e.g. Table 1). However, in an aspect, treatment can refer to a cure or complete ablation or eradication of an LSD. [0053] 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 an LSD is intended. The words “prevent” and “preventing” and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given an LSD-related complication from progressing to that complication. [0054] In an aspect, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject.

[0055] In an aspect, a therapeutically effective amount of disclosed vector can be delivered intravenously and can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴vg/kg.

[0056] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof so as to treat or prevent an LSD. 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 of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof.

[0057] In an aspect, “modifying the 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 of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject, or by changing the duration of time one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination are administered to a subject.

[0058] In an aspect, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0059] In an aspect, the term “contacting” refers to bringing one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof together with a target area or intended target area in such a manner that the one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof exert an effect on the intended target or targeted area either directly or indirectly. A target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, muscle, kidney, brain, etc.). In an aspect, a target area or intended target area can be any cell or any organ infected by an LSD. In an aspect, a target area or intended target area can be the liver.

[0060] In an aspect, “determining” can refer to measuring or ascertaining the presence and severity of an LSD. Methods and techniques used to determine the presence and/or severity of an LSD 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 an LSD.

[0061] 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 an LSD or a suspected a glycogen storage disease. 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., an LSD). 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 polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition (such as an LSD), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., an LSD). 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 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 well within the skill of the art to start doses of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations at levels lower than those 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 glycogen storage disease.

[0062] 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 antibacterial and 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.

[0063] 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.

[0064] 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’-0-Me-RNA, 2’-ME0-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, a long 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 a RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form 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 of liver or muscle glycogen synthase (e.g., GYSI and/or GYS2). 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 base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0065] In an aspect, “small molecule” can refer to any organic or inorganic material that is not a polymer. Small molecules exclude large macromolecules, such as large proteins (e.g., proteins with molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weight of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). In an aspect, a “small molecule”, for example, can be a drug that can enter cells easily because it has a low molecular weight.

[0066] In an aspect, “CpG-free” can mean completely free of CpGs or partially free of CpGs. In an aspect, “CpG-free” can mean “CpG-depleted”. In an aspect, “CpG-depleted” can mean completely depleted of CpGs or partially depleted of CpGs. In an aspect, “CpG-free” can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art.

[0067] In an aspect, “guaiacol” refers to a small molecule having a MW of 124.14. Guaiacol is a monomethoxybenzene comprising phenol with a methoxy substituent at the ortho position (C7H8O2). In an aspect, guaiacol can increase inactivating GYSI phosphorylation and/or can increase phosphorylation of the master activator of catabolism, AMP-dependent protein kinase. In an aspect, guaiacol can be a competitive inhibitor of purified GYSI and GYS2 and a mixed inhibitor of the enzymes in cell lysates. In an aspect, guaiacol can reduce the expression level and/or activity level of glycogen synthase (such as GYSI and/or GYS2).

[0068] As known to the art, miRNAs are small non-coding RNAs that are about 17 to about 25 nucleotide bases (nt) in length in their biologically active form. In an aspect, a disclosed miRNA can regulate gene expression post transcriptionally by decreasing target mRNA translation. In an aspect, a disclosed miRNA can function as a negative regulator. In an aspect, a disclosed miRNA is about 17 to about 25, about 17 to about 24, about 17 to about 23, about 17 to about 22, about 17 to about 21, about 17 to about 20, about 17 to about 19, about 18 to about 25, about 18 to about 24, about 18 to about 23, about 18 to about 22, about 18 to about 21, about 18 to about 20, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 19 to about 22, about 19 to about 21, about 20 to about 25, about 20 to about 24, about 20 to about 23, about 20 to about 22, about 21 to about 25, about 21 to about 24, about 21 to about 23, about 22 to about 25, about 22 to about 24, or about 22 nucleotides in length. Generally, there are three forms of miRNAs: primary miRNAs (pri-miRNAs), premature miRNAs (pre-miRNAs), and mature miRNAs, all of which are within the scope of the present disclosure.

[0069] 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 between 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.

[0070] In an aspect, an “enhancer” such as a transcription or transcriptional enhancer refers to regulatory DNA segment that is typically found in multicellular eukaryotes. An enhancer can strongly stimulate (“enhance”) the transcription of a linked transcription unit, z.e., it acts in cis. An enhancer can activate transcription over very long distances of many thousand base pairs, and from a position upstream or downstream of the site of transcription initiation. An enhancers can have a modular structure by being composed of multiple binding sites for transcriptional activator proteins. Many enhancers control gene expression in a cell type-specific fashion. Several remote enhancers can control the expression of a singular gene while a singular enhance can stimulate the transcription of one or more genes.

[0071 ] In an aspect, “expression cassette” or “transgene cassette” can refer to a distinct component of vector DNA comprising a transgene and one or more regulatory sequences to be expressed by a transfected cell. Generally, an expression cassette or transgene cassette can comprise a promoter sequence, an open reading frame (i.e., the transgene), and a 3’ untranslated region (e.g., in eukaryotes a polyadenylation site).

[0072] 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.

[0073] “Tissue-specific promoters” are known to the art and include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal musclespecific promoters, and heart-specific promoters.

[0074] “Liver-specific promoters” are known to the art and include, but are not limited to, the thyroxin binding globulin (TBG) promoter, the al-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone- binding globulin promoter, the a- 1 -anti -trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human al -antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC 172 promoter comprising the hAAT promoter and the al -microglobulin enhancer, the DC 190 promoter comprising the human albumin promoter and the prothrombin enhancer, or any other natural or synthetic liver-specific promoter.

[0075] In an aspect, a disclosed liver-specific promoter can comprise any liver-specific promoter known to the art. In an aspect, a liver specific promoter can comprise about 845-bp and comprise the thyroid hormone-binding globulin promoter sequences (2382 to 13), two copies of al- microglobulinybikunin enhancer sequences (22,804 through 22,704), and a 71 -bp leader sequence (Ill CR, et al. (1997) Blood Coagul Fibrinolysis. 8 Suppl 2:S23-S30). In an aspect, a disclosed liver-specific promoter can comprise the sequence set forth in SEQ ID NO:97 or SEQ ID NO: 161. In an aspect, a disclosed liver-specific promoter can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 161. In an aspect, a disclosed liver-specific promoter can comprise a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in SEQ ID NO:97 or SEQ ID NO: 161. [0076] In an aspect, a disclosed ubiquitous promoter can be a CMV enhancer/chicken P-actin promoter (CB promoter).

[0077] In an aspect, an “inducible promoter” refers to a promoter that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[0078] In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, the term promoter/enhancer can refer to a segment of DNA that contains nucleotide sequences capable of providing both promoter and enhancer functions.

[0079] As discussed above, a disclosed promoter can be an endogenous promoter. Endogenous refers to a disclosed promoter or disclosed promoter/enhancer that is naturally linked with its gene. In an aspect, a disclosed endogenous promoter can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene (such as, for example, a disclosed phosphorylase kinase, phosphorylase, or some other enzyme involved in the glycogen metabolic pathway). In an aspect, a disclosed endogenous promoter can be used for constitutive and efficient expression of a disclosed transgene (e.g., a nucleic acid sequence encoding a polypeptide capable of preventing glycogen accumulation and/or degrading accumulated glycogen).

[0080] As discussed above, a disclosed promoter can be an exogenous promoter. Exogenous (or heterologous) refers to a disclosed promoter or a disclosed promoter/enhancer that can be placed in juxtaposition to a gene by means of molecular biology techniques such that the transcription of that gene can be directed by the linked promoter or linked promoter/enhancer. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer.

[0081] In an aspect, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness can be determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).

[0082] In an aspect, “tropism” refers to the specificity of an AAV capsid protein present in an AAV viral particle, for infecting a particular type of cell or tissue. The tropism of an AAV capsid for a particular type of cell or tissue may be determined by measuring the ability of AAV vector particles comprising the hybrid AAV capsid protein to infect or to transduce a particular type of cell or tissue, using standard assays that are well- known in the art such as those disclosed in the examples of the present application. In an aspect, the term “liver tropism” or “hepatic tropism” refers to the tropism for liver or hepatic tissue and cells, including hepatocytes. [0083] “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 glycogen branching enzymes and amylases. 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.

[0084] 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/).

[0085] In an aspect, “GYSI” refers to glycogen synthase (muscle), which is an enzyme that transfers the glycosyl residue from UDP-Glc to the non-reducing end of alpha- 1,4-glucan while “GYS2” refers to glycogen synthase (liver), which is an enzyme that transfers the glycosyl residue from UDP-Glc to the non-reducing end of alpha- 1,4-glucan.

[0086] In an aspect, “substrate reduction therapy” or “SRT” refers to methods of reducing the level of the substrate to a point where residual degradative activity of one or more enzymes is sufficient to prevent substrate accumulation. Generally, SRT aims to use small molecule inhibitors of biosynthesis to reduce the concentration of accumulating substrate to a level where the residual degradative enzymes can maintain homeostasis. For example, in an aspect, SRT refers to a method of inhibiting glycogen synthase (i.e., GYSI and/or GYS2) in a cell or a subject to reduce glycogen synthesis and/or glycogen accumulation in cells and tissues. In an aspect, SRT can comprise siRNA-based therapies, shRNA-based therapies, antisense therapies, gene-editing therapies, and therapies using one or more small molecules or peptide drugs. In an aspect, SRT can comprise administration of one or more small molecules that can traverse the blood-brain barrier in quantities that are therapeutic for a subject having neuropathic glycogen storage disease. In an aspect, SRT can comprise administration of one or more small molecules that do not traverse the blood-brain barrier in quantities but are nonetheless therapeutic for a subject having neuropathic glycogen storage disease. In an aspect, a disclosed small molecule that inhibits glycogen synthase (GYSI) in SRT can be orally delivered.

[0087] 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., a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed transgene product, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, 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 or by liver-restricted transgene expression with an AAV vector. Low or absent antibody titers over time is an indicator of immune tolerance. For example, in some embodiments, 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 within following gene therapy (such as the administration of the transgene encoding, for example, a lysosomal enzyme.

[0088] 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.

[0089] In an aspect, also disclosed herein are partial self-complementary parvovirus (e.g., a disclosed AAV) genomes, plasmid vectors encoding the parvovirus genomes, and parvovirus (e.g., a disclosed AAV) particles including such genomes. In an aspect, provided herein is a plasmid vector comprising a nucleotide sequence encoding a disclosed parvovirus genome such as for example, a disclosed AAV. In an aspect, provided herein is a partial self-complementary parvovirus genome including a payload construct, parvovirus ITRs flanking the payload construct, and a self-complementary region flanking one of the ITRs. A self-complementary region can comprise a nucleotide sequence that is complementary to the payload construct. A disclosed self- complementary region can have a length that is less the entire length of the payload construct. [0090] In an aspect, a disclosed self-complementary region of a disclosed parvovirus genome can comprise a minimum length, while still having a length that is less the entire length of the payload construct. In an aspect, a disclosed self-complementary region can comprise at least 50 bases in length, at least 100 bases in length, at least 200 in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, at least 900 bases in length, or at least 1,000 bases in length. [0091] In an aspect, a “self-complementary parvovirus genome” can be a single stranded polynucleotide having, in the 5' to 3' direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct comprising, for example, lysosomal enzyme), a second parvovirus ITR sequence, a second heterologous sequence, wherein the second heterologous sequence is complementary to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a “partial self-complementary genome” does not include three parvovirus ITRs and the second heterologous sequence that is complementary to the first heterologous sequence has a length that is less than the entire length of the first heterologous sequence (e.g., payload construct). Accordingly, a partial self- complementary genome is a single stranded polynucleotide having, in the 5' to 3' direction or the 3' to 5' direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct), a second parvovirus ITR sequence, and a self-complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence.

[0092] In an aspect, “immune-modulating” refers to the ability of a disclosed polypeptide, 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.

[0093] 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, mycophenolate 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 -IGF 1R antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an 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, a disclosed immune modulator can be Tacrolimus. 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.

[0094] 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.

[0095] 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.

[0096] 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., a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a 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) the administration of a second therapy (e.g., agent) to a subject having or diagnosed with an LSD.

[0097] Disclosed are the components to be used to prepare the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as 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. Lysosomal Storage Diseases

[0098] Lysosomal storage diseases (LSDs) are heritable (inborn) errors of metabolism that affect the function of the lysosome. LSDs comprise a group of 70 monogenic disorders of lysosomal catabolism, most of which are inherited as autosomal recessive traits, but three are X- linked. These disorders are caused by mutations in genes encoding lysosomal proteins, such as lysosomal glycosidases, proteases, integral membrane proteins, transporters, enzyme modifiers or activators. Mutations in lysosomal genes affect the function of the encoded protein, resulting in lysosomal malfunction and the gradual accumulation of substrates inside the lysosome (that is, ‘storage’), which ultimately leads to cell dysfunction and cell death. Of the -1,300 genes involved in lysosomal function, many monogenic disorders have been described, including 50 enzyme deficiencies, which can be subclassified according to the biochemical type of stored material (for example, the sphingolipidoses, mucopolysaccharidoses and glycoproteinoses) into 7 disorders involving integral membrane proteins, 12 dis orders of lysosome-related organelles (LROs) and 14 disorders involving the production of lipofuscin.

Table 1 - Listing of Lysosomal Storage Diseases and Presenting Symptoms

C. Compositions for Treating and/or Preventing LSD Progression

1. Polypeptides

[0099] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0100] In an aspect, a disclosed lysosomal targeting peptide can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions. In an aspect, a disclosed insulin-like growth factor 2 (IGF2) can comprise one substitution. In an aspect, a disclosed insulin-like growth factor

2 (IGF2) can comprise two substitutions. In an aspect, a disclosed insulin-like growth factor 2

(IGF2) can comprise three substitutions. In an aspect, a disclosed insulin-like growth factor 2

(IGF2) can comprise four substitutions. In an aspect, a disclosed insulin-like growth factor 2

(IGF2) can comprise five substitutions. In an aspect, a disclosed insulin-like growth factor 2

(IGF2) can comprise more than five substitutions. In an aspect, one or more substitutions can occur at Glu6, Phel9, Phe26, Tyr27, Val43, or any combination thereof. In an aspect, one or more substitutions can occur at Glu6, Phel9, Phe26, Tyr27, Val43, or at any other position, or any combination thereof. Exemplary substitutions are set forth below in Table 2 and Table 3.

Table 2 - IGF2 Mutants and Relevant Sequences Table 3 - Protein and DNA sequences of the residues mutated in the IGF2 mutants

[0101] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leu27 substitution. In an aspect, a disclosed IGF2 having a Leu27 substitution can comprise the sequence set forth in SEQ ID NO:03. In an aspect, a disclosed IGF2 having a Leu27 substitution can be encoded by the sequence set forth in SEQ ID NO:37. In an aspect, a disclosed IGF2 having a Leu27 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:03. In an aspect, a disclosed IGF2 having a Leu27 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:37.

[0102] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Met43 substitution. In an aspect, a disclosed IGF2 having a Met43 substitution can comprise the sequence set forth in SEQ ID NO:04. In an aspect, a disclosed IGF2 having a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:38. In an aspect, a disclosed IGF2 having a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:04. In an aspect, a disclosed IGF2 having a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:38.

[0103] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leu43 substitution. In an aspect, a disclosed IGF2 having a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:05. In an aspect, a disclosed IGF2 having a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:39. In an aspect, a disclosed IGF2 having a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:05. In an aspect, a disclosed IGF2 having a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:39.

[0104] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution and a Leu27 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu27 substitution can comprise the sequence set forth in SEQ ID NO:06. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu27 substitution can be encoded by the sequence set forth in SEQ ID NO:40. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu27 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:06. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu27 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:40.

[0105] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution and a Leu43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:07. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:41. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:07. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:41.

[0106] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution and a Ser26 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Ser26 substitution can comprise the sequence set forth in SEQ ID NO:08. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Ser26 substitution can be encoded by the sequence set forth in SEQ ID NO:42. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Ser26 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:08. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Ser26 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:42.

[0107] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution and a Met43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Met43 substitution can comprise the sequence set forth in SEQ ID NO:09. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:43. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:09. In an aspect, a disclosed IGF2 having an Arg6 substitution and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:43.

[0108] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leu27 substitution and a Met43 substitution. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Met43 substitution on can comprise the sequence set forth in SEQ ID NO: 10. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:44. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 10. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:44.

[0109] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, aLeu27 substitution, and aMet43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Met43 substitution can comprise the sequence set forth in SEQ ID NO: 11. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:45. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 11. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:45.

[0110] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Ser26 substitution. In an aspect, a disclosed IGF2 having a Ser26 substitution can comprise the sequence set forth in SEQ ID NO: 12. In an aspect, a disclosed IGF2 having a Ser26 substitution can be encoded by the sequence set forth in SEQ ID NO:46. In an aspect, a disclosed IGF2 having a Ser26 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 12. In an aspect, a disclosed IGF2 having a Ser26 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:46.

[0111] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Ser26 substitution and a Met43 substitution. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Met43 substitution can comprise the sequence set forth in SEQ ID NO: 13. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:47. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 13. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:47.

[0112] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, and a Met43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Met43 substitution can comprise the sequence set forth in SEQ ID NO: 14. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:48. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 14. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:48. [0113] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Ser26 substitution and a Leu27 substitution. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Leu27 substitution can comprise the sequence set forth in SEQ ID NO: 15. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Leu27 substitution can be encoded by the sequence set forth in SEQ ID NO:49. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Leu27 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:15. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Leu27 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:49.

[0114] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, and a Leu27 substitution. In an aspect, a disclosed IGF2 having comprise an Arg6 substitution, a Ser26 substitution, and a Leu27 substitution can comprise the sequence set forth in SEQ ID NO: 16. In an aspect, a disclosed IGF2 having a comprise an Arg6 substitution, a Ser26 substitution, and a Leu27 substitution can be encoded by the sequence set forth in SEQ ID NO:50. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Leu27 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 16. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Leu27 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:50.

[0115] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Ser26 substitution, aLeu27 substitution, and aMet43 substitution. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can comprise the sequence set forth in SEQ ID NO: 17. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:51. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 17. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:51. [0116] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Met43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can comprise the sequence set forth in SEQ ID NO: 18. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:52. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 18. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:52.

[0117] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leu27 substitution and a Leu43 substitution. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO: 19. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:53. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 19. In an aspect, a disclosed IGF2 having a Leu27 substitution and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO 53.

[0118] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, aLeu27 substitution, and aLeu43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:20. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:54. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:20. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:54.

[0119] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Ser26 substitution and a Leu43 substitution. In an aspect, a disclosed IGF2 having comprise a Ser26 substitution and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:21. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:55. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:21. In an aspect, a disclosed IGF2 having a Ser26 substitution and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:55.

[0120] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, and a Leu43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:22. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:56. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:22. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:56.

[0121] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Ser26 substitution, aLeu27 substitution, and aLeu43 substitution. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:23. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:57. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:23. In an aspect, a disclosed IGF2 having a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:57.

[0122] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:24. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:58. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:24. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:58.

[0123] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leul9 substitution and a Ser26 substitution. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Ser26 substitution can comprise the sequence set forth in SEQ ID NO:25. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Ser26 substitution can be encoded by the sequence set forth in SEQ ID NO:59. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Ser26 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:25. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Ser26 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:59.

[0124] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leul9 substitution and a Leu27 substitution. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu27 substitution can comprise the sequence set forth in SEQ ID NO:26. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu27 substitution can be encoded by the sequence set forth in SEQ ID NO:60. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu27 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:26. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu27 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:60.

[0125] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leul9 substitution and a Leu43 substitution. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:27. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:61. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:27. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:61.

[0126] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leul9 substitution and a Met43 substitution. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Met43 substitution can comprise the sequence set forth in SEQ ID NO:28. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:62. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:28. In an aspect, a disclosed IGF2 having a Leul9 substitution and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:62.

[0127] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, aLeul9 substitution, and aLeu27 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu27 substitution can comprise the sequence set forth in SEQ ID NO:29. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu27 substitution can be encoded by the sequence set forth in SEQ ID NO:63. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu27 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:29. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu27 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:63.

[0128] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, aLeul9 substitution, and aLeu43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:30. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:64. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:30. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:64.

[0129] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, and a Ser26 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Ser26 substitution can comprise the sequence set forth in SEQ ID NO:31. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Ser26 substitution can be encoded by the sequence set forth in SEQ ID NO:65. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Ser26 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:31. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Ser26 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:65.

[0130] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, aLeul9 substitution, and aMet43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Met43 substitution can comprise the sequence set forth in SEQ ID NO:32. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:66. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:32. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:66.

[0131] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Met43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can comprise the sequence set forth in SEQ ID NO:33. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:67. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:33. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:67.

[0132] In an aspect, a disclosed IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:34. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:68. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:34. In an aspect, a disclosed IGF2 having an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:68.

[0133] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leul9 substitution, aLeu27 substitution, and aMet43 substitution. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can comprise the sequence set forth in SEQ ID NO:35. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by the sequence set forth in SEQ ID NO:69. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:35. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Met43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:69.

[0134] In an aspect, a disclosed IGF2 having one or more substitutions can comprise a Leul9 substitution, aLeu27 substitution, and aLeu43 substitution. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can comprise the sequence set forth in SEQ ID NO:36. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by the sequence set forth in SEQ ID NO:70. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:36. In an aspect, a disclosed IGF2 having a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:70.

[0135] In an aspect, a disclosed IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36. In an aspect, a disclosed IGF2 having one or more substitutions can be encoded by the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70. In an aspect, a disclosed IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36. In an aspect, a disclosed IGF2 having one or more substitutions can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70. [0136] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise acid alpha glucosidase (GAA), acid ceramidase, acid sphingomyelinase, alpha-galactosidase A, alpha-glucosaminide acetyltransferase, alpha-l-fucosidase, alpha-L-iduronidase, alpha-N-acetylgalactosaminidase, alpha-N-acetylglucosaminidase, arylsulfatase A, arylsulfatase B, aspartoglucosaminidase, betagalactosidase, beta-glucocerebrosidase, beta-glucuronidase, beta-hexosaminidase, beta- mannosidase, cystinosin, formylglycine-generating enzyme, galactocerebrosidase, galactosylceramidase, heparan-a-glucosaminide-N-acetyltransferase, hyaluronidase 1, iduronate 2-sulfatase, iduronate sulfate sulfatase, LAMP2, lysosomal acid glucosylceramidase, lysosomal acid lipase / cholesteryl ester hydrolase, lysosomal a-glucosidase, lysosomal a-mannosidase, mucolipin 1, N-acetylgalactosamine-4-sulfatase, N-acetylgalactosamine-6-sulfatase, N- Acetylglucosamine-1 -phosphotransferase subunits a/p, N-acetylglucosamine-6-sulfatase, N- Acetyl-a-glucosaminidase, neuraminidase- 1, NPC intracellular cholesterol transporter 1 and 2, N- sulfoglucosamine, N-sulphoglucosamine sulphohydrolase, sialidase-1, sialin, or sphingomyelin phosphodiesterase.

[0137] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise an enzyme listed in Table 4 (below). Table 4 - Exemplary Lysosomal Enzymes with Amino Acid Sequence

[0138] In an aspect, a disclosed enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise an enzyme associated with or connected to any lysosomal disease or disorder listed in Table 5.

Table 5 - Selected LSDs and the Defective Enzymes [0139] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise any enzyme associated with Pompe Disease, Fabry Disease, Gaucher Disease, Niemann-Pick Disease, Farber Disease, Krabbe Disease, Lysosomal Acid Lipase Deficiency, GM1 Gangliosidosis, GM2 Gangliosidosis, Mucolipidosis Type I (ML1), Metachromatic Leukodystrophy, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX.

[0140] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0141] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise the sequence set forth in SEQ ID NO:71. In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:71.

[0142] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0143] Disclosed herein is polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0144] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. [0145] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0146] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0147] Disclosed herein is a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0148] In an aspect, a disclosed polypeptide can comprise a signal peptide that is cleaved during post-translation processing. In an aspect, a disclosed polypeptide can comprise a signal peptide that is not cleaved during post-translation processing. In an aspect, a disclosed signal peptide can comprise the sequence set forth in SEQ ID NO:95. In an aspect, a disclosed signal peptide can be encoded by the sequence set forth in SEQ ID NO:63. In an aspect, a disclosed polypeptide can have reduced off-target binding. In an aspect, a disclosed polypeptide can demonstrate a lower incidence of off-target binding.

[0149] In an aspect, a disclosed polypeptide can reduce off-target binding. In an aspect, a disclosed polypeptide can reduce off-target binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed polypeptide can reduce off-target binding and can increase binding to the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (CI-M6PR/IGF2R).

[0150] In an aspect, a disclosed cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (CI-M6P/IGF2R or IGF2R) is a type-1 transmembrane glycoprotein consisting of a large N-terminal extracytoplasmic domain, which allows it to bind to a wide variety of ligands. It has two major types of ligands: insulin-like growth factor 2 (IGF-2) and mannose 6- phosphate (M6P)-labeled glycosylated proteins, both of which have distinct but important roles in normal development and homeostasis in mammals.

[0151] In an aspect, a disclosed polypeptide can have reduced binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed polypeptide can have reduced binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof when compared to a polypeptide have wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, a disclosed polypeptide can have increased binding to the cation-independent mannose 6- phosphate/insulin-like growth factor 2 receptor (CI-M6PR/IGF2R). In an aspect, a disclosed polypeptide can have increased binding to CI-M6PR/IGF2R over binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed polypeptide can increase clearance of glycogen from one or more cells or tissues. In an aspect, a disclosed binding affinity to CI-M6PR/IGF2R of a disclosed polypeptide can be higher than that a polypeptide having wildtype IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, a disclosed polypeptide can experience stronger binding affinity to CI-M6PR/IGF2R than to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed polypeptide can increase enzyme uptake efficiency when compared to a polypeptide have wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, a disclosed can increase clearance of glycogen from one or more cells or tissues.

[0152] In an aspect, a disclosed lysosomal targeting peptide or fragment thereof can be operably linked to a lysosomal enzyme or a fragment thereof via a disclosed linker. In an aspect, a disclosed linker can comprise a flexible linker. In an aspect, a disclosed linker can comprise a rigid linker. [0153] In an aspect, a disclosed flexible polypeptide linker can comprise a poly-glycine (G8) linker. In an aspect, a disclosed poly-glycine (G8) linker can comprise the sequence of SEQ ID NO: 111. In an aspect, a disclosed poly-glycine (G8) linker can be encoded by the sequence of SEQ ID NO: 112. [0154] In an aspect, a disclosed flexible linker can comprise the sequence set forth in any one of SEQ ID NO: 113 - SEQ ID NO: 128. In an aspect, a disclosed rigid linker can comprise the sequence set forth in any one of SEQ ID NO: 129 - SEQ ID NO: 136. In an aspect, a disclosed linker can comprise the sequence set forth in any one of SEQ ID NO: 137 - SEQ ID NO: 152.

[0155] In an aspect, a disclosed polypeptide can restore one or more aspects of cellular homeostasis and/or cellular functionality. In an aspect, a disclosed restoration of one or more aspects of cellular homeostasis and/or cellular functionality can comprise (i) correction of cell starvation in one or more cell types; (ii) normalization of aspects of the autophagy pathway; (iii) improvement and/or restoration of mitochondrial functionality and/or structural integrity; (iv) improvement and/or restoration of organelle functionality and/or structural integrity; (v) prevention and/or slowing of hypoglycemia, ketosis, and/or other liver abnormalities; (vi) correction of liver enzyme dysregulation; (vii) prevention and/or slowing of the rate of progression of the multi-systemic manifestations an LSD; (viii) prevention and/or slowing of the rate of progression of liver disease including fibrosis, cirrhosis, hepatic adenomas, and/or liver hepatocellular carcinoma, (ix) restoration of the balance of glycogen metabolism, including glycogen synthesis and breakdown, (x) restoration of a lysosomal enzyme functionality and/or structural integrity, (xi) prevention and/or slowing of the rate of progression of the multi -systemic manifestations of any LSD listed in Table 1 (supra); or (xii) any combination thereof. In an aspect, a disclosed restoration of one or more aspects of cellular homeostasis and/or cellular functionality can comprise correction and/or mitigation of one or more presenting symptoms associated with one or more LSDs as presented in Table 1 (supra).

[0156] In an aspect, a disclosed peptide can be encapsulated in lipid nanoparticles. In an aspect, lipid nanoparticles or LNPs can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA- guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be used interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH. [0157] In an aspect, a disclosed peptide can improve one or more symptoms of an LSD. Symptoms of an LSD can comprise, for example, a delay in intellectual and physical development; seizures; facial and other bone deformities; joint stiffness and pain; difficulty breathing; problems with vision and hearing; anemia, nosebleeds, and easy bleeding or bruising; swollen abdomen due to enlarged spleen or liver; severe intellectual problems including mental retardation; behavioral problems including aggressive behavior and hyperactivity, or any combination thereof. In an aspect, an improvement in one or more symptoms of an LSD can be subjective and/or objective. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s motor skills. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s quality of life.

[0158] In an aspect, a disclosed peptide can ameliorate and/or mitigate the negative and/or deleterious effect that an LSD has on one or more organs in a subject including, for example, the brain, the spleen, the liver, the bones, the muscle, the lungs, or any combination thereof.

2. Nucleic Acid Molecules

[0159] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0160] In an aspect, encoded wild-type insulin-like growth factor 2 (IGF2) can comprise the sequence set forth in SEQ ID NO:OL In an aspect, wild-type insulin-like growth factor 2 (IGF2) can comprise the sequence set forth in SEQ ID NO:02. In an aspect, a disclosed encoded lysosomal targeting peptide can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions. In an aspect, a disclosed encoded insulin-like growth factor 2 (IGF2) can comprise one substitution. In an aspect, a disclosed encoded insulin-like growth factor 2 (IGF2) can comprise two substitutions. In an aspect, a disclosed encoded insulin-like growth factor 2 (IGF2) can comprise three substitutions. In an aspect, a disclosed encoded insulin-like growth factor 2 (IGF2) can comprise four substitutions. In an aspect, a disclosed encoded insulin-like growth factor 2 (IGF2) can comprise five substitutions. In an aspect, a disclosed encoded insulin-like growth factor 2 (IGF2) can comprise more than five substitutions. In an aspect, a disclosed substitution can occur at Glu6, Phel9, Phe26, Tyr27, Val43, or any combination thereof in a disclosed encoded IGF2. In an aspect, a disclosed substitution can occur at Glu6, Phel9, Phe26, Tyr27, Val43, or at any other position, or any combination thereof in a disclosed encoded IGF2.

[0161] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70.

[0162] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70. [0163] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can encode the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36.

[0164] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can encode a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of of SEQ ID NO: 03 - SEQ ID NO:36.

[0165] In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leu27 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution and a Leu27 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution and a Ser26 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leu27 substitution and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leu27 substitution, and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Ser26 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Ser26 substitution and aMet43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Ser26 substitution and aLeu27 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, and a Leu27 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Ser26 substitution, a Leu27 substitution, and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leu27 substitution and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leu27 substitution, and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Ser26 substitution and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Ser26 substitution, a Leu27 substitution, and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leul9 substitution and a Ser26 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leul9 substitution and a Leu27 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leul9 substitution and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leul9 substitution and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, and a Leu27 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, and a Ser26 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise an Arg6 substitution, a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leul9 substitution, a Leu27 substitution, and a Met43 substitution. In an aspect, a disclosed encoded IGF2 having one or more substitutions can comprise a Leul9 substitution, a Leu27 substitution, and a Leu43 substitution.

[0166] In an aspect, a disclosed IGF2 having one or more substitutions can be encoded by the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70. In an aspect, a disclosed IGF2 having one or more substitutions can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70.

[0167] In an aspect, a disclosed encoded lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise acid alpha glucosidase (GAA), acid ceramidase, acid sphingomyelinase, alpha-galactosidase A, alpha-glucosaminide acetyltransferase, alpha-l-fucosidase, alpha-L-iduronidase, alpha-N-acetylgalactosaminidase, alpha-N-acetylglucosaminidase, arylsulfatase A, arylsulfatase B, aspartoglucosaminidase, betagalactosidase, beta-glucocerebrosidase, beta-glucuronidase, beta-hexosaminidase, beta- mannosidase, cystinosin, formylglycine-generating enzyme, galactocerebrosidase, galactosylceramidase, heparan-a-glucosaminide-N-acetyltransferase, hyaluronidase 1, iduronate 2-sulfatase, iduronate sulfate sulfatase, LAMP2, lysosomal acid glucosylceramidase, lysosomal acid lipase / cholesteryl ester hydrolase, lysosomal a-glucosidase, lysosomal a-mannosidase, mucolipin 1, N-acetylgalactosamine-4-sulfatase, N-acetylgalactosamine-6-sulfatase, N- Acetylglucosamine-1 -phosphotransferase subunits a/p, N-acetylglucosamine-6-sulfatase, N- Acetyl-a-glucosaminidase, neuraminidase- 1, NPC intracellular cholesterol transporter 1 and 2, N- sulfoglucosamine, N-sulphoglucosamine sulphohydrolase, sialidase-1, sialin, or sphingomyelin phosphodiesterase.

[0168] In an aspect, a disclosed encoded lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise an enzyme listed in Table 4 or Table 5.

[0169] In an aspect, a disclosed encoded lysosomal enzyme can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0170] In an aspect, a disclosed enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise an enzyme associated with or connected to any lysosomal disease or disorder listed in Table 1, Table 4, Table 5. In an aspect, a disclosed encoded lysosomal enzyme can be identified by UniProtKB # P10253, P06280, P04062, P17405, Q13510, P54803, P38571, P16278, P07686, Q99519, P15289, P35475, P22304, P51688, P54802, Q68CP4, P15586, P34059.1, P16278, P15848, P08236, or Q12794.2.

[0171] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment thereof can comprise any enzyme associated with Pompe Disease, Fabry Disease, Gaucher Disease, Niemann-Pick Disease, Farber Disease, Krabbe Disease, Lysosomal Acid Lipase Deficiency, GM1 Gangliosidosis, GM2 Gangliosidosis, Mucolipidosis Type I (ML1), Metachromatic Leukodystrophy, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX.

[0172] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. [0173] In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise the sequence set forth in SEQ ID NO:71. In an aspect, a disclosed lysosomal enzyme or a fragment thereof operably linked to the lysosomal targeting peptide or fragment can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:71.

[0174] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0175] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0176] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0177] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0178] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0179] Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0180] In an aspect, a disclosed nucleic acid molecule can comprise a nucleotide sequence encoding a signal peptide that is cleaved during post-translation processing. In an aspect, a disclosed nucleic acid molecule can comprise a nucleotide sequence encoding a signal peptide that is not cleaved during post-translation processing. In an aspect, a disclosed encoded signal peptide can comprise the sequence set forth in SEQ ID NO:95. In an aspect, a disclosed nucleotide sequence encoding a signal peptide can comprise the sequence set forth in SEQ ID NO:63.

[0181] In an aspect, a disclosed encoded signal / leader peptide can be the signal / leader peptide of Human Oncostatin (OSM), Human IgKV III, Human Chymotrypsinogen, Human Trypsinogen- 2, Human Insulin, Human IL-2, Human BM40 (osteonectin SPARC), Human Serum Albumin, Human Tissue Plasminogen Activator, Secrecon, CD33, Vesicular stomatitis virus G protein (VSV-G), Gaussia luc, Influenza Haemagglutinin, Silkworm Fibroin LC, Mouse Ig Kappa, or Mouse Ig Heavy.

[0182] In an aspect, a disclosed encoded polypeptide can have reduced off-target binding. In an aspect, a disclosed encoded polypeptide can demonstrate a lower incidence of off-target binding. In an aspect, a disclosed encoded polypeptide can reduce off-target binding. In an aspect, a disclosed encoded polypeptide can reduce off-target binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed encoded polypeptide can reduce off-target binding and can increase binding to the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (CI-M6PR/IGF2R). In an aspect, a disclosed encoded polypeptide can have reduced binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed encoded polypeptide can have reduced binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof when compared to a polypeptide have wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, a disclosed encoded polypeptide can have increased binding to the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (CI-M6PR/IGF2R). In an aspect, a disclosed encoded polypeptide can have increased binding to CI-M6PR/IGF2R over binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed encoded polypeptide can increase clearance of glycogen from one or more cells or tissues. In an aspect, a disclosed binding affinity to CI-M6PR/IGF2R of a disclosed polypeptide can be higher than that a polypeptide having wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, a disclosed encoded polypeptide can experience stronger binding affinity to CI-M6PR/IGF2R than to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, a disclosed encoded polypeptide can increase enzyme uptake efficiency when compared to a polypeptide have wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, a disclosed encoded polypeptide can increase clearance of glycogen from one or more cells or tissues.

[0183] In an aspect, a disclosed encoded lysosomal targeting peptide or fragment thereof can be operably linked to a lysosomal enzyme or a fragment thereof via a disclosed linker. In an aspect, a disclosed linker can comprise a flexible linker. In an aspect, a disclosed linker can comprise a rigid linker. In an aspect, a disclosed flexible polypeptide linker can comprise a poly-glycine (G8) linker. In an aspect, a disclosed encoded poly-glycine (G8) linker can comprise the sequence of SEQ ID NO: 111. In an aspect, a disclosed poly-glycine (G8) linker can be encoded by the sequence of SEQ ID NO: 112. In an aspect, a disclosed encoded flexible linker can comprise the sequence set forth in any one of SEQ ID NO: 113 - SEQ ID NO: 128. In an aspect, a disclosed encoded rigid linker can comprise the sequence set forth in any one of SEQ ID NO: 129 - SEQ ID NO: 136. In an aspect, a disclosed encoded linker can comprise the sequence set forth in any one of SEQ ID NO: 137 - SEQ ID NO: 152.

[0184] In an aspect, a disclosed encoded polypeptide can restore one or more aspects of cellular homeostasis and/or cellular functionality. In an aspect, a disclosed restoration of one or more aspects of cellular homeostasis and/or cellular functionality can comprise (i) correction of cell starvation in one or more cell types; (ii) normalization of aspects of the autophagy pathway; (iii) improvement and/or restoration of mitochondrial functionality and/or structural integrity; (iv) improvement and/or restoration of organelle functionality and/or structural integrity; (v) prevention and/or slowing of hypoglycemia, ketosis, and/or other liver abnormalities; (vi) correction of liver enzyme dysregulation; (vii) prevention and/or slowing of the rate of progression of the multi -systemic manifestations of an LSD; (viii) prevention and/or slowing of the rate of progression of liver disease including fibrosis, cirrhosis, hepatic adenomas, and/or liver hepatocellular carcinoma, (ix) restoration of the balance of glycogen metabolism, including glycogen synthesis and breakdown, (x) restoration of a lysosomal enzyme functionality and/or structural integrity, (xi) prevention and/or slowing of the rate of progression of the multi -systemic manifestations of any LSD listed in Table 1 (supra); or (xii) any combination thereof. In an aspect, a disclosed restoration of one or more aspects of cellular homeostasis and/or cellular functionality can comprise correction and/or mitigation of one or more presenting symptoms associated with one or more LSDs as presented in Table 1 (supra).

[0185] In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that has reduced off-target binding. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can demonstrate a lower incidence of off-target binding. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can reduce off-target binding. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can reduce off-target binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can reduce off-target binding and can increase binding to the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (CLM6PR/IGF2R). In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can have reduced binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can have reduced binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof when compared to a polypeptide have wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can have increased binding to the cation-independent mannose 6- phosphate/insulin-like growth factor 2 receptor (CI-M6PR/IGF2R). In an aspect, a disclosed encoded polypeptide can generate a polypeptide that can have increased binding to CI- M6PR/IGF2R over binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can increase clearance of glycogen from one or more cells or tissues. In an aspect, a disclosed binding affinity to CI-M6PR/IGF2R of a disclosed encoded polypeptide can be higher than that a polypeptide having wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, a disclosed encoded polypeptide can experience stronger binding affinity to CI-M6PR/IGF2R than to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that can increase enzyme uptake efficiency when compared to a polypeptide have wild-type IGF2 as the lysosomal targeting peptide or fragment thereof. In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide that increase clearance of glycogen from one or more cells or tissues. In an aspect, a disclosed encoded lysosomal targeting peptide or fragment thereof can be operably linked to a lysosomal enzyme or a fragment thereof via a disclosed linker.

[0186] In an aspect, when expressed, a disclosed nucleic acid can generate a polypeptide can restore one or more aspects of cellular homeostasis and/or cellular functionality. In an aspect, a disclosed restoration of one or more aspects of cellular homeostasis and/or cellular functionality can comprise (i) correction of cell starvation in one or more cell types; (ii) normalization of aspects of the autophagy pathway; (iii) improvement and/or restoration of mitochondrial functionality and/or structural integrity; (iv) improvement and/or restoration of organelle functionality and/or structural integrity; (v) prevention and/or slowing of hypoglycemia, ketosis, and/or other liver abnormalities; (vi) correction of liver enzyme dysregulation; (vii) prevention and/or slowing of the rate of progression of the multi -systemic manifestations of an LSD; (viii) prevention and/or slowing of the rate of progression of liver disease including fibrosis, cirrhosis, hepatic adenomas, and/or liver hepatocellular carcinoma, (ix) restoration of the balance of glycogen metabolism, including glycogen synthesis and breakdown, (x) restoration of a lysosomal enzyme functionality and/or structural integrity, (xi) prevention and/or slowing of the rate of progression of the multi- systemic manifestations of any LSD listed in Table 4 (supra); or (xii) any combination thereof. In an aspect, a disclosed restoration of one or more aspects of cellular homeostasis and/or cellular functionality can comprise correction and/or mitigation of one or more presenting symptoms associated with one or more LSDs as presented in Table 1 (supra).

[0187] In an aspect, a disclosed nucleic acid sequence can be CpG depleted and codon-optimized for expression in a mammalian cell or cells or in a human cell or cells.

[0188] Disclosed herein is a nucleic acid sequence for a G6PC minimal promoter. Disclosed herein is a nucleic acid sequence for a human G6PC minimal promoter. Disclosed herein is a nucleic acid sequence for a non-human G6PC minimal promoter. In an aspect, a non-human G6PC minimal promoter can comprise a mouse promoter. [0189] In an aspect, a disclosed human G6PC min promoter can preferentially drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof.

[0190] In an aspect, a disclosed human G6PC min promoter can preferentially drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof.

[0191] In an aspect, a disclosed human G6PC min promoter can comprise the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 and that can preferentially drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof. In an aspect, a disclosed human G6PC min promoter can comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 and that can preferentially drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof.

[0192] In an aspect, a disclosed promoter can be part of a hybrid promoter. In an aspect, a disclosed hybrid promoter can comprise a human skeletal muscle-specific transcriptional cis- regulatory module. In an aspect, a disclosed hybrid promoter can comprise a human skeletal muscle-specific enhancer. In an aspect, a disclosed human skeletal muscle-specific transcriptional cis-regulatory module or enhancer can comprise Sk-CRM4. In an aspect, a disclosed Sk-CRM4 can comprise the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In an aspect, a disclosed Sk-CRM4 can comprise a fragment of the sequence set forth in SEQ ID NO: 1014 or SEQ ID NO: 102. In an aspect, a disclosed Sk-CRM4 can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102.

[0193] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can be used in a disclosed method. In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 can be used to drive the expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0194] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can be used to generate a depot of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof. In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to generate a depot of the therapeutic expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof in a subj ect’ s liver and/or in a subj ect’ s skeletal muscle.

[0195] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used in a disclosed method of mitigating the effect a defective gene (e.g., see Table 1, Table 4, and Table 5) can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof preferentially in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof.

[0196] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, (ii) a fragment of the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can be used increase (protein or gene) expression of GAA in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof.

[0197] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to decrease glycogen content in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof.

[0198] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to decrease the level of triglycerides in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof. In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to improve hepatocyte morphology and/or to decrease hepatocyte vacuolation.

[0199] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to restore normal blood glucose levels in a patient or a subject in need thereof.

[0200] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to reduce the subject’s or patient’s risk of developing long-term adverse effects associated with a lysosomal storage disease.

[0201] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to increase survival of a patient or a subject in need thereof.

[0202] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as, for example, any enzyme in Table 1, Table 4, and Table 5).

[0203] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation (such as, for example, glycogen accumulation). In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as, for example, any enzyme in Table 1, Table 4, and Table 5).

[0204] In an aspect, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO: 159 or SEQ ID NO: 160, (ii) a fragment of the sequence set forth in SEQ ID NO: 159 or SEQ ID NO: 160, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 159 or SEQ ID NO: 160 can drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof and can be used to restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation (such as, for example, glycogen accumulation). In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as, for example, any enzyme in Table 1, Table 4, and Table 5).

[0205] In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iii) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (iv) correcting enzyme dysregulation; (v) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi- systemic manifestations of a genetic disease or disorder; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, (vii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy), or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. [0206] In an aspect, a disclosed G6PC promoter can drive supraphysiologic GAA expression in a subject’s liver and/or a subject’s skeletal muscle. In an aspect, a disclosed G6PC promoter can drive supraphysiologic G6PC expression in a subject’s liver and/or a subject’s skeletal muscle. In an aspect, a disclosed G6PC promoter can restore normal glycogen content in a subject’s heart, diaphragm, quadriceps, or any combination thereof. In an aspect, a disclosed G6PC promoter can improve a subject’s muscle strength. In an aspect, a disclosed G6PC promoter can drive supraphysiologic expression of a gene having one or more pathogenetic defects in a subject’s liver and/or a subject’s skeletal muscle. In an aspect, a disclosed G6PC promoter can restore normal gene expression in a subject’s heart, diaphragm, quadriceps, or any combination thereof. In an aspect, a disclosed G6PC promoter can improve a subject’s muscle strength.

[0207] In an aspect, a disclosed promoter can ensure persistent expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof. In an aspect, a disclosed hybrid promoter can comprise a G6PC min promoter and a human skeletal muscle-specific transcriptional cis-regulatory module. In an aspect, a disclosed hybrid promoter can comprise a G6PC min promoter and a human skeletal muscle-specific enhancer. In an aspect, a disclosed hybrid promoter can comprise a G6PC min promoter and a hSk-CRM4 regulatory module.

[0208] In an aspect of a disclosed hybrid promoter, a disclosed G6PC min promoter can comprise the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100. In an aspect of a disclosed hybrid promoter, a disclosed G6PC min promoter can comprise a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100. In an aspect of a disclosed hybrid promoter, a disclosed G6PC min promoter can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100.

[0209] In an aspect of a disclosed hybrid promoter, a disclosed human skeletal muscle-specific transcriptional cis-regulatory module or enhancer can comprise Sk-CRM4. In an aspect of a disclosed hybrid promoter, a disclosed Sk-CRM4 regulatory module can comprise the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In an aspect of a disclosed hybrid promoter, a disclosed Sk-CRM4 regulatory module can comprise a fragment of the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In an aspect of a disclosed hybrid promoter, a disclosed Sk- CRM4 regulatory module can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102.

[0210] In an aspect of a disclosed hybrid promoter comprising a G6PC min promoter and a hSk- CRM4 regulatory module can comprise a spacer or a linker. In an aspect, a disclosed hybrid promoter can comprise the sequence set forth in any one of SEQ ID NO: 103 - SEQ ID NO: 106 or a fragment thereof. In an aspect, a disclosed hybrid promoter can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in any one of SEQ ID NO: 103 - SEQ ID NO: 106 or a fragment thereof.

[0211] In an aspect, a disclosed hybrid promoter can comprise the sequence set forth in any one of SEQ ID NO: 107 - SEQ ID NO: 110 or a fragment thereof. In an aspect, a disclosed hybrid promoter can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in any one of SEQ ID NO: 107 - SEQ ID NO: 110 or a fragment thereof.

[0212] In an aspect, a disclosed nucleic acid molecule can comprise a CpG-free sequence. In an aspect, “CpG-free” can mean completely free of CpGs or partially free of CpGs. In an aspect, “CpG-free” can mean “CpG-depleted”. In an aspect, “CpG-depleted” can mean “CpG-free”. In an aspect, “CpG-depleted” can mean completely depleted of CpGs or partially depleted of CpGs. In an aspect, “CpG-free” can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art.

[0213] In an aspect, a disclosed nucleic acid molecule can comprise the sequence for one or more regulatory elements. In an aspect, a disclosed regulatory element can comprise 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). In an aspect, a disclosed regulatory element can comprise Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), an iron response element, or any combination thereof. For example, a disclosed regulatory element can comprise a promoter operably linked to a disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed encoded polypeptide.

[0214] In an aspect, a disclosed nucleic acid molecule can be encapsulated in lipid nanoparticles. In an aspect, lipid nanoparticles or LNPs can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA-guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-

(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be used interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH.

[0215] In an aspect, a disclosed nucleic acid, when expressed, can improve one or more symptoms of an LSD. Symptoms of an LSD can comprise, for example, a delay in intellectual and physical development; seizures; facial and other bone deformities; joint stiffness and pain; difficulty breathing; problems with vision and hearing; anemia, nosebleeds, and easy bleeding or bruising; swollen abdomen due to enlarged spleen or liver; severe intellectual problems including mental retardation; behavioral problems including aggressive behavior and hyperactivity, or any combination thereof. In an aspect, an improvement in one or more symptoms of an LSD can be subjective and/or objective. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s motor skills. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s quality of life.

[0216] In an aspect, a disclosed nucleic acid, when expressed, can ameliorate and/or mitigate the negative and/or deleterious effect that an LSD has on one or more organs in a subject including, for example, the brain, the spleen, the liver, the bones, the muscle, the lungs, or any combination thereof.

[0217] Disclosed herein is an expression cassette comprising a disclosed promoter operably linked to a disclosed nucleic acid. Disclosed herein is an expression cassette comprising a disclosed promoter operably linked to a disclosed nucleic acid and comprising one or more disclosed regulatory elements. Disclosed herein is an expression cassette comprising a disclosed promoter operably linked a nucleic acid molecule comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70, and wherein the expression cassette comprises one or more disclosed regulatory elements. Disclosed herein is an expression cassette comprising a disclosed promoter operably linked to a nucleic acid molecule comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70„ and wherein the expression cassette comprises one or more disclosed regulatory elements. Disclosed herein is an expression cassette comprising a disclosed promoter operably linked to a nucleic acid molecule comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can encode the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36, and wherein the expression cassette comprises one or more disclosed regulatory elements. Disclosed herein is an expression cassette comprising a disclosed promoter operably linked to a nucleic acid molecule comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can encode a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of of SEQ ID NO:03 - SEQ ID NO:36, and wherein the expression cassette comprises one or more disclosed regulatory elements. Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, and wherein the expression cassette comprises one or more disclosed regulatory elements. In an aspect, a disclosed expression cassette can be incorporated into a disclosed vector (discussed below).

[0218] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0219] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70.

[0220] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:70.

[0221] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can encode the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36.

[0222] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can encode a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of of SEQ ID NO:03 - SEQ ID NO:36.

[0223] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0224] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0225] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0226] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0227] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. [0228] Disclosed herein is a nucleic acid molecule, comprising: a promoter operably linked to a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

3. Vectors

[0229] Disclosed herein is a vector comprising a disclosed nucleic acid molecule. Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding one or more disclosed encoded polypeptides. Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof. Polypeptides comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof are disclosed supra.

[0230] Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0231] Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0232] Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0233] Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0234] Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. [0235] Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0236] In an aspect, a disclosed vector can be 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 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.

[0237] In an aspect, a disclosed viral vector can be an adeno-associated virus (AAV) vector In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV- DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, MyoAAV4A, MyoAAV4E, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be AAV8. In an aspect, a disclosed AAV vector can be AAVhum.8. In an aspect, a disclosed AAV vector can be a self-complementary AAV as disclosed herein.

[0238] In an aspect, a disclosed nucleic acid sequence can have a coding sequence that is less than about 4.0 kilobases.

[0239] In an aspect, a disclosed promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art.

[0240] In an aspect, a disclosed vector can comprise a liver-specific promoter operably linked to the isolated nucleic acid molecule. In an aspect, a disclosed liver-specific promoter can be the thyroxin binding globulin (TBG) promoter, the al-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone- binding globulin promoter, the thyroxin binding globulin promoter, the a-l-anti-trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human al- antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC 172 promoter consisting of the hAAT promoter and the al -microglobulin enhancer, the DC 190 promoter containing the human albumin promoter and the prothrombin enhancer, or any one of other natural and synthetic liver-specific promoter.

[0241] In an aspect, a disclosed liver-specific promoter can comprise any liver-specific promoter known to the art. In an aspect, a disclosed encoded liver-specific promoter can comprise the sequence set forth in SEQ ID NO:97 or SEQ ID NO: 161. In an aspect, a disclosed encoded liverspecific promoter can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO:97 or SEQ ID NO: 161. In an aspect, a disclosed encoded liverspecific promoter can comprise a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 161.

[0242] In an aspect, a disclosed promoter can comprise an immunotolerant dual promoter. In an aspect, a disclosed immunotolerant dual promoter can comprise a LSP-CB promoter. In an aspect, a disclosed immunotolerant dual promoter can comprise the sequence set forth in SEQ ID NO: 159. In an aspect, a disclosed immunotolerant dual promoter can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 159. In an aspect, a disclosed immunotolerant dual promoter can comprise a sequence having at least 40%-60%, at least 60%- 80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in SEQ ID NO: 159.

[0243] In an aspect, a disclosed promoter can comprise a dual promoter. In an aspect, a disclosed dual promoter can comprise an LSP-CMV/hEFla promoter. In an aspect, a disclosed LSP- CMV/hEFla promoter can comprise the sequence set forth in SEQ ID NO: 160. In an aspect, a disclosed LSP-CMV/hEFla promoter can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 160. In an aspect, a disclosed LSP- CMV/hEFla promoter can comprise a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in SEQ ID NO: 160.

[0244] In an aspect, a disclosed ubiquitous promoter can be a CMV enhancer/chicken [Lactin promoter (CB promoter). In an aspect, a disclosed promoter can be a disclosed G6PC promoter or disclosed G6PC min promoter. In an aspect of a disclosed vector, a disclosed human G6PC min promoter can comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 and that can preferentially drive expression of a disclosed encoded polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof in kidney tissue, heart tissue, skeletal muscle tissue, liver tissue, or any combination thereof. In an aspect of a disclosed vector, a disclosed promoter comprising (i) the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, (ii) a fragment of the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, or (iii) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100 can be used in a disclosed method.

[0245] In an aspect of a disclosed vector, a disclosed promoter can be part of a hybrid promoter. In an aspect of a disclosed vector, a disclosed hybrid promoter can comprise a human skeletal muscle-specific transcriptional cis-regulatory module. In an aspect of a disclosed vector, a disclosed hybrid promoter can comprise a human skeletal muscle-specific enhancer. In an aspect of a disclosed vector, a disclosed human skeletal muscle-specific transcriptional cis-regulatory module or enhancer can comprise Sk-CRM4. In an aspect, a disclosed Sk-CRM4 can comprise the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In an aspect, a disclosed Sk-CRM4 can comprise a fragment of the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In an aspect, a disclosed Sk-CRM4 can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102.

[0246] In an aspect of a disclosed vector, a disclosed hybrid promoter can comprise the sequence set forth in any one of SEQ ID NO: 103 - SEQ ID NO: 106 or a fragment thereof. In an aspect, a disclosed hybrid promoter can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in any one of SEQ ID NO: 103 - SEQ ID NO: 106 or a fragment thereof.

[0247] In an aspect of a disclosed vector, a disclosed hybrid promoter can comprise the sequence set forth in any one of SEQ ID NO: 107 - SEQ ID NO:110 or a fragment thereof. In an aspect, a disclosed hybrid promoter can comprise a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than at least 95% identity to the sequence set forth in any one of SEQ ID NO: 107 - SEQ ID NO: 110 or a fragment thereof.

[0248] In an aspect, a disclosed nucleic acid molecule can comprise the sequence for one or more regulatory elements. In an aspect, a disclosed regulatory element can comprise 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). In an aspect, a disclosed regulatory element can comprise Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), an iron response element, or any combination thereof. For example, a disclosed regulatory element can comprise a promoter operably linked to a disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed encoded polypeptide.

[0249] Disclosed herein is a vector comprising a promoter operably linked to a disclosed nucleic acid molecule that can drive the expression of the encoded polypeptide in one or more target tissues (e.g., liver, muscle, heart, brain, etc., or any combination thereof).

[0250] In an aspect, a disclosed vector can improve one or more symptoms of an LSD. Symptoms of an LSD can comprise, for example, a delay in intellectual and physical development; seizures; facial and other bone deformities; joint stiffness and pain; difficulty breathing; problems with vision and hearing; anemia, nosebleeds, and easy bleeding or bruising; swollen abdomen due to enlarged spleen or liver; severe intellectual problems including mental retardation; behavioral problems including aggressive behavior and hyperactivity, or any combination thereof. In an aspect, an improvement in one or more symptoms of an LSD can be subjective and/or objective. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s motor skills. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s quality of life.

[0251] In an aspect, a disclosed vector can ameliorate and/or mitigate the negative and/or deleterious effect that an LSD has on one or more organs in a subject including, for example, the brain, the spleen, the liver, the bones, the muscle, the lungs, or any combination thereof.

4. Pharmaceutical Formulations

[0252] Disclosed herein is a pharmaceutical formulation comprising a disclosed polypeptide.

[0253] Disclosed herein is a pharmaceutical formulation comprising a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0254] Disclosed herein is a pharmaceutical formulation comprising a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0255] Disclosed herein is a pharmaceutical formulation comprising polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0256] Disclosed herein is a pharmaceutical formulation comprising a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0257] Disclosed herein is a pharmaceutical formulation comprising a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0258] Disclosed herein is a pharmaceutical formulation comprising a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0259] Disclosed herein is a pharmaceutical formulation comprising a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0260] Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule. Disclosed herein is a nucleic acid molecule, comprising: a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof. Nucleic acid molecules are discussed supra.

[0261] Disclosed herein is a pharmaceutical formulation comprising a disclosed vector. Vectors are discussed supra.

[0262] 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 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), mycophenolate 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).

[0263] In an aspect, a disclosed formulation can comprise an RNA therapeutic. A RNA therapeutic can comprise RNA-mediated interference (RNAi) and/or antisense oligonucleotides (ASO). A disclosed RNA therapeutic can comprise therapy delivered via LNPs. In an aspect, a disclosed pharmaceutical formulation can comprise an enzyme or enzyme precursor for enzyme replacement therapy (ERT).

[0264] In an aspect, a disclosed pharmaceutical formulation can improve one or more symptoms of an LSD. Symptoms of an LSD can comprise, for example, a delay in intellectual and physical development; seizures; facial and other bone deformities; joint stiffness and pain; difficulty breathing; problems with vision and hearing; anemia, nosebleeds, and easy bleeding or bruising; swollen abdomen due to enlarged spleen or liver; severe intellectual problems including mental retardation; behavioral problems including aggressive behavior and hyperactivity, or any combination thereof. In an aspect, an improvement in one or more symptoms of an LSD can be subjective and/or objective. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s motor skills. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s quality of life.

[0265] In an aspect, a disclosed pharmaceutical formulation can ameliorate and/or mitigate the negative and/or deleterious effect that an LSD has on one or more organs in a subject including, for example, the brain, the spleen, the liver, the bones, the muscle, the lungs, or any combination thereof.

5. Plasmids

[0266] Disclosed herein is a plasmid comprising one or more disclosed nucleic acids molecules and one or more disclosed promoters. Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof.

[0267] Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0268] Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0269] Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0270] Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0271] Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0272] Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

6. Cells

[0273] Disclosed herein are cells comprising a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed plasmid. Disclosed herein are cells contacted with one or more disclosed polypeptides, one or more disclosed nucleic acid molecule, one or more disclosed vectors, and/or one or more disclosed plasmids. Cells are known to the art.

7. Animals

[0274] Disclosed herein are animals treated with a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed plasmid. Disclosed herein are animals contacted with one or more disclosed polypeptides, one or more disclosed nucleic acid molecule, one or more disclosed vectors, and/or one or more disclosed plasmids.

8. Kits

[0275] Disclosed herein is a kit comprising a disclosed polypeptide, 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 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 LSD or an LSD- related complication.

[0276] 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 an LSD).

[0277] 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.

[0278] 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 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). [0279] The label or package insert can indicate that 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 LSD or complications and/or symptoms associated with a disclosed LSD. A kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes.

D. Methods of Treating and/or Preventing Disease Progression

1. Polypeptide-Based Methods

[0280] Disclosed herein is a method of treating and/or preventing disease progression, the method comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof.

[0281] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, and modulating the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0282] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, and increasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0283] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, and decreasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0284] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is modulated.

[0285] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is increased.

[0286] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is decreased.

[0287] Disclosed herein is a method of treating and/or preventing disease progression, the method comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof.

[0288] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, and modulating the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0289] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, and increasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0290] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, and decreasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0291] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is modulated.

[0292] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is increased.

[0293] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is decreased.

[0294] In an aspect, a disclosed polypeptide can comprise a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0295] In an aspect, a disclosed polypeptide can comprise a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0296] In an aspect, a disclosed polypeptide can comprise a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0297] In an aspect, a disclosed polypeptide can comprise a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0298] In an aspect, a disclosed polypeptide can comprise a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0299] In an aspect, a disclosed polypeptide can comprise a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0300] In an aspect, a disclosed polypeptide can be any disclosed polypeptide. In an aspect, a disclosed pharmaceutical formulation can be any disclosed pharmaceutical formulation.

[0301] Disclosed herein is a method of treating and/or preventing disease progression, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more disclosed polypeptides or a pharmaceutical formulation thereof.

[0302] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of one or more disclosed polypeptides or a pharmaceutical formulation thereof, and modulating the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0303] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of one or more disclosed polypeptides or a pharmaceutical formulation thereof, and increasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0304] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of one or more disclosed polypeptides or a pharmaceutical formulation thereof, and decreasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0305] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of one or more disclosed polypeptides or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is modulated.

[0306] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of one or more disclosed polypeptides or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is increased.

[0307] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of one or more disclosed polypeptides or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is decreased.

[0308] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed mutant IGF2-hGAA polypeptide or a pharmaceutical formulation thereof, and increasing the expression level and/or activity level of GAA.

[0309] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed mutant IGF2-hGAA polypeptide or a pharmaceutical formulation thereof, wherein the expression level and/or activity level of GAA is increased.

[0310] In an aspect, a subject can have a disclosed lysosomal storage disease. LSDs, for example, are disclosed supra in Table 1. Lysosomal enzymes are disclosed, for example, in Table 1, Table 4, and Table 5. [0311] In an aspect, a therapeutically effective amount of a disclosed polypeptide can comprise at least about 0.1 mg/kg body weight to at least about 100 mg/kg body weight. In an aspect, a therapeutically effective amount of a disclosed polypeptide can comprise at least about 0.1 mg/kg body weight to at least about 1 mg/kg body weight, at least about 1 mg/kg body weight to at least about 10 mg/kg body weight, at least about 10 mg/kg body weight to at least about 20 mg/kg body weight, at least about 20 mg/kg body weight to at least about 30 mg/kg body weight, at least about 30 mg/kg body weight to at least about 40 mg/kg body weight, at least about 40 mg/kg body weight to at least about 50 mg/kg body weight, at least about 50 mg/kg body weight to at least about 60 mg/kg body weight, at least about 60 mg/kg body weight to at least about 70 mg/kg body weight, at least about 70 mg/kg body weight to at least about 80 mg/kg body weight, at least about 80 mg/kg body weight to at least about 90 mg/kg body weight, or at least about 90 mg/kg body weight to at least about 100 mg/kg body weight.

[0312] In an aspect, regular interval of administration of a disclosed polypeptide can comprise bimonthly, monthly, triweekly, biweekly, weekly, twice weekly, daily, or at variable intervals.

[0313] In an aspect, administration of a disclosed polypeptide to a subject can comprise intravenous injection, intramuscular injection, intracoronary injection, intracerebroventricular injection, intracisterna magna injection, intracerebral injection, intrathecal injection, or any combination thereof.

[0314] In an aspect, a subject can be an adult, a child, or an infant. In an aspect, a subject can be treatment-naive.

[0315] In an aspect, a disclosed LSD can be Pompe disease, and the lysosomal enzyme can be a- glucosidase. In an aspect, a disclosed LSD can be Fabry disease (GLA), and the lysosomal enzyme can be a- Galactosidase A. In an aspect, a disclosed LSD can be Gaucher Disease, and the lysosomal enzyme can be Lysosomal Acid Gucosylceramidase. In an aspect, a disclosed LSD can be Niemann-Pick Disease, and the lysosomal enzyme can be Acid Sphingomyelinase. In an aspect, a disclosed LSD can be Farber Disease, and the lysosomal enzyme can be Acid Ceramidase. In an aspect, a disclosed LSD can be Krabbe Disease, and the lysosomal enzyme can be Galactocerebrosidase. In an aspect, a disclosed LSD can be Lysosomal Acid Lipase Deficiency, and the lysosomal enzyme can be Lysosomal Acid Lipase. In an aspect, a disclosed LSD can be GM1 Gangliosidosis, and the lysosomal enzyme can be Acid Beta-Galactosidase. In an aspect, a disclosed LSD can be GM2 Gangliosidosis, and the lysosomal enzyme can be Beta- Hexosaminidase. In an aspect, a disclosed LSD can be Mucolipidosis Type I (ML1), and the lysosomal enzyme can be Sialidase-1. In an aspect, a disclosed LSD can be Metachromatic Leukodystrophy, and the lysosomal enzyme can be Arylsulfatase A. In an aspect, a disclosed LSD can be MPS I, and the lysosomal enzyme is Alpha-L-Iduronidase. wherein the lysosomal storage disease is MPS II, and the lysosomal enzyme can be Iduronate Sulfate Sulfatase. In an aspect, a disclosed LSD can be MPS IIIA, and the lysosomal enzyme can be N- Sulphoglucosamine sulphohydrolase. In an aspect, a disclosed LSD can be MPS IIIB, and the lysosomal enzyme can be Alpha-N-Acetylglucosaminidase. In an aspect, a disclosed LSD can be MPS IIIC, and the lysosomal enzyme can be Alpha-Alucosaminide Acetyltransferase. In an aspect, a disclosed LSD can be MPS IIID, and the lysosomal enzyme can be N- Acetylglucosaminine-6-Sulfatase. In an aspect, a disclosed LSD can be MPS IVA, and the lysosomal enzyme can be N-Acetylgalactosamine-6-sulfatase. In an aspect, a disclosed LSD can be MPS IVB, and the lysosomal enzyme can be Beta-Galactosidase. In an aspect, a disclosed LSD can be MPS VI, and the lysosomal enzyme can be N-Acetylgalactosamine-4-Sulfatase. In an aspect, a disclosed LSD can be MPS VII, and the lysosomal enzyme can be Beta- Glucuronidase. wherein the lysosomal storage disease is MPS IX, and the lysosomal enzyme can be Hyaluronidase. In an aspect, a disclosed LSD can be a-Mannosidosis (type I mild, type II moderate, and type III severe and the lysosomal enzyme can be Lysosomal a-mannosidase. In an aspect, a disclosed LSD can be P-Mannosidosis, and the lysosomal enzyme can be P-Mannosidase. In an aspect, a disclosed LSD can be Fucosidosis, and the lysosomal enzyme can be a-1- Fucosidase. In an aspect, a disclosed LSD can be Aspartylglucosaminuria, and the lysosomal enzyme can be Aspartoglucosaminidase. In an aspect, a disclosed LSD can be Schindler disease (Kanzaki disease), and the lysosomal enzyme can be a-A-Acetyl-galactosaminidase. In an aspect, a disclosed LSD can be Sialidosis type I, and the lysosomal enzyme can be Neuraminidase- 1. In an aspect, a disclosed LSD can be Sialidosis type II, and the lysosomal enzyme can be Neuraminidase- 1. In an aspect, a disclosed LSD can be Galactosialidosis, and the lysosomal enzyme can be cathepsin A and/or P-galactosidase. In an aspect, a disclosed LSD can be Acid lipase deficiency (Wolman disease) (LIPA), and the lysosomal enzyme can be Lysosomal acid lipase/cholesteryl ester hydrolase. In an aspect, a disclosed LSD can be Multiple sulfatase deficiency (SUMF1), and the lysosomal enzyme can be Formylgly cine-generating enzyme. In an aspect, a disclosed LSD can be Cystinosis, and the lysosomal enzyme can be Cystinosin. In an aspect, a disclosed LSD can be Danon disease, and the lysosomal enzyme can be LAMP2.

[0316] In an aspect, a disclosed method can restore the balance of lysosomal metabolism. In an aspect, a disclosed method can restore one or more aspects of lysosomal metabolism. In an aspect, a disclosed method can correct or restore one or more aspects of the lysosomal metabolism. In an aspect, a disclosed method can correct, restore, supplement, and/or replenish the enzymatic activity of one or more disclosed lysosomal enzymes. In an aspect, a disclosed method can correct, restore, supplement, and/or replenish the enzymatic activity of one or more disclosed lysosomal enzymes (see, for example, those disclosed in Table 5). [0317] In an aspect, restoring the activity and/or expression and/or functionality of a disclosed lysosomal enzyme can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration 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, for example, a pretreatment level. In an aspect, restoration can be measured against a control level (e.g., a level in a subject not having a LSD). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity and/or functionality is similar to that of a wild-type or control level.

[0318] In an aspect, a disclosed method can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types (such as, for example, liver cells and muscle cells); (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) preventing, slowing, and/or eliminating hypoglycemia, ketosis, and/or other liver abnormalities; (vi) correcting liver enzyme dysregulation; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi -systemic manifestations of a disclosed LSD; (viii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of liver disease including fibrosis, cirrhosis, hepatic adenomas, and/or liver hepatocellular carcinoma, or (ix) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0319] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, hypoglycemia can be sporadically and/or continuously measured and monitored. Methods and techniques for measuring and monitoring hypoglycemia are known to the skilled person and include, but not limited to, by continuous glucose monitoring (CGM) methods and capillary blood glucose sticks.

[0320] In an aspect, a disclosed method can comprise measuring the level or amount of one or more biomarkers (e.g., glucose, ALT, creatinine, glycogen, hepatocellular vacuolation, etc.), one or more indicators of the subject’s metabolomic health, or any combination thereof. [0321] In an aspect, a disclosed method can comprise administering to the subject one or more additional therapeutic agents. In an aspect, a disclosed therapeutic agent can comprise enzyme replacement therapy, gene therapy, mRNA therapy, small molecule therapy, substrate reduction therapy, or any combination thereof.

[0322] In an aspect, a disclosed method can comprise treating the subject. In an aspect, treating the subject can comprise administering to the subject one or more agents that modulate the level of one or more differentially present cellular metabolites. In an aspect, treating the subject can comprise implementing a change in the subject’s dietary intake of carbohydrates. Implementing a change in the subject’s dietary intake of carbohydrates can comprise adding carbohydrates to the subject’s diet, or removing carbohydrates from the subject’s diet, or changing the type of carbohydrates in the subject’s diet, or changing the frequency of carbohydrates consumed by the subject. In an aspect, treating the subject can comprise administering cornstarch to the subject, or administering glycoside to the subject, or administering one or more anaplerotic agents to the subject.

[0323] In an aspect, a disclosed method can comprise validating the efficacy of the administered polypeptide. In an aspect, validating the efficacy of the administered polypeptide can comprise administering to the subject a disclosed polypeptide, measuring the activity or expression of one or more biomarkers related to cellular function; and comparing the resulting activity or expression level of the one or more biomarkers to a control level, wherein the administered polypeptide is effective when the activity or expression level of the one or more biomarkers following treatment is modulated compared to the control level. In an aspect, modulated can comprise increasing the activity or expression level of the one or more biomarkers. In an aspect, modulated can comprise increasing the activity or expression level of the one or more biomarkers. In an aspect, modulated can comprise decreasing the activity or expression level of the one or more biomarkers. In an aspect, based on the pathology of the LSD listed in Table 1, Table 4, and Table 5, whether the modulated activity or expression level of the one or more biomarkers can be determined.

[0324] In an aspect, a disclosed method can comprise measuring one or more biomarkers prior to the administering of one or more disclosed polypeptides. In an aspect, a disclosed method can comprise measuring one or more biomarkers during the administering of one or more disclosed polypeptides. In an aspect, a disclosed method can comprise measuring one or more biomarkers after the administering of one or more disclosed polypeptides.

[0325] Thus, in an aspect, the increase or decrease in expression and/or activity level postadministration 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 (e.g., pre-administration level). In an aspect, the increase or decrease in expression and/or activity level post-administration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to a native or control level (e.g., pre-administration level). 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 (e.g., preadministration level). In an aspect, a native or control level can be a pre-disease or pre-disorder level or pre-treatment level).

[0326] In an aspect, measuring the expression of one or more disclosed biomarkers can comprise measuring the protein concentration of the transgene and/or the reporter gene or measuring the mRNA level of transgene and/or the reporter gene. For example, in an aspect, measuring the protein concentration of transgene and/or the reporter gene comprises a protein chip analysis, an immunoassay, a ligand binding assay, a MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, a SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, a radioimmunoassay, a radioimmunodiffusion assay, an octeroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, a complement fixation assay, 2D by electrophoretic analysis, liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography -Mass Spectrometry/Mass Spectrometry (LC-MS/MS), Western blotting, ELISA (enzyme linked immunosorbent assay), or any combination thereof. Similarly, in an aspect, measuring the mRNA level of a disclosed lysosomal enzyme and/or the reporter gene comprises a reverse transcription polymerase reaction (RT-PCR), a competitive reverse transcription polymerase reaction (Competitive RT-PCR), a real-time reverse transcription polymerization, an enzyme reaction (Real-time RT-PCR), an RNase protection assay (RPA), Northern blotting, a DNA chip, or any combination thereof.

[0327] In an aspect, a disclosed method 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. In an aspect, a disclosed method can further comprise repeating a monitoring step.

[0328] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, 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 days per 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.

[0329] 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 polypeptide, 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 polypeptide, 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 polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0330] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering 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. [0331] In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.

[0332] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, antithymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0333] In an aspect, a disclosed method of repairing a defective gene can further comprise administering 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 CD 19 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).

[0334] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response. [0335] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering a [32 agonist. For example, in an aspect, a disclosed method can comprises administering a P2 agonist to increase the expression of one or more receptors for a lysosomal enzyme. In an aspect, P2 agonists include but are not limited to albuterol, clenbuterol, formoterol, indacaterol, olodaterol, salmeterol, vilanterol, and any combination thereof, growth hormones (e.g., human growth hormone), autocrine glycoprotein (e.g., Follistatin), or any combination thereof (see, e.g., U.S. Patent No. 8,679,478 for a discussion of appropriate P2 agonists, which patent is incorporated by reference it its entirety for these teachings).

[0336] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering to a subject or patient one or more fibrates. In an aspect, a disclosed fibrate can comprise bezafibrate, fenofibrate, ciprofibrate, gemfibrozil, clofibrate, an analog thereof, or a combination thereof. In an aspect, a disclosed method can comprise repeating the administering of one or more fibrates one or more times. In an aspect, a therapeutically effective amount of one or more fibrates can comprise at least about 20 mg/day to at least 500 mg/day. In an aspect, a therapeutically effective amount of one or more fibrates can comprise at least about 20 mg/day, at least about 20 mg/day, about 30 mg/day, about 40 mg/day, at least about 50 mg/day, at least about 60 mg/day, at least about 70 mg/day, at least about 80 mg/day, at least about 90 mg/day, at least about 100 mg/day, at least about 120 mg/day, at least about 140 mg/day, at least about 160 mg/day, at least about 180 mg/day, at least about 200 mg/day, at least about 220 mg/day, at least about 240 mg/day, at least about 260 mg/day, at least about 280 mg/day, at least about 300 mg/day, at least about 320 mg/day, at least about 340 mg/day, at least about 360 mg/day, at least about 380 mg/day, at least about 400 mg/day, at least about 420 mg/day, at least about 440 mg/day, at least about 460 mg/day, at least about 480 mg/day, or at least about 500 mg/day.

[0337] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise modifying one or more of the disclosed steps. For example, modifying one or more of 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 a disclosed polypeptide or a pharmaceutical formulation thereof administered to a subject, or by changing the frequency of administration of a disclosed polypeptide or a pharmaceutical formulation thereof to a subject, or by changing the duration of time a disclosed polypeptide or a pharmaceutical formulation thereof is administered to a subject. In an aspect, one or more disclosed polypeptides can be administered concurrently or sequentially.

[0338] In an aspect, a disclosed method can further comprise diagnosing a subject with a genetic defect using one or more known methods to the skilled person, such as, for example, genotyping. In an aspect, a disclosed polypeptide or a pharmaceutical formulation thereof does not elicit an immune response. In an aspect, a disclosed method can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof.

[0339] In an aspect, a disclosed method can comprise generating one or more disclosed polypeptides, purifying one or more disclosed polypeptides, formulating one or more disclosed polypeptides, packaging one or more disclosed polypeptides, or any combination thereof.

[0340] In an aspect of a disclosed method, a disclosed nucleic acid molecule can be encapsulated in lipid nanoparticles. In an aspect, lipid nanoparticles or LNPs can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA-guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-di enoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be used interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH.

[0341] In an aspect, a disclosed method can further comprise administering to the subject one or more disclosed nucleic acid molecules or one or more disclosed vectors.

[0342] In an aspect, a disclosed method can improve one or more symptoms of an LSD. Symptoms of an LSD can comprise, for example, a delay in intellectual and physical development; seizures; facial and other bone deformities; joint stiffness and pain; difficulty breathing; problems with vision and hearing; anemia, nosebleeds, and easy bleeding or bruising; swollen abdomen due to enlarged spleen or liver; severe intellectual problems including mental retardation; behavioral problems including aggressive behavior and hyperactivity, or any combination thereof. In an aspect, an improvement in one or more symptoms of an LSD can be subjective and/or objective. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s motor skills. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s quality of life.

[0343] In an aspect, a disclosed method can ameliorate and/or mitigate the negative and/or deleterious effect that an LSD has on one or more organs in a subject including, for example, the brain, the spleen, the liver, the bones, the muscle, the lungs, or any combination thereof.

2. Nucleic Acid-Based Methods

[0344] Disclosed herein is a method of treating and/or preventing disease progression, the method comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and modulating the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and increasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and decreasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is modulated.

[0345] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is increased.

[0346] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is decreased. Disclosed herein is a method of treating and/or preventing disease progression, the method comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed polypeptide or a pharmaceutical formulation thereof. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and modulating the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and increasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0347] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and decreasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is modulated. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is increased. Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is decreased.

[0348] In an aspect, a disclosed nucleic acid molecule can comprise a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. In an aspect, a disclosed nucleic acid molecule can comprise a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. In an aspect, a disclosed nucleic acid molecule can comprise a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. [0349] In an aspect, a disclosed nucleic acid molecule can comprise a nucleic acid sequence encoding a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. In an aspect, a disclosed nucleic acid molecule can comprise a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. [0350] In an aspect, a disclosed nucleic acid molecule can comprise a nucleic acid sequence encoding a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0351] In an aspect, a disclosed nucleic acid molecule can be any disclosed nucleic acid molecule. In an aspect, a disclosed vector can be any disclosed vector. In an aspect of a disclosed method, following transduction, the encoded polypeptide is expressed in one or more cells of the subject. In an aspect of a disclosed method, following transfection, the encoded polypeptide is expressed in one or more cells of the subject.

[0352] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule encoding a mutant IGF2-hGAA polypeptide or a disclosed vector comprising the disclosed nucleic acid, and increasing the expression level and/or activity level of GAA. [0353] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule encoding a mutant IGF2-hGAA polypeptide or a disclosed vector comprising the disclosed nucleic acid, wherein the expression level and/or activity level of GAA is increased.

[0354] Disclosed herein is a method of treating and/or preventing disease progression, the method comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector.

[0355] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of o a disclosed nucleic acid molecule or a disclosed vector, and modulating the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0356] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and increasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0357] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, and decreasing the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD.

[0358] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is modulated.

[0359] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is increased.

[0360] Disclosed herein is a method of treating and/or preventing disease progression of a lysosomal storage disease (LSD) comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed nucleic acid molecule or a disclosed vector, wherein the expression level and/or activity level of one or more enzymes associated with and/or related to the LSD is decreased.

[0361] In an aspect, a subject can have a disclosed lysosomal storage disease. LSDs, for example, are disclosed supra in Table 1. Lysosomal enzymes are disclosed, for example, in Table 4.

[0362] In an aspect, a subject can be an adult, a child, or an infant. In an aspect, a subject can be treatment-naive.

[0363] In an aspect, a disclosed LSD can be Pompe disease, and the lysosomal enzyme can be a- glucosidase. In an aspect, a disclosed LSD can be Fabry disease (GLA), and the lysosomal enzyme can be a- Galactosidase A. In an aspect, a disclosed LSD can be Gaucher Disease, and the lysosomal enzyme can be Lysosomal Acid Gucosylceramidase. In an aspect, a disclosed LSD can be Niemann-Pick Disease, and the lysosomal enzyme can be Acid Sphingomyelinase. In an aspect, a disclosed LSD can be Farber Disease, and the lysosomal enzyme can be Acid Ceramidase. In an aspect, a disclosed LSD can be Krabbe Disease, and the lysosomal enzyme can be Galactocerebrosidase. In an aspect, a disclosed LSD can be Lysosomal Acid Lipase Deficiency, and the lysosomal enzyme can be Lysosomal Acid Lipase. In an aspect, a disclosed LSD can be GM1 Gangliosidosis, and the lysosomal enzyme can be Acid Beta-Galactosidase. In an aspect, a disclosed LSD can be GM2 Gangliosidosis, and the lysosomal enzyme can be Beta- Hexosaminidase. In an aspect, a disclosed LSD can be Mucolipidosis Type I (ML1), and the lysosomal enzyme can be Sialidase-1. In an aspect, a disclosed LSD can be Metachromatic Leukodystrophy, and the lysosomal enzyme can be Arylsulfatase A. In an aspect, a disclosed LSD can be MPS I, and the lysosomal enzyme is Alpha-L-Iduronidase. wherein the lysosomal storage disease is MPS II, and the lysosomal enzyme can be Iduronate Sulfate Sulfatase. In an aspect, a disclosed LSD can be MPS IIIA, and the lysosomal enzyme can be N- Sulphoglucosamine sulphohydrolase. In an aspect, a disclosed LSD can be MPS IIIB, and the lysosomal enzyme can be Alpha-N-Acetylglucosaminidase. In an aspect, a disclosed LSD can be MPS IIIC, and the lysosomal enzyme can be Alpha-Alucosaminide Acetyltransferase. In an aspect, a disclosed LSD can be MPS IIID, and the lysosomal enzyme can be N- Acetylglucosaminine-6-Sulfatase. In an aspect, a disclosed LSD can be MPS IVA, and the lysosomal enzyme can be N-Acetylgalactosamine-6-sulfatase. In an aspect, a disclosed LSD can be MPS IVB, and the lysosomal enzyme can be Beta-Galactosidase. In an aspect, a disclosed LSD can be MPS VI, and the lysosomal enzyme can be N-Acetylgalactosamine-4-Sulfatase. In an aspect, a disclosed LSD can be MPS VII, and the lysosomal enzyme can be Beta- Glucuronidase. wherein the lysosomal storage disease is MPS IX, and the lysosomal enzyme can be Hyaluronidase. In an aspect, a disclosed LSD can be a-Mannosidosis (type I mild, type II moderate, and type III severe and the lysosomal enzyme can be Lysosomal a-mannosidase. In an aspect, a disclosed LSD can be P-Mannosidosis, and the lysosomal enzyme can be P-Mannosidase. In an aspect, a disclosed LSD can be Fucosidosis, and the lysosomal enzyme can be a-1- Fucosidase. In an aspect, a disclosed LSD can be Aspartylglucosaminuria, and the lysosomal enzyme can be Aspartoglucosaminidase. In an aspect, a disclosed LSD can be Schindler disease (Kanzaki disease), and the lysosomal enzyme can be a-A-Acetyl-galactosaminidase. In an aspect, a disclosed LSD can be Sialidosis type I, and the lysosomal enzyme can be Neuraminidase- 1. In an aspect, a disclosed LSD can be Sialidosis type II, and the lysosomal enzyme can be Neuraminidase- 1. In an aspect, a disclosed LSD can be Galactosialidosis, and the lysosomal enzyme can be cathepsin A and/or P-galactosidase. In an aspect, a disclosed LSD can be Acid lipase deficiency (Wolman disease) (LIPA), and the lysosomal enzyme can be Lysosomal acid lipase/cholesteryl ester hydrolase. In an aspect, a disclosed LSD can be Multiple sulfatase deficiency (SUMF1), and the lysosomal enzyme can be Formylgly cine-generating enzyme. In an aspect, a disclosed LSD can be Cystinosis, and the lysosomal enzyme can be Cystinosin. In an aspect, a disclosed LSD can be Danon disease, and the lysosomal enzyme can be LAMP2.

[0364] In an aspect, a disclosed method can restore the balance of lysosomal metabolism. In an aspect, a disclosed method can restore one or more aspects of lysosomal metabolism. In an aspect, a disclosed method can correct or restore one or more aspects of the lysosomal metabolism. In an aspect, a disclosed method can correct, restore, supplement, and/or replenish the enzymatic activity of one or more disclosed lysosomal enzymes. In an aspect, a disclosed method can correct, restore, supplement, and/or replenish the enzymatic activity of one or more disclosed lysosomal enzymes (see, for example, those disclosed in Table 5).

[0365] In an aspect, restoring the activity and/or expression and/or functionality of a disclosed lysosomal enzyme can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration 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, for example, a pretreatment level. In an aspect, restoration can be measured against a control level (e.g., a level in a subject not having a LSD). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity and/or functionality is similar to that of a wild-type or control level.

[0366] Thus, in an aspect, the increase or decrease in expression and/or activity level postadministration 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 (e.g., pre-administration level). In an aspect, the increase or decrease in expression and/or activity level post-administration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to a native or control level (e.g., pre-administration level). 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 (e.g., preadministration level). In an aspect, a native or control level can be a pre-disease or pre-disorder level or pre-treatment level).

[0367] In an aspect, a disclosed method can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types (such as, for example, liver cells and muscle cells); (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) preventing, slowing, and/or eliminating hypoglycemia, ketosis, and/or other liver abnormalities; (vi) correcting liver enzyme dysregulation; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a disclosed LSD; (viii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of liver disease including fibrosis, cirrhosis, hepatic adenomas, and/or liver hepatocellular carcinoma, or (ix) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0368] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, hypoglycemia can be sporadically and/or continuously measured and monitored. Methods and techniques for measuring and monitoring hypoglycemia are known to the skilled person and include, but not limited to, by continuous glucose monitoring (CGM) methods and capillary blood glucose sticks.

[0369] In an aspect, a disclosed method can comprise measuring the level or amount of one or more biomarkers (e.g., glucose, ALT, creatinine, glycogen, hepatocellular vacuolation, etc.), one or more indicators of the subject’s metabolomic health, or any combination thereof.

[0370] In an aspect, a disclosed method can comprise administering to the subject one or more additional therapeutic agents. In an aspect, a disclosed therapeutic agent can comprise enzyme replacement therapy, gene therapy, mRNA therapy, small molecule therapy, substrate reduction therapy, or any combination thereof. [0371] In an aspect, a disclosed method can comprise treating the subject. In an aspect, treating the subject can comprise administering to the subject one or more agents that modulate the level of one or more differentially present cellular metabolites. In an aspect, treating the subject can comprise implementing a change in the subject’s dietary intake of carbohydrates. Implementing a change in the subject’s dietary intake of carbohydrates can comprise adding carbohydrates to the subject’s diet, or removing carbohydrates from the subject’s diet, or changing the type of carbohydrates in the subject’s diet, or changing the frequency of carbohydrates consumed by the subject. In an aspect, treating the subject can comprise administering cornstarch to the subject, or administering glycoside to the subject, or administering one or more anaplerotic agents to the subject.

[0372] In an aspect, a disclosed method can comprise validating the efficacy of the administered nucleic acid molecule or vector. In an aspect, validating the efficacy of the administered nucleic acid molecule or vector can comprise administering to the subject a disclosed nucleic acid molecule or a disclosed vector, measuring the activity or expression of one or more biomarkers related to cellular function; and comparing the resulting activity or expression level of the one or more biomarkers to a control level, wherein the administered nucleic acid molecule or vector is effective when the activity or expression level of the one or more biomarkers following treatment is modulated compared to the control level. In an aspect, modulated can comprise increasing the activity or expression level of the one or more biomarkers. In an aspect, modulated can comprise increasing the activity or expression level of the one or more biomarkers. In an aspect, modulated can comprise decreasing the activity or expression level of the one or more biomarkers. In an aspect, based on the pathology of the LSD listed in Table 1, Table 4, and Table 5, whether the modulated activity or expression level of the one or more biomarkers can be determined. In an aspect, a disclosed method can comprise measuring one or more biomarkers prior to the administering of one or more disclosed polypeptides. In an aspect, a disclosed method can comprise measuring one or more biomarkers during the administering of one or more disclosed polypeptides. In an aspect, a disclosed method can comprise measuring one or more biomarkers after the administering of a disclosed nucleic acid molecule or a disclosed vector. In an aspect of a disclosed method, the expression and/or activity level of a disclosed lysosomal enzyme can be modulated.

[0373] In an aspect, measuring the expression of one or more disclosed biomarkers can comprise measuring the protein concentration of the encoded polypeptide and/or the reporter gene or measuring the mRNA level of encoded polypeptide and/or the reporter gene. For example, in an aspect, measuring the protein concentration of encoded polypeptide and/or the reporter gene comprises a protein chip analysis, an immunoassay, a ligand binding assay, a MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, a SELDLTOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, a radioimmunoassay, a radioimmunodiffusion assay, an octeroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, a complement fixation assay, 2D by electrophoretic analysis, liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography -Mass Spectrometry/Mass Spectrometry (LC-MS/MS), Western blotting, ELISA (enzyme linked immunosorbent assay), or any combination thereof. Similarly, in an aspect, measuring the mRNA level of a disclosed encoded polypeptide and/or the reporter gene comprises a reverse transcription polymerase reaction (RT-PCR), a competitive reverse transcription polymerase reaction (Competitive RT-PCR), a real-time reverse transcription polymerization, an enzyme reaction (Real-time RT-PCR), an RNase protection assay (RPA), Northern blotting, a DNA chip, or any combination thereof.

[0374] In an aspect, a disclosed method 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. In an aspect, a disclosed method can further comprise repeating a monitoring step.

[0375] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, 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 days per 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.

[0376] 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.

[0377] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering 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. [0378] In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.

[0379] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD further comprise administering 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), my cophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0380] In an aspect, a disclosed method of repairing a defective gene can further comprise administering 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 CD 19 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).

[0381] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0382] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering a [32 agonist. For example, in an aspect, a disclosed method can comprises administering a [32 agonist to increase the expression of one or more receptors for a lysosomal enzyme. In an aspect, [32 agonists include but are not limited to albuterol, clenbuterol, formoterol, indacaterol, olodaterol, salmeterol, vilanterol, and any combination thereof, growth hormones (e.g., human growth hormone), autocrine glycoprotein (e.g., Follistatin), or any combination thereof (see, e.g., U.S. Patent No. 8,679,478 for a discussion of appropriate [32 agonists, which patent is incorporated by reference it its entirety for these teachings).

[0383] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise administering to a subject or patient one or more fibrates. In an aspect, a disclosed fibrate can comprise bezafibrate, fenofibrate, ciprofibrate, gemfibrozil, clofibrate, an analog thereof, or a combination thereof. In an aspect, a disclosed method can comprise repeating the administering of one or more fibrates one or more times. In an aspect, a therapeutically effective amount of one or more fibrates can comprise at least about 20 mg/day to at least 500 mg/day. In an aspect, a therapeutically effective amount of one or more fibrates can comprise at least about 20 mg/day, at least about 20 mg/day, about 30 mg/day, about 40 mg/day, at least about 50 mg/day, at least about 60 mg/day, at least about 70 mg/day, at least about 80 mg/day, at least about 90 mg/day, at least about 100 mg/day, at least about 120 mg/day, at least about 140 mg/day, at least about 160 mg/day, at least about 180 mg/day, at least about 200 mg/day, at least about 220 mg/day, at least about 240 mg/day, at least about 260 mg/day, at least about 280 mg/day, at least about 300 mg/day, at least about 320 mg/day, at least about 340 mg/day, at least about 360 mg/day, at least about 380 mg/day, at least about 400 mg/day, at least about 420 mg/day, at least about 440 mg/day, at least about 460 mg/day, at least about 480 mg/day, or at least about 500 mg/day.

[0384] In an aspect, a disclosed method of treating and/or preventing disease progression of an LSD can further comprise modifying one or more of the disclosed steps. For example, modifying one or more of 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 a disclosed nucleic acid molecule or a disclosed vector administered to a subject, or by changing the frequency of administration of a disclosed nucleic acid molecule or a disclosed vector to a subject, or by changing the duration of time a disclosed nucleic acid molecule or a disclosed vector is administered to a subject.

[0385] In an aspect, one or more disclosed nucleic acid molecules or disclosed vectors can be administered concurrently or sequentially.

[0386] In an aspect, a disclosed method can further comprise diagnosing a subject with a genetic defect using one or more known methods to the skilled person, such as, for example, genotyping. [0387] In an aspect, a disclosed polypeptide does not elicit an immune response.

[0388] In an aspect, a disclosed method can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof.

[0389] In an aspect, a disclosed method can ensure persistent expression of a disclosed polypeptide. In an aspect, a disclosed method can ensure persistent expression of a disclosed polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof. In an aspect, a disclosed method can ensure persistent expression of a disclosed polypeptide. In an aspect, a disclosed method can ensure persistent expression of a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. In an aspect, a disclosed method can ensure persistent expression of polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92. In an aspect, a disclosed method can ensure persistent expression of a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO:36 and operably linked to a lysosomal enzyme or a fragment thereof comprising the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0390] In an aspect, a disclosed method can ensure persistent expression of a polypeptide comprising a lysosomal targeting peptide or a fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide or fragment thereof can comprise insulin-like growth factor 2 (IGF2) having one or more substitutions; wherein the IGF2 having one or more substitutions can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0391] In an aspect, a disclosed method can ensure persistent expression of a polypeptide comprising a lysosomal targeting peptide or fragment thereof operably linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide of fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO:36; and wherein the lysosomal enzyme or fragment thereof can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0392] In an aspect, a disclosed method can ensure persistent expression of a polypeptide comprising a lysosomal targeting peptide or fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO: 03 - SEQ ID NO: 36; and operably linked to a lysosomal enzyme or a fragment thereof comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

[0393] In an aspect, a disclosed method can drive supraphysiologic expression of a disclosed lysosomal enzyme in a subject’s liver and/or a subject’s skeletal muscle. In an aspect, a disclosed method can restore normal lysosomal enzyme content in a subject’s heart, diaphragm, quadriceps, or any combination thereof. In an aspect, a disclosed method can improve a subject’s muscle strength.

[0394] In an aspect, a disclosed vector can be delivered to the subject’s liver. In an aspect, a disclosed vector can be administered via intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic, hepatic intra-arterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed vector can be administered via intra-CSF administration in combination with a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation. In an aspect, 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 vector can be administered via LNP administration. In an aspect, a subject can be a human subject. In an aspect, a disclosed vector can be delivered to the subject’s liver, heart, skeletal muscle, smooth muscle, CNS, PNS, or a combination thereof. In an aspect, a disclosed vector can be concurrently and/or serially administered to a subject via multiple routes of administration.

[0395] In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ to about 8 x 10¹³ vg/kg or about 1 x 10¹² to about 8 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹³ to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x 10¹⁰, at least about 5 x 10¹⁰, at least about 1 x 10¹¹, at least about 5 x 10¹¹, at least about 1 x 10¹², at least about 5 x 10¹², at least about 1 x 10¹³, at least about 5 x 10¹³, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x 10¹⁰, no more than about 5 x 10¹⁰, no more than about 1 x 10¹¹, no more than about 5 x 10¹¹, no more than about 1 x 10¹², no more than about 5 x 10¹², no more than about 1 x 10¹³, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results. [0396] In an aspect of a disclosed method, a disclosed vector can be encapsulated in lipid nanoparticles. In an aspect, lipid nanoparticles or LNPs can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA-guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-

[0397] In an aspect, a disclosed LSD can be Pompe disease, and the lysosomal enzyme can be a- glucosidase. In an aspect, a disclosed method can restore one or more aspects of the glycogen metabolic pathway, restore one or more aspects of the glycogenolysis metabolic pathway, can restore one or more aspects of the glycogenesis metabolic pathway, or any combination thereof. For example, in an aspect, a disclosed method can restore the balance of glycogen synthesis and degradation. In an aspect, a disclosed method can restore the balance of glycogen metabolism, wherein glycogen metabolism comprises glycogen synthesis and breakdown. In an aspect, a disclosed method can further comprise reducing glycogen levels by administering a glycogen synthase inhibitor (e.g., RNAi, ASO, etc.) to the subject, or modifying the subject’s diet, for example, by using cornstarch or another slow release starch to prevent hypoglycemia, or modifying the subject’s diet, for example, by consuming a high amount of protein, fat, or other anaplerotic agents (such as, for example, C7 compounds like triheptanoin or MCT), exercise or a combination thereof. In an aspect, a disclosed method can comprise reducing the expression level, activity level, or both of glycogen synthase. In an aspect, a glycogen synthase can be GYSI (muscle glycogen synthase) or GYS2 (liver glycogen synthase) or both. In an aspect of a disclosed method, reducing the expression level and/or activity level of glycogen synthase can comprise SRT. For example, in an aspect, SRT can comprise inhibiting glycogen synthase (i.e., GYSI and/or GYS2) in a cell or a subject to reduce glycogen synthesis and/or glycogen accumulation in cells and tissues (e.g., skeletal muscle, lung tissue, liver tissue, brain tissue, or any other tissue having glycogen accumulation) when GAA and/or GBE activity and/or expression levels are reduced. In an aspect, SRT can comprise siRNA-based therapies, shRNA-based therapies, antisense therapies, gene-editing therapies, and therapies using one or more small molecules or peptide drugs. In an aspect of a disclosed method, reducing the expression level and/or activity level of glycogen synthase can comprise administering a small molecule. In an aspect, a disclosed small molecule can reduce activity and/or expression of GYSI in view of the reduced activity and/or expression level of GAA, GBE, or one or more other enzymes in the metabolic pathways of glycogen synthesis and breakdown. In an aspect, a disclosed small molecule can traverse the blood-brain-barrier. In an aspect, a disclosed small molecule can be guaiacol.

[0398] In an aspect, a disclosed method can further comprise administering to the subject one or more disclosed polypeptides or one or more pharmaceutical formulations thereof.

[0399] In an aspect, a disclosed method can improve one or more symptoms of an LSD. Symptoms of an LSD can comprise, for example, a delay in intellectual and physical development; seizures; facial and other bone deformities; joint stiffness and pain; difficulty breathing; problems with vision and hearing; anemia, nosebleeds, and easy bleeding or bruising; swollen abdomen due to enlarged spleen or liver; severe intellectual problems including mental retardation; behavioral problems including aggressive behavior and hyperactivity, or any combination thereof. In an aspect, an improvement in one or more symptoms of an LSD can be subjective and/or objective. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s motor skills. In an aspect, an improvement in one or more symptoms of an LSD can improve a subject’s quality of life.

[0400] In an aspect, a disclosed method can ameliorate and/or mitigate the negative and/or deleterious effect that an LSD has on one or more organs in a subject including, for example, the brain, the spleen, the liver, the bones, the muscle, the lungs, or any combination thereof.

VII. EXAMPLES

[0401] Pompe Disease or PD (also known as glycogen storage disease type II) is a rare neuromuscular disorder with an incidence of approximately one in every 16,000 births. The disease is caused by acid-a-glucosidase (GAA) deficiency, which leads to pathological buildup of lysosomal glycogen primarily in heart, skeletal muscles, respiratory system, and the central nervous system (CNS). Patients with infantile-onset PD (IOPD) typically develop both cardiomyopathy and skeletal muscle myopathy and die within the first year of life from cardiorespiratory failure if not treated; some individuals may also present with CNS involvement such as neuroinflammation, neurodegeneration, and cognitive impairment. Patients with late- onset PD (LOPD) have milder phenotypes including progressive skeletal muscle myopathy and impaired motor function, with respiratory failure being the most common cause of mortality; morphological and functional brain alterations with mild neuropsychological dysfunction have been reported in some cases.

[0402] Cation-independent mannose 6-phosphate receptor (CI-M6PR)-mediated ERT with rhGAA (alglucosidase alfa) is the current standard of care for PD and seems to be effective in slowing disease progression and improving survival, but it has not been shown to halt or reverse disease for the majority of patients, due to poor enzyme uptake in skeletal muscles and the inability of the therapeutic enzyme to cross the blood-brain barrier to correct neuronal deficits in the CNS. [0403] Next generation ERT using chemically modified hGAA containing a higher content of the M6P moi eties (avalglucosidase alfa and cipaglucosidase alfa) have recently been approved for the treatment of LOPD, however, neither has shown clear superiority over standard ERT in patients, despite promising outcomes in pre-clinical studies. Other notable limitations of ERT include the requirement for lifetime intravenous enzyme administration, high drug cost, and, most importantly, the development of antibody responses and allergic reactions from repeated infusions of high-dose enzyme that limit therapeutic efficacy and cause safety issues. Thus, significant unmet medical needs for patients with PD remain.

[0404] Gene therapy with adeno-associated virus (AAV) vectors is an alternative approach with the potential to provide a curative solution for PD. Current AAV vectors, AAV serotype 9 (AAV9) in particular, can reliably transduce liver, muscle, and the brain. AAV9 has been extensively used in preclinical studies and clinical trials for treatment of numerous diseases, and sufficient safety and efficacy profiles have been established. In the past two decades, several AAV gene therapy approaches for PD have been advanced with successful translation to early phase clinical trials. As PD is a multiple-organ disorder, a systemic gene therapy approach is preferred. However, intravenous administration of an AAV vector containing the ubiquitous CMV enhancer/chicken P-actin (CB) promoter provoked both cellular and antibody responses against hGAA, leading to a quick loss of transgene expression in GAA-KO mice.

[0405] Muscle directed gene therapy by systemic injection of an AAV vector containing a muscle specific promoter can evade hGAA-specific cellular (but not humoral) immune response and achieve long-term, stable hGAA expression and glycogen reduction in the cardiac and skeletal muscles of GAA-KO mice. This approach has major limitations, though, including administration of very high doses of AAV vectors that can cause significant hepatotoxicity and genotoxicity in patients, the lack of cross-correction among muscle cells due to the development of anti-hGAA antibodies, and the inability to correct disease abnormalities in the CNS where the muscle-specific promoter is not active. Liver-depot gene therapy using an AAV vector containing a liver-specific promoter can induce immune tolerance to hGAA in GAA-KO mice. This liver targeted gene therapy, which functions as an constitutive ERT using liver as a depot to continuously produce and secret hGAA into blood circulation for cross-correction of muscle tissues, has achieved broad correction of muscles in GAA-KO mice. However, clinical studies suggested that this liver-depot approach has limited effect on skeletal muscle in human patients with LOPD (similar to ERT with rhGAA). Liver gene therapy with AAV vectors expressing highly secretable hGAA (containing a heterologous signal peptide from secretory proteins) can significantly increase the amount of hGAA in blood circulation, leading to improved capability of reversing biochemical abnormalities in skeletal muscle, heart, and even the brain in GAA-KO mice. However, the alternative signal peptide changes the route of hGAA sorting and trafficking from lysosomal to the secretory pathway, resulting in poorly glycosylated (low M6P content) and phosphorylated hGAA, which reduces its affinity to the CI-M6PR and limits efficacy of this approach.

[0406] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only and are not to be taken as limiting the invention.

Example 1

Site-Specific Mutagenesis of the IGF2 Tag to Reduced Off-Target Binding of the IGF2-hGAA Chimeric Enzyme to the IR and IGF1R

[0407] Based on the critical sites on human IGF2 for binding to IR, IGF1R, and IGF binding proteins (IGFBPs), mutagenesis of specific residues was undertaken. Specifically, mutations at the residues of Phe26, Tyr27, Val43, and Glu6 were made to reduce the IGF2 off-target binding to IR, IGFR1, and IGFBPs while not affecting its affinity to the CI-M6PR/IGF2R (FIG. 1).

[0408] The regeneration IGF2-hGAA variants were generated by making a series of DNA plasmids expressing the highly secretable unmodified IGF2-hGAA and six variants (ml -hGAA to m6-hGAA) containing either a single or double mutations at the positions of Phe26, Tyr27, and/or Val43, or with an additional mutation at the position of Glu6. The variants were driven by the ubiquitous CMV enhancer/chicken P-actin (CB) promoter (FIG. 2; Table 1).

Table 6 - Protein and DNA Sequences of the Residues Mutated in the IGF2 Mutants

Table 7 - IGF2 Mutants and Relevant Sequences

[0409] To produce IGF2-hGAA chimeric enzymes, HEK293 cells in 150-mm plates were transfected with 30 pg DNA plasmid for each fusion enzyme using standard calcium-phosphate method. A plasmid expressing the untagged hGAA was used to generate the control enzyme. The next day, cells were switched to low serum medium (3% FBS) and culture media were collected every 24 hr. (and fresh medium replenished) for 3 days. The combined media were concentrated using Centricon plus-70 filters (cut-off 100 kDa) and stored at -80 °C until use.

Example 2

Mutagenesis of the IGF2 Tag in l^st-Generation IGF2-hGAA Variants Remarkably Increased Enzyme Uptake Efficiency in Cultured Muscle Cells

[0410] Rat L6 myoblasts in 12-well plates were incubated with each of the concentrated enzymes (1000 U/mL) with or without the competitor M6P (5mM) or human IGF2 (2.4 mM). After 24 hr., cells were washed twice with phosphate-buffered saline (PBS), lysed in 100 pL NP-40 lysis buffer, sonicated twice, and centrifuged for 10 min at 4 °C. GAA activity in cell lysates was analyzed and fusion proteins were visualized by Western blotting. As shown in FIG. 3A, the unmodified IGF2-hGAA and all the six IGF2-hGAA variants displayed remarkably greater enzyme uptake efficiency than the untagged hGAA in the L6 cells. The addition of IGF2 in the culture medium almost completely blocked the uptake of all the IGF2-hGAA fusion proteins; in contrast, M6P only blocked the uptake of the untagged hGAA. Western blot analysis revealed that all the IGF2-hGAA fusion proteins, including the unmodified IGF2-hGAA and its variants, were correctly processed into the normal 67-kDa mature form GAA protein, like the untagged hGAA, in the L6 cells (FIG. 3B).

Example 3

Mutagenesis of the IGF2 Tag Remarkably Reduced the Binding of l^st-Generation IGF2-hGAA Variants to the IR and IGF1R Over-Expressed in the HEK293 Cells

[0411] HEK293 cells were seeded in 12-well plates and transfected next day with 0.8 pg of the pCMV-IR plasmid (Addgene cat. #24049) or pCMV-IGFIR plasmid (Addgene cat #201979), to overexpress IR and IGF1R. After 48 hr., the culture medium was replaced with fresh medium containing the concentrated enzymes (300 U/mL). After incubation for 2 hr., cell lysates were prepared as described above. Binding of unmodified IGF2-GAA to HEK293 cells was enhanced by over-expression of IR or IGF1R (FIG. 4A). In comparison with the unmodified IGF2-hGAA, all the IGF2-hGAA variants demonstrated significantly lowered binding affinity to the IR (FIG. 4B) and IGF1R (FIG. 4C), as determined by GAA activity assay.

Example 4

The Modification of the IGF2 Tag Did Not Affect the Efficacy of l^st-Generation IGF2-hGAA Variants in Pompe Disease (GAA-KO) Mice

[0412] A proof-of concept in vivo experiment to compare the treatment efficacy between the unmodified IGF2-hGAA and IGF2-hGAA variants was performed. AAV plasmid vectors expressing the unmodified IGF2-hGAA (AAV-IGF2-hGAA) and two regeneration IGF2-hGAA variants (AAV-IGF2^ml-hGAA and AAV-IGF2^m2-hGAA) under control of the 448-bp liverspecific LP1 promoter were constructed (FIG. 5) and packaged into AAV9 capsid. These AAV vectors were intravenously injected at the same dose (1 x 10¹³ vg/kg) into 4-month-old GAA-KO mice. Mice were euthanized after 4 weeks for collection of tissues and blood. Similar high-level GAA activities were observed in the livers of all AAV treatment groups (FIG. 6A) but plasma GAA activity in the AAV-IGF2-hGAA treated mice was slightly higher than that in the AAV9- IGF2^ml-hGAA or AAV9-IGF2^m2-hGAA treated mice (FIG. 6B). Increased GAA activity (via enzyme uptake) was observed in the heart and skeletal muscles, but not the brain, of all AAV- treated mice when compared with untreated (UT) control mice (FIG. 7A). Age-matched wildtype (WT) mice were included as normal controls. Correspondingly, glycogen content was almost cleared in the heart, and remarkably decreased in skeletal muscles, but not in the brain, of all AAV-treated mice (FIG. 7B). The blood glucose levels (non- fasting) were within normal ranges in all AAV-treated and untreated GAA-KO mice, indicating that other parameters are needed for evaluation of the adverse effects related to the IGF2 off-target binding.

Example 5 2^nd-Generation IGF2-hGAA Variants Remarkably Increased Enzyme Uptake Efficiency in Cultured Muscle Cells Compared to l^st-Generation Variants

[0413] To further increase enzyme uptake efficiency, the l^st-generation IGF2-hGAA variants were modified by adding an additional mutation at the position of Phel9, to generate our 2^nd- generation variants (m7-hGAA to mlO-hGAA) (Table 1). DNA plasmid construction and fusion enzyme production were done as described above (e.g., Example 1).

[0414] For enzyme uptake study, rat L6 myoblasts in 12-well plates were incubated with each of the concentrated enzymes (1000 U/mL) with or without the competitor M6P (5 mM) or human IGF2 (2.4 mM). After 24 hr., cells were washed twice with PBS, lysed in 100 pL NP-40 lysis buffer, sonicated twice, and centrifuged for 10 min at 4 °C. GAA activity in cell lysates was analyzed and fusion proteins were visualized by Western blotting. All the four 2^nd-generation IGF2-hGAA variants (m7-hGAA to mlO-hGAA) displayed remarkably greater enzyme uptake efficiency than the unmodified IGF2-hGAA and the two l^st-generation variants (m5-hGAA & m6-hGAA) in L6 cells (FIG. 8A). IGF2 almost completely blocked the uptake of all the IGF2- hGAA fusion proteins as well as the untagged hGAA; in contrast, M6P only blocked the uptake of the untagged hGAA (FIG. 8A). Western blot analysis revealed that all the IGF2-hGAA fusion proteins were correctly processed into the 67-kDa mature GAA protein, like the untagged hGAA, in L6 cells (FIG. 8B).

Example 6 2nd-Generation IGF2-hGAA Variants Showed Similar Reduced the Binding Affinity to the IR and IGF1R as Ist-Generation Variants

[0415] A similar IR/IGF1R receptor binding assay was performed as described above. HEK293 cells were seeded in 12-well plates and transfected next day with 0.8 pg of the pCMV-IR plasmid (Addgene cat. #24049) or pCMV-IGFIR plasmid (Addgene cat. #201979), to overexpress IR and IGF1R. After 48 hr., the culture medium was replaced with fresh medium containing the concentrated enzymes (300 U/mL). After incubation for 2 hr., cell lysates were prepared as described above. All the l^st-generation IGF2-hGAA variants and 2^nd-generation IGF2-hGAA variants demonstrated greatly lowered binding affinity to the IR (FIG. 9A) and IGF1R (FIG. 9B).

Example 7

2nd-Generation IGF2-hGAA Variant Enhanced Glycogen Reduction in Skeletal Muscles of GAA-KO Mice

[0416] An in vivo experiment to compare the treatment efficacy among the untagged hGAA, unmodified IGF2-hGAA, and IGF2^ml0-hGAA (this 2nd-generation variant was chosen because it displayed the lowest binding affinity to the IR and IGF1R as shown in FIG. 9A and FIG. 9B). Three recombinant AAV9 vectors (i.e., AAV-hGAA, AAV-IGF2-hGAA, and AAV-IGF2^m10- hGAA) under the control of the 448-bp liver-specific LP1 promoter (SEQ ID NO:XX). Then, intravenously injected these AAV vectors at the same dose (5 x 10¹² vg/kg) into 4-month-old GAA-KO mice and collected blood and tissues after 6 weeks.

[0417] Both AAV-IGF2-hGAA and AAV-IGF2^ml0-hGAA treatments resulted in significantly higher GAA activity and greater glycogen clearance in the heart, skeletal muscles, and lung than those in the AAV-hGAA treated mice (FIG. 10A). AAV-IGF2^ml0-hGAA treatment showed better glycogen clearance (to WT level) than AAV-IGF2-hGAA in hindlimb muscles (quadriceps and gastrocnemius) (FIG. 10B), despite that a similar level of GAA activity was observed in these tissues (FIG. 10A). This indicates that IGF2^ml0-hGAA has a better enzyme uptake profile (can transduce more muscle cells) than the unmodified IGF2-hGAA in the type II skeletal muscle fibers. Similarly high levels of GAA activities were observed in the livers of all treatment groups (FIG. 10A), but, surprisingly, plasma GAA activity was remarkably higher in the AAV-IGF2- hGAA treated mice (6993 ± 125 nmol/h/mL) than that in the AAV-IGF2^ml0-hGAA treated mice (1412 ± 303 nmol/h/mL) or the AAV-hGAA treated mice (966 ± 308 nmol/h/mL). The plasma GAA activity result was further confirmed by Western blotting (FIG. 11). This phenomenon was likely caused by the binding of the unmodified IGF2-hGAA to the IGFBPs, resulting in the accumulation of the fusion protein in blood. This indicates that IGF2^ml0-hGAA fusion enzyme does not compete with human IGF2 for binding to the IGFBPs in blood circulation of human patients. Blood glucose levels (fasting and non-fasting) were within normal ranges in all AAV- treated and untreated GAA-KO mice.

Example 8 Identification of a Lead Clinical Candidate AAV-IGF2-hGAA Vector for Liver-Depot Gene Therapy in GAA-KO Mice

[0418] Correction of skeletal muscle remains a major challenge for the treatment of Pompe disease, an inherited lysosomal storage disorder (LSD) caused by GAA deficiency. Enzyme replacement therapy with rhGAA (Alglucosidase alfa) is the current standard of care but has little effect on skeletal muscles. IGF2-tagged hGAA (IGF2-hGAA, reveglucosidase alfa) greatly increased the efficiency of enzyme uptake mediated by IGF2 receptor (IGF2R) in skeletal muscles, however, hypoglycemia caused by the cross-binding of the IGF2 moiety to the IR and IGF1R were frequently observed in patients. AAV gene therapy has shown promise for the treatment of PD with successful translation to early phase clinical trials. Current muscle gene therapy using a muscle-specific promoter requires administration of very high doses of AAV vectors that can cause significant hepatotoxicity and genotoxicity. Liver-depot gene therapy relying on secretion of hGAA from liver-specific transgene expression requires lower vector doses but has limited effect on skeletal muscles (similar to ERT). Hence, there is a critical unmet need for an improved therapy for patients with Pompe disease that can correct the genetic defects in skeletal muscles. Site-specific mutagenesis of IGF2-hGAA is expected to prevent its off-target binding thereby reducing the adverse effects, and increasing its safety and clinical translatability for the treatment of Pompe disease and other LSDs. Based on the critical sites on human IGF2 for binding to IR, IGF1R, and IGF binding proteins (IGFBPs), a series of IGF2-hGAA variants that display remarkably higher enzyme uptake efficiency than the untagged hGAA and significantly lower binding affinity to the IR and IGF1R than the unmodified IGF2-hGAA in cultured cells have been engineered. First, the lead clinical candidate AAV-IGF2-hGAA vector in GAA-KO mice is identified. Liver-directed AAV gene therapy with the modified IGF2-hGAA variants increases the therapeutic efficacy in skeletal muscle and the brain with minimized side effects, given that IGF2 can increase receptor-mediated hGAA uptake and can cross the bloodbrain barrier.

[0419] First, identification of the lead AAV-IGF2-hGAA vector in GAA-KO mice. AAV9 vectors expressing the unmodified and mutated IGF2-hGAA transgenes from a liver-specific LP1 promoter are constructed and compared these candidate vectors with regard to safety and efficacy in adult GAA-KO mice. Research design (Experimental groups of mice are summarized in Table 8).

Table 8 - Identification the Lead AAV-IGF2-hGAA Vector in GAA-KO Mice

[0420] Here, AAV9 vectors expressing the untagged hGAA, the unmodified IGF2-hGAA, and the top four IGF2-hGAA variants selected from the preliminary study using a 448-bp liver- specific LP1 promoter are constructed. This LP1 promoter comprises core liver-specific elements from the human apolipoprotein hepatic control region (HCR) and the human alpha- 1 -antitrypsin (hAAT) gene promoter. These vectors are intravenously injected at the same dose (1 x 10¹³ vg/kg) into 3-month-old GAA-KO mice (Groups 2-7 in Table 8) based on the dosage used in the preliminary experiment. Gender-matched and age-matched GAA-KO mice (Group 1) and wildtype (WT) mice (Group 8) are mock-treated with vehicle as controls. Blood is drawn at 3 weeks and 6 weeks following vector administration for testing the levels of secreted hGAA enzymes and the titers of anti-hGAA and anti-IGF2 antibodies. A panel of comprehensive functional testing (as indicated below) will be performed at 6 weeks to evaluate the improvement of neuromuscular and neurological functions. All mice are euthanized at 6 weeks following vector administration. Urine samples are collected for analyzing the concentrations of glucose tetrasaccharide Glc4, a well-recognized disease biomarker for Pompe disease, by mass spectrometry. Fresh tissue specimens including the liver, heart, diaphragm, quadriceps, gastrocnemius, tongue, lung, spinal cord, and the brain are frozen on dry ice and stored at -80 °C until use, or fixed immediately for histopathology and immunohistochemistry (H4C).

[0421] The results are evaluated for correction of biochemical abnormalities. Biochemical analyses including GAA activity and protein expression (Western blot), glycogen content, biodistribution of AAV genomes are performed with heart, diaphragm, quadriceps, gastrocnemius, tongue, lung, and the brain tissues using established methods. PAS-staining of glycogen and H&E staining for pathological changes will be perform with tissue sections indicated above, to define the correction of histopathology in these tissues. Correction of abnormalities of autophagy in skeletal muscles are evaluated by Western blot detection of LC3-II and p62 in tissue homogenates and by IHC staining of a single muscle fiber with anti-LC3 and anti -LAMP 1 antibodies using established methods.

[0422] Results are also evaluated for correction of neuromuscular and neurological functional deficits. Comprehensive functional testing is conducted at pre-dose and 6 weeks following vector administration. These behavioral tests have been successfully used in our previous studies in GAA-KO mice, to assess the functional improvements in muscle strength (inverted wire-hang test), motor coordination and balance (rota-rod test, cylinder test, and beam-walking test), gait impairment (footprint test), and sensory impairment/peripheral neuropathy (von Frey test) by AAV gene therapy.

[0423] Results are also be evaluated for potential adverse effects related to the off-target binding of IGF2 to IR and IGF1R. The potential adverse effects related to IGF-2 off-target binding to the IGF1R and IR include the overgrowth of body/tissues, hypoglycemia, and liver and heart damages. Body-weight is measured weekly to monitor abnormal body weight gain. Organs including the liver, heart, gastrocnemius and quadriceps muscles, kidney, spleen, and the brain will be weighed at euthanasia, to evaluate the overgrowth of these tissues. A comprehensive metabolic panel (CMP) test that includes ALT, ALP, AST, CK, glucose, albumin, bilirubin, and total protein will be performed with blood samples collected at 3 and 6 weeks following AAV injection, to evaluate for hypoglycemia and liver or muscle toxicity. Hypoglycemia is be detected by an 8-hour fasting challenge. Tissue histology (H & E) is used to evaluate the pathological changes in the liver, heart, and skeletal muscles.

[0424] Upon completion of the experiment, the lead AAV-IGF2-hGAA (AAV-LPl-IGF2mut- hGAA) vector is identified by comparing the adverse effects and treatment outcomes among Groups 4-7 using Group 3 as control (Table 8).

[0425] Second, evaluation of the long-term efficacy of the lead AAV-IGF2-hGAA vector in GAA-KO mice. It is important to determine the effective doses and examine the treatment outcomes of the lead AAV-IGF2-hGAA vector in GAA-KO mice prior to clinical translation. Gene therapy with the lead AAV-LPl-IGF2mut-hGAA vector are age and sex-dependent, given that AAV vectors transduce tissues less efficiently in female mice and aged mice. Age-dependent and gender-dependent, dose-ranging studies are conducted with the lead AAV-IGF2-hGAA vector in young adult and old GAA-KO mice, to examine the effective doses, safety, and longterm efficacy in muscles and the brain. Experimental groups are summarized in Table 9.

Table 9 - Evaluation of Long-Term Efficacy of Lead AAV-IGF2-hGAA Vector in GAA-KO mice [0426] A dose-ranging experiment is performed to determine the effective doses and long-term efficacy of the lead AAV vector in young adult mice. The lead AAV-LPl-IGF2mut-hGAA vector is intravenously administered at three escalating doses (4 x 10¹² vg/kg, 1 x 10¹³ vg/kg, and 2.5 x 10¹³ vg/kg) into 3-month-old male and female GAA- KO mice (Table 9, Groups 2-4).

[0427] Gender- and age-matched GAA-KO (Group 1) and WT mice (Group 5) are mock-treated with vehicle as controls. Functional testing including rota-rod test, wire-hang test, cylinder test, beam-walking test, footprint test, and von Frey test are performed at pre-dose, 3 months, 6 months, 9 months, and 12 months following vector administration to determine the improvement of neuromuscular and neurological functions (described supra). Body weight is measured monthly to monitor abnormal body growth. Blood is drawn at 3 months, 6 months, and 12 months postvector injection for testing enzyme secretion, anti-hGAA and anti-IGF2 antibodies, and blood chemistry panel (CMP test) as described supra. Urine is collected at 3 months, 6 months, and 12 months for analyzing disease biomarker urinary Glc4. Six mice from each group are euthanized at 3 months (short-term study) and the remaining mice are euthanized at 12 months (long-term study), following vector administration. Organ weight measurement, tissue collection and analyses (for correction of biochemical, histopathological, and autophagic abnormalities) are done as described above.

[0428] A similar experiment is performed to examine the ability of the lead AAV vector to reverse PD in old GAA- KO mice. The lead AAV-LPl-IGF2mut-hGAA vector is injected at two doses (1 x 10¹³ vg/kg and 2.5 x 10¹³ vg/kg) into 8-old-month male and female GAA-KO mice using mock-treated GAA-KO and WT mice as controls (Groups 6-9 in Table 9). Functional testing, sample collection, and analyses are done at 3 months following vector administration (as described above). Dose-dependent correction of heart and skeletal muscles is expected in the AAV-treated mice in comparison with the untreated control mice (Groups 2-4 vs. Group 1; Groups 7 and 8 vs. Group 6). It is expected that correction of the brain is achieved only by the high-dose vector treatment (2.5 x 10¹³ vg/kg, Groups 4 and 8). It is expected that better treatment efficacy occurs in male mice than in female mice and in young mice than in old mice, when administered with the same dose of the AAV vector. Gene therapy related immune responses and adverse effects in any of the AAV-treated mice are not expected.

[0429] Major limitations of cation -independent mannose 6-phosphate receptor (CI-M6PR)- mediated enzyme replace therapy (ERT) for lysosomal storage diseases (LSDs) include the poor therapeutic efficacy in some target tissues (such as skeletal muscles, the respiratory system, and the central nervous system), high cost for drug production, and, most importantly, the development of antibody responses and allergic reactions from repeated infusions of high-dose enzyme. Adding a mutated IGF2 with multiple substitutions to thethe therapeutic lysosomal enzymes will greatly (1) increase the treatment efficacy in these difficult tissues; (2) lower the cost for enzyme production; and (3) reduces the effective doses without causing adverse effects and thus increase the safety, efficacy, and clinical translatability. This treatment approach can be broadly applied for LSDs in the context of ERT and gene therapy.

Table 10 - Table of Sequences

Claims

VIII. CLAIMS What is claimed is:

1. A polypeptide, comprising: a lysosomal targeting peptide or fragment thereof linked to a lysosomal enzyme or a fragment thereof, wherein the lysosomal targeting peptide comprises insulin-like growth factor 2 (IGF2) having one or more substitutions.

2. The polypeptide of Claim 1, wherein the one or more substitutions occur at Glu6, Phel9, Phe26,

Tyr27, Val43, or at any other position, or any combination thereof.

3. The polypeptide of Claim 1, wherein the IGF2 having one or more substitutions comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:03 - SEQ ID NO: 36; and

4. The polypeptide of Claim 1, wherein the lysosomal enzyme or fragment thereof comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in any one of SEQ ID NO:71 - SEQ ID NO:92.

5. The polypeptide of Claim 1, wherein the polypeptide demonstrates reduced off-target binding to the insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R), insulin growth factor binding proteins (IGFBPs), or any combination thereof.

6. The polypeptide of Claim 1, wherein the polypeptide demonstrates increased binding affinity to the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (CI- M6PR/IGF2R).

7. The polypeptide of Claim 1, wherein the polypeptide demonstrates an increased enzyme uptake efficiency when compared to a polypeptide have wild-type IGF2 as the lysosomal targeting peptide or fragment thereof.

8. A nucleic acid molecule encoding the polypeptide of any one of Claims 1 - 7.

9. A vector, comprising: the isolated nucleic acid molecule of Claim 8.

10. The vector of Claim 9, further comprising a liver-specific promoter operably linked to the nucleic acid molecule.

11. The vector of Claim 10, wherein the liver-specific promoter comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 161.

12. The vector of Claim 9, further comprising a ubiquitous promoter operably linked to the nucleic acid molecule.

13. The vector of Claim 12, wherein the ubiquitous promoter is a CMV enhancer/chicken P-actin promoter.

14. The vector of Claim 9, further comprising an immunotolerant dual promoter comprising a liver-specific promoter and a ubiquitous promoter operably linked to the nucleic acid molecule.

15. The vector of Claim 14, wherein the immunotolerant dual promoter comprises the sequence set forth in SEQ ID NO: 159.

16. The vector of Claim 9, wherein the vector is a viral vector or non-viral vector.

17. The vector of Claim 16, wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus (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.

18. The vector of Claim 17, wherein the viral vector is an adeno-associated virus (AAV) vector.

19. The vector of Claim 16, wherein the non-viral vector is a polymer-based vector, a peptide- based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid based vector.

20. A lipid nanoparticle (LNP), comprising: the polypeptide of any one of Claims 1 - 7 or the nucleic acid molecule of Claim 8.

21. A pharmaceutical formulation comprising the polypeptide of any one of Claims 1 - 7, the vector of any one of Claims 9 - 19, or the LNP of Claim 20 and a pharmaceutically acceptable carrier.

22. A method of treating and/or preventing disease progression, the method comprising: administering to a subject having a lysosomal storage disease a therapeutically effective amount of the pharmaceutical formulation of Claim 21.

23. The method of Claim 22, wherein administering comprises an intravenous injection, intramuscular injection, intracoronary injection, intracerebroventricular injection, intracistema magna injection, intracerebral injection, intrathecal injection, or any combination thereof.

24. The method of Claim 22, wherein the lysosome storage disorder is Pompe disease and the encoded lysosomal enzyme is a-glucosidase; Fabry disease (GLA) and the lysosomal enzyme can be a-Galactosidase A; Gaucher Disease and the lysosomal enzyme is Lysosomal Acid Gucosylceramidase; Niemann-Pick Disease and the lysosomal enzyme is Acid Sphingomyelinase; Farber Disease and the lysosomal enzyme is Acid Ceramidase; Krabbe Disease and the lysosomal enzyme is Galactocerebrosidase; Lysosomal Acid Lipase Deficiency and the lysosomal enzyme is Lysosomal Acid Lipase; GM1 Gangliosidosis and the lysosomal enzyme is Acid Beta-Galactosidase; GM2 Gangliosidosis and the lysosomal enzyme is Beta-Hexosaminidase; Mucolipidosis Type I (ML1) and the lysosomal enzyme is Sialidase-1; Metachromatic Leukodystrophy and the lysosomal enzyme is Arylsulfatase A; MPS I and the lysosomal enzyme is Alpha-L-Iduronidase; MPS II, and the lysosomal enzyme is Iduronate Sulfate Sulfatase; MPS IIIA and the lysosomal enzyme is N-Sulphoglucosamine sulphohydrolase; MPS IIIB and the lysosomal enzyme is Alpha-N-Acetylglucosaminidase; MPS IIIC and the lysosomal enzyme is Alpha-Alucosaminide Acetyltransferase; MPS IIID and the lysosomal enzyme is N-Acetylglucosaminine-6-Sulfatase; MPS IVA and the lysosomal enzyme is N-Acetylgalactosamine-6-sulfatase; MPS IVB and the lysosomal enzyme is Beta-Galactosidase; MPS VI and the lysosomal enzyme is N-Acetylgalactosamine- 4-Sulfatase; MPS VII and the lysosomal enzyme is Beta-Glucuronidase; MPS IX and the lysosomal enzyme is Hyaluronidase; a-Mannosidosis (type I mild, type II moderate and type III severe and the lysosomal enzyme is Lysosomal a-mannosidase; P-Mannosidosis and the lysosomal enzyme is P-Mannosidase; Fucosidosis and the lysosomal enzyme is a-1- Fucosidase; Aspartylglucosaminuria and the lysosomal enzyme is Aspartoglucosaminidase; Schindler disease (Kanzaki disease) and the lysosomal enzyme is a-7V-Acetyl- galactosaminidase; Sialidosis type I, and the lysosomal enzyme is Neuraminidase- 1; Sialidosis type II and the lysosomal enzyme is Neuraminidase- 1; Galactosialidosis and the lysosomal enzyme is cathepsin A and/or P-galactosidase; Acid lipase deficiency (Wolman disease) (LIPA), and the lysosomal enzyme is Lysosomal acid lipase/cholesteryl ester hydrolase; Multiple sulfatase deficiency (SUMF1) and the lysosomal enzyme is Formylgly cine- generating enzyme; Cystinosis and the lysosomal enzyme is Cystinosin; or Danon disease and the lysosomal enzyme is LAMP2.

25. The method of any one of Claims 22 - 24, wherein following the administering step one or more aspects of cellular homeostasis and/or cellular functionality are restored.

26. The method of Claim 25, wherein restoration of one or more aspects of cellular homeostasis and/or cellular functionality comprises (i) correction of cell starvation in one or more cell types; (ii) normalization of aspects of the autophagy pathway; (iii) improvement and/or restoration of mitochondrial functionality and/or structural integrity; (iv) improvement and/or restoration of organelle functionality and/or structural integrity; (v) prevention and/or slowing of hypoglycemia, ketosis, and/or other liver abnormalities; (vi) correction of liver enzyme dysregulation; (vii) prevention and/or slowing of the rate of progression of the multi -systemic manifestations of a LSD; (viii) prevention and/or slowing of the rate of progression of liver disease including fibrosis, cirrhosis, hepatic adenomas, and/or liver hepatocellular carcinoma, (ix) restoration of the balance of glycogen, lipids, and glycoproteins metabolism, including synthesis and breakdown, (x) restoration of lysosomal enzyme functionality and/or structural integrity, or (xi) any combination thereof.