Compositions and methods using dual promoter vectors for the treatment of lysosomal storage disorders

Abstract

A composition is provided that includes a vector for co-expression of a modified GlcNAc-1 phosphotransferase gene and a lysosomal enzyme. The gene encoding the lysosomal enzyme is operably linked to a first promoter, and the gene encoding the GlcNAc-1 phosphotransferase is operably linked to a second promoter. Also provided herein is a method for treating a lysosomal storage disorder, comprising administering a composition of the present disclosure to a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 306,615, filed February 4, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Lysosomal storage disorders (LSDs) relate to inherited metabolic disorders resulting from defective lysosomal function. Currently, about 50 different LSDs have been identified, but only a small number of these (less than 10) are reported to have treatments. Thus, there is an unmet need in the art for safe and effective treatment of LSDs. The present disclosure provides two solutions to this unmet need, either by enzyme replacement therapy (ERT) or by gene therapy. Summary of the Invention

[0003] The present disclosure provides a composition comprising a vector comprising a sequence encoding a first promoter operably linked to a first polynucleotide encoding a lysosomal enzyme and a second promoter operably linked to a second polynucleotide encoding a modified GlcNAc-1 phosphotransferase (PTase).

[0004] In some embodiments, the vector of the present disclosure is a viral vector or a non-viral vector. In some embodiments, the vector of the present disclosure is an adenoviral vector or an adeno-associated virus (AAV) vector or a plasmid.

[0005] In some embodiments, the first promoter of the vector of the present disclosure is CBH and the second promoter is selected from EFS or JeT.

[0006] In some embodiments, the engineered GlcNAc-1 phosphotransferase of the vector of the disclosure comprises an S1S3 PTase.

[0007] In some embodiments, the lysosomal enzyme of the vector of the present disclosure is selected from the group consisting of b-glucocebrosidase (GBA), galactosylceremidase (GALC), a-galactosidase (GLA), a-N-acetylglucosaminidase (NAGLU), acid a-glucosidase (GAA), b-hexosaminidase (HexM), and lysosomal acid a-mannosidase (LAMAN).

[0008] In any of the foregoing embodiments, the vector of the disclosure further comprises a 3'UTR. In some embodiments, the 3'UTR is selected from SV40 and bGH polyA.

[0009] In any of the foregoing embodiments, the composition of the present disclosure further comprises a pharma- ceutically acceptable carrier.

[0010] The present disclosure also provides a method of treating a lysosomal storage disorder (LSD), comprising administering to a subject an effective amount of any of the aforementioned compositions, wherein the composition increases phosphorylation of a lysosomal enzyme involved in the LSD, thereby treating the LSD.

[0011] In some embodiments, the subject has been diagnosed with LSD and / or the subject exhibits signs or symptoms of LSD.

[0012] The present disclosure also provides a method for preventing the occurrence or development of a lysosomal storage disorder (LSD), comprising administering to a subject an effective amount of any of the aforementioned compositions, wherein the composition increases phosphorylation of a lysosomal enzyme involved in LSD, thereby preventing the occurrence of LSD in the subject.

[0013] In some embodiments, the subject is at risk for developing or developing an LSD and / or the subject exhibits signs or symptoms of an LSD.

[0014] The present disclosure also provides a method for restoring phosphorylation of a lysosomal enzyme involved in a lysosomal storage disorder (LSD), comprising contacting a cell with an effective amount of any of the aforementioned compositions, wherein the composition increases phosphorylation of the lysosomal enzyme.

[0015] In some embodiments, the cell is an in vitro or ex vivo cell.

[0016] In some embodiments, the subject comprises the cell. In some embodiments, the subject exhibits signs or symptoms of an LSD, the subject is at risk of developing or developing an LSD, and / or the subject has been diagnosed with an LSD. In any of the foregoing embodiments, the subject is a human.

[0017] In any of the methods of the present disclosure above, administering comprises a systemic or local route of administration. In some embodiments, the systemic route of administration is enteral, parenteral, oral, intramuscular (IM), subcutaneous (SC), intravenous (IV), intraarterial (IA), intrathecal, intraspinal, or intraventricular.

[0018] For the purpose of illustrating the present disclosure, there are shown in the drawings certain specific embodiments of the present disclosure. However, the present disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0019] [Figure 1] 1 is a graphical representation of dual promoter gene expression cassettes for expression of S1S3 PTase and GLA, including inside-out, outside-in and back-to-back configurations. [Figure 2A]1 is a graphical representation of a dual promoter gene expression cassette for expression of S1S3 PTase and GLA in an inside / outside configuration transfected into HEK293T cells. [Figure 2B] 1 is a graph showing GLA activity in lysates of HEK293T cells transfected with gene expression cassettes of the inside / outside configuration. [Figure 2C] FIG. 1 shows a Western blot showing GLA, S1S3 PTase, and GAPDH proteins in lysates of HEK293T cells transfected with the inside-outside gene expression cassettes. [Figure 3A] FIG. 1 is a graphical representation of a dual promoter gene expression cassette for expression of S1S3 PTase and GLA in an exogenous configuration transfected into HEK293T cells. [Figure 3B] 1 is a graph showing GLA activity in lysates of HEK293T cells transfected with gene expression cassettes in exogenous and endogenous configurations. [Figure 3C] FIG. 1 shows a Western blot showing GLA, S1S3 PTase, and GAPDH proteins in lysates of HEK293T cells transfected with exogenous and endogenous gene expression cassettes. [Figure 4A] 1 is a graph showing GLA and S1S3 PTase gene copy levels in the liver. [Figure 4B] 1 is a graph showing GLA and S1S3 PTase gene copy levels in the kidney. [Figure 4C] 1 is a graph showing GLA and S1S3 PTase mRNA levels in the liver. [Figure 4D] 1 is a graph showing GLA and S1S3 PTase mRNA levels in the kidney. [Figure 5A] 1 is a graphical representation of the dual promoter plasmid design for expression of S1S3 PTase and PPT1. [Figure 5B] FIG. 5B shows SDS-PAGE and Coomassie staining of lysates of Expi293 cells transfected with the plasmid designs shown in FIG. 5A. [Figure 5C] FIG. 5B shows a Western blot demonstrating expression of S1S3 PTase in Expi293 cells transfected with the plasmid designs shown in FIG. 5A. [Figure 5D] FIG. 5B is a graph showing PPT1 CI-MPR binding in conditioned medium of Expi293 cells transfected with the plasmid designs shown in FIG. 5A. [Figure 6A] Graphical representation of the dual promoter AAV vector design for expression of S1S3 PTase and GBA1. [Figure 6B] FIG. 6B is a graph showing GCase activity of Expi293 cells transfected with the plasmid designs shown in FIG. 6A. [Figure 6C] FIG. 6B shows SDS-PAGE and Western blots showing GCase and GAPDH expression in Expi293 cells transfected with the plasmid designs shown in FIG. 6A. [Figure 6D] FIG. 6B is a graph showing GCase CI-MPR binding in conditioned medium of Expi293 cells transfected with the plasmid designs shown in FIG. 6A. [Figure 7] FIG. 6B is a graph showing GCase activity in the liver, heart, and brain of mice injected with AAV9 viral vectors generated from the plasmid design shown in FIG. 6A. [Figure 8A] FIG. 6B is a graph showing GBA1 and S1S3 PTase gene copy numbers in the liver, heart, brain, bone marrow, and spleen of mice injected with AAV9 viral vectors generated from the plasmid design shown in FIG. 6A. [Figure 8B] FIG. 6B is a graph showing GBA1 and S1S3 PTase mRNA copy numbers in the liver, heart, brain, bone marrow, and spleen of mice injected with AAV9 viral vectors generated from the plasmid design shown in FIG. 6A. [Figure 9] FIG. 6B shows GBA1 mRNA expression in the striatum of mice injected with AAV9 viral vectors generated from the plasmid design shown in FIG. 6A. [Figure 10]FIG. 6B shows GCase protein expression in the somatosensory cortex of mice injected with AAV9 viral vectors generated from the plasmid design shown in FIG. 6A. [Figure 11A] 1 is a graphical representation of the dual promoter AAV vector design for expression of S1S3 PTase and HexM. [Figure 11B] FIG. 11B is a graph showing HexM activity in conditioned medium of Expi293 cells transfected with the plasmid designs shown in FIG. 11A. [Figure 11C] FIG. 11B is a graph showing HexM activity in cell lysates of Expi293 cells transfected with the plasmid designs shown in FIG. 11A. [Figure 11D] FIG. 11B is a graph showing PTase activity in cell lysates of Expi293 cells transfected with the plasmid designs shown in FIG. 11A. [Figure 11E] FIG. 11B is a graph showing HexM CI-MPR binding in conditioned medium of Expi293 cells transfected with the plasmid designs shown in FIG. 11A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention is directed to novel compositions, including expression vectors and gene therapy vectors, and methods for producing lysosomal enzymes operably linked to S1S3 variants of GlcNAc-1 phosphotransferase (S1S3 PTase). The present disclosure provides novel dual promoter vectors, in which the lysosomal enzyme is under the control of one promoter and the S1S3 PTase is under the control of a second promoter. When co-expressed, the dual promoter vectors produce lysosomal enzymes with appropriately phosphorylated N-linked oligosaccharides.

[0021] The expression of S1S3 PTase and lysosomal enzymes significantly increases the M6P content of the expressed lysosomal enzymes. Having a fully phosphorylated enzyme allows for efficient uptake and lysosomal delivery of the enzyme. This allows for better tissue distribution, cellular uptake, lysosomal targeting, and substrate reduction. The dual promoter vector allows the expression of lysosomal enzymes and S1S3 PTase to be separately regulated in gene transcription and translation (which can increase the lysosomal enzyme expression level in cells), avoiding the complications caused by using longer bicistronic messages and allowing the expression of only the coding sequence (avoiding the inclusion of additional amino acid sequences). The expression of S1S3 PTase significantly increases the M6P content level in lysosomal enzymes.

[0022] Definition. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the present invention, the following terminology is used:

[0023] The term "expression vector" as used herein refers to a vector that comprises a recombinant polynucleotide that comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). In some embodiments, the vectors of the present disclosure are referred to herein as viral vectors. In some embodiments, the vectors of the present disclosure are referred to herein as expression vectors.

[0024] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0025] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable substances, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating substances, which are involved in carrying or transporting the compounds of the present invention into or to a subject so that the compounds can perform their intended function. Typically, such compounds are carried or transported from one organ or part of the body to another organ or part of the body. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of substances which may function as pharma- ceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol. and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; diluents; granulating agents; lubricants; binders; disintegrating agents; wetting agents; emulsifying agents; coloring agents; release agents; coating agents; sweeteners; flavoring agents; flavoring agents; preservatives; antioxidants; plasticizers; gelling agents; viscosifying agents; hardening agents; solidifying agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic and compatible substances used in pharmaceutical preparations, or any combination thereof. As used herein, "pharmaceutical acceptable carriers" also include any coatings, antibacterial and antifungal agents, and absorption retardants that are compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0026] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of viral particles or infectious units produced from a vector of the invention that is required to prevent a particular disease state or that reduces the severity and / or ameliorates the disease state or at least one symptom thereof or a condition associated therewith.

[0027] Compositions of the present disclosure. The disclosure provides a composition comprising a vector comprising a polynucleotide encoding a lysosomal enzyme controlled by a first promoter and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase controlled by a second promoter.

[0028] In some embodiments, the dual promoter vector comprises a particular nucleic acid sequence, hi other embodiments, the dual promoter vector comprises a nucleic acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to SEQ ID NO:1.

[0029] In some embodiments, the polynucleotide encoding the modified GlcNAc-1 PTase comprises a nucleic acid sequence corresponding to SEQ ID NO: 4 as disclosed in WO2021003442, the contents of which are incorporated herein in their entirety. In other embodiments, the GlcNAc-1 PTase is encoded by a polynucleotide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% similarity to SEQ ID NO: 4 as disclosed in WO2021003442.

[0030] In some embodiments, the polynucleotide encoding the lysosomal enzyme encodes a lysosomal enzyme involved in at least one LSD. Such lysosomal enzymes are known in the art and include, but are not limited to, those listed in Tables 1A-1C of WO2021003442, the contents of which are fully incorporated herein. By way of example and not limitation, in some embodiments, the lysosomal enzyme is selected from the group consisting of b-glucosebrosidase (GCase, GBA), galactosylceremidase (GALC), a-galactosidase (GLA), a-N-acetylglucosaminidase (NAGLU), acid a-glucosidase (GAA), b-hexosaminidase (HexM), and lysosomal acid a-mannosidase (LAMAN). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NOs: 5-10 of WO2021003442, the contents of which are fully incorporated herein.

[0031] In some embodiments, the dual promoter vector comprises an outside-inside configuration, where the two promoters are in a distal configuration and are flanked by the 3'UTR regions of each gene.

[0032] In some embodiments, a dual promoter vector comprises an inside-outside configuration in which the 5' ends of both promoters are adjacent to drive gene expression outward.

[0033] promoter. The promoter of the dual promoter vector can be a constitutive, inducible / repressible, and / or cell type specific promoter. In certain embodiments, the promoter can be a constitutive promoter. Non-limiting examples of constitutive promoters for mammalian cells include cytomegalovirus (CMV), UBC, EF1 alpha (EFS), JeT, SV40, PGK, CAG, CBA / CAGGS / ACTB, CBh, MeCP2, U6, and HI promoters, including wild type and known modifications thereof. Non-limiting examples of inducible promoters for mammalian cells include tetracycline, heat shock, steroid hormone, heavy metal, phorbol ester, adenovirus E1 A element, interferon, and serum inducible promoters. Without excluding other cell type specific promoters known in the art, some non-limiting examples of cell type specific promoters for neurons (e.g. synapsin), astrocytes (e.g. GFAP), oligodendrocytes (e.g. myelin basic protein), microglia (e.g. CX3CR1), neuroendocrine cells (e.g. chromogranin A), muscle cells (e.g. desmin, Mb), or cardiomyocytes (e.g. alpha myosin heavy chain promoter) can be used. In one embodiment, the cell type specific promoter can be the Nrl (rod photoreceptor specific) promoter or the HBB (hemoglobin beta) promoter.

[0034] The promoters of the dual promoter vector may both be constitutive, inducible / repressible, and / or cell type specific promoters. Alternatively, the promoters of the dual promoter may be different types of promoters. By way of example and not limitation, the first promoter may comprise a constitutive promoter and the second promoter may comprise an inducible / repressible promoter. Alternatively, the first promoter may comprise a constitutive promoter and the second promoter may comprise a cell type specific promoter. Alternatively, the first promoter may comprise an inducible / repressible promoter and the second promoter may comprise a constitutive promoter. Alternatively, the first promoter may comprise an inducible / repressible promoter and the second promoter may comprise a cell type specific promoter. Alternatively, the first promoter may comprise a cell type specific promoter and the second promoter may comprise a constitutive promoter. Alternatively, the first promoter may comprise a cell type specific promoter and the second promoter may comprise a cell type specific promoter.

[0035] By way of example, and not limitation, the promoter of the S1S3 PTase gene can be EFS or JeT, and the promoter of the lysosomal enzyme gene can be CBH.

[0036] The vector of the present disclosure may further comprise one or more specific 3' untranslated regions (3'UTRs, also referred to as polyA sequences). In some embodiments, the 3'UTRs may be bidirectional. Non-limiting examples of 3'UTRs include sequences from CMV, SP1, SV40 polyA, bovine growth hormone polyA (bGH polyA), rabbit beta globin polyA, and bidirectional UTRs, such as those from SV40.

[0037] The 3'UTR of the dual promoter vector may be the same. Alternatively, the 3'UTR of the dual promoter vector may be different types of 3'UTR. For example, but not limited to, the S1S3 PTase gene may be enhanced by SV40, and the lysosomal enzyme gene may be enhanced by bGH polyA.

[0038] Reporter / selectable marker genes. To evaluate the expression of a polypeptide in a dual promoter vector, the vector can also include either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population that is being transfected or infected with a viral vector. In some embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selection markers include those known in the art, such as antibiotic resistance genes, such as neo.

[0039] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene encoding a polypeptide whose expression is indicated by some easily detectable property, such as enzymatic activity, that is not present in or expressed by the recipient organism or tissue. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Non-limiting examples of reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0040] Introduction of genes into cells. Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian cell, a bacterial cell, a yeast cell, or an insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0041] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle guns, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0042] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0043] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0044] In some embodiments where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. In some embodiments, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in lipid as a suspension, contained in or complexed with micelles, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be dispersed in the solution, and may sometimes form aggregates that are not uniform in size or shape.Lipids are fatty substances that may be naturally occurring lipids or synthetic lipids.For example, lipids include lipid droplets that naturally occur in cytoplasm, as well as classes of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0045] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO, dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids may be in micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0046] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm that the recombinant DNA sequence is present in the host cell. Such assays include, but are not limited to, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or assays described herein that identify agents within the scope of the present disclosure.

[0047] vector. The vectors disclosed herein for use in treating or preventing a LSD in a subject are replication-competent and, optionally, suitable for integration into eukaryotic cells.

[0048] Typical vectors contain transcription and translation termination elements, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0049] The vector of the present disclosure can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present disclosure provides a gene therapy vector.

[0050] The isolated nucleic acids of the present disclosure can be cloned into several types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0051] Additionally, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0052] Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.Generally, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0053] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0054] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, since they allow long-term and stable integration of transgenes into daughter cells and proliferation of transgenes in daughter cells. Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In a preferred embodiment, the composition comprises a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become a powerful gene delivery tool for the treatment of various disorders. AAV vectors have several characteristics that make them ideally suited for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to stably and efficiently transduce postmitotic cells. Expression of a particular gene contained in an AAV vector can be specifically targeted to one or more types of cells by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0055] In some embodiments, the dual promoter viral vector of the present disclosure is an adenovirus (e.g., Ad-SYE, AdSur-SYE, Ad5 / 3-MDA7 / IL-24, Ad-SB, Ad-CRISPR, oncolytic Ad), adeno-associated virus, AAV (e.g., AAV-MeCP2, AAV1, AAV5, dual AAV9 AAV8, AAV9, AAVrhlO, AAVhu37), herpes simplex virus, HSV (e.g., HSV1, HSV2, HSV-1, HF10 oncolytic HSV-2), retrovirus (e.g., RRV / Toca, 511, GRV), lentivirus (e.g., HIV-1, HIV-2), alphavirus (SFV, Ml), flavivirus (Kunjin virus), rhabdovirus (VSV), measles virus (e.g., MV-Edm), Newcastle disease virus (e.g., NDV90), anhinga picornavirus, coxsackievirus (e.g., CVB3, CAV21, EV1), or poxvirus (e.g., PANVAC, VV, VV-GLV-lhl53, CPXV).

[0056] In some embodiments, the dual promoter vector of the present disclosure is a viral vector comprising adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus (HSV), measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus.In some embodiments, the dual promoter vector is a viral vector comprising adenovirus, adeno-associated virus (AAV), retrovirus, or lentivirus.

[0057] In some embodiments, the polynucleotide encoding lysosomal enzyme and the polynucleotide encoding modified GlcNAc-1 PTase are contained in an AAV vector. More than 30 naturally occurring serotypes of AAV are available. Many natural variants in AAV capsids exist, allowing the identification and use of AAV with properties that are particularly suitable for skeletal muscle. AAV viruses can be engineered using conventional molecular biology techniques, which allows these particles to be optimized for cell-specific delivery of nucleic acid sequences, minimizing immunogenicity, adjusting stability and particle lifespan, efficient degradation, and precise delivery to the nucleus, to name a few.

[0058] The use of AAV is a common mode of exogenous delivery of DNA, which is relatively non-toxic, achieves efficient gene transfer, and can be easily optimized for specific purposes. Among the serotypes of AAV isolated and well-characterized from humans or non-human primates (NHPs), human serotype 2 was the first AAV developed as a gene transfer vector and has been widely used for efficient gene transfer experiments into various target tissues and animal models. Clinical trials for the experimental application of AAV2-based vectors to some human disease models are ongoing, including therapies for diseases such as cystic fibrosis and hemophilia B. Other useful AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0059] Desirable AAV fragments for vector assembly include cap proteins including vpl, vp2, vp3 and hypervariable regions, rep proteins including rep78, rep68, rep52, and rep40, and sequences encoding these proteins. These fragments can be readily utilized in a variety of vector systems and host cells. Such fragments may be used alone or in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of vpl capsid protein) in combination with heterologous sequences that can be obtained from different selected AAV serotypes, non-contiguous portions of the same AAV serotype, non-AAV viral sources, or non-viral sources. The artificial AAV serotype may be, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Thus, exemplary AAVs or artificial AAVs suitable for expressing the lysosomal enzyme of interest and the modified GlcNAc-1 PTase include, among others, AAV2 / 8 (see U.S. Pat. No. 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (International Patent Publication No. WO2005 / 033321), AAV2 / 6 (U.S. Pat. No. 6,156,303), and AAVrh8 (International Patent Publication No. WO2003 / 042397).

[0060] In some embodiments, the dual promoter vectors described herein contain sequences encoding a selected AAV serotype capsid, e.g., AAV8 capsid, or fragments thereof. In another embodiment, the dual promoter vectors described herein contain sequences encoding a selected AAV serotype rep protein, e.g., AAV8 rep protein, or fragments thereof. Optionally, such vectors may contain both AAV cap protein and AAV rep protein. In vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences may both be of one serotype origin, e.g., all of AAV8 origin. Alternatively, the dual promoter vectors described herein may contain rep sequences from an AAV serotype different from the AAV serotype providing the cap sequence. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or host cell and vector). In another embodiment, these rep sequences are fused in frame with cap sequences of different AAV serotypes to form chimeric AAV vectors, e.g., AAV2 / 8, as described in U.S. Pat. No. 7,282,199.

[0061] A suitable recombinant adeno-associated virus (AAV) is produced by culturing a host cell that contains a nucleic acid sequence encoding an adeno-associated virus (AAV) serotype capsid protein or a fragment thereof as defined herein; a functional rep gene; a minigene composed of at least an AAV inverted terminal repeat (ITR) sequence and a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 PTase; and sufficient helper functions to allow packaging of the minigene into an AAV capsid protein. The components required to be cultured in the host cell to package the AAV minigene into an AAV capsid can be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., the minigene, the rep sequence, the cap sequence, and / or the helper functions) can be provided by a stable host cell that has been engineered to contain one or more of the required components using methods known to those skilled in the art.

[0062] Most preferably, such a stable host cell may contain the required components under the control of a constitutive promoter. However, the required components may also be under the control of an inducible promoter. Examples of suitable inducible and constitutive promoters are provided elsewhere herein and are well known in the art. In yet another alternative, the selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated that is derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contains rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be generated by those skilled in the art.

[0063] The minigene, rep sequences, cap sequences, and helper functions required to produce the rAAV of the present disclosure can be delivered to the packaging host cell in the form of any genetic element that transfers the sequences carried. The selected genetic element can be delivered using any suitable method, including the methods described herein and any other methods available in the art. The methods used to construct any embodiment of the present disclosure are known to those skilled in the art of nucleic acid processing and include genetic engineering, recombinant engineering, and synthetic techniques (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Similarly, methods of generating rAAV virions are well known, and the selection of a suitable method is not a limitation of the present disclosure (see, e.g., K. Fisher et al., 1993 J. Virol., 70:520-532 and U.S. Pat. No. 5,478,745, among others).

[0064] Unless otherwise specified, the AAV ITRs and other selected AAV components described herein can be readily selected from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or other known or as yet unknown AAV serotypes. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those of skill in the art. Such AAV can be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Va.). Alternatively, AAV sequences may be obtained by synthesis or other suitable means by reference to published sequences such as those available in the literature or databases, e.g., GenBank, PubMed, etc.

[0065] Modified cells. In some embodiments, the disclosure provides a cell comprising a vector of the disclosure.

[0066] The cell can be a prokaryotic or eukaryotic cell. Suitable cells include, but are not limited to, mammalian cells, bacterial cells, yeast cells, fungal cells, and insect cells.

[0067] In some embodiments, the disclosure provides a mammalian cell comprising the dual promoter vector of the disclosure. In some embodiments, the cell is a mammalian cell capable of expressing a human sequence and / or producing a human protein. In some embodiments, the mammalian cell is isolated or derived from a mouse, rat, guinea pig, rabbit, cat, dog, or non-human primate. In some embodiments, the cell is a human cell capable of expressing a human sequence and / or producing a human protein. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is an immortalized or stabilized cell line. Non-limiting examples of mammalian cells that can be used for protein expression include Chinese hamster ovary (CHO) cells, HeLa cells, human embryonic kidney 293 (HEK293) cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells, aurochs (Bos primigenius), and Mus musculus. In a specific embodiment, the cell is a CHO cell. In addition, the mammalian cells can be established commercially available cell lines (e.g., American Type Culture Collection (ATCC), Manassas, VA). The cells can be immortalized cells. Alternatively, the cells can be primary cells.

[0068] In some embodiments, the cell is a bacterial cell. Non-limiting examples of suitable bacterial cells include E. coli and other Enterobacteriaceae, Escherichia sp., Campylobacter sp., Wolinella sp., Desulfovibrio sp., Vibrio sp., Pseudomonas sp., Bacillus sp., Listeria sp., Staphylococcus sp., Streptococcus sp., Peptostreptococcus sp., Megasphaera sp., Pectinatus sp., Selenomonas sp., and the like. sp., Zymophilus sp., Actinomyces sp., Arthrobacter sp., Frankia sp., Micromonospora sp., Nocardia sp., Propionibacterium sp., Streptomyces sp., Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Acetobacterium sp., Eubacterium sp., Heliobacterium sp. sp., Heliospirillum sp., Sporomusa sp., Spiroplasma sp., Ureaplasma sp., Erysipelothrix sp.), Corynebacterium sp., Enterococcus sp., Clostridium sp., Mycoplasma sp., Mycobacterium sp., Actinobacteria sp., Salmonella sp., Shigella sp., Moraxella sp., Helicobacter sp., Stenotrophomonas sp., Micrococcus sp., Neisseria sp., Bdellovibrio sp., Hemophilus sp., Klebsiella sp. sp., Proteus mirabilis, Enterobacter cloacae, Serratia sp., Citrobacter sp., Proteus sp., Serratia sp., Yersinia sp., Acinetobacter sp., Actinobacillus sp., Bordetella sp., Brucella sp., Capnocytophaga sp., Cardiobacterium sp., Eikenella sp., Francisella sp., Haemophilus sp., Kingella sp. sp., Pasteurella sp., Flavobacterium sp., Xanthomonas sp., Burkholderia sp., Aeromonas sp., Plesiomonas sp.), Legionella sp., and alpha-proteobaeteria, e.g., Wolbachia sp., cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria, gram-negative cocci, fastidious gram-negative bacilli, Enterobacteriaceae glucose-fermenting gram-negative bacilli, glucose non-fermenting gram-negative bacilli, glucose-fermenting oxidase-positive gram-negative bacilli. Particularly useful bacterial cells for protein expression include gram-negative bacteria such as Escherichia coli, Pseudomonas fluorescens, Pseudomonas haloplanctis, Pseudomonas putida AC 10, Pseudomonas pseudof lava, Bartonella henselae, Pseudomonas syringae, Caulobacter crescentus, Zymomonas mobilis, Rhizobium meliloti, Myxococcus xanthus, as well as gram-positive bacteria such as Bacillus subtilis. Examples of expression cells include Escherichia coli, Corynebacterium subtilis, Streptococcus cremoris, Streptococcus lividans, and Streptomyces lividans. Escherichia coli is one of the most widely used expression cells. Thus, techniques for overexpression in E. coli are well developed and readily available to those skilled in the art. In addition, Pseudomonas fluorescens is commonly used for high-level production of recombinant proteins (i.e., development of biological therapeutics and vaccines).

[0069] In some embodiments, the cell is a yeast cell or a fungal cell. Particularly useful fungal cells for protein expression include Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, Aspergillus nidulans, and Fusarium graminearum. Particularly useful yeast cells for protein expression include Candida albicans, Candida maltose, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Yarrowia lipolytica.

[0070] In some embodiments, the cell is an insect cell, non-limiting examples of which include a Spodoptera frugiperda cell line (e.g., Sf9 or Sf21), a Drosophila cell line, or a mosquito cell line (e.g., a cell line from Aedes albopictus).

[0071] In some embodiments, the cells are primary cells that are modified to express a vector of the present disclosure and cultured ex vivo, hi some embodiments, the cultured cells are immortalized or otherwise modified to promote the growth of the cells in vitro indefinitely, generating a cultured cell line.

[0072] Cells containing the dual promoter vectors of the present disclosure can be used for protein expression and, optionally, purification. Methods for expressing proteins from cells and, optionally, purifying the expressed proteins are standard methods in the art.

[0073] In some embodiments, cells containing the dual promoter vector of the present disclosure can be used to produce a polypeptide encoded by the enzyme construct of the present disclosure. In general, the production of a polypeptide of the present disclosure involves transfecting a cell with a vector containing the enzyme construct, and then culturing the cell so that the cell transcribes and translates the desired polypeptide. The isolated cells can then be lysed and the expressed polypeptide extracted for subsequent purification.

[0074] Pharmaceutical compositions and formulations. Also provided herein are pharmaceutical compositions comprising a lysosomal enzyme expressed by the dual promoter vector of the present disclosure.

[0075] Such pharmaceutical compositions are in a form suitable for administration to a subject, or the pharmaceutical compositions may further comprise one or more pharma- ceutically acceptable carriers, one or more additional ingredients, or any combination thereof. Various components of the pharmaceutical compositions may be present in the form of physiologically acceptable salts, e.g., in combination with physiologically acceptable cations or anions, as is well known in the art.

[0076] The relative amounts of the active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the present disclosure may vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered.

[0077] Pharmaceutical compositions useful in the methods of the present disclosure can be developed to be suitable for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous, or another route of administration. Other contemplated formulations include planned nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route of administration will be readily apparent to one of skill in the art and will depend on many factors, including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0078] The pharmaceutical composition formulation described herein can be prepared by any method known or hereafter developed in the field of pharmacology.In general, such preparation method includes the step of bringing active ingredient into association with carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into desired single or multiple dose units.In some embodiments, the composition of the present disclosure can be formulated as naked RNA into natural capsid, modified capsid, or can be enclosed in a protective coat.

[0079] The amount of active ingredient is generally equal to a dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or for one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0080] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans according to certified standards, those skilled in the art will understand that such compositions are generally suitable for administration to all kinds of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and a veterinary pharmacologist of ordinary skill can design and implement such modifications, if necessary, with no more than routine experimentation. Subjects to which the pharmaceutical compositions of the present disclosure are contemplated include, but are not limited to, humans and other primates, mammals including commercially suitable mammals, such as cows, pigs, horses, sheep, cats, and dogs. In one embodiment, the subject is a human or non-human mammal, such as, but not limited to, horses, sheep, cows, pigs, dogs, cats, and mice. In one embodiment, the subject is a human.

[0081] In one embodiment, the composition is formulated using one or more pharma- ceutically acceptable excipients or carriers.In some embodiments, the present disclosure provides a pharmaceutical composition for treating a subject suffering from LSD.In some embodiments, the present disclosure provides a pharmaceutical composition comprising the lysosomal enzyme expressed by the dual promoter vector of the present disclosure and a pharma- ceutically acceptable carrier.

[0082] Useful pharma- ceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharma-ceutically acceptable salt solutions, such as phosphates and salts of organic acids. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it is preferable to include an isotonic agent, such as sugar, sodium chloride, or a polyalcohol, such as mannitol and sorbitol, in the composition. Prolonged absorption of an injectable composition can be achieved by including an agent that delays absorption, such as aluminum monostearate or gelatin, in the composition.

[0083] The formulations can be used as mixtures with conventional excipients, i.e., pharma- ceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art. The pharmaceutical preparations can be sterilized and, if desired, can be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffers, coloring substances, flavoring substances, and / or aromatic substances. The pharmaceutical preparations can also be combined, if desired, with other active agents, such as other analgesics.

[0084] The compositions of the present disclosure may include a preservative at about 0.005% to 2.0% of the total weight of the composition. Preservatives are used to prevent deterioration when exposed to environmental contaminants. Examples of preservatives useful in the present disclosure include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. In some embodiments, the preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0085] The compositions may include antioxidants and chelating agents that inhibit degradation of the compounds. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol, and ascorbic acid in the preferred range of about 0.01%-0.3%, more preferably BHT in the range of 0.03%-0.1% by weight of the total weight of the composition. Preferably, the chelating agent is present in an amount of 0.01%-0.5% by weight of the total weight of the composition. Particularly preferred chelating agents include edetate (e.g., disodium edetate) and citric acid in the weight range of about 0.01%-0.20%, more preferably in the range of 0.02%-0.10% by weight of the total weight of the composition. Chelating agents are useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. In some embodiments, BHT and disodium edetate are antioxidants and chelating agents, respectively, for some compounds, although other suitable and equivalent antioxidants and chelating agents may be substituted therefor, as may be known to those skilled in the art.

[0086] The present disclosure provides a pharmaceutical composition comprising a lysosomal enzyme expressed by a vector of the present disclosure and a pharma- ceutically acceptable carrier.

[0087] The compositions of the present disclosure may be used for enzyme replacement therapy (ERT). Alternatively, or in addition, the compositions of the present disclosure may be used for gene therapy.

[0088] The method of the present disclosure. The present disclosure provides a method for treating defective lysosomal enzymes in a subject diagnosed with or at risk of developing LSD. The method improves phosphorylation of the lysosomal enzymes, thereby treating or preventing the occurrence of LSD in the subject. Additionally, the method improves the quality of life of the patient.

[0089] The present disclosure provides a method of treating LSD comprising administering to a subject a composition of the present disclosure, thereby treating the LSD.

[0090] The present disclosure provides a method of treating LSD, comprising administering to a subject a therapeutically effective amount of a composition of the present disclosure, wherein the composition increases phosphorylation of a lysosomal enzyme, thereby treating the LSD.

[0091] The present disclosure provides a method of treating a subject suffering from an LSD, comprising administering to the subject a pharmaceutical composition of the present disclosure, thereby increasing phosphorylation of a lysosomal enzyme and treating the subject.

[0092] The present disclosure provides a method of preventing the onset of LSD in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present disclosure, thereby increasing phosphorylation of a lysosomal enzyme and preventing the onset of LSD in the subject.

[0093] The present disclosure provides a method of restoring phosphorylation of a lysosomal enzyme involved in LSD in a subject in need thereof, comprising administering to the subject a composition of the present disclosure, wherein the composition increases phosphorylation of the lysosomal enzyme.

[0094] Administration / dosage. The administration schedule (regimen) can affect what constitutes an effective amount. For example, the therapeutic formulation can be administered to the patient subject either before or after a surgical intervention related to LSD, or immediately after the patient is diagnosed with LSD. Furthermore, several divided doses and dispersed doses can be administered daily or sequentially, or the dose can be continuously infused or bolus injected. Furthermore, the dosage of the therapeutic formulation can be proportionally increased or decreased as required by the exigencies of the treatment or prophylactic situation.

[0095] Administration of the compositions of the present disclosure to a patient subject, preferably a mammal, more preferably a human, can be carried out using known procedures at dosages and durations effective to treat LSD in the subject. The effective amount of the therapeutic compound required to achieve a therapeutic effect can vary depending on several factors, such as the activity of the particular compound used, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds or substances used in combination with the compound, the state of the disease or disorder, age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field. The dosing regimen can be adjusted to achieve an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced if required by the exigencies of the therapeutic situation. One non-limiting example of an effective dose range for the therapeutic compounds of the present disclosure is about 0.01 and 50 mg / kg body weight / day.

[0096] The compound may be administered to a subject as frequently as several times daily, or may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less. It is understood that the amount of compound administered per day may be administered, in non-limiting examples, every day, every other day, every second day, every third day, every fourth day, or every fifth day. For example, in every other day administration, a dose of 5 mg per day is administered starting on Monday, the first subsequent dose of 5 mg per day is administered on Wednesday, the second subsequent dose of 5 mg per day is administered on Friday, and so on. The frequency of administration is readily apparent to those skilled in the art and depends on many factors, including but not limited to the type and severity of the disease being treated, and the type and age of the animal. The actual dosage level of the active ingredient in the pharmaceutical composition of the present disclosure may be varied to obtain the amount, composition, and administration mode of the active ingredient that is effective for achieving the desired therapeutic response in a particular patient without being toxic to the patient.A medical doctor, such as a physician or veterinarian, with ordinary skill can easily determine and prescribe the effective amount of the pharmaceutical composition required.For example, a physician or veterinarian may start the dosage of the compound of the present disclosure used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0097] In some embodiments, it is particularly advantageous to formulate the compound into dosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically separate unit suitable as a unitary dosage for the patient to be treated, each unit containing a predetermined amount of therapeutic compound calculated to produce a desired therapeutic effect, associated with a required pharmaceutical vehicle.The dosage unit form of the present disclosure is determined by and directly depends on (a) the inherent characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technical field of compounding / formulating such therapeutic compound for the treatment of LSD.

[0098] Route of administration. One of skill in the art will recognize that, although more than one route of administration is available, a particular route may provide a more immediate and effective response than another route.

[0099] Routes of administration of the compositions of the present disclosure include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​intravaginal (e.g., vaginal and perivaginal), nasal, and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, intracisternoma (ICM), intraspinal, intraventricular, intraventricular, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel capsules, troches, dispersions, suspensions, liquids, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, disks, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like. It should be understood that the formulations and compositions that may be useful in the present disclosure are not limited to the specific formulations and compositions described herein. In one embodiment, the treatment of LSD comprises a route of administration selected from the group consisting of inhalation, oral, rectal, intravaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrahepatic artery, intrathoracic, intrathecal, intratumoral, intravenous, and any combination thereof.

[0100] Those skilled in the art will recognize that a variety of delivery methods are available for administering vectors to cells, including (1) physical means such as electroporation (electricity), using a gene gun (physical force) or applying large volumes of liquid (pressure), and (2) methods in which the vector is complexed with another entity such as a liposome, an aggregation protein, or a transporter molecule.

[0101] Furthermore, the actual dose and schedule may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, the amount may vary depending on the particular cell line utilized in in vitro applications (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector used for gene transfer to replicate in that cell line). Furthermore, the amount of vector added per cell will likely vary depending on the length and stability of the therapeutic gene inserted into the vector, as well as the nature of the sequence, in particular parameters that must be empirically determined and may be subject to change due to factors not inherent to the method of the present disclosure (e.g., costs associated with synthesis). Those skilled in the art can easily make any necessary adjustments according to the exigencies of a particular situation.

[0102] The cells containing the therapeutic agent may also contain a suicide gene, i.e., a gene encoding a product that can be used to destroy the cell. In many gene therapy situations, it is desirable to be able to express a gene of therapeutic interest in the host, i.e., the cell, but it is also desirable to have the ability to destroy the host cell at will. The therapeutic agent may be linked to a suicide gene whose expression is not activated in the absence of an activating compound. When death of a cell into which both the drug and the suicide gene have been introduced is desired, an activating compound is administered to the cell, thereby activating expression of the suicide gene and killing the cell. Examples of suicide gene / prodrug combinations that can be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

[0103] [Example] The present disclosure will now be described with reference to the following examples, which are provided for illustrative purposes only, and the present disclosure should in no way be construed as being limited to these examples, but rather as encompassing any variations that become evident as a result of the present disclosure provided herein.

[0104] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the disclosed compounds and practice the claimed methods. The following examples should not be construed as limiting the disclosure in any way.

[0105] [Example 1] Construction of S1S3-GLA dual promoter vector. To generate a vector containing two promoters, DNA fragments were assembled to generate gene expression cassettes that could express S1S3 PTase and the lysosomal enzyme GLA from separate promoters and separate 3'UTRs. The fragments were assembled in various compositions such that the lysosomal enzyme was driven by a strong promoter (i.e., Cbh promoter) with a standard 3'UTR (i.e., bovine growth hormone polyA) and S1S3 PTase was driven by a separate promoter (i.e., EF-1-alpha core (Efs) or modified JeT promoter) with a separate 3'UTR (i.e., sv40 polyA). These two gene expression cassettes were then assembled in a plasmid for the generation of AAV (see FIG. 1 ) such that (1) the 5' ends of both promoters were adjacent (inner-outer), (2) the 3' UTR regions were adjacent (outer-inner), or (3) the two cassettes were arranged consecutively (the 3' UTR of cassette 1 was adjacent to the 5' region of the second promoter) (contiguous).

[0106] DNA fragments containing promoters (e.g., EFs or JeT) and UTRs (e.g., sv40, bGH polyA) were ordered from IDT or Twist Biosciences (https: / / www.idtdna.com / pages / products / genes-and-gene-fragments / double-stranded-dna-fragments / gblocks-gene-fragments), a leader in DNA synthesis innovation, Twist Bioscience (https: / / www.twistbioscience.com / ). These fragments were assembled using restriction enzymes and standard molecular biology techniques that allowed the creation of cassettes containing lysosomal enzymes or S1S3 PTase. Restriction sites (i.e., SacII, BamHI, NheI) were placed to allow replacement of lysosomal enzymes or modify the S1S3 PTase sequence (XbaI / PmeI). Using standard molecular biology techniques (restriction digestion, ligation, bacterial transformation), the DNA fragments were assembled to generate the final plasmid containing the ITR sequences from AAV.

[0107] The gene expression cassettes were placed in three different orientations (Figure 1). The first orientation is described as Inside-Out, where the promoters are flanked to drive gene expression outward with the 3'UTR sequence on the opposite side. The promoters used can be two different promoters, e.g., Cbh, EFs, JeT or bidirectional promoters. The second orientation, Outside-In, features the promoters in a distal configuration, flanked by the 3'UTR regions of the cassette. This allows the genes to be driven by separate promoters, e.g., Cbh, EFs, JeT. The 3'UTR can be bovine growth hormone polyA, sv40 polyA, rabbit beta globin polyA, or a sequence from a bidirectional UTR, e.g., sv40. The third orientation is the sequential orientation, where the gene expression cassettes are placed consecutively on the same strand. The sequential orientation has the 5' promoter, first gene, then the 3'UTR, followed by the second promoter, gene and second UTR consecutively. These different orientations allow for the expression of mRNA from individual promoters, which can be used to generate proteins of S1S3 PTase with lysosomal enzymes. This configuration allows a specific set of promoters and UTRs to individually provide controlled gene expression of each gene by using separate gene cassettes. Figure 1 shows how GLA and S1S3 PTase can be expressed using two promoters. Figure 1 shows the configuration of the AAV expression cassette. These dual promoter arrangements for co-expression of S1S3 PTase can be used for protein expression in enzyme replacement therapy, AAV-based gene therapy, and nanoparticle-based technologies.

[0108] [Example 2] Transfection of the inside-out dual promoter vector into HEK293T cells. First, the dual promoter inside-out design for co-expression of S1S3 PTase and lysosomal enzymes (disclosed in Example 1) was tested using transient transfection into HEK293T cells. First, cells were transfected with the inside-out plasmid ((G0543, Efs-S1S3-sv40 / Cbh-GLA-bGH polyA), (G0547, JeT-S1S3-sv40 / Cbh-GLA-bGH polyA)) or control (G0500, Cbh-GLA-sv40) (Figure 2A). Cell lysates were assayed for GLA activity. An increase in GLA activity by plasmid transfection compared to mock transfection control was detectable in the control (G0500) and the inside-out dual promoter system for GLA / S1S3 PTase expression (G0543 or G0547) (Figure 2B). Expression of GLA protein and S1S3 PTase protein was confirmed by Western blot (Figure 2C). These panels demonstrate the functionality of the inside-outside dual promoter construct.

[0109] Next, a dual promoter exo-intra design for co-expression of S1S3 PTase and lysosomal enzymes was performed using transient transfection into HEK293T cells. First, cells were transfected with exo-intra plasmid G0544 (Efs-S1S3-sv40 / Cbh-GLA-bGH polyA), G0595 (JeT-S1S3-sv40 / Cbh-GLA-bGH polyA), intra-intra construct G0543 (Efs-S1S3-sv40 / Cbh-GLA-bGH polyA), or control (G0500, Cbh-GLA-sv40) (Figure 3A). Cell lysates were assayed for GLA activity. Again, an increase in GLA activity by plasmid transfection compared to mock transfection controls was detectable in the control (G0500), the inside-out dual promoter system (G0543), and using the outside-in dual promoter system for GLA / S1S3 expression (G0544 or G0595) (Figure 3B). Expression of GLA protein and S1S3 PTase protein was confirmed by Western blot (Figure 3C). These panels demonstrate the functionality of the inside-out dual promoter configuration.

[0110] method. HEK-293T cells were transfected with plasmids for AAV cassettes containing dual promoters, one (e.g., Cbh) driving GLA expression and the other (e.g., EFs) driving S1S3 PTase expression, using lipofectamine 3000. Cells were harvested 48 hours after transfection, and conditioned media and cell lysates were stored for analysis by enzyme activity assays, CIMPR binding, and Western blot.

[0111] α-GAL activity was measured according to established protocols (see Mayes, Scheerer et al., 1981; Yasuda, Huston et al., 2020).

[0112] Briefly, the assay was performed in 0.1M-0.2M citrate-phosphate buffer, pH 4.5, 0.25% TX-100. The α-GAL substrate was 4-methylumbelliferyl α-D-galactopyranoside, Cat:M7633, Millipore-Sigma) dissolved in DMSO, diluted to 1-5 mM in assay buffer. The assay buffer contained Gal-B inhibitor (GalNAc) N-acetyl-galactosamine, Cat:A2795, Millipore-Sigma 90 mM. 45ul of substrate solution was aliquoted into a 96-well plate and 5ul of sample (cell lysate, conditioned media, purified protein, etc.) was added in duplicate (2 wells per sample). To compare activity, serial dilutions of 4-MU (4-methylumbelliferone sodium salt, Sigma, M1381) were added to the same plate during the assay. The plate was then sealed and incubated for 1 hour at 37°C with shaking / agitation. The reaction was stopped by the addition of 150ul of stop buffer (0.4M glycine-NaOH, pH 10.8). α-GAL activity was then measured using substrate fluorescence at Ex 360, Em 460 using a Molecular Devices Spectramax Id3 plate reader and Softmax Pro 7.1 software. Background fluorescence was subtracted based on substrate alone or mock transfection conditions. Analysis was performed in Excel and graphs generated using GraphPad Prism software. Protein concentration of samples was measured using the BCA protein assay (ThermoFisher 23227) according to the manufacturer's protocol. Absorbance at 260 was then measured using a Molecular Devices Spectramax Id3 plate reader and Softmax Pro 7.1 software was used to measure concentrations compared to BSA standards.

[0113] [Example 3] Dual promoter vector expression in vivo. To determine the expression of AAV9 gene therapy using dual promoter transgenes of GLA and S1S3 PTase, 8-week-old wild-type C57BL / 6J mice were dosed with a medium dose of GLA-only AAV9 or GLA with S1S3 PTase in dual promoter inside-out configuration. Two weeks after AAV9 injection, tissues were harvested and homogenized. Gene copy and its mRNA levels were measured by digital droplet PCR. Compared with A0500-GLA-only AAV9, the two dual promoter AAV9 vectors show higher GLA and S1S3 PTase gene copy levels (Figure 4A and Figure 4B) and mRNA levels (Figure 4C and Figure 4D) in the liver and kidney.

[0114] [Example 4] Contiguous promoter design for co-expression of PPT1 and S1S3 PTase A sequential promoter design for co-expression of PPT1 and S1S3 phosphotransferase was used for transfection in Expi293 suspension cells. (Figure 5A) shows the sequential plasmid design with a CMV promoter for expression of PPT1 and a second promoter (PGK, JeT, EF1-alpha) for S1S3 expression, and G0802 is a plasmid for expression of PPT1 alone. Expi293 cells were transfected with the indicated plasmids, conditioned media was harvested 48 hours after transfection, and expression of PPT1 was examined by SDS-PAGE and Coomassie staining (Figure 5B). All PPT1-containing plasmids demonstrate comparable PPT1 enzyme / protein expression. The effect of S1S3 expression was examined in cell lysates by Western blotting against a V5 tag fused to the C-terminus of the S1S3 gene. As shown in Figure 5C, all promoters of S1S3 result in good expression of S1S3, with EF1-alpha resulting in the best expression. Cation-independent mannose 6-phosphate receptor (CI-MPR) binding assays were also performed using conditioned medium from transfected cells. Binding was determined using a PPT1 activity assay. Plasmids containing the contiguous promoters for S1S3 expression demonstrate enhanced binding of PPT1 to the CI-MPR (G0813, G0816, G0817) compared to PPT1 expression alone (G0802) (Figure 5D).

[0115] [Example 5] Dual promoter design for AAV vector-mediated expression of GBA1 In vitro. The effect of the dual promoter design was evaluated using AAV vectors. A plasmid was designed containing the AAV ITR sequence and a cassette for the expression of the GBA1 gene using the Cbh promoter. In addition, an outside-inside design was used to co-express S1S3 using the short EF1-a promoter (EFs) (G0158 and G0194). The co-expression plasmid was compared to a plasmid expressing only GBA1 (G0101) (Figure 6A).

[0116] The exogenous AAV plasmids were transfected into Expi293 suspension cells. Cells were transfected with a plasmid expressing GBA1 alone (G0101) or GBA1 / S1S3 co-expressing plasmids (G0158 and G0194) and conditioned media was harvested 48 hours post-transfection. Conditioned media was assayed for GCase enzyme activity using an in vitro activity assay. Minimal activity is detectable in vehicle transfected cells and there is comparable GCase activity in (G0101, G0158 or G0194) transfected cells (Figure 6B). Conditioned media (from Figure 6B) was examined by SDS-PAGE and Western blot using anti-GCase (top) or anti-GAPDH loading control (bottom) (Figure 6C). There is similar amount of GCase in G0101, G0158 or G0194 and minimal GCase in media from vehicle control. (Note the size shift due to the K360N mutation in G0158 / G0194). Conditioned media (from FIG. 6B) was assessed for binding to the CI-MPR. CI-MPR was prebound to 96-well dishes and incubated with conditioned media from G0101- or G0194-expressing cells. Binding was determined using a GCase activity assay (FIG. 6D). G0194 (orange) demonstrates enhanced binding to the CI-MPR compared to G0101 (grey).

[0117] In vivo animal studies. Plasmids described in (Figure 5A) were used to generate AAV9 viral vectors for expression of GBA1 alone (G0101) or for co-expression with S1S3 (G0158 and G0194). Wild-type mice were intravenously (IV) injected via the tail vein with 2e13vg / kg AAV9 or formulation buffer as a control. 21 days after injection, animal tissues were harvested, homogenized, and GCase activity was measured in the lysates. GCase activity was detected in the liver, heart, and brain (Figure 7).

[0118] Tissues were also processed for analysis of GBA1 and S1S3 genomic DNA and mRNA from liver, heart, brain, bone marrow, and spleen. DNA and mRNA were analyzed by ddPCR using specific primers and probes to detect GBA1 and S1S3. As shown in Figures 8A-8B, increased GBA1 DNA (Figure 8A) and mRNA (Figure 8B) copies were detected in all tissues of animals treated with AAV (G0101, G0158, G0194; blue). Increased S1S3 DNA (Figure 8A) and mRNA (Figure 8B) copies were detected in all tissues of animals treated with AAV (G0158, G0194; orange). This demonstrates that AAV9 can co-express both GBA1 and S1S3 (G0158 and G0194).

[0119] To better understand AAV9 transduction into mouse brain via systemic injection (IV), mouse brain was examined by Basescope, which evaluates GBA1 transcription in situ. The striatum was examined by microscopy to visualize GBA1 mRNA expression and distribution in the striatum from mouse brain. The panel demonstrates similar GBA1 mRNA expression for all mouse brains treated with AAV9 containing the GBA1 gene (Figure 9). GBA1 is undetectable in vehicle-treated controls.

[0120] Mouse brains were also examined by immunohistochemical staining using a human GCase-specific antibody to detect GCase enzyme / protein in the somatosensory cortex of mouse brains. The panel demonstrates enhanced GCase staining for mice treated with GBA1-S1S3 co-expressing AAV9 (G0158 and G0194) (Figure 10). This suggests that the tissue distribution of GCase is enhanced when co-expressed with S1S3 using our dual promoter AAV design.

[0121] [Example 6] Dual promoter design for AAV vector-mediated expression of HexM A plasmid was designed containing AAV ITR sequences and a cassette for expression of HexM with a C-tailed FLAG tag using the Cbh promoter (for a description of HexM, see Tropak, MB, Yonekawa, S, Karumuthil-Melethil, S, Thompson, P, Wakarchuk, W, Gray, SJ, et al. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo. Mol. Ther. - Methods Clin. Dev. 3: 15057 (2016)) (Figure 11A). In addition, an outside-inside design was used to co-express S1S3 using the short EF1-a promoter (EFs) (G0720). The co-expression plasmid was compared to a plasmid expressing only FLAG-tagged HexM (G0718).

[0122] The exogenous AAV plasmids were transfected into Expi293 suspension cells. Cells were transfected with a plasmid expressing HexM alone (G0718) or a HexM / S1S3 co-expressing plasmid (G720), and conditioned media and cell lysates were harvested 48 hours post-transfection. Conditioned media was assayed for HexM enzyme activity using 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside) sodium salt (MUGS, sigma, M0662). Minimal activity was detectable in vehicle-transfected cells, and comparable HexM activity was present in (G0718 or G0720)-transfected cells (Figure 11B). Cell lysates from transfected Expi293 cells were assayed for HexM activity by the 4-MU substrate MUGS (Figure 11C) and for phosphotransferase (PTase) activity (Lin Liu et al., Mol Ther Methods Clin Dev. 2017 Mar 29;5:59-65) (Figure 11D). Similar HexM activity is detected in cell lysates from cells transfected with HexM plasmids (G0718 or G0720). Only cells transfected with the S1S3 expression plasmid (G0720) show enhanced PTase activity compared to vehicle or cells expressing HexM alone (Figure 11D). Conditioned media (from Figure 11B) was assessed for binding to the CI-MPR. CI-MPR was prebound to 96-well dishes and incubated with conditioned media from G0718- or G0720-expressing cells. Binding was determined using a HexM activity assay (FIG. 11E). G0720 (orange) demonstrates enhanced binding of HexM to the CI-MPR compared to G0718 (grey).

[0123] Methods: Wild-type mice were purchased from Jackson laboratory and transported to Sanford Research center for animal studies. Mice were maintained on a 12-hour light / 12-hour dark cycle. At 8 weeks of age, animals were dosed intravenously (IV) via the tail vein, with 5 animals per group. Three weeks after injection, animals were sacrificed and tissues were harvested after perfusion with 10 mL of cold PBS buffer. Consistent areas from each tissue were collected for either quick freezing or fixation in 4% formaldehyde. Viability observations for pain, moribundity, and death were performed at Sanford research center, and body weights were assessed daily. No animals showed any significant changes during the study.

Claims

1. A composition comprising a vector comprising an sequence encoding a first promoter operably linked to a first polynucleotide encoding a lysosomal enzyme and a second promoter operably linked to a second polynucleotide encoding a modified GlcNAc-1 phosphotransferase.

2. The composition of claim 1 , wherein the vector is a viral vector.

3. The composition of claim 1 , wherein the vector is a non-viral vector.

4. The composition of claim 1, wherein the vector is an adenovirus vector or an adeno-associated virus (AAV) vector.

5. The composition of claim 1 , wherein the vector is a plasmid.

6. 6. The composition of claim 1, wherein the first promoter is CBH and the second promoter is selected from EFS or JeT.

7. 6. The composition of any one of claims 1 to 5, wherein the first promoter is CMV and the second promoter is selected from PGK, JeT, or EF1-α.

8. 8. The composition of claim 1, wherein the modified GlcNAc-1 phosphotransferase comprises an S1S3 PTase.

9. The composition of any one of claims 1 to 8, wherein the lysosomal enzyme is selected from the group consisting of b-glucosebrosidase (GBA), galactosylceremidase (GALC), a-galactosidase (GLA), a-N-acetylglucosaminidase (NAGLU), acid a-glucosidase (GAA), lysosomal acid a-mannosidase (LAMAN), and HexM.

10. 10. The composition of any one of claims 1 to 9, wherein the vector further comprises a 3'UTR.

11. The composition of claim 10, wherein the 3'UTR is selected from SV40 and bGH polyA.

12. 12. The composition of any one of claims 1 to 11, further comprising a pharma- ceutically acceptable carrier.

13. A method for treating a lysosomal storage disorder (LSD), comprising administering to a subject an effective amount of a composition described in any one of claims 1 to 12, wherein the composition increases phosphorylation of a lysosomal enzyme involved in the LSD, thereby treating the LSD.

14. 14. The method of claim 13, wherein the subject has been diagnosed with LSD.

15. 14. The method of claim 13, wherein the subject exhibits signs or symptoms of LSD.

16. A method for preventing the occurrence or onset of a lysosomal storage disorder (LSD), comprising administering to a subject an effective amount of a composition described in any one of claims 1 to 12, wherein the composition increases phosphorylation of a lysosomal enzyme involved in LSD, thereby preventing the occurrence of LSD in the subject.

17. 17. The method of claim 16, wherein the subject is at risk of developing or developing an LSD.

18. 17. The method of claim 16, wherein the subject exhibits signs or symptoms of LSD.

19. A method for restoring phosphorylation of a lysosomal enzyme involved in a lysosomal storage disorder (LSD), comprising contacting a cell with an effective amount of a composition described in any one of claims 1 to 12, wherein the composition increases phosphorylation of the lysosomal enzyme.

20. 20. The method of claim 19, wherein the cell is an in vitro or ex vivo cell.

21. The method of claim 19, wherein the subject comprises a cell.

22. 22. The method of claim 21, wherein the subject exhibits signs or symptoms of LSD.

23. 22. The method of claim 21, wherein the subject is at risk of developing or developing an LSD.

24. 22. The method of claim 21, wherein the subject has been diagnosed with LSD.

25. 25. The method of any one of claims 13 to 24, wherein administering comprises a systemic route of administration.

26. 26. The method of claim 25, wherein the systemic administration route is enteral, parenteral, oral, intramuscular (IM), subcutaneous (SC), intravenous (IV), intraarterial (IA), intrathecal, intraspinal, or intraventricular.

27. 25. The method of any one of claims 13 to 24, wherein administering comprises a topical route of administration.

28. 25. The method of any one of claims 13 to 24, wherein the subject is a human.

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