Adeno-associated virus titer assay and uses thereof

Abstract

The present invention provides methods and kits for detecting and monitoring the efficacy of enzymes encoded by adeno-associated viruses. Exemplary enzymes that may be used in conjunction with the compositions and methods of the present disclosure include, among others, myotubularin 1 (MTM1) and myotubularin-related proteins, such as myotubularin-related protein 1 (MTMR1), MTMR2, MTMR3, MTMR4, MTMR5, MTMR6, MTMR7, MTMR8, MTMR9, MTMR10, MTMR11, MTMR12, MTMR12, MTMR13, and MTMR14.

Description

[Technical field]

[0001] SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on Mar. 30, 2023, is "51037-061 WO2_Sequence_Listing_3_30_23" and is 20,293 bytes in size. TECHNICAL FIELD TO WHICH THEINVENTION BELONGS The present invention relates to an adeno-associated virus (AAV) efficacy assay, and more particularly to methods, uses and kits for monitoring the activity of enzymes encoded by AAV. CROSS-REFERENCE TO RELATED APPLICATIONS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 327,574, filed April 5, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] One of the most important classes of intracellular activities is the post-translational modification of proteins. Post-translational modification leads to changes in the biological activity and / or function of the putatively modified protein. Post-translational modification can be achieved by enzymes (e.g., phosphatases). For example, protein phosphatases, a type of enzyme, remove phosphate groups from proteins, and changes in the phosphorylation state of proteins can affect protein functions such as protein localization or protein-protein interactions. Such changes in protein function can then regulate cell metabolism, regulation, proliferation, differentiation, but in some cases, the altered protein function can become abnormal and result in pathology. Researchers have found many human diseases and disorders resulting from genetic defects in protein phosphatases. For example, X-linked myotubular myopathy (XLMTM) is a fatal monogenic disease of skeletal muscle disease, resulting from loss-of-function mutations in the protein phosphatase myotubularin 1 (MTM1). Approximately 1 in 50,000 newborns has XLMTM, which typically presents with significant hypotonia and respiratory failure. Recently, a viral vector-mediated (e.g., adeno-associated virus (AAV)-mediated) gene therapy approach involving delivery of MTM1 has been developed for the treatment of XLMTM. However, there is a need in the art for improved methods to evaluate the efficacy of viral vectors (e.g., AAV) encoding enzymes (e.g., phosphatases), such as AAV encoding MTM1, that are sensitive and adaptable to high-throughput screening methods. Summary of the Invention

[0003] The present disclosure provides methods and uses for determining the efficacy of an adeno-associated virus (AAV) vector encoding an enzyme (e.g., myotubularin 1 (MTM1)) by assessing the amount or concentration of the product resulting from enzymatic cleavage of a substrate, for example, by high performance liquid chromatography (HPLC) in AAV-permissive cells, in the surrounding extracellular medium, or from cell lysates. In certain embodiments, after a change in the amount or concentration of the cleavage product is determined, the patient is administered a therapeutically effective amount of the AAV encoding the enzyme.

[0004] In one aspect, the disclosure provides a method for determining efficacy of an adeno-associated viral (AAV) vector encoding an enzyme for the treatment of a loss-of-function disease associated with a deleterious mutation in a patient, the method comprising: The present invention provides a method of treating a loss-of-function disease associated with a deleterious mutation, comprising: a) contacting an AAV vector with an AAV-permissive cell that expresses a substrate that is specifically cleaved by an enzyme for a period of time sufficient for the AAV-permissive cell to express the enzyme; and b) evaluating by chromatography the amount or concentration of the substrate or a product resulting from cleavage of the substrate by the enzyme in the cell or surrounding extracellular medium; wherein the AAV vector is identified as having efficacy for treating a loss-of-function disease associated with a deleterious mutation upon a finding that (i) the amount or concentration of the cleavage product in the cell or surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the cell or surrounding extracellular medium is decreased compared to a reference amount or concentration of the substrate.

[0005] In another aspect, the disclosure provides a method for determining efficacy of an adeno-associated virus (AAV) vector encoding an enzyme for the treatment of a loss-of-function disease associated with a deleterious mutation in a patient, the method comprising: The present invention provides a method for treating a loss-of-function disease associated with a deleterious mutation in an AAV vector, the method comprising: a) contacting an AAV vector with an AAV-permissive cell for a sufficient period of time for the AAV-permissive cell to express the enzyme; b) lysing the AAV-permissive cell line and exposing the expressed enzyme to a substrate that is specifically cleaved by the enzyme in a reaction medium; and c) evaluating by chromatography the amount or concentration of the substrate or a product resulting from cleavage of the substrate by the enzyme in the reaction medium, wherein the AAV vector is identified as having efficacy for treating a loss-of-function disease associated with a deleterious mutation upon a finding that (i) the amount or concentration of the cleavage product in the reaction medium is increased relative to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the reaction medium is decreased relative to a reference amount or concentration of the substrate.

[0006] In another aspect, the disclosure provides a method of producing an adeno-associated virus (AAV) vector encoding an enzyme in a gene encoding the enzyme for the treatment of a loss-of-function disease associated with a deleterious mutation in a patient in need of treatment, the method comprising: a) contacting the AAV vector with an AAV-permissive cell line for a sufficient time for the AAV-permissive cell line to express the enzyme; b) evaluating by chromatography the amount or concentration of a substrate or a product resulting from cleavage of the substrate by the enzyme in the cell or surrounding extracellular medium; and c) releasing the AAV vector for treating the disease if (i) the amount or concentration of the cleavage product in the cell or surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the cell or surrounding extracellular medium is decreased compared to a reference amount or concentration of the substrate.

[0007] In a further aspect, the disclosure provides a method of producing an adeno-associated virus (AAV) vector encoding an enzyme in a gene encoding the enzyme for the treatment of a loss-of-function disease associated with a deleterious mutation in a patient in need of treatment, comprising: a) contacting the AAV vector with an AAV-permissive cell line for a time sufficient for the AAV-permissive cell line to express the enzyme; b) lysing the AAV-permissive cell line and exposing the expressed enzyme to a substrate that is specifically cleaved by the enzyme in a reaction medium; c) assessing by chromatography the amount or concentration of the substrate or a product resulting from cleavage of the substrate by the enzyme in the reaction medium; and d) determining whether (i) the amount or concentration of the cleavage product in the reaction medium is increased relative to a reference amount or concentration of the cleavage product, or (ii) releasing an AAV vector for treating a loss-of-function disease associated with a deleterious mutation when the amount or concentration of the substrate in the reaction medium is decreased relative to a reference amount or concentration of the substrate.

[0008] In another aspect, the disclosure provides a method of producing an adeno-associated virus (AAV) vector encoding an enzyme in a gene encoding the enzyme for treatment of a loss-of-function disease associated with a deleterious mutation in a patient in need of treatment, the method comprising: a) contacting the AAV vector with an AAV-permissive cell expressing a substrate that is specifically cleaved by the enzyme for a period of time sufficient for the AAV-permissive cell to express the enzyme; b) determining by chromatography that (i) the amount or concentration of the cleavage product in the cell or surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the cell or surrounding extracellular medium is decreased compared to a reference amount or concentration of the substrate; and c) releasing the AAV vector to treat the disease.

[0009] In another aspect, the disclosure provides a method of producing an adeno-associated virus (AAV) vector encoding an enzyme in a gene encoding the enzyme for treatment of a loss-of-function disease associated with a deleterious mutation in a patient in need of treatment, the method comprising: a) contacting the AAV vector with an AAV-permissive cell for a period of time sufficient for the AAV-permissive cell to express the enzyme; b) lysing the cells and exposing the expressed enzyme to a substrate that is specifically cleaved by the enzyme, in a reaction medium; c) determining by chromatography that (i) the amount or concentration of cleavage product in the reaction medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the reaction medium is decreased compared to a reference amount or concentration of the substrate; and d) releasing the AAV vector to treat the disease.

[0010] In another aspect, the disclosure provides a method for treating a loss-of-function disease associated with a deleterious mutation in a gene encoding an enzyme in a patient in need of treatment, comprising: a) contacting an AAV vector encoding the enzyme with an AAV-permissive cell expressing a cleavable substrate specific for the enzyme for a period of time sufficient for the AAV-permissive cell to express the enzyme; b) determining by chromatography that (i) the amount or concentration of the cleavage product in the cell or surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the cell or surrounding extracellular medium is decreased compared to a reference amount or concentration of the substrate; and c) administering a therapeutically effective amount of the AAV vector to the patient.

[0011] In another aspect, the disclosure provides a method for treating a loss-of-function disease associated with a deleterious mutation in a gene encoding an enzyme in a patient in need of treatment, comprising: a) contacting an AAV vector with an AAV-permissive cell for a period of time sufficient for the AAV-permissive cell to express the enzyme; b) lysing the cell and exposing the expressed enzyme to a substrate that is specifically cleaved by the enzyme, in a reaction medium; c) determining by chromatography that (i) the amount or concentration of the cleavage product in the reaction medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the reaction medium is decreased compared to a reference amount or concentration of the substrate; and d) administering a therapeutically effective amount of the AAV vector to the patient.

[0012] In another aspect, the disclosure provides a method for treating a loss-of-function disease associated with a deleterious mutation in a gene encoding an enzyme in a patient in need of treatment, comprising administering to the patient an AAV vector encoding the gene, wherein the AAV vector has been identified as efficacious for treating the disease by a method comprising: a) contacting the AAV vector with an AAV-permissive cell expressing a substrate that is specifically cleaved by the enzyme for a period of time sufficient for the AAV-permissive cell to express the enzyme; and b) determining by chromatography that (i) the amount or concentration of a product resulting from cleavage of the substrate by the enzyme in the cell or a surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the cell or a surrounding extracellular medium is decreased compared to a reference amount or concentration of the substrate.

[0013] In another aspect, the disclosure provides a method for treating a loss-of-function disease associated with a deleterious mutation in a gene encoding an enzyme in a patient in need of treatment, comprising administering to the patient an AAV vector encoding the gene, wherein the AAV vector has been identified as efficacious for treating the disease by a method comprising: a) contacting the AAV vector with an AAV-permissive cell for a period of time sufficient for the AAV-permissive cell to express the enzyme; b) lysing the cell and exposing the expressed enzyme, in a reaction medium, to a substrate that is specifically cleaved by the enzyme; and c) determining by chromatography: (i) an amount or concentration of the cleavage product in the reaction medium is increased compared to a reference amount or concentration of the cleavage product, or (ii) an amount or concentration of the substrate in the reaction medium is decreased compared to a reference amount or concentration of the substrate.

[0014] In certain embodiments of any of the above aspects, the expressed enzyme is not purified from a cell lysate obtained from a lysis step prior to exposing the enzyme to the cleavable substrate.

[0015] In certain embodiments of any of the above aspects, the enzyme is a phosphatase. For example, in certain embodiments, the phosphatase is a lipid phosphatase (e.g., MTM1, myotubularin-related protein 1 (MTMR1), MTMR2, MTMR3, MTMR4, MTMR5, MTMR6, MTMR7, MTMR8, MTMR9, MTMR10, MTMR11, MTMR12, MTMR12, MTMR13, MTMR14, synaptojanin 1, or phosphatase and tensin homolog deleted on chromosome 10). In certain embodiments, the lipid phosphatase is MTM1.

[0016] In some embodiments of any of the above aspects, determining the amount or concentration of the product resulting from enzymatic cleavage of the substrate further comprises determining the amount or concentration of a marker. For example, in some embodiments, the marker is a fluorescently labeled lipid (e.g., a fluorescently labeled phospholipid), molecule, or protein. In some embodiments, the fluorescently labeled lipid is a fluorescently labeled phospholipid (e.g., phosphatidylinositol 3-phosphate). In some embodiments, the fluorescently labeled phospholipid is phosphatidylinositol 3-phosphate. In some embodiments, the marker is a genetically encoded fluorescent marker (e.g., green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein, red fluorescent protein, mCherry, dsRed, luciferase, β-galactosidase, or chloramphenicol acetyltransferase).

[0017] In some embodiments of any of the above aspects, the AAV-permissive cells are C2C12 cells, Huh7 cells, Hela cells, HelaS3 cells, Hepa1-6 cells, HEK293 cells, HepG2 cells, or IMY-N9 cells. For example, in some embodiments, the AAV-permissive cells are C2C12 cells. In some embodiments, the AAV-permissive cells (e.g., C2C12 cells) are lysed.

[0018] In certain embodiments of any of the above aspects, the chromatography is high performance liquid chromatography (HPLC), thin layer chromatography, column chromatography, ion exchange chromatography, gel permeation chromatography, affinity chromatography, paper chromatography, gas chromatography, hydrophobic interaction chromatography, or pseudo-affinity chromatography. For example, in certain embodiments, the chromatography is HPLC.

[0019] In certain embodiments of any of the above aspects, the HPLC is normal phase HPLC, reverse phase HPLC, phase HPLC, hydrophobic interaction chromatography, or ion chromatography. For example, in certain embodiments, the HPLC is normal phase HPLC.

[0020] In certain embodiments of any of the above aspects, the reference amount or concentration is the expression level of the cleavage product in the AAV-permissive cell line prior to contacting the AAV-permissive cell line with the AAV vector.

[0021] In certain embodiments of any of the above aspects, the reference amount or concentration is the level of cleavage product or substrate in the reaction medium prior to exposing the expressed enzyme to the substrate.

[0022] In certain embodiments of any of the above aspects, the AAV vector comprises a capsid protein from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74.

[0023] In some embodiments of any of the above aspects, the AAV vector is a pseudotyped AAV. In some embodiments, the pseudotyped AAV is AAV2 / 9. In some embodiments, the pseudotyped AAV is AAV2 / 8.

[0024] In certain embodiments of any of the above aspects, the AAV vector comprises a recombinant capsid protein.

[0025] In certain embodiments of any of the above aspects, the enzyme is MTM1.

[0026] In certain embodiments of any of the above aspects, MTM1 has a nucleic acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid of the sequence set forth in SEQ ID NO:3.

[0027] In certain embodiments of any of the above aspects, the AAV vector further comprises a muscle-specific promoter (e.g., a desmin promoter, a phosphoglycerate kinase promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of eye pair-like homeodomain 3). For example, in certain embodiments, the muscle-specific promoter is a desmin promoter. In certain embodiments, the desmin promoter is a human desmin promoter. In certain embodiments, the promoter is located 5' to a polynucleotide encoding an enzyme.

[0028] In some embodiments of the above aspects, the desmin promoter has a nucleic acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid of the sequence set forth in SEQ ID NO:1.

[0029] In certain embodiments of any of the above aspects, the AAV vector further comprises a polyadenylation site (pA, e.g., the simian virus 40 (SV40) late polyadenylation site, the SV40 early polyadenylation site, the human β-globin polyadenylation site, or the bovine growth hormone polyadenylation site). For example, in certain embodiments, the pA site comprises the SV40 late polyadenylation site. In certain embodiments, pA is located 3' to the polynucleotide encoding the enzyme.

[0030] In some embodiments of the above aspects, the AAV vector further comprises an intron (e.g., an SV40 intron). For example, in some embodiments, the intron is an SV40 intron. In some embodiments, the intron is located 3' to the promoter and 5' to the polynucleotide encoding the enzyme.

[0031] In some embodiments of any of the above aspects, the AAV vector comprises a nucleic acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the AAV vector comprises a nucleic acid set forth in SEQ ID NO:2.

[0032] In certain embodiments of any of the above aspects, the AAV vector is resamirigene bilparvovec.

[0033] In certain embodiments of the above aspects, the patient in need thereof is 5 years old or younger (e.g., 5 years old or younger, 4 years old or younger, 3 years old or younger, 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger) at the time of administration of the AAV vector. For example, in certain embodiments, the patient is about 1 month to about 5 years old (e.g., about 1 month to about 5 years old, about 2 months to about 5 years old, about 3 months to about 5 years old, about 4 months to about 5 years old, about 5 months to about 5 years old, about 6 months to about 5 years old, about 1 year to about 5 years old, about 2 years to about 5 years old, about 3 years to about 5 years old, or about 4 years to about 5 years old).

[0034] In some embodiments of the above aspects, the patient is in need of mechanical ventilation support, for example, in some embodiments, mechanical ventilation support includes invasive mechanical ventilation support and non-invasive mechanical ventilation support.

[0035] In certain embodiments of the above aspects, the AAV vector is about 3×10 13 vg / kg ~ approx. 2.3×10 14 vg / kg (e.g., 3 × 10 13 vg / kg, 3.1×10 13 vg / kg, 3.2 × 10 13 vg / kg, 3.3 × 10 13 vg / kg, 3.4×10 13 vg / kg, 3.5×10 13 vg / kg, 3.6×10 13 vg / kg, 3.7×10 13 vg / kg, 3.8×10 13 vg / kg, 3.9 × 10 13 vg / kg, 4 × 10 13 vg / kg, 4.1×10 13 vg / kg, 4.2 × 10 13 vg / kg, 4.3 × 10 13 vg / kg, 4.4×10 13 vg / kg, 4.5×10 13 vg / kg, 4.6×10 13 vg / kg, 4.7×10 13 vg / kg, 4.8×10 13vg / kg、4.9×10 13 vg / kg、5×10 13 vg / kg、5.1×10 13 vg / kg、5.2×10 13 vg / kg、5.3×10 13 vg / kg、5.4×10 13 vg / kg、5.5×10 13 vg / kg、5.6×10 13 vg / kg、5.7×10 13 vg / kg、5.8×10 13 vg / kg、5.9×10 13 vg / kg、6×10 13 vg / kg、6.1×10 13 vg / kg、6.2×10 13 vg / kg、6.3×10 13 vg / kg、6.4×10 13 vg / kg、6.5×10 13 vg / kg、6.6×10 13 vg / kg、6.7×10 13 vg / kg、6.8×10 13 vg / kg、6.9×10 13 vg / kg、7×10 13 vg / kg、7.1×10 13 vg / kg、7.2×10 13 vg / kg、7.3×10 13 vg / kg、7.4×10 13 vg / kg、7.5×10 13 vg / kg、7.6×10 13 vg / kg、7.7×10 13 vg / kg、7.8×10 13 vg / kg、7.9×10 13 vg / kg、8×10 13 vg / kg、8.1×10 13 vg / kg、8.2×10 13 vg / kg、8.3×10 13 vg / kg、8.4×10 13 vg / kg、8.5×10 13 vg / kg、8.6×10 13 vg / kg、8.7×10 13 vg / kg、8.8×10 13vg / kg, 8.9×10 13 vg / kg, 9×10 13 vg / kg, 9.1×10 13 vg / kg, 9.2 × 10 13 vg / kg, 9.3 × 10 13 vg / kg, 9.4 × 10 13 vg / kg, 9.5×10 13 vg / kg, 9.6×10 13 vg / kg, 9.7×10 13 vg / kg, 9.8×10 13 vg / kg, 9.9×10 13 vg / kg, 1×10 14 vg / kg, 1.1×10 14 vg / kg, 1.2×10 14 vg / kg, 1.3×10 14 vg / kg, 1.4×10 14 vg / kg, 1.5×10 14 vg / kg, 1.6×10 14 vg / kg, 1.7×10 14 vg / kg, 1.8×10 14 vg / kg, 1.9×10 14 vg / kg, 2×10 14 vg / kg, 2.1×10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.3 × 10 14 For example, in one embodiment, the AAV vector is about 1.3×10 14 The drug is administered to the patient in an amount of vg / kg.

[0036] In some embodiments of the above aspects, the AAV vector is administered to the patient in a single dose containing that amount. In some embodiments of the above aspects, the AAV vector is administered to the patient in two or more doses (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) that together contain that amount. In some embodiments, the AAV vector is administered to the patient in two or more doses (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) that each individually contain that amount.

[0037] In some embodiments of the above aspects, the two or more doses are separated from each other by one year or more, hi some embodiments, the two or more doses are administered to the patient within about 12 months (e.g., about 11 months, about 10 months, about 9 months, about 8 months, about 7 months, about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, or about 1 month) of each other.

[0038] In some embodiments of the above aspects, the AAV vector is administered to the patient by intravenous, intramuscular, intradermal, or subcutaneous administration.

[0039] In some embodiments of the above aspects, upon administration of the AAV vector to a patient, the patient exhibits a change from baseline in quantitative analysis of enzyme (e.g., MTM1) expression in muscle biopsies. For example, in some embodiments, the patient exhibits a change from baseline in quantitative analysis of enzyme expression in muscle biopsies by about 24 weeks after administration of the AAV vector to the patient.

[0040] In certain embodiments of any of the above aspects, the loss-of-function disease is a congenital loss-of-function disease (e.g., X-linked myotubular myopathy (XLMTM)). For example, in certain embodiments, the congenital loss-of-function disease is XLMTM.

[0041] In other aspects, the disclosure provides a kit comprising an AAV vector encoding an enzyme and a package insert instructing a user of the kit to determine the potency of the AAV vector according to the method of any one of the above aspects. [Brief description of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram showing cleavage of a phosphate group (circled) from labeled (e.g., BODIPY fluorescently labeled ("FL")) phosphatidylinositol 3-phosphate (PI(3)P) by the phosphatase myotubularin 1 (MTM1), resulting in FL phosphatidylinositol (PI; FL-PI). [Diagram 2]FIG. 1 depicts on-column chromatography (e.g., high performance liquid chromatography) of chloroform / methanolextracted PI(3)P and PI. [Diagram 3] 3A and 3B are schematics and graphs of experiments in which the amount of PI (ng) was varied as a function of the titer of adeno-associated virus (AAV) encoding MTM1, respectively, transfected into cells (e.g., C2C12 cells). Figure 3A is an experimental schematic showing, from right to left, AAV encoding the phosphatase MTM1 transfected into AAV-permissive C2C12 cells for a time sufficient for the AAV-permissive cells to express MTM1, lysing the cells and contacting them with FL-PI(3)P as described in Figure 1, allowing MTM1 to cleave FL-PI(3)P into FL-PI. FL-PI(3)P or FL-PI is then detected and quantified by high performance liquid chromatography (HPLC). FIG. 3B is a quantification of the concentration of FL-PI as described in FIG. 3A as a function of the titer of the transfected AAV (e.g., 5×10 (“E.00E+11”)) and payload (AAV-DES-MTM1 encodes MTM1, while AAV-MCK-GAA encodes acid α-glucosidase), respectively. [Figure 4] FIG. 3B depicts quantification of the concentration of PI(3)P as described in FIG. 3A as a function of the titer (e.g., E.00E+11) and payload of AAV transfected, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] definition As used herein, the term "about" refers to a value within 10% above or below the stated value. For example, "100 pounds" used in the context of masses described herein includes amounts within 10% above or below 100 lbs. Additionally, when used in the context of a list of numerical quantities, the term "about," when preceding a list of numerical quantities, should be understood to apply to each individual amount recited in the list.

[0044] The term "AAV permissive cell" refers to one or more cells into which adeno-associated virus (AAV) can efficiently transfer genes. For example, the cell may include cells derived from muscle, brain, and / or liver. Exemplary AAV permissive cells include, but are not limited to, C2C12 cells, Huh7 cells, Hela cells, HelaS3 cells, Hepa1-6 cells, HEK293 cells, HepG2 cells, or IMY-N9 cells.

[0045] The term "adeno-associated virus" (AAV) as used herein includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, and any other AAV now known or discovered in the future. See, for example, Fields et al. Virology, 4 thed. Lippincott-Raven Publishers, Philadelphia, 1996. Additional AAV serotypes and clades have been identified recently. (See, e.g., Gao et al. J. Virol. 78:6381 (2004); Morris et al. Virol. 33:375 (2004)). The genomic sequences of the various serotypes of AAV, as well as the sequences represented by the native ITRs, Rep proteins, and capsid subunits, are known in the art. The sequences can be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, and J0190 See, e.g., J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_001358, NC_001540, AF513851, AF513852, and AY530579; the disclosures of which are incorporated by reference herein for their teaching of AAV nucleic acid and amino acid sequences.For example, Bantel-Schaal et al. J. Virol. 73:939 (1999); Chiorini et al. J. Virol. 71:6823 (1997); Chiorini et al. J. Virol. 73: 1309 (1999); Gao et al. al.Virol.33:375(2004);Muramatsu et al., Virol.221:208(1996);Ruffing et al., J.Gen.Virol.75:3385(1994);Rutledge et al., J.Virol.72:309(1998);Schmidt et al. al.J.Virol.82:8911(2008);Shade et al. See, e.g., Srivastava et al. J. Virol. 45:555 (1983); Xiao et al. J. Virol. 73:3994 (1999); WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Pat. No. 6,156,303; the disclosures of which are incorporated by reference herein for their teaching of AAV nucleic acid and amino acid sequences.

[0046] As used herein, the term "activity" in relation to an enzyme of interest, such as myotubularin 1 (MTM1), refers to the biological functionality associated with the wild-type enzyme. For example, the term "activity" refers to the function of the enzyme to accomplish substrate turnover in a manner that results in the cleavage product of the corresponding chemical reaction. Activity levels of enzymes, such as MTM1, can be detected and quantified, for example, using the methods described herein, as well as substrate turnover assays known in the art.

[0047] As used herein, the terms "administering," "administration," and the like refer to providing a therapeutic agent (e.g., a pharmaceutical composition including an AAV encoding an enzyme) directly to a patient by any effective route. Exemplary routes of administration are described herein and include, among others, systemic routes, such as intravenous injection, as well as direct routes of administration into the patient's central nervous system, such as by intrathecal or intraventricular injection.

[0048] The term "chromatography" as used herein refers to a technique for separating mixtures by passing the mixture in solution or suspension, or as a vapor, through a medium in which the components move at different rates. Exemplary types of chromatography include, but are not limited to, high performance liquid chromatography, thin layer chromatography, column chromatography, ion exchange chromatography, gel permeation chromatography, affinity chromatography, paper chromatography, gas chromatography, hydrophobic interaction chromatography, or pseudo-affinity chromatography.

[0049] As used herein, the terms "effective amount," "therapeutically effective amount," and the like, when used in reference to a therapeutic composition, such as an AAV vector, described herein, refer to an amount sufficient to effect a beneficial or desired result, such as a clinical result, when administered to a subject, including a mammal, e.g., a human. For example, in the context of treating a loss-of-function disease (e.g., a congenital loss-of-function disease), such as XLMTM, the term refers to an amount of the composition sufficient to effect a therapeutic response compared to the response obtained without administration of the composition of interest. An "effective amount," "therapeutically effective amount," and the like, of a composition, such as an AAV vector, of the present disclosure also includes an amount that effects a beneficial or desired result in a subject compared to a control.

[0050] As used herein, the terms "encode" and "encoding," in the context of a gene (e.g., encoding gene), refer to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. In the context of a gene that encodes a protein product, terms such as "gene expression" are used interchangeably with terms such as "protein expression." Expression of a gene or protein of interest in a subject may be revealed, for example, by detecting an increase in the amount or concentration of mRNA encoding the corresponding protein (e.g., assessed using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the amount or concentration of the corresponding protein (e.g., assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), among others), and / or an increase in the activity of the corresponding protein (e.g., in the case of an enzyme, assessed using an enzyme activity assay described herein or known in the art) in a sample obtained from the subject. As used herein, a cell is considered to "express" a gene or protein of interest if one or more or all of the above events can be detected in the cell or in the medium in which the cell is present. For example, a gene or protein of interest is considered to be "expressed" if it is possible to detect: (i) production of a corresponding RNA transcript, such as an mRNA template (e.g., using an RNA detection procedure described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5' capping, and / or 3' end processing) (e.g., using an RNA detection procedure described herein); (iii) translation of the RNA template into a protein product (e.g., using a protein detection procedure described herein); and / or (iv) post-translational modification of the protein product (e.g., using a protein detection procedure described herein).

[0051] As used herein, the term "extracellular medium" refers to the collection of endogenous factors such as collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial agents, chemoattractants, cytokines, growth factors, and other molecules that are located outside the cell membrane. As used herein, extracellular medium also refers to the extracellular contents that surround a cell after lysis.

[0052] As used herein, the terms "high performance liquid chromatography" and "HPLC" are used interchangeably and refer to a technique in analytical chemistry used to separate, identify, and quantify components in a mixture, as known to those of skill in the art. Exemplary HPLC include, but are not limited to, normal phase HPLC, reverse phase HPLC, hydrophobic interaction chromatography HPLC, and ion chromatography.

[0053] The term "marker" refers to a chemical moiety useful as a label, indicator, or imaging agent. Markers may be detected by liquid chromatography (e.g., high performance liquid chromatography), absorbance spectroscopy, emission spectroscopy, fluorescence spectroscopy, magnetic resonance spectroscopy (e.g., magnetic resonance imaging), or radioisotope detection. The terms "fluorescent marker" and "fluorescent label" refer interchangeably to a marker that can absorb electromagnetic energy and at least partially re-emit a portion of that energy as electromagnetic radiation. Suitable fluorescent moieties include, but are not limited to, Bodipy™ FL, coumarin and related dyes, xanthene dyes such as fluorescein, rhodol, and rhodamine, resorufins, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic acid hydrazides such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminoenzofurans, aminoquinolines, dicyanohydroquinones, fluorescent nanocrystals, fluorescent proteins, and fluorescent europium and terbium complexes and related compounds. In certain embodiments, the marker may be a member of a specific binding pair or may be associated (e.g., covalently) with a member of a specific binding pair. A specific binding pair is a pair of molecules that can specifically interact with each other, e.g., have affinity for each other. For example, a specific binding pair may be a ligand-protein binding pair, e.g., an enzyme substrate, biotin-streptavidin, or an epitope-antibody binding pair. A binding pair that includes a marker has a larger apparent size than a corresponding binding pair that does not include a marker, and a larger apparent size than either member of the binding pair alone. The binding pair complex may be detected by the methods described herein or by other methods known to those skilled in the art, e.g., chromatographic assays, immunoassay formats, or gel shift assays.

[0054] The term "loss-of-function disease" as used herein refers to a disease caused by a loss-of-function mutation, which is a type of mutation in which the modified gene product does not have the molecular function of the wild-type gene. For example, a congenital loss-of-function disease may include, but is not limited to, X-linked myotubular myopathy (XLMTM). In some embodiments, the loss-of-function disease may be congenital.

[0055] As used herein, the term "sample" refers to a specimen isolated from a cell or its surrounding extracellular medium. The cell can be, for example, a cell that has been contacted with an AAV vector encoding an enzyme (e.g., MTM1).

[0056] As used herein, the term "substrate" refers to a substance that an enzyme (e.g., Myotubularin 1) acts on (e.g., hydrolyzes). For example, a "non-phosphorylated substrate" is distinguished from a "phosphorylated substrate" primarily by the presence of a phosphate group that has or has not been hydrolyzed, respectively, by a phosphatase.

[0057] The term "level" as used herein refers to the level of a molecule or protein compared to a reference. The reference can be any useful reference as defined herein. "Decreased level" and "increased level" of a protein refers to a decrease or increase in protein level compared to a reference (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more decrease or increase; a decrease or increase of about 1% or more compared to a reference). By "a decrease or increase" is meant a decrease or increase of more than 0%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%; a decrease or increase of about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase of about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold, about 1000-fold, or more. The level of a molecule or protein may be expressed as mass / volume (e.g., g / dL, mg / mL, pg / mL, or ng / mL) or as a ratio of the total molecule or protein, respectively, in the sample.

[0058] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic compounds that is administered to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition that affects or may affect the subject.

[0059] As used herein, the term "pharmaceutical acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response and other problematic complications, commensurate with a reasonable benefit / risk ratio.

[0060] The term "promoter" as used herein refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the transgene. Exemplary promoters suitable for use with the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which relates to nucleic acid regulatory elements and is incorporated herein by reference. Additionally, the term "promoter" may refer to synthetic promoters, which are regulatory DNA sequences that do not naturally occur in biological systems. Synthetic promoters contain parts of natural promoters combined with non-natural polynucleotide sequences and can be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types.

[0061] As used herein, the terms "patient" and "subject" refer to an organism receiving treatment for a particular disease or condition described herein (e.g., a loss of function disease, e.g., XLMTM). Examples of subjects and patients include mammals, such as humans, receiving treatment for a disease or condition described herein.

[0062] "Reference" refers to any useful reference used to compare the cleavage product level in a sample containing a substrate cleaved by an enzyme (e.g., an enzyme encoded by a vector). The reference can be any sample, standard, standard curve, or level used for comparison purposes. The reference can be a normal reference sample or a reference standard or level. A "reference sample" can be, for example, a control, e.g., a predetermined negative control value such as a "normal control", or a previous sample taken from the same sample (e.g., a cell or tissue) that has not been exposed to the enzyme; a sample contacted with an enzyme that does not cleave the substrate; or a sample of a known normal concentration of purified substrate (e.g., any of those described herein). "Reference standard or level" refers to a value or number obtained from a reference sample. A standard curve of a purified substrate within a normal reference range, e.g., any of the levels described herein, can also be used as a reference. As described herein, the reference can be an amount or concentration. In an embodiment, the reference amount or concentration is the expression level of the enzyme cleavage product in an AAV permissive cell before the AAV permissive cell is contacted with an AAV vector.

[0063] As used herein, the terms "treat" and "treatment" refer to therapeutic treatment, the purpose of which is, inter alia, to prevent or slow (alleviate) undesirable physiological changes or disorders, such as the progression of a loss-of-function disease (e.g., a congenital loss-of-function disease) such as XLMTM. Beneficial or desired clinical results include, but are not limited to, relief of symptoms (e.g., stiffness and / or joint contracture), reduction in the extent of disease, a stable (e.g., non-worsening) state of disease, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and remission (either partial or total) (either detectable or undetectable). In the context of a loss-of-function disorder (e.g., a congenital loss-of-function disorder) such as MTM1, treatment of a patient may be manifested as one or more detectable changes, such as an increase in the concentration of MTM1 protein or nucleic acid (e.g., DNA or RNA such as mRNA) encoding MTM1, or an increase in MTM1 activity (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or more). The concentration of MTM1 protein can be determined using protein detection assays known in the art, including the ELISA assays described herein. The concentration of nucleic acid encoding MTM1 can be determined using nucleic acid detection assays (e.g., RNA Seq assays) described herein. Furthermore, treatment of patients suffering from loss-of-function diseases (e.g., congenital loss-of-function diseases) such as XLMTM can reveal improvement in the patient's muscle function (e.g., skeletal muscle function), as well as improvement in muscle coordination. For example, signs of improvement can include increased progression of diaphragm and / or respiratory muscles.

[0064] As used herein, the terms "X-linked myotubular myopathy" and "XLMTM" refer to a congenital loss-of-function disorder caused by mutations in the MTM1 gene and characterized by symptoms including mild to severe muscle weakness, hypotonia (low muscle tone), feeding difficulties, and / or severe respiratory complications. The NCBI gene number for human MTM1 is 4534. An exemplary wild-type human MTM1 nucleic acid sequence is provided in NCBI RefSeq Acc. SEQ ID NO: NM_000252.3, and an exemplary wild-type myotubularin 1 amino acid sequence is NCBI RefSeq Acc. SEQ ID NO: NP_000243.1 (SEQ ID NO: 4).

[0065] [Detailed Description of the Invention] The present disclosure provides a method that can be used to determine the efficacy of an AAV vector, for example, for the treatment of a loss-of-function disease (e.g., a congenital loss-of-function disease, e.g., X-linked myotubular myopathy). According to the methods and uses described herein, a patient (e.g., a human patient) with a loss-of-function disease (e.g., a congenital loss-of-function disease, e.g., XLMTM) can be administered an AAV encoding an enzyme (e.g., myotubularin 1 (MTM1)). The method can include, for example, contacting the AAV vector with an AAV-permissive cell expressing a substrate specifically cleaved by the enzyme for a sufficient time for the AAV-permissive cell to express the enzyme, followed by assessing or determining the amount or concentration of the product resulting from cleavage of the substrate by the enzyme in the cell by high performance liquid chromatography (HPLC). Alternatively, the method may include contacting the AAV vector with AAV-permissive cells for a time sufficient for the AAV-permissive cells to express the enzyme, lysing the cells, and exposing the expressed enzyme in a reaction medium to a substrate that is specifically cleaved by the enzyme, and then evaluating or determining the amount or concentration of the product resulting from cleavage of the substrate by the enzyme in the reaction medium by HPLC. In an embodiment, a finding that the amount or concentration of the cleavage product in the cell, surrounding extracellular medium, or reaction medium is increased compared to a reference amount or concentration of the cleavage product identifies the AAV vector as efficacious for treating a disease. In an embodiment, a finding that the amount or concentration of the substrate in the cell, surrounding extracellular medium, or reaction medium is decreased compared to a reference amount or concentration of the substrate identifies the AAV vector as efficacious for treating a disease. An exemplary AAV vector that may be used in conjunction with the methods and uses of the present disclosure is resamirigene bilparvovec.

[0066] The present disclosure is based, at least in part, on the discovery of a method that is surprisingly useful in determining AAV potency, for example, in a batch release assay. Thus, the disclosed method addresses important needs associated with existing methods of AAV potency assays, such as the significant need for high-throughput quantitative measurement of potency. For example, existing AAV potency assays using thin layer chromatography may qualitatively analyze the number of components in a mixture, whereas the HPLC method described herein is highly sensitive to detection and quantification, which is an essential output measure for determining the potency of AAV payloads. Surprisingly, detection and quantification of products resulting from substrates specifically cleaved by the expressed enzymes was successfully performed directly on cell lysate samples. For example, detection and quantification of products resulting from substrates specifically cleaved by the expressed enzymes was successfully performed using cell lysate samples without purifying the expressed enzymes prior to exposure of the enzymes to the cleavable substrates. One aspect of the applicant's teachings is to provide a method for assaying AAV potency, comprising contacting AAV-permissive cells with AAV encoding an enzyme for a period of time sufficient for the AAV-permissive cells to express the enzyme; lysing the cells; incubating the cells or the expressed enzyme with a phosphorylated substrate labeled with a marker; and assessing by HPLC the amount or concentration of the labeled cleavage product compared to the amount or concentration of the phosphorylated substrate (e.g., uncleaved) labeled with the marker.

[0067] In one embodiment, the disclosure describes a method for producing an AAV vector encoding an enzyme for the treatment of a loss-of-function disease in a patient associated with a deleterious mutation in a gene encoding the enzyme, comprising determining the efficacy of the AAV vector encoding the enzyme (e.g., MTM1) by assessing the amount or concentration of product resulting from enzymatic cleavage of a substrate by HPLC in AAV-permissive cells, the surrounding extracellular medium, or the reaction medium.

[0068] In one embodiment, the disclosure describes a method for treating a loss-of-function disease associated with a deleterious mutation in a gene encoding an enzyme in a patient, comprising determining the efficacy of an AAV vector encoding an enzyme (e.g., MTM1) by assessing the amount or concentration of product produced by enzymatic cleavage of a substrate by HPLC in AAV-permissive cells, the surrounding extracellular medium, or a reaction medium, and administering a therapeutically effective amount of the AAV vector to the patient.

[0069] The following sections provide descriptions of therapeutic enzymes (e.g., phosphatases), exemplary loss-of-function diseases associated with deleterious mutations in genes encoding the enzymes, exemplary AAVs, and HPLC methods for assessing the amount or concentration of products resulting from cleavage of substrates by the enzymes described herein. The following sections also describe various transduction agents, pharmaceutical compositions, and routes of administration that can be used in conjunction with the compositions and methods of the present disclosure.

[0070] therapeutic enzymes Enzymes that can be encoded by AAV and whose respective cleavage products can be evaluated according to the methods described herein include therapeutic enzymes, such as those that can be transferred to a subject (e.g., a human patient) suffering from a disease or condition characterized by an enzyme deficiency.For example, enzymes that can be encoded by the AAVs described herein include myotubularin 1 (MTM1), myotubularin-related protein 1 (MTMR1), MTMR2, MTMR3, MTMR4, MTMR5, MTMR6, MTMR7, MTMR8, MTMR9, MTMR10, MTMR11, MTMR12, MTMR13, MTMR14, synaptojanin 1, phosphatase and tensin homolog deleted on chromosome 10, acid alpha-glucosidase (GAA), and phosphodiesterase (GPA). , carbamoyl synthetase 1, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, α-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VII, factor IX, thrombin, factor X, factor XI, superoxide dismutase, glutathione peroxidase and reductase, hemoglobin kinase, β-glucosidase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (also called P-protein), Cu / Zn superprotein, β-glucosidase, hepatic phosphorylase, hepatic phosphorylase kinase, hepatic glycine decarboxylase (also called P-protein), hepatic phospholipase, ... These include suicide genes such as oxidodismutase, aromatic amino acid decarboxylase, tyrosine hydroxylase, acetylcholine synthetase, prohormone convertase, lactase, lipase, gastrointestinal enzymes including trypsin, chymotrypsin, and pepsin, adenosine deaminase, alpha-1 antitrypsin, tissue inhibitor of metalloproteinases, trehalose phosphate synthase, hexokinase I, II, and III, glucokinase, and thymidine kinase and cytosine deaminase.Other useful enzymes include those involved in lysosomal storage disorders, such as acid p-glucosidase, alpha-galactosidase a, alpha 1-iduronidase, iduroate sulfatase, lysosomal acid alpha-glucosidase, sphingomyelinase, hexosaminidase A, hexosaminidases A and B, arylsulfatase A, acid lipase, acid ceramidase, galactosylceramidase, afucosidase, aspartylglucosidase, and glycerylglucosidase. Examples of enzymes include GAA, neuraminidase, galactosylceramidase, heparan-N-sulfatase, N-acetylglucosaminidase, acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase B, p-glucuoronidase, and hexosaminidase A and B. In one embodiment, the enzyme is GAA.

[0071] Representative enzymes associated with diseases or conditions characterized by enzymatic deficiencies include those listed in Table 1 below. Table 1. Exemplary disorders associated with enzyme deficiencies

[0072] [Table 1] In some embodiments, it is assessed that there is an increase or decrease in the amount or concentration of the cleavage product of the enzyme described herein compared to a reference amount or concentration. In some embodiments, this finding that the amount or concentration of the cleavage product of the enzyme is altered compared to the reference amount or concentration identifies the AAV described herein as having efficacy for treating a loss-of-function disease, such as those exemplified in Table 1 above. In some embodiments, it is determined that there is an increase or decrease in the amount or concentration of a cleavage product of an enzyme described herein compared to a reference amount or concentration, in some embodiments, this determination identifies the AAV described herein as having efficacy for treating a loss-of-function disease, such as those exemplified in Table 1 above.

[0073] Therapeutic phosphatases Phosphatases are a type of enzyme that dephosphorylates a substrate, for example, they hydrolyze a phosphate monoester into a phosphate ion and a molecule with a free hydroxyl group. Phosphatases can be classified into two main categories: cysteine-dependent phosphatases (CDPs) and metallophosphatases. Metallophosphatases depend on the presence of one or more metal ions at their active site for activity.

[0074] CDP catalyzes the hydrolysis of phosphoester bonds via a phospho-cysteine ​​intermediate. The free cysteine ​​nucleophile forms a bond with the phosphorus atom of the phosphate moiety, and the PO bond linking the phosphate group to the tyrosine is protonated either by an appropriately placed acidic amino acid residue or by a water molecule. The phospho-cysteine ​​intermediate is then hydrolyzed by another water molecule, thus regenerating the active site for another dephosphorylation reaction.

[0075] In contrast, metallophosphatases catalyze the catalytic activity of essential metal ions, such as magnesium, manganese, iron, zinc, or any combination thereof, within their active site, in which a hydroxyl ion bridging two metal ions is involved in a nucleophilic attack on the phosphate group.

[0076] Exemplary phosphatases whose cleavage products can be assessed according to the methods described herein include MTM1, MTMR1, MTMR2, MTMR3, MTMR4, MTMR5, MTMR6, MTMR7, MTMR8, MTMR9, MTMR10, MTMR11, MTMR12, MTMR12, MTMR13, MTMR14, synaptojanin 1, phosphatase, and tensin homolog deleted on chromosome 10, and lipid phosphatases such as glucose-6-phosphatase. In one embodiment, the phosphatase is MTM1.

[0077] Exemplary disorders associated with phosphatase deficiency: X-linked myotubular myopathy XLMTM is a rare, life-threatening congenital myopathy caused by loss-of-function mutations in the gene encoding the phosphatase MTM1 and is characterized by severe muscle weakness and hypotonia at birth that leads to severe respiratory insufficiency, inability to sit, stand, or walk, and early death in most patients.

[0078] The myopathy associated with XLMTM impairs the development of motor skills such as sitting, standing, and walking. Affected infants may also have difficulty feeding due to muscle weakness. Individuals with the condition often do not have the muscle strength to breathe on their own and must be supported with mechanical ventilation. Some affected individuals only require mechanical ventilation periodically, such as during sleep, while others require mechanical ventilation continuously. Patients with XLMTM may also have weakness of the muscles that control eye movement (ophthalmoplegia), weakness of other muscles in the face, and lack of reflexes (areflexia).

[0079] In XLMTM, muscle weakness often prevents normal bone development and can result in brittle bones, abnormal curvature of the spine (scoliosis), and deformed hip and knee joints (contractures). Patients with XLMTM may have narrow, elongated faces, high-arched palates (roof of the mouth), and large heads. Patients may also have liver disease, recurrent ear and respiratory infections, or seizures.

[0080] As a result of their severe respiratory distress, patients with XLMTM usually only survive into childhood, however, some patients with this condition survive into adulthood. The compositions and methods of the present disclosure provide an important medical benefit by restoring functional MTM1 expression, which can extend the lifespan of such patients.

[0081] Detectable Markers The methods and uses described herein include determining the amount or concentration of a marker (e.g. a substrate for an enzyme described herein that is associated with a marker such that a putative cleavage product is also associated with said marker).

[0082] The choice of marker is governed by many factors, including the detection format, availability of specific equipment, and ease of binding of the marker to the substrate. Other factors that may be relevant to a particular use include the effect of the marker on the solubility of the substrate, the kinetics of post-translational or protease activity on the substrate, and the desired detection sensitivity of the AAV potency assay.

[0083] Many markers are commercially available or can be easily produced. In general, a marker can exhibit optical, magnetic, or radioactive properties. Thus, once bound to a substrate, the marker can exhibit similar or identical optical, magnetic, or radioactive properties to the resulting substrate as the marker alone. In some embodiments, the association of the marker with the substrate can change the detectable properties of the marker to a greater or lesser extent. For example, conjugation of a fluorophore to a substrate can result in a composition with a different emission maximum than the fluorophore alone in solution. In other embodiments, the marker can be a member of a specific binding pair. For example, the marker can be the ligand member of a ligand-protein binding pair, such as the biotin member of the biotin-streptavidin binding pair.

[0084] For fluorescent markers, preferred fluorophores should usually exhibit good quantum yields, long excited state lifetimes, and large extinction coefficients; be resistant to collisional quenching and bleaching; and be easily conjugated to peptides. Exemplary fluorophores include cyanines, oxazines, thiazines, porphyrins, phthalocyanines, fluorescent infrared-emitting polynuclear aromatic hydrocarbons such as violanthrone, fluorescent proteins, and near-IR squaraine dyes.

[0085] Specific fluorophores include, but are not limited to, fluorescein isothiocyanate, 5-FAM (5-carboxyfluorescein), 6-FAM (6-carboxyfluorescein), 5,6-FAM, 7-hydroxycoumarin-3-carboxamide, 6-chloro7-hydroxycoumarin-3-carboxamide, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5(and 6)-isothiocyanate, 1,3-bis(2-dialkylamino-5-thienyl)-substituted squaraines, 5(and 6)-carboxyfluorescein, 5(and 6)-carboxytetramethylrhodamine, and succinimidyl ester of 7-amino-4-methylcoumarin-3-acetic acid. Semiconductor fluorescent nanocrystals are available with a range of emission spectra and are highly fluorescent and useful (see Bruchez et al., Science 281:2013-2016).

[0086] Genetically encoded fluorescent markers that may be used in the methods and uses of the present disclosure include, but are not limited to, any one of the following: green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), mCherry, dsRed, luciferase (Luc), β-galactosidase (lacZ), chloramphenicol acetyltransferase (CAT), mTagBFP2, Mtuquoise 2, mCerulean3, EGFP, mWasabi, Superfolder GFP, mNeonGreen, mClover3, Venus, Citrine, mKOK, tdTomato, TagRFP-T, mRuby3, mScarlet, FusionRed, mCherry, mStable, mKate2, mMaroonl, mCardinal, T-Sapphire, mCyRFPI, LSSmOrange, or mBeRFP.

[0087] Lanthanide complexes (e.g., metal chelates of Eu or Tb) are also useful. Their long lifetimes can easily suppress autofluorescence of biological samples, so that the fluorescence signal can be measured after the background signal has decayed. Therefore, lanthanide complexes such as Eu or Tb metal chelates can be useful.

[0088] Exemplary light-emitting moieties include chemiluminescent compounds, electroluminescent compounds, and bioluminescent compounds. Exemplary bioluminescent compounds include bioluminescent proteins such as firefly, bacterial, or click beetle luciferase, aequorin, and other photoproteins. Alternative electroluminescent moieties include ruthenium complexes, and alternative chemiluminescent moieties include those based on 1,2-dioxetanes.

[0089] Magnetically detectable moieties include magnetic resonance contrast agents, such as paramagnetic, ferromagnetic, or diamagnetic metal ion chelates, or magnetic particles.

[0090] In some applications, it may be desirable to derivatize the marker to make it more hydrophobic and permeable to the cell membrane. The derivatization group can undergo hydrolysis in the cell to regenerate the composition and thereby trap them inside the cell. For this purpose, the phenolic hydroxyl or free amine in the structure is preferably acylated with C1-C4 acyl groups (e.g., formylacetyl, n-butyl) or converted, for example, to esters and carbonates, as described in Bundgaard, H., Design of Prodrugs, Elsevier Science Publishers (1985), Chapter 1, page 3 et seq. Further modification of the fluorescent moiety can also be achieved, for example, as described in US Pat. No. 5,741,657, incorporated herein by reference.

[0091] The marker may be attached to the substrate by a linker that provides a spacer between the marker and the substrate, thereby preventing, for example, steric or charge interference of the marker with the interaction between the enzyme and the substrate. Preferred linkers are substantially stable under cellular conditions and are easily attached to the substrate and the marker. Examples include flexible aliphatic linkers such as gamma-amino-n-butyric acid (GABA), diaminopentane, and aminohexanoyl, as well as rigid aromatic linkers. Such linkers are known in the art and are described, for example, in Handbook of Fluorescent Probes and Research Chemicals (Richard Hangland, published by Molecular Probes), which is incorporated herein by reference.

[0092] Non-covalent attachment methods can also be used to associate substrate with marker.For example, substrate can be designed to include specific binding site for marker, as described in pending U.S. Patent Nos. 6,054,271; 6,008,378 and 5,932,474.Labeling can then be achieved by incubating substrate with membrane-permeable fluorescent binding partner, which allows substrate to be expressed in intact living cells, and then these peptides can be labeled in situ to produce the composition of the present invention in intact living cells (e.g., AAV-permissive cells).

[0093] In some embodiments, the marker is a fluorescently labeled lipid. In some embodiments, the fluorescently labeled lipid is a fluorescently labeled phospholipid. In some embodiments, the fluorescently labeled phospholipid is phosphatidylinositol 3-phosphate.

[0094] Chromatography Chromatography is a technique for separating mixtures by passing the mixture in solution or suspension, or as a vapor, through a medium in which the components move at different rates. Chromatography in its broadest sense refers to a process that can separate a mixture of components as a result of differences in the rates at which the individual components of the mixture move through what is called a stationary phase or medium under the influence of the mobile phase. Chromatography is used to separate mixtures of chemicals into their individual components. Exemplary types of chromatography include, but are not limited to, high performance liquid chromatography (HPLC), thin layer chromatography, column chromatography, ion exchange chromatography, gel permeation chromatography, affinity chromatography, paper chromatography, gas chromatography, hydrophobic interaction chromatography, or pseudo-affinity chromatography.

[0095] Ion exchange chromatography is a technique for separating molecules, usually ions or polar molecules, based on their ionic charge. Thus, the functionalized chromatographic media for use in the method contain one or more moieties that are positively or negatively charged. The positive and / or negative charges in the functionalized chromatographic media are usually balanced with one or more counterions. Ion exchange chromatography includes one or more of cation exchange chromatography and anion exchange chromatography.

[0096] Affinity chromatography is a technique for separating molecules based on their affinity for a particular ligand, usually, but always, a biological ligand. The method may rely, for example, on the attraction between an antibody and an antigen, or between an enzyme and a substrate. Functionalized chromatographic media for use in affinity capture chromatography usually contain one or more moieties selected from one or more proteins, peptides, antibodies or fragments thereof, dyes, histidine, or groups containing metal cations. Alternatively, functionalized chromatographic media for use in affinity chromatography may contain mimetic or synthetic ligands that mimic the action of protein ligands.

[0097] Paper chromatography is a separation method in which a mobile phase is passed through a filter paper material. A mixture, such as a line or spot of dye, is placed on the filter paper. When the edge of the filter paper is exposed to a suitable fluid mobile phase, such as a liquid solvent like water, the solvent will flow by capillary action.

[0098] Hydrophobic interaction chromatography is a method for separating proteins based on the strength of their relative hydrophobic interactions with a hydrophobic adsorbent. Hydrophobicity is generally defined as the repulsion between a non-polar compound and a polar environment such as water. Hydrophobic "interactions" are essentially the tendency of a polar environment to exclude non-polar (i.e., hydrophobic) compounds from the polar environment and to force aggregation of hydrophobic compounds with each other. The phenomenon of hydrophobic interactions is applied to the separation of proteins by using aqueous salt solutions to aggregate or absorptively bind hydrophobic proteins in a sample to hydrophobic functional groups (adsorbents) immobilized on a solid support. The adsorbed proteins are released from the adsorbent by elution with decreasing salt concentrations, which reverses the environment that promotes hydrophobic interactions, resulting in the loss of hydrophobic interactions between the proteins and the support and the release of the proteins from the support in order of increasing hydrophobicity (the least hydrophobic proteins are released first).

[0099] In gel permeation or size-exclusion chromatography, the separation of components is a function of their molecular size, and the stationary phase typically does not attract the components. Separation depends on the amount of time the material spends in the porous stationary phase compared to the time in the fluid. The probability that a molecule will be present in a pore depends on the size of the molecule and the pore. Furthermore, the ability of a material to penetrate the pores is determined by the diffusion mobility of the macromolecules, which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all, and the permeability of very small macromolecules is close to 1. Components with larger molecular sizes pass through the stationary phase more quickly, while components with smaller molecular sizes have a longer path through the pores of the stationary phase and are therefore retained in the stationary phase longer. Size-exclusion chromatography is widely used in various scientific fields. In biological sciences, size-exclusion chromatography is used for the isolation and purification of biomolecules such as peptides, hormones, or DNA.

[0100] Thin layer chromatography is a technique in which small amounts of various solutions of the mixture of substances to be separated are applied to a thin porous layer in the form of spots on a starting line. The edge of the support plate below the starting line is then contacted with a liquid eluent, where the capillary rise of the eluent in the thin porous layer carries the individual substances with it. Due to different adsorption or distribution coefficients, the substances are transported at different rates and are therefore separated.

[0101] High-performance liquid chromatography HPLC, formerly known as high pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify components in a mixture. In a conventional liquid chromatography system, a liquid solvent (called the mobile phase) is introduced from a reservoir and pumped through the liquid chromatography system. The mobile phase exits the pump under pressure. The mobile phase then proceeds through tubing to a sample injection valve. The sample injection valve allows the operator to inject a sample into the liquid chromatography system, where the sample is carried along with the mobile phase.

[0102] Additionally, in conventional LC systems, the sample and mobile phase pass through one or more filters and often guard columns before reaching the column. In certain embodiments, any suitable column may be used. In certain embodiments, the column is chloroform / methanol. A typical column usually consists of pieces of steel tubing packed with a packing material. The packing consists of a granular material packed inside the column. It usually consists of silica or polymer-based particles, often chemically bonded with chemical functional groups. The packing material is also known as the stationary phase. One of the fundamental principles of separation is that the mobile phase passes continuously through the stationary phase. As the sample is carried through the column (along with the mobile phase), the various components (solutes) in the sample move at different rates (e.g., there is differential migration of the solutes) through the packing in the column. Due to the different migration rates, the components gradually separate as they move through the column. The differential migration is influenced by factors such as the composition of the mobile phase, the composition of the stationary phase (e.g., the material packed into the column), and the temperature at which the separation occurs. Thus, such factors affect the separation of the various components of the sample. As the sample (with its components now separated) exits the column, it flows past the detector along with the mobile phase. The detector detects the presence of a particular molecule or compound. In liquid chromatography applications, two general types of detectors are used. One type measures the change in some gross physical property of the mobile phase and the sample (e.g., their refractive index). The other type measures only some property of the sample (such as absorption of ultraviolet light). Typical detectors in LC systems can measure and provide an output in terms of mass per unit volume (e.g., grams per milliliter) or mass per unit time (e.g., grams per second) of the components of the sample. From the output signal, a chromatogram is provided, which can then be used by the operator to determine the chemical components present in the sample.

[0103] The difference between traditional liquid chromatography and HPLC is that the solvent in liquid chromatography is moved by gravity, so HPLC relies on a pump to pass a pressurized liquid solvent containing the sample mixture through a column packed with a solid adsorbent material. HPLC techniques used in the present invention include, but are not limited to, normal phase HPLC, reversed phase HPLC, hydrophobic interaction chromatography, and ion chromatography. In some embodiments, any suitable method of HPLC can be used in the methods described herein.

[0104] Normal phase HPLC separates analytes based on their affinity to polar stationary surfaces such as silica, and thus the ability of the analytes to participate in polar interactions (such as hydrogen bonding or dipole-dipole type interactions) with the sorbent surface. Normal phase HPLC uses a non-polar, non-polar HPLC. This separation works effectively to separate analytes that are readily soluble in non-polar solvents. In contrast, reversed phase HPLC has a non-polar stationary phase and an aqueous, moderately polar mobile phase. In addition, other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts increases the surface tension of aqueous solutions in a gentle linear manner, increasing retention times. This technique is called hydrophobic interaction chromatography and is used to gently separate and recover proteins as well as protect their biological activity in protein analysis. Another method of HPLC, called ion chromatography, separates ions and polar molecules based on their affinity to ion exchangers. The two types of ion chromatography are anion exchange and cation exchange. In both cases, charged molecules bind to oppositely charged moieties by forming ionic bonds to the insoluble stationary phase. The equilibrated stationary phase consists of ionizable functional groups, where target molecules of the mixture to be separated and quantified can bind during passage through the column. Cation exchange chromatography is used when the molecules of interest are positively charged. In this chromatography, the stationary phase is negatively charged and loaded such that positively charged molecules are attracted to it. Anion exchange chromatography is when the stationary phase is positively charged and loaded such that negatively charged molecules are attracted to it. The bound molecules can then be eluted and collected using an eluent containing anions and cations, by passing a higher concentration of ions through the column, or by changing the pH of the column.

[0105] In some embodiments, the HPLC is normal phase HPLC. In some embodiments, the HPLC is reverse phase HPLC. In some embodiments, the HPLC is hydrophobic interaction chromatography. In some embodiments, the HPLC is ion chromatography.

[0106] In some embodiments, an increase in the amount or concentration of an enzymatic cleavage product compared to a reference amount or concentration is assessed by HPLC, hi some embodiments, a decrease in the amount or concentration of an enzymatic cleavage product compared to a reference amount or concentration is assessed by HPLC.

[0107] In some embodiments, there is an increase in the amount or concentration of an enzymatic cleavage product compared to a reference amount or concentration, as determined by HPLC, In some embodiments, there is a decrease in the amount or concentration of an enzymatic cleavage product compared to a reference amount or concentration, as determined by HPLC.

[0108] AAV Vectors for Delivery of Exogenous Nucleic Acids to Target Cells In certain embodiments, nucleic acids encoding the enzymes of the methods described herein are incorporated into recombinant AAV (rAAV) vectors and / or virions to facilitate their introduction into cells (e.g., AAV-permissive cells).

[0109] In some embodiments, the AAV permissive cell is a C2C12 cell, a Huh7 cell, a Hela cell, a HelaS3 cell, a Nepal-6 cell, a HEK293 cell, a HepG2 cell, or an IMY-N9 cell. For example, in some embodiments, the AAV permissive cell is a C2C12 cell. In some embodiments, the AAV permissive cell is a Huh7 cell. In some embodiments, the AAV permissive cell is a Hela cell. In some embodiments, the AAV permissive cell is a HelaS3 cell. In some embodiments, the AAV permissive cell is a Hepa1-6 cell. In some embodiments, the AAV permissive cell is a HEK293 cell. In some embodiments, the AAV permissive cell is a HepG2 cell. In some embodiments, the AAV permissive cell is an IMY-N9 cell. The AAV permissive cell can be lysed.

[0110] A rAAV vector useful in the present invention is a recombinant nucleic acid construct that includes (1) a transgene to be expressed (e.g., a polynucleotide encoding an MTM1 protein) and (2) viral nucleic acid that facilitates the integration and expression of a heterologous gene. The viral nucleic acid may include sequences represented by AAV required in cis for DNA replication and packaging into virions (e.g., functional inverted terminal repeats (ITRs)). In a typical application, the transgene encodes MTM1, which is useful for correcting MTM1 mutations in patients suffering from loss-of-function diseases such as XLMTM (e.g., congenital loss-of-function diseases). The rAAV vector may also include a marker or reporter gene. Useful rAAV vectors have one or more AAV wild-type genes deleted in whole or in part but retaining functional adjacent ITR sequences. The AAV ITRs may be of any serotype (e.g., from serotype 2) appropriate for the particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000) and Monahan and Samulski, Gene Delivery 7:24-30 (2000), the disclosures of which each relate to AAV vectors for gene delivery and are incorporated herein by reference.

[0111] The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate the introduction of the nucleic acid or vector into cells. The capsid protein of AAV constitutes the external, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virion assembly. For construction of rAAV virions, see, e.g., U.S. Pat. Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as Rabinowitz et al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of which each relate to AAV vectors for gene delivery and are incorporated herein by reference.

[0112] The rAAV virions useful in conjunction with the compositions and methods described herein are derived from various AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8 and 9. For targeting muscle cells, rAAV virions containing at least one serotype 1 capsid protein may be particularly useful. Since serotype 6 capsid protein is structurally similar to serotype 1 capsid protein, it is expected to also lead to high expression of MTM1 in muscle cells, and rAAV virions containing at least one serotype 6 capsid protein may also be particularly useful. rAAV serotype 9 has also been found to be an efficient transducer of muscle cells. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol. 72:2224-2232 (1998); Halbert et al., J. Virol. 74:1524-1532 (2000); Halbert et al., J. Virol. 75:6615-6624 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001), the disclosures of which each relate to AAV vectors for gene delivery and are incorporated herein by reference.

[0113] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV8 vector encoding a therapeutic protein pseudotyped with a capsid gene from AAV serotype 2. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art, see, e.g., Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001).

[0114] AAV virions with mutations in the virion capsid can be used to infect specific cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations to facilitate targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635-45 (2000). Other rAAV virions that can be used in the methods of the invention include capsid hybrids generated by molecular breeding of the virus and by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001).

[0115] The AAV vectors described herein may contain polynucleotide sequences and additional sequence elements that are used, for example, for the expression of enzymes and / or for the integration of these enzyme sequences into the genome of mammalian cells. Certain AAV vectors that can be used to express transgenes described herein contain regulatory sequences (e.g., promoter and enhancer regions) that direct gene transcription. Other useful AAV vectors for the expression of enzymes contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the AAV vector.

[0116] In one embodiment, the promoter is a desmin promoter, a PGK promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of eye pair-like homeodomain 3. In one embodiment, the promoter is a desmin promoter.

[0117] The disclosure herein provides methods of producing an AAV encoding an enzyme for treating a loss-of-function disease associated with a deleterious mutation in a gene encoding the enzyme in a patient in need of treatment, for example, by contacting an AAV vector with an AAV-permissive cell expressing a cleavable substrate specific for the enzyme for a period of time sufficient for the AAV-permissive cell to express the enzyme; assessing the amount or concentration of product resulting from cleavage of the substrate by the enzyme in the cell or surrounding extracellular medium by HPLC; and releasing the AAV vector for treating the disease if the amount or concentration of the cleavage product in the cell or surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product or decreased compared to a reference amount or concentration of the cleavage product.

[0118] The disclosure herein also provides methods of producing AAV, for example, by contacting an AAV vector with an AAV-permissive cell that expresses a cleavable substrate specific for the enzyme for a period of time sufficient for the AAV-permissive cell to express the enzyme; determining by HPLC that the amount or concentration of product resulting from cleavage of the substrate by the enzyme in the cell or surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product or decreased compared to a reference amount or concentration of the cleavage product; and releasing the AAV vector to treat a loss-of-function disease.

[0119] Exemplary AAV:Resamirigene Bilparvovec As described herein, the AAV vector can include a pseudotyped AAV vector that includes a nucleic acid sequence encoding the MTM1 gene (SEQ ID NO:3) operably linked to the desmin promoter (SEQ ID NO:1) flanked by AAV2 ITRs and packaged within a capsid protein from AAV8 (AAV2 / 8), as well as other genetic components listed in Table 2. The exemplary vector is known by the International Nonproprietary Name (INN) "resamirigene bilparvovec." Resamirigene bilparvovec is a non-replicating recombinant AAV8 vector expressing non-codon-optimized human MTM1 cDNA under the control of the muscle-specific human desmin promoter. The MTM1 expression cassette was constructed by cloning a synthetic DNA sequence complementary to the coding portion (nucleotides 43-1864) of the wild-type human MTM1 transcript (NCBI reference sequence number_000252.3) downstream of the 1.05 kb human desmin enhancer / promoter region. The second intron and polyadenylation sequence of the human β-globin gene (HBB) were inserted upstream and downstream of the MTM1 synthetic cDNA, respectively, to mediate RNA processing. The expression cassette was flanked by AAV serotype-2 (AAV2) inverted terminal repeats (ITRs). The vector was produced in AAV8 capsids in a full GMP process by two-plasmid transfection in HEK293 cells in suspension culture in a bioreactor. The components of resamirigene bilparvovec are shown in Table 2 below. Table 2. Resamirigene Bilparvovec nucleic acid sequence (SEQ ID NO:2)

[0120] [Table 2] As described herein, resamirigene bilparvovec refers to an AAV vector having the nucleic acid sequence represented by SEQ ID NO:2, shown below.

[0121] [ka] TIFF2025512955000007.tif249161TIFF2025512955000008.tif249161TIFF2025512955000009.tif249161 TIFF2025512955000010.tif249161TIFF2025512955000011.tif249161TIFF2025512955000012.tif219169 Methods for delivery of exogenous nucleic acids into target cells Transfection technology Techniques that can be used to introduce AAV vectors, such as AAV vectors encoding the enzymes described herein, into target cells are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the cells of interest. Mammalian cells, such as human cells, that are subjected to an external electric field in this manner are then predisposed to the uptake of exogenous nucleic acids (e.g., nucleic acids that can be expressed in neurons, glial cells, or non-neuronal cells, such as colon and kidney cells). Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. As a similar technique, NUCLEOFECTION™ utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. NUCLEOFECTION™ and protocols useful for carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and U.S. Patent Application Publication No. 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0122] Another technique useful for transfection of target cells is squeeze perforation. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that are formed in response to applied stress. This technique is advantageous in that no vector is required to deliver nucleic acid to cells, such as human target cells. Squeeze perforation is described in detail, for example, in Sharei et al., J.Vis.Exp.81:e50980(2013) (the disclosure of which is incorporated herein by reference).

[0123] Lipofection represents another technique useful for transfection of target cells. This method involves loading nucleic acid into liposomes, which often present cationic functional groups such as quaternary amines or protonated amines directed toward the outside of the liposome. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, and ultimately uptakes the exogenous nucleic acid, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce uptake of exogenous nucleic acid is contacting the cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that favors interaction with cell membranes include activated dendrimers (e.g., Dennig, Top. Curr. Chern. 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and DEAE-dextran (the use of which as a transfection agent is described in detail, e.g., in Gulick et al., Curr Protoc Mol Biol. 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference).

[0124] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laser transfection, also known as optical transfection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique has been found to be similar to, and in some cases superior to, electroporation.

[0125] Impalefection is another technique that can be used to deliver genetic material to target cells. It depends on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene intended for intracellular delivery is attached to the nanostructure surface. The chip with the needle array is then pressed against a cell or tissue. Cells penetrated by the nanostructures are able to express the delivered gene. An example of this technique is described in Shalek et al., PNAS107:25 1870 (2010), the disclosure of which is incorporated herein by reference.

[0126] MAGNETOFECTION™ can also be used to deliver nucleic acids to target cells. The principle of MAGNETOFECTION™ is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and are coated with specific cationic proprietary molecules that vary depending on the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by a magnet. This technique is described in Scherer et al., Gene Ther. 9:102 (2002), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to slowly and efficiently transfect target cells, since the method utilizes an applied magnetic field to direct the uptake of nucleic acids. This technique is described in detail, for example, in U.S. Patent Application Publication No. 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0127] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) to modify the permeability of the plasma membrane of a cell, permeabilizing the cell and allowing polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods Cell Biol. 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0128] Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and genome-modifying proteins, such as nucleases, can be used to efficiently deliver proteins to cells, which then catalyze the site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of the cells for covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also called gesicles, for genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract], Methylation changes in early embryonic genes in cancer [abstract] and Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0129] Targeted gene integration using gene editing technology Additionally, various tools have been developed that can be used to integrate a gene of interest into a target cell, such as a human cell. One method that can be used to integrate a polynucleotide encoding a target gene into a target cell is the use of a transposon. A transposon is a polynucleotide that encodes a transposase enzyme and contains a polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered into the cell, expression of the transposase gene is initiated, resulting in an active enzyme that excises the gene of interest from the transposon. This activity is mediated by site-specific recognition of the transposon excision sites by the transposase. In some instances, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of similar excision sites present in the nuclear genome of the cell. This allows the gene of interest to be inserted into the nuclear DNA that has been cut at the complementary excision site, and the integration process is then completed by covalent ligation of a phosphodiester bond that links the gene of interest to the DNA of the mammalian cell genome. In some cases, the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA before being integrated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail, for example, in WO 2010 / 085699) and the sleeping beauty transposon (described in detail, for example, in US 2005 / 0112764), the disclosures of which are each incorporated herein by reference as they relate to transposons for use in gene delivery to cells of interest.

[0130] Another tool for integrating target genes into the genome of target cells is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR / Cas system comprises palindromic repeat sequences in plasmid DNA and the associated Cas9 nuclease. This DNA and protein ensemble directs site-specific DNA cleavage of a target sequence by first integrating foreign DNA into the CRISPR locus. Polynucleotides containing these foreign sequences and the repeat spacer elements of the CRISPR locus are then transcribed in the host cell to generate guide RNAs that can subsequently anneal to the target sequence and localize the Cas9 nuclease to this site. Thus, highly site-specific cas9-mediated DNA cleavage can be generated in foreign polynucleotides, since the interactions that bring cas9 into the vicinity of the target DNA molecule are governed by RNA:DNA hybridization. As a result, CRISPR / Cas systems can be designed to cleave any target DNA molecule of interest. This technology has been utilized to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an efficient means of site-specifically editing target cell genomes to cleave DNA prior to integration of a gene encoding a target gene. The use of CRISPR / Cas to regulate gene expression is described, for example, in U.S. Pat. No. 8,697,359, the disclosure of which is incorporated herein by reference as it relates to the use of CRISPR / Cas systems for genome editing. Alternative methods for site-specifically cleaving genomic DNA prior to integration of a gene of interest in a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike CRISPR / Cas systems, these enzymes do not contain guide polynucleotides to localize to specific target sequences. Target specificity is instead controlled by DNA binding domains within these enzymes.The use of ZFNs and TALENs in genome editing applications is described, for example, in Umov et al. 11:636 (2010); and Joung et al., Nat. Rev. Mol. Cell Biol. 14:49 (2013), the disclosures of which are incorporated by reference herein as they relate to compositions and methods for genome editing, respectively.

[0131] Further genome editing techniques that can be used to integrate a polynucleotide encoding a target gene into a target genome include the use of ARCUS™ meganucleases, which can be rationally designed to site-specifically cleave genomic DNA. The use of these enzymes for the integration of a gene encoding a target gene into the genome of a mammalian cell is advantageous in view of the defined structure-activity relationship that has been established for the enzymes. Single-stranded meganucleases can be modified at specific amino acid positions to create nucleases that selectively cleave DNA at desired positions, allowing site-specific integration of the target gene into the nuclear DNA of the target cell. These single-stranded nucleases are extensively described, for example, in U.S. Patent No. 8,021,867 and U.S. Patent No. 8,445,251, the disclosures of which are incorporated herein by reference as they relate to compositions and methods for genome editing, respectively.

[0132] Pharmaceutical Compositions and Routes of Administration The AAV vectors described herein may encode an enzyme, such as an AAV encoding MTM1, which may be incorporated into a vehicle for administration to a patient, such as a human patient suffering from a congenital loss-of-function disease (e.g., XLMTM). Pharmaceutical compositions comprising viral vectors comprising a transcriptional regulatory element (e.g., desmin promoter) described herein operably linked to a therapeutic enzyme may be prepared using methods known in the art. For example, the compositions may be prepared in a desired form, such as a lyophilized formulation or an aqueous solution, using, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference).

[0133] The AAV vectors described herein, including a transcriptional regulatory element operably linked to a therapeutic enzyme, can be administered to a patient (e.g., a human patient) by a variety of routes of administration. The routes of administration can vary, for example, depending on the onset and severity of the disease, and can include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. Intravascular administration includes delivery to the vasculature of a patient. In some embodiments, administration is to a blood vessel that is considered a vein (intravenous), and in some administrations, administration is to a blood vessel that is considered an artery (intraarterial). Veins include, but are not limited to, the internal jugular vein, peripheral veins, coronary veins, hepatic veins, portal vein, great saphenous vein, pulmonary veins, superior vena cava, inferior vena cava, gastric vein, splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and / or femoral vein. The arteries may include, but are not limited to, coronary arteries, pulmonary arteries, brachial arteries, internal carotid arteries, aortic arches, femoral arteries, peripheral arteries, and / or ciliary arteries. Delivery is contemplated to be through or directed to arterioles or capillaries.

[0134] Mixtures of the AAV vectors described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, the preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the formulation may be sterile and fluid to the extent that easy syringability exists. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. The action of microorganisms can also be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Agents that delay absorption, for example, aluminum monostearate and gelatin, can be used in the composition to prolong the absorption of the injectable composition.

[0135] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered if necessary, and liquid diluents are first rendered isotonic with sufficient saline or glucose. Such aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion fluid or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. Moreover, for human administration, preparations can meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0136] In some embodiments, administration of the AAV vector to a patient results in a change from baseline in a quantitative analysis of AAV-encoded enzyme expression (e.g., in a muscle biopsy). In some embodiments, the patient results in a change from baseline in a quantitative analysis of enzyme expression in a muscle biopsy by about 24 weeks after administration of the AAV vector to the patient.

[0137] kit The compositions described herein can be provided in a kit for determining the efficacy of AAV vectors according to the methods described herein.In an embodiment, the kit can include one or more AAV vectors encoding the enzymes described herein.The kit can include a package insert that instructs a user of the kit, such as a person skilled in the art, to carry out any one of the methods described herein.

[0138] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used and evaluated and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

[0139] Viral vectors encoding enzyme activity assays The objective of this study was to test the efficacy of an adeno-associated virus (AAV) vector encoding an enzyme (e.g., phosphatase) as determined by the amount or concentration of substrate cleaved by the enzyme (e.g., phosphatase activity) encoded by the enzyme relative to a reference amount or concentration of the cleavage product using a novel efficacy assay protocol. Materials and Methods Infection of host cells with AAV and preparation of cell lysates On day 0, host cells (e.g., C2C12 cells; ATCC® CRL1772) are seeded onto collagen-coated 24-well clear plates (50,000 cells / 0.5 mL / well) in growth medium (e.g., DMEM, 20% FBS, penicillin-streptomycin). Incubate at 37° C., 5% CO. 2 On day 1, first, discard the medium and add 0.5 mL of differentiation medium (e.g., DMEM, 2% FBS, penicillin-streptomycin) to each well. Second, add 5 × 109 vg / well to 5 × 1011 vg / well of an AAV vector encoding an enzyme (e.g., resamirigene bilparvovec). Third, incubate at 37 °C, 5% CO. 2 Incubate at 4°C for 3 days. On the fourth day, first discard the medium and wash with PBS. Then add 500 μL of lysis buffer* to each well and pipette. The lysis buffer may contain 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 5 mM EDTA, 0.5% TritonX-100, 1 mM dithiothreitol, and 1×Halt™ protease inhibitor cocktail (Thermo Fisher #78437). Then transfer the cell lysate to a 1.5 mL tube. Then centrifuge at 15,000 rpm for 10 minutes at 4°C, transfer the supernatant to a new 1.5 mL tube (e.g., divide into two or three aliquots) and store at -80°C.

[0140] Enzyme cleavage assay First, thaw the tubes and measure the protein concentration of the samples with the Pierce BCA Protein Assay kit (Thermo Fisher #23227). The assay involves adding 10 μL of standards and samples to a 96-well plate. Then, add 10 μL of lysis buffer to the standard wells. In some embodiments, different formulations of lysis buffer will affect the results. Then, add 200 μL of BCA working solution (A:B=1:50) to each well. Shake for 30 seconds, incubate at 37°C for 30 minutes, shake again for 30 seconds, and measure the optical density at 562 nm.

[0141] Add substrate and lysate to a 1.5 mL tube. Specifically, this contains 50 mM ammonium acetate (pH 6.0), 100 ng BODIPY FL phosphatidylinositol 3-phosphate (hereafter "BODIPY FL PI(3)P"; FIG. 1) (Echelon Bioscience #C-03F6), 10 μL cell lysate (300 ng, dilute cell lysate in lysis buffer) for a total reaction volume of 100 μL.

[0142] This is followed by incubation at 30°C for 60 minutes. Then, 2 μL of 10% SDS (final concentration 0.2%) is added and incubated at 75°C for 10 minutes. The purpose of this step is to inactivate the AAV. During the incubation, standards are prepared in 1.5 mL tubes. These include 0.3 ng to 100 ng of BODIPY FL PI(3)P, a water-soluble analogue of PI(3)P labeled with a green fluorophore at the sn-1 position, Bodipy®-FL, and Bodipy®-FL (Echelon Bioscience #C-00F6) in 100 μL of 50 mM ammonium acetate (pH 6.0), 0.1× lysis buffer, and 0.2% SDS. Extraction of cleavage products First, 100 μL of chloroform / methanol (1:1) is added to the samples and standards. Then, the samples are vortexed and centrifuged at 15,000 rpm at 25°C for 5 minutes. Then, 120 μL of fluorescent lipids (upper phase) are collected and transferred to a 96 deep-well plate (BM EQUIPMENT #BM6019). The aggregates are blown dry with gas (e.g., N2 gas). The samples can be stored at -20°C.

[0143] High-performance liquid chromatography Sample preparation Prior to analysis, the dried samples were reconstituted in 50 μL of starting LC eluent mobile phase A / B (25:75 v / v), followed by mixing and centrifugation (3,000 rpm) for 10 min at 4° C. The supernatant was transferred to an autosampler vial. Equipment and conditions The equipment and conditions were as follows: Acquity UPLC H-Class Bio (WATERS), Bio-inert, Acquity UPLC FLR (fluorescence) (WATERS) HPLC. Detector, excitation / excitation was 358 / 505 (nm), gain was 1, column consisted of L-column 2 HPLC column, 2.0×150 mm, 3 μm (CERI#731021), mobile phase A consisted of 5 mM ammonium formate / acetonitrile=70 / 30, mobile phase B consisted of 2-propanol / acetonitrile=9 / 1, injection volume was 1 μL, and run time was 10 min. Liquid chromatography conditions: In particular, the liquid chromatography binary gradient conditions listed in Table 3 were as follows: Table 3: Liquid chromatography binary gradient conditions

[0144] [Table 3] result As a result, the chromatograms demonstrated the extraction of both PI(3)P and PI on the chloroform / methanol column (Figure 2). The amount (ng) of extracted fluorescently labeled (FL) phosphatidylinositol (PI) increased as a function of increasing titers of AAV encoding MTM1 (AAV-DES-MTM1) for C2C12 transfected cells. In contrast, when AAV encoding acid α-glucosidase (GAA;:AAV-MCK-GAA) was transfected into C2C12 cells, the amount (ng) of extracted phosphatidylinositol 3-phosphate (PI(3)P) was significantly decreased (Figure 3). Consistent with the above results, the amount (ng) of extracted PI(3)P decreased as a function of increasing titers of AAV encoding MTM1 (AAV-DES-MTM1) for C2C12 transfected cells (Figure 4). Collectively, these results illustrate the development of a novel AAV potency assay that provides a high-throughput, quantitative, and sensitive measure of the potency of AAV encoding enzymes (eg, MTM1). EXAMPLES

[0145] Myotubularin 1-encoding AAV potency assay This example describes a method to determine the efficacy of an AAV vector encoding MTM1 for the treatment of a loss-of-function disease associated with a deleterious mutation in the gene in a patient, such as X-linked myotubular myopathy (XLMTM). According to the materials and methods described in Example 1, an AAV vector encoding an enzyme is contacted with AAV permissive cells (e.g., C2C12 cells) expressing a substrate specifically cleaved by the enzyme for a time sufficient for the AAV permissive cells to express the enzyme. An exemplary AAV vector for transfection is resamirigene bilparvovec, a non-replicating recombinant AAV8 vector expressing a non-codon-optimized human MTM1 cDNA under the control of the muscle-specific human desmin promoter. The MTM1 expression cassette was constructed by cloning a synthetic DNA sequence complementary to the coding portion (nucleotides 43-1864) of the wild-type human MTM1 transcript (NCBI reference sequence number 000252.3) downstream of the 1.05 kb human desmin enhancer / promoter region. The second intron and polyadenylation sequence of the human β-globin gene (HBB) were inserted upstream and downstream of the MTM1 synthetic cDNA, respectively, to mediate RNA processing. The expression cassette was flanked by AAV2 inverted terminal repeats (ITRs). The vector was produced in AAV8 capsids in a full GMP process by two-plasmid transfection in HEK293 cells in suspension culture in a bioreactor.

[0146] The amount or concentration of the product resulting from cleavage of the substrate by the MTM1 enzyme in the surrounding extracellular medium can then be assessed by HPLC.

[0147] In the materials and methods described in Example 1, resamirigene bilparvovec is identified as being effective in treating loss-of-function diseases (e.g., XLMTM) by the finding that the amount or concentration of FL-PI, a cleavage product of MTM1, is increased compared to a reference amount or concentration of FL-PI. EXAMPLES

[0148] Methods for Treating Loss of Function Diseases This example describes a method of treatment comprising determining the efficacy of an AAV vector encoding a putative enzyme in a patient in need of treatment for a loss-of-function disease associated with a deleterious mutation in a gene encoding the enzyme, and administering a therapeutically effective amount of the AAV vector to the patient. According to the materials and methods described in Example 1, an AAV vector encoding an enzyme was contacted with AAV permissive cells (e.g., C2C12 cells) expressing a substrate specifically cleaved by the enzyme for a time sufficient for the AAV permissive cells to express the enzyme. An exemplary AAV vector for transfection is resamirigene bilparvovec, a non-replicating recombinant AAV8 vector expressing a non-codon-optimized human MTM1 cDNA under the control of the muscle-specific human desmin promoter. An MTM1 expression cassette was constructed by cloning a synthetic DNA sequence complementary to the coding portion (nucleotides 43-1864) of the wild-type human MTM1 transcript (NCBI reference sequence number_000252.3) downstream of the 1.05 kb human desmin enhancer / promoter region. The second intron and polyadenylation sequence of the human β-globin gene (HBB) were inserted upstream and downstream of the MTM1 synthetic cDNA, respectively, to mediate RNA processing. The expression cassette was flanked by AAV2 inverted terminal repeats (ITRs). The vectors were produced in AAV8 capsids in a full GMP process by two-plasmid transfection in HEK293 cells in suspension culture in a bioreactor.

[0149] The amount or concentration of the product resulting from enzymatic cleavage of the substrate in the surrounding extracellular medium can then be assessed by HPLC. A finding that the amount or concentration of FL-PI, a cleavage product of MTM1, in the cell or surrounding extracellular medium is increased relative to a reference amount or concentration of FL-PI identifies the AAV vector as having efficacy for treating a loss-of-function disease.

[0150] After determining that the amount or concentration of the cleavage product in the cells or the surrounding extracellular medium is increased compared to a reference amount or concentration of the cleavage product, the patient is administered a therapeutically effective amount of an AAV vector (e.g., about 3×10 13 vg / kg ~ approx. 2.3×10 14 At a dose of 10 vg / kg, in some cases, the AAV vector was approximately 8 × 10 13 vg / kg ~ approx. 1.8×10 14 vg / kg, approx. 1×10 14 vg / kg ~ approx. 1.6×10 14 vg / kg, approximately 1.1×10 14 vg / kg ~ approx. 1.5×10 14 vg / kg, or approximately 1.2 × 10 14 vg / kg ~ approx. 1.4×10 14 vg / kg administered to the patient).

[0151] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. While the invention has been described in relation to particular embodiments thereof, it will be understood that the invention can be further modified, and this application is intended to cover any variation, use, or adaptation of the invention generally in accordance with the principles of the invention, which is within known or customary practice in the art to which the invention pertains, and which applies to the essential features hereinbefore described, including departures from the invention as defined by the claims. Other embodiments are within the scope of the claims.

Claims

1. 1. A method for determining efficacy of an adeno-associated viral (AAV) vector encoding an enzyme for the treatment of a loss-of-function disease associated with a deleterious mutation in a patient, the method comprising: a) contacting an AAV vector with an AAV permissive cell line for a period of time sufficient for said AAV permissive cell line to express said enzyme; b) lysing the AAV permissive cell line and exposing, in a reaction medium, the expressed enzyme to a substrate that is specifically cleaved by the enzyme; and c) evaluating the amount or concentration of the substrate or a product resulting from cleavage of the substrate by the enzyme in the reaction medium by chromatography, wherein (i) the amount or concentration of the cleavage product in the reaction medium is increased relative to a reference amount or concentration of the cleavage product; or (ii) the finding that the amount or concentration of the substrate in the reaction medium is decreased relative to a reference amount or concentration of the substrate identifies the method as having efficacy for treating a loss-of-function disease associated with a deleterious mutation in the AAV vector.

2. 1. A method for producing an adeno-associated virus (AAV) vector encoding an enzyme in a gene encoding said enzyme for the treatment of a loss-of-function disease associated with a deleterious mutation in a patient in need of such treatment, comprising: a) contacting an AAV vector with an AAV permissive cell line for a period of time sufficient for said AAV permissive cell line to express said enzyme; b) lysing the AAV permissive cell line and exposing, in a reaction medium, the expressed enzyme to a substrate that is specifically cleaved by the enzyme; c) evaluating the amount or concentration of the substrate or the product resulting from cleavage of the substrate by the enzyme in the reaction medium by chromatography; and d) (i) the amount or concentration of the cleavage product in the reaction medium is increased relative to a reference amount or concentration of the cleavage product; or (ii) releasing an AAV vector for treating a loss-of-function disorder associated with a deleterious mutation when the amount or concentration of the substrate in the reaction medium is decreased relative to a reference amount or concentration of the substrate; The method includes:

3. 1. A method for treating a loss-of-function disease associated with a deleterious mutation in a gene encoding an enzyme in a patient in need of such treatment, comprising: a) contacting an AAV vector with an AAV permissive cell line for a period of time sufficient for said AAV permissive cell line to express said enzyme; b) lysing the AAV permissive cell line and exposing, in a reaction medium, the expressed enzyme to a substrate that is specifically cleaved by the enzyme; c) determining by chromatography whether (i) the amount or concentration of the cleavage product in the reaction medium is increased relative to a reference amount or concentration of the cleavage product, or (ii) the amount or concentration of the substrate in the reaction medium is decreased relative to a reference amount or concentration of the substrate; and d) administering to a patient a therapeutically effective amount of said AAV vector.

4. 2. The method of claim 1, wherein the expressed enzyme is not purified from the cell lysate obtained from b) prior to exposure to the substrate capable of cleaving the enzyme.

5. The method of claim 1 , wherein the enzyme is myotubularin 1 (MTM1).

6. 2. The method of claim 1, wherein the AAV vector further comprises a muscle-specific promoter, optionally wherein the muscle-specific promoter is located 5' to the polynucleotide encoding the enzyme.

7. 7. The method of claim 6, wherein the muscle-specific promoter is a desmin promoter, a phosphoglycerate kinase promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of eye pair-like homeodomain 3.

8. 2. The method of claim 1, wherein the AAV vector has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO:

2.

9. The AAV vector is about 3×10 １３ vg / kg ~ approx. 2.3 x 10 １４ vg / kg of AAV vector, and in some cases, the AAV vector is administered to the patient in an amount of about 8×10 １３ vg / kg ~ approx. 1.8 x 10 １４ vg / kg, approximately 1×10 １４ vg / kg ~ approx. 1.6×10 １４ vg / kg, approximately 1.1×10 １４ vg / kg ~ approx. 1.5×10 １４ vg / kg, or about 1.2 x 10 １４ vg / kg ~ approx. 1.4×10 １４ 4. The method of claim 3, wherein the amount of the compound administered to the patient is in the range of 0.5 to 100 mg / kg.

10. 10. The method of claim 9, wherein the loss of function disease is X-linked myotubular myopathy (XLMTM).

11. 11. A kit comprising an AAV vector encoding an enzyme and a package insert, the package insert instructing a user of the kit to determine the potency of the AAV vector according to the method of claim 10.

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