Vector composition for treating lysosomal storage disorders and method for using the same

Abstract

Provided herein are compositions and methods using a bicistronic vector for treating or preventing a lysosomal storage disease (LSD) in a subject. The disclosed compositions include a bicistronic vector comprising a promoter, an internal ribosome entry site (IRES), a polynucleotide encoding a lysosomal enzyme, and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). The method includes administering to a subject a pharmaceutical composition comprising the bicistronic vector disclosed herein.

Description

[Technical Field]

[0001] Related applications This application claims the benefits of Provisional Applications USSN62 / 869,781 and USSN62 / 869,808, filed 2 July 2019, which are incorporated herein by reference in their entirety.

[0002] Incorporation of sequence lists The entire contents of the text file named "M6PT-002 / 01WO_SeqList.txt", which was created on July 1, 2020, and has a size of 611KB, are incorporated into this specification by reference.

[0003] Technical field The disclosed information pertains to compositions and methods for treating lysosomal storage disorders. More specifically, the disclosed information pertains to the field of treating lysosomal disorders using improved gene therapy and improved enzyme replacement therapy (ERT). [Background technology]

[0004] background Lysosomal storage disorders (LSDs) relate to hereditary metabolic disorders resulting from defects in lysosomal function. Currently, approximately 50 distinct LSDs have been identified, but only a small number (less than 10) have been reported to be treated. Therefore, there is an unaddressed need in the art for safe and effective treatments for LSDs. This disclosure offers two solutions to this unaddressed need: either enzyme replacement therapy (ERT) or gene therapy. [Overview of the project] [Means for solving the problem]

[0005] Abstract This disclosure provides a composition comprising a vector containing a promoter, a first polynucleotide sequence encoding a lysosomal enzyme, and a second polynucleotide sequence encoding a modified N-acetylglucosamine-1-phosphotransferase (GlcNAc-1 PTase, PTase), wherein the promoter is capable of driving expression in mammalian cells, and the promoter is operably linked to the first polynucleotide and the second polynucleotide.

[0006] In some embodiments of the compositions of this disclosure, the vector further comprises a sequence encoding an intra-sequence ribosome entry site (IRES). In some embodiments, the sequence encoding the IRES is positioned between the sequence encoding the lysosomal enzyme and the sequence encoding the modified GlcNAc-1 PTase. In some embodiments, the vector comprises, from 5' to 3', a sequence encoding the modified GlcNAc-1 PTase, a sequence encoding the IRES, and a sequence encoding the lysosomal enzyme. In some embodiments, the vector comprises, from 5' to 3', a sequence encoding the lysosomal enzyme, a sequence encoding the IRES, and a sequence encoding the modified GlcNAc-1 PTase.

[0007] In some embodiments of the compositions of this disclosure, the vector further comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site comprises a sequence encoding a 2A self-cleaving peptide.

[0008] In some embodiments of the compositions of this disclosure, the vector is an expression vector. In some embodiments, the expression vector comprises a plasmid.

[0009] In some embodiments of the compositions of this disclosure, the vector is a delivery vector. In some embodiments, the delivery vector includes a viral vector. In some embodiments, the viral vector includes an AAV vector or a lentiviral vector. In some embodiments, the AAV vector includes a sequence isolated from or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments, the delivery vector includes a nonviral vector. In some embodiments, the nonviral vector includes liposomes, lipid nanoparticles (LNPs), micelles, polymerosomes, nanoparticles, polymer nanoparticles, or exosomes.

[0010] In some embodiments of the compositions of this disclosure, the vector is a viral vector. In some embodiments, the viral vector includes an AAV vector or a lentiviral vector. In some embodiments, the AAV vector includes a sequence isolated from or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments of the compositions of this disclosure, the vector is a nonviral vector. In some embodiments, the nonviral vector includes liposomes, lipid nanoparticles (LNPs), micelles, polymerosomes, nanoparticles, polymer nanoparticles, or exosomes.

[0011] In some embodiments of the compositions of this disclosure, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adenovirus vector or an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAV vector comprises a sequence encoding a capsid isolated from or derived from one or more serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAV vector comprises a sequence encoding at least one terminal inversion sequence (ITR) isolated from or derived from one or more serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0012] In some embodiments of the compositions of this disclosure, the vector is a bicistronic vector.

[0013] In some embodiments of the compositions of this disclosure, the vector is a multi-cistronic vector.

[0014] In some embodiments of the compositions of this disclosure, the promoter includes a ubiquitous promoter. In some embodiments, the promoter is capable of driving expression in mammalian cells. In some embodiments, the promoter is capable of driving expression in human cells.

[0015] In some embodiments of the compositions of the present disclosure, the promoter includes a cell-type specific promoter. In some embodiments, the promoter is capable of driving expression in mammalian cells. In some embodiments, the promoter is capable of driving expression in human cells. In some embodiments, the promoter is capable of driving expression in nervous system cells including, but not limited to, neurons or glial cells. In some embodiments, the promoter is capable of driving expression in muscle cells including, but not limited to, smooth muscle cells, striated muscle cells or cardiac muscle cells. In some embodiments, the promoter is capable of driving expression in lung cells. In some embodiments, the promoter is capable of driving expression in bone cells. In some embodiments, the promoter is capable of driving expression in blood cells including, but not limited to, red blood cells, white blood cells, their precursors or hematopoietic stem cells. In some embodiments, the promoter is capable of driving expression in immune cells including, but not limited to, T cells, B cells or macrophages. In some embodiments, the promoter is capable of driving expression in cells of the spleen or pancreas. In some embodiments, the promoter is capable of driving expression in cells of the kidney.

[0016] In some embodiments of the compositions of the present disclosure, the promoter is the human T-lymphotropic virus type I (HTLV-I) promoter.

[0017] In some embodiments of the compositions of the present disclosure, the promoter is the CBh promoter. In some embodiments, the CBh promoter includes a CMV early enhancer fused to a modified chicken β-actin promoter.

[0018] In some embodiments of the compositions of the present disclosure, the promoter is the CEF or hCEFI promoter. In some embodiments, the hCEFI promoter comprises a human CMV enhancer operably linked to a human EF1a promoter. In some embodiments, the hCEFI promoter comprises the sequence of SEQ ID NO: 161.

[0019] In some embodiments of the compositions of the present disclosure, the promoter comprises a constitutive promoter. In some embodiments, the constitutive promoter comprises a cytomegalovirus (CMV) promoter.

[0020] In some embodiments of the compositions of the present disclosure, the vector comprises the nucleic acid sequence of SEQ ID NO: 1.

[0021] In some embodiments of the compositions of the present disclosure, the polynucleotide encoding the modified GlcNAc-1 PTase comprises the nucleic acid sequence of SEQ ID NO: 4.

[0022] In some embodiments of the compositions of the present disclosure, the lysosomal enzyme is involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B or Table 1C.

[0023] In some embodiments of the compositions of the present disclosure, the lysosomal enzyme comprises at least one lysosomal enzyme listed in Table 1A, Table 1B or Table 1C.

[0024] In some embodiments of the compositions of the present disclosure, the lysosomal enzyme is selected from the group consisting of β-glucosylceramidase (glucocebrosidase) (GCase / GBA, encoded by the GBA gene), galactosylceramidase (GALC), α-galactosidase (encoded by the GLA gene), α-N-acetylglucosaminidase (NAGLU), acid α-glucosidase (GAA) and lysosomal acid α-mannosidase (LAMAN).

[0025] In some embodiments of the compositions of this disclosure, the lysosomal enzyme comprises β-glucocerebrosidase (GCase / GBA). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 5.

[0026] In some embodiments of the compositions of this disclosure, the lysosomal enzyme comprises galactosylceramidase (GALC). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 23.

[0027] In some embodiments of the compositions of this disclosure, the lysosomal enzyme comprises α-galactosidase (GLA). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 7.

[0028] In some embodiments of the compositions of this disclosure, the lysosomal enzyme comprises α-N-acetylglucosaminidase (NAGLU). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 8.

[0029] In some embodiments of the compositions of this disclosure, the lysosomal enzyme comprises acid α-glucosidase (GAA). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 9.

[0030] In some embodiments of the compositions of this disclosure, the lysosomal enzyme comprises lysosomal acid α-mannosidase (LAMAN). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 10.

[0031] This disclosure provides a method for treating lysosomal storage disorders (LSD), comprising the step of administering an effective amount of a composition of this disclosure to a subject, wherein the composition increases the phosphorylation of lysosomal enzymes that cause LSD, thereby treating the LSD. This disclosure provides a method for treating lysosomal storage disorders (LSD), comprising the step of administering an effective amount of a composition of this disclosure to a subject, wherein the composition increases the phosphorylation of N-linked oligosaccharides that cause LSD, thereby treating the LSD. In some embodiments, the subject exhibits signs or symptoms of LSD. In some embodiments, the subject has been diagnosed with LSD.

[0032] This disclosure provides a method for preventing the appearance or development of lysosomal storage disorders (LSDs), comprising the step of administering an effective amount of the composition of this disclosure to a subject, wherein the composition increases the phosphorylation of lysosomal enzymes that cause LSDs, thereby preventing the appearance of LSDs in the subject. In some embodiments, the subject is at risk of developing or developing LSDs. In some embodiments, the subject exhibits signs or symptoms of LSDs.

[0033] This disclosure provides a method for improving the phosphorylation of lysosomal enzymes that cause lysosomal storage disorders (LSD), comprising the step of administering an effective amount of the composition of this disclosure to a subject, wherein the composition increases the phosphorylation of lysosomal enzymes. In some embodiments, the subject exhibits signs or symptoms of LSD. In some embodiments, the subject is at risk of developing or having LSD. In some embodiments, the subject has been diagnosed with LSD.

[0034] This disclosure provides a method for improving the phosphorylation of lysosomal enzymes that cause lysosomal storage disorders (LSDs), comprising the step of contacting cells with an effective amount of the composition of this disclosure, wherein the composition increases the phosphorylation of lysosomal enzymes. In some embodiments, the cells are in vitro or ex vivo. In some embodiments, the cells are in vivo. In some embodiments, the subjects include cells. In some embodiments, the subjects exhibit signs or symptoms of LSD. In some embodiments, the subjects are at risk of developing or having LSD. In some embodiments, the subjects have been diagnosed with LSD.

[0035] In some embodiments of the methods of this disclosure, lysosomal enzymes are involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C.

[0036] In some embodiments of the methods of this disclosure, the lysosomal enzyme is at least one of those listed in Table 1A, Table 1B, or Table 1C.

[0037] In some embodiments of the methods of the present disclosure, the lysosomal enzyme includes one or more of the following: β-glucocerebrosidase (GCase / GBA), galactosylceramidase (GALC), α-galactosidase (GLA), α-N-acetylglucosaminidase (NAGLU), acid α-glucosidase (GAA), and lysosomal acid α-mannosidase (LAMAN).

[0038] In some embodiments of the methods of this disclosure, the administration step includes a systemic route of administration. In some embodiments, the systemic route of administration is enteral, parenteral, oral, intramuscular (IM), subcutaneous (SC), intravenous (IV), intra-arterial (IA), intraspinal, intraventricular, subarachnoid, or intraventricular.

[0039] In some embodiments of the methods of this disclosure, the administration step includes a local administration route.

[0040] In some embodiments of the methods disclosed herein, the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female.

[0041] For illustrative purposes, certain embodiments of this disclosure are shown in the figures. However, this disclosure is not limited to the exact combinations and means of the embodiments shown in the figures.

[0042] The patent or application file contains at least one color drawing. A copy of the published patent or application accompanied by the color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0043] [Figure 1A-1C] Figures 1A-1C are a series of figures and graphs illustrating S1-S3 bicistronic vectors. Figure 1A: CMV-S1S3 vector. Figure 1B: pLL01: pCMV-MCS-IRES-S1S3 vector. Figure 1C: Graph illustrating the expression levels of CMV-S1S3 and pLL01 (CPM: counts / minute).

[0044] [Figure 2A-2C] Figures 2A–2C are a series of figures and histograms illustrating the generation of GBA bicistronic expression plasmids in the S1-S3 bicistronic vector. Figure 2A: pLL11: pCMV-hGBA-IRES-S1S3 vector. Figure 2B: GBA activity in conditional medium. Figure 1C: Histogram illustrating percentage of PTase activity.

[0045] [Figure 3A-3C] Figures 3A–3C are a series of graphs and histograms showing that bicistronic expression increases the phosphorylation of the GBA enzyme.

[0046] [Figure 4A-4D]Figures 4A–4D are a series of figures, graphs, and histograms showing that bicistronic expression increases the phosphorylation of the GAA enzyme.

[0047] [Figures 5A-5D] Figures 5A–5D are a series of figures, graphs, and histograms showing that bicistronic expression increases the phosphorylation of GALC enzymes.

[0048] [Figures 6A-6D] Figures 6A–6D are a series of figures, graphs, and histograms showing that bicistronic expression increases phosphorylation of the NAGLU enzyme.

[0049] [Figures 7A-7D] Figures 7A–7D are a series of figures, graphs, and histograms showing that bicistronic expression increases the phosphorylation of the GLA enzyme.

[0050] [Figures 8A-8D] Figures 8A–8D are a series of figures, graphs, and histograms showing that bicistronic expression increases the phosphorylation of the LAMAN enzyme.

[0051] [Figures 9A-9E] Figures 9A–9E are a series of graphs (A–C) demonstrating that the S1-S3 PTase bicistronic vector of this disclosure significantly increases the binding of the GBA enzyme to CI-MPR and its cellular uptake in the treatment of Gaucher disease. Panels D and E demonstrate that a single point mutation in the GBA enzyme increases its stability without affecting its binding to CI-MPR.

[0052] [Figure 10A-10C] Figures 10A–10C are a series of graphs demonstrating that the S1-S3 PTase bicistronic vector of this disclosure significantly increases the binding of GAA enzyme to CI-MPR and its cellular uptake in the treatment of Pompe disease.

[0053] [Figure 11A-11C] Figures 11A–11C are a series of graphs demonstrating that the S1-S3 PTase bicistronic vector of this disclosure significantly increases the binding of GALC enzymes to CI-MPR and their cellular uptake in the treatment of Krabbe disease.

[0054] [Figure 12A-12C] Figures 12A–12C are a series of graphs demonstrating that the S1-S3 PTase bicistronic vector of this disclosure significantly increases the binding of the NAGLU enzyme to CI-MPR and its cellular uptake in the treatment of MPS IIIB disease.

[0055] [Figure 13A-13C] Figures 13A–13C are a series of graphs demonstrating that the S1-S3 PTase bicistronic vector of this disclosure significantly increases the binding of GLA enzymes to CI-MPR and their cellular uptake in the treatment of Fabry disease.

[0056] [Figure 14A-14C] Figures 14A–14C are a series of graphs demonstrating that the S1-S3 PTase bicistronic vector of this disclosure significantly increases the binding of LAMAN enzymes to CI-MPR and their cellular uptake in the treatment of α-mannosidosis.

[0057] [Figures 15A-15B] Figures 15A–15B are schematic diagrams and graphs demonstrating that the S1-S3 PTase bicistronic vector of this disclosure, delivered by the AAV9 vector, can be used as gene therapy in the treatment of mucolipidosis.

[0058] [Figures 16A-16B]Figures 16A–16B are pairs of graphs showing elevated glucosylceramide levels observed in the liver, lungs, and spleen of 20-week-old GaucherD409V / null mice. Accumulation of glucocerebroside, a natural substrate of GBA, was determined in tissue homogenates. Accumulation of GC in the lungs is a statistically and therapeutically valuable result, representing a known but unaddressed need for current standard therapeutics. Glucosylceramide was extracted from 20 μL of constant-volume tissue homogenates and a suitable control by adding 200 μL of methanol / ACN / H2O (v:v:v=85:10:5), mixing at 800 rpm for 5 minutes, and then centrifugation at 3220 g at 4°C for 15 minutes;3). 50 μL of the supernatant was collected, dried with nitrogen, resuspended in methanol / ACN / H2O (v:v:v=85:10:5), and directly injected for LC-MS / MS analysis.

[0059] [Figure 17A-17C]Figures 17A–17C are a series of graphs demonstrating that GCaseM6P has a longer half-life and greater tissue uptake compared to imiglucerase in the GBAD409V / null mouse model. PK / PD studies in the Gaucher D409V / null mouse model were performed using the standard therapeutic agent, imiglucerase, and purified GBA produced by transient co-expression of S1-S3 PTase and a native variant of GBA in Expi293 cells using a bicistronic vector. This GCase variant exhibits greater stability under neutral and slightly alkaline conditions. Briefly, three animals were injected via tail vein at approximately 1.5 mg / kg of recombinant GCase. For serum pharmacokinetic data, plasma samples were collected at 2, 10, 20, 40, and 60 minutes. Activity was measured using the synthetic substrate 4-methylumbelliferyl-beta-D-glucopyranoside (4MU-Glc). Activity in individual animals was normalized by setting the 2-minute time point as 100% activity and expressing subsequent time points as percentages relative to t=2 minutes. Stabilized GCase expressed in the presence of S1-S3 PTase appears to have a longer half-life. This longer half-life is a combination of the enzyme's greater stability and a different clearance pathway. To determine how much GCase was taken up by tissue, tissue was collected, homogenized, and activity measured using 4MU-Glc substrate 2 hours after enzyme injection. Activity was normalized to total protein in the homogenate determined by BCA for protein determination. The true benefit of stable GCase with reasonable phosphorylation is evident in the shown tissue uptake data. Greater activity is found for stabilized GCase expressed using the bicistronic S1-S3 PTase vector platform S1'S3 PTase in all tissues evaluated. This is most dramatic in the lung, muscle, and brain, where imiglucerase activity is minimal.Combining tissue and serum data reveals the advantage of more stable GCases with greater N-linked oligosaccharide phosphorylation in terms of delivering more enzyme to affected tissue. This is the first time that significant amounts of GCase have been delivered to the lungs, muscles, and heart at these doses.

[0060] [Figures 18A-18C] Figures 18A–18E are a series of photographs and bar graphs demonstrating that GCaseM6PERT better reduced tissue macrophages than imiglucerase (anti-CD68 staining) in the GBAD409V / null mouse model. Efficacy studies in the D409V Gaucher mouse model were performed using Cerezyme, a standard therapeutic agent, and purified GBA (M0111) transiently co-expressed in Expi293 cells using a bicistronic vector encoding S1S3 PTase and a native variant of GBA reported to be more stable under neutral and slightly alkaline conditions. Approximately 20-week-old Gaucher mice were treated with approximately 1.5 mg / kg of the enzyme once a week for 4 weeks. After 4 weeks, liver and lung tissues were collected for immunohistochemical examination using CD68 antibody and fixed in 4% paraformaldehyde-PBS, pH 7.4. As evidenced by the reduction in macrophages in the affected tissue visualized by CD68 Ab, M0111 has significantly greater efficacy compared to current standard therapies. [Figures 18D-18E]Figures 18A–18E are a series of photographs and bar graphs demonstrating that GCaseM6PERT better reduced tissue macrophages than imiglucerase (anti-CD68 staining) in the GBAD409V / null mouse model. Efficacy studies in the D409V Gaucher mouse model were performed using Cerezyme, a standard therapeutic agent, and purified GBA (M0111) transiently co-expressed in Expi293 cells using a bicistronic vector encoding S1S3 PTase and a native variant of GBA reported to be more stable under neutral and slightly alkaline conditions. Approximately 20-week-old Gaucher mice were treated with approximately 1.5 mg / kg of the enzyme once a week for 4 weeks. After 4 weeks, liver and lung tissues were collected for immunohistochemical examination using CD68 antibody and fixed in 4% paraformaldehyde-PBS, pH 7.4. As evidenced by the reduction in macrophages in the affected tissue visualized by CD68 Ab, M0111 has significantly greater efficacy compared to current standard therapies.

[0061] [Figures 19A-19C]Figures 19A–19C are a series of photographs demonstrating that GCaseM6PERT better reduced the number and size of Gaucher storage cells than imiglucerase in the GBAD409V / null mouse model (hematoxylin-eosin (H&E) staining). Efficacy studies in the D409A Gaucher mouse model were performed using Cerezyme, the standard therapeutic agent, and purified GBA transiently co-expressed in Expi293 cells using a bicistronic vector encoding S1-S3 PTase and a native variant of GBA that has been reported to be more stable under neutral and slightly alkaline conditions. Approximately 20-week-old Gaucher mice were treated with approximately 1.5 mg / kg of the enzyme once a week for 4 weeks. After 4 weeks, liver and lung tissues were collected and fixed in 4% paraformaldehyde-PBS, pH 7.4 for formalin treatment for hematoxylin-eosin (H&E) staining. As evidenced by the reduction in storage cells in the affected tissue visualized by H&E staining, GCaseM6P exhibits significant efficacy compared to current standard therapies.

[0062] [Figures 20A-20B]Figures 20A and 20B are pairs of graphs demonstrating that GCaseM6PERT reduced the accumulated substrate more effectively than imiglucerase in the GBAD409V / null mouse model. Gaucher mice approximately 20 weeks old were treated with approximately 1.5 mg / kg of enzyme once a week for 4 weeks. Tissue samples were collected and homogenized for glycosylceramide analysis. The accumulation of glucocerebroside, a natural substrate of GCase, was determined in tissue homogenates. The accumulation of GC in the lungs is a known unaddressed need for current standard therapeutics and is of great value. From 20 μL of a constant volume of tissue homogenate and a suitable control, glucosylceramide was extracted by adding 200 μL of methanol / ACN / H2O (v:v:v=85:10:5), mixing at 800 rpm for 5 minutes, and then centrifugation at 3220 g at 4°C for 15 minutes;3). 50 μL of the supernatant was collected, dried with nitrogen, resuspended in methanol / ACN / H2O (v:v:v=85:10:5), and directly injected for LC-MS / MS analysis. Regarding the two ceramides measured, animals treated with GCaseM6P had lower levels than imiglucerase after ERT therapy.

[0063] [Figures 21A-21D] Figures 21A–21D are a series of graphs showing the results of in vivo AAV-mediated gene therapy trials for treating Gaucher disease. To determine the efficacy of AAV9 gene therapy using a bicistronic transgene of stable GBA+S1-S3 PTase with three different promoters, 15-week-old GBAD409V / null mice were administered a moderate dose of AAV9-stable GBA+S1-S3 PTase, 5E11vg. To determine how much GBA was produced by the tissue, tissue was collected, homogenized, and its activity was measured using a 4MU-Glc substrate two weeks after AAV9 injection. The activity was normalized to the total protein in the homogenate, which was determined by the BCA method for protein determination.

[0064] [Figures 22A-22C]Figures 22A–22C are a series of graphs showing the results of in vitro studies on the use of lysosomal alpha-mannosidase (LAMAN) as an ERT (Endoscopic Transdermal Retention Therapy).

[0065] [Figures 23A-23B] Figures 23A-23B show photographs and corresponding data tables illustrating the expression, purification, and characterization of the LAMAN enzyme. Two preparations of LAMAN were transiently co-expressed in Expi293 cells, with or without a bicistronic vector encoding S1-S3 PTase (M0611). Both were purified using an HPC4 affinity tag. A significant increase in phosphorylation was demonstrated by measuring the amount of LAMAN dose-dependently bound to an immobilized cation-independent mannose 6-phosphate receptor. The amount of bound LAMAN was based on the activity of LAMAN using 4-methylumbelliferyl-α-D-mannopyranoside (4MU-Man), a LAMAN synthesis substrate. The specificity of binding via phosphorylated oligosaccharides was confirmed by the ability to block the binding of added mannose 6-phosphate. It should be noted that LAMANM6P (M0611) can bind to the receptor even in the presence of M6P. LAMANM6P (M0611, P-0030) and LAMAN (P-0031) were selected for in vivo animal testing.

[0066] [Figure 23C]Figure 23C is a graph showing the expression, purification, and characterization of the LAMANM6P (M0611) enzyme. Two preparations of LAMAN were transiently co-expressed in Expi293 cells with or without a bicistronic vector encoding S1-S3 variants of PTase. Both were purified using an HPC4 tag. A significant increase in phosphorylation was demonstrated by measuring the amount of LAMAN dose-dependently bound to an immobilized cation-independent mannose 6-phosphate receptor. The amount of bound LAMAN was determined by activity using the synthetic substrate 4-methylumbelliferyl-α-D-mannopyranoside (4MU-Man). The specificity of binding via phosphorylated oligosaccharides was confirmed by the ability to block the binding of added mannose 6-phosphate. It should be noted that M0611 can bind to the receptor even in the presence of M6P. LAMANM6P (M0611, P-0030) and LAMAN (P-0031) were selected for in vivo animal studies.

[0067] [Figures 24A-24B]Figures 24A–24B are pairs of graphs demonstrating the in vivo distribution of LAMAN and LAMANM6P enzymes in wild-type mice in relation to enzyme replacement therapy. To evaluate the difference in tissue uptake between LAMAN and LAMANM6P (LAMAN co-expressed with S1-S3 PTase), each preparation was injected via tail vein into wild-type mice (n=4) at 2 mg / kg. Tissues were collected, homogenized, and activity measured using 4MU-Man substrate at 2 and 8 hours after administration. Activity was normalized to total protein in the homogenate determined by BCA for protein determination. The advantage of LAMANM6P (LAMAN co-expressed with S1-S3 PTase) was observed in the tissue uptake data. In the liver, spleen, heart, lung, and brain, tissue activity was higher at 2 hours. This trend remained true at 8 hours, with the exception of the lung. This may be a result of the high variability observed in the analysis of this tissue. The only exception to the finding of higher activity was the kidney. Endogenous LAMAN activity was subtracted from all samples. Higher LAMAN enzyme activity was detected in most tissues of mice injected with our LAMANM6P enzyme.

[0068] [Figures 25A-25B]Figures 25A-25B are a pair of graphs demonstrating the in vivo distribution of αLAMAN and LAMANM6P enzymes in wild-type mice in relation to enzyme replacement therapy. To evaluate the difference in tissue uptake between LAMAN and LAMANM6P (LAMAN co-expressed with S1-S3 PTase), each preparation was injected via tail vein into wild-type mice (n=4) at 10 mg / kg. Tissues were collected, homogenized, and activity measured using 4MU-Man substrate at 2 and 8 hours after administration. Activity was normalized to total protein in the homogenate determined by BCA for protein determination. The advantage of LAMANM6P (LAMAN co-expressed with S1-S3 PTase) was observed in the tissue uptake data. In the liver, spleen, heart, lungs, and brain, tissue activity was higher at 2 hours. This trend remained true at 8 hours, with the exception of the kidney. This may be a result of the high variability observed in the analysis of this tissue.

[0069] [Figures 26A-26B] Figures 26A-26B are schematic diagrams and graphs showing the design and in vitro testing of AAV9 gene therapy (GTx) for mucolipidosis. 293T cells were transduced using various M0021 (AAV9-CAGp-S1-S3) viruses, cultured for 2 days, and then subjected to a PTase activity assay.

[0070] [Figures 27A-27B]Figures 27A and 27B are a pair of graphs demonstrating that M0021 treatment reduces serum lysosomal enzyme levels in ML II mice. To determine the efficacy of S1-S3 PTase gene therapy, 34-week-old female mice were administered a moderate dose of M0021 (AAV9-CAGp-S1-S3), 4e12vg (2e13vg / kg). One of the ML II phenotypes is elevated serum levels of lysosomal enzymes due to the inability to target lysosomal enzymes to intracellular lysosomes. Promising results were observed when serum LAMAN and ManB activity decreased exactly one week after treatment. This result is significant as it demonstrates that the ML II mouse model can produce the described phenotype.

[0071] [Figures 28A-28C] Figures 28A–28C are a series of graphs demonstrating that M0021 treatment increases lysosomal enzyme phosphorylation in ML II mice. To further understand the effect of S1-S3 PTase gene therapy on the decrease in serum activity of LAMAN and ManB, we evaluated the binding of enzymes found in serum to CI-MPR using a previously described immobilized receptor binding assay. Briefly, a known amount of enzyme is added to immobilized CI-MPR in increments. Unbound enzymes are washed away, and the remaining bound enzymes are measured using a suitable synthetic substrate; Man-b-4MU(ManB, LAMAN 4MU-Man(LAMAN)). AAV9-S1S3 gene therapy increases glycan phosphorylation of lysosomal enzymes in ML II mice. Total phosphorylated lysosomal enzymes in serum normalized to normal or slightly elevated levels after 3 weeks.

[0072] [Figures 29A-29C]Figures 29A–29C are a series of graphs showing enzyme activity and GCase substrate selection in the lungs and liver two weeks after injection of AAV9-hTLV-GBAM6P gene therapy in Gaucher mice. AAV9-hTLV-GBA-S1S3 is also known as AAV9-hTLV-GBAM6P, where M6P refers to the S1S3 construct. Two weeks after AAV9 hTLV-GBA or AAV9 hTLV-GBAM6P (transgene with a bicistronic vector containing GBA and S1-S3 PTase), the expression of both constructs was elevated in the liver (Figure 29A). When hepatic glucosyl-β-ceramide levels were measured (Figures 29B, C), animals treated with AAV9 hTLV-GBAM6P showed the greatest reduction in accumulated substrates compared to animals treated with AAV9 hTLV-GBA, despite lower hepatic GCase activity. This greater substrate reduction at low activity suggests that N-linked oligosaccharide phosphorylation is important for gene therapy in terms of cellular uptake and lysosome targeting. In the lungs, GCase activity was low in animals treated with AAV9. However, animals treated with AAV9-hTLV-GBAM6P showed a significant reduction in accumulated glucosyl-β-ceramide levels in the lungs (Figure 29B, C). A slight reduction was observed in animals treated with AAV9-hTLV-GBA. This demonstrates that phosphorylated transgene products with high affinity for CI-MPR can lead to effective therapy, even at low activity levels, due to efficient cellular uptake and lysosome targeting. [Modes for carrying out the invention]

[0073] Detailed explanation Lysosomal storage disorders (LSDs) are hereditary metabolic disorders resulting from defects in lysosome function. Currently, approximately 50 distinct LSDs have been identified, but only a few (less than 10) have been reported to be treated. Patients are currently treated with intravenous enzyme replacement therapy (ERT), which addresses the disease symptoms by supplementing the deficient enzyme in the patient. The goal of ERT is to introduce sufficient amounts of normal enzyme into the lysosomes of deficient cells to clear storage material and restore lysosomal function. To ensure efficient uptake of ERT into affected lysosomes, it is essential that the ERT contains high levels of mannose 6-phosphate (M6P). Ideally, patients with LSD should be treated by administering a deficient enzyme with high-saturation levels of M6P to enable effective delivery to lysosomes. However, this process is extremely difficult because the phosphorylation process that enables the addition of M6P to lysosomes is inherently inefficient. Recent discoveries of S1-S3 variants of GlcNAc-1PTase significantly improve the phosphorylation process of lysosomal enzymes. Furthermore, there is a need for gene therapy techniques that can provide long-term cures for LSD in patients.

[0074] This disclosure provides expression vectors, compositions, and methods for generating lysosomal enzymes operably ligated to S1-S3 variants of GlcNAc-1-phosphotransferase. The S1-S3 variants of GlcNAc-1-phosphotransferase significantly increase the intracellular and blood serum or renal transport of the operably ligated lysosomal enzyme for increased uptake, distribution, and lysosomal enzyme activity.

[0075] This disclosure provides gene therapy vectors, compositions, and methods for generating lysosomal enzymes operably linked to S1-S3 variants of GlcNAc-1-phosphotransferase. This disclosure demonstrates that expression of the S1-S3 variants increases the uptake, distribution, and activity of endogenous lysosomal enzymes.

[0076] This disclosure provides an ERT, vector, composition, and method for generating a lysosomal enzyme with a reasonable phosphorylated N-linked oligosaccharide by co-expression with S1-S3 PTase via a novel bicistronic vector. Bicistronic expression of S1-S3 PTase and the lysosomal enzyme significantly increases the M6P content of the expressed lysosomal enzyme. The well-phosphorylated enzyme enables efficient uptake and delivery to the lysosome. This allows for better tissue distribution, cellular uptake, lysosome targeting, and substrate reduction. This disclosure provides a gene therapy vector, composition, and method for generating a high-level expression or high-level activity M6P lysosomal enzyme by co-expression with S1-S3 PTase. Bicistronic expression of the S1-S3 variant of PTase significantly increases the M6P content level of the lysosomal enzyme. High M6P on the surface of lysosomal enzymes allows for delivery of the enzymes to tissue cells, increasing their uptake, distribution, and efficacy in vitro and in vivo.

[0077] The vectors, compositions, and methods of this disclosure can be used in enzyme replacement therapy (ERT).

[0078] Alternatively, or in addition to the above, the vectors, compositions, and methods of this disclosure may be used for gene therapy.

[0079] Numerous lysosomal enzymes have been described, and their use in both ERT and gene therapy has been demonstrated. Importantly, the vectors, compositions, and methods of this disclosure can be used with any lysosomal enzyme to increase the cellular uptake of the lysosomal enzyme and, therefore, to increase the activity of the lysosomal enzyme in one or more body tissues.

[0080] In some embodiments, compositions and methods of the present disclosure, comprising S1-S3 PTase operably linked to lysosomal proteins, increase the uptake and activity of lysosomal proteins in one or more of the spleen, brain, one or more lungs, or one or more muscles of a target.

[0081] In some embodiments, the vectors, compositions, and methods of the Disclosure comprising S1-S3 GlcNAc-1 phosphotransferase, including embodiments in which the bicistronic vector comprises a sequence encoding S1-S3 GlcNAc-1 phosphotransferase and a sequence encoding a lysosomal protein, enhance the uptake and activity of the encoded lysosomal protein in one or more of the target spleen, brain, one or more lungs, or one or more muscles. Exemplary Embodiments

[0082] This disclosure provides a composition comprising a vector containing a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase).

[0083] This disclosure provides a composition comprising a bicistronic vector containing a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase).

[0084] In some embodiments of the compositions of this disclosure, the bicistronic vector includes an intrasequence ribosome entry site (IRES) located before the polynucleotide encoding the modified GlcNAc-1 PTase and after the polynucleotide encoding the lysosomal enzyme.

[0085] In some embodiments of the compositions of this disclosure, the bicistronic vector includes a promoter. In some embodiments, the bicistronic vector includes a constitutive promoter. In some embodiments, the constitutive promoter includes a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is operably ligated to a polynucleotide encoding a lysosomal enzyme or a polynucleotide encoding a modified GlcNAc-1 PTase. In some embodiments, the promoter is operably ligated to a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 PTase.

[0086] In some embodiments of the compositions of this disclosure, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 1.

[0087] In some embodiments of the compositions disclosed herein, the polynucleotide encoding the modified GlcNAc-1 phosphotransferase comprises the nucleic acid sequence of SEQ ID NO: 4.

[0088] In some embodiments of the compositions of this disclosure, the encoded lysosomal enzyme is involved in at least one lysosomal storage disorder (LSD) listed in Table 1. In some embodiments, the encoded lysosomal enzyme or a variant thereof causes at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C. In some embodiments, the activity or function of the encoded lysosomal enzyme or a variant thereof is reduced, inhibited, or deregulated in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C.

[0089] In some embodiments of the compositions of this disclosure, the lysosomal enzyme comprises the lysosomal enzymes listed in Table 1A, Table 1B, or Table 1C. In some embodiments, the lysosomal enzymes listed in Table 1A, Table 1B, or Table 1C comprises at least one lysosomal enzyme. In some embodiments, the lysosomal enzyme comprises one or more lysosomal enzymes listed in Table 1A, Table 1B, or Table 1C. In some embodiments, the lysosomal enzyme is selected from the group consisting of β-glucocerebrosidase (GCase, GBA), galactosylceramidase (GALC), α-galactosidase (GLA), α-N-acetylglucosaminidase (NAGLU), acid α-glucosidase (GAA), and lysosomal acid α-mannosidase (LAMAN). In some embodiments, the lysosomal enzyme comprises β-glucocerebrosidase (GCase, GBA). In some embodiments, the lysosomal enzyme comprises galactosylceramidase (GALC). In some embodiments, the lysosomal enzyme comprises α-galactosidase (GLA). In some embodiments, the lysosomal enzyme comprises α-N-acetylglucosaminidase (NAGLU). In some embodiments, the lysosomal enzyme comprises acidic α-glucosidase (GAA). In some embodiments, the lysosomal enzyme comprises lysosomal acidic α-mannosidase (LAMAN). In some embodiments, the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequences of SEQ ID NOs. 5 to 10.

[0090] This disclosure provides a composition comprising a bicistronic vector containing a polynucleotide encoding a constitutive promoter, an intrasequence ribosome entry site (IRES), and a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase).

[0091] In some embodiments of the compositions of this disclosure, the compositions further comprise a pharmaceutically acceptable carrier.

[0092] In some embodiments of the vectors of this disclosure, the vector is a viral vector. In some embodiments, the viral vector is an adenovirus, an adeno-associated virus (AAV), a retrovirus, or a lentivirus. In some embodiments, the viral vector includes an adenovirus. In some embodiments, the viral vector includes an AAV vector. In some embodiments, the AAV vector includes a sequence isolated from or derived from one or more AAVs of serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAV vector includes a sequence isolated from or derived from an AAV of serotype 1 (AAV1). In some embodiments, the AAV vector includes a sequence isolated from or derived from an AAV of serotype 2 (AAV2). In some embodiments, the AAV vector includes a sequence isolated from or derived from an AAV of serotype 3 (AAV3). In some embodiments, the AAV vector includes a sequence isolated from or derived from an AAV of serotype 4 (AAV4). In some embodiments, the AAV vector includes a sequence isolated from or derived from a serotype 5 (AAV5) AAV. In some embodiments, the AAV vector includes a sequence isolated from or derived from a serotype 6 (AAV6) AAV. In some embodiments, the AAV vector includes a sequence isolated from or derived from a serotype 7 (AAV7) AAV. In some embodiments, the AAV vector includes a sequence isolated from or derived from a serotype 8 (AAV8) AAV. In some embodiments, the AAV vector includes a sequence isolated from or derived from a serotype 9 (AAV9) AAV.

[0093] In some embodiments of the vectors of this disclosure, the vector is an expression vector. In some embodiments, the expression vector includes the polynucleotide sequence of SEQ ID NO: 1.

[0094] This disclosure provides cells comprising the vector of this disclosure. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are primate cells. In some embodiments, the cells are human cells. In some embodiments, the cells are cultured cells. In some embodiments, the cells are immortalized or stabilized cell lines. In some embodiments, the cells are Chinese hamster ovary (CHO) cells. In some embodiments, the cells are human fetal kidney 293 (HEK293) cells.

[0095] This disclosure provides cells comprising the bicistronic vector of this disclosure. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are primate cells. In some embodiments, the cells are human cells. In some embodiments, the cells are cultured cells. In some embodiments, the cells are immortalized or stabilized cell lines. In some embodiments, the cells are Chinese hamster ovary (CHO) cells. In some embodiments, the cells are human fetal kidney 293 (HEK293) cells.

[0096] This disclosure provides cells comprising the compositions of this disclosure. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are primate cells. In some embodiments, the cells are human cells. In some embodiments, the cells are cultured cells. In some embodiments, the cells are immortalized or stabilized cell lines. In some embodiments, the cells are Chinese hamster ovary (CHO) cells. In some embodiments, the cells are human fetal kidney 293 (HEK293) cells.

[0097] This disclosure provides a pharmaceutical composition comprising a lysosomal enzyme expressed by the vector of this disclosure and a pharmaceutically acceptable carrier.

[0098] This disclosure provides a method for treating lysosomal storage disorders (LSDs), comprising the step of administering a composition of the disclosure to a subject, thereby treating the LSD.

[0099] This disclosure provides a method for treating lysosomal storage disorders (LSDs), comprising the step of administering a therapeutically effective dose of the composition of this disclosure to a subject, wherein the composition increases the phosphorylation of lysosomal enzymes, thereby treating the LSD.

[0100] This disclosure provides a method for treating a subject suffering from a lysosomal storage disorder (LSD), comprising the step of administering the subject a pharmaceutical composition of this disclosure, thereby increasing the phosphorylation of lysosomal enzymes and treating the subject.

[0101] This disclosure provides a method for preventing the development of lysosomal storage disorders (LSDs) in subjects requiring such treatment, comprising the step of administering the pharmaceutical composition of this disclosure to a subject, thereby increasing the phosphorylation of lysosomal enzymes and preventing the development of LSDs in the subject.

[0102] This disclosure provides a method for improving the phosphorylation of lysosomal enzymes that cause lysosomal storage disorders (LSDs) in subjects requiring such improvement, comprising the step of administering a composition of this disclosure to a subject, wherein the composition increases the phosphorylation of lysosomal enzymes.

[0103] In some embodiments of the methods of this disclosure, lysosomal enzymes are involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C.

[0104] In some embodiments of the methods of this disclosure, the lysosomal enzyme comprises a lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C. In some embodiments, the lysosomal enzyme comprises at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C. In some embodiments, the lysosomal enzyme comprises one or more lysosomal storage disorders (LSDs) listed in Table 1A, Table 1B, or Table 1C. Enzyme replacement therapy (ERT)

[0105] The present invention provides a composition comprising a bicistronic expression vector containing a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). In some embodiments, the disclosed bicistronic expression vector includes an intrasequence ribosome entry site (IRES) located before the polynucleotide encoding the modified GlcNAc-1 PTase and after the polynucleotide encoding the lysosomal enzyme. In other embodiments, the disclosed bicistronic expression vector includes an IRES located after the polynucleotide encoding the modified GlcNAc-1 PTase and before the polynucleotide encoding the lysosomal enzyme.

[0106] Mammalian cells containing the disclosed bicistronic expression vector are provided in the present invention.

[0107] The present invention provides a pharmaceutical composition comprising a lysosomal enzyme expressed by a bicistronic vector disclosed herein and a pharmaceutically acceptable carrier.

[0108] The present invention provides a method for treating subjects suffering from lysosomal storage disorders (LSDs) and a method for preventing the onset of lysosomal storage disorders (LSDs) in subjects requiring such treatment. gene therapy

[0109] The present invention provides a composition comprising a bicistronic viral vector containing a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). In some embodiments, the disclosed bicistronic viral vector includes an intrasequence ribosome entry site (IRES) located before the polynucleotide encoding the modified GlcNAc-1 PTase and after the polynucleotide encoding the lysosomal enzyme. In other embodiments, the disclosed bicistronic viral vector includes an IRES located after the polynucleotide encoding the modified GlcNAc-1 PTase and before the polynucleotide encoding the lysosomal enzyme. In some embodiments, the viral vector is an adenovirus, adeno-associated virus (AAV), retrovirus, or lentivirus.

[0110] The present invention provides a method for treating subjects suffering from lysosomal storage disorders (LSDs) and a method for preventing the development of lysosomal storage disorders (LSDs) in subjects requiring such treatment, by administering a disclosed bicistronic viral vector to the subject.

[0111] The present invention further provides a method for improving the phosphorylation of lysosomal enzymes that cause LSD in subjects requiring it.

[0112] The present invention provides compositions and methods using bicistronic vectors for treating or preventing lysosomal storage disorders (LSDs) in subjects.

[0113] This disclosure provides a composition comprising a bicistronic vector containing a promoter, an intrasequence ribosome entry site (IRES), a polynucleotide encoding a lysosomal enzyme, and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). The method of this disclosure comprises the step of administering a pharmaceutical composition comprising the bicistronic vector disclosed herein to a subject.

[0114] definition Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art relating to this invention. Any methods and materials similar to or equivalent to those described herein may be used in carrying out the tests of the present invention, but preferred materials and methods are described herein. The following terms are used in the description and claims of this invention:

[0115] It should be understood that the terminology used herein is merely for the purpose of describing certain embodiments and is not limited thereto.

[0116] As used herein, the articles “a” and “an” are used to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means one element or more elements.

[0117] As used herein, when referring to measurable values ​​such as quantity or duration, the term “about” means to include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, and such variation is therefore reasonable for carrying out the disclosed method.

[0118] The terms "2A," "2A peptide," or "2A-like peptide" refer to self-processing viral peptides. A 2A peptide can be a distinct protein-coding sequence within a single ORF transcription unit (Ryan et al., 1991, J Gen Virol 72: 2727-2732). While referred to as a "self-cleaving" peptide or protease site, the mechanism by which a 2A sequence generates two proteins from a single transcript occurs via ribosome skipping, where normal peptide bonds are disrupted at 2A, resulting in two discontinuous protein fragments from a single translation event. Linking with the 2A peptide sequence leads to the cellular expression of numerous distinct proteins (essentially equimolar amounts) derived from a single ORF (de Felipe et al., 2006, Trends Biotechnol 24: 68-75).

[0119] The terms “biological” or “biological specimen” refer to a specimen obtained from a living organism or from a component of a living organism (e.g., cells). A specimen may be a specimen of any biological tissue or biological fluid. More frequently, specimens are “clinical specimens” that are patient-derived specimens. Such specimens include, but are not limited to, bone marrow, cardiac tissue, sputum, blood, lymph, blood cells (e.g., white blood cells), tissue or fine-needle biopsy specimens, urine, peritoneal fluid, and pleural fluid, or cells derived therefrom. Biological specimens may also include tissue sections, such as frozen sections obtained for histological purposes.

[0120] As used herein, the term “derivative” indicates that a derivative of a virus may have differences in nucleic acid or amino acid sequence with respect to the template viral nucleic acid or amino acid sequence.

[0121] A "disease" is a state of animal health in which the animal is unable to maintain homeostasis, and if the disease does not improve, the animal's health continues to deteriorate.

[0122] In contrast, a "disorder" in animals is a state of health in which the animal can maintain homeostasis, but its health is less favorable than when the disorder is absent. If left untreated, a disorder does not necessarily lead to a further deterioration of the animal's health.

[0123] An "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by host cells or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or liposome-containing) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), into which the recombinant polynucleotide is incorporated. In some embodiments, the disclosed vectors are referred to herein as viral vectors. In some embodiments, the disclosed vectors are referred to herein as expression vectors.

[0124] As used herein, “higher” means that the expression level is at least 10% or greater than the control reference, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or greater, and / or 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2.0 times higher or greater, as well as any total or partial change between these. An expression level higher than the reference value disclosed herein means an expression level (mRNA or protein) that is higher than the normal or control level derived from expression (mRNA or protein) measured in a healthy subject or defined or used in the art.

[0125] As used herein, “lower” means an expression level that is at least 10% or less lower than a control reference, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or less lower, and / or 1 / 1.1, 1 / 1.2, 1 / 1.4, 1 / 1.6, 1 / 1.8, 1 / 2.0 or less lower, as well as any amount between these and any whole or partial change. An expression level lower than the reference values ​​disclosed herein means an expression level (mRNA or protein) that is lower than normal or control levels derived from expression (mRNA or protein) measured in a healthy subject or defined or used in the art.

[0126] As used herein, the terms “comparison” and “reference” may be used interchangeably and refer to the value used as the standard of comparison.

[0127] As used herein, “combination therapy” means administering a first agent in combination with another agent. “In combination with” or “in combination with” means administering one treatment modality in addition to one treatment modality. Thus, “in combination with” means administering one treatment modality to an individual before, during, or after the delivery of another treatment modality. Such combination is considered part of a single treatment regimen or regime. For example, a vector or a composition containing a vector of the Disclosure may be delivered or administered to a subject in combination with a second therapeutic agent. In some embodiments, the vector and composition of the Disclosure are delivered or administered to the subject simultaneously with or sequentially with the second therapeutic agent. In some embodiments, the vector and composition of the Disclosure are delivered or administered to the subject simultaneously with the second therapeutic agent. In some embodiments, the vector and composition of the Disclosure are delivered or administered to the subject sequentially with the second therapeutic agent. In some embodiments, the vector and composition of the Disclosure are delivered or administered to the subject before the administration of the second therapeutic agent. In some embodiments, the vector and composition of the Disclosure are delivered or administered to the subject after the administration of the second therapeutic agent. In some embodiments, the second therapeutic agent comprises a composition of the second vector of the Disclosure. In some embodiments, the second therapeutic agent comprises the vector or composition of the Disclosure encoding a variant form of the lysosomal enzyme of the Disclosure. In some embodiments, the second therapeutic agent comprises one or more agents for alleviating signs or symptoms of lysosomal storage disease. In some embodiments, the second therapeutic agent comprises one or more anti-inflammatory or immunosuppressant agents.

[0128] As used herein, the term "operably linked" means that the expression of a nucleic acid sequence is under the control of a promoter that is spatially connected to the nucleic acid sequence. The promoter may be positioned 5' (upstream) of the nucleic acid sequence under its control.

[0129] As used herein, “primary cells” refers to cells obtained directly from living tissue (i.e., biopsy material) and whose growth has been established in vitro, having undergone very little population doubling, and therefore more clearly representing the major functional components and characteristics of the tissue from which they originate compared to continuous tumorigenic or artificially immortalized cell lines.

[0130] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein composed of two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and longer chains, also commonly called proteins in the art, of which many types exist. Examples of “polypeptides” include, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0131] As used herein, the term “promoter” may mean a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of a nucleic acid. As used herein, a promoter is defined as a DNA sequence recognized by a cellular synthetic mechanism, or introduced synthetic mechanism, which is required to initiate the specific transcription of the polynucleotide sequence.

[0132] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence required for the expression of a gene product operably ligated to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, it may also include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses a gene product in a tissue-specific manner.

[0133] A "constitutive" promoter is a nucleotide sequence that, when operably ligated to a polynucleotide encoding or designating a gene product, causes the cell to produce that gene product under most or all physiological conditions.

[0134] An "inducible" promoter is a nucleotide sequence that, when operably ligated to a polynucleotide encoding or designating a gene product, causes the cell to produce the gene product only if the corresponding inducer is present within the cell.

[0135] As used herein, the term "RNA" is defined as ribonucleic acid.

[0136] When used in connection with the present invention, the term “treatment” encompasses therapeutic treatments, as well as preventive or suppressive measures, for a disease or disorder. As used herein, the term “treatment” and related terms such as “to treat” and “to treat” mean a reduction in the exacerbation, severity, and / or duration of a disease condition or at least one of its symptoms. Therefore, the term “treatment” refers to any regimen that may benefit the subject. Treatment may relate to an existing condition or be preventive (preventive treatment). Treatment may include curative, mitigating, or preventive effects. References to “therapeutic” and “preventive” treatments herein should be understood in their broadest context. The term “therapeutic” does not necessarily mean that the subject was treated until complete recovery. Similarly, “preventive” does not necessarily mean that the subject will never ultimately contract the disease condition. Therefore, for example, the term “treatment” includes administering a drug before or after the onset of a disease or disorder to prevent or eliminate all signs of the disease or disorder. As another example, administering medication after the clinical manifestation of a disease in order to combat its symptoms constitutes "treatment" of the disease.

[0137] As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). It should also be understood that the term includes analogues of either RNA or DNA derived from nucleotide analogues, and, where applicable to the embodiments described, single-stranded (sense or antisense) and double-stranded polynucleotides as equivalents. ESTs, chromosomes, cDNA, mRNA, and rRNA are typical examples of molecules that may be referred to as nucleic acids.

[0138] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the scope of the present invention and other chemical components such as carriers, stabilizers, diluents, adjuvants, dispersants, suspending agents, thickeners, and / or excipients. Pharmaceutical compositions facilitate the administration of compounds to living organisms. Numerous techniques exist in the art for administering compounds, including, but not limited to, intratumoral, intravenous, intrapleural, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0139] The term “pharmaceutically acceptable carrier” includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in transporting or carrying the compound(s) of the present invention into or to a subject so that it may perform its intended function. Generally, such compounds are transported or carried from one organ or part of the body to another organ or part of the body. Each salt or carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, e.g., cocoa butter and suppository wax; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol Examples include: recalls, etc.; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; diluents; granulators; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic, suitable substances used in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” includes any coatings, antibacterial and antifungal agents, as well as absorption retarders, etc., that are suitable for the activity of the compound and physiologically acceptable to the subject. Supplemental active compounds can also be incorporated into the composition.

[0140] As used herein, the terms “effective dose” or “therapeutic dose” mean the amount of viral particles or infectious units produced by the vector of the present invention that is necessary to prevent a particular disease condition, or to reduce the severity of a disease condition or at least one symptom or related condition, and / or to improve a disease condition or at least one symptom or related condition.

[0141] "Subject" or "patient," as used herein, may be human or non-human mammal. Examples of non-human mammals include livestock and pets, such as sheep, cattle, pigs, dogs, cats, and mice. The subject is preferably human.

[0142] Scope: Throughout this disclosure, some embodiments may be described in range form. It should be understood that range descriptions are for convenience and brevity only and should not be interpreted as inflexible limitations on the scope of this disclosure. Therefore, range descriptions should be considered to have all possible subranges specifically disclosed and the individual values ​​that fall within those ranges. For example, a range description such as 1 to 6 should be considered to have all specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers that fall within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0143] composition The present invention provides compositions and methods for treating or preventing lysosomal storage disorders (LSDs) in subjects by administering a pharmaceutical product containing a bicistronic expression vector to the subjects.

[0144] In some embodiments, the disclosure provides a composition comprising a bicistronic vector containing a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). In one embodiment, the polynucleotide encoding the lysosomal enzyme and the polynucleotide encoding the modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase) are operably linked.

[0145] In some embodiments, the disclosure provides compositions comprising a bicistronic vector containing a constitutive promoter, an intra-sequence ribosome entry site (IRES), and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase).

[0146] In some embodiments, the bicistronic vector includes an IRES located before the polynucleotide encoding the modified GlcNAc-1 PTase and after the polynucleotide encoding the lysosomal enzyme. In other embodiments, the bicistronic vector includes an IRES located after the polynucleotide encoding the modified GlcNAc-1 PTase and before the polynucleotide encoding the lysosomal enzyme.

[0147] The sequence of IRES may be a sequence known in the art or a variant thereof. The IRES variant may be modified or mutated. In one embodiment, the sequence of IRES includes sequence number 3. In other embodiments, the sequence of IRES is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to sequence number 3.

[0148] In one embodiment, the polynucleotide of a lysosomal enzyme is operably ligated to a 2A DNA encoding a 2A peptide, which in turn is operably ligated to the polynucleotide of a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). Various 2A peptides known in the art, including but not limited to T2A, P2A, E2A, and F2A, can be used in the disclosed bicistronic vector. In some embodiments, a GSG residue may be added to the 5' end of the peptide to improve cleavage efficiency.

[0149] In some embodiments, the bicistronic viral vector includes a promoter operably ligated to a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 PTase.

[0150] In some embodiments, the bicistronic expression vector includes a promoter.

[0151] The promoter may be a constitutive promoter, an inducible / repressible promoter, or a cell type-specific promoter. In certain embodiments, the promoter may be a constitutive promoter. Non-limiting examples of constitutive promoters for mammalian cells include CMV, UBC, EF1a, SV40, PGK, CAG, CBA / CAGGS / ACTB, CBh, MeCP2, U6, and H1. In some embodiments, the bicistronic vectors of this disclosure include a constitutive promoter. In some embodiments, the constitutive promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the polynucleotide of the CMV promoter includes the nucleic acid sequence of SEQ ID NO: 2.

[0152] In other embodiments, the promoter may be an inductive promoter. The inductive promoter can be selected from the group consisting of tetracycline, heat shock, steroid hormones, heavy metals, phorbol esters, adenovirus E1A element, interferon, and serum inductive promoters.

[0153] In different embodiments, the promoter may be a cell type-specific promoter. For example, a cell type-specific promoter for neurons (e.g., synapsin), a cell type-specific promoter for astrocytes (e.g., GFAP), a cell type-specific promoter for oligodendrocytes (e.g., myelin basic protein), a cell type-specific promoter for microglia (e.g., CX3CR1), a cell type-specific promoter for neuroendocrine cells (e.g., chromogranin A), a cell type-specific promoter for muscle cells (e.g., desmin, Mb), or a cell type-specific promoter for cardiomyocytes (e.g., alpha-myosin heavy chain promoter) may be used. In exemplary embodiments, the promoter may be an Nrl (rod photoreceptor-specific) promoter or an HBB (hemoglobin beta) promoter. The promoter may further include one or more specific transcriptional regulatory sequences to further enhance nucleic acid expression and / or to modify the spatial and / or temporal expression of nucleic acids.

[0154] The expression of genes contained in a vector can also be regulated by enhancer sequences found in the vector. Generally, enhancers are bound to protein factors to enhance gene transcription. Enhancers can be located upstream or downstream of the gene they regulate. Enhancers may be tissue-specific to enhance transcription in a particular cell or tissue type. In one embodiment, the bicistronic vector contains one or more enhancers to boost the transcription of genes present in the vector. Non-limiting examples of enhancers include CMV enhancers and SP1 enhancers.

[0155] In some embodiments, more than one promoter can be operably ligated to each polynucleotide encoding a polypeptide, and the promoters may be the same or different. The distance between the promoter and the expressed nucleic acid sequence may be approximately the same as the distance between the promoter and the native nucleic acid sequence it controls. As is known in the art, variations in this distance can be adapted without loss of promoter function.

[0156] To evaluate polypeptide expression within a bicistronic vector, the vector may also include either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells being sought for transfecting or infection by the viral vector. In some embodiments, the selection marker can be placed on a separate piece of DNA and used in a simultaneous transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0157] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is manifested by several readily detectable characteristics, such as enzymatic activity. Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with the smallest 5' facile region exhibiting the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be ligated to the reporter gene and used to evaluate the ability of a drug to modulate promoter-driven transcription.

[0158] Methods for introducing and expressing genes in cells are well known in the art. Regarding expression vectors, they can be easily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0159] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0160] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0161] Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0162] In some embodiments utilizing nonviral delivery systems, the exemplary delivery vehicle is a liposome. The use of lipid formulations for introducing nucleic acids into host cells is intended (in vitro, ex vivo, or in vivo). In some embodiments, the nucleic acid may be bound to a lipid. Lipid-bound nucleic acids may be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via linking molecules bound to both the liposome and the oligonucleotide, confined within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension within a lipid, contained in a micelle or complexed with a micelle, or otherwise bound to a lipid. Compositions involving lipids, lipid / DNA, or lipid / expression vectors are not limited to any specific structure in solution. For example, such compositions may exist in a bilayer structure, as micelles, or in a "broken-down" structure. The composition may also simply be dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic. For example, lipids include a class of compounds containing naturally occurring fatty droplets in the cytoplasm, as well as long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0163] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing various single and multi-membrane lipid vehicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer and an internal aqueous medium. Multilayer liposomes have numerous lipid layers separated by an aqueous medium. These form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reconfiguration to form a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with structures different from normal vesicle structures in solution are also included. For example, lipids may exist as micellar structures or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also intended.

[0164] Regardless of the method used to introduce exogenous nucleic acids into host cells, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; and “biochemical” assays, such as detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blotting) or by assays described herein for identifying drugs that fall within the scope of this disclosure. Gene therapy vectors

[0165] The vectors used to treat or prevent LSD in the subjects disclosed herein are suitable for replication and, if necessary, integration into eukaryotic cells. Typical vectors contain transcription and translation terminators, start sequences, and promoters useful for regulating the expression of desired nucleic acid sequences.

[0166] The vectors of this disclosure can also be used for nucleic acid immunotherapy and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, this disclosure provides a vector for gene therapy.

[0167] The isolated nucleic acids of this disclosure can be cloned into a number of vector types. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0168] Furthermore, vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a functional replication origin, promoter sequence, convenient restriction endonuclease site, and one or more selection markers in at least one organism (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0169] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors using techniques known in the art and packaged into retroviral particles. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Numerous adenovirus vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0170] For example, retrovirus-derived vectors, such as lentiviruses, are suitable tools for long-term gene transfer because they enable stable integration and transmission of the introduced gene to daughter cells over extended periods. Lentiviral vectors have further advantages over vectors derived from onchoretroviruses, such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. Lentiviral vectors also have the further advantage of low immunogenicity. In preferred embodiments, the composition includes a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become a powerful gene delivery tool for treating a variety of disorders. AAV vectors have numerous features that make them ideally suited for gene therapy, including their non-pathogenicity, minimal immunogenicity, and ability to transduce into terminating cells in a stable and efficient manner. By selecting a suitable combination of AAV serotype, promoter, and delivery method, the expression of specific genes contained in the AAV vector can be specifically targeted to one or more types of cells.

[0171] In some embodiments, the disclosed bicistronic virus vectors include adenoviruses (e.g., Ad-SYE, AdSur-SYE, Ad5 / 3-MDA7 / IL-24, Ad-SB, Ad-CRISPR, oncolytic Ad); adeno-associated viruses, AAVs (e.g., AAV-MeCP2, AAV1, AAV5, Dual AAV9, AAV8, AAV9, AAVrh10, AAVhu37); herpes simplex viruses, HSVs (e.g., HSV1, HSV2, HSV-1, HF10, oncolytic HSV-2); and retroviruses (e.g., RRV / Toca). Including 511, GRV); lentiviruses (e.g., HIV-1, HIV-2); alphaviruses (SFV, M1); flaviviruses (Kunjin virus); rhabdoviruses (VSV); measles virus (e.g., MV-Edm); Newcastle disease virus (e.g., NDV90); snakeupicornavirus coxsackievirus (e.g., CVB3, CAV21, EV1); or poxviruses (e.g., PANVAC, VV, VV-GLV-1h153, CPXV).

[0172] In one embodiment, the disclosed bicistronic viral vector is an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus (HSV), measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus. In one embodiment, the disclosed bicistronic viral vector is an adenovirus, adeno-associated virus (AAV), retrovirus, or lentivirus.

[0173] In one embodiment, a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 PTase are contained in the AAV vector. More than 30 naturally occurring AAV serotypes are available. Many natural variants exist in AAV capsids, which allows for the identification and use of AAVs with properties particularly suitable for skeletal muscle. AAV viruses can be engineered using conventional molecular biology techniques, which allows for the optimization of these particles to, to name a few, cell-specific delivery of nucleic acid sequences, minimize immunogenicity, adjust stability and particle lifetime, enable efficient degradation, and deliver accurately to the nucleus.

[0174] The use of AAVs is a common method of exogenous DNA delivery because AAVs are relatively non-toxic, result in efficient gene transfer, and can be easily optimized for specific purposes. Among the well-characterized AAV serotypes isolated from humans or non-human primates (NHPs), the first AAV developed as a gene transfer vector is human serotype 2; this has been widely used in efficient gene transfer experiments in different target tissues and animal models. Clinical trials of experimental application of AAV2-based vectors to several human disease models are underway, including, for example, the treatment of diseases such as cystic fibrosis and hemophilia B. Other useful AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0175] Desired AAV fragments for assembly in vectors include cap proteins including vp1, vp2, vp3 and the hypervariable region, rep proteins including rep78, rep68, rep52, and rep40, and sequences encoding these proteins. These fragments can be readily used in various vector systems and host cells. Such fragments can be used alone, in combination with sequences or fragments of other AAV serotypes, or in combination with elements derived from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs having capsid proteins that do not exist in nature. Such artificial capsids can be generated by any suitable technique using a combination of a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) and heterologous sequences that can be obtained from different selected AAV serotypes, from non-contiguous portions of the same AAV serotype, from non-AAV viral sources, or from non-viral sources. Artificial AAV serotypes may be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Therefore, exemplary AAVs or artificial AAVs suitable for the expression of the target lysosomal enzyme and modified GlcNAc-1 PTase include, in particular, AAV2 / 8 (see U.S. Patent No. 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (International Patent Publication WO2005 / 033321), AAV2 / 6 (U.S. Patent No. 6,156,303), and AAVrh8 (International Patent Publication WO2003 / 042397).

[0176] In one embodiment, a vector useful for the compositions and methods described herein contains at least a sequence encoding the capsid of a selected AAV serotype, for example, the capsid or a fragment thereof of AAV8. In another embodiment, a useful vector contains at least a sequence encoding the rep protein of a selected AAV serotype, for example, the rep protein or a fragment thereof of AAV8. If necessary, such a vector may contain both the cap protein and the rep protein of AAV. In a vector providing both AAV rep and cap, both the AAV rep sequence and the AAV cap sequence may originate from a single serotype, for example, both from AAV8. Alternatively, a vector may be used in which the rep sequence originates from a different AAV serotype than the AAV serotype providing the cap sequence. In one embodiment, the rep sequence and the cap sequence are expressed from separate sources (e.g., separate vectors, or host cells and a vector). In another embodiment, these rep sequences are fused in-frame with cap sequences of different AAV serotypes to form a chimeric AAV vector, such as AAV2 / 8 as described in U.S. Patent No. 7,282,199.

[0177] A suitable recombinant adeno-associated virus (AAV) is produced by culturing a host cell containing a capsid protein or fragment thereof of an adeno-associated virus (AAV) serotype as defined herein; a functional rep gene; a minigene consisting of at least an AAV terminal inversion sequence (ITR) and a polynucleotide encoding a lysosomal enzyme and a modified GlcNAc-1 PTase; and a nucleic acid sequence encoding sufficient helper function to enable the packaging of the minigene into the AAV capsid protein. The components that need to be cultured in the host cell to package the AAV minigene into the AAV capsid can be provided trans to the host cell. Alternatively, any one or more of the required components (e.g., minigene, rep sequence, cap sequence, and / or helper function) can be provided by a stable host cell engineered to contain one or more of the required components using methods known to those skilled in the art.

[0178] Such stable host cells are most appropriately characterized by containing the necessary components(s) under the control of a constitutive promoter. However, the necessary components(s) may also be under the control of an inductive promoter. Suitable examples of inductive and constitutive promoters are presented elsewhere in this specification and are well known in the art. Alternatively, selected stable host cells may contain selected components(s) under the control of a constitutive promoter and other selected components(s) under the control of one or more inductive promoters. For example, stable host cells derived from 293 cells (containing E1 helper function under the control of a constitutive promoter) can be generated, which contain rep and / or cap proteins under the control of an inductive promoter. Those skilled in the art can generate other stable host cells.

[0179] The minigenes, rep sequences, cap sequences, and helper functions necessary for constructing the rAAVs of this disclosure can be delivered to a packaging host cell in the form of any gene element that carries and transfers these sequences. The selected gene element can be delivered using any suitable method, including those described herein and any others available in the art. The methods used to construct any embodiment of this disclosure are known to those skilled in the art of nucleic acid manipulation, and include genetic engineering, recombination, and synthetic techniques (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY). Similarly, methods for generating rAAV virions are well known, and the selection of a suitable method is not limited to this disclosure (see, for example, K. Fisher et al, 1993 J. Virol., 70: 520-532 and U.S. Patent No. 5,478,745).

[0180] Unless otherwise specified, AAV ITRs and other selected AAV components described herein can be readily selected from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or other known or unknown AAV serotypes. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAVs can be isolated or obtained from scientific, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Va.). Alternatively, AAV sequences can be obtained by synthesis or other suitable means by referring to publicly available sequences, such as those available in the literature or databases, e.g., GenBank, PubMed, etc.

[0181] In some embodiments, the bicistronic vector contains the nucleic acid sequence of SEQ ID NO: 1. In other embodiments, the bicistronic vector contains a nucleic acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to SEQ ID NO: 1.

[0182] In some embodiments, the encoded lysosomal enzyme is involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C below. In other embodiments, the lysosomal enzyme is at least one of those listed in Table 1A, Table 1B, or Table 1C below.

[0183] [Table 1A-1] [Table 1A-2] [Table 1A-3]

[0184] [Table 1B-1] [Table 1B-2] [Table 1B-3]

[0185] [Table 1C-1] [Table 1C-2] [Table 1C-3] [Table 1C-4] [Table 1C-5] [Table 1C-6] [Table 1C-7] [Table 1C-8]

[0186] In some embodiments, the lysosomal enzyme is selected from the group consisting of β-glucocerebrosidase (GBA), galactosylceramidase (GALC), α-galactosidase (GLA), α-N-acetylglucosaminidase (NAGLU), acid α-glucosidase (GAA), and lysosomal acid α-mannosidase (LAMAN). In yet other embodiments, the polynucleotide encoding the lysosomal enzyme includes the nucleic acid sequences of SEQ ID NOs. 5-10. In other embodiments, the lysosomal enzyme is encoded by a polynucleotide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to SEQ ID NOs. 5-10.

[0187] In some embodiments, the S1-S3 PTase is encoded by a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 4. In other embodiments, the GlcNAc-1 PTase is encoded by a polynucleotide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to SEQ ID NO: 4.

[0188] This disclosure should also be construed to include any form of polypeptide or polynucleotide having substantial homology to those disclosed herein.

[0189] A "substantially homologous" polypeptide is preferably about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably about 95% homologous, and even more preferably about 99% homologous to the amino acid sequence of the peptide disclosed herein.

[0190] Alternatively, polypeptides can be produced by recombinant means or by cleavage from longer polypeptides. The composition of the peptide can be confirmed by amino acid analysis or sequencing. Variants of polypeptides according to this disclosure may include: (i) variants in which one or more amino acid residues are substituted with conserved or unconserved amino acid residues (conserved amino acid residues are preferred), such substituted amino acid residues may or may not be encoded by the genetic code; (ii) variants in which one or more modified amino acid residues, e.g., residues modified by substituent attachment; (iii) variants in which the polypeptide is an alternative splice variant of the polypeptide of this disclosure; (iv) a polypeptide fragment; and / or (v) a polypeptide fused with another polypeptide, such as a reader sequence or secretion sequence or a sequence used for purification (e.g., a His tag) or a sequence used for detection (e.g., an Sv5 epitope tag). Fragments include polypeptides produced by proteolytic cleavage (including multi-site proteolytic) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to fall within the scope of the skills of those skilled in the art based on the teachings herein.

[0191] As is well known in the art, “similarity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of the second polypeptide. A variant is defined as a polypeptide sequence that differs from the original sequence, preferably by less than 40% of residues per target segment from the original sequence, more preferably by less than 25% of residues per target segment from the original sequence, more preferably by less than 10% of residues per target segment from the original sequence, most preferably by only a few residues per target segment from the original protein sequence, and at the same time is homologous to the original sequence so that the functionality and / or ability to bind to ubiquitin or ubiquitinated proteins of the original sequence is conserved. This disclosure includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two polypeptides is determined using computer algorithms and methods widely known to those skilled in the art. It is preferable to determine the identity between two amino acid sequences using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0192] Polypeptides disclosed herein can be post-translationally modified. Examples of post-translational modifications within the scope of this disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and protein processing. Some modifications or processing events require the introduction of additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation can be tested by adding canine microsome membranes or African clawed frog egg extracts to a standard translation reaction.

[0193] The polypeptides of this disclosure may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. Various methods are available for introducing unnatural amino acids during protein translation.

[0194] When used herein, the term "functionally equivalent" refers to a polypeptide that is preferred to retain at least one biological function or activity of a particular amino acid sequence of the lysosomal enzyme of this disclosure.

[0195] Polypeptides can be conjugated with other molecules, such as proteins, to prepare fusion proteins. This can be achieved, for example, by synthesizing an N-terminal or C-terminal fusion protein, provided that the resulting fusion protein retains the functionality of the lysosomal enzyme of this disclosure.

[0196] Polypeptides can be phosphorylated using conventional methods. In one embodiment, the lysosomal enzyme of the Disclosure can be phosphorylated by a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase) of the Disclosure.

[0197] Cyclic derivatives of peptides or chimeric proteins are also intended herein. Cyclization may allow peptides or chimeric proteins to assume a more favorable conformation for bonding with other molecules. Cyclization can be achieved using techniques known in the art. For example, a disulfide bond can be formed between two reasonably spaced components having free sulfhydryl groups, or an amide bond can be formed between an amino group of one component and a carboxyl group of another component.

[0198] Cyclization can also be achieved using azobenzene-containing amino acids. The components forming the bond can be amino acid side chains, non-amino acid components, or a combination of the two. In one embodiment, the cyclic peptide may contain a beta turn at the right position. The beta turn can be introduced into the peptide of this disclosure by adding the amino acid Pro-Gly to the right position. It is desirable to produce cyclic peptides that are more flexible than those containing the above-described peptide bond linkage. More flexible peptides can be prepared by introducing cysteines at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are positioned so that the beta-sheet and turn do not deform. The peptide becomes more flexible as a result of the length of the disulfide linkage and the number of hydrogen bonds in the beta-sheet portion. The relative flexibility of the cyclic peptide can be determined by molecular dynamics simulation. tag

[0199] In one embodiment, the polypeptide disclosed herein further comprises the amino acid sequence of a tag. The tags include, but are not limited to, polyhistidine tags (His tags) (e.g., H6 and H10) or other tags for use in IMAC systems, e.g., Ni2+ affinity columns, GST fusions, MBP fusions, streptavidin tags, BSP biotinylation target sequences of the bacterial enzyme BIRA, and tag epitopes targeted by antibodies (e.g., c-myc tags, FLAG tags, HPC4- tags). As will be recognized to those skilled in the art, tag peptides can be used for the purification, inspection, selection, and / or visualization of the fusion proteins of this disclosure. In one embodiment, the tag is a detection tag and / or a purification tag. It will be understood that the tag sequence does not interfere with the function of the proteins of this disclosure. Leader sequence and secretion sequence

[0200] Therefore, the polypeptides of the Disclosure can be fused with another polypeptide or tag, such as a leader sequence or secretion sequence or a sequence used for purification or detection. In some embodiments, the polypeptides of the Disclosure include a glutathione-S-transferase protein tag, which provides a basis for rapid high-affinity purification of the polypeptides of the Disclosure. In fact, this GST-fusion protein can then be purified from cells by high affinity for glutathione. Agarose beads can be coupled to glutathione, and such glutathione-agarose beads bind to the GST protein. Therefore, in certain embodiments, the polypeptides can be bound to a solid support. In some embodiments, if the polypeptide contains a GST moiety, the polypeptide is coupled to a glutathione-modified support. In some embodiments, the glutathione-modified support is a glutathione-agarose bead. Furthermore, a sequence encoding a protease cleavage site can be included between the affinity tag and the polypeptide sequence, thus making it possible to remove the bound tag after incubation with this particular enzyme, and thus facilitating the purification of the corresponding protein of interest.

[0201] Polypeptides disclosed herein may also be fused to or incorporated with a targeting domain that can orient a target protein and / or chimeric protein to a desired cellular component, cell type, or tissue. Chimeric proteins may also contain additional amino acid sequences or domains. Chimeric proteins are recombinant in the sense that their various components originate from different sources and are therefore not found together in nature (i.e., heterogeneous).

[0202] In some embodiments of the compositions of the present disclosure, the polypeptide comprises a peptide mimetic of the lysosomal protein of the present disclosure or a vector encoding a peptide mimetic of the lysosomal protein of the present disclosure. The peptide mimetic is a compound based on or derived from peptides and proteins.

[0203] N-terminal or C-terminal fusion proteins, comprising the peptides or chimeric proteins of this disclosure conjugated with other molecules, can be prepared by fusion of the N-terminus or C-terminus of the peptide or chimeric protein with the sequence of a selected protein or selected marker having a desired biological function using a recombination technique. The resulting fusion protein contains a lysosomal enzyme comprising the peptide or chimeric protein fused with the selected protein or marker protein described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and the truncated myc.

[0204] The polypeptides and chimeric proteins of this disclosure can be converted into pharmaceutical salts by reacting them with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, and phosphoric acid, or with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid. Modified cells

[0205] In some embodiments, the Disclosure provides cells containing the vector of the Disclosure. In some embodiments, the vector is a viral vector (e.g., AAV or lentiviral vector). In some embodiments, the vector is a nonviral vector (e.g., liposomes, nanoparticles, lipid nanoparticles, micelles, polymerosomes, or exosomes). In some embodiments, the vector is an expression vector. In some embodiments, the vector contains at least one element that enables bicistronic, polycistronic, or multicistronic expression of at least two sequences. In some embodiments, the vector contains a sequence encoding a lysosomal enzyme of the Disclosure. Alternatively, or in addition thereto, in some embodiments, the vector contains a sequence encoding the S1S3 construct of the Disclosure. In some embodiments, the lysosomal enzyme is one or more of the enzymes listed in Table 1A, Table 1B, or Table 1C. In some embodiments, the vector contains a nucleic acid or amino acid sequence encoding a lysosomal enzyme that is one or more of the enzymes listed in Table 1A, Table 1B, or Table 1C.

[0206] In some embodiments, cells containing the vectors of the Disclosure are modified cells of the Disclosure. In some embodiments, cells containing the vectors of the Disclosure do not exist in nature.

[0207] In some embodiments, the cells are mammalian cells capable of expressing human sequences and / or producing human proteins. In some embodiments, the mammalian cells are isolated from or derived from mice, rats, guinea pigs, rabbits, cats, dogs, or non-human primates.

[0208] In some embodiments, the cells are human cells capable of expressing human sequences and / or producing human proteins.

[0209] In some embodiments, the cells are primary cells that have been modified to express the vector of this disclosure and cultured ex vivo. In some embodiments, the cultured cells are immortalized or otherwise modified to facilitate unrestricted cell proliferation in vitro, thereby generating a cultured cell line. host cell

[0210] In some embodiments, the Disclosure provides cells containing the bicistronic vector of the Disclosure. The cells may be prokaryotic or eukaryotic. Suitable cells include, but are not limited to, bacterial cells, yeast cells, fungal cells, insect cells, and mammalian cells.

[0211] In some embodiments, the Disclosure provides mammalian cells containing the bicistronic vector of the Disclosure.

[0212] Host cells containing the disclosed bicistronic vector can be used for protein expression and, if necessary, purification. Methods for expressing proteins and, if necessary, purifying the expressed proteins from the host are standard in the art.

[0213] In some embodiments, host cells containing the vector of the Disclosure can be used to produce polypeptides encoded by the enzyme construct of the Disclosure. Generally, the production of polypeptides of the Disclosure involves transfecting host cells with a vector containing the enzyme construct, then culturing the cells, resulting in the transcription and translation of the desired polypeptide by the cells. The isolated host cells can then be lysed to extract the expressed polypeptide for subsequent purification.

[0214] In some embodiments, the host cell is a prokaryotic cell. Non-limiting examples of suitable prokaryotic cells include E. coli and other Enterobacteriaceae, Escherichia sp., Campylobacter sp., Wolinella sp., Desulfovibrio sp., Vibrio sp., Pseudomonas sp., Bacillus sp., Listeria sp., Staphylococcus sp., Streptococcus sp., Peptostreptococcus sp., Megasphaera sp., Pectinatus sp., Selenomonas sp., Zymophilus sp., Actinomyces sp., Arthrobacter sp., Frankia sp., Micromonospora sp., Nocardia sp., Propionibacterium sp., Streptomyces sp., Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Pediococcus sp., Acetobacterium sp., Eubacterium sp., Heliobacterium sp., Heliospirillum sp., Sporomusa sp., Spiroplasma sp., Ureaplasma sp., Erysipelothrix sp., Corynebacterium sp., Enterococcus sp., Clostridium sp., Mycoplasma sp., Mycobacterium sp., Actinobacteria sp., Salmonella sp., Shigella sp., Moraxella sp., Helicobacter sp., Stenotrophomonas sp., Micrococcus sp., Neisseria sp., Bdellovibrio sp., Hemophilus sp., Klebsiella sp., Proteus mirabilis, Enterobacter cloacae, Serratia sp., Citrobacter sp., Proteus sp., Serratia sp., Yersinia sp.Examples include alpha-proteobacteria such as Acinetobacter sp., Actinobacillus sp., Bordetella sp., Brucella sp., Capnocytophaga sp., Cardiobacterium sp., Eikenella sp., Francisella sp., Haemophilus sp., Kingella sp., Pasteurella sp., Flavobacterium sp., Xanthomonas sp., Burkholderia sp., Aeromonas sp., Plesiomonas sp., Legionella sp., and Wolbachia sp., as well as cyanobacteria, spirochetes, green sulfur bacteria and green non-sulfur bacteria, gram-negative cocci, preferred gram-negative bacilli, Enterobacteriaceae glucose-fermenting gram-negative bacilli, non-glucose-fermenting gram-negative bacilli, and oxidase-positive glucose-fermenting gram-negative bacilli. Particularly useful bacterial host cells for protein expression include Gram-negative bacteria such as Escherichia coli, Pseudomonas fiuorescens, Pseudomonas haloplanctis, Pseudomonas putida AC 10, Pseudomonas pseudof lava, Bartonella henselae, Pseudomonas syringae, Caulobacter crescentus, Zymomonas mobilis, Rhizobium meliloti, and Myxococcus xanthus, and Gram-positive bacteria such as Bacillus subtilis, Corynebacterium, Streptococcus cremoris, Streptococcus lividans, and Streptomyces lividans. E. coli is one of the most widely used expression hosts. Therefore, techniques for overexpression in E. coli are well-developed and readily available to those skilled in the art.

[0215] Furthermore, Pseudomonas fiuorescens is commonly used for high-level production of recombinant proteins (i.e., for developing biopharmaceuticals and vaccines).

[0216] In some embodiments, the host cell is a yeast or fungal cell. Fungal host cells particularly useful for protein expression include Aspergillis oryzae, Aspergillis niger, Trichoderma reesei, Aspergillus nidulans, and Fusarium graminearum. Yeast host cells particularly useful for protein expression include Candida albicans, Candida maltose, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Yarrowia lipolytica.

[0217] In some embodiments, the host cell is an insect cell. Non-limiting examples include Spodoptera frugiperda cell lines (e.g., Sf9 or Sf21), Drosophila cell lines, or mosquito cell lines (e.g., Aedes albopictus cell lines).

[0218] In some embodiments, the host cell is a mammalian cell. Mammalian host cells useful for protein expression include Chinese hamster ovary (CHO) cells, HeLa cells, human fetal kidney 293 (HEK293) cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human fetal kidney cells, Bos primigenius, and Mus musculus. In certain embodiments, the host cell is a CHO cell. Furthermore, the mammalian host cell may be an established, commercially available cell line (e.g., American Type Culture Collection (ATCC), Manassas, VA). The host cell may be an immortalized cell. Alternatively, the host cell may be a primary cell.

[0219] In some embodiments, host cells are engineered to produce high levels of the target protein. Method of Disclosure

[0220] In some embodiments, the Disclosure provides a method for treating subjects suffering from lysosomal storage disorders (LSDs). The method comprises administering to a subject a pharmaceutical composition comprising a lysosomal enzyme expressed by a bicistronic vector as disclosed elsewhere herein, thereby increasing the phosphorylation of the lysosomal enzyme and treating the subject.

[0221] In some embodiments, the Disclosure provides a method for preventing the development of lysosomal storage disorders (LSDs) in subjects requiring such prevention. The method comprises administering to a subject a pharmaceutical composition comprising a lysosomal enzyme expressed by a bicistronic vector as disclosed elsewhere herein, thereby increasing the phosphorylation of the lysosomal enzyme and preventing the development of LSDs in the subject.

[0222] In some embodiments, the lysosomal enzyme is involved in at least one of the lysosomal storage disorders (LSDs) listed in Table 1. In other embodiments, the lysosomal enzyme is at least one of those listed in Table 1.

[0223] In further embodiments, the administration step includes a route of administration selected from the group consisting of enteral, parenteral, oral, intramuscular (IM), subcutaneous (SC), intravenous (IV), and intra-arterial (IA). Additional routes of administration that can be used in the disclosed method are described in detail elsewhere in this specification. Combination therapy

[0224] The compositions and methods for treating or preventing LSD described herein may be useful in combination with at least one additional compound useful for treating LSD. The additional compound may include commercially available compounds known to treat, prevent, or reduce the symptoms of LSD. The compound may be, but is not limited to, ERTs known in the art. Pharmaceutical compositions and formulations

[0225] The present invention also provides a pharmaceutical composition comprising a lysosomal enzyme expressed by the bicistronic vector of the present disclosure.

[0226] Such pharmaceutical compositions are in a form suitable for administration to a subject, or the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers, one or more additional components, or some combination thereof. The various components of the pharmaceutical composition may exist in the form of physiologically acceptable salts, for example, in combination with physiologically acceptable cations or anions well known in the art.

[0227] In some embodiments of this disclosure, a pharmaceutical composition useful for carrying out the method of this disclosure may be administered at a delivery dose between 1 ng / kg / day and 100 mg / kg / day. In some embodiments of this disclosure, a pharmaceutical composition useful for carrying out this disclosure may be administered at a delivery dose between 1 ng / kg / day and 500 mg / kg / day. The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical composition of this disclosure will vary depending on the identity, size, and condition of the subject being treated, and further depending on the route through which the composition is administered. For example, a composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0228] In some embodiments of this disclosure, a pharmaceutical composition useful for carrying out the method of this disclosure may be administered in a delivery dose between 1 ng / kg and 100 mg / kg. In some embodiments of this disclosure, a pharmaceutical composition useful for carrying out this disclosure may be administered in a delivery dose between 1 ng / kg and 500 mg / kg. In some embodiments of this disclosure, the pharmaceutical composition may be provided daily, weekly, bi-weekly, monthly, or annually. The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical composition of this disclosure will vary depending on the identity, size, and condition of the subject being treated, and further depending on the route of administration of the composition. For example, a composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0229] Pharmaceutical compositions useful in the methods disclosed herein can be appropriately developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, subarachnoid, intravenous, or other routes of administration. Other intended formulations include protruding nanoparticles, liposome preparations, resealed red blood cells containing active ingredients, and immunotherapy formulations. The route(s) of administration will be readily apparent to those skilled in the art and will depend on any number of factors, including the type and severity of the disease being treated, and the type and age of the animal or human patient being treated.

[0230] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the field of pharmacology. Generally, such preliminary methods include the steps of binding the active ingredient to a carrier or one or more other minor components, and then, if necessary or desirable, shaping or packaging the product into desired single-dose or multi-dose doses. In some embodiments, the compositions of this disclosure can be formulated in natural capsids, modified capsids, as naked RNA, or encapsulated in a protective coating.

[0231] The amount of the active ingredient is generally equal to the total dose of the active ingredient to be administered to the subject, or a convenient portion of such a dose, such as half or one-third of such a dose. A unit dosage form may be a single daily dose or one dose from a group of daily doses (e.g., about 1 to 4 times per day or more). When using a group of daily doses, each dose in the unit dosage form may be the same or different.

[0232] The description of the pharmaceutical compositions provided in this invention primarily concerns pharmaceutical compositions suitable for ethical administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any animal. Modifications of compositions to make pharmaceutical compositions suitable for administration to humans suitable for administration to various animals are well understood, and such modifications, if made, can be designed and carried out by a veterinary pharmacologist of ordinary art, simply by ordinary experiments. Subjects intended to receive the pharmaceutical compositions of this disclosure include, but are not limited to, humans and other primates, and mammals including commercially suitable mammals such as cattle, pigs, horses, sheep, cats, and dogs. In one embodiment, the subject is a human, or, but is not limited to, a non-human mammal such as a horse, sheep, cattle, pig, dog, cat, and mouse. In one embodiment, the subject is a human.

[0233] In one embodiment, the composition is formulated using one or more pharmaceutically acceptable excipients or carriers. In some embodiments, the Disclosure provides a pharmaceutical composition for treating a subject suffering from LSD. In some embodiments, the Disclosure provides a pharmaceutical composition comprising a lysosomal enzyme expressed by a bicistronic vector of the Disclosure and a pharmaceutically acceptable carrier.

[0234] Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, physiological saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and organic acid salts. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Reasonable 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. Prevention of microbial action can be achieved with various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, it is preferable to include isotonic agents, such as sugars, sodium chloride, or polyhydric alcohols such as mannitol and sorbitol, in the composition. The inclusion of absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition can result in sustained absorption of the injectable composition.

[0235] The formulation can be used in combination with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances known in the art that are suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration. The pharmaceutical preparation can be sterilized and, if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, buffers, salts that affect osmotic pressure, colorants, flavoring agents and / or aromatic agents. The pharmaceutical preparation can also be combined with other active agents, such as other analgesics, if desired.

[0236] The disclosed compositions may contain preservatives in an amount ranging from about 0.005% to 2.0% of the total weight of the composition. The preservatives are used to prevent spoilage when exposed to contaminants in the environment. Examples of preservatives useful in accordance with this disclosure, but not limited to, are selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidourea, and combinations thereof. In some embodiments, the preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0237] The composition may contain antioxidants and chelating agents that inhibit the degradation of the compounds. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol, and ascorbic acid in a preferred range of about 0.01% to 0.3%, with BHT being more preferred in a range of 0.03% to 0.1% by weight of the total weight of the composition. Chelating agents are preferably present in amounts from 0.01% to 0.5% by weight of the total weight of the composition. Particularly preferred chelating agents include EDTA salts (e.g., disodium EDTA) and citric acid in a weight range of about 0.01% to 0.20% and more preferably 0.02% to 0.10% by weight of the total weight of the composition. Chelating agents are useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. In some embodiments, BHT and disodium EDTA are the antioxidant and chelating agents for some compounds, but other suitable and equivalent antioxidants and chelating agents known to those skilled in the art may be substituted. Administration / Medication

[0238] The administration regimen may affect what constitutes the effective dose. For example, the therapeutic formulation may be administered to a patient either before or after surgical intervention related to lysosomal storage disorders (LSD), or immediately after the patient is diagnosed with a lysosomal storage disorder (LSD). Furthermore, several divided doses, as well as staggered doses, may be administered daily or sequentially, or the dose may be administered by continuous infusion or bolus injection. In addition, the dosage of the therapeutic formulation may be increased or decreased proportionally, as indicated by the urgency of the therapeutic or prophylactic situation.

[0239] The compositions of this disclosure are administered to patient subjects, preferably mammals, more preferably humans, using known procedures, in doses effective for treating lysosomal storage disorders (LSDs) in the subject, for a duration effective for that purpose. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary depending on factors such as the activity of the particular compound used; the time of administration; the elimination rate of the compound; the duration of treatment; other drugs, compounds, or materials used in combination with the compound; the state of the disease or disorder, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and similar factors well known in the medical field. The dosage regimen can be adjusted to bring about an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the urgency of the treatment situation. Non-limiting examples of the effective dose range of the therapeutic compounds of this disclosure range from about 0.01 mg per kg of body weight per day to 50 mg per kg of body weight per day.

[0240] The compound can also be administered several times a day, or at a lower frequency, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less frequently. It is understood that the amount of the compound administered per day can be administered, in non-limiting examples, daily, every other day, every two days, every three days, every four days, or every five days. For example, when administered every other day, a dose of 5 mg per day can be started on Monday, the first subsequent dose of 5 mg per day can be administered on Wednesday, the second subsequent dose of 5 mg per day can be administered on Friday, and so on. The dosing frequency will readily be apparent to those skilled in the art and depends on any number of factors, including but not limited to the type and severity of the disease being treated, as well as the type and age of the animal. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure can be varied so that an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient can be obtained without the composition and the dosage form being toxic to the patient. A medical doctor having ordinary skill in the art, such as a physician or veterinarian, can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start the dosing of the compounds of the present disclosure used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0241] In some embodiments, it is particularly advantageous to formulate the compounds in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to physically discrete units suitable as unit dosages for the patient to be treated; each unit containing a predetermined quantity of the therapeutic compound calculated to produce the desired therapeutic effect, accompanied by the necessary pharmaceutical vehicle. The dosage unit forms of the present disclosure are defined by, and directly depend on, (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such therapeutic compounds for the treatment of LSD. Route of administration

[0242] More than one route can be used for administration, but it will be understood by those skilled in the art that a particular route may provide a more immediate and effective response than another route.

[0243] Routes of administration of the disclosed compositions include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, tongue, (trans)buccal, (trans)urethral, ​​vagina (e.g., transvaginal and perivaginal), nasal (intra) and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, subarachnoid, intracisional (ICM), intraspinal, intraventricular, intraventricular, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and local administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel capsules, lozenges, dispersants, suspensions, liquids, syrups, granules, beads, transdermal patches, gels, powders, pellets, mud, lozenges, creams, pastes, transdermal patches, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, and compositions and formulations for intravesical administration. It should be understood that useful formulations and compositions in this disclosure are not limited to the specific formulations and compositions described herein. In one embodiment, the administration of LSD includes routes of administration selected from the group consisting of inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmoscopy, intrahepatic artery, intrapleural, subarachnoid, intratumoral, intravenous, and any combination thereof. Administration of gene therapy

[0244] Those skilled in the art will understand that vectors can be administered to cells using various delivery methods. Examples include (1) methods utilizing physical means such as electroporation (electrical), gene guns (physical force), or the application of a large volume of liquid (pressure); and (2) methods of complexing the vector with another entity such as liposomes, aggregated proteins, or transporter molecules.

[0245] Furthermore, the actual dose and schedule may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug elimination, and metabolism. Similarly, in in vitro applications, the amount may be varied depending on the specific cell line used (e.g., based on the number of vector receptors present on the cell surface, or the ability of the specific vector used for gene transfer to replicate in that cell line). Moreover, the amount of vector added per cell is likely to vary depending on the length and stability of the therapeutic gene inserted into the vector, as well as the properties of the sequence, and is a parameter that needs to be determined particularly empirically and may be altered due to factors not specific to the method of this disclosure (e.g., the cost of synthesis). Those skilled in the art can easily make any necessary adjustments according to the urgency of the particular situation.

[0246] Cells containing a therapeutic agent may also contain a suicide gene, i.e., a gene that codes for a product that can be used to destroy the cell. In many gene therapy scenarios, it is desirable that the therapeutic gene be able to be expressed in the host cell, but it is also desirable that it has the ability to destroy the host cell at will. The therapeutic agent can be linked to a suicide gene whose expression is not activated in the absence of an activating compound. If it is desired that the cells into which both the drug and the suicide gene have been introduced be killed, an activating compound is administered to the cells, thereby activating the expression of the suicide gene and killing the cells. Examples of usable suicide gene / prodrug combinations include herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Treatment drugs

[0247] This disclosure comprises methods for treating lysosomal enzyme deficiencies in subjects diagnosed with LSD or at risk of developing LSD. These methods improve the phosphorylation of lysosomal enzymes, thereby treating the subject or preventing the development of LSD in the subject. Furthermore, these methods improve the patient's quality of life. In one embodiment, the method of this disclosure includes administering to a subject a composition comprising a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding GlcNAc-1 PTase.

[0248] Nucleic acid sequence:

[0249] pLL01 bicistronic vector sequence (SEQ ID NO: 1) (CMV promoter: italics and underlined. IRES: bold and italics. S1-S3: bold and underlined.) [ka] [ka] [ka]

[0250] CMV sequence (sequence number 2) [ka]

[0251] IRES sequence (sequence number 3) [ka]

[0252] Modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase), S1-S3 sequence (SEQ ID NO: 4) [ka] [Chemical formula]

[0253] Wild-type hGBA sequence (SEQ ID NO: 5): [Chemical formula]

[0254] Natural variant sequence of hGBA (SEQ ID NO: 162): hGBA (K360N) sequence. The nucleotides at the mutation site are bolded and underlined. [Chemical formula] [Chemical formula]

[0255] Engineered variant sequence of hGBA (SEQ ID NO: 163): hGBA (C165S) sequence. The nucleotides at the mutation site are bolded and underlined. [Chemical formula]

[0256] mGALC sequence (SEQ ID NO: 6): [Chemical formula] [Chemical formula]

[0257] hGLA sequence (SEQ ID NO: 7): [Chemical formula]

[0258] hNAGLU sequence (SEQ ID NO: 8): [Chemical formula] [ka]

[0259] hGAA array (sequence number 9): [ka] [ka]

[0260] hGAA (Sequence ID 164; UniProt accession number P10253-1) [ka]

[0261] hLAMAN sequence (sequence number 10): [ka] [ka]

[0262] hGALC sequence (SEQ ID NO: 23; GenBank accession number: BC036518.2): [ka] [ka]

[0263] CEF promoter sequence (SEQ ID NO: 161): [ka]

[0264] [Table 2] [Examples]

[0265] The present disclosure will now be described with reference to the following examples. These examples are presented for illustrative purposes only and should not be construed as limiting the disclosure to these examples, but rather as encompassing all possible variations that may become apparent as a result of the teachings presented herein.

[0266] Without further explanation, those skilled in the art will likely be able to use the above description and the following examples to create, utilize, and carry out the claimed methods of the compounds of the Disclosure. Therefore, the following examples are intended to illustrate preferred embodiments of the Disclosure and are not intended to limit the remainder of the Disclosure.

[0267] The materials and methods used in these experiments are described here.

[0268] Cell line: HEK293T cells were maintained in DMEM (Corning) containing 0.11 g / L sodium pyruvate and 4.5 g / L glucose, supplemented with 10% (vol / vol) FBS (Gibco), 100,000 U / L penicillin, 100 mg / L streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). Expi293 cells (Invitrogen) were grown as a suspension in Expi293 expression medium (Invitrogen).

[0269] DNA construct: The CMV-S1S3 plasmid was provided by Prof. Stuart Kornfeld of the Washington University School of Medicine in St. Louis. The bicistronic vector pLL01 was created in two steps as follows: In the first step, a 486 bp IRES sequence was amplified from Ptase α / β and γ bicistronic constructs (provided by Prof. Stuart Kornfeld), and the S1-S3 gene fragment was obtained from plasmid CMV-S1S3 by PCR. These two fragments were then ligated by overlap extension PCR in the second step to form the IRES-S1S3 fragment. The IRES-S1S3 fragment was digested with HpaI and PmeI restriction enzymes (NEB) and ligated into a pcDNA3.1(+) vector. To generate pLL11, pLL21, pLL31, pLL41, pLL51, and pLL61 bicistronic plasmids, the hGBA, hGAA, mGALC, hNAGLU, hGLA, and hLAMAN genes were amplified using their specific primers (Table 1) and inserted into a bicistronic vector (pLL01).

[0270] Phosphotransferase assay: HEK293T cells or Expi293 cells were harvested and lysed in a lysis buffer (25 mM Tris-Cl, pH 7.2, 150 mM NaCl, 1% Triton® X-100, and a protease inhibitor cocktail). 5 μl of cell extract was incubated in phosphotransferase assay buffer (50 mM Tris-Cl, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 2 mg / mL BSA, 2 mM ATP) in the presence of 75 mM UDP-GlcNAc, 1 mCi UDP-[3H]GlcNAc, and 100 mM aMM, to a final volume of 50 μL, at 37°C for 0.5 hours. The reaction was stopped by adding 1 mL of 2 mM EDTA, pH 8.0, and the sample was subjected to QAE-Sephadex chromatography.

[0271] Enzyme production: Expi293 cells were transfected with an empty vector, a bicistronic plasmid, or a single-expression plasmid. The culture medium was collected after 2-3 days. To stabilize the secreted enzyme for GBA production, the culture medium containing 30 μM isofagomine in the cell culture was dialyzed overnight at 4°C in PBS buffer to remove isofagomine for enzyme activity assays.

[0272] Enzyme activity assay: The following substrates are used for the enzyme activity assay: 4-methylumbelliferyl[3-D-glucopyranoside (GCase / GBA enzyme substrate, M3633, Sigma), 4-methylumbelliferyl α-D-glucopyranoside (GAA enzyme substrate, M9766, Sigma), 6-hexadecanoylamino-4-methylumbelliferyl[3-D-galactopyranoside (GALC enzyme substrate, EH05989, Carbosynth), 4-methylumbelliferyl-N-acetyl-α-D-glucosaminid (NAGLU enzyme substrate, 474500, Millipore), 4-methylumbelliferyl α-D-galactopyranoside (GLA enzyme substrate, M7633, Sigma), and 4-methylumbelliferyl α-D-mannopyranoside (LAMAN enzyme substrate, M3657, Sigma). GBA enzyme activity was assayed using 1 mM GBA substrate in citrate-phosphate buffer, pH 5.0, 0.25% TX-100, and 0.25% sodium taurocholate. GAA enzyme activity was assayed using 1 mM GAA substrate in citrate buffer, pH 4.0, and 0.25% TX-100. GALC enzyme activity was assayed using 0.1 mM GALC substrate in citrate-phosphate buffer, pH 4.0, 0.25% TX-100, 0.6% sodium taurocholate, and 0.2% oleic acid. NAGLU enzyme activity was assayed using 1 mM NAGLU substrate in citrate buffer, pH 4.0, and 0.25% TX-100. GLA enzyme activity was assayed using 1 mM GLA substrate in citrate buffer, pH 4.5, and 0.25% TX-100. LAMAN enzyme activity was assayed using 1 mM LAMAN substrate in 0.25% TX-100 at pH 4.0 with citrate buffer.

[0273] CI-MPR binding assay: Binding to CI-MPR was performed in a high-binding 96-well plate (Costar 3601). The plate was immobilized with 50 μl of purified bovine CI-MPR at 10 μg / ml for 1 hour at room temperature (RT), and then blocked with 2% BSA at room temperature for another 1 hour. A fixed volume of the transfected Expi293 cell-derived condition medium was diluted with Hepes buffer (40 mM Hepes, pH 6.8, 150 mM NaCl, 0.05% Tween®-20) and incubated with the immobilized CI-MPR at room temperature for 1 hour to bind the phosphorylated lysosomal enzyme. After three washes, lysosomal enzyme activity was assayed using the 4-methylumbelliferone method.

[0274] (Example 1) Generation of an empty bicistronic vector containing phosphotransferases (S1-S3) for lysosomal enzyme expression. GlcNAc-1-phosphotransferase (GlcNAc-1-PTase, also known as Ptase), an α2β2γ2 hexamer encoded by two genes (GNPTAB and GNPTG), is involved in the production of phosphorylated oligosaccharides necessary for targeting lysosomes via the cation-independent mannose 6-phosphate receptor (CI-MPR). Phosphorylation of expressed lysosomal enzymes is significantly increased by simultaneous transfection with engineered truncated Ptase (S1-S3). This study utilizes the S1-S3 construct for the production of phosphorylated lysosomal enzymes to treat lysosomal storage diseases (LSD, but not limited to Gaucher disease, Pompe disease, and α-mannosidosis).

[0275] To produce a highly phosphorylated therapeutic lysosomal enzyme for enzyme replacement therapy (ERT), the therapeutic lysosomal enzyme and S1-S3 were co-expressed in the same cells. Since S1-S3 and the lysosomal enzyme are expressed in different vectors, stable cell lines expressing the lysosomal enzyme and S1-S3 were generated in two steps to produce a highly phosphorylated therapeutic lysosomal enzyme: (a) create a stable cell line expressing Ptase S1-S3; (b) generate a second cell line based on the S1-S3 stable cell line in which the expression of the therapeutic lysosomal enzyme is added to the S1-S3 stable cell line. To avoid this time-consuming two-step procedure, a bicistronic vector is disclosed herein that allows the expression of two separate genes under a single promoter by introducing an intrasequence ribosome entry site (IRES).

[0276] Bicistronic expression can also be applied to gene therapy for lysosomal storage disease (LSD). An empty bicistronic vector, pLL01 (Figure 1B), contains a 486 bp IRES sequence and S1-S3 genes under a cytomegalovirus (CMV) promoter in the pcDNA3.1(+) plasmid vector. Bicistronic vector pLL01 has three unique restriction enzyme cleavage sites within a multi-cloning site located prior to the IRES sequence, enabling insertion of therapeutic lysosomal enzyme genes. To investigate S1-S3 expression using bicistronic vector pLL01, HEK293 cells were transfected with equivalent amounts of plasmid pcDNA3.1(+), CMV-S1S3 (Figure 1A), or pLL01. After 48 hours, the cells were harvested and lysed in a lysis buffer (25 mM Tris buffer with a protease inhibitor cocktail, pH 7.4, 150 mM NaCl, 1% TX-100). Phosphotransferase activity analysis was performed on whole cell extracts expressing pcDNA3.1(+), CMV-S1S3, or pLL01 to determine S1-S3 expression. As shown in Figure 1C, phosphotransferase activity in the pcDNA3.1(+) sample was negligible compared to the CMV-S1S3 sample, while 9.3% activity was maintained with the bicistronic vector pLL01.

[0277] (Example 2) Bicistronic expression enhances the phosphorylation of therapeutic lysosomal enzymes. Since S1-S3 expression was low in the bicistronic vector (9.3%) (see Example 1), this study was designed to determine whether low S1-S3 activity was sufficient for phosphorylation of lysosomal enzymes. Six different lysosomal enzymes were tested in this bicistronic vector. The enzymes were: acid β-glucosidase (GBA), acid α-glucosidase (GAA), galactosylceramidase (GALC), α-N-acetylglucosaminidase (NAGLU), α-galactosidase (GLA), and acid α-mannosidase (LAMAN).

[0278] Acid β-glucosidase (GBA): GBA is a lysosomal enzyme that degrades its substrate, glucocerebroside, in lysosomes. Deficiency of GBA in lysosomes leads to Gaucher disease, the most common lysosomal storage disease (LSD). To test the phosphorylation of GBA in the bicistronic vectors of this disclosure, the GBA bicistronic plasmid-pLL11 was generated by inserting a 1611 bp human GBA cDNA sequence with a stop codon into a bicistronic empty vector-pLL01 through NheI and NotI restriction sites (Figure 2A). Expi293 cells were transfected with or without the pLL11 and CMV-S1S3 plasmids in equal amounts. After 48 hours, cells and conditional media were harvested separately. Surprisingly, GBA activity in the pLL11 condition medium was 240 nmol / hour / ml, which was more than twice that of medium prepared with GBA alone (96 nmol / hour / ml) or co-transfection with GBA and S1-S3 (90 nmol / hour / ml, Figure 2B). In addition to GBA expression, S1-S3 expression was quantified by phosphotransferase assay using cell extracts. Similar to the bicistronic vector pLL01 lacking GBA, phosphotransferase expression in the pLL11 sample was 7.5% compared to the co-transfection sample with GBA and S1-S3 (Figure 2C).

[0279] Since S1-S3 expression was reduced with the bicistronic vector, the results of low phosphotransferase expression on GBA phosphorylation were determined. For this purpose, conditional media of pLL11, GBA alone, and GBA co-transfected with S1-S3 were collected, and the degree of phosphorylation was quantified by performing cation-independent mannose 6-phosphate receptor (CI-MPR) binding experiments. Binding of GBA produced with the bicistronic vector of this disclosure to CI-MPR was higher in the plateau phase (Figure 3A). Nevertheless, when the percentage of receptor binding was calculated using a linear range point, 44% of the GBA produced with the disclosed bicistronic vector bound to CI-MPR, which is the same as GBA produced by co-transfection with S1-S3 (43%) and 10 times higher than GBA produced with endogenous phosphotransferase (4.5%, Figure 3B).

[0280] Titer measurement is widely used in the art to determine the concentration of identified analytes. Titer measurement was performed on the concentration of CI-MPR in binding experiments. For receptor binding assays, serially diluted CI-MPR was immobilized in 96-well plates, and GBA enzyme produced by the bicistronic vector of this disclosure or endogenous phosphatase (Ptase) was added to the plates in similar amounts. As shown in Figure 3C, binding of GBA from pLL11 samples was dependent on the concentration of CI-MPR, saturating when the receptor concentration reached 15 μg / ml, while binding of GBA produced by endogenous Ptase remained at low levels. These data demonstrate that the disclosed bicistronic vector significantly increases the phosphorylation level of the GBA enzyme.

[0281] Acid α-glucosidase (GAA): The lysosomal enzyme GAA is essential for the breakdown of glycogen into glucose in lysosomes. Mutations in the GAA gene are associated with Pompe disease, a lysosomal storage disorder. To create the GAA bicistronic plasmid pLL21, a 2859 base pair (bp) human GAA gene fragment containing a stop codon was amplified and inserted into the bicistronic vector pLL01 after digestion with restriction enzymes NheI and NotI (Figure 4A). The sequenced pLL21 and GAA plasmids were transfected into Expi293 cells. After 48 hours, the conditional medium was collected for GAA activity and CI-MPR binding experiments. Similar to GBA, GAA activity in pLL21 conditional medium was higher than in GAA monoexpression (Figure 4B). Binding in pLL21 conditional medium was faster and more pronounced than in GAA monoexpression medium (Figure 4C). During a one-hour incubation period, 72.5% of GAA derived from pLL21 medium bound to CI-MPR, compared to only 21.5% of GAA from single-expression GAA (Figure 4D). These data suggest that the bicistronic expression platform of this disclosure can significantly increase the phosphorylation of the GAA enzyme.

[0282] Galactosylceramidase (GALC): In lysosomes, the GALC enzyme is responsible for the catabolism of galactosylceramide by removing galactose from ceramide derivatives. Genetic deficiency of the GALC enzyme is the cause of Krabbe disease. To test the GALC enzyme in bicistronic expression as disclosed herein, the bicistronic plasmid pLL31 was generated by inserting the mouse GALC gene into the vector pLL01 (Figure 5A). GALC enzyme activity in pLL31-conditioned medium collected from Expi293 cells transfected with pLL31 was similar to that in GALC alone (0.86 nmol / μl / hour vs. 0.62 nmol / μl / hour, Figure 5B). Binding to the CI-MPR receptor showed that bicistronic expression of GALC with S1-S3 increased GALC binding to CI-MPR from 28.4% to 56.8% (Figure 5C&D).

[0283] α-N-acetylglucosaminidase (NAGLU): The NAGLU gene encodes an enzyme that degrades heparin sulfate in lysosomes. Deficiencies in the NAGLU enzyme lead to Sanfilippo syndrome type B, also known as mucopolysaccharidosis (MPS) IIIB. The NAGLU enzyme produced in cell lines for ERT lacks any phosphate group at the mannose residue. Clinical trials of the NAGLU enzyme for ERT also failed earlier this year. The same procedure as described above was used to express NAGLU in the bicistronic vector of this disclosure. A 2229 bp human NAGLU gene was inserted into the pLL01 bicistronic vector (Figure 6A), and Expi293 cells were transfected with the NAGLU bicistronic plasmid-pLL41 and the NAGLU single-expression plasmid. Using conditional medium, NAGLU activity in sample pLL41 was shown to be higher than in the NAGLU single-expression sample (Figure 6B). Regarding binding to CI-MPR, almost no NAGLU binding was detected in NAGLU monoexpression samples, even when large amounts of enzyme (up to 9 nmol / hour, Figures 6C-6D) were introduced. However, NAGLU produced by a bicistronic vector bound to CI-MPR by up to 25% (Figures 6C-6D).

[0284] α-Galactosidase (GLA): The lysosomal enzyme GLA hydrolyzes melibiose to galactose and glucose and can also metabolize globotriaosylceramide (GL-3). Deficiency in GLA enzyme activity leads to Fabry disease, an X-linked disorder. To create the GLA bicistronic plasmid pLL51, human GLA gene fragments and the bicistronic vector pLL01 were digested with BamHI and NotI and ligated with T4 ligase (Figure 7A). Accurate pLL51 clones and single GLA plasmids were transfected into Expi293 cells and expressed. GLA activity assays and binding experiments to CI-MPR were performed using these conditional media. As shown in Figure 7B, GLA activity was similar in both GLA alone and in the pLL51 conditional medium. Titeral curves using these two media suggest that the pLL51 sample binds to CI-MPR more and faster than the GLA sample (Figure 7C). The overall binding percentage for the pLL51 sample was 62.1%, which is almost double that of the GLA sample (33.1%, Figure 7D).

[0285] Acid α-mannosidase (LAMAN): The genetic disorder α-mannosidosis is caused by a defect in LAMAN, a lysosomal enzyme encoded by the MAN2B1 gene. Since the human LAMAN enzyme is largely unphosphorylated, hLAMAN is a good candidate for disclosed bicistronic expression. A 3033 bp human LAMAN gene was inserted into the pLL01 bicistronic vector for later testing (Figure 8A) and expressed in Expi293 cells. LAMAN activity in the LAMAN bicistronic plasmid pLL61 condition medium was slightly lower than that of LAMAN monoexpression (Figure 8B). Titer analysis was performed for LAMAN binding to CI-MPR. Binding of the LAMAN enzyme to CI-MPR was barely detectable using the LAMAN monoexpression sample, but a large amount of LAMAN enzyme from the pLL61 sample was found to interact with CI-MPR (Figure 8C). Using S1-S3 bicistronic expression, the binding of LAMAN to CI-MPR increased from 1.6% to 75.2% (Figure 8D).

[0286] The six enzymes described above can be categorized into two groups based on their basal phosphorylation levels. Group 1 consists of low-phosphorylated lysosomal enzymes (GBA, NAGLU, and LAMAN), which are poor substrates for wild-type Ptase during enzyme production. Group 2 consists of high-phosphorylated enzymes (GAA, GALC, and GLA). These enzymes are considered good substrates for wild-type Ptase and receive a substantial amount of phosphate. Bi-cistronic expression of S1-S3 of the Disclosure has been shown to significantly increase the phosphorylation of the six lysosomal enzymes, regardless of their basal phosphorylation levels. Considering these findings, the bi-cistronic vector pLL01 disclosed herein can be used to produce highly phosphorylated lysosomal enzymes to treat all lysosomal storage diseases. Clearly, the bi-cistronic vector of the Disclosure offers significant benefits to ERT and gene therapy for treating lysosomal storage diseases.

[0287] (Example 3) Treatment for Gaucher disease Enzyme replacement therapy (ERT)

[0288] Expression vectors containing sequences encoding GBA and S1-S3 Ptase can be used to treat or prevent the signs or symptoms of Gaucher disease. The following tests demonstrate that expression of (GCase / GBA)-S1-S3 in a standard mouse model of Gaucher disease understood in the art leads to (GCase / GBA)-S1-S3 expression, transport of v-S1-S3 into cells from the circulating bloodstream, and increased activity of v in cells that have taken up the v-S1-S3 complex. A small increase in (GCase / GBA) activity resulting from the expression and uptake of the GBA-S1-S3 complex leads to significant functional recovery in the mouse model.

[0289] Expression of GBA using a bicistronic expression vector containing S1-S3 PTase generates a recombinant protein with high levels of phosphorylated oligosaccharides that can be used to treat or prevent the signs or symptoms of Gaucher disease. The following studies demonstrate that ERT using recombinant protein expressed with a bicistronic expression vector containing S1-S3 PTase in a standard mouse model of Gaucher disease understood in the art leads to a longer half-life, greater tissue uptake, greater substrate reduction, and better correction of histopathological values ​​compared to current standard therapeutics.

[0290] Figures 16A-16B show a 20-week-old Gaucher D409V / ヌルThis is a pair of graphs showing elevated glucosylceramide levels observed in the liver, lungs, and spleen of mice. The accumulation of glucocerebroside, a natural substrate of GBA, was determined in tissue homogenates. The accumulation of GC in the lungs is a statistically and therapeutically valuable result, representing a known yet unaddressed need for current standard therapeutics. From 20 μL of a constant volume of tissue homogenate and a suitable control, glucosylceramide was extracted by adding 200 μL of methanol / ACN / H2O (v:v:v=85:10:5), mixing at 800 rpm for 5 minutes, and then centrifugation at 3220 g at 4°C for 15 minutes;3). 50 μL of the supernatant was collected, dried with nitrogen, resuspended in methanol / ACN / H2O (v:v:v=85:10:5), and directly injected for LC-MS / MS analysis.

[0291] Figures 17A-17C show the GBA D409V / ヌル In mouse models, GCase M6PThis is a series of graphs demonstrating that has a longer half-life and greater tissue uptake compared to imiglucerase. PK / PD studies were performed in a Gaucher D409V / null mouse model using imiglucerase, the standard therapeutic agent, and purified GBA produced by transient co-expression of S1-S3 PTase and a bicistronic vector encoding a native variant of GBA in Expi293 cells. This GCase variant exhibits greater stability under neutral and slightly alkaline conditions. Briefly, three animals were injected via tail vein at approximately 1.5 mg / kg of recombinant GCase. For serum pharmacokinetic data, plasma samples were collected at 2, 10, 20, 40, and 60 minutes. Activity was measured using the synthetic substrate 4-methylumbelliferyl-beta-D-glucopyranoside (4MU-Glc). Activity in individual animals was normalized by setting the 2-minute time point as 100% activity and expressing subsequent time points as percentages relative to t=2 minutes. Stabilized GCase expressed in the presence of S1-S3 PTase appears to have a longer half-life. This longer half-life is a combination of the enzyme's greater stability and a different clearance pathway. To determine how much GCase was taken up by tissue, tissue was collected, homogenized, and activity measured using 4MU-Glc substrate 2 hours after enzyme injection. Activity was normalized to total protein in the homogenate determined by BCA for protein determination. The true benefit of stable GCase with reasonable phosphorylation is evident in the shown tissue uptake data. Greater activity is found for stabilized GCase expressed using the bicistronic S1-S3 PTase vector platform S1'S3 PTase in all tissues evaluated. This is most dramatic in the lung, muscle, and brain, where imiglucerase activity is minimal. When combining tissue and serum data, the advantages of more stable GCases with greater N-linked oligosaccharide phosphorylation are evident in terms of delivering more enzymes to affected tissue.This marks the first time that significant amounts of GCase have been delivered to the lungs, muscles, and heart at these doses.

[0292] Figures 18A to 18E show the GBA D409V / ヌル In the mouse model, GCase M6P These are a series of photographs and bar graphs demonstrating that ERT reduced tissue macrophages more effectively than imiglucerase (anti-CD68 staining). Efficacy testing in a D409V Gaucher mouse model was performed using the standard therapeutic agent Cerezyme and purified GBA (M0111) transiently co-expressed in Expi293 cells using a bicistronic vector encoding S1S3 PTase and a native variant of GBA reported to be more stable under neutral and slightly alkaline conditions. Approximately 20-week-old Gaucher mice were treated with approximately 1.5 mg / kg of the enzyme once a week for 4 weeks. After 4 weeks, liver and lung tissues were collected for immunohistochemical examination using CD68 antibody and fixed in 4% paraformaldehyde-PBS, pH 7.4. M0111 exhibits greater efficacy compared to the current standard therapeutic agent, as evidenced by the reduction in macrophages in the affected tissue visualized by CD68 Ab.

[0293] Figures 19A-19C show the GBA D409V / ヌル In the mouse model, GCase M6PA series of photographs demonstrating that ERT reduced the number and size of Gaucher storage cells better than imiglucerase (hematoxylin and eosin (H&E) staining). The efficacy test in the D409A Gaucher mouse model was performed using purified GBA transiently co-expressed in Expi293 cells using a bicistronic vector encoding a natural variant of GBA that has been reported to have greater stability under neutral and slightly alkaline conditions compared to the standard therapeutic agent Cerezyme and S1-S3 PTase. Gaucher mice of approximately 20 weeks of age were treated once a week for 4 weeks with approximately 1.5 mg / kg of the enzyme. After 4 weeks, liver and lung tissues were collected and fixed in 4% paraformaldehyde-PBS, pH 7.4 for formalin treatment for hematoxylin and eosin (H&E) staining. As demonstrated by the decrease in storage cells in the affected tissues visualized by H&E staining, GCase M6P has greater efficacy compared to the current standard therapeutic agent.

[0294] Figures 20A - 20B show that in the GBA D409V / ヌル mouse model, GCase M6PThis is a pair of graphs demonstrating that ERT reduced the accumulated substrate more effectively than imiglucerase. Gaucher mice, approximately 20 weeks old, were treated with approximately 1.5 mg / kg of enzyme once a week for 4 weeks. Tissue samples were collected and homogenized for glycosylceramide analysis. The accumulation of glucocerebroside, a natural substrate of GCase, was determined in tissue homogenates. The accumulation of GC in the lungs is a known unaddressed need for current standard therapeutics and is of great value. From 20 μL of a constant volume of tissue homogenate and a suitable control, glucosylceramide was extracted by adding 200 μL of methanol / ACN / H2O (v:v:v=85:10:5), mixing at 800 rpm for 5 minutes, and then centrifugation at 3220 g at 4°C for 15 minutes;3). 50 μL of the supernatant was collected, dried with nitrogen, resuspended in methanol / ACN / H2O (v:v:v=85:10:5), and directly injected for LC-MS / MS analysis. For the two ceramides measured, GCase M6P Animals treated with ERT therapy had lower levels of imiglucerase than other enzymes after ERT therapy.

[0295] gene therapy

[0296] The signs or symptoms of Gaucher disease can be treated or prevented using a delivery vector having a bicistronic vector containing a sequence encoding GBA and a sequence encoding S1-S3 PTase. In some embodiments, the delivery vector is a viral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector is a nonviral vector. In some embodiments, the nonviral vector is a liposome, LNP, polymer nanoparticle, nanoparticle, micelle, polymerosome, or exosome. The following tests demonstrate the expression of GBA and S1-S3 PTase using a bicistronic vector in a standard mouse model of Gaucher disease known in the art, thereby improving GBA M6P This demonstrates that increased expression, increased activity in tissues and serum, and decreased substrate levels are achieved. This shows that phosphorylated transgene products with high affinity for CI-MPR can lead to effective therapy, even at low activity levels, due to efficient cellular uptake and targeting to lysosomes.

[0297] Figures 21A–21D are a series of graphs showing the results of in vivo AAV-mediated gene therapy trials to treat Gaucher disease. To determine the efficacy of AAV9 gene therapy using a bicistronic transgene of a stable GBA+S1-S3 PTase with three different promoters, 15-week-old GBA D409V / ヌル Mice were administered a moderate dose of AAV9-stable GBA+S1-S3 PTase, 5E11vg. To determine how much GBA was generated in the tissue, tissue was collected, homogenized, and its activity was measured using a 4MU-Glc substrate two weeks after AAV9 injection. The activity was normalized to the total protein in the homogenate, which was determined by the BCA method for protein determination.

[0298] Figures 29A-29C show AAV9-hTLV-GBA in Gaucher mice. M6P This is a series of graphs showing enzyme activity and GCase substrate selection in the lungs and liver two weeks after gene therapy injection. AAV9-hTLV-GBA-S1S3 is also shown as AAV9-hTLV-GBA M6P It is publicly known as, where M6P refers to the S1S3 construct. AAV9 hTLV-GBA or AAV9 hTLV-GBA M6P Two weeks after transgene administration (with a bicistronic vector containing GBA and S1-S3 PTase), the expression of both constructs increased in the liver (Figure 29A). Liver glucosyl-β-ceramide levels were measured (Figures 29B and C), and AAV9 hTLV-GBA M6P In animals treated with AAV9, the greatest reduction in accumulated substrate was observed, despite lower GCase activity in the liver compared to animals treated with AAV9 hTLV-GBA. This greater substrate reduction at low activity suggests that N-linked oligosaccharide phosphorylation is important for gene therapy in terms of cellular uptake and targeting to lysosomes. In the lungs, GCase activity was low in animals treated with AAV9. However, AAV9-hTLV-GBA M6P In animals treated with [the specified agent], a significant reduction in accumulated glucosyl-β-ceramide levels in the lungs was observed (Figure 29B, C). A slight reduction was observed in animals treated with AAV9-hTLV-GBA. This demonstrates that phosphorylated transgene products with high affinity for CI-MPR can lead to effective treatment, even at low activity levels, due to efficient cellular uptake and targeting to lysosomes.

[0299] (Example 4) Treatment of α-mannosidosis Enzyme replacement therapy (ERT)

[0300] Expression vectors containing sequences encoding LAMAN and S1-S3 Ptase can be used to treat or prevent signs or symptoms of α-mannosidosis. The following tests demonstrate that expression of LAMAN-S1-S3 in a mouse model leads to increased LAMAN activity in cells that have taken up the LAMAN-S1-S3 complex, as well as increased LAMAN expression, transport of LAMAN-S1-S3 into cells from the circulating bloodstream, and uptake of the LAMAN-S1-S3 complex. A small increase in LAMAN resulting from the expression and uptake of the LAMAN-S1-S3 complex leads to significant functional recovery in the mouse model.

[0301] Expression of LAMAN using a bicistronic expression vector containing S1-S3 PTase generates a recombinant protein with high levels of phosphorylated oligosaccharides that can be used to treat or prevent signs or symptoms of α-mannosidosis. The following tests demonstrate that ERT using recombinant LAMAN protein expressed using a bicistronic expression vector containing S1-S3 PTase in wild-type mice leads to greater tissue uptake and wider distribution in tissues.

[0302] Figures 22A–22C are a series of graphs showing the results of in vitro studies on the use of lysosomal alpha-mannosidase (LAMAN) as an ERT (Endoscopic Transdermal Retention Therapy).

[0303] Figures 23A-23B show photographs and corresponding data tables illustrating the expression, purification, and characterization of the LAMAN enzyme. Two preparations of LAMAN were transiently co-expressed in Expi293 cells with or without a bicistronic vector encoding S1-S3 PTase (M0611). Both were purified using an HPC4 affinity tag. A significant increase in phosphorylation was demonstrated by measuring the amount of LAMAN dose-dependently bound to an immobilized cation-independent mannose 6-phosphate receptor. The amount of bound LAMAN was based on the activity of LAMAN using the LAMAN synthesis substrate 4-methylumbelliferyl-α-D-mannopyranoside (4MU-Man). The specificity of binding via phosphorylated oligosaccharides was confirmed by the ability to block the binding of added mannose 6-phosphate. M6P It should be noted that (M0611) can bind to the receptor even in the presence of M6P. LAMAN M6P (M0611, P-0030) and LAMAN (P-0031) were selected for in vivo animal testing.

[0304] Figure 23C shows LAMAN M6P This graph shows the expression, purification, and characterization of the (M0611) enzyme. Two preparations of LAMAN were transiently co-expressed in Expi293 cells, with or without a bicistronic vector encoding S1-S3 variants of PTase. Both were purified using an HPC4 tag. A significant increase in phosphorylation was demonstrated by measuring the amount of LAMAN that dose-dependently bound to an immobilized cation-independent mannose 6-phosphate receptor. The amount of bound LAMAN was determined by activity using the synthetic substrate 4-methylumbelliferyl-α-D-mannopyranoside (4MU-Man). The specificity of binding via phosphorylated oligosaccharides was confirmed by the ability to block the binding of added mannose 6-phosphate. It should be noted that M0611 can bind to the receptor even in the presence of M6P. LAMAN M6P(M0611, P-0030) and LAMAN (P-0031) were selected for in vivo animal testing.

[0305] Figures 24A-24B show the relationship between LAMAN and LAMAN in wild-type mice regarding enzyme replacement therapy. M6P A pair of graphs demonstrating the distribution of enzymes in the body. LAMAN and LAMAN M6P To evaluate the differences in tissue uptake between LAMAN (co-expressed with S1-S3 PTase), each preparation at 2 mg / kg was injected into wild-type mice (n=4) via the tail vein. Tissues were collected, homogenized, and activity measured using 4MU-Man substrate 2 and 8 hours after administration. Activity was normalized to total protein in the homogenate determined by BCA for protein determination. LAMAN M6P The advantages of (LAMAN co-expressed with S1S3 PTase) were observed in tissue uptake data. Higher activity was observed in the liver, spleen, heart, lungs, and brain at 2 hours. This trend remained true at 8 hours, with the exception of the lungs. This may be a result of the high variability observed in the analysis of this tissue. The only exception to this observation was the kidney. Endogenous LAMAN activity was subtracted from all samples. Our LAMAN M6P Higher LAMAN enzyme activity was detected in most tissues of mice injected with the enzyme.

[0306] Figures 25A-25B show the relationship between αLAMAN and LAMAN in wild-type mice regarding enzyme replacement therapy. M6P A pair of graphs demonstrating the distribution of enzymes in the body. LAMAN and LAMAN M6P To evaluate the differences in tissue uptake between LAMAN (co-expressed with S1-S3 PTase), each preparation was injected via tail vein into wild-type mice (n=4) at a concentration of 10 mg / kg. Tissues were collected, homogenized, and activity was measured using 4MU-Man substrate 2 and 8 hours after administration. Activity was normalized to the total protein in the homogenate, determined by BCA for protein determination. LAMANM6P The advantages of LAMAN (co-expressed with S1-S3 PTase) were observed in tissue uptake data. Higher activity was observed in the liver, spleen, heart, lungs, and brain at 2 hours. This trend remained true at 8 hours, with the exception of the kidney. This may be a result of the high variability observed in the analysis of this tissue.

[0307] gene therapy

[0308] A delivery vector containing a sequence encoding LAMAN and a sequence encoding S1-S3 modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase) can be used to treat or prevent signs or symptoms of α-mannosidosis. In some embodiments, the delivery vector is a viral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector is a nonviral vector. In some embodiments, the nonviral vector is a liposome, LNP, polymer nanoparticle, nanoparticle, micelle, polymerosome, or exosome. The following studies demonstrate that expression of LAMAN-S1-S3 in a mouse model of α-mannosidosis leads to increased LAMAN-S1-S3 expression, transport of LAMAN-S1-S3 into cells from the circulating bloodstream, and increased LAMAN activity in cells that have taken up the LAMAN-S1-S3 complex. A small increase in v resulting from the expression and uptake of the LAMAN-S1-S3 complex leads to significant functional recovery in the mouse model.

[0309] Alternatively, or in addition to the above, signs or symptoms of α-mannosidosis can be treated or prevented using a delivery vector containing sequences encoding S1-S3 modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). Expression of S1-S3 can increase the uptake of endogenous LAMAN by somatic tissues, thereby inducing significant functional recovery in mouse models.

[0310] (Example 5) Treatment of mucolipidosis Enzyme replacement therapy (ERT)

[0311] An expression vector containing a sequence encoding a modified S1-S3 GlcNAc-1 phosphotransferase (GlcNAc-1 PTase) can be used to treat or prevent signs or symptoms of mucolipidosis. The following tests demonstrate that S1-S3 expression leads to S1-S3 expression, transport of S1-S3 and one or more lysosomal enzymes into cells from the circulating bloodstream, and increased activity of one or more lysosomal enzymes in cells that have taken up the S1-S3 complex. A small increase in the S1-S3 complex resulting from the expression and uptake of the S1-S3 complex and one or more lysosomal enzymes leads to a significant functional recovery.

[0312] gene therapy

[0313] A delivery vector containing a sequence encoding S1-S3 modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase) can be used to treat or prevent signs or symptoms of mucolipidosis. In some embodiments, the delivery vector is a viral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector is a nonviral vector. In some embodiments, the nonviral vector is a liposome, LNP, polymer nanoparticle, nanoparticle, micelle, polymerosome, or exosome. A delivery vector containing a sequence encoding soluble S1-S3 modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase) can be used to treat or prevent signs or symptoms of mucolipidosis. A delivery vector containing a sequence encoding a modified S1-S3 GlcNAc-1 phosphotransferase (GlcNAc-1 PTase) can be used to treat or prevent signs or symptoms of mucolipidosis. The following tests demonstrate that expression of S1-S3 PTase leads to S1-S3 intracellular activity, resulting in the correction of misdelivered serum levels of lysosomal enzymes by increasing their N-linked oligosaccharide phosphorylation, thereby enabling efficient targeting to lysosomes.

[0314] Alternatively, or in addition to the above, signs or symptoms of mucolipidosis can be treated or prevented using a delivery vector containing a sequence encoding S1-S3 modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). Expression of S1-S3 PTase can increase the uptake of one or more endogenous lysosomal enzymes by body tissues, thereby inducing significant functional recovery in mouse models.

[0315] Figures 26A-26B are schematic diagrams and graphs showing the design and in vitro testing of AAV9 gene therapy (GTx) for mucolipidosis. 293T cells were transduced using various M0021 (AAV9-CAGp-S1-S3) viruses, cultured for 2 days, and then subjected to a PTase activity assay.

[0316] Figures 27A and 27B are a pair of graphs demonstrating that M0021 treatment reduces serum lysosomal enzyme levels in ML II mice. To determine the efficacy of S1-S3 PTase gene therapy, 34-week-old female mice were administered a moderate dose of M0021 (AAV9-CAGp-S1-S3), 4e12vg (2e13vg / kg). One of the ML II phenotypes is elevated serum levels of lysosomal enzymes due to the inability to target lysosomal enzymes to intracellular lysosomes. Promising results were observed when serum LAMAN and ManB activity decreased exactly one week after treatment. This result is significant as it demonstrates the ability of the MLII mouse model to exhibit the described phenotype.

[0317] Figures 28A–28C are a series of graphs demonstrating that M0021 treatment increases lysosomal enzyme phosphorylation in ML II mice. To further understand the effect of S1-S3 PTase gene therapy on the decrease in serum activity of LAMAN and ManB, we evaluated the binding of enzymes found in serum to CI-MPR using a previously described immobilized receptor binding assay. Briefly, known amounts of enzyme are added to immobilized CI-MPR in escalating doses. Unbound enzymes are washed away, and the remaining bound enzymes are measured using a suitable synthetic substrate; Man-b-4MU(ManB, LAMAN 4MU-Man(LAMAN)). AAV9-S1S3 gene therapy increases glycan phosphorylation of lysosomal enzymes in ML II mice. Total phosphorylated lysosomal enzymes in serum normalized to normal or slightly elevated levels after 3 weeks.

[0318] All patents, patent applications, and publications cited herein are thus incorporated herein by reference in their entirety. While this disclosure is made with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of this disclosure can be conceived without departing from the true spirit and scope of this disclosure. The appended claims are intended to be construed as encompassing all such embodiments and equivalent variations. In certain embodiments, for example, the following are provided: (Item 1) A composition comprising a vector containing a promoter, a sequence encoding a first polynucleotide encoding a lysosomal enzyme, and a sequence encoding a second polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase), wherein the promoter is capable of driving expression in mammalian cells, and the promoter is operably linked to the first polynucleotide and the second polynucleotide. (Item 2) The composition according to item 1, wherein the vector further comprises a sequence encoding an intra-sequence ribosome entry site (IRES). (Item 3) The composition according to item 2, wherein the sequence encoding the IRES is located between the sequence encoding the lysosomal enzyme and the sequence encoding the modified GlcNAc-1 PTase. (Item 4) The composition according to item 2 or 3, wherein the vector comprises, from 5' to 3', a sequence encoding the modified GlcNAc-1 PTase, a sequence encoding the IRES, and a sequence encoding the lysosomal enzyme. (Item 5) The composition according to item 2 or 3, wherein the vector comprises, from 5' to 3', a sequence encoding the lysosomal enzyme, a sequence encoding the IRES, and a sequence encoding the modified GlcNAc-1 PTase. (Item 6) The composition according to item 1, wherein the vector further comprises an array encoding a cleavage site. (Item 7) The composition according to item 6, wherein the cleavage site comprises a sequence encoding a 2A self-cleaving peptide. (Item 8) The composition according to any one of items 1 to 7, wherein the vector is an expression vector. (Item 9) The composition according to any one of items 1 to 7, wherein the vector is a delivery vector. (Item 10) The composition according to any one of items 1 to 9, wherein the vector is a nonviral vector. (Item 11) The composition according to any one of items 1 to 10, wherein the vector is a viral vector. (Item 12) The composition according to item 11, wherein the vector is a lentiviral vector. (Item 13) The composition according to item 11, wherein the vector is an adenovirus vector or an adeno-associated virus (AAV) vector. (Item 14) The composition according to item 13, wherein the AAV vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. (Item 15) The composition according to item 13 or 14, wherein the AAV vector comprises a sequence encoding a capsid isolated from or derived from one or more serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. (Item 16) The composition according to any one of items 13 to 15, wherein the AAV vector comprises a sequence encoding at least one terminal inversion sequence (ITR) isolated from or derived from one or more serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. (Item 17) The composition according to any one of items 1 to 16, wherein the vector is a bicistronic vector. (Item 18) The composition according to any one of items 1 to 16, wherein the vector is a multi-cistronic vector. (Item 19) The composition according to any one of items 1 to 18, wherein the promoter includes a constitutive promoter. (Item 20) The composition according to item 19, wherein the constitutive promoter comprises a cytomegalovirus (CMV) promoter. (Item 21) The composition according to any one of items 1 to 20, wherein the vector comprises the nucleic acid sequence of sequence number 1. (Item 22) The composition according to any one of items 1 to 21, wherein the polynucleotide encoding the modified GlcNAc-1 phosphotransferase comprises the nucleic acid sequence of SEQ ID NO: 4. (Item 23) The composition according to any one of items 1 to 22, wherein the lysosomal enzyme is involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C. (Item 24) The composition according to item 23, wherein the lysosomal enzyme comprises at least one lysosomal enzyme listed in Table 1A, Table 1B, or Table 1C. (Item 25) The composition according to any one of items 1 to 21 or item 24, wherein the lysosomal enzyme is selected from the group consisting of β-glucocerebrosidase (GBA), galactosylceramidase (GALC), α-galactosidase (GLA), α-N-acetylglucosaminidase (NAGLU), acid α-glucosidase (GAA), and lysosomal acid α-mannosidase (LAMAN). (Item 26) The composition according to any one of items 1 to 21 or item 24, wherein the lysosomal enzyme comprises β-glucocerebrosidase (GBA). (Item 27) The composition according to item 26, wherein the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of Sequence ID No. 5. (Item 28) The composition according to any one of items 1 to 21 or 24, wherein the lysosomal enzyme comprises galactosylceramidase (GALC). (Item 29) The composition according to item 28, wherein the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 6. (Item 30) The composition according to item 29, wherein the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of SEQ ID NO: 23. (Item 31) The composition according to any one of items 1 to 21 or item 24, wherein the lysosomal enzyme comprises α-galactosidase (GLA). (Item 32) The composition according to item 31, wherein the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of Sequence ID No. 7. (Item 33) The composition according to any one of items 1 to 21 or 24, wherein the lysosomal enzyme comprises α-N-acetylglucosaminidase (NAGLU). (Item 34) The composition according to item 33, wherein the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of Sequence ID No. 8. (Item 35) The composition according to any one of items 1 to 21 or item 24, wherein the lysosomal enzyme comprises acid α-glucosidase (GAA). (Item 36) The composition according to item 35, wherein the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of Sequence ID No. 9. (Item 37) The composition according to any one of items 1 to 21 or 24, wherein the lysosomal enzyme comprises lysosomal acidic α-mannosidase (LAMAN). (Item 38) The composition according to item 37, wherein the polynucleotide encoding the lysosomal enzyme comprises the nucleic acid sequence of Sequence ID No. 10. (Item 39) A method for treating lysosomal storage disorder (LSD), comprising the step of administering to a subject an effective amount of a composition described in any one of items 1 to 38, wherein the composition increases the phosphorylation of lysosomal enzymes that cause the LSD, thereby treating the LSD. (Item 40) The method according to item 39, wherein the subject exhibits signs or symptoms of LSD. (Item 41) The method according to item 39 or 40, wherein the subject has been diagnosed with LSD. (Item 42) A method for preventing the appearance or onset of lysosomal storage disorder (LSD), comprising the step of administering to a subject an effective amount of a composition described in any one of items 1 to 38, wherein the composition increases the phosphorylation of lysosomal enzymes that cause the LSD, thereby preventing the appearance of the LSD in the subject. (Item 43) The method described in item 42, wherein the subject is at risk of developing or experiencing LSD. (Item 44) The method according to item 42 or 43, wherein the subject exhibits signs or symptoms of LSD. (Item 45) A method for improving the phosphorylation of lysosomal enzymes that cause lysosomal storage disorders (LSD), comprising the step of administering to a subject an effective amount of a composition described in any one of items 1 to 38, wherein the phosphorylation of the lysosomal enzymes is increased by the composition. (Item 46) The method according to item 45, wherein the subject exhibits signs or symptoms of LSD. (Item 47) The method described in item 45 or 46, wherein the subject is at risk of developing or experiencing LSD. (Item 48) The method according to item 45 or 46, wherein the subject has received the diagnosis of LSD. (Item 49) A method for improving the phosphorylation of lysosomal enzymes that cause lysosomal storage disorders (LSDs), comprising the step of contacting cells with an effective amount of a composition described in any one of items 1 to 38, wherein the composition increases the phosphorylation of the lysosomal enzymes. (Item 50) The method according to item 49, wherein the cells are present in vitro or ex vivo. (Item 51) The method described in item 49, wherein the aforementioned cells are present in vivo. (Item 52) The method according to any one of items 49 to 51, wherein the subject includes the aforementioned cells. (Item 53) The method according to item 52, wherein the subject exhibits signs or symptoms of LSD. (Item 54) The method described in item 52 or 53, wherein the subject is at risk of developing or experiencing LSD. (Item 55) The method according to item 52 or 53, wherein the subject has been diagnosed with LSD. (Item 56) The method according to any one of items 39 to 55, wherein the lysosomal enzyme is involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C. (Item 57) The method according to any one of items 39 to 56, wherein the lysosomal enzyme is at least one of those listed in Table 1A, Table 1B, or Table 1C. (Item 58) The method according to any one of items 39 to 56, wherein the lysosomal enzyme comprises one or more of β-glucocerebrosidase (GBA), galactosylceramidase (GALC), α-galactosidase (GLA), α-N-acetylglucosaminidase (NAGLU), acid α-glucosidase (GAA), and lysosomal acid α-mannosidase (LAMAN). (Item 59) The method according to any one of items 39 to 58, wherein the administration step includes a systemic route of administration. (Item 60) The method according to item 59, wherein the systemic administration route is enteral, parenteral, oral, intramuscular (IM), subcutaneous (SC), intravenous (IV), intra-arterial (IA), subarachnoid, intraspinal, or intraventricular. (Item 61) The method according to any one of items 39 to 58, wherein the administration step includes a local route of administration. (Item 62) The method according to any one of items 39 to 61, wherein the subject is a human. (Item 63) The method according to any one of items 39 to 62, wherein the subject is male. (Item 64) The method according to any one of items 39 to 62, wherein the subject is female.

Claims

1. A composition comprising a vector, wherein the vector comprises, from 5' to 3', a sequence encoding a promoter, a first polynucleotide encoding a lysosomal enzyme, a sequence encoding an intrasequence ribosome entry site (IRES), and a second polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase), wherein the promoter is capable of driving expression in mammalian cells and is operably linked to the first polynucleotide and the second polynucleotide, the lysosomal enzyme is selected from the group consisting of β-glucocerebrosidase (GBA), galactosylceramidase (GALC), α-galactosidase (GLA), α-N-acetylglucosaminidase (NAGLU), acid α-glucosidase (GAA), and lysosomal acid α-mannosidase (LAMAN), and the modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). A composition wherein PTase) is encoded by a polynucleotide containing the nucleic acid sequence of Sequence ID No. 4, and is capable of phosphorylating the lysosomal enzyme.

2. The composition according to claim 1, wherein the vector further comprises an array encoding a cleavage site.

3. The composition according to claim 1, wherein the vector is an expression vector or a delivery vector.

4. The vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. (a) comprising a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9; (b) A sequence comprising a sequence encoding a capsid isolated from or derived from one or more serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9; and / or (c) The composition according to claim 3, comprising a sequence encoding at least one terminal inversion (ITR) isolated from or derived from one or more serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

5. The composition according to claim 1, wherein the vector is a bicistronic vector or a multicistronic vector.

6. The composition according to claim 1, wherein the promoter includes a constitutive promoter.

7. The composition according to claim 1, wherein the second polynucleotide encoding the modified GlcNAc-1 PTase comprises the nucleic acid sequence of SEQ ID NO:

4.

8. The composition according to claim 1, wherein the vector comprises the nucleic acid sequence of sequence number 1.

9. The composition according to claim 1, wherein the lysosomal enzyme is involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C.

10. The aforementioned lysosomal enzyme, (a) Contains β-glucocerebrosidase (GBA); (b) containing galactosylceramidase (GALC); (c) Contains α-galactosidase (GLA); (d) Contains α-N-acetylglucosaminidase (NAGLU); (e) containing acid α-glucosidase (GAA); or (f) The composition according to claim 9, comprising lysosomal acidic α-mannosidase (LAMAN).

11. The composition according to claim 1 for use in the following method: (a) A method for treating lysosomal storage disease (LSD), comprising the step of administering an effective amount of the composition to a subject, wherein the composition increases the phosphorylation of lysosomal enzymes that cause the LSD, thereby treating the LSD; (b) A method for preventing the appearance or development of lysosomal storage disease (LSD), comprising the step of administering an effective amount of the composition to a subject, wherein the composition increases the phosphorylation of lysosomal enzymes that cause the LSD, thereby preventing the appearance or development of the LSD; (c) A method for improving the phosphorylation of lysosomal enzymes that cause lysosomal storage disorders (LSD), comprising the step of administering the composition to a subject in an effective amount, wherein the phosphorylation of the lysosomal enzymes increases with the composition; or (d) A method for improving the phosphorylation of lysosomal enzymes that cause lysosomal storage disorders (LSDs), comprising the step of contacting cells with an effective amount of the composition, wherein the phosphorylation of the lysosomal enzymes is increased by the composition.

12. The composition for use according to claim 11, wherein the lysosomal enzyme is involved in at least one lysosomal storage disorder (LSD) listed in Table 1A, Table 1B, or Table 1C.

13. The aforementioned administration step is (a) including systemic routes of administration; or (b) Including local administration routes, The composition for use according to claim 11.

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