Methods of using viral vector constructs for the treatment of Fabry disease

Abstract

The present disclosure provides a dose of approximately 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The present invention relates to a method for treating or ameliorating one or more symptoms of Fabry disease, reducing the amount of glycosphingolipids, and / or increasing the activity of α-galactosidase A (α-Gal A) protein in a subject in need thereof by administering an expression vector (e.g., an AAV expression vector) comprising an α-galactosidase A (α-Gal A) expression cassette that includes an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein at a dose of 100 mg / kg.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing dates of U.S. Provisional Patent Application No. 63 / 275,390, filed November 3, 2021, and U.S. Provisional Patent Application No. 63 / 373,826, filed August 29, 2022, the entire contents of both of which are incorporated herein by reference.

[0002] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING The contents of the electronically submitted sequence listing in ASCII text file format (Name: 4341_024PC02_Seqlisting_ST26, Size: 16,280 bytes, Creation Date: November 2, 2022) submitted at the time of filing is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to methods for treating or ameliorating one or more symptoms of Fabry disease, reducing the amount of glycosphingolipids, and / or increasing the activity of α-galactosidase A (α-Gal A) protein in a subject in need thereof by administering a therapeutically effective amount of an expression vector (e.g., an adeno-associated virus (AAV) expression vector) comprising an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein. [Background technology]

[0004] Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of the enzyme α-galactosidase A (GLA). As a result, the terminal α-galactosyl moiety of globotriaosylceramide (Gb3) is not hydrolyzed, leading to accumulation of Gb3 in lysosomes and elsewhere within the cell. Early characteristic clinical symptoms include severe neuropathic pain (acral paresthesia), skin lesions (angiokeratoma), and ocular manifestations (corneal verticillate opacities). In later life, cardiac, renal, and cerebrovascular complications cause severe morbidity and shorten life expectancy. Desnick et al., α-Galactosidase A Deficiency: Fabry Disease, in: Beaudet et al. (Ed.), The Online Metabolic and Molecular Bases of Inherited Disease, The McGraw-Hill Companies, Inc., New York, NY (2014);Van der Veen SJ et al., Mol Genet Metab. 126(2):162-168 (2019).

[0005] Since 2001, Fabry disease patients have been treated with two different enzyme replacement therapies (ERT) based on infusion of recombinant enzymes (agalsidase α and agalsidase β). Eng et al., N Engl J Med 345:9-16(2001); Schiffmann et al., JAMA285:2743-2749(2001); Lenders et al., J Am Soc Nephrol.27(1):256-64(2016). These treatments only treat symptoms, but do not provide a cure, so patients must receive repeated doses of these proteins throughout their lives.

[0006] Studies suggest that infusion of recombinant enzyme may lead to the formation of anti-GLA neutralizing antibodies, which may result in short-term acute complications, as well as long-term adverse effects due to treatment inhibition, leading to a significant decrease in Gb3 and lyso-Gb3. Lenders et al.,J Am Soc Nephrol.27(1):256-64(2016). In typical male patients with Fabry disease, treatment with ERT delays the development of complications, especially if treatment is started before the onset of irreversible organ damage. However, more than half of typically affected male patients treated with ERT develop anti-GLA neutralizing antibodies. In female patients and in patients with atypical disease phenotypes, antibody formation against the administered recombinant enzyme is rarely observed. Van der Veen SJ et al.,Mol Genet Metab. 126(2):162-168 (2019).

[0007] Thus, there remains a need for non-ERT methods and compositions that can be used to treat Fabry disease, including, for example, treatment by genome editing to deliver therapeutically relevant levels of an expressed transgene-encoded gene product.

[0008] In some aspects, provided herein is a method of treating Fabry disease or ameliorating one or more symptoms associated with Fabry disease in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, and a bovine growth hormone polyA signal sequence, wherein the AAV expression vector is administered to the subject at a concentration of about 5×10 12 Vector genome (vg / kg) ~ approx. 5 × 10 13The drug is administered at a dose of 100 mg / kg.

[0009] In some embodiments, provided herein is a method of treating Fabry disease or ameliorating one or more symptoms associated with Fabry disease in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an α-Gal A transgene encoding at least one α-Gal A protein, wherein the α-Gal A transgene comprises the nucleotide sequence set forth in SEQ ID NO:5, and the AAV expression vector is administered at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The drug is administered at a dose of 100 mg / kg.

[0010] In some aspects, provided herein is a method of decreasing the amount of glycosphingolipids in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, and a bovine growth hormone polyA signal sequence, wherein the AAV expression vector is administered to the subject at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The amount of glycosphingolipid that is administered is at a dose of vg / kg and is reduced relative to the amount of glycosphingolipid in the subject prior to administration.

[0011] In some embodiments, provided herein is a method of reducing the amount of glycosphingolipids in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an α-Gal A transgene encoding at least one α-Gal A protein, wherein the α-Gal A transgene comprises the nucleotide sequence set forth in SEQ ID NO:5, and the AAV expression vector is administered at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The amount of glycosphingolipid that is administered is at a dose of vg / kg and is reduced relative to the amount of glycosphingolipid in the subject prior to administration.

[0012] In some aspects, provided herein is a method of increasing activity of an alpha-galactosidase A (α-Gal A) protein in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an alpha-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-Gal A transgene encoding at least one α-Gal A protein, and a bovine growth hormone polyA signal sequence, wherein the AAV expression vector is administered to the subject at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The increased activity of α-Gal A protein is relative to the activity of α-Gal A protein in the subject prior to administration, when administered at a dose of 1000 mg / kg.

[0013] In some embodiments, provided herein is a method of increasing activity of an α-galactosidase A (α-Gal A) protein in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an α-Gal A transgene encoding at least one α-Gal A protein, the α-Gal A transgene comprising the nucleotide sequence set forth in SEQ ID NO:5, the AAV expression vector being administered to the subject at a concentration of about 5×10 ... 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The increased activity of α-Gal A protein is relative to the activity of α-Gal A protein in the subject prior to administration, when administered at a dose of 1000 mg / kg.

[0014] In some embodiments, the subject is afflicted with Fabry disease.

[0015] In some embodiments, the a-Gal A expression cassette further comprises a mutated Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) sequence.

[0016] In some embodiments, the mutated WPRE sequence comprises a mut6 mutated WPRE sequence.

[0017] In some embodiments, the a-Gal A expression cassette further comprises an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, and a bovine growth hormone polyA signal sequence.

[0018] In some embodiments, the transgene comprises a wild-type or codon-optimized a-Gal A sequence.

[0019] In some embodiments, the signal peptide is an a-Gal A signal peptide.

[0020] In some embodiments, the enhancer comprises a nucleotide sequence set forth in SEQ ID NO:2, the promoter comprises a nucleotide sequence set forth in SEQ ID NO:3, the intron comprises a nucleotide sequence set forth in SEQ ID NO:4, the α-Gal A transgene comprises a nucleotide sequence set forth in SEQ ID NO:5, the mutated WPRE sequence comprises a nucleotide sequence set forth in SEQ ID NO:6, and the polyA signal sequence comprises a nucleotide sequence set forth in SEQ ID NO:7.

[0021] In some embodiments, the enhancer comprises a nucleotide sequence set forth in SEQ ID NO:2, the promoter comprises a nucleotide sequence set forth in SEQ ID NO:3, the intron comprises a nucleotide sequence set forth in SEQ ID NO:4, the mutated WPRE sequence comprises a nucleotide sequence set forth in SEQ ID NO:6, and the polyA signal sequence comprises a nucleotide sequence set forth in SEQ ID NO:7.

[0022] In some embodiments, the a-Gal A expression cassette comprises the nucleotide sequence set forth in SEQ ID NO:9.

[0023] In some embodiments, the AAV expression vector serotype is AAV2 / 6.

[0024] In some embodiments, the α-Gal A expression cassette is flanked on both sides by inverted terminal repeats (ITRs). In some embodiments, the ITRs are derived from adeno-associated virus type 2 (AAV2). In some embodiments, the AAV expression vector further comprises an α-Gal A expression cassette flanked on both sides by ITRs from AAV2, and the α-Gal A expression cassette is packaged with a capsid derived from adeno-associated virus type 6 (AAV6).

[0025] In some embodiments, the subject has one or more of the following symptoms: globotriaosylceramide (Gb3) levels above normal, globotriaosylsphingosine (lyso-Gb3) levels above normal, kidney disease, heart disease, anhidrosis, acroparesthesia, angiokeratoma, gastrointestinal (GI) tract pain, corneal and lenticular opacities, or cerebrovascular disease. In some embodiments, the angiokeratoma is periumbilical angiokeratoma.

[0026] In some embodiments, the subject has less than about 5% a-Gal A protein activity.

[0027] In some embodiments, α-Gal A protein activity is measured in the subject's plasma and / or white blood cells.

[0028] In some embodiments, the subject is a male subject. In some embodiments, the subject is a female subject.

[0029] In some embodiments, the subject has an α-Gal A gene mutation indicative of Fabry disease. In some embodiments, the α-Gal A gene mutation results in the amino acid mutation G261D, C422T, W340R, S297Y, Q283X, D215S, IVS5 / c.801+3A>G, P362L, C422T, or N34S.

[0030] In some embodiments, the subject has pre-existing anti-α-Gal A antibodies as determined by enzyme-linked immunosorbent assay (ELISA) prior to administration.

[0031] In some embodiments, upon analysis of the subject's biological sample, the subject is an anti-α-Gal A neutralizing antibody positive subject, where an anti-α-Gal A neutralizing antibody positive subject has a biological sample that has greater than about 9.6% inhibition of α-galactosidase A activity as measured by an anti-α-Gal A neutralizing antibody assay.

[0032] In some embodiments, the α-Gal A protein expressed from the transgene reduces the amount of glycosphingolipid in the subject by at least about two-fold compared to the amount of glycosphingolipid in the subject prior to administration.

[0033] In some embodiments, the α-Gal A protein expressed from the transgene increases the amount of glycosphingolipid in the subject by about 10 percent (%), about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 0%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76% %, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0034] In some embodiments, the α-Gal A protein expressed from the transgene maintains the amount of glycosphingolipids in the subject at the same level as before administration.

[0035] In some embodiments, the glycosphingolipid comprises globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), galabiosylceramide, or any combination thereof.

[0036] In some embodiments, Gb3 and / or lyso-Gb3 levels are measured in the subject's plasma and / or urine.

[0037] In some embodiments, Gb3 and / or lyso-Gb3 levels are measured in tissue of the subject.

[0038] In some embodiments, the a-Gal A protein expressed from the transgene reduces the amount of glycosphingolipids in one or more of plasma, liver, heart, kidney, urine, skin, or spleen.

[0039] In some embodiments, the α-Gal A protein activity in the subject is about 0 fold higher to about 2 fold higher, about 2 fold higher to about 5 fold higher, about 5 fold higher to about 10 fold higher, about 10 fold higher to about 20 fold higher, about 20 fold higher to about 30 fold higher, about 30 fold higher to about 40 fold higher, about 30 fold higher to about 40 fold higher, about 40 fold higher to about 50 fold higher, about 50 fold higher to about 60 fold higher, about 60 fold higher to about 70 fold higher, about 70 fold higher to about 80 fold higher, about 80 fold higher to about 90 fold higher, about 90 fold higher to about 100 fold higher, about 100 fold higher to about 200 fold higher, about 200 fold higher to about 300 fold higher, about 300 fold higher to about 400 fold higher, or about 400 fold higher to about 500 fold higher than average normal α-Gal A protein activity.

[0040] In some embodiments, the α-Gal A protein activity in the subject is about 0 fold higher than average normal α-Gal A protein activity, compared to the α-Gal A protein activity in the subject prior to administration, about 2 fold higher, about 2 fold higher, about 3 fold higher, about 4 fold higher, about 5 fold higher, about 6 fold higher, about 7 fold higher, about 8 fold higher, about 9 fold higher, about 10 fold higher, about 11 fold higher, about 12 fold higher, about 13 fold higher, about 14 fold higher, about 15 fold higher, about 16 fold higher, about 17 fold higher, about 18 fold higher, about 19 fold higher, about 20 fold higher, about 21 fold higher, about 22 fold higher, about 23 fold higher, about 24 fold higher, about 25 fold higher, about 26 fold higher, about 27 fold higher, about 28 fold higher, about 29 fold higher, about 30 fold higher, about 31 fold higher, about 32 fold higher, about 33 fold higher, about 34 fold higher, about 35 fold higher, about 36 fold higher, about 37 fold higher, about 38 fold higher, about 39 fold higher, about 40 fold higher, about 41 fold higher, about 42 fold higher, about 43 fold higher, about 44 fold higher, about 45 fold higher, about 46 fold higher, about 47 fold higher, about 48 fold higher, about 49 fold higher, about 50 fold higher, about 51 fold higher, about 52 fold higher, about 53 fold higher, about 54 fold higher, about 55 fold higher, about 56 fold higher, about 57 fold higher, about 58 fold higher, about 5 5 times higher, 26 times higher, 27 times higher, 28 times higher, 29 times higher, 30 times higher, 31 times higher, 32 times higher, 33 times higher, 34 times higher, 35 times higher, 36 times higher, 37 times higher, 38 times higher, 39 times higher, 40 times higher, 41 times higher, 42 times higher, 43 times higher, 44 times higher, 45 times higher, 46 times higher, 47 times higher, 48 times higher, 49 times higher, 50 times higher 51 times higher, 52 times higher, 53 times higher, 54 times higher, 55 times higher, 56 times higher, 57 times higher, 58 times higher, 59 times higher, 60 times higher, 61 times higher, 62 times higher, 63 times higher, 64 times higher, 65 times higher, 66 times higher, 67 times higher, 68 times higher, 69 times higher, 70 times higher, 71 times higher, 72 times higher, 73 times higher, 74 times higher, 75 times higher, 76 times higher, about 77 times higher, about 78 times higher, about 79 times higher, about 80 times higher, about 81 times higher, about 82 times higher, about 83 times higher, about 84 times higher, about 85 times higher, about 86 times higher, about 87 times higher, about 88 times higher, about 89 times higher, about 90 times higher, about 91 times higher, about 92 times higher, about 93 times higher, about 94 times higher, about 95 times higher, about 96 times higher, about 97 times higher, about 98 times higher, about 99 times higher, or about 100 times higher.

[0041] In some embodiments, levels of a-Gal A protein expressed from the transgene are measured in one or more of the subject's plasma, serum, whole blood, dried blood spots, white blood cells, or other blood components.

[0042] In some embodiments, the a-Gal A protein expressed from the transgene is active in the kidney, liver, skin, and heart of the subject.

[0043] In some embodiments, the AAV expression vector is administered parenterally. In some embodiments, the AAV expression vector is administered intravenously.

[0044] In some embodiments, the AAV expression vector is administered in a pharma- ceutically acceptable carrier. In some embodiments, the pharma- ceutically acceptable carrier comprises phosphate buffered saline containing CaCl2, MgCl2, NaCl, sucrose, and Kolliphor (poloxamer) P188.

[0045] In some embodiments, only one dose of the AAV expression vector is administered to the subject. In some embodiments, the AAV expression vector is administered in a dose of about 5×10 12 In some embodiments, the AAV expression vector is administered at a dose of about 1×10 13 In some embodiments, the AAV expression vector is administered at a dose of about 3×10 13 In some embodiments, the AAV expression vector is administered at a dose of about 5×10 13 The drug is administered at a dose of 100 mg / kg.

[0046] In some embodiments, the subject is administered an immunosuppressant prior to and / or during administration of the AAV expression vector. In some embodiments, the immunosuppressant comprises prednisone.

[0047] In some embodiments, the subject is not administered an immunosuppressant prior to and / or during administration of the AAV expression vector.

[0048] In some embodiments, the subject is not administered a preconditioning therapy prior to administration of the AAV expression vector.

[0049] In some embodiments, expression of at least one α-Gal A protein is sustained for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months.

[0050] In some embodiments, after administration, the subject has an estimated glomerular filtration rate (eGFR) (ml / min / 1.73 m ) calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. 2 In some embodiments, the annual rate of decline in eGFR is less than that of comparable untreated subjects with Fabry disease.

[0051] In some embodiments, after administration, the subject is measured for ejection fraction (EF) as stroke volume (SV) / left ventricular end diastolic volume (LVEDV). In some embodiments, the annual rate of EF decline is less than that of comparable untreated subjects with Fabry disease.

[0052] In some embodiments, after administration, the subject is measured for global longitudinal strain (GLS) by two-dimensional (2D) strain echocardiography or cardiac magnetic resonance imaging (cardiac MRI or CMR). In some embodiments, the annual shortening progression of cardiac muscle contractility is lower than that of comparable untreated subjects with Fabry disease.

[0053] In some embodiments, after administration, the subject measures myocardial relaxation time by native T1 mapping on cardiac magnetic resonance imaging (cardiac MRI or CMR).In some embodiments, the annual decrease in relaxation time is lower than that of a comparable untreated subject with Fabry disease.

[0054] In some embodiments, after administration, the subject is measured for edema as an increase in water content in the myocardium by T2 mapping of cardiac magnetic resonance imaging (cardiac MRI or CMR).In some embodiments, the annual increase in water content is lower than that of untreated subjects with comparable Fabry disease.

[0055] In some embodiments, after administration, the subject is measured for left ventricular mass index (LVMI) as left ventricular mass (LVM) / body surface area. In some embodiments, the annual LVMI increase is lower than that of comparable untreated subjects with Fabry disease.

[0056] In some embodiments, after administration, one or more auditory symptoms in the subject are improved. In some embodiments, the one or more auditory symptoms are tinnitus, dizziness, or progressive hearing loss.

[0057] In some embodiments, the subject's sweating levels are reversed from anhidrosis to hypohidrosis or normal sweating.

[0058] In some embodiments, the subject is undergoing enzyme replacement therapy (ERT) for Fabry disease prior to administration ("pre-treatment"). In some embodiments, the enzyme replacement therapy comprises recombinant alpha-galactosidase A (GLA) protein or a gene expressing GAL. In some embodiments, the enzyme replacement therapy for pre-treatment comprises administering Galafold, AVR-RD-01, FLT-190, pegnigalusidase alpha, 4D-310, or any combination thereof.

[0059] In some embodiments, the enzyme replacement therapy for pretreatment comprises recombinant alpha-galactosidase A (GLA) protein in combination with an active site-specific chaperone (ASSC) for GLA. In some embodiments, the ASSC is 1-deoxygalactonojirimycin.

[0060] In some embodiments, the enzyme replacement therapy for pre-treatment comprises agalsidase alpha and / or beta, or genes expressing agalsidase alpha and / or beta. In some embodiments, the enzyme replacement therapy for pre-treatment comprises Fabrazyme, Replagal, PRX-102, or any combination thereof.

[0061] In some embodiments, the enzyme replacement therapy for pretreatment comprises gene therapy. In some embodiments, the gene therapy comprises a vector encoding an enzyme. In some embodiments, the gene therapy comprises administering AVR-RD-01, FLT-190, pegniglucidase alfa, 4D-310, or any combination thereof.

[0062] In some embodiments, the vector comprises an mRNA encoding human GLA protein or agalsidase alpha and / or beta. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector comprises an adeno-associated viral (AAV) vector or a lentiviral vector. In some embodiments, the gene therapy is delivered by lipid nanoparticles.

[0063] In some embodiments, the subject has been administered a non-enzyme replacement therapy for Fabry disease prior to administration ("pre-treatment"). In some embodiments, the pre-treatment therapy for Fabry disease comprises lucerastat, benglustat, apabetalone, or any combination thereof.

[0064] In some embodiments, Fabry disease is the type 1 classical phenotype or the type 2 late-onset phenotype. [Brief description of the drawings]

[0065] [Figure 1]FIG. 1 shows a schematic diagram of the AAV-001 human alpha galactosidase A (hGLA) AAV cassette, which contains liver-specific regulatory elements (e.g., enhancers, promoters, introns), an alpha-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein (human alpha galactosidase A), a mutated form of the Woodchuck Hepatitis Virus (WHV) posttranscriptional regulatory element (WPREmut6), and ITR sequences and flanking polyadenylation (polyA) signal sequences. SP refers to the endogenous hGLA signal peptide. The sizes of the various elements in the overall cassette size (3321 bp) are indicated. [Diagram 2] FIG. 1 is a schematic diagram of the Phase 1 / 2, global, open-label, single-dose, dose-ranging, multicenter study to evaluate the safety and tolerability of AAV-001 (AAV2 / 6 human α-Gal A gene therapy) in patients with Fabry disease as described in Example 1. [Diagram 3] Baseline patient characteristics (age (years), ERT status, plasma α-Gal A protein activity (nmol / h / ml), plasma lyso-Gb3 (ng / ml), major disease signs and symptoms, renal function (eGFR), pre-existing α-Gal A antibodies, and α-Gal A amino acid mutations) are shown for patients 1–9, cohorts 1–4, and patient 10 in the expansion cohort. Ab = antibody, CKD-EPI = Chronic Kidney Disease Epidemiology Collaboration, eGFR = estimated glomerular filtration rate, LOD = limit of detection. *Baseline values ​​were considered to be the time point immediately prior to AAV-001 administration. †eGFR was measured as CKD-EPI (mL / min / 1.73 m2). [Figure 4A] Safety and tolerability data associated with AAV-001 treatment are presented. All safety data were evaluated from 4 patients in the first 2 dose cohorts (0.5e13vg / kg and 1e13vg / kg) as of the cutoff date (e.g., date of last measurement point). [Figure 4B]Safety and tolerability data associated with AAV-001 treatment are shown. All safety data were evaluated from 5 patients in dose cohorts 1-3 (0.5e13vg / kg, 1.0e13vg / kg, and 3.0e13vg / kg) as of the cutoff date. Follow-up periods ranged from 4 to 52 weeks (subjects 1 and 2, 52 weeks; subject 3, 40 weeks; subject 4, 25 weeks; and subject 5, 3 weeks). MedDRA = Medical Device Regulatory Restriction Act; vg / kg = vector genomes per kilogram of body weight. [Figure 4C] Safety and tolerability data associated with AAV-001 treatment are presented. All safety data were evaluated from 9 patients in dose cohorts 1-4 (0.5e13vg / kg, 1.0e13vg / kg, 3.0e13vg / kg, and 5.0e13vg / kg, respectively) as of the cutoff date. Follow-up ranged from 4 weeks to 15 months. MedDRA = Medical Device Regulatory Restriction Act; vg / kg = vector genomes per kilogram of body weight. [Figure 5A] Plasma α-Gal A protein activity (nmol / h / ml) measured in patients 1-4 as described in Example 2. ERT = enzyme replacement therapy, vg / kg = vector genomes per kilogram of body weight. [Figure 5B] Plasma α-Gal A protein activity (nmol / h / ml) measured in patients 1 to 5 as described in Example 2 is shown. ERT = enzyme replacement therapy, vg / kg = vector genomes per kilogram of body weight. [Figure 5C] Plasma α-Gal A protein activity (nmol / h / ml) measured in patients 1 to 8 as described in Example 2. ERT = enzyme replacement therapy, vg / kg = vector genomes per kilogram of body weight. [Figure 6A] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentration (ng / ml) measured at weeks 12 and 48 after AAV-001 administration in patient 1, as described in Example 2. [Figure 6B]1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentration (ng / ml) measured 52 weeks after AAV-001 administration in patient 1, as described in Example 2. [Figure 6C] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 1, as described in Example 2. [Figure 6D] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentration (ng / ml) measured at weeks 12 and 48 after AAV-001 administration in patient 2, as described in Example 2. [Figure 6E] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentration (ng / ml) measured 52 weeks after AAV-001 administration in patient 2, as described in Example 2. [Figure 6F] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 2, as described in Example 2. [Figure 6G] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentration (ng / ml) measured at weeks 12 and 28 after AAV-001 administration in patient 3, as described in Example 2. [Figure 6H] 1 shows plasma α-Gal A protein activity (nmol / h / ml) measured at week 40 and Lyso-Gb3 concentration (ng / ml) measured at week 36 after AAV-001 administration in patient 3, as described in Example 2. [Figure 6I] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 3, as described in Example 2. [Figure 6J]1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentration (ng / ml) measured 12 weeks after AAV-001 administration in patient 4, as described in Example 2. [Figure 6K] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 4, as described in Example 2. [Figure 6L] 1 shows plasma α-Gal A protein activity (nmol / h / ml) measured at 25 weeks and Lyso-Gb3 concentration (ng / ml) measured at 20 weeks after AAV-001 administration in patient 4, as described in Example 2. [Figure 6M] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentration (ng / ml) measured over time following AAV-001 administration in patient 5, as described in Example 2. [Figure 6N] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 6, as described in Example 2. [Figure 6O] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 7, as described in Example 2. [Figure 6P] 1 shows plasma α-Gal A protein activity (nmol / h / ml) and Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 8, as described in Example 2. [Figure 6Q] 1 shows Lyso-Gb3 concentrations (ng / ml) measured over time following AAV-001 administration in patient 9, as described in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The present disclosure provides a dose of approximately 5×10 per kilogram of body weight. 12Vector genome (vg / kg) ~ approx. 5 × 10 13 The present invention relates to a method for treating or ameliorating one or more symptoms of Fabry disease, reducing the amount of glycosphingolipids, and / or increasing the activity of α-galactosidase A (α-Gal A) protein in a subject in need thereof by administering an expression vector (e.g., an AAV expression vector) comprising an α-galactosidase A (α-Gal A) expression cassette that includes an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein at a dose of 100 mg / kg.

[0067] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0068] It should be noted that the terms "a" or "an" refer to one or more of that entity; for example, a "nucleic acid sequence" is understood to refer to one or more nucleic acid sequences, unless otherwise specified. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0069] Furthermore, "and / or" as used herein is to be construed as specifically disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0070] Where an embodiment is described herein using the term "comprising," it is understood that similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0071] As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which the word is used. If there are uses of the term that are not clear to a person of ordinary skill in the art, taking into account the context in which the term is used, "about" will mean up to plus or minus 10% of the particular value.

[0072] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a series of numbers or ranges, it is understood that "at least" can modify each number in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without considering the number of significant digits).

[0073] As used herein, "less than" or "below" is to be understood as a logical value or integer that is logically lower than zero given the value adjacent to the phrase and the context. When "less than" precedes a series of numbers or a range, it is understood that the "less than" can modify each number in the series or range.

[0074] As used herein, the term "immune response" refers to a biological response within an organism to foreign agents or abnormal cells (e.g., Fabry disease cells) that protects the organism from such agents / cells and the diseases caused by them. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver to selectively target, bind to, damage, destroy, and / or eliminate from the organism's body invading pathogens, pathogen-infected cells or tissues, Fabry disease cells, or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. In some embodiments, the immune response includes, for example, activation or inhibition of T cells, such as effector T cells or Th cells (e.g., CD4+ T cells or CD8+ T cells), or inhibition of regulatory T cells (Treg cells).

[0075] The term "vector" or "delivery vector" as used herein is intended to refer to a nucleic acid molecule that is capable of transporting another nucleic acid to which it is linked. The term "vector" or "delivery vector" includes both viral and non-viral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Additionally, some vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors", otherwise known as "expression constructs"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, adeno-associated viruses ("AAV"), and lentiviruses), which serve equivalent functions, are also included.

[0076] The term "expression vector" or "expression construct" refers to any type of genetic construct that contains a nucleic acid from which part or all of a nucleic acid coding sequence can be transcribed.

[0077] "Viral vector" refers to a sequence that includes one or more polynucleotide regions that code for or include a molecule of interest, such as, for example, a protein, a peptide, and an oligonucleotide, or a plurality thereof. Viral vectors can be used to deliver genetic material to cells. Viral vectors can be modified for specific applications. In some embodiments, the delivery vector of the present disclosure is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

[0078] The term "adeno-associated virus vector" or "AAV vector" as used herein refers to any vector that comprises or is derived from an adeno-associated virus component and is suitable for infecting mammalian cells, preferably human cells. The term AAV vector generally refers to an AAV-type virus particle or virion that contains a payload. AAV vectors can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV), or from various genomes (e.g., single-stranded or self-complementary).

[0079] As used herein, the term "AAV2 / 6," "rAAV2 / 6," or "pseudotyped AAV2 / 6" vector refers to an AAV expression vector of the disclosure (e.g., an AAV-001 rAAV vector) that contains an a-Gal A expression cassette flanked on both sides by ITRs from AAV2, where the a-Gal A expression cassette is packaged in a capsid derived from adeno-associated virus type 6 (AAV6).

[0080] Additionally, the AAV vector can be replication defective and / or targeted. As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh8, AAVrh10, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, the AAV serotypes and phylogenetic groups disclosed in Gao et al. (J. Virol. 78:6381 (2004)) and Morris et al. (Virol. 33:375 (2004)), and other AAVs now known or hereafter discovered. See, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV vectors" include derivatives of known AAV vectors. In some embodiments, "AAV vectors" include modified AAV vectors or artificial AAV vectors. In some embodiments, "AAV vectors" include recombinant adeno-associated viruses (rAAV). In some embodiments, the AAV expression vector serotype is AAV2 / 6.

[0081] In some embodiments, the AAV vector is modified or mutated relative to the wild-type AAV serotype sequence.

[0082] Insertion of a polynucleotide into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector with complementary cohesive termini. The vector can be designed to encode a selection marker or reporter that allows for the selection or identification of cells that have incorporated the vector. Expression of the selection marker or reporter allows for the identification and / or selection of host cells that incorporate and express other coding regions contained in the vector. Examples of selection marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selection markers can also be considered reporters. In some embodiments, the delivery vector is selected from the group consisting of a viral vector (e.g., an AAV vector), a plasmid, a lipid, a protein particle, a bacterial vector, a lysosome, a virus-like particle, a polymeric particle, an exosome, or a vault particle.

[0083] Some embodiments of the present disclosure are directed to biological vectors, which may include viruses, particularly attenuated and / or replication-defective viruses.

[0084] As used herein, the term "promoter" refers to a DNA sequence that is recognized by the cellular machinery, or an introduced synthetic machinery, that is required to initiate transcription of a gene. The term "promoter" is also meant to encompass nucleic acid elements sufficient for promoter-dependent gene expression that can be controlled in a cell type-specific, tissue-specific, or inducible by external signals or factors, and such elements can be located in the 5' or 3' region of the native gene. In some embodiments, the promoter is a constitutively active promoter, a cell type-specific promoter, or an inducible promoter. In some embodiments, the promoter is an alpha 1-antitrypsin (hAAT) promoter.

[0085] In some embodiments, microRNA target sequences are included to increase the specificity of vector-mediated transgene expression. See, e.g., Anja Geisler and Henry Fechner, World J Exp Med., 20;6(2):37-54(2016).

[0086] As used herein, the term "enhancer" refers to a cis-acting element that stimulates or inhibits the transcription of adjacent genes. Enhancers that inhibit transcription are also referred to as "silencers". Enhancers can function in either direction (e.g., can be associated with coding sequences) over distances of up to several kilobase pairs (kb) from the coding sequence and from a position downstream of the transcribed region. In some embodiments, the enhancer is an apolipoprotein E (APOE) enhancer. In some embodiments, the APOE enhancer is operably linked to the hAAT promoter.

[0087] As used herein, the term "regulatable promoter" is any promoter whose activity is affected by cis- or trans-acting factors (e.g., an inducible promoter such as an external signal or agent).

[0088] As used herein, the term "constitutive promoter" refers to any promoter that will direct RNA production in most or all tissues / cell types, e.g., the human CMV immediate-early enhancer / promoter region, which promotes constitutive expression of a cloned DNA insert in mammalian cells.

[0089] The terms "transcriptional regulatory protein," "transcriptional regulator," and "transcription factor" are used interchangeably herein to refer to nuclear proteins that bind to DNA response elements, thereby transcriptionally regulating the expression of associated genes. Transcriptional regulatory proteins usually bind directly to DNA response elements, although in some cases they may bind indirectly to DNA, by binding to another protein that either binds to the DNA response element or is bound to the DNA response element.

[0090] As used herein, the term "termination signal sequence" may be any genetic element that causes an RNA polymerase to terminate transcription, such as, for example, a polyadenylation (polyA or pA) signal sequence. A polyadenylation signal sequence is a recognition region required for endonuclease cleavage of an RNA transcript, followed by the polyadenylation consensus sequence AATAAA. A polyadenylation signal sequence provides a "polyA site," i.e., a site on an RNA transcript where adenine residues are added by posttranscriptional polyadenylation.

[0091] The terms "operably linked," "operably inserted," "operably positioned," "under control," or "under transcriptional control" mean that a promoter is in the correct position and orientation relative to the nucleic acid to control the initiation of RNA polymerase and expression of the gene. In some embodiments, the term "operably linked" means that a DNA sequence and a regulatory sequence are linked in such a way that gene expression is permitted when the appropriate molecule (e.g., a transcriptional activator protein) is bound to the regulatory sequence. In some embodiments, the term "operably inserted" means that a DNA of interest introduced into a cell is positioned adjacent to a DNA sequence that directs the transcription and translation of the introduced DNA (i.e., promotes the production of a polypeptide, etc., encoded by the DNA of interest).

[0092] A "coding sequence" or a sequence "encoding" a particular molecule (e.g., at least one α-Gal A protein) is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into a polypeptide in vitro or in vivo when operably linked to appropriate regulatory sequences, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is usually located 3' to the coding sequence.

[0093] As used herein, the term "derived from" refers to a component isolated from or made using a particular molecule or organism, or information (e.g., an amino acid or nucleic acid sequence) from a particular molecule or organism. For example, an amino acid sequence (e.g., an AAV vector) derived from a second nucleic acid sequence (e.g., another AAV vector) can include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence.

[0094] In the case of the polynucleotide disclosed herein, the derived species can be obtained, for example, by natural mutagenesis, artificially induced mutagenesis, or artificial random mutagenesis.The mutagenesis used to derive polynucleotide can be intentionally directed, intentionally random, or a mixture thereof.The mutagenesis of polynucleotide to generate a different polynucleotide derived from a first polynucleotide can be a random event (e.g., caused by polymerase mismatch), and the identification of the induced polynucleotide can be performed by suitable screening methods.

[0095] As used herein, the term "mutation" refers to a change in the structure of a gene resulting in a variant (also called a "mutant") that can be transmitted to subsequent generations. Genetic mutations can be caused by the substitution of a single base in DNA or by the deletion, insertion, or rearrangement of larger portions of genes or chromosomes.

[0096] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide or ribonucleotide polymers in linear or circular structure and in either single- or double-stranded form. For purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. These terms can encompass known analogs of natural nucleotides as well as nucleotides modified at the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). Generally, an analog of a particular nucleotide has the same base-pairing specificity, i.e., an analog of A will base pair with T. In some embodiments, a nucleic acid molecule can be complementary DNA (cDNA). Polynucleotides can be produced recombinantly, enzymatically, or synthetically, e.g., by solid-phase chemical synthesis followed by purification. When referring to a polynucleotide or nucleic acid sequence, reference is made to the sequence or order of the nucleobase moieties or modifications of the covalently linked nucleotides or nucleosides.

[0097] As used herein, the term "cDNA" refers to a DNA copy of a messenger RNA (mRNA) molecule produced by reverse transcriptase, a DNA polymerase that can use either DNA or RNA as a template.

[0098] As used herein, the term "mRNA" refers to a single-stranded RNA that codes for the amino acid sequences of one or more polypeptide chains.

[0099] Nucleic acids can be present in whole cells, cell lysates, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when they have been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques such as alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, or other techniques well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0100] Nucleic acids, e.g., cDNAs, can be mutated according to standard techniques to provide gene sequences. In the case of coding sequences, these mutations can affect the amino acid sequence as necessary. In particular, DNA sequences that are substantially homologous to or derived from the naturally occurring V, D, J, constant, switch, and other sequences described herein are contemplated ("derived" indicates that the sequence is identical to or modified from another sequence).

[0101] The term "antisense" as used herein refers to a nucleic acid that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA to prevent expression of an endogenous gene. A "complementary" polynucleotide is one that can base pair according to standard Watson-Crick complementarity rules. Specifically, purines base pair with pyrimidines, forming combinations of guanine pairing with cytosine (G:C) and adenine pairing with thymine (A:T) in the case of DNA, or adenine pairing with uracil (A:U) in the case of RNA. It is understood that two polynucleotides can hybridize to each other without being completely complementary, so long as each has at least one region that is substantially complementary to the other.

[0102] The terms "antisense strand" and "guide strand" refer to the strand of dsRNA, e.g., shRNA, that comprises a region that is substantially complementary to a target sequence, e.g., mRNA.Antisense strand has a sequence that is sufficiently complementary to the desired target mRNA sequence to induce target-specific silencing, e.g., has sufficient complementarity to induce the destruction of the desired target mRNA by the RNAi mechanism or process.

[0103] The terms "sense strand" and "passenger strand" as used herein refer to a strand of a dsRNA, such as an shRNA, that comprises a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein. For example, the antisense strand and the sense strand of a dsRNA, such as an shRNA, hybridize to form a duplex structure.

[0104] As used herein, the term "gene" includes the region of DNA that encodes a gene product, as well as all regions of DNA that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0105] The term "transgene" as used herein refers to a segment of DNA from one organism that is introduced into the genome of another organism. Transgenes may be delivered to cells in a variety of ways, whereby the transgene is integrated into the transgene cell's own genome and maintained there. Recently, strategies for transgene integration have been developed that use site-specific nuclease cleavage to target insertion into selected genomic loci (see, for example, U.S. Pat. No. 7,888,121). Nuclease systems such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or RNA-guided CRISPR / Cas systems (using designed guide RNAs) can be utilized to insert transgene constructs by either homology-directed repair (HDR) or end capture during a non-homologous end joining (NHEJ) driven process, specific to the target gene. For example, U.S. Patent Nos. 9,394,545, 9,255,250, 9,200,266, 9,045,763, 9,005,973, 9,150,847, 8,956,828, 8,945,868, 8,703,489, 8,586,526, 6,534,261, Nos. 6,599,692, 6,503,717, 6,689,558, 7,067,317, 7,262,054, 7,888,121, 7,972,854, 7,914,796, 7,951,925, 8,110,379, 8,409,861, and U.S. Patent Publication No. 20030232410, 20050208489, 20050026157, 20050064474, 20060063231, 20080159996, 201000218264, 20120017290, 20110265198, 20130137104, See Nos. 20130122591, 20130177983, 20130196373, 20140120622, 20150056705, 20150335708, 20160030477, and 20160024474, the disclosures of which are incorporated by reference in their entireties.

[0106] Transgenes can be introduced and maintained in cells in a variety of ways. According to the "cDNA" approach, transgenes are introduced into cells so that they are maintained extrachromosomally, rather than integrated into the chromatin of the cell. Transgenes can be maintained on circular vectors (e.g., plasmids or non-integrating viral vectors such as AAV or lentivirus), and the vectors can include transcriptional regulatory sequences such as promoters, enhancers, polyA signal sequences, introns, and splicing signals (US Patent Application Publication No. 20170119906). In some embodiments, the AAV expression vector of the present disclosure comprises an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein. In some embodiments, the transgene of the present disclosure comprises a wild-type α-Gal A sequence or a codon-optimized α-Gal A sequence. In some embodiments, the α-Gal A transgene of the present disclosure comprises the nucleotide sequence set forth in SEQ ID NO:5.

[0107] The term "gene expression" as used herein refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNA that is modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristyrylation, and glycosylation.

[0108] As used herein, the term "GLA gene" encodes at least one α-galactosidase A (α-Gal A) protein, as described herein.

[0109] As used herein, the term "modulation" of gene expression refers to a change in the activity of a gene. Modulation of expression includes, but is not limited to, gene activation and gene suppression. Genome editing (e.g., truncation, modification, inactivation, random mutation) can be used to regulate expression. Gene inactivation refers to a decrease in gene expression, for example, compared to cells that do not contain ZFP, TALE, or CRISPR / Cas system. Thus, gene inactivation can be partial or complete.

[0110] The term "region of interest" refers to any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desired to bind an exogenous molecule. Binding may be for the purposes of targeted DNA cleavage and / or targeted recombination. A region of interest may be present, for example, in a chromosome, episome, organelle genome (e.g., mitochondria, chloroplasts), or in an infecting viral genome. A region of interest may be present within the coding region of a gene, within a transcribed non-coding region (e.g., leader sequence, trailer sequence, or intron, etc.), or within a non-transcribed region either upstream or downstream of a coding region. A region of interest may be as small as a single nucleotide pair or up to 2,000 nucleotide pairs long, or any integer value of nucleotide pairs long.

[0111] As used herein, the term "eukaryotic cells" includes, but is not limited to, fungal cells (such as yeast cells), plant cells, animal cells, mammalian cells, and human cells (e.g., liver cells, muscle cells, red blood cells, etc.), including stem cells (pluripotent and multipotent).

[0112] The term "secretory tissue" as used herein refers to tissue in animals that secrete products from individual cells into some type of lumen, usually of epithelial origin. Examples of secretory tissues localized to the digestive tract include cells lining the intestine, pancreas, and gallbladder. Other secretory tissues include tissues associated with the liver, eye, and mucous membranes, such as salivary glands, mammary glands, prostate, pituitary gland, and other members of the endocrine system. Additionally, secretory tissues include individual cells of tissue types capable of secretion.

[0113] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides," and includes any chain of amino acids or a chain of two or more amino acids. Thus, as used herein, "peptide," "peptide subunit," "protein," "amino acid chain," "amino acid sequence," or other terms used to refer to a chain of two or more amino acids are included in the definition of "polypeptide," even though each may have a more specific meaning. The term "polypeptide" may be used in place of or synonymously with any of these terms. The term further includes polypeptides that have been post-translationally or post-synthetically modified, such as conjugation of palmitoyl groups, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. As used herein, the term "peptide" encompasses full-length peptides and fragments, variants, or derivatives thereof. The "peptides" disclosed herein may be part of a fusion polypeptide that includes additional components, such as, for example, an Fc domain or an albumin domain, to extend half-life. The peptides described herein may also be derivatized in various ways. The peptides described herein can include modifications including, for example, the attachment of a palmitoyl group.

[0114] As used herein, the term "functional fragment thereof" refers to a fragment or portion of a protein, such as α-galactosidase A protein, that is still capable of performing one or more functions associated with the intact protein (e.g., stimulating, regulating, controlling, or modifying an immune response).

[0115] The terms "α-galactosidase A," "α-Gal A," and "GAL" are used interchangeably and refer to a protein having an enzymatic activity that involves hydrolysis of terminal non-reducing α-D-galactose residues of α-D-galactosides, including galactose oligosaccharides, galactomannans, and galactolipids. In some embodiments, α-Gal A includes enzymes described in IUBMB Enzyme Nomenclature EC 3.2.1.22 (e.g., Suzuki et al., J. Biol. Chem. 245:781-786 (1970); Wiederschain, G. and Beyer, E. Dokl. Akad. Nauk SSSR 231:486-488 (1976)). In some embodiments, α-Gal A includes a protein encoded by a nucleic acid that includes a human GLA gene, e.g., the human α-Gal A gene as defined in GenBank Accession No. NM_000169. In some embodiments, a-Gal A comprises a protein comprising the amino acid sequence defined by GenBank Accession No. NP_000160.

[0116] In some embodiments, the GAL can be obtained from a cell that endogenously expresses a-Gal A or the a-Gal A can be recombinant human a-Gal A (rha-Gal A). In some embodiments, the rha-Gal A is full-length wild-type a-Gal A. In some embodiments, the rha-Gal A comprises a subset of amino acid residues present in wild-type a-Gal A, including amino acid residues of wild-type a-Gal A that form an active site for substrate binding and / or substrate reduction. In some embodiments, the rha-Gal A is a fusion protein that comprises the wild-type a-Gal A active site for substrate binding and / or substrate reduction as well as other amino acid residues that may or may not be present in wild-type a-Gal A.

[0117] α-Gal A can be obtained from commercial sources or by synthetic techniques well known to those of skill in the art. The wild-type enzyme can be purified from recombinant cell expression systems (e.g., mammalian cells such as CHO cells, or insect cells, see, e.g., U.S. Pat. Nos. 5,580,757, 6,395,884, 6,458,574, 6,461,609, 6,210,666, 6,083,725), human placenta, or animal milk.

[0118] Other synthetic techniques for obtaining α-Gal A suitable for pharmaceutical use are described, for example, in U.S. Pat. Nos. 7,560,424, 7,396,811, 423,135, 6,534,300, and 6,537,785; U.S. Published Application Nos. 2009 / 0203575, 2009 / 0029467, 2008 / 0299640, 2008 / 0241118, 2006 / 0121018, 2005 / 0244400, 2007 / 0280925, and 2004 / 0029779, and International Published Application No. WO 2005 / 077093.

[0119] In some embodiments, the α-Gal A is agalsidase alpha produced by genetic engineering in a human cell line. Agalsidase alpha is available as Replagal® from Shire Plc (Dublin, Ireland). In some embodiments, the α-Gal A is agalsidase beta produced by recombinant DNA technology in a Chinese Hamster Ovary (CHO) cell line. Agalsidase beta is available as Fabrazyme® from Sanofi Genzyme (Cambridge, Mass.). In some embodiments, the α-Gal A is recombinant human α-Gal A produced in CHO cells transformed with an expression vector encoding the human α-Gal A gene (JCR Pharmaceuticals Co. Ltd, (Japan)), identified as JR-051.

[0120] In addition to proteins that contain amino acid sequences identical to the human α-Gal A protein described herein, the present disclosure also encompasses α-Gal A proteins that are "substantially similar" thereto. Proteins described herein as being "substantially similar" to a reference protein include proteins that retain some of the structural and functional characteristics of the native protein, but that differ from the native amino acid sequence at one or more amino acid positions (i.e., by amino acid substitution).

[0121] Proteins that are altered from the native sequence can be prepared by substituting amino acid residues in the native protein and selecting proteins with the desired activity. For example, amino acid residues in the α-Gal A protein can be systematically substituted with other residues and the substituted proteins tested in standard assays to assess the effect of such substitutions on the protein's ability to hydrolyze terminal non-reducing α-D-galactose residues in α-D-galactosides, including galactose oligosaccharides, galactomannans, and galactolipids, and / or to treat or prevent Fabry disease.

[0122] In some embodiments, conservative amino acid substitutions are made to retain functional activity. As used herein, "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in the α-Gal A protein is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0123] In some embodiments, an α-Gal A protein of the present disclosure is about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of an α-Gal A protein described herein or known in the art.

[0124] The percent identity between two sequences is a function of the number of identical positions shared by the sequences taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0125] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0126] The nucleic acid and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, for example to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, they can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0127] "Antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region comprises three constant domains, C H1 , C H2 , and C H3 Each light chain comprises a light chain variable region (herein referred to as V L The light chain constant region comprises one constant domain, C L Includes: V H and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). For example, the term "anti-GAL antibody" includes a complete antibody having two heavy chains and two light chains that specifically bind α-Gal A, as well as the antigen-binding portion of the complete antibody.

[0128] Immunoglobulins may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the class or subclass of antibody (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce immunogenicity in humans. Unless expressly stated and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, and single chain antibodies.

[0129] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a-Gal A is substantially free of antibodies that specifically bind antigens other than a-Gal A). However, an isolated antibody that specifically binds a-Gal A may exhibit cross-reactivity to other antigens, such as a-Gal A molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0130] "Anti-antigen antibody" refers to an antibody that specifically binds to an antigen. For example, an anti-GAL antibody specifically binds to GAL.

[0131] The term "anti-GLA neutralizing antibody", "anti-GLA NAb", "anti-drug antibody", "ADA", "neutralizing anti-drug antibody", or "neutralizing ADA" refers to an antibody that binds to and inactivates (neutralizes) the α-Gal A enzyme. In some embodiments, when anti-GLA neutralizing antibodies are present, enzyme replacement therapy is directly inactivated (neutralized) by anti-GLA neutralizing antibodies in plasma (Linthorst et al., Kidny Int 66:1589-1595(2004); Lenders et al., J Allergy Clin Immunol 141:2289-2292.e7(2018)).

[0132] An "antigen-binding portion" (also called an "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen to which the whole antibody binds. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, such as the anti-GLA3 antibody described herein, include (i) a Fab fragment (a fragment from papain cleavage) or a V L , V H (ii) a F(ab')2 fragment (a fragment resulting from papain cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V Hand an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody. L and V H (v) an Fv fragment consisting of a V H (vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. In addition, the two domains V of the Fv fragment may be combined with each other to form a dAb fragment. L and V H Although the V are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods. L and V H The domains pair to form a monovalent molecule known as a single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0133] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ), equilibrium binding constant (K A) can be measured and / or expressed in a variety of ways known in the art, including, but not limited to, K D is k off / k on It is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on For example, k refers to the binding rate constant between an antibody and an antigen, off refers to, for example, the dissociation of an antibody and an antigen. on and k off can be determined by techniques well known to those skilled in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIAcore®, BLI (BioLayer Interferometry), or kinetic exclusion assays (KinExA®).

[0134] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are similar terms in the context of antibodies and refer to a molecule (e.g., an antibody) that binds to an antigen (e.g., an epitope or immune complex) according to binding understood by one of skill in the art. For example, a molecule that specifically binds to an antigen can generally bind other peptides or polypeptides with low affinity, as determined, for example, by immunoassays, BIAcore®, KinExA® 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays well known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K A At least 2 log, 2.5 log, 3 log, 4 log or K A It binds to antigens at ultra high temperatures.

[0135] Antibodies are usually -5 ~10 -11 The dissociation constant (K D ) specifically binds to its cognate antigen with high affinity. -4 K over MD As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds with high affinity to the antigen and to a substantially identical antigen, as measured, for example, by immunoassays (e.g., ELISA) surface plasmon resonance (SPR) technology in a BIACORE™ 2000 instrument using a given antigen, or BLI (biolayer interferometry), with a binding affinity of 10 or more. -7 M or less, preferably 10 -8 M or less, and even more preferably 10 -9 M or less, most preferably 10 -8 M to 10 -10 K below M D This means that the antibody has a high affinity for the antigen but does not bind with high affinity to unrelated antigens.

[0136] The term "recombinant host cell" (or simply "host cell") as used herein is intended to refer to a cell that contains a nucleic acid that is not naturally occurring in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because some modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0137] As used herein, the term "linkage" refers to the attachment of two or more molecules. The linkage may be covalent or non-covalent. The linkage may also be genetic (i.e., recombinant fusion). Such linkages may be achieved using a variety of art-recognized techniques, including chemical conjugation and recombinant protein production.

[0138] The term "Fabry disease" refers to classic Fabry disease, late-onset Fabry disease, and hemizygous females with a mutation in the gene encoding α-Gal A. As used herein, the term "Fabry disease" further includes any situation in which a subject exhibits lower than normal endogenous α-Gal A activity. Fabry disease is also known by many other names, such as alpha-galactosidase A deficiency, Anderson-Fabry disease, diffuse angiokeratoma corporis, diffuse angiokeratoma corporis, ceramide trihexosidase deficiency, Fabry disease, GLA deficiency, hereditary ectopic lipidosis, etc. In some embodiments, Fabry disease is a classic phenotype of type 1, or a late-onset phenotype of type 2.

[0139] The term "enzyme replacement therapy" or "ERT" refers to the introduction of a non-naturally occurring purified enzyme into an individual who is deficient in such an enzyme (e.g., α-Gal A). The administered enzyme can be obtained from a natural source or by recombinant expression. The term also refers to the introduction of a purified enzyme into an individual who requires or would benefit from administration of the purified enzyme, e.g., who suffers from a protein deficiency. The introduced enzyme can be a purified recombinant enzyme produced in vitro, or an enzyme purified from an isolated tissue or fluid, e.g., placenta or animal milk, or a plant.

[0140] The term "co-formulation" refers to a composition that includes an enzyme, such as an enzyme used in ERT (e.g., human recombinant α-Gal A enzyme (rhα-Gal A)), that is formulated with an active site-specific chaperone (ASSC) of α-Gal A enzyme (e.g., 1-deoxygalactonojirimycin (DGJ)). In some embodiments, the ASSC is 1-deoxygalactonojirimycin (DGJ) or a pharma- ceutically acceptable salt, ester, or prodrug of 1-deoxygalactonojirimycin. In some embodiments, the salt is a hydrochloride salt (i.e., 1-deoxygalactonojirimycin-HCl). In some embodiments, treating a subject with a co-formulation includes administering the co-formulation to the subject such that the α-Gal A enzyme and the ASSC are administered simultaneously as part of the co-formulation.

[0141] The term "combination therapy" refers to therapy that provides improved results compared to the effects of each therapy administered individually. Individual therapies in the combination therapy can be administered simultaneously or sequentially.

[0142] Enhancement includes the improvement of the effect of various therapies, which may provide advantageous results compared to the results achieved by the therapies when performed alone. Enhanced effect and the determination of enhanced effect can be measured by various parameters, including but not limited to, temporal parameters (e.g., duration of treatment, recovery time, long-term effect of treatment, or reversibility of treatment); biological parameters (e.g., cell number, cell volume, cell composition, tissue volume, tissue size, tissue composition); spatial parameters (e.g., tissue strength, tissue size, or tissue accessibility); and physiological parameters (e.g., body contour, pain, discomfort, recovery time, or visible traces). Enhanced effect can include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy when performed alone. Enhanced effect can also include additive enhancement, where the enhanced effect is substantially equivalent to the additive effect of each therapy when performed alone. The enhanced effect can also include an effect that is less than synergistic, that is, the enhanced effect is less than the additive effect of each therapy performed alone, but is still greater than the effect of each therapy performed alone.

[0143] The term "stabilizing proper conformation" refers to the ability of a compound, peptide, or other molecule to bind to a wild-type protein, or a mutant protein capable of performing wild-type functions in vitro and in vivo, and maintain the structure of the wild-type or mutant protein in its native or proper form. This effect can be manifested in practice through one or more of the following: (i) an increased shelf life of the protein; (ii) a higher activity per unit / amount of protein; (iii) a greater in vivo efficacy. This can be observed experimentally by similar means, such as increased yield from the ER during expression, increased resistance to unfolding with increasing temperature (e.g., as determined by a thermostability assay), or the presence of chaotropic agents.

[0144] As used herein, the term "active site" refers to a region of a protein that has a specific biological activity. For example, it may be a site that binds a substrate or other binding partner and provides amino acid residues that are directly involved in the formation and cleavage of chemical bonds. Active sites in this application may include the catalytic site of an enzyme, the antigen-binding site of an antibody, the ligand-binding domain of a receptor, the binding domain of a regulatory factor, or the receptor-binding domain of a secreted protein. Active sites may also include transcriptional activation, protein-protein interactions, or DNA-binding domains of transcription factors and regulatory factors.

[0145] As used herein, the term "active site-specific chaperone" refers to any molecule, including proteins, peptides, nucleic acids, carbohydrates, etc., that specifically and reversibly interacts with the active site of a protein and promotes the formation of a stable molecular structure. As used herein, "active site-specific chaperone" does not include endogenous general chaperones present in the ER of cells, such as Bip, calnexin, calreticulin, or general non-specific chemical chaperones, such as deuterated water, DMSO, TMAO, etc.

[0146] The term "non-enzyme replacement therapy" refers to a therapy that is not an enzyme replacement therapy (e.g., Fabry disease therapy). Non-enzyme replacement therapy may include small molecule therapy. Some new drug development strategies for small molecule therapy of Fabry disease include, but are not limited to, substrate reduction therapy (SRT), residual enzyme activation, GLA promoter activation, protein homeostasis regulation (proteostasis), and chemical chaperone therapy (CCT).

[0147] The term "immunotherapy" refers to treating a subject suffering from a disease or at risk of developing or recurring a disease by methods that involve inducing, enhancing, suppressing, or modifying an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject or the administration of an active agent to a subject with the intent of reversing, mitigating, ameliorating, inhibiting, delaying, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical signs associated with a disease (such as Fabry disease). "Immunosuppressive therapy" refers to a therapy that reduces (mitigates) the immune response of a subject.

[0148] As used herein, the phrase "contacting a cell" (e.g., contacting a cell with an AAV expression vector or composition of the present disclosure) includes contacting a cell directly or indirectly. In some embodiments, contacting a cell with an AAV expression vector or composition of the present disclosure includes contacting a cell with a composition or AAV vector in vitro, or contacting a cell with an AAV vector or composition of the present disclosure in vivo. Thus, for example, an AAV vector or composition of the present disclosure can be physically contacted with a cell by the individual performing the method, or alternatively, an AAV vector or composition of the present disclosure can be placed in a situation that allows or allows it to contact a cell thereafter.

[0149] In some embodiments, contacting the cells in vitro can be performed, for example, by incubating the cells with the AAV vector. In some embodiments, contacting the cells in vivo can be performed, for example, by injecting the AAV vector or composition of the present disclosure into or near the tissue in which the cells reside, or by injecting the AAV vector or composition of the present disclosure into another area, for example, the bloodstream or subcutaneous space, so that the agent then reaches the tissue in which the contacted cells reside. For example, the AAV vector can be encapsulated and / or bound to a ligand that directs the AAV vector to the desired site. A combination of in vitro and in vivo contact methods is also possible. For example, the cells can be contacted in vitro with the AAV vector or composition of the present disclosure and then transplanted into a subject.

[0150] In some embodiments, contacting a cell with an AAV vector or composition of the present disclosure includes "introducing" or "delivering" (directly or indirectly) an AAV vector or composition of the present disclosure to a cell by promoting or effecting uptake or absorption into the cell. Introducing an AAV vector or composition of the present disclosure into a cell can be in vitro and / or in vivo. For example, in the case of in vivo introduction, an AAV vector or composition of the present disclosure can be injected into a specific tissue site (e.g., a site where a therapeutic effect is desired) or administered systemically (e.g., administration of an AAV vector targeted to a site where a therapeutic effect is desired). In vitro methods of introduction into a cell include methods well known in the art, such as electroporation and lipofection.

[0151] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount," for example, of an AAV vector or composition disclosed herein, refer to an amount sufficient to effect beneficial or desired results, including clinical results, when administered to a subject, including a human; thus, "effective amount" or its synonyms will vary depending on the context in which it is applied. In some embodiments, a therapeutically effective amount of an agent (e.g., an AAV vector or composition disclosed herein) is an amount that effects beneficial or desired results in a subject compared to a control.

[0152] The amount of a given agent (e.g., an AAV vector or composition disclosed herein) will depend on a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, sex, and / or weight) or host being treated.

[0153] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a polynucleotide comprising a promoter operably linked to a nucleic acid encoding an immunomodulatory protein (e.g., a cytokine or subunit thereof) disclosed herein or a functional fragment thereof) into the cells and / or tissues of an individual to treat a disease, alleviate the symptoms of a disease, or reduce the likelihood of a disease. Gene therapy also includes the insertion of a transgene that inhibits, reduces or decreases the expression, activity or function of an endogenous gene or protein, such as an inherently inhibitory, i.e., undesirable or abnormal (e.g., pathogenic) gene or protein. Such a transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that is not normally present in the cells, tissues, and / or individual to be treated. Both acquired and congenital diseases are amenable to gene therapy.

[0154] As used herein, the term "prophylactically effective amount" includes an amount of an agent (e.g., an AAV vector or composition disclosed herein) sufficient to prevent, reduce, or ameliorate a disease or disorder, or one or more symptoms of a disease or disorder, when administered to a subject suffering from or predisposed to a disease or disorder (e.g., Fabry disease). Ameliorating a disease or disorder includes slowing the progression of a disease or disorder or reducing the severity of a later-onset disease or disorder. A "prophylactically effective amount" can vary depending on the characteristics of the agent (e.g., an AAV expression vector or composition disclosed herein), the method of administration of the agent, the degree of risk of the disease, and the medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment (if any), and other individual characteristics of the patient being treated.

[0155] As used herein, "off-target" refers to an unintended effect on one or more targets, genes, or cellular transcripts.

[0156] As used herein, the term "in vitro" refers to events that take place not in a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a Petri dish, etc.

[0157] As used herein, the term "in vivo" refers to events that take place within a living organism (e.g., an animal, plant, or microorganism, or the cells and tissues thereof).

[0158] As used herein, the term "transfection" refers to a method of introducing exogenous nucleic acid into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments, cationic lipids or mixtures thereof. The list of substances that can be transfected into cells is vast, including, for example, siRNA, shRNA, sense and / or antisense sequences, DNA that encodes one or more genes and is organized into an expression plasmid such as a vector, etc.

[0159] "Determining the level of a protein" means detecting the protein or the mRNA encoding the protein, directly or indirectly, by methods well known in the art. "Directly determining" means performing a process to obtain a physical entity or value (e.g., performing an assay or test on a sample, or "analyzing a sample" as that term is defined herein). "Indirectly determining" refers to receiving a physical entity or value from another party or source (e.g., a third-party laboratory that obtains the physical entity or value directly). Methods for measuring protein levels generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC) mass spectrometry, microcytometry, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry, and assays based on the properties of the protein, including, but not limited to, enzyme activity or interaction with other protein partners. Methods for measuring mRNA levels are well known in the art.

[0160] The term "level" as used herein refers to the level / amount or activity of a protein or an mRNA encoding a protein, optionally compared to a reference. The reference can be any useful reference, as defined herein. The level of a protein can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or as a percentage of the total protein or mRNA in a sample.

[0161] As used herein, the term "reduced level" or "reduced level" of a protein refers to a decrease / reduction in protein levels compared to a reference. As used herein, the term "reducing the amount of glycosphingolipids" in a subject refers to a decrease / reduction in the amount (ng / ml) of glycosphingolipids (e.g., globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), galabiosylceramide, or any combination thereof) compared to the amount (ng / ml) of glycosphingolipids in the same subject before administration. The decrease / reduction in the amount (ng / ml) of glycosphingolipids is measured from a baseline point to the last measurement point, as described in Example 2 herein below. In some embodiments, the amount of glycosphingolipids (e.g., lyso-Gb3 concentration (ng / ml)) is measured from a baseline point to the last measurement point, as described in Example 2 herein below. In some embodiments, the amount of glycosphingolipid (e.g., Gb3 concentration (ng / ml)) is measured from a baseline point to the last measurement point, as described herein below in Example 2. As used herein, the term "baseline point" refers to the time point immediately prior to administration of an AAV expression vector or pharmaceutical composition of the present disclosure.

[0162] As used herein, the term "increased levels" of a protein refers to an increase in protein levels compared to a reference. As used herein, the term "increased α-galactosidase A (α-Gal A) protein activity" in a subject refers to an increase in α-Gal A protein (enzyme) activity (nmol / h / ml) relative to the α-Gal A protein activity in the subject prior to administration, where the increase in α-Gal A protein activity is measured as described in Example 2 herein below.

[0163] As used herein, the term "maintained levels" of a protein means that there was no significant decrease / reduction or increase in the protein level compared to the baseline. As used herein, the term "maintaining the amount of glycosphingolipids" in a subject means that there was no statistically significant decrease / reduction or increase in the amount of glycosphingolipids (e.g., globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), galabiosylceramide, or any combination thereof) compared to the amount of glycosphingolipids in the same subject before administration. In some embodiments, the subject is an enzyme replacement therapy (ERT) pre-treated subject. ERT must have been administered at a stable dose (defined as not missing 4 or more doses of ERT in the 6 months prior to consent) and regimen (14 days ± 1 day for at least 3 months prior to enrollment). Whether a portion is statistically significant can be determined in a simple manner by those skilled in the art by using various well-known statistical evaluation tools, such as, for example, determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details are described in Dowdy and Wearden, Statistics for Research, John Wiley and Sons, New York 1983. Preferred confidence intervals are at least about 90%, at least about 95%, at least about 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.

[0164] As used herein, the term "pharmaceutical composition" refers to a composition that contains a compound or molecule described herein, such as an AAV vector disclosed herein, formulated with a pharma- ceutical acceptable excipient, and that can be manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal.

[0165] As used herein, "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has the properties of being substantially non-toxic and non-inflammatory in a patient.

[0166] "Reference" refers to a useful standard used to compare protein or mRNA levels or activity. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample, or a reference standard or level. A "reference sample" can be, for example, a control, such as a predefined negative control value, such as a "normal control," or a previous sample taken from the same subject, a sample from a normal, healthy subject, such as a normal cell or normal tissue, a sample (e.g., cell or tissue) from a subject without a disease, a sample from a subject diagnosed with a disease but not yet treated with a compound described herein, a sample from a subject treated with a compound described herein, or a sample of a known normal concentration of purified protein (e.g., as described herein).

[0167] As used herein, the term "subject" refers to any organism to which a composition disclosed herein, e.g., an AAV expression vector or composition of the present disclosure, can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include any animal (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). A subject can be a human or animal seeking treatment, in need of treatment, requesting treatment, undergoing treatment, will undergo treatment in the future, or receiving care from a trained professional for a particular disease or condition. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0168] As used herein, the term "comparable untreated subjects with Fabry disease" refers to human subjects who are matched to the treated subjects by, for example, age, sex, race, and / or disease manifestations. (See, e.g., Giugliani et al., Journal of Inborn Errors of Metabolism & Screening Volume 4:1-12 (2016)).

[0169] As used herein, the term "vector genomes per kilogram of body weight (vg / kg)" refers to copies of an expression cassette (e.g., an α-Gal A expression cassette of the present disclosure) administered to a subject. The number of copies of vector genomes per kilogram of body weight can be measured by quantitative PCR and / or droplet digital PCR.

[0170] The term "fixed dose," when used in connection with the methods and dosages of the present disclosure, refers to a dosage administered to a patient regardless of the patient's weight or body surface area (BSA). Thus, a fixed dose is provided not as a mg / kg dose, but rather as an absolute amount of agent (e.g., recombinant α-Gal A protein). For example, a person weighing 60 kg and a person weighing 100 kg would receive the same amount of antibody (e.g., 12 mg of recombinant α-Gal A protein).

[0171] The term "weight-based dose" as referred to herein means that the dose administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires 0.2 mg / kg of recombinant α-Gal A protein, then an appropriate amount of recombinant α-Gal A protein (i.e., 12 mg) can be calculated and used for administration.

[0172] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is that amount of any drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to the disease. The ability of a therapeutic agent to promote regression of a disease can be assessed using a variety of methods known to the skilled practitioner, such as assaying the activity of the therapeutic agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0173] As used herein, the terms "treat", "treating" and "treatment" refer to any type of intervention or process performed on a subject or administering an active agent to a subject for the purpose of reversing, mitigating, ameliorating, inhibiting, or slowing or preventing the progression, onset, severity or recurrence of symptoms, complications, conditions or biochemical signs associated with a disease, or improving overall survival. Treatment can be performed on subjects with a disease or subjects without a disease (such as prophylaxis).

[0174] As used herein, the term "prevention" in reference to a condition refers to the administration of a composition that reduces the frequency or delays the onset of symptoms of a condition in a subject compared to a subject not administered the composition.

[0175] The term "amelioration" refers to a therapeutically beneficial outcome in the treatment of a disease state, for example, Fabry disease, including prevention, reduction in severity or progression, remission, or cure. In some embodiments, the disclosed method is to ameliorate one or more symptoms associated with Fabry disease in a human subject in need thereof.

[0176] The term "effective amount" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is an amount of drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time that a patient diagnosed with a disease, such as Fabry disease, is still alive from the date of diagnosis or the date of initiation of treatment), or prevention of disability or disabling due to the affliction of the disease. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage," which is an amount of drug that inhibits the onset or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or at risk of recurrence of a disease. The ability of a therapeutic agent to promote regression of a disease or inhibit the onset or recurrence of a disease can be evaluated using a variety of methods known to the skilled practitioner, such as assaying the activity of a therapeutic agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0177] A "sample" or "biological sample" of the present disclosure, in some embodiments, is of biological origin, such as from a eukaryotic organism. In some embodiments, the sample is a human sample, although animal samples can also be used. Non-limiting sources of samples for use in the present disclosure include, for example, solid tissue, biopsy aspirate, ascites, fluid extract, blood, plasma, serum, spinal fluid, lymphatic fluid, external portions of the skin, respiratory tract, intestinal tract, genitourinary tract, tears, saliva, milk, tumors, organs, cell cultures, and / or cell culture components.

[0178] "Administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration for recombinant α-Gal A protein or genetically expressed α-Gal A include, for example, intravenous or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration" as used herein refers to methods of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, intrasternal, and in vivo electroporation. Other parenteral routes include oral, topical, epidermal or mucosal routes of administration, e.g., intranasal, intravaginal, intrarectal, sublingual or topical. In some embodiments, the AAV expression vectors of the disclosure are administered intravenously.

[0179] Also, administration can be performed, for example, once, multiple times, and / or over one or more extended periods of time, hi some embodiments, only one dose of the AAV expression vector of the disclosure is administered.

[0180] As used herein, the terms "about once a week," "about once every two weeks," or other similar dosing intervals refer to approximate numbers. "About once a week" can include every 7 days ± 1 day, i.e., every 6 to 8 days. "About once every two weeks" can include every 14 days ± 3 days, i.e., every 11 to 17 days. Similar approximations apply, for example, to about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, and about once every 12 weeks. In some embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose can be administered on any day in the first week, and the next dose can be administered on any day in the sixth or twelfth week, respectively. In other embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that a first dose is administered on a particular day of the week (e.g., Monday) in the first week, and a subsequent dose is administered on the same day of the week (i.e., Monday) in the sixth or twelfth week, respectively.

[0181] The term "essentially containing" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, depending in part on the method of measuring or determining the value or composition, i.e., the limitations of the measurement system. For example, "essentially containing" can mean within one standard deviation or more than one standard deviation, as is customary in the art. Alternatively, "essentially containing" can mean a range of up to 10%. Moreover, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in the present application and claims, unless otherwise stated, the meaning of "essentially containing" should be considered to be within an acceptable error range for the particular value or composition.

[0182] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0183] Units, prefixes, and symbols are indicated in the format accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in a 5' to 3' orientation. Amino acid sequences are written left to right in an amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure which may be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0184] As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0185] Various aspects of the disclosure are described in further detail in the following subsections.

[0186] II. Methods of the Disclosure Provided herein are methods for treating or ameliorating one or more symptoms of Fabry disease, reducing the amount of glycosphingolipids, and / or increasing the activity of α-galactosidase A (α-Gal A) protein in a subject in need thereof by administering an expression vector (e.g., an adeno-associated virus (AAV) expression vector) comprising an α-galactosidase A (α-Gal A) expression cassette that includes an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein.

[0187] In some embodiments, the disclosure is directed to a method of treating Fabry disease or ameliorating one or more symptoms associated with Fabry disease in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an alpha-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, and a bovine growth hormone polyA signal sequence, wherein the AAV expression vector is administered to the subject at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The drug is administered at a dose of 100 mg / kg.

[0188] In some embodiments, the disclosure is directed to a method of treating Fabry disease or ameliorating one or more symptoms associated with Fabry disease in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an α-Gal A transgene encoding at least one α-Gal A protein, the α-Gal A transgene comprising the nucleotide sequence set forth in SEQ ID NO:5, the AAV expression vector being administered at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The drug is administered at a dose of 100 mg / kg.

[0189] In some aspects, the disclosure relates to a method of reducing the amount of glycosphingolipids in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an alpha-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, and a bovine growth hormone polyA signal sequence, wherein the AAV expression vector is administered to the subject at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The amount of glycosphingolipid that is administered is at a dose of vg / kg and is reduced relative to the amount of glycosphingolipid in the subject prior to administration.

[0190] In some embodiments, the disclosure is directed to a method of reducing the amount of glycosphingolipids in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an α-Gal A transgene encoding at least one α-Gal A protein, the α-Gal A transgene comprising the nucleotide sequence set forth in SEQ ID NO:5, the AAV expression vector comprising at least about 5×10 α-Gal A expression cassette per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The amount of glycosphingolipid that is administered is at a dose of vg / kg and is reduced relative to the amount of glycosphingolipid in the subject prior to administration.

[0191] In some embodiments, the disclosure is directed to a method of increasing activity of an alpha-galactosidase A (α-Gal A) protein in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an alpha-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-Gal A transgene encoding at least one α-Gal A protein, and a bovine growth hormone polyA signal sequence, wherein the AAV expression vector is administered to the subject at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The increased activity of α-Gal A protein is relative to the activity of α-Gal A protein in the subject prior to administration, when administered at a dose of 1000 mg / kg.

[0192] In some embodiments, the disclosure is directed to a method of increasing activity of an α-galactosidase A (α-Gal A) protein in a human subject in need thereof, the method comprising administering to the subject an adeno-associated virus (AAV) expression vector comprising an α-galactosidase A (α-Gal A) expression cassette comprising an α-Gal A transgene encoding at least one α-Gal A protein, the α-Gal A transgene comprising the nucleotide sequence set forth in SEQ ID NO:5, the AAV expression vector being administered to the subject at a concentration of about 5×10 per kilogram of body weight. 12 Vector genome (vg / kg) ~ approx. 5 × 10 13 The increased activity of α-Gal A protein is relative to the activity of α-Gal A protein in the subject prior to administration, when administered at a dose of 1000 mg / kg.

[0193] In some embodiments, the AAV expression vector of the disclosure comprises about 1×10 11 vg / kg, approx. 2×10 11 vg / kg, approx. 3×10 11 vg / kg, approx. 4×10 11 vg / kg, approx. 5×10 11 vg / kg, approximately 6×10 11 vg / kg, approximately 7×10 11 vg / kg, approx. 8×10 11 vg / kg, approx. 9×10 11 vg / kg, approximately 1×10 12 vg / kg, approx. 2×10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 4×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 6×10 12 vg / kg, approximately 7×10 12 vg / kg, approx. 8×10 12 vg / kg, approx. 9×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 2×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 4×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 6×10 14 vg / kg, approximately 7×10 14 vg / kg, approx. 8×10 14 vg / kg, or approximately 9 × 10 14 The drug is administered at a dose of 100 mg / kg.

[0194] In some embodiments, the AAV expression vector of the disclosure comprises about 5×10 12 In some embodiments, the AAV expression vector of the disclosure is administered at a dose of about 1×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered at a dose of about 3×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered at a dose of about 5×10 13 The drug is administered at a dose of 100 mg / kg.

[0195] In some embodiments, the AAV expression vector of the disclosure comprises about 1×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 2×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 3×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 4×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 5×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 6×10 11In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 7×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 8×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 9×10 11 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 1×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 2×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 3×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 4×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 5×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 6×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 7×10 12 vg / kg, approx. 8×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 9×10 12 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 1×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 2×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 3×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 4×10 13In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 5×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 6×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 7×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 8×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 9×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 1×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 2×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 3×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 4×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 5×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 6×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 7×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 8×10 14 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 9×10 14 It is given in only one dose of vg / kg.

[0196] In some embodiments, the AAV expression vector of the disclosure comprises about 5×10 12In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 1×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 3×10 13 In some embodiments, the AAV expression vector of the present disclosure is administered in a single dose of about 5×10 13 It is given in only one dose of vg / kg.

[0197] In some embodiments, the AAV expression vector disclosed herein is administered to a subject, for example, a human subject, using various methods that depend in part on the route of administration.In some embodiments, the AAV expression vector disclosed herein is administered parenterally.In some embodiments, the AAV expression vector disclosed herein is administered intravenously.In some embodiments, the AAV expression vector disclosed herein is administered to a subject by intravenous infusion.

[0198] In some embodiments, the subject is afflicted with Fabry disease. In some embodiments, the subject has one or more of the following symptoms associated with Fabry disease: globotriaosylceramide (Gb3) levels above normal, globotriaosylsphingosine (lyso-Gb3) levels above normal, kidney disease, heart disease, anhidrosis, acroparesthesia, angiokeratoma, gastrointestinal (GI) tract pain, corneal and lenticular opacities, or cerebrovascular disease. In some embodiments, the angiokeratoma is periumbilical angiokeratoma. In some embodiments, the Fabry disease is a classical phenotype of type 1 or a late-onset phenotype of type 2.

[0199] In some embodiments, the subject has less than about 5% α-Gal A protein activity. In some embodiments, the subject is an enzyme replacement therapy (ERT) naive subject. In some embodiments, the ERT naive subject is an ERT naive male with classic Fabry disease.

[0200] In some embodiments, α-Gal A protein activity is measured in plasma skin and / or white blood cells of a subject, as described in Example 2 below.

[0201] In some embodiments, the subject is a male subject. In some embodiments, the subject is a female subject.

[0202] In some embodiments, the subject has an α-Gal A gene mutation indicative of Fabry disease (e.g., classic Fabry disease), e.g., as listed in a database such as the International Database of Fabry Disease Genotypes and Phenotypes (dbFGP). In some embodiments, the α-Gal A gene mutation can result in, but is not limited to, the amino acid mutations G261D, C422T, W340R, S297Y, Q283X, D215S, IVS5 / c.801+3A>G, P362L, C422T, or N34S.

[0203] In some embodiments, the subject has pre-existing anti-α-Gal A antibodies as determined by enzyme-linked immunosorbent assay (ELISA) prior to administration.

[0204] As used herein, the term "pre-existing anti-α-Gal A antibodies" refers to total anti-drug antibodies (ADA) to α-Gal A, for example, in human serum, where ADA antibodies are detected using an enzyme-linked immunosorbent assay (ELISA). Samples with % inhibition ≧46.1 are reported as positive for the presence of antibodies to α-Gal A ("anti-α-Gal A antibody positive").

[0205] Anti-GLA neutralizing antibody Total anti-drug antibodies (ADA) against α-Gal A in human serum are detected and titrated in human serum in a fluorescent enzyme inhibition assay. This assay determines the presence of anti-α-Gal A neutralizing antibodies (NAb) by assessing the neutralizing capacity of human serum against α-Gal A activity. Samples with % inhibition equal to or greater than a cutoff point of 9.6% are identified as NAb positive, while samples below the cutoff point are considered negative.

[0206] In some embodiments, upon analysis of a biological sample from the subject, the subject is an anti-α-Gal A neutralizing antibody positive subject, an anti-α-Gal A neutralizing antibody positive subject has a biological sample that has greater than about 9.6% inhibition of α-galactosidase A activity as measured by the anti-α-Gal A neutralizing antibody assay described above.

[0207] In some embodiments, anti-GLA neutralizing antibodies bind to and inactivate (neutralize) α-Gal A enzyme. In some embodiments, when anti-GLA neutralizing antibodies are present, enzyme replacement therapy is directly inactivated (neutralized) by anti-GLA neutralizing antibodies in plasma. In some embodiments, when anti-GLA neutralizing antibodies are not present, enzyme replacement therapy (e.g., recombinant α-Gal A enzyme) enters cells (e.g., endothelial cells) via the M6P receptor, resulting in Gb3 clearance from lysosomes. In some embodiments, when anti-GLA neutralizing antibodies are present, they can neutralize ERT activity by binding to the enzyme (e.g., recombinant α-Gal A).

[0208] In some embodiments, anti-GLA neutralizing IgG antibody tagged ERT molecules are internalized and digested by macrophages. If there are more anti-GLA neutralizing antibodies than ERT, this can result in reduced cellular Gb3 clearance. If ERT dose exceeds anti-GLA neutralizing antibody titer, more ERT can enter the lysosomes of target cells, resulting in increased Gb3 clearance. (Lenders et al., J Am Soc Nephrol 29:2265-2278 (2018)).

[0209] The neutralizing activity of anti-GLA antibodies is described, for example, in Rombach et al., PLoS One7:e47805(2012); Lenders et al., J Am Soc Nephrol27:256-264(2016); Smid et al., Mol Genet Metab108:132-137(2013).

[0210] In some embodiments, the anti-GLA neutralizing antibody is an IgG antibody. In some embodiments, the anti-GLA neutralizing antibody is an IgG4 antibody. In some embodiments, the anti-GLA neutralizing antibody is an IgG2 antibody. In some embodiments, the anti-GLA neutralizing antibody is an IgG1 antibody.

[0211] In some embodiments, anti-GLA neutralizing antibodies may develop within about 1 month, within about 2 months, within about 3 months, within about 4 months, within about 5 months, within about 6 months, within about 7 months, within about 8 months, within about 9 months, within about 10 months, within about 11 months, or within about 12 months after initiation of enzyme replacement therapy.

[0212] In some embodiments, a human subject capable of developing anti-GLA neutralizing antibodies is, for example, a male patient with classic Fabry disease, as described, for example, in Van der Veen et al., Mol Genet Metab. 126(2):162-168(2019); Wilcox et al., Mol Genet Metab. 105(3):443-449(2012).

[0213] In some embodiments, the α-Gal A protein expressed from the transgene reduces the amount of glycosphingolipid in the subject by at least about two-fold compared to the amount of glycosphingolipid in the subject prior to administration.

[0214] In some embodiments, the α-Gal A protein expressed from the transgene increases the amount of glycosphingolipid in the subject by about 10 percent (%), about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 0%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76% %, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0215] In some embodiments, the α-Gal A protein expressed from the transgene maintains the amount of glycosphingolipids in the subject at the same level as before administration. In some embodiments, in subjects receiving a stable dose of enzyme replacement therapy (ERT) pretreatment as described herein, the amount of glycosphingolipids is maintained at the same level as before administration. The term "stable dose of enzyme replacement therapy" or "stable dose of ERT" is defined as not missing 4 or more doses of ERT in the 6 months prior to consent and in the regimen (14 days ± 1 day for at least 3 months prior to enrollment). In some embodiments, subjects with low baseline levels of lyso-Gb3 (e.g., ERT-treated subjects) can maintain the same lyso-Gb3 levels during the observation period (e.g., lyso-Gb3 levels remain stable or show a slight decrease that is not statistically significant).

[0216] The terms "ERT-naive subject," "ERT naive subject," or "enzyme replacement therapy (ERT) naive subject" refer to a subject (e.g., a human subject) who has not undergone enzyme replacement therapy (ERT).

[0217] The terms "ERT-pseudo-naive subject," "ERT sham-naive subject," or "ERT sham-naive subject" refer to a subject (e.g., a human subject) who has previously received ERT but has not received ERT treatment within the past 6 months prior to the initiation of a clinical trial described herein.

[0218] In some embodiments, the glycosphingolipid comprises globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), galabiosylceramide, or any combination thereof. In some embodiments, Gb3 and / or lyso-Gb3 levels are measured in the plasma and / or urine of the subject, as described in Example 2 below. In some embodiments, Gb3 and / or lyso-Gb3 levels are measured in the tissue of the subject.

[0219] In some embodiments, the a-Gal A protein expressed from the transgene reduces the amount of glycosphingolipids in one or more of plasma, liver, heart, kidney, urine, skin, or spleen.

[0220] In some embodiments, the α-Gal A protein activity in the subject is about 0 fold higher to about 2 fold higher, about 2 fold higher to about 5 fold higher, about 5 fold higher to about 10 fold higher, about 10 fold higher to about 20 fold higher, about 20 fold higher to about 30 fold higher, about 30 fold higher to about 40 fold higher, about 30 fold higher to about 40 fold higher, about 40 fold higher to about 50 fold higher, about 50 fold higher to about 60 fold higher, about 60 fold higher to about 70 fold higher, about 70 fold higher to about 80 fold higher, about 80 fold higher to about 90 fold higher, about 90 fold higher to about 100 fold higher, about 100 fold higher to about 200 fold higher, about 200 fold higher to about 300 fold higher, about 300 fold higher to about 400 fold higher, or about 400 fold higher to about 500 fold higher than average normal α-Gal A protein activity.

[0221] In some embodiments, the α-Gal A protein activity in the subject is about 0 fold higher than average normal α-Gal A protein activity, compared to the α-Gal A protein activity in the subject prior to administration, about 2 fold higher, about 2 fold higher, about 3 fold higher, about 4 fold higher, about 5 fold higher, about 6 fold higher, about 7 fold higher, about 8 fold higher, about 9 fold higher, about 10 fold higher, about 11 fold higher, about 12 fold higher, about 13 fold higher, about 14 fold higher, about 15 fold higher, about 16 fold higher, about 17 fold higher, about 18 fold higher, about 19 fold higher, about 20 fold higher, about 21 fold higher, about 22 fold higher, about 23 fold higher, about 24 fold higher, about 25 fold higher, about 26 fold higher, about 27 fold higher, about 28 fold higher, about 29 fold higher, about 30 fold higher, about 31 fold higher, about 32 fold higher, about 33 fold higher, about 34 fold higher, about 35 fold higher, about 36 fold higher, about 37 fold higher, about 38 fold higher, about 39 fold higher, about 40 fold higher, about 41 fold higher, about 42 fold higher, about 43 fold higher, about 44 fold higher, about 45 fold higher, about 46 fold higher, about 47 fold higher, about 48 fold higher, about 49 fold higher, about 50 fold higher, about 51 fold higher, about 52 fold higher, about 53 fold higher, about 54 fold higher, about 55 fold higher, about 56 fold higher, about 57 fold higher, about 58 fold higher, about 5 5 times higher, 26 times higher, 27 times higher, 28 times higher, 29 times higher, 30 times higher, 31 times higher, 32 times higher, 33 times higher, 34 times higher, 35 times higher, 36 times higher, 37 times higher, 38 times higher, 39 times higher, 40 times higher, 41 times higher, 42 times higher, 43 times higher, 44 times higher, 45 times higher, 46 times higher, 47 times higher, 48 times higher, 49 times higher, 50 times higher 51 times higher, 52 times higher, 53 times higher, 54 times higher, 55 times higher, 56 times higher, 57 times higher, 58 times higher, 59 times higher, 60 times higher, 61 times higher, 62 times higher, 63 times higher, 64 times higher, 65 times higher, 66 times higher, 67 times higher, 68 times higher, 69 times higher, 70 times higher, 71 times higher, 72 times higher, 73 times higher, 74 times higher, 75 times higher, 76 times higher, about 77 times higher, about 78 times higher, about 79 times higher, about 80 times higher, about 81 times higher, about 82 times higher, about 83 times higher, about 84 times higher, about 85 times higher, about 86 times higher, about 87 times higher, about 88 times higher, about 89 times higher, about 90 times higher, about 91 times higher, about 92 times higher, about 93 times higher, about 94 times higher, about 95 times higher, about 96 times higher, about 97 times higher, about 98 times higher, about 99 times higher, or about 100 times higher.

[0222] In some embodiments, the level of the a-Gal A protein expressed from the transgene is measured in one or more of the subject's plasma, serum, whole blood, dried blood spots, white blood cells, or other blood components, hi some embodiments, the a-Gal A protein expressed from the transgene is active in the kidney, liver, skin, and heart of the subject.

[0223] In some embodiments, expression of at least one α-Gal A protein is sustained for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months.

[0224] In some embodiments, the subject is administered an immunosuppressant (e.g., prophylactic steroid therapy) prior to and / or during administration of the AAV expression vector. In some embodiments, the immunosuppressant comprises prednisone.

[0225] In some embodiments, the subject is not administered an immunosuppressant prior to and / or during administration of the AAV expression vector.

[0226] In some embodiments, the subject is not administered a preconditioning therapy (e.g., a conditioning agent) prior to administration of the AAV expression vector. The term "precondition" or "preconditioning" as used herein refers to the use of a chemotherapy conditioning agent, such as, for example, busulfan (Myleran®, GlaxoSmithKline, Busulfex®, Otsuka America Pharmaceutical, Inc.). The conditioning agent can be used, for example, in ex vivo lentiviral gene therapy to deplete / kill bone marrow cells to create space in the patient's bone marrow. When the patient undergoes gene therapy, it is expected that the therapeutic stem cells will engraft in the bone marrow and generate cells containing the therapeutic gene. The conditioning agent can cause side effects associated with chemotherapy (e.g., serious effects on fertility).

[0227] In some embodiments, the subject is not administered a conditioning or immunosuppressant agent (e.g., prophylactic steroid therapy) prior to and / or during administration of the AAV expression vector.

[0228] In some embodiments, the AAV expression vectors and pharmaceutical compositions of the disclosure eliminate the need for biweekly enzyme replacement therapy (ERT) infusions.

[0229] In some embodiments, the AAV expression vectors and pharmaceutical compositions of the present disclosure can preserve renal function in subjects with Fabry disease. In some embodiments, after administration, the subject has a reduced estimated glomerular filtration rate (eGFR) (ml / min / 1.73 m using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. 2 (units) are measured. (See, e.g., Rombach SM et al., Nephrol Dial Transplant. 25(8):2549-2556 (2010)). In some embodiments, the annual rate of decline in eGFR is less than that of comparable untreated subjects with Fabry disease.

[0230] In some embodiments, the AAV expression vectors and pharmaceutical compositions of the present disclosure can reduce cardiac disease in subjects with Fabry disease. In some embodiments, after administration, the subject is measured for ejection fraction (EF) as stroke volume (SV) / left ventricular end-diastolic volume (LVEDV). (See, e.g., Pieroni et al., Journal of the American College of Cardiology, 77(7):922-936 (2021); Linhart A. The heart in Fabry disease. In: Mehta A, Beck M, Sunder-Plassmann G, editors. Fabry Disease: Perspectives from 5 Years of FOS. Oxford: Oxford PharmaGenesis; 2006. Chapter 20). In some embodiments, the annual rate of EF decline is lower than in comparable untreated subjects with Fabry disease.

[0231] In some embodiments, after administration, the subject measures global longitudinal strain (GLS) by 2D strain echocardiography or cardiac magnetic resonance imaging (cardiac MRI or CMR). (See, e.g., Pieroni et al., Journal of the American College of Cardiology, 77(7):922-936(2021)). In some embodiments, the annual shortening progression of cardiac muscle contractility is lower than that of comparable untreated subjects with Fabry disease.

[0232] In some embodiments, after administration, the subject is measured for left ventricular mass index (LVMI) as left ventricular mass (LVM) / body surface area. (See, e.g., Pieroni et al., Journal of the American College of Cardiology, 77(7):922-936(2021)). In some embodiments, the annual LVMI increase is lower than that of comparable untreated subjects with Fabry disease.

[0233] In some embodiments, the AAV expression vectors and pharmaceutical compositions of the present disclosure can improve one or more auditory symptoms in a subject suffering from Fabry disease. In some embodiments, after administration, one or more auditory symptoms in the subject are improved. In some embodiments, the one or more auditory symptoms are tinnitus, vertigo, or progressive hearing loss.

[0234] In some embodiments, the AAV expression vectors and pharmaceutical compositions of the present disclosure can improve sweating in subjects with Fabry disease, hi some embodiments, the subject's sweating levels can be reversed from anhidrosis to hypohidrosis or normal sweating.

[0235] III. Adeno-associated Virus (AAV) Expression Vectors AAV, a parvovirus belonging to the Dependovirus genus, has several attractive features not found in other viruses. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells from a variety of species. Importantly, AAV has not been associated with any disease in humans or animals, and is not believed to alter the physiological properties of host cells upon integration. Finally, AAV is stable over a wide range of physical and chemical conditions, making it suitable for manufacturing, storage, and transportation requirements.

[0236] The AAV genome is a linear, single-stranded DNA molecule containing approximately 4700 nucleotides (the AAV-2 genome consists of 4681 nucleotides) and typically contains inverted terminal repeats (ITRs) flanked at each end by internal non-repeated segments. The ITRs are approximately 145 nucleotides in length (AAV-1 has an ITR of 143 nucleotides) and have multiple functions, including acting as origins of replication and as packaging signals for the viral genome.

[0237] The internal, non-repetitive portion of the genome contains two large open reading frames (ORFs) known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode the replication and capsid gene products, respectively, that allow replication, assembly, and packaging of complete AAV virions. More specifically, at least four viral protein families are expressed from the AAV rep region: Rep78, Rep68, Rep52, and Rep40, all of which are named based on their apparent molecular weight. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0238] AAV is a helper-dependent virus and requires coinfection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) to form functionally complete AAV virions. In the absence of coinfection with a helper virus, AAV establishes a latent state, and the viral genome is inserted into the host cell chromosome or present in the form of an episome, but no infectious virions are produced. Subsequent infection with a helper virus allows the integrated genome to be "rescued," replicated, and packaged into viral capsids, reconstituting infectious viral particles. Although AAV can infect cells from different species, the helper virus must be the same species as the host cell. Thus, for example, human AAV replicates in canine cells coinfected with canine adenovirus.

[0239] To generate recombinant AAV (rAAV) virions containing HNA, a suitable host cell line is transfected with an AAV vector that contains HNA but lacks rep and cap. The host cells are then infected with wild-type (wt) AAV and a suitable helper virus to form rAAV virions. Alternatively, the wtAAV genes (known as helper function genes, including rep and cap) and the helper virus function genes (known as accessory function genes) can be provided on one or more plasmids, thereby eliminating the need for wtAAV and helper virus in the generation of rAAV virions. The helper function gene products and accessory function gene products are expressed in the host cell and act in trans on the rAAV vector containing the heterologous gene. The heterologous gene is then replicated and packaged as if it were a wtAAV genome, forming recombinant AAV virions. When the patient's cells are transduced with the resulting rAAV virions, the HNA enters and is expressed in the patient's cells. Because the patient's cells lack the rep and cap genes, as well as accessory function genes, the rAAV virion cannot further replicate and package its genome. Furthermore, without a source of the rep and cap genes, wtAAV virions cannot form in the patient's cells. See, e.g., U.S. Patent Application Publication No. 2003 / 0147853.

[0240] In some embodiments, the AAV expression vector of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype.According to the present disclosure, the AAV serotype may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV2 / 6.In some embodiments, the AAV serotype is AAV2 / 6.In some embodiments, the AAV serotype is AAV2.In some embodiments, the AAV serotype is AAV6.

[0241] The AAV expression vector of the present disclosure may include an expression cassette that may include any promoter, enhancer, intron, signal peptide, GLA code, polyA sequence, or woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) sequence. In some embodiments, the enhancer and / or promoter is liver-specific, e.g., composed of the human apolipoprotein E (APOE) enhancer and the human alpha 1-antitrypsin (hAAT) promoter (Miao CH et al., Mol. Ther. 1(6):522-532(2000)). In some embodiments, the liver-specific promoter includes one or more ApoE enhancer sequences (e.g., 1, 2, 3, and / or 4; see Okuyama et al., Hum Gen Ther 7(5):637-645(1996)). In some embodiments, the promoter is linked to an intron. In some embodiments, the intron is a human hemoglobin beta (HBB)-IGG chimeric intron that includes a 5' donor site from the first intron of the human beta globin gene and a branch and 3' acceptor site from an intron of the immunoglobulin gene heavy chain variable region. In some embodiments, the ApoE / hAAT promoter is specifically and highly active in the intended target tissue, hepatocytes, but is inactive in non-hepatic cells and tissue types, thereby reducing or preventing expression and activity in non-target tissues. In some embodiments, the signal peptide includes an α-Gal A signal peptide (e.g., human α-Gal A signal peptide) and the polyadenylation signal includes a bovine growth hormone (bGH) polyA signal sequence. The WPRE sequence can be any wild-type or mutant WPRE sequence. See, e.g., U.S. Patent No. 10,179,918. In some embodiments, the WPRE sequence includes a mutated WPRE, such as a mut6WPRE sequence.

[0242] In some embodiments, the AAV expression vector of the present disclosure comprises an a-Gal A expression cassette flanked by two ITRs, which are at the 5' and 3' ends of the a-Gal A expression cassette.

[0243] In some embodiments, the AAV expression vector of the disclosure comprises an α-Gal A expression cassette comprising an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein. In some embodiments, the α-Gal A transgene comprises a wild-type sequence of a functional α-Gal A gene. In some embodiments, the AAV expression vector of the disclosure comprises a modified α-Gal A transgene. The sequence of the modified α-Gal A transgene is altered in some way to enhance biological activity (e.g., codon optimized to enhance biological activity, and / or altered transcriptional and translational regulatory sequences to improve gene expression). In some embodiments, the α-Gal A gene is modified to improve expression characteristics. Such modifications include, but are not limited to, the insertion of a translation initiation site (e.g., methionine), the addition of an optimized Kozak sequence, the insertion of a signal peptide, and / or codon optimization.

[0244] In some embodiments, the AAV expression vector described herein is AAV-001, also referred to as variant #21 in International Publication No. WO / 2020 / 142752, the entirety of which is incorporated herein by reference.

[0245] The rAAV vector of the present disclosure (e.g., AAV-001 rAAV vector) contains a human α-Gal A (hGLA) expression cassette (3321 bp) that contains liver-specific regulatory elements that drive expression of the hGLA transgene (see FIG. 1). The hGLA transgene is controlled by the enhancer and liver control region of the human apolipoprotein E (ApoE) gene and the human α-1-antitrypsin (hAAT) promoter. The engineered chimeric intron (HBB-IghGLA transgene) contains the codon-optimized hGLA α-Gal A enzyme with the same amino acid sequence as the native hGLA protein and the approved recombinant α-Gal A (Fabrazyme®).

[0246] The α-Gal A expression cassette of the present disclosure contains a mutated version of the Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPREmut6). WPREmut6 is a 592-bp DNA sequence that contains the promoter region of the WHV X protein followed by a truncated version of the X protein itself (WPRE, Zufferey et al., J Virol. 73(4):2886-2892(1999)), with a point mutation in the putative promoter region and initiation codon of the X protein open reading frame, preventing expression of the X protein (mut6, Zanta-Boussif et al., Gene Therapy 16(5):605-619(2009)). The polyA sequence is a derivative of the bovine growth hormone polyadenylation signal. Addition of the WPREmut6 element increased the production of α-Gal A protein. Indeed, compared to AAV-001PC (lacking the WPREmut6 element), the potency of AAV-001 is higher. The AAV-001rAAV vector contains an α-Gal A expression cassette flanked at both ends by ITRs from AAV2, which is packaged together with a capsid derived from adeno-associated virus type 6 (AAV6) using the Sf9 insect cell / recombinant baculovirus (Sf9 / rBV) expression system.

[0247] In some embodiments, the AAV expression vector (e.g., AAV-001rAAV vector) sequence comprises the elements and sequences of an a-Gal A expression cassette, as shown in Table 1 below. Table 1: α-Gal AcDNA elements and complete sequences [Table 1] TIFF2024542023000002.tif210159 SEQ ID NO:9 (complete transgene sequence) [Table 2] TIFF2024542023000004.tif53159

[0248] In some embodiments, an AAV expression vector of the disclosure (e.g., an AAV-001rAAV vector) comprises an alpha-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, and a bovine growth hormone polyA signal sequence. In some embodiments, the enhancer comprises the nucleotide sequence set forth in SEQ ID NO:2, the promoter comprises the nucleotide sequence set forth in SEQ ID NO:3, the intron comprises the nucleotide sequence set forth in SEQ ID NO:4, the α-Gal A transgene comprises the nucleotide sequence set forth in SEQ ID NO:5, the mutated WPRE sequence comprises the nucleotide sequence set forth in SEQ ID NO:6, and the polyA signal sequence comprises the nucleotide sequence set forth in SEQ ID NO:7.

[0249] In some embodiments, an a-Gal A expression cassette of the present disclosure comprises the nucleotide sequence set forth in SEQ ID NO:9.

[0250] In some embodiments, the signal peptide of human α-galactosidase A comprises the amino acid sequence set forth in SEQ ID NO:10.

[0251] Human α-galactosidase A signal peptide (SEQ ID NO: 10) MQLRNPELHLGCALALRFLALVSWDIPGARA

[0252] IV. Pharmaceutical Compositions and Formulations In some embodiments, the AAV expression vectors useful in the methods and compositions disclosed herein are present in a pharmaceutical composition. Thus, some embodiments of the present disclosure are directed to pharmaceutical compositions comprising the AAV expression vectors of the present disclosure together with pharma- ceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants.

[0253] In some embodiments, the pharma- ceutically acceptable carrier comprises phosphate buffered saline containing CaCl2, MgCl2, NaCl, sucrose, and Kolliphor (poloxamer) P188.

[0254] In some embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used. In some embodiments, the formulation material(s) are for subcutaneous and / or intravenous administration. In some embodiments, pharmaceutical compositions include formulation materials to alter, maintain or preserve, for example, pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition.In some embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming agents. These include, but are not limited to, ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic®, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate 80, etc.), triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents, excipients, and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, ARGennaro, ed., Mack Publishing Company (1995).

[0255] In some embodiments, optimal pharmaceutical compositions will be determined by one of skill in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions affect the physical state, stability, in vivo release rate, and / or in vivo clearance rate of the AAV expression vector.

[0256] In some embodiments, the primary vehicle or carrier in a pharmaceutical composition is either aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier is water for injection, saline, or artificial cerebrospinal fluid, which may be supplemented with other materials common to compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate buffered saline. In some embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In some embodiments, the pharmaceutical composition comprises a Tris buffer at about pH 7.0-8.5, or an acetate buffer at about pH 4.0-5.5. In some embodiments, the pharmaceutical composition further comprises sorbitol or a suitable substitute thereof. In some embodiments, the composition comprising the AAV expression vector is prepared for storage by mixing the selected composition having the desired purity with any formulation drug in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, supra). Additionally, in some embodiments, the composition comprising the AAV expression vector is formulated as a lyophilizate using a suitable excipient such as sucrose.

[0257] In some embodiments, the pharmaceutical composition is selected for parenteral delivery.

[0258] In some embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In some embodiments, a buffer is used to maintain the composition at physiological pH or a slightly lower pH, usually within the range of about 5 to about 8.

[0259] In some embodiments, when parenteral administration is intended, the therapeutic composition is in the form of a pyrogen-free parenterally acceptable aqueous solution that contains the AAV expression vector of the present disclosure in a pharma- ceutically acceptable vehicle. In some embodiments, the vehicle for parenteral injection is sterile distilled water, in which the AAV expression vector is formulated as a sterile isotonic solution and appropriately preserved. In some embodiments, the preparation includes formulating the desired molecule with agents such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, which provide controlled or sustained release of the product, which can then be delivered by depot injection. In some embodiments, hyaluronic acid is also used. The presence of hyaluronic acid has the effect of promoting sustained duration in the circulation. In some embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0260] In some embodiments, pharmaceutical composition comprises an effective amount of AAV expression vector in a mixture with non-toxic excipient suitable for tablet manufacture.In some embodiments, tablet is dissolved in sterile water or another suitable medium to prepare a solution in unit dose form.In some embodiments, suitable excipients include but are not limited to inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0261] Additional pharmaceutical compositions, including formulations involving AAV expression vectors in sustained or controlled delivery formulations, will be apparent to those skilled in the art. In some embodiments, the techniques of formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, depot injections, etc., are also well known to those skilled in the art. For example, see PCT Application No. PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. In some embodiments, sustained release formulations can include semipermeable polymer matrices, for example, in the form of molded articles of films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra), or poly-D(-)-3-hydroxybutyrate (EP 133,988). In some embodiments, sustained release compositions also include liposomes, which can be prepared by any of several methods well known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0262] Pharmaceutical compositions for use in in vivo administration are usually sterile. In some embodiments, this is accomplished by filtration through sterile filtration membranes. In some embodiments, if the composition is lyophilized, sterilization using this method is performed before or after lyophilization and reconstitution. In some embodiments, compositions for parenteral administration are stored in lyophilized form or in solution. In some embodiments, parenteral compositions are generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0263] In some embodiments, once the pharmaceutical composition is formulated, it is stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. In some embodiments, such formulations are stored either in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) prior to administration.

[0264] V. Fabry Disease Treatment In some embodiments, the disclosure is directed to a method of treating Fabry disease in a human subject using an AAV expression vector of the disclosure, where the subject has been administered enzyme replacement therapy (ERT) for Fabry disease prior to administration ("pre-treatment") or has been administered a non-enzyme replacement therapy for Fabry disease prior to administration ("pre-treatment").

[0265] VA enzyme replacement therapy In some embodiments, the pre-treatment ERT comprises enzyme replacement therapy.

[0266] In some embodiments, the pretreatment ERT comprises a recombinant alpha-galactosidase A (GLA) protein or a gene expressing GAL. In some embodiments, the pretreatment ERT comprises agalsidase alpha and / or beta, or a gene expressing agalsidase alpha and / or beta. In some embodiments, the enzyme replacement therapy comprises administering agalsidase alpha (Replagal®, Shire Human Genetic Therapies), agalsidase beta (Fabrazyme®, Sanofi Genzyme), pegnigalsidase alpha (PRX-102, Protalix BioTherapeutics), or any combination thereof. These forms of ERT aim to compensate for insufficient alpha-Gal A activity in patients with a recombinant form of the enzyme administered intravenously. ERT has been demonstrated to reduce Gb3 deposition in renal capillary endothelium and several other cell types. Although ERT is effective in many situations, the treatment also has limitations. ERT has not been demonstrated to reduce the risk of stroke, the myocardial response is slow, and clearance of Gb3 from some cell types in the kidney is limited. Some patients develop an immune response to ERT. See, e.g., U.S. Patent No. 10,155,027.

[0267] In some embodiments, about 0.2 mg / kg body weight of agalsidase alfa is injected as an intravenous infusion every two weeks.

[0268] In some embodiments, about 0.3 mg / kg of body weight of agalsidase beta is injected as an intravenous infusion every two weeks. In some embodiments, about 1 mg / kg of body weight of agalsidase beta is injected as an intravenous infusion every two weeks.

[0269] In some embodiments, saturating higher anti-GLA neutralizing antibody levels in a subject via higher doses of ERT administration can provide clinical benefit (e.g., lower plasma lyso-Gb3 levels).See, e.g., Lenders et al., Orphanet Journal of Rare Diseases, 13(171)(2018).

[0270] In some embodiments, the pretreatment ERT comprises gene therapy. In some embodiments, the gene therapy comprises a vector encoding an enzyme. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector comprises an adeno-associated viral (AAV) vector or a lentiviral vector. In some embodiments, the gene therapy comprises administering AVR-RD-01 (AvroBio), FLT-190 (Freeline Therapeutics), pegniglucidase alfa (PRX-102, Protalix BioTherapeutics), and 4D-310 (4D Molecular Therapeutics), or any combination thereof.

[0271] The AVR-RD-01 drug product contains an autologous CD34+ cell-enriched fraction containing cells transduced with a lentiviral vector / alpha-galactosidase A (AGA) encoding the human AGA complementary deoxyribonucleic acid (cDNA) sequence. (ClinicalTrials.gov; Identifier: NCT03454893).

[0272] FLT190 is a single-stranded (ss) AAV gene therapy construct containing codon-optimized human GLAc DNA driven by a liver-specific promoter (FRE1) and pseudotyped in an AAV8 capsid (ssAAV8-FRE1-GLAco). (Nephron Clinical Practice, Abstracts: 6th Update on Fabry Disease: Biomarkers, Progression and Treatment Opportunities, May 26-28, 2019, Prague, Czech Republic).

[0273] Pegnigalsidase alfa (PRX-102) is an investigational, stabilized version of recombinant α-galactosidase-A enzyme expressed in plant cell culture and chemically modified. Protein subunits are covalently linked via chemical cross-linking using short PEG moieties, resulting in a molecule with unique pharmacokinetic parameters. In clinical trials, PRX-102 has been observed to have a circulating half-life of approximately 80 hours.

[0274] 4D-310 is an adeno-associated virus (AAV) gene therapy that consists of two active components: a capsid (4D-C102) and a transgene cassette encoding a codon-optimized full-length human GLA transgene driven by a CAG promoter. 4D-310 is engineered to be replication-incompetent (replication-defective). (ClinicalTrials.gov; Identifier: NCT04519749).

[0275] In some embodiments, the gene therapy comprises a vector encoding an enzyme. In some embodiments, the vector comprises an mRNA encoding human GLA protein or agalsidase alpha and / or beta, for example as described in U.S. Pat. No. 9,308,281. In some embodiments, the mRNA can comprise one or more modifications that confer stability to the mRNA (e.g., compared to a wild-type or native version of the mRNA) and can also comprise one or more modifications relative to the wild-type that correct defects responsible for the associated aberrant expression of the protein. For example, the nucleic acid of the present disclosure can comprise modifications to one or both of the 5' and 3' untranslated regions. Such modifications can include, but are not limited to, incorporation of a partial sequence of the cytomegalovirus (CMV) immediate early 1 (IE1) gene, a polyA tail, a Cap1 structure, or a sequence encoding human growth hormone (hGH). In some embodiments, the mRNA is modified to reduce mRNA immunogenicity.

[0276] In some embodiments, the gene therapy is delivered by a delivery vehicle. In some embodiments, the delivery vehicle is a liposomal delivery vehicle, e.g., lipid nanoparticles, as described in U.S. Pat. No. 9,308,281. In some embodiments, the mRNA encoding human GLA protein or agalsidase alpha and / or beta is formulated into a liposomal delivery vehicle to facilitate delivery to target cells. The delivery vehicle contemplated may include one or more cationic lipids, non-cationic lipids, and / or PEG-modified lipids. For example, the delivery vehicle may include at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, and HGT5001. In some embodiments, the delivery vehicle includes cholesterol (chol) and / or PEG-modified lipids. In some embodiments, the delivery vehicle includes DMG-PEG2K. In some embodiments, the delivery vehicle comprises one of the following lipid formulations: C12-200, DOPE, chol, DMG-PEG2K, DODAP, DOPE, cholesterol, DMG-PEG2K, HGT5000, DOPE, chol, DMG-PEG2K, HGT5001, DOPE, chol, and DMG-PEG2K.

[0277] In some embodiments, the pretreatment ERT comprises administering Galafold® (migalastat; Amicus Therapeutics). Galafold® is an alpha-galactosidase A (alpha-Gal A) pharmacological chaperone. In some embodiments, 123 mg of Galafold® is administered orally at the same time every other day, e.g., as described in Lenders et al., J Am Soc Nephrol, 29:2265-2278 (2018).

[0278] VB Enzyme replacement therapy and active site-specific chaperones In some embodiments, the pretreatment ERT of the present disclosure comprises a combination of an α-galactosidase A protein (e.g., recombinant α-Gal A (rhα-Gal A)) with an active site-specific chaperone (ASSC) of α-Gal A, such as migastat (1-deoxygalactonojirimycin (DGJ)), e.g., as described in U.S. Pat. No. 10,155,027.

[0279] In some embodiments, the disclosure provides a combination therapy of a-Gal A (e.g., rha-Gal AERT) with ASSC for a-Gal A enzyme (e.g., (DGJ)). In some embodiments, a-Gal A and ASSC are co-formulated together and administered to a subject simultaneously as a co-formulation. In some embodiments, ASSC1-deoxygalactonojirimycin is co-formulated with a-Gal A as a pharmaceutical composition. Such compositions can increase the stability of a-Gal A both during storage (i.e., in vitro) and in vivo after administration to a subject, thereby increasing circulating half-life, tissue uptake, and increasing the therapeutic effect of a-Gal A (e.g., increasing the reduction in tissue GL-3 levels). In some embodiments, the route of administration is intravenous. Administration can be by periodic bolus injection of the formulation or can be as a sustained release dosage form over an extended period of time, e.g., intravenous administration from an external reservoir (e.g., IV bag).

[0280] Suitable co-formulations for intravenous use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that puncture resistance exists. In some embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. In some embodiments, the co-formulation comprises a carrier such as a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, benzyl alcohol, sorbic acid, etc.).

[0281] In some embodiments, isotonic agents such as sugars or sodium chloride are added. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions can be prepared by incorporating α-Gal A and ASSC (e.g., DGJ) in the required amount in an appropriate solvent with various other ingredients as described above, followed by filtration or terminal sterilization as required. In general, dispersions can be prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0282] In some embodiments, the co-formulation may include an excipient. Pharmaceutically acceptable excipients that may be included in the co-formulation include buffers such as citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer, amino acids, urea, alcohol, ascorbic acid, phospholipids; proteins such as serum albumin, collagen, and gelatin, salts (such as EDTA, EGTA, and sodium chloride), liposomes, polyvinylpyrrolidone, sugars (such as dextran, mannitol, sorbitol, and glycerol), propylene glycol, and polyethylene glycol (e.g., PEG-4000, PEG-6000), glycerol, glycine (or other amino acids), and lipids. Buffer systems used with the co-formulation may include citrate buffer, acetate buffer, bicarbonate buffer, and phosphate buffer.

[0283] The co-formulation may also include non-ionic detergents, including, but not limited to, polysorbate 20, polysorbate 80, Triton X-100, Triton X-114, Nonidet® P-40, octyl alpha-glucoside, octyl beta-glucoside, Bridge 35, Pluronic®, and Tween 20.

[0284] For lyophilization of protein and chaperone formulations, the protein concentration may be from about 0.1 mg / mL to about 10 mg / mL. Bulking agents such as glycine, mannitol, albumin, and dextran may be added to the lyophilization mixture. Additionally, cryoprotectants such as disaccharides, amino acids, and PEG may be added to the lyophilization mixture. Any of the buffers, excipients, and detergents listed above may also be added.

[0285] In some embodiments, the co-formulation comprises α-Gal A at a concentration of about 0.05 to about 100 μM, about 0.1 to about 75 μM, about 0.2 to about 50 μM, about 0.3 to about 40 μM, about 0.4 to about 30 μM, about 0.5 to about 20 μM, about 0.6 to about 15 μM, about 0.7 to about 10 μM, about 0.8 to about 9 μM, about 0.9 to about 8 μM, about 1 to about 7 μM, about 2 to about 6 μM, or about 3 to about 5 μM.

[0286] In some embodiments, the co-formulation comprises a-Gal A at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 μM.

[0287] In some embodiments, the co-formulation comprises α-Gal A at a concentration of about 0.0025 to about 5 mg / ml, about 0.005 to about 4.5 mg / ml, about 0.025 to about 4 mg / ml, about 0.05 to about 3.5 mg / ml, about 0.25 to about 3 mg / ml, about 0.5 to about 2.5 mg / ml, about 0.75 to about 2 mg / ml, or about 1 to about 1.5 mg / ml.

[0288] In some embodiments, the co-formulation comprises DGJ at a concentration of about 10 to about 25,000 μM, about 50 to about 20,000 μM, about 100 to about 15,000 μM, about 150 to about 10,000 μM, about 200 to about 5,000 μM, about 250 to about 1,500 μM, about 300 to about 1,000 μM, about 350 to about 550 μM, or about 400 to about 500 μM.

[0289] In some embodiments, the co-formulation comprises DGJ at a concentration of about 0.002 to about 5 mg / ml, about 0.005 to about 4.5 mg / ml, about 0.02 to about 4 mg / ml, about 0.05 to about 3.5 mg / ml, about 0.2 to about 3 mg / ml, about 0.5 to about 2.5 mg / ml, or about 1 to about 2 mg / ml.

[0290] In some embodiments, the co-formulation comprises a concentration of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 μM DGJ.

[0291] In some embodiments, the α-Gal A enzyme and DGJ are combined to form a co-formulation for administration to a subject, and the dose of the α-Gal A enzyme in the co-formulation administered to the subject is about 0.05 to about 10 mg / kg, about 0.1 to about 5 mg / kg, about 0.2 to about 4 mg / kg, about 0.3 to about 3 mg / kg, about 0.4 to about 2 mg / kg, about 0.5 to about 1.5 mg / kg, or about 0.5 to about 1 mg / kg.

[0292] In some embodiments, the dosage of the a-Gal A enzyme of the co-formulation administered to a subject is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mg / kg.

[0293] In some embodiments, the α-Gal A enzyme and DGJ are combined to form a co-formulation for administration to a subject, and the dose of DGJ in the co-formulation administered to the subject is about 0.05-20 mg / kg, about 0.1-15 mg / kg, about 0.2-10 mg / kg, about 0.3-10 mg / kg, about 0.4-9 mg / kg, about 0.5-8 mg / kg, about 0.6-7 mg / kg, about 0.7-6 mg / kg, about 0.8-5 mg / kg, about 0.9-4 mg / kg, about 1-3 mg / kg, or about 1.5-2 mg / kg.

[0294] In some embodiments, the dosage of DGJ of the co-formulation administered to a subject is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

[0295] In some embodiments, a co-formulation of α-Gal A and DGJ can be administered intravenously to a subject in an amount effective to achieve a plasma AUC concentration that is about 0.5 to 10 times, about 1 to about 8 times, about 1.5 to about 6 times, about 2 to about 5.5 times, about 2.5 to about 5 times, or about 3 to about 4.5 times the plasma AUC concentration achieved when α-Gal A is administered to a subject at the same dose as the co-formulation, but without DGJ.

[0296] As used herein, the term "AUC" refers to a mathematical calculation to assess the body's total exposure to a given drug over time. In a graph plotting blood concentrations after administration, the drug concentration variable is on the y-axis and time is on the x-axis. The area between the drug concentration curve and the x-axis at a specified time interval is the AUC. AUC is used as a guide for administration schedules and to compare the availability of different drugs in the body.

[0297] In some embodiments, a co-formulation of α-Gal A and DGJ can be administered intravenously to a subject in an amount effective to achieve a level of α-Gal A tissue uptake that is about 0.5 to 10 times, about 1 to about 8 times, about 1.5 to about 6 times, about 2 to about 5.5 times, about 2.5 to about 5 times, or about 3 to about 4.5 times the level of α-Gal A tissue uptake achieved when α-Gal A is administered to a subject at the same dose as the co-formulation, but without DGJ.

[0298] Delivery of the co-formulation can continue for a preselected administration period ranging from a few hours, one to several weeks, one to several months, or up to a year or more. In some embodiments, the dosage form is suitable for delivering a-Gal A for an extended period of time. Such a delivery device can be adapted to administer a-Gal A for a few hours (e.g., 2 hours, 12 hours, or 24 hours to 48 hours or more), a few days (e.g., 2 to 5 days or more, about 100 days or more), a few months, or a few years. In some embodiments, the device is suitable for delivery over a period ranging from about 1 month to about 12 months or more. The α-Gal A delivery device can be adapted to administer α-Gal A to an individual for a period of time ranging from about 2 hours to about 72 hours, from about 4 hours to about 36 hours, from about 12 hours to about 24 hours, from about 2 days to about 30 days, from about 5 days to about 20 days, from about 7 days to about 100 days or more, from about 10 days to about 50 days; from about 1 week to about 4 weeks, from about 1 month to about 24 months or more, from about 2 months to about 12 months, from about 3 months to about 9 months, or other ranges of time including incremental ranges within these ranges as appropriate.

[0299] In some embodiments, the dose of α-Gal A present in the co-formulation with DGJ is administered intravenously once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, or once every 6 days. In some embodiments, the dose does not result in toxic levels of α-Gal A in the liver of the individual. In some embodiments, the co-formulation composition of α-Gal A and DGJ is administered at a dose sufficient to result in a peak concentration of α-Gal A in the tissue of the subject within about 24 hours after administration. In some embodiments, the co-formulation composition is administered at a dose sufficient to result in a peak concentration of α-Gal A in the tissue of the subject within about 0.2 to about 50 hours, about 0.2 to about 24 hours, about 0.2 to about 5 hours, about 0.2 to about 1 hour, about 0.2 to about 0.5 hours, or about 40, 30, 20, 10, 5, 1, 0.5 or less hours after administration of the dose. In some embodiments, the co-formulation is administered as a single dose. In some embodiments, the co-formulation is administered as multiple doses.

[0300] VC non-enzyme replacement therapy In some embodiments, the therapy for Fabry disease is enzyme replacement therapy.

[0301] In some embodiments, non-enzyme replacement therapy comprises small molecule therapy.Some new drug development strategies for small molecule therapy of Fabry disease include, but are not limited to, substrate reduction therapy (SRT), residual enzyme activation, GLA promoter activation, protein homeostasis regulation (proteostasis), and chemical chaperone therapy (CCT), for example, as described in Motabar et al., Curr Chem Genomics 4:50-56 (2010).

[0302] In some embodiments, the small molecule therapy comprises administering lucerastat (Idorsia Pharmaceuticals Ltd), benglustat (Sanofi Genzyme), or apabetalone (development codes RVX208, RVX-208, and RVX000222, Resverlogix Corp.), or any combination thereof.

[0303] VI. Methods for Producing AAV Expression Vectors Also included within the scope of the present disclosure is a method of producing AAV particles by viral genome replication in a viral replicating cell, comprising contacting the viral replicating cell with an AAV polynucleotide or AAV genome (e.g., an AAV vector of the present disclosure).In the context of the present disclosure, the AAV expression vectors disclosed herein are considered to be AAV payload construct vectors.

[0304] In some embodiments, the AAV particles are produced by a method comprising the steps of: (1) co-transfecting a bacmid vector and either a viral construct vector and / or an AAV payload construct vector into competent bacterial cells; (2) isolating the resulting viral construct expression vector and the AAV payload construct expression vector and separately transfecting viral replicating cells; (3) isolating and purifying the resulting payload and viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector; (4) co-infecting viral replicating cells with both the AAV payload and the viral construct expression vector or the viral construct particles comprising the AAV payload construct expression vector; and (5) collecting and purifying the viral particles comprising the parvovirus genome.

[0305] In one aspect, the disclosure provides a method for producing AAV particles, the method comprising: (1) simultaneously co-transfecting a payload region (e.g., a polynucleotide encoding a therapeutic molecule of the disclosure), a construct expressing the rep and cap genes, and a helper construct into a mammalian cell, such as, but not limited to, HEK293 cells; and (2) harvesting and purifying the AAV particles containing the viral genome.

[0306] In some embodiments, AAV particles can be produced in virus-replicating cells, including insect cells. Growth conditions for cultured insect cells and production of heterologous products in cultured insect cells are well known in the art, see, e.g., U.S. Patent No. 6,204,059.

[0307] The viral replicating cells can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. The viral replicating cells can include mammalian cells such as A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, etc.

[0308] W138, HeLa, HEK293, Saos, C2C12, L cells, HT1080, HepG2, and primary fibroblasts, hepatocytes, myoblasts of mammalian origin. Viral replicating cells include cells from mammalian species, including but not limited to, human, monkey, mouse, rat, rabbit, and hamster, or cell types including but not limited to, fibroblasts, hepatocytes, tumor cells, cell line transformed cells.

[0309] The viral production disclosed herein describes processes and methods for generating AAV particles that contact target cells and deliver a payload (e.g., a recombinant viral construct comprising a polynucleotide sequence encoding a payload, such as an α-Gal A protein).

[0310] In some embodiments, AAV particles can be produced in viral replicating cells, including mammalian cells.Viral replicating cells commonly used for producing recombinant AAV particles include, but are not limited to, 293 cells, COS cells, HeLa cells, and KB cells.

[0311] In some embodiments, AAV particles are produced in mammalian cells and all three VP proteins are expressed with a stoichiometry close to 1:1:10 (VP1:VP2:VP3). The regulatory mechanisms that allow for this controlled level of expression include the production of two mRNAs, one for VP1 and one for VP2 and VP3, that are generated by differential splicing.

[0312] In some embodiments, AAV particles are produced in mammalian cells using a triple transfection method, where the payload construct, the parvoviral Rep and parvoviral Cap, and the helper construct are contained within three different constructs. The triple transfection method of the three components of AAV particle production can be utilized to produce small lots of virus for assays such as transduction efficiency, target tissue (tropism) assessment, stability, etc.

[0313] In some embodiments, the viral construct vector and the AAV payload construct vector can each be incorporated into a bacmid (also known as a baculovirus plasmid) by a transposon donor / acceptor system, by standard molecular biology techniques well known and practiced by those skilled in the art. By transfection of separate viral replicating cell populations, two baculoviruses are generated, one containing the viral construct expression vector and the other containing the AAV payload construct expression vector. The two baculoviruses can be used to infect a single viral replicating cell population to generate AAV particles.

[0314] Baculovirus expression vectors for providing viral particle products in insect cells, including but not limited to Spodoptera frugiperda (Sf9) cells, provide high titer viral particle production. Recombinant baculoviruses encoding viral construct expression vectors and AAV payload construct expression vectors initiate productive infection of virus replicating cells. Infectious baculovirus particles released from the primary infection secondarily infect additional cells in culture, exponentially infecting the entire cell culture population with a number of infection cycles that is a function of the initial multiplicity of infection, see e.g., Urabe, M. et al., J Virol. 2006 Feb;80(4):1874-85, the contents of which are incorporated herein by reference in their entirety. In some embodiments, an AAV expression vector of the disclosure (e.g., AAV-001rAAV vector) comprises an a-Gal A expression cassette flanked on both sides by ITRs from AAV2, which is packaged with a capsid from adeno-associated virus type 6 (AAV6) using an Sf9 insect cell / recombinant baculovirus (Sf9 / rBV) expression system. In some embodiments, an AAV expression vector of the disclosure (e.g., AAV-001rAAV vector) comprises an a-Gal A expression cassette flanked on both sides by ITRs from AAV2, which is packaged with a capsid from AAV6 using a mammalian expression system, e.g., HEK293.

[0315] The use of baculovirus in insect cell systems to produce AAV particles addresses the well-known genetic and physical instability of baculovirus. Virus-producing cells infected with baculovirus are harvested in aliquots that can be frozen and stored in liquid nitrogen. The aliquots retain viability and infectivity for infection of large-scale virus-producing cell cultures (Wasilko DJ et al., Protein Expr Purif. 2009 Jun;65(2):122-32).

[0316] In some embodiments, stable viral replication cells permissive to baculovirus infection are engineered with at least one stable integrated copy of any of the elements required for AAV replication and viral particle production, including, but not limited to, the entire AAV genome, the Rep and Cap genes, the Rep gene, the Cap gene, each Rep protein as a separate transcription cassette, each VP protein as a separate transcription cassette, AAP (assembly activating protein), or at least one of the baculovirus helper genes with native or non-native promoters.

[0317] In some embodiments, AAV particle production can be modified to scale up production. Transfection of replicating cells in large-scale culture formats can be performed according to any method known in the art.

[0318] In some embodiments, cell culture bioreactors can be used for large-scale virus production. In some cases, the bioreactor comprises a stirred tank reactor.

[0319] Cell lysis The cells of the present disclosure (including but not limited to virus-producing cells) can be subjected to cell lysis according to any method known in the art. Cell lysis can be performed to obtain one or more factors (e.g., virus particles) present within any cell of the present disclosure.

[0320] Cell lysis methods can be chemical or mechanical. Chemical cell lysis methods typically involve contacting one or more cells with one or more lysis agents. Mechanical lysis typically involves subjecting one or more cells to one or more lysis conditions and / or one or more lytic forces. In some embodiments, chemical lysis can be used to lyse cells. As used herein, the term "lysis agent" refers to any agent that can aid in the destruction of cells. In some cases, the lysis agent is introduced into a solution called a lysis solution or lysis buffer. As used herein, the term "lysis solution" refers to a solution (usually aqueous) that contains one or more lysis agents. In addition to the lysis agent, the lysis solution can include one or more buffers, solubilizing agents, detergents, preservatives, cryoprotectants, enzymes, enzyme inhibitors, and / or chelating agents.

[0321] The concentration of salt can be increased or decreased to obtain a concentration effective for disrupting cell membranes. Lysing agents, including detergents, can include ionic detergents or non-ionic detergents. Detergents can function to disrupt or dissolve cell structures, including but not limited to cell membranes, cell walls, lipids, carbohydrates, lipoproteins, and glycoproteins.

[0322] In some embodiments, mechanical cell lysis is performed. Mechanical cell lysis methods may include the use of one or more lysis conditions and / or one or more lysis powers. As used herein, the term "lysis conditions" refers to a state or situation that promotes cell disruption. Lysis conditions may include a particular temperature, pressure, osmotic purity, salinity, etc. In some embodiments, the lysis conditions include an increase or decrease in temperature. In some embodiments, the lysis conditions include a temperature change to promote cell disruption. Cell lysis performed according to such embodiments may include freeze-thaw lysis.

[0323] As used herein, the term "lytic force" refers to physical activity used to disrupt cells. Lytic forces may include, but are not limited to, mechanical, sonic, gravitational, optical, electrical, etc. Cell lysis performed by mechanical force is referred to herein as "mechanical lysis." Mechanical forces that can be used according to mechanical lysis may include high shear fluid forces.

[0324] In some embodiments, the method of harvesting AAV particles without dissolution can be used for efficient and scalable AAV particle production.As a non-limiting example, AAV particles can be produced by culturing AAV particles that lack heparin binding sites, thereby passing AAV particles through the supernatant of cell culture, collecting the supernatant from culture, and separating AAV particles from the supernatant, as described in US Patent Application 20090275107.

[0325] AAV purification The cell lysate containing the viral particles may be subjected to clarification. Clarification refers to the first step taken in purifying viral particles from the cell lysate. Clarification serves to remove larger insoluble debris to prepare the lysate for further purification. Clarification steps may include, but are not limited to, centrifugation and filtration.

[0326] In some embodiments, AAV particles can be purified from clarified cell lysates by one or more chromatography methods. Chromatography refers to a variety of methods well known in the art for separating one or more components from a mixture. Such methods include, but are not limited to, ion exchange chromatography (e.g., cation exchange chromatography and anion exchange chromatography), immunoaffinity chromatography, and size exclusion chromatography.

[0327] All references cited above, and all references cited herein, are incorporated herein by reference in their entirety.

[0328] The following examples are offered by way of illustration and not by way of limitation.

[0329] Working Example Although the present disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. Furthermore, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure.

[0330] Example 1 Clinical Evaluation of AAV-001 in Adults with Fabry Disease AAV-001 was evaluated in a Phase 1 / 2, multicenter, open-label, single-dose, dose-ranging study to assess the safety and tolerability of AAV-001, a rAAV2 / 6 human α-Gal A gene therapy, in subjects with Fabry disease. The study design is presented in Figure 2.

[0331] The primary objective of this study is to evaluate the safety and tolerability of AAV-001. Secondary objectives of this study are to evaluate the pharmacodynamics of α-Gal A and the presence of its substrates in plasma over time, to evaluate the effect of AAV-001 on the required ERT dosing in subjects receiving ERT, to evaluate the effect of AAV-001 on renal and cardiac function, to evaluate the clinical impact of AAV-001 on Fabry disease, including quality of life (QoL), and to evaluate the shedding of rAAV2 / 6 vector DNA over time. Additional objectives of this study are to evaluate the pharmacodynamics of α-Gal A and the presence of its substrates in urine and tissues over time, to evaluate the pharmacokinetics of α-Gal A over time, and to evaluate the immune response to rAAV2 / 6 and α-Gal A.

[0332] Test evaluation Evaluations will include the occurrence of treatment-emergent adverse events (TEAEs), periodic hematology, chemistry, liver function, vital signs, ECO and ECHO, serial alpha-fetoprotein (AFP) tests, and MRI (or equivalent) of the liver to monitor for the formation of liver masses. Additionally, plasma α-Gal A activity, plasma Gb3 levels, plasma Lyso-Gb3 levels, frequency of FABRAZYME® (or equivalent ERT) infusions, estimated glomerular filtration rate (eGFR) calculated from blood creatinine levels, left ventricular mass measured by cardiac magnetic resonance imaging (MRI), urinary total protein and albumin-to-creatinine ratio, α-Gal A and Gb3 levels measured in tissue, substrate levels measured in tissue and urine, urinary biomarkers of renal function, neuropathic pain measured by Brief Pain Inventory (BPI), frequency of analgesic use, gastrointestinal (GI) symptoms measured by the GI Symptom Rating Scale, Mainz Severity Score Index (MSSI), patient-reported quality of life (QOL) outcomes measured by the SF-36 questionnaire, and the association of rAAV2 / 6 and α-Gal with α-Gal-α-Glucocorticoids (ACGs) at specific time points over a one-year period. Changes from baseline in immune responses to A can be measured, and clearance of rAAV vectors can be measured by levels of vector genome in blood, plasma, saliva, urine, stool, and semen.

[0333] Target population and eligibility criteria Male and female subjects aged 18 years or older with classic Fabry disease. Subjects may be undergoing an enzyme replacement therapy (ERT) regimen as described herein, or may be ERT naïve, or ERT sham naïve and have not received ERT treatment in the past 6 months.

[0334] Subject inclusion criteria included the following: (1) subjects with a documented diagnosis of classic Fabry disease, defined as either plasma or leukocyte α-Gal A activity less than 5%, and one or more of the following symptomatic characteristics of classic Fabry disease: i) corneal verticillate opacity, ii) acroparesthesia, iii) anhidrosis, or iv) angiokeratoma (if clustering of periumbilical angiokeratoma is documented, this symptom alone is sufficient as this is the pathognomonic sign of classic Fabry disease); (2) subjects receiving ERT (14-day [± 1-day] regimen) or subjects receiving ERT but not α-Gal A activity. (3) For subjects receiving ERT, ERT must have been administered at a stable dose (defined as no more than four missed doses of ERT in the six months preceding consent) and regimen (14 days ± 1 day for at least three months prior to enrollment); (4) subjects with a mutation indicative of classic Fabry disease (i.e., listed in a database such as the International Database of Fabry Disease Genotypes and Phenotypes (dbFGP)); (5) subjects with trough α-Gal A activity below the lower limit of the normal range for the assay; (6) subjects > approximately 18 years of age; (7) sexually mature subjects must agree to use condoms and refrain from sperm donation from the time of administration of the expression construct, with at least three consecutive semen samples that are AAV negative after administration of the investigational treatment, and until at least 90 days after administration of the investigational treatment; and (8) the subject's signed, written, informed consent.

[0335] For subjects without a documented diagnostic α-Gal A activity level, a blood sample is drawn to measure α-Gal A activity levels (in plasma and / or leukocytes). For subjects receiving ERT, this blood draw is performed at least 13 days after the last ERT infusion (trough), i. If the subject's α-Gal A activity level is greater than 5% and the subject is receiving ERT, this enzyme activity level may be due to residual α-Gal A activity from the last ERT infusion. In this case, a diagnosis of classic Fabry disease is confirmed if the following three criteria are met:

[0336] Two or more of the following diagnostic features of classic Fabry disease are documented: corneal verticillate opacity, acroparesis, anhidrosis, and angiokeratoma. If clustered periumbilical angiokeratoma is documented, this symptom alone is sufficient as it is the pathognomonic sign of classic Fabry disease. b. Mutations indicative of classic Fabry disease (i.e., those listed in databases such as www.dbfgp.org); and c. Trough α-Gal A activity is below the lower limit of the normal range for the test.

[0337] Fabry disease gene sequencing was performed at screening to determine whether subjects had a mutation in the α-Gal A gene. The assay was performed on blood or saliva samples. Genetic sequencing results obtained prior to testing could be used if available. Screening included testing for HIV, HAV, HBV, HCV, and TB. Subjects with a diagnosis of HIV or evidence of active HAV, HBV, HCV, or TB infection may not be eligible to participate in this study.

[0338] The level of neutralizing antibodies against AAV6 was measured at screening to assess the subject's pre-existing immune response to AAV6. Subjects with high pre-existing neutralizing antibodies against AAV6 may not be eligible to participate in this study. If dosing is not completed within 3 months of screening, the serum neutralization assay against AAV6 will be repeated.

[0339] If available, results from diagnostic α-Gal A activity levels in plasma or leukocytes collected prior to the study will be used. For subjects without documented diagnostic α-Gal A activity levels, a blood sample will be collected to measure α-Gal A activity levels (in plasma and / or leukocytes). For subjects receiving ERT, this blood collection will occur at least 13 days after the last ERT infusion.

[0340] A chest x-ray (also known as a chest PA radiograph) may be obtained to assess the subject's overall health and eligibility for the study. Unless medically indicated, a chest x-ray taken within 6 months of study enrollment will be used to determine subject eligibility. A physical examination will be performed on each subject and will include, at a minimum, a general appearance, head, eyes, ears, nose, and throat (HEENT), as well as cardiovascular, dermatological, respiratory, gastrointestinal, musculoskeletal, and neurological examinations.

[0341] Exclusion criteria may include subjects with: (1) a known non-response to ERT as determined by the investigator and medical monitor (e.g., no documented reduction in substrate levels with ERT); (2) current treatment with migalastat (Galafold™) or prior treatment within 3 months of informed consent; (3) a positive neutralizing antibody response to AAV (e.g., AAV6); (4) concurrent illness that would be expected to compromise safety or efficacy evaluations during the observation period of the study as determined by the investigator or medical monitor; and (5) an eGFR of 60 ml / min / 1.73 m 2(6) New York Heart Association class PI or higher; (7) active infection with hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) (HCV-DNA negative), or human immunodeficiency virus (HIV) as measured by quantitative polymerase chain reaction (qPCR), or active infection with tuberculosis (TB); (8) history of liver disease within 6 months of informed consent, including secondary fatty liver, nonalcoholic steatohepatitis (NASH), cirrhosis, cholangitis, or biliary tract disease (Gilbert syndrome). ; excluding abnormalities in circulating AFP), (9) for subjects receiving ERT, recent or ongoing hypersensitivity reaction to ERT treatment within 6 months prior to consent, manifested as a significant infusion reaction to ERT as judged by the investigator and medical monitor, (10) markers of hepatitis or cause of overt or underlying liver dysfunction as confirmed by one or more of the following: (i) albumin < 3.5 g / dL, (ii) total bilirubin > upper limit of normal (ULN) and direct bilirubin > 0.5 mg / dL, (iii) alkaline phosphatase (ALP) > 2.0 x ULN. (iv) alanine aminotransferase (ALT) >1.5xULN; (11) current or prior use within the past 6 months of systemic (IV or oral) immunomodulators or steroids (topical treatments such as for asthma and eczema are permitted) (occasional use of systemic steroids may be permitted after consultation with the Medical Monitor); (12) contraindication to the use of corticosteroids for immunosuppression; (13) history of malignancy other than non-melanoma skin cancer; (14) history of alcohol or drug abuse; (15) participation in a previous interventional drug or device trial within the past 3 months prior to consent (excluding implantable loop recorders such as the RalLRoAD trial); (16) previous treatment with a gene therapy product; (17) known hypersensitivity to any component of the AAV-001 formulation; or (18) any other reason that would make the subject unsuitable for participation in the study, as determined by the Investigator or Medical Monitor.

[0342] Baseline Period Baseline assessments and procedures were performed within 12 weeks prior to AAV-001 infusion. For subjects receiving ERT, assessments will include α-Gal A levels, Gb3, and lyso-Gb3 at ERT trough levels, defined as 14 days (± 1 day) after the last ERT dose. Two samples will be collected at baseline, in the morning on two different days. For subjects not receiving ERT, two samples will be collected twice at baseline, 14 days (± 1 day) apart.

[0343] Concomitant medications All medications will be permitted except those with potential hepatotoxicity. Hepatotoxic drugs such as diclofenac, amiodarone, cloipromazine, fluconazole, isoniazid, rifampicin, valproic acid, high dose acetaminophen (4-8 g / day), and hepatotoxic herbal supplements such as Senecio / Crotalaria, Germander in tea, Chaparral, Jin bu huan, and Ma-huang (Chinese herbs) should not be taken during the study. For subjects receiving ERT, ERT must be administered at a stable dose (defined as not missing 4 or more doses of ERT in the 6 months prior to consent) and regimen (14 days ± 1 day for at least 3 months prior to enrollment). Subjects must continue to receive ERT at a stable dose and regimen (14 days ± 1 day) during the study period, as per standard of care, unless ERT is discontinued.

[0344] Dose cohorts Doses deemed to be tolerated and safe will be utilized in dose expansion cohorts. After dose escalation is complete, dose expansion will commence with up to six subjects enrolled per dose expansion cohort, including α-Gal A antibody-positive classic Fabry disease patients, α-Gal A antibody-negative classic Fabry disease patients, female patients (female cohort), and patients who meet renal (renal cohort) and cardiac (cardiac cohort) inclusion and exclusion criteria. Doses in expansion cohorts may be reevaluated if new safety considerations arise.

[0345] Anti-α-Gal AAb positive cohort: Up to 6 male subjects with classic Fabry disease who are α-Gal A antibody positive will be enrolled.

[0346] Anti-α-Gal AAb negative cohort: Up to six male subjects with α-Gal A antibody negative classical Fabry disease will be enrolled.

[0347] Female cohort: Up to six female subjects with classic Fabry disease will be enrolled.

[0348] Renal cohort: Linear negative eGFR slope (estimated from at least three previous serum creatinine values ​​within 18 months, including the value obtained at the screening visit) ≥ 2 mL / min / 1.73 m 2 Up to six male or female subjects with symptomatic Fabry disease per year will be enrolled.

[0349] Cardiac cohort: Up to six male or female subjects with symptomatic Fabry disease with cardiac involvement who have left ventricular hypertrophy (LVH) on 2D echocardiography or CMR (end-diastolic septal and posterior wall thickness ≥ 12 mm) and no other explanation for LVH or cardiac changes indicative of disease progression, such as decreased global longitudinal strain on 2D strain echocardiography or declining native T1 mapping on CMR, will be enrolled.

[0350] Subjects who have experienced a cardiovascular event within 6 months prior to screening may be excluded at the investigator's discretion.

[0351] The starting dose is 5.0E+12vg / kg, and dose escalation to the next dose level will be based on the recommendation of a Safety Monitoring Committee (SMC), which is comprised of external experts, study medical monitors, and investigators, who review data from previous cohorts and / or other clinical trials using in vivo rAAV2 / 6-based therapy, as appropriate. As used herein, SMC members have appropriate medical and scientific expertise and provide safety oversight of the study. Additionally, depending on the observed enzyme activity levels and safety profile of dosed subjects, the SMC may recommend dose escalation to an intermediate dose level of 3.0E+13vg / kg (a three-fold increase over the dose in cohort 2) in cohort 3, instead of increasing the dose in cohort 3 five-fold. The protocol also allows for the inclusion of an additional dose level of 5.0E+13vg / kg as an additional dose in cohort 4; however, the dose administered to a subject will not exceed 5.0E+13 without significant modification. Dose cohorts are shown in Table 2. (See also Figure 3) Table 2. Dosage by cohort [Table 3] *rAAV - recombinant adeno-associated virus

[0352] Treatment planning Subjects >18 years of age who meet all inclusion / exclusion criteria will be enrolled. At least two subjects will be assigned to each dose cohort, with each cohort potentially augmented by four additional adult subjects for a total of up to 18 subjects after review by the SMC. Expression vectors were administered by intravenous infusion. Within each cohort, treatment will be stepped, so that subsequent subjects will not be infused until at least approximately 2 weeks have passed since the previous subject was dosed. Dose escalation to the next dose level cannot be performed until at least approximately 4 weeks have passed since the last subject in the previous cohort was dosed and the safety data for the entire previous cohort have been reviewed by the SMC.

[0353] Subjects receiving ERT prior to study enrollment should continue to receive ERT during the study and maintain their current dose and regimen (14 days ± 1 day) according to standard of care unless ERT is discontinued. For subjects receiving ERT, baseline testing of enzyme and substrate levels will be timed to allow for two separate samples to be taken at trough morning, defined as 14 days (+ / - 1 day) after the last ERT infusion. Because additional time points were taken up front during the screening period, there are three time points to assess residual levels of α-Gal A at trough prior to gene therapy administration. These three samples will be taken at trough, preferably at the same time of day (e.g., morning) to minimize non-specific factors that may affect enzyme levels.

[0354] To minimize potential immune responses to rAAV capsid proteins, avoid loss of transgene expression in case of liver injury, and maintain liver function, prednisone or an equivalent corticosteroid can be administered prophylactically starting approximately 2 days prior to expression vector infusion and tapered over up to approximately 20 weeks.

[0355] The expression vector can be infused using a syringe pump or IV infusion pump (see Study Pharmacy Manual). The total amount will vary depending on the subject's cohort assignment and baseline body weight (kg). The expression vector can be administered through an IV catheter at a controlled rate while the subject is in the hospital or acute care facility, with monitoring of the subject's vital signs (temperature, heart rate, respiratory rate, blood pressure), and the subject can remain for observation for at least 24 hours after the expression vector infusion is completed. The subject can be discharged when all vital signs have stabilized and adverse events (AEs) have resolved, or when the subject is deemed stable by the investigator's judgment.

[0356] Figure 4A shows safety data assessed from four patients in the first two dose cohorts (0.5e13vg / kg and 1e13vg / kg) as of the cutoff date. There were no liver enzyme elevations requiring steroid treatment. There were no adverse events (AEs) leading to study discontinuation, hospitalization, or death.

[0357] Figure 4B shows safety data evaluated from five patients in dose cohorts 1-3 (0.5e13vg / kg, 1.0e13vg / kg, and 3.0e13vg / kg) as of the cutoff date. There were no liver enzyme elevations requiring steroid treatment. There were no adverse events (AEs) leading to study discontinuation, hospitalization, or death.

[0358] Figure 4C shows safety data assessed from five patients in dose cohorts 1-4 (0.5e13vg / kg, 1.0e13vg / kg, 3.0e13vg / kg, and 5.0e13vg / kg) as of the cutoff date. There were no liver enzyme elevations requiring steroid treatment. There were no adverse events (AEs) leading to study discontinuation, hospitalization, or death.

[0359] After AAV-001 infusion, study visits may be conducted at Day 8, Week 2, Week 4, Week 6, Week 8, Week 12, Week 16, Week 20, Week 24, Week 28, Week 32, Week 36, Week 40, Week 44, Week 48, and Week 52. Study visits at Weeks 28, 32, 40, 44, and 48 may be conducted remotely as some evaluations do not require on-site evaluation. Assessments of AEs and concomitant medications may be conducted remotely via telephone.

[0360] Liver tests (AST, ALT, GOT, total and direct bilirubin, ALP, LDH, albumin, total protein levels) will be performed twice weekly to monitor AAV-mediated immunogenicity for approximately the first 20 weeks after expression vector infusion while subjects are taking prednisone or equivalent corticosteroids, which may be performed remotely. Blood samples for liver tests will be collected at 2-4 day intervals when possible, except for the first week, when they can be collected at the day 2 and day 8 visits. Liver tests will be performed weekly (weeks 21-24) for 4 weeks after stopping immunosuppressants, and then monthly (weeks 28-52) in conjunction with study visits.

[0361] If there is evidence of ALT elevation despite prior treatment with prednisone or equivalent corticosteroids, prednisone or equivalent corticosteroids may be continued (prednisone 1 mg / kg [maximum 60 mg] or equivalent orally or intravenously and / or increased on a case-by-case basis, assessing liver enzymes twice weekly until liver enzymes normalize, then follow protocol).

[0362] Dose escalation For the first two subjects in each cohort, treatment will be stepped up such that each subsequent subject will not be infused until the previous subject has been observed for at least 2 weeks. Dose escalation to the next dose level cannot occur until at least 4 weeks have elapsed since the last two subjects in the previous cohort were dosed and safety data for the two subjects in the previous cohort have been reviewed by the SMC. Dosing and dose escalation may be paused if any stopping rules are met.

[0363] ERT discontinued Treatment with AAV-001 may result in long-term, liver-specific expression of a-Gal A in subjects with Fabry disease, obviating the need for ERT, by using a rAAV vector encoding the cDNA for human α-Gal A. Subjects undergoing discontinuation of ERT will be closely monitored for AEs, changes in vital signs, safety laboratory values, and levels of α-Gal A and substrates compared to baseline. Discontinuation of ERT should be considered after 4 weeks to allow sufficient time for transduction of targeted hepatocytes. Subjects discontinuing ERT will be closely monitored for clinical symptoms such as fatigue, neuropathic pain, changes in AEs, vital signs, and safety laboratory values ​​(e.g., liver function tests, levels of α-Gal A and substrates (Gb3 and Lyso-Gb3)) compared to baseline. Discontinuation of ERT may be decided at the discretion of the investigator after consultation with the sponsor and will be considered for subjects willing to participate who meet the following criteria: (1) >4 weeks have passed since administration of AAV-001, (2) medically stable and able to tolerate temporary discontinuation of ERT at the discretion of the investigator; (3) consent to enhanced safety surveillance and additional laboratory testing until the ERT discontinuation follow-up visit; (4) There was no need to restart ERT after the ERT discontinuation follow-up visit. However, ERT may be resumed at any time based on clinical circumstances or investigator discretion.

[0364] Discontinuation of ERT may be repeated if previously unsuccessful, at the investigator's discretion and in consultation with the sponsor, only if the subject consents and at least 12 weeks have passed since the previous attempt.

[0365] ERT discontinuation monitoring (± 2 days) ERT discontinuation monitoring visits will occur weekly for the first 4 weeks after the ERT discontinuation visit, then every other week for the final 8 weeks after the ERT discontinuation visit until the ERT discontinuation follow-up visit. To reduce study burden, ERT discontinuation monitoring visits should be combined with regularly scheduled visits whenever possible. ERT discontinuation monitoring visits can be conducted remotely.

[0366] ERT discontinuation follow-up (up to 12 weeks after ERT discontinuation) The ERT discontinuation follow-up visit will occur 12 weeks after the ERT discontinuation visit, but may occur earlier at the investigator's discretion if clinically indicated. If clinically indicated, ERT may be resumed at any time based on clinical circumstances or investigator judgment.

[0367] End-of-study visit and long-term follow-up survey An end-of-study (EOS) visit for final evaluation will be conducted at week 52. At the EOS visit, subjects will be invited to participate in a separate long-term follow-up study, with further follow-up for up to 4 years.

[0368] Test period The study participation period may be up to 76 weeks per subject, divided into up to 8 weeks for screening, up to 12 weeks for baseline, and 52 weeks for post-treatment follow-up. The accrual period is planned to be 9 to 12 months.

[0369] Safety Monitoring Committee and Suspension Rules If any of the following criteria are met, enrollment in the study may be paused and the SMC may convene to make recommendations regarding an appropriate course of action: (1) the occurrence of one grade 3 or greater adverse event that is at least reasonably likely to be causally related to the expression vector product, (2) the occurrence of a serious adverse event (SAE) that is at least reasonably likely to be causally related to the expression vector product, (3) death of a human subject, or (4) the occurrence of a malignancy.

[0370] The trial may also be terminated for any of the following reasons: (1) if the Sponsor, in consultation with the SMC or regulatory authorities, determines that continuation of the trial may for any reason jeopardize the safety of the subjects; or (2) if the Sponsor decides to discontinue development of AAV-001.

[0371] All data will be evaluated to determine whether any changes need to be made to the study or whether data collection should continue or be stopped. If stopping criteria are met, a substantial amendment will be submitted to regulatory authorities for review and no subjects will be dosed at that dose level or higher until the amendment has been approved by the site's Institutional Review Board (IRB) / Independent Ethics Committee (IEC) or equivalent.

[0372] Example 2 Plasma α-Gal A activity and Lyso-Gb3 To test the effect of AAV-001 treatment in adults with Fabry disease, plasma α-Gal A activity and lyso-Gb3 were evaluated in nine patients (Patient 1, Patient 2, Patient 3, Patient 4, Patient 5, Patient 6, Patient 7, Patient 8, and 9) across four dose cohorts (Cohort 1 (n=2; Patients 1 and 2); 0.5e13vg / kg, Cohort 2 (n=2; Patients 3 and 4); 1e13vg / kg, Cohort 3 (n=3; Patients 5, 6, and 7); 3.0e13vg / kg, Cohort 4 (n=2; Patients 8 and 9); 5.0e13vg / kg). Baseline characteristics of patients 1-9 are shown in Figure 3.

[0373] Plasma α-Gal A activity α-Gal A activity in K2-EDTA plasma was determined by measuring the production of 4-methylumbelliferone (4-MU) from the degradation of the artificial substrate 4-methylumbelliferyl α-D galactopyranoside (4-MU-α-Gal) at 37°C and is shown below: 4-MU-α-Gal (substrate, non-fluorescent) + α-Gal A → 4-MU (fluorescent) Activity is expressed as the amount of 4-MU (nmol / mL) produced during a 3-h reaction time and is given in nmol / hr / mL.

[0374] Normal male ranges for α-Gal A plasma activity and fold change The normal male range for α-Gal A was determined from 40 healthy male donors using a validated plasma α-Gal A activity assay with a 3-h incubation time and is expressed in nmol / hr / mL. Normal range for men: 2.45-11.37 nmol / h / mL, mean 5.70 nmol / h / mL. Fold change relative to the mean was calculated relative to the mean normal range determined at 5.70 nmol / h / mL.

[0375] Normal male range for lyso-Gb3: Using a validated LC-MS / MS and lyso-Gb3-d7 as an internal standard, the normal male range for lyso-Gb3 in K2-EDTA plasma was determined from 40 healthy male donors. The method utilized a protein precipitation extraction procedure prior to LC-MS / MS analysis. Male normal range: 0.323-0.625 ng / mL, with a mean concentration of 0.473 ng / mL.

[0376] FIG. 5C shows that all subjects with data available by the data cutoff exhibited above-normal levels of α-Gal activity.

[0377] Biomarker results were evaluated from eight subjects across four dose cohorts (0.5e13vg / kg, 1.0e13vg / kg, 3.0e13vg / kg, and 5.0e13vg / kg) at the cutoff date. Fold change at the last measurement point was calculated. α-Gal A activity was measured using a 3-hour reaction time and is expressed in nmol / hr / mL. For patients 1, 4, 5, 6, and 7, this was sampled at ERT trough. Normal ranges and means were determined based on healthy male individuals. Figure 5C shows that through the data cutoff for subjects 1-8, elevated α-Gal A activity was observed for up to 18 months for the first two subjects treated. For subjects 7 and 8, α-Gal A activity is within normal range at week 2. Subjects 1 and 4 discontinued ERT.

[0378] Activity levels up to 21-fold above mean normal levels were observed.

[0379] FIG. 6A shows that patient 1 exhibited above-normal levels of α-Gal activity that persisted for 1 year until the end of the study. Lyso-Gb3 remained within 10% of baseline until the end of the study. FIG. 6B shows that patient 1 exhibited above-normal levels of α-Gal activity that persisted for 48 weeks. FIG. 6C shows that patient 1 exhibited above-normal levels of α-Gal activity that persisted for 18 months. Left ventricular hypertrophy (ptotic ventricular hypertrophy) increased during the observation period and stabilized on MRI after 1 year of treatment. The low baseline levels of plasma lyso-Gb3 remained stable over time. Patient 1 reported improvement in leg edema and sweating ability. Patient 1 transitioned to long-term follow-up (3-monthly follow-up for an additional 4 years).

[0380] Figure 6D shows that patient 2 had above-normal levels of α-Gal activity that persisted for 48 weeks. Figure 6E shows that patient 2 had above-normal levels of α-Gal activity that persisted for 1 year. Figure 6F shows that patient 2 had above-normal levels of α-Gal activity that persisted for 18 months. The patient's baseline mild biventricular dilation improved on MRI 1 year later. The low baseline levels of plasma lyso-Gb3 remained stable over time. Patient 2 reported improved sweating ability. Patient 2 progressed to long-term follow-up (3-monthly follow-up for an additional 4 years).

[0381] FIG. 6G shows that patient 3 exhibited above-normal levels of α-Gal activity that persisted until the last measurement point at week 28. Patient 2 also showed a 40% decrease in lyso-Gb3 from baseline to week 28. A decrease in lyso-Gb3 was observed over 12 weeks after AAV-001 administration. FIG. 6H shows that patient 3 exhibited above-normal levels of α-Gal activity that persisted until the last measurement point at week 40. FIG. 6I shows that patient 3 exhibited above-normal levels of α-Gal activity that persisted until the last measurement point at week 52. The patient's cardiac MRI was normal at baseline and at week 24. The patient's plasma lyso-Gb3 levels were elevated at baseline. Patient 3 showed a 40% decrease in plasma lyso-Gb3 within 10 weeks after administration from baseline that persisted until week 52. Patient 3 reported improved sweating ability.

[0382] FIG. 6J shows that patient 4 exhibited above normal levels of α-Gal activity that were sustained for 12 weeks. FIG. 6K shows that patient 4 exhibited above normal levels of α-Gal activity that were sustained for 25 weeks. FIG. 6L shows that patient 4 exhibited above normal levels of α-Gal activity that were sustained for 40 weeks. Patient 4 had mild LVH at baseline. Patient 4 discontinued ERT at week 24 (based on dosing frequency every 2 weeks).

[0383] FIG. 6M shows that patient 5 had above normal levels of α-Gal activity that was sustained over 24 weeks. Patient 5 had mild LVH at baseline. FIG. 6N shows that patient 6 had above normal levels of α-Gal activity that was sustained over 12 weeks. Patient 6 had mild LVH at baseline. FIG. 6O shows baseline and α-Gal dosing data for patient 7. Patient 7 had mild to moderate LVH at baseline. FIG. 6P shows baseline and α-Gal dosing data for patient 8. Patient 8 had a normal cardiac MRI at baseline. FIG. 6Q shows that patient 9 has no α-Gal data at the time of this data cut. Patient 9 had moderate LVH at baseline.

Claims

1. A composition for treating Fabry disease or ameliorating one or more symptoms associated with Fabry disease in a human subject in need thereof, comprising an adeno-associated virus (AAV) expression vector; the vector comprises an α-galactosidase A (α-Gal A) expression cassette, the α-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, and a bovine growth hormone poly(A) signal sequence; The composition is administered in an amount of about 5×10 per kilogram of body weight. １２ Vector genome (vg / kg) ~ approximately 5 x 10 １３ vg / kg of the AAV expression vector is administered. composition.

2. A composition for treating Fabry disease or ameliorating one or more symptoms associated with Fabry disease in a human subject in need thereof, comprising an adeno-associated virus (AAV) expression vector; the vector comprises an α-galactosidase A (α-Gal A) expression cassette, the α-galactosidase A (α-Gal A) expression cassette comprising an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, the α-Gal A transgene comprising the nucleotide sequence set forth in SEQ ID NO:5; The composition is administered in an amount of about 5×10 per kilogram of body weight. １２ Vector genome (vg / kg) ~ approximately 5 x 10 １３ vg / kg of the AAV expression vector is administered. composition. Claim 3: A composition for reducing the amount of glycosphingolipids in a human subject in need thereof, comprising an adeno-associated virus (AAV) expression vector, the vector comprises an α-galactosidase A (α-Gal A) expression cassette, the α-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-galactosidase A (α-Gal A) transgene encoding at least one α-Gal A protein, and a bovine growth hormone poly(A) signal sequence; The composition is administered in an amount of about 5×10 per kilogram of body weight. １２ Vector genome (vg / kg) ~ approximately 5 x 10 １３ vg / kg of the AAV expression vector is administered; the amount of glycosphingolipid is reduced compared to the amount of glycosphingolipid in the subject before said administration. composition.

4. A composition for increasing the activity of α-galactosidase A (α-Gal A) protein in a human subject in need thereof, comprising an adeno-associated virus (AAV) expression vector; the vector comprises an α-galactosidase A (α-Gal A) expression cassette, the α-galactosidase A (α-Gal A) expression cassette comprising an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, an α-Gal A transgene encoding at least one α-Gal A protein, and a bovine growth hormone poly(A) signal sequence; The composition is administered in an amount of about 5×10 per kilogram of body weight. １２ Vector genome (vg / kg) ~ approximately 5 x 10 １３ vg / kg of the AAV expression vector is administered; the activity of the α-Gal A protein is increased compared to the activity of the α-Gal A protein in the subject prior to the administration. composition.

5. 3. The composition of claim 1, wherein the α-Gal A expression cassette further comprises a mutated woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE) sequence, and the mutated WPRE sequence may comprise a mut6 mutated WPRE sequence.

6. 3. The composition of claim 2, wherein the α-Gal A expression cassette further comprises an apolipoprotein E (APOE) enhancer operably linked to an alpha 1-antitrypsin (hAAT) promoter, a human hemoglobin beta (HBB)-IGG intron, a sequence encoding a signal peptide, and a bovine growth hormone polyA signal sequence.

7. 7. The composition of any one of claims 1, 3, 4 and 6, wherein the transgene comprises a wild-type α-Gal A sequence or a codon-optimized α-Gal A sequence.

8. The composition of any one of claims 1, 3, 4 and 6, wherein the signal peptide is an α-Gal A signal peptide.

9. 6. The composition of claim 5, wherein the enhancer comprises the nucleotide sequence set forth in SEQ ID NO:2, the promoter comprises the nucleotide sequence set forth in SEQ ID NO:3, the intron comprises the nucleotide sequence set forth in SEQ ID NO:4, the α-Gal A transgene comprises the nucleotide sequence set forth in SEQ ID NO:5, the mutated WPRE sequence comprises the nucleotide sequence set forth in SEQ ID NO:6, and the poly A signal sequence comprises the nucleotide sequence set forth in SEQ ID NO:

7.

10. The composition of claim 5, wherein the enhancer comprises a nucleotide sequence set forth in SEQ ID NO: 2, the promoter comprises a nucleotide sequence set forth in SEQ ID NO: 3, the intron comprises a nucleotide sequence set forth in SEQ ID NO: 4, the mutated WPRE sequence comprises a nucleotide sequence set forth in SEQ ID NO: 6, and the polyA signal sequence comprises a nucleotide sequence set forth in SEQ ID NO:

7.

11. The composition of any one of claims 1 to 4 and 6, wherein the α-Gal A expression cassette comprises the nucleotide sequence set forth in SEQ ID NO:

9.

12. The composition according to any one of claims 1 to 4 and 6, wherein the serotype of the AAV expression vector is AAV2 / 6.

13. 7. The composition of any one of claims 1 to 4 and 6, wherein the subject has one or more of the following symptoms: above-normal globotriaosylceramide (Gb3) levels, above-normal globotriaosylsphingosine (lyso-Gb3) levels, kidney disease, heart disease, anhidrosis, acroparesthesia, angiokeratoma, gastrointestinal (GI) tract pain, corneal and lens opacities, or cerebrovascular disease.

14. The composition of any one of claims 1 to 4 and 6, wherein the subject has pre-existing anti-α-Gal A antibodies as determined by enzyme-linked immunosorbent assay (ELISA) prior to administration of the composition.

15. 7. The composition of any one of claims 1 to 4 and 6, wherein analysis of a biological sample from the subject reveals that the subject is an anti-α-Gal A neutralizing antibody positive subject, and the anti-α-Gal A neutralizing antibody positive subject has a biological sample that exhibits greater than about 9.6% inhibition of α-galactosidase A activity as measured by an anti-α-Gal A neutralizing antibody assay.

16. 7. The composition of any one of claims 1-4 and 6, wherein the α-Gal A protein expressed from the transgene reduces the amount of the glycosphingolipid in the subject by at least about two-fold compared to the amount of the glycosphingolipid in the subject before administration of the composition.

17. The α-Gal expressed from the transgene about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, or About 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79% 8. The composition of any one of claims 1 to 4 and 6, wherein the composition reduces the amount of erythrocyte proliferation and secretion of erythrocytes by about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

18. 7. The composition of any one of claims 1-4 and 6, wherein the α-Gal A protein expressed from the transgene reduces the amount of glycosphingolipids in one or more of plasma, liver, heart, kidney, urine, skin, or spleen.

19. The composition of any one of claims 1 to 4 and 6, wherein the composition is administered parenterally.

20. The composition of any one of claims 1 to 4 and 6, wherein the composition is administered intravenously.

21. The composition of any one of claims 1 to 4 and 6, wherein the subject is administered an immunosuppressant prior to and / or during administration of the composition.

22. The composition of any one of claims 1 to 4 and 6, wherein the subject is not receiving an immunosuppressant prior to and / or during administration of the composition.

23. The composition of any one of claims 1 to 4 and 6, wherein the subject is not subjected to a preconditioning therapy prior to administration of the composition.

24. 7. The composition of any one of claims 1-4 and 6, wherein expression of the at least one α-Gal A protein is sustained for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months.

25. 7. The composition of any one of claims 1 to 4 and 6, wherein the subject is receiving enzyme replacement therapy (ERT) for Fabry disease prior to administration of the composition.

26. 7. The composition of any one of claims 1 to 4 and 6, wherein the subject is receiving a non-enzyme replacement therapy for Fabry disease prior to administration of the composition.

Images