Compositions and methods for the treatment of Fabry disease

Abstract

The present disclosure provides, inter alia, a method of treating Fabry disease in a subject, comprising administering to a subject in need thereof a recombinant adeno-associated viral vector (rAAV) packaged within an AAV capsid with broad tissue tropism, the vector comprising: (a) a 5' inverted repeat, (b) a ubiquitous promoter, (c) a nucleotide sequence encoding a wild-type α-GAL enzyme or a variant thereof, (d) optionally, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (e) polyA, and (d) a 3' ITR.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 154,485, filed February 26, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing This specification references a Sequence Listing, which has been submitted electronically as a .txt file named MIL-014WO_ST25 on Feb. 25, 2022. The .txt file was created on Feb. 24, 2022 and is 177 KB in size. The entire contents of the Sequence Listing are incorporated herein by reference. [Background technology]

[0003] Fabry disease is a rare, progressive, inborn error of metabolism caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-GAL) as a result of mutations in the GLA gene. If left untreated, people with Fabry disease have a short life expectancy, often dying around age 40 or 50 due to vascular disease affecting the kidneys, heart and / or central nervous system.

[0004] Lack of α-GAL enzyme activity leads to a progressive systematic accumulation of its main substrate, globotriaosylceramide (GB3), and its deacetylated soluble form, globotriaosylsphingosine (lysoGb3), resulting in a wide variety of health problems and reduced life expectancy, including one or more of renal, cardiac, and / or cerebrovascular disease. The disease manifests as early childhood / adolescence-onset classic Fabry disease or a milder (adult) form later in life, depending on the mutation and the amount of remaining α-GAL enzyme. Classic Fabry disease occurs when there is less than 5% residual enzyme activity (Arends M, et al. (2017) PLoS ONE 12(8): e0182379.), typically affecting males.

[0005] Fabry disease is also associated with the development of pain. Pain may be caused by lipid deposition in the dorsal root and sympathetic ganglia or by small fiber neuropathy. In general, the pain is chronic or intermittent. The intermittent pain of Fabry disease, called "Fabry crises," typically begins in the extremities and radiates proximally, and may be triggered by movement, illness, temperature changes, or other physical and emotional stresses. This neuropathic pain is also associated with a lack of temperature sensation. Summary of the Invention [Problem to be solved by the invention]

[0006] The specific currently approved treatment for Fabry disease is enzyme replacement therapy ("ERT"), which involves treating patients with one of two versions of recombinant human α-GAL: agalsidase alpha produced by a cultured human cell line, and agalsidase beta produced by Chinese hamster ovary cells transduced with the GLA gene. While ERT is often effective, it requires lifelong intravenous administration of α-GAL every two weeks. Although ERT eliminates symptoms associated with Fabry disease, it is not curative and does not halt disease progression. For example, the two aforementioned α-GAL products have not been shown to substantially reduce the risk of stroke, the myocardium responds slowly to treatment, and there is limited clearance of lipid deposits from some cell types in the kidney. The poor pharmacological response is largely due to the short circulating half-life of the enzyme and suboptimal intracellular delivery. Thus, there remains a need for therapies to treat Fabry disease that can halt disease progression and potentially be curative. [Means for solving the problem]

[0007] SUMMARY OF THEINVENTION This application discloses methods and compositions for the treatment and / or prevention of Fabry disease. This disclosure provides, in part, a gene therapy method using recombinant adeno-associated virus vectors (rAAV) to mediate the introduction and expression of the GLA gene. This application is based on the discovery that gene delivery vehicles such as rAAV vectors, which have broad tissue tropism and utilize ubiquitous promoters to drive broad gene expression, result in sustained high levels of protein expression and robust protein exposure to a wide range of tissues and / or reduced amounts of GB3 or lysoGb3. This application is also based on the discovery that codon-optimized or engineered variants of GLA delivered using broad or tissue-specific rAAV vectors and utilizing ubiquitous or tissue-specific promoters result in increased α-GAL activity in vivo and / or reduced amounts of lysoGb3 or GB3 in vivo. Furthermore, this gene delivery method for inducing the expression of GLA variants encoding α-GAL proteins with extended half-life and improved cellular uptake leads to further increase in α-GAL exposure in key target tissues. Collectively, these findings allow the delivery vehicle described herein to achieve broad tissue distribution of administered transgenes, and also allow for better therapeutic outcomes. The delivery vehicle comprising the GLA sequence described herein is particularly useful for treating Fabry disease.

[0008] Described herein are methods and compositions for the effective delivery of the alpha-galactosidase A (GLA) gene to cells of a subject in need thereof. The delivered GLA transgene results in the expression of alpha-GAL protein. The present disclosure is based, in part, on the creation of a recombinant adeno-associated virus (rAAV) vector that contains, inter alia, the alpha-galactosidase A (GLA) gene and exhibits robust alpha-GAL protein expression once present in a cell. The present disclosure is based, at least in part, on the surprising discovery that a construct containing the GLA gene under a ubiquitous promoter and packaged within a rAAV capsid with broad tissue tropism results in sustained alpha-GAL protein expression and robust tissue biodistribution in the kidney, heart, gastrointestinal tract, brain, and peripheral neurons even at low doses. The broad distribution achieved using this vector allows for efficient delivery of alpha-GAL to tissues affected by Fabry disease, resulting in robust therapeutic results.

[0009] The rAAV vectors described herein can be used with either a GLA gene having a wild-type sequence (SEQ ID NO:3) or a GLA gene having a modified sequence as described herein. Such modified GLA sequences include, for example, codon-optimized GLA and / or engineered variants of GLA. The rAAV vectors described herein enable substrate clearance of globotriaosylsphingosine (lysoGb3) and / or globotriaosylceramide (GB3) in various tissues.

[0010] As detailed below, the gene therapy system described herein results in overall improved health as evidenced by weight gain, improved renal function, and neurological symptoms in mouse models of Fabry disease, and is further expected to show similar improvements in humans. The methods and compositions provided herein can be used to achieve sustained expression of GLA in a wide variety of tissues affected by Fabry disease. Thus, the present application provides highly effective compositions and methods for treating Fabry disease and alleviating associated symptoms.

[0011] In some aspects, a recombinant adeno-associated virus (rAAV) vector having broad tissue tropism is provided, the vector comprising (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter, (c) a nucleotide sequence encoding an α-GAL enzyme, (d) polyA, and (e) a 3' ITR.

[0012] In some aspects, a recombinant adeno-associated virus (rAAV) vector having broad tissue tropism is provided, the vector comprising (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter, (c) a nucleotide sequence encoding an α-GAL enzyme, (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (e) polyA, and (f) a 3' ITR.

[0013] Various types of AAV capsids with broad tissue tropism (the terms "broad tissue tropism" and "wide-tropism" are used interchangeably herein) can be used in the rAAV vectors described herein. For example, in some embodiments, the AAV capsid is a broad-tropism AAV capsid selected from AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAV9 capsid, AAV11, 12, 13, AAVhu.37, AAVrh.8, AAVrh.10, and AAVrh.39, AAV-DJ, or AAV-DJ / 8.

[0014] Thus, in some embodiments, the AAV capsid with broad tropism is an AAV that is AAV1. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV2. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV3. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV4. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV5. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV6. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV7. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV8. In some embodiments, the AAV capsid with broad tropism is an AAV that is AAV9.

[0015] Various types of capsids and associated tropisms are described in Curr Opin Vir. 2016 December 21:75-80, the contents of which are incorporated herein by reference. By "broad tissue tropism" it is meant that the capsid can allow gene transfer to two or more, three or more, four or more, five or more, six or more, seven or more, eight or more tissue types. For example, in some embodiments, a capsid with broad tissue tropism allows gene transfer to one or more of the tissues, i.e., liver, kidney, heart, gastrointestinal tract, and / or peripheral neurons of a subject.

[0016] In some embodiments, the ubiquitous promoter is selected from chicken beta-actin (CBA) promoter, CAG promoter, EF-1α promoter, PGK promoter, UBC promoter, LSE beta-glucuronidase (GUSB) promoter, or ubiquitous chromatin opening element (UCOE) promoter. In some embodiments, the ubiquitous promoter comprises CBh (CMV enhancer, chicken beta-actin promoter, chicken-beta actin-MVM hybrid intron). Thus, in some embodiments, the ubiquitous promoter is chicken beta-actin (CBA) promoter. In some embodiments, the ubiquitous promoter is EF-1α promoter. In some embodiments, the EF-1α promoter is combined with a chimeric intron from chicken beta-actin gene and rabbit beta-globin gene. In some embodiments, the ubiquitous promoter is a UBC promoter. In some embodiments, the ubiquitous promoter is a LSE beta-glucuronidase (GUSB) promoter. In some embodiments, the ubiquitous promoter is a ubiquitous chromatin opening element (UCOE) promoter. (Powell SK, et al. Discov Med. 2015 Jan;19(102):49-57.)

[0017] In some embodiments, the ubiquitous promoter comprises a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron.

[0018] In some embodiments, the ubiquitous promoter comprises a truncated EF-1α promoter and one or more introns.

[0019] In some embodiments, the one or more introns are from a chicken β-actin gene and / or a rabbit β-globin gene.

[0020] In some embodiments, the AAV9 capsid is naturally occurring or modified.

[0021] In some embodiments, the WPRE sequence is optional or modified.

[0022] In some embodiments, the WPRE sequence is WPRE mut6delATG.

[0023] Exemplary polyA sequences that may be included in gene therapy vectors encompassed by the present disclosure include human growth hormone polyA (hGHpA), synthetic polyA (SPA), Simian Virus 40 late polyA (SV 40pA), and bovine growth hormone (BGH) polyA. In certain embodiments, the polyA is bovine growth hormone (BGH) polyA.

[0024] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is codon optimized.

[0025] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is codon-optimized for human cells.

[0026] In some embodiments, the α-GAL enzyme has an unmodified sequence.

[0027] In some embodiments, the α-GAL enzyme has a modified sequence.

[0028] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is engineered.

[0029] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme has been engineered and codon optimized.

[0030] In some embodiments, the modified sequence contains one or more amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30).

[0031] In some embodiments, the modified sequence comprises 1 to 25 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). For example, in some embodiments, the modified sequence comprises 5 to 25 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 5 to 20 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 5 to 15 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 5 to 10 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 10 to 25 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 10 to 20 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence contains between 10 and 15 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30).

[0032] In some embodiments, the modified sequence comprises 1 to 10 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). For example, in some embodiments, the modified sequence comprises 1 to 9 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 1 to 8 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 1 to 7 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 1 to 6 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 1 to 5 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 1 to 4 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30). In some embodiments, the modified sequence comprises 1 to 3 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30).

[0033] In some embodiments, the modified sequence contains 10 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 30).

[0034] In some embodiments, a recombinant alpha galactosidase A is provided. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:30, or a functional fragment thereof. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 85% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 86% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 87% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 88% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 89% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 90% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 91% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 92% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 93% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 94% sequence identity to SEQ ID NO:30. In some embodiments, the recombinant alpha galactosidase A comprises a polypeptide sequence that has at least 95% sequence identity to SEQ ID NO:30.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence that has at least 96% sequence identity to SEQ ID NO: 30. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence that has at least 97% sequence identity to SEQ ID NO: 30. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence that has at least 98% sequence identity to SEQ ID NO: 30. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence that has at least 99% sequence identity to SEQ ID NO: 30.

[0035] In some embodiments, the recombinant alpha galactosidase A comprises at least one substitution or substitutions in SEQ ID NO: 30 at one or more positions selected from T41 / M70 / L75 / S78 / E79 / Y123 / R193 / S197 / K237 / F248 / N247 / N278 / L286 / A292 / H302 / Q333 / K314 / L347 / M353 / S364 / A368 / S371 / K374 / K393 / F396 / E398 / W399 / R404 / M423.

[0036] Additional exemplary GLA transgenes and α-GAL enzyme sequences can be found in PCT Publication Nos. PCT / US2021 / 019811, PCT / US2019 / 067493, and PCT / US2015 / 063329, each of which is incorporated by reference in its entirety herein.

[0037] In some embodiments, the modified α-GAL enzyme is selected from one of SEQ ID NOs: 7-17, 33, 34, and 46-60.

[0038] In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:15. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 57.In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the modified α-GAL enzyme comprises the amino acid sequence of SEQ ID NO: 60.

[0039] In some embodiments, the modified α-GAL enzyme has improved stability as compared to the wild-type α-GAL enzyme (SEQ ID NO: 30).

[0040] In some embodiments, the modified α-GLA enzyme has increased intracellular activity compared to the wild-type α-GLA enzyme (SEQ ID NO: 30).

[0041] In some embodiments, the modified α-GLA enzyme has improved serum stability compared to the wild-type α-GLA enzyme (SEQ ID NO: 30).

[0042] In some embodiments, the modified α-GLA enzyme has improved lysosomal stability compared to the wild-type α-GLA enzyme (SEQ ID NO: 30).

[0043] In some embodiments, the modified α-GLA enzyme has increased specific catalytic activity compared to the wild-type α-GLA enzyme (SEQ ID NO:30).

[0044] In some aspects, methods of treating Fabry disease in a subject are provided, comprising administering to a subject in need thereof a recombinant adeno-associated viral vector (rAAV) as described herein.

[0045] In some aspects, pharmaceutical compositions are provided, such compositions comprising a rAAV vector as described herein.

[0046] In some aspects, a cell is provided, such cell comprising a rAAV vector described herein. The cell can be any type of mammalian cell. For example, in some embodiments, the cell is a cardiac cell. In some embodiments, the cell is a kidney cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a skeletal muscle cell. In some embodiments, the cell is a gastrointestinal tract cell. In some embodiments, the cell is a brain cell, such as, for example, a neuron or a glial cell. In some embodiments, the cell is a peripheral neuron.

[0047] In some aspects, methods of treating Fabry disease in a subject are provided, the methods comprising administering to a subject in need thereof a recombinant adeno-associated virus vector (rAAV) packaged within a rAAV capsid with broad tissue tropism, the vector comprising (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme, (d) polyA, and (e) a 3' ITR.

[0048] In some aspects, a method of treating Fabry disease in a subject is provided, the method comprising administering to a subject in need thereof a recombinant adeno-associated virus vector (rAAV) packaged within a rAAV capsid with broad tissue tropism, the vector comprising (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme, (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (e) polyA, and (f) a 3' ITR.

[0049] In some embodiments, the AAV capsid is a broad tropism AAV capsid selected from AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, or AAV9 capsid. In some embodiments, the broad tropism AAV capsid is an AAV1 capsid. In some embodiments, the broad tropism AAV capsid is an AAV2 capsid. In some embodiments, the broad tropism AAV capsid is an AAV3 capsid. In some embodiments, the broad tropism AAV capsid is an AAV4 capsid. In some embodiments, the broad tropism AAV capsid is an AAV5 capsid. In some embodiments, the broad tropism AAV capsid is an AAV6 capsid. In some embodiments, the broad tropism AAV capsid is an AAV7 capsid. In some embodiments, the broad tropism AAV capsid is an AAV8 capsid. In some embodiments, the broad tropism AAV capsid is an AAV9 capsid.

[0050] In some embodiments, the rAAV vector is administered intravenously, subcutaneously, or transdermally.Thus, in some embodiments, the rAAV vector is administered intravenously to the subject in need thereof.In some embodiments, the rAAV vector is administered subcutaneously to the subject in need thereof.In some embodiments, the rAAV vector is administered transdermally to the subject in need thereof.

[0051] In some embodiments, transdermal administration is by gene gun.

[0052] In some embodiments, the rAAV vector exists as an episome following administration.

[0053] In some embodiments, the rAAV described herein is administered to a subject in need thereof at a lower dose than would be expected to be used with an AAV vector that targets the liver for expression of α-GAL. In other embodiments, the rAAV described herein exhibits higher α-GAL exposure in serum and tissues when administered at an equivalent dose to that of a liver-targeted rAAV.

[0054] In some embodiments, the rAAV vector with broad tissue tropism and ubiquitous promoter achieves a therapeutic effect for treating Fabry disease at a lower dose than the dose of the rAAV vector containing AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, or AAV8 capsid using a liver-specific promoter. Thus, in some embodiments, the rAAV vector with broad tissue tropism and ubiquitous promoter achieves a therapeutic effect at a lower dose than the dose of the rAAV vector containing AAV1 capsid with a liver-specific promoter. In some embodiments, the rAAV vector with broad tissue tropism and ubiquitous promoter achieves a therapeutic effect at a lower dose than the dose of the rAAV vector containing AAV2 capsid with a liver-specific promoter. In some embodiments, a rAAV vector with broad tissue tropism and a ubiquitous promoter achieves a therapeutic effect at a lower dose than that of a rAAV vector comprising an AAV3 capsid with a liver-specific promoter. In some embodiments, a rAAV vector with broad tissue tropism and a ubiquitous promoter achieves a therapeutic effect at a lower dose than that of a rAAV vector comprising an AAV3 capsid with a liver-specific promoter. In some embodiments, a rAAV vector with broad tissue tropism and a ubiquitous promoter achieves a therapeutic effect at a lower dose than that of a rAAV vector comprising an AAV4 capsid with a liver-specific promoter. In some embodiments, a rAAV vector with broad tissue tropism and a ubiquitous promoter achieves a therapeutic effect at a lower dose than that of a rAAV vector comprising an AAV5 capsid with a liver-specific promoter. In some embodiments, a rAAV vector with broad tissue tropism and a ubiquitous promoter achieves a therapeutic effect at a lower dose than that of a rAAV vector comprising an AAV6 capsid with a liver-specific promoter.In some embodiments, a rAAV vector with broad tissue tropism and a ubiquitous promoter achieves a therapeutic effect at a lower dose than a rAAV vector comprising an AAV7 capsid with a liver-specific promoter, hi some embodiments, a rAAV vector with broad tissue tropism and a ubiquitous promoter achieves a therapeutic effect at a lower dose than a rAAV vector comprising an AAV8 capsid with a liver-specific promoter.

[0055] In some embodiments, the rAAV vector capable of expressing the α-GAL enzyme can include a tissue-specific promoter, such as a liver-specific promoter. Exemplary liver-specific promoters include, but are not limited to, the transthyretin promoter (TTR), the thyroxine-binding globulin (TBG) promoter, the hybrid liver-specific promoter (HLP), and the alpha-1-antitrypsin (AAT) promoter.

[0056] In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 5 weeks, 10 weeks, 15 weeks, 26 weeks, 1 year, 5 years, 10 years, or 20 years. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 5 weeks. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 10 weeks. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 15 weeks. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 26 weeks. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 1 year. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 5 years. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 10 years. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 15 years. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for at least 20 years. In some embodiments, after administration of the rAAV vector, the subject has detectable α-GAL in the serum for the lifetime of the subject.

[0057] In some embodiments, expression of the modified α-GAL enzyme results in 3-fold, 10-fold, 30-fold, 100-fold, or 300-fold higher serum α-GAL concentrations compared to expression of WT α-GAL, hi some embodiments, expression of the modified α-GAL enzyme results in 3-fold, 10-fold, 30-fold, 100-fold, or 300-fold higher intracellular enzyme amounts compared to expression of WT α-GAL.

[0058] In some embodiments, administration results in α-GAL enzyme exposure in one or more of the subject's liver, kidney, heart, gastrointestinal tract, brain, and / or peripheral neurons. Thus, in some embodiments, administration results in α-GAL enzyme exposure in the liver. In some embodiments, administration results in α-GAL enzyme exposure in the kidney. In some embodiments, administration results in α-GAL enzyme exposure in the heart. In some embodiments, administration results in α-GAL enzyme exposure in the gastrointestinal tract and cells associated with the gastrointestinal tract. In some embodiments, administration results in α-GAL enzyme exposure in the brain. In some embodiments, administration results in α-GAL enzyme exposure in peripheral neurons.

[0059] In some embodiments, administration of the rAAV vector provides a survival benefit to mice / patients with Fabry disease. In some embodiments, administration of the rAAV vector reduces the amount of globotriaosylceramide (GB3) in one or more of the liver, heart, kidney, and gastrointestinal tract of the subject. Thus, in some embodiments, administration of the rAAV vector reduces the amount of GB3 in the heart. In some embodiments, administration of the rAAV vector reduces the amount of GB3 in skeletal muscle. In some embodiments, administration of the rAAV vector reduces the amount of GB3 in the kidney. In some embodiments, administration of the rAAV vector reduces the amount of GB3 in the gastrointestinal tract. GB3 levels can be assessed by any means known in the art, such as by chromatographic methods, such as by liquid chromatography-tandem mass spectrometry.

[0060] In some aspects, the disclosure encompasses a method of expressing an α-GAL enzyme in a cell, the method comprising administering a rAAV vector packaged within an AAV9 capsid, the vector comprising (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme, (d) a bovine growth hormone (BGH) polyA, and (f) a 3' ITR.

[0061] In some aspects, the disclosure encompasses a method of expressing an α-GAL enzyme in a cell, the method comprising administering a rAAV vector packaged within an AAV9 capsid, the vector comprising (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme, (d) optionally a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having a mut6delATG mutation, (e) a bovine growth hormone (BGH) polyA, and (f) a 3' ITR.

[0062] In some aspects, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a 5' inverted repeat (ITR), a liver-specific promoter, a nucleotide sequence encoding an α-GAL enzyme, polyA, and a 3' ITR.

[0063] In some aspects, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a 5' inverted repeat (ITR), a liver-specific promoter, a nucleotide sequence encoding an alpha-GAL enzyme, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), polyA, and a 3' ITR. [Brief description of the drawings]

[0064] [Figure 1] FIG. 1 is a vector schematic diagram of an exemplary rAAV9 (commonly referred to herein as "rAAV9") comprising a wild-type GAL under the control of a ubiquitous promoter as described herein. [Diagram 2] 1 is a graph showing serum alpha-galactosidase concentrations over a 12-week period following injection of rAAV9 in a Fabry disease (GLAko) mouse model, as described herein. [Figure 3A] 1 is a series of graphs showing alpha-galactosidase expression in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice.Alpha-galactosidase expression in the liver following administration of rAAV9 compared to rAAV9-null ("null" refers to AAV9 that does not contain any transgene) and untreated controls. [Figure 3B]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice. Alpha-galactosidase expression in kidney following administration of rAAV9-WT compared to rAAV9-null and untreated controls. [Figure 3C] 1 is a series of graphs showing alpha-galactosidase expression in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice, and alpha-galactosidase expression in the heart following administration of rAAV9-WT compared to untreated controls. [Figure 3D]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry disease GLAko mice. Alpha-galactosidase expression in the duodenum following administration of rAAV9-WT compared to rAAV9-null (null vector) and untreated controls. [Figure 3E]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice. Alpha-galactosidase expression in the colon following administration of rAAV9 compared to rAAV9-null and untreated controls. [Figure 3F]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice. GB3 concentrations in serum are shown compared to ERT and untreated controls. [Figure 3G]

[0023] Figure 1 is a series of graphs showing alpha-galactosidase expression in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice, showing GB3 concentrations in the liver compared to ERT and untreated controls. [Figure 3H]

[0023] Figure 1 is a series of graphs showing the expression of alpha-galactosidase in various tissues and the reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice, showing GB3 concentrations in the kidney compared to ERT and untreated controls. [Figure 3I]

[0023] Figure 1 is a series of graphs showing alpha-galactosidase expression in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice, showing GB3 concentrations in the heart compared to ERT and untreated controls. [Figure 3J]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice. Serum lysoGB3 levels are shown compared to ERT and untreated controls. [Figure 3K]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice, showing lysoGB3 concentrations in the liver compared to ERT and untreated controls. [Figure 3L]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice, showing lysoGb3 concentrations in kidney compared to ERT and untreated controls. [Figure 3M]

[0023] Figure 1 is a series of graphs showing expression of alpha-galactosidase in various tissues and reduction of GB3 in various tissues following administration of rAAV9-WT in Fabry GLAko mice, showing lysoGb3 levels in the heart compared to ERT and untreated controls. [Figure 4A] 1 is a series of graphs showing alpha-galactosidase expression in various tissues following administration of rAAV9 in severe Fabry disease model (G3Stg / GLAko) mice. Dose-dependent alpha-galactosidase activity in serum compared to vehicle (i.e., formulation buffer alone) is shown over 18 weeks. [Figure 4B] 1 is a series of graphs showing expression of alpha-galactosidase in various tissues following administration of rAAV9 in a severe Fabry disease model (G3Stg / GLAko) mouse, showing a dose-dependent increase in alpha-galactosidase activity in the liver compared to null vector and vehicle (i.e., formulation buffer alone). [Figure 4C] 1 is a series of graphs showing expression of alpha-galactosidase in various tissues following administration of rAAV9 in a severe Fabry disease model (G3Stg / GLAko) mouse, showing a dose-dependent increase in alpha-galactosidase activity in the kidney compared to null vector and vehicle (i.e., formulation buffer alone). [Figure 4D] 1 is a series of graphs showing expression of alpha-galactosidase in various tissues following administration of rAAV9 in a severe Fabry disease model (G3Stg / GLAko) mouse, showing a dose-dependent increase in alpha-galactosidase activity in the heart compared to null vector and vehicle (i.e., formulation buffer alone). [Figure 4E]1 is a series of graphs showing expression of alpha-galactosidase in various tissues following administration of rAAV9 in a severe Fabry disease model (G3Stg / GLAko) mouse, showing a dose-dependent increase in alpha-galactosidase activity in the duodenum compared to null vector and vehicle (i.e., formulation buffer alone). [Figure 4F] 1 is a series of graphs showing expression of alpha-galactosidase in various tissues following administration of rAAV9 in a severe Fabry disease model (G3Stg / GLAko) mouse, showing a dose-dependent increase in alpha-galactosidase activity in the colon compared to null vector and vehicle (formulation buffer). [Figure 4G] 1 is a series of graphs showing expression of alpha-galactosidase in various tissues following administration of rAAV9 in a severe Fabry disease model (G3Stg / GLAko) mouse, showing a dose-dependent increase in alpha-galactosidase activity in the brain compared to null vector and vehicle (i.e., formulation buffer alone). [Figure 5A] 1 is a series of graphs showing reduction in GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease. Showing reduction in GB3 in the liver following administration of rAAV9-WT compared to rAAV9-null and vehicle (i.e., formulation buffer alone). [Figure 5B] 1 is a series of graphs showing reduction in GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease. Shown is reduction in GB3 in the kidney following administration of rAAV9-WT compared to rAAV9-null and vehicle (i.e., formulation buffer alone). [Figure 5C] 1 is a series of graphs showing reduction in GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease. Shown is reduction in GB3 in the heart following administration of rAAV9-WT compared to rAAV9-null and vehicle (i.e., formulation buffer alone). [Figure 5D]1 is a series of graphs showing the reduction of GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease. Shown is the reduction of GB3 in the duodenum following administration of rAAV9-WT compared to rAAV9-null and vehicle (i.e., formulation buffer alone). [Figure 5E] 1 is a series of graphs showing reduction in GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease. Shown is reduction in GB3 in the colon following administration of rAAV9-WT compared to rAAV9-null and vehicle (i.e., formulation buffer alone). [Figure 5F] 1 is a series of graphs showing reduction in GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease. Shown is reduction in GB3 in the brain following administration of rAAV9-WT compared to rAAV9-null and vehicle (i.e., formulation buffer alone). [Figure 5G] 1 is a series of graphs showing reduction of GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease, showing a dose-dependent reduction of lysoGb3 in the liver compared to rAAV9-null. [Figure 5H] 1 is a series of graphs showing reduction of Gb3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease, showing a dose-dependent reduction of lysoGb3 in the kidney compared to rAAV9-null. [Figure 5I] 1 is a series of graphs showing reduction of GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease, showing a dose-dependent reduction of lysoGb3 in the heart compared to rAAV9-null. [Figure 5J]

[0023] Figure 1 is a series of graphs showing reduction of Gb3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease, showing a dose-dependent reduction of lysoGb3 in the duodenum compared to rAAV9-null. [Figure 5K] 1 is a series of graphs showing reduction of Gb3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease, showing a dose-dependent reduction of lysoGb3 in the colon compared to rAAV9-null. [Figure 5L] 1 is a series of graphs showing reduction of GB3 in various tissues following administration of rAAV9 in a mouse model of severe Fabry disease, showing a dose-dependent reduction of lysoGb3 in the brain compared to rAAV9-null. [Figure 6] Graph showing weight gain following administration of increasing concentrations of rAAV9-WT compared to null vector (rAAV9-null) and vehicle (i.e., formulation buffer alone) in severe Fabry disease mice. [Figure 7] A series of graphs showing improvement of renal function in 27-28 week old severe Fabry disease model mice after administration of rAAV9-WT. (A) Dose-dependent normalization of serum BUN. (B) Normalization of urinary albumin after administration of rAAV9-WT. [Figure 8A] Fig. 1 shows immunohistochemical staining of the footpad of a mouse model of severe Fabry disease (G3Stg / GLAko). Fig. 2 shows immunohistochemical staining slides of the footpad of a Fabry disease model mouse, showing reduced vacuolization in the dorsal root nerve. [Figure 8B] Fig. 1 shows immunohistochemical staining of the footpad of a mouse model of severe Fabry disease (G3Stg / GLAko). Immunohistochemical staining slides showing dose-dependent normalization of PGP9.5 (neuronal marker) staining in a Fabry disease model mouse. [Figure 8C] Fig. 1 shows immunohistochemical staining of the footpad of a mouse model of severe Fabry disease (G3Stg / GLAko). Immunohistochemical staining slides showing a dose-dependent increase in MPZ (Schwann cell marker) staining in a Fabry disease model mouse. [Figure 9A] Images showing histopathological characteristics of the kidney, heart, and dorsal root ganglion (DRG) are shown. p62 (autophagy marker) staining observed in the kidney collecting duct of a wild-type mouse is shown. [Figure 9B] Images showing histopathological features of the kidney, heart and dorsal root ganglion (DRG) are shown. p62 staining observed in the kidney collecting duct of an untreated Fabry mouse. [Figure 9C]Images of histopathological features of kidney, heart and dorsal root ganglion (DRG) are shown. p62 staining observed in kidney collecting duct of Fabry mice treated with 6.25e12vg / kg rAAV9-WT. [Figure 9D] Images of histopathological features of kidney, heart and dorsal root ganglion (DRG) are shown. p62 staining observed in wild type mouse heart. [Figure 9E] Images of histopathological features of kidney, heart and dorsal root ganglion (DRG) are shown. p62 staining observed in the heart of an untreated Fabry mouse. [Figure 9F] Images of histopathological features of kidney, heart and dorsal root ganglion (DRG) are shown. p62 staining observed in Fabry mouse hearts when treated with 6.25e12vg / kg rAAV9-WT. [Figure 9G] Images of histopathological features of kidney, heart and dorsal root ganglion (DRG) are shown, showing CD68 staining observed in wild type mouse DRG. [Figure 9H] 1 shows images of histopathological features of kidney, heart and dorsal root ganglion (DRG), CD68 staining observed in DRG of untreated Fabry mice. [Figure 9I] 1 shows images of the histopathological features of the kidney, heart and dorsal root ganglion (DRG), CD68 staining observed in the DRG of Fabry mice treated with 6.25e12vg / kg rAAV9-WT. [Figure 10A] 1 shows the exposure of α-GAL variants in serum and tissues in Fabry mice 2 days after hydrodynamic tail vein injection of a plasmid expressing the α-GAL variant. [Figure 10B] 1 shows the presence of α-GAL variants in serum and tissues of Fabry mice 2 days after hydrodynamic tail vein injection of a plasmid expressing the α-GAL variant. [Figure 10C]1 shows the presence of α-GAL variants in serum and tissues of Fabry mice 2 days after hydrodynamic tail vein injection of a plasmid expressing the α-GAL variant. 1 shows the presence of α-GAL variants in the heart. [Figure 10D] 1 shows the exposure of α-GAL variants in serum and tissues in Fabry mice 2 days after hydrodynamic tail vein injection of a plasmid expressing the α-GAL variant. [Figure 10E] 1 shows the presence of α-GAL variants in serum and tissues of Fabry mice 2 days after hydrodynamic tail vein injection of a plasmid expressing the α-GAL variant. [Figure 10F] 1 shows the presence of α-GAL variants in serum and tissues of Fabry mice 2 days after hydrodynamic tail vein injection of a plasmid expressing the α-GAL variant. 1 shows the presence of α-GAL variants in the heart. [Figure 11A] Graph showing serum alpha-galactosidase concentrations over a 12 week period following injection with either a rAAV9-based construct with a ubiquitous promoter or a rAAV8-based construct with a liver-specific promoter. [Figure 11B] Shows alpha-galactosidase expression in the kidney following administration of rAAV9-WT compared to rAAV8-WT, as well as alpha-galactosidase protein administered at 1 mg / kg and untreated controls. [Figure 11C] Shown is the percentage of GB3 reduction in the kidney following administration of rAAV9-WT compared to rAAV8-WT, as well as alpha-galactosidase protein administered at 1 mg / kg and untreated controls. [Figure 12]A series of graphs showing renal function in 27-28 week old Fabry disease model mice after administration of rAAV9-WT and rAAV8-WT. (A) shows dose-dependent normalization of blood urea nitrogen (BUN) in the case of rAAV9-WT. (B) shows changes in serum BUN in the case of rAAV8-WT. [Figure 13A] FIG. 1 is a series of graphs showing serum and tissue alpha-GAL variant galactosidase activity following treatment of severe Fabry disease mice with rAAV9 expressing the alpha-GAL variant. Serum alpha-galactosidase activity at the 5.0×10 vg / kg dose from Study 1 is shown. [Figure 13B] 1 is a series of graphs showing serum and tissue alpha-GAL variant galactosidase activity following treatment of severe Fabry mice with rAAV9 expressing the alpha-GAL variant. [Figure 13C] 1 is a series of graphs showing serum and tissue α-GAL variant galactosidase activity following treatment of severe Fabry disease mice with rAAV9 expressing the α-GAL variant. 2 shows kidney alpha-galactosidase activity at two different doses from Study 1. [Figure 13D] 1 is a series of graphs showing serum and tissue α-GAL variant galactosidase activity following treatment of severe Fabry disease mice with rAAV9 expressing the α-GAL variant. FIG. 2 shows cardiac alpha-galactosidase activity at two different doses from Study 1. [Figure 13E] 1 is a series of graphs showing serum and tissue α-GAL variant galactosidase activity following treatment of severe Fabry mice with rAAV9 expressing the α-GAL variant. 2 shows liver alpha-galactosidase activity at two different doses from Study 1. [Figure 14A]1 is a series of graphs showing serum and tissue alpha-galactosidase concentrations of α-GAL variants following treatment of severe Fabry mice with rAAV9 expressing the α-GAL variant from Study 2. Serum alpha-galactosidase activity at the 2.5×10 vg / kg dose from Study 2 is shown. [Figure 14B] 1 is a series of graphs showing serum and tissue alpha-galactosidase concentrations of α-GAL variants following treatment of severe Fabry mice with rAAV9 expressing the α-GAL variant from Study 2. 2 shows kidney alpha-galactosidase activity at two different doses from Study 2. [Figure 14C] 1 is a series of graphs showing serum and tissue alpha-galactosidase concentrations of α-GAL variants following treatment of severe Fabry mice with rAAV9 expressing the α-GAL variant from Study 2. FIG. 1 shows cardiac alpha-galactosidase activity at two different doses from Study 2. [Figure 14D] 1 is a series of graphs showing serum and tissue alpha-galactosidase concentrations of α-GAL variants following treatment of severe Fabry mice with rAAV9 expressing the α-GAL variant from Study 2. FIG. 1 shows liver alpha-galactosidase activity at two different doses from Study 2. [Figure 15] 1 is a series of graphs showing the presence of GB3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 1. A shows serum GB3 in response to treatment with rAAV9 expressing an α-GAL variant. B shows kidney GB3 in response to treatment with rAAV9 expressing an α-GAL variant. C shows heart GB3 in response to treatment with rAAV9 expressing an α-GAL variant. D shows liver GB3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 16]1 is a series of graphs showing the presence of lysoGb3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 1. A shows serum lysoGb3 in response to treatment with rAAV9 expressing an α-GAL variant. B shows kidney lysoGb3 in response to treatment with rAAV9 expressing an α-GAL variant. C shows heart lysoGb3 in response to treatment with the variant. D shows liver lysoGb3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 17A] 1 is a series of graphs showing the presence of GB3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. Serum GB3 in response to treatment with the variants. [Figure 17B] 1 is a series of graphs showing the presence of GB3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. 2 shows kidney GB3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 17C] 1 is a series of graphs showing the presence of GB3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. FIG. 1 shows cardiac GB3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 17D] 1 is a series of graphs showing the presence of GB3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. FIG. 1 shows hepatic GB3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 18A] 1 is a series of graphs showing the presence of lysoGb3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. FIG. 1 shows serum lysoGb3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 18B]1 is a series of graphs showing the presence of lysoGb3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. FIG. 1 shows kidney lysoGb3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 18C] 1 is a series of graphs showing the presence of lysoGb3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. FIG. 1 shows cardiac lysoGb3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 18D] 1 is a series of graphs showing the presence of lysoGb3 in serum and tissues in mice treated with rAAV9 expressing an α-GAL variant from Study 2. FIG. 1 shows liver lysoGb3 in response to treatment with rAAV9 expressing an α-GAL variant. [Figure 19] FIG. 1 is a series of graphs showing in vitro α-GAL activity in HUH-7 and HEK293 cells following transfection with plasmids expressing codon-optimized α-GAL variants 004 and D. A is a histogram showing in vitro α-GAL activity in HUH-7 cells transfected with variants of the plasmid including D, D1-D6 compared to WT α-GAL and no plasmid. B is a histogram showing in vitro α-GAL activity in HEK293 cells transfected with variants of the plasmid including D, D1-D6 compared to WT α-GAL and no plasmid. C is a histogram showing in vitro α-GAL activity in HUH-7 cells transfected with variants of the plasmid including 004, 004-1-004-5 compared to WT α-GAL and no plasmid. D is a histogram showing in vitro α-GAL activity in HEK293 cells transfected with plasmid variants including 004, 004-1 to 004-5 compared to WT α-GAL and no plasmid. [Figure 20A]1 is a series of graphs showing dose-dependent α-GAL activity in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a histogram of dose-dependent α-GAL activity in serum in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 20B] 1 is a series of graphs showing dose-dependent α-GAL activity in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a histogram of dose-dependent α-GAL activity in the kidney in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 20C] 1 is a series of graphs showing dose-dependent α-GAL activity in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a histogram of dose-dependent α-GAL activity in the heart in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 20D] 1 is a series of graphs showing dose-dependent α-GAL activity in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a histogram of dose-dependent α-GAL activity in the liver in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 21A] 1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of GB3 in serum in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 21B]1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of lysoGb3 in serum in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 21C] 1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of GB3 in kidney in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 21D] 1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of lysoGb3 in kidney in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 21E] 1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of GB3 in the heart in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 21F] 1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of lysoGb3 in the heart in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Figure 21G]1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of GB3 in the liver in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. [Fig. 21H] 1 is a series of graphs showing dose-dependent reduction of substrates in various tissues in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3. FIG. 2 is a graph showing dose-dependent reduction of lysoGb3 in the liver in severe Fabry disease model mice treated with rAAV9-D3 and rAAV9-004-3.

[0065] definition Approximately or about: As used herein, the term "approximately" refers to a value similar to the stated reference value when applied to one or more values ​​of interest. In certain embodiments, the term "approximately" refers to a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or smaller) from the stated reference value, unless otherwise stated or otherwise clear from the context (except when such number exceeds 100% of possible values). When the term "about" or "approximately" is used to modify a stated reference value, it is understood that the stated reference value itself is encompassed along with values ​​that are in the vicinity of the stated reference value on either side of the stated reference value.

[0066] Administering: The terms "administer", "administration" and "administering" refer to providing a composition of the invention (e.g., a recombinant gene therapy vector expressing alpha-galactosidase) to a subject in need thereof (e.g., a person suffering from the effects of Fabry disease).

[0067] Administered in combination: As used herein, the term "administered in combination" or "co-administration" means that two or more agents are administered to a subject either simultaneously or within a period of time such that there may be overlap in the effect of each agent on the patient. In some embodiments, the administration of the agents is spaced sufficiently close to each other that a combined (e.g., synergistic) effect is achieved.

[0068] Allogeneic: As used herein, allogeneic refers to any material that is derived from a different animal of the same species as the individual to which the material is introduced. Two or more individuals are said to be allogeneic to each other if their genes are not identical at one or more loci. In some embodiments, allogeneic material from individuals of the same species may be genetically sufficiently different to interact as an antigen.

[0069] Amino acid substitution: The term "amino acid substitution" refers to the replacement of an amino acid residue present in a parent or reference sequence (e.g., a wild-type GLA sequence) with another amino acid residue. An amino acid can be replaced in a parent or reference sequence (e.g., a wild-type GLA polypeptide sequence) for example, via chemical peptide synthesis or via recombinant methods known in the art. Thus, reference to a "substitution at position X" refers to the replacement of the amino acid present at position X with an alternative amino acid residue. In some embodiments, the substitution pattern can be described according to the scheme AnY, where A is the one-letter code corresponding to the amino acid naturally occurring or naturally occurring at position n, and Y is the alternative amino acid residue. In other embodiments, the substitution pattern can be described according to the scheme An(YZ), where A is the one-letter code corresponding to the amino acid residue that is substituted for the amino acid naturally occurring or naturally occurring at position X, and Y and Z are the alternative amino acid residues that are substituted.

[0070] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamate (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When a three-letter abbreviation is used, the amino acid can be in either the L- or D-configuration about the α-carbon (Cα) unless specifically preceded by "L" or "D" or otherwise clear from the context in which the abbreviation is used. In various embodiments described herein, one or more amino acids in the wild-type GLA sequence can be replaced with a different amino acid, thereby resulting in a variant of the α-GAL protein.

[0071] Substitutions in the amino acid sequence of a protein or polypeptide can be of a conservative or non-conservative nature. A conservative amino acid substitution refers to the replacement of a residue with a different residue having a similar side chain, and thus typically involves replacing an amino acid in a polypeptide (e.g., an α-GAL amino acid sequence) with an amino acid within the same or a similarly defined class of amino acids. By way of example and not limitation, an amino acid having an aliphatic side chain may be replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid having a hydroxyl side chain may be replaced with another amino acid having a hydroxyl side chain (e.g., serine and threonine), an amino acid having an aromatic side chain may be replaced with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid having a basic side chain may be replaced with another amino acid having a basic side chain (e.g., lysine and arginine), an amino acid having an acidic side chain may be replaced with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid), and / or a hydrophobic or hydrophilic amino acid may be replaced with another hydrophobic or hydrophilic amino acid, respectively. A non-conservative substitution refers to the replacement of an amino acid in a polypeptide (e.g., an α-GAL amino acid sequence) with an amino acid having significantly different side chain properties. By way of example and not limitation, exemplary non-conservative substitutions would be an acidic amino acid replaced with a basic or aliphatic amino acid, an aromatic amino acid replaced with a small amino acid, and a hydrophilic amino acid replaced with a hydrophobic amino acid.

[0072] In the context of the present disclosure, substitutions (even when referred to as amino acid substitutions) are made at the nucleic acid level, i.e., the replacement of an amino acid residue with an alternative amino acid residue is made by replacing a codon encoding a first amino acid with a codon encoding a second amino acid.

[0073] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and insects. In some embodiments, the animal is a transgenic animal, a genetically modified animal, or a clone.

[0074] Blood urea nitrogen: As used herein, the term "blood urea nitrogen" or "BUN" refers to the amount of urea in the blood. Kidney-related pathologies have elevated blood urea nitrogen. Chronic kidney disease is one of the major features of Fabry disease, leading to end-stage renal failure. Gb-3 deposition in glomerular podocytes is believed to contribute, at least in part, to the rate or severity of proteinuria or kidney damage in Fabry disease. Blood urea nitrogen measures how efficiently the kidneys remove urea from the blood. High BUN indicates poor kidney function.

[0075] Chimera: As used herein, a "chimera" is an entity having two or more mismatched or heterologous parts or regions. For example, a chimeric molecule can include a first part that includes a GLA polypeptide and a second part (e.g., fused to the first part by genetic engineering) that includes a second therapeutic protein (e.g., a protein with a distinct enzymatic activity, an antigen-binding portion, or a portion capable of extending the plasma half-life of α-GAL, e.g., the Fc region of an antibody).

[0076] Codon substitution: As used herein, the term "codon substitution" or "codon replacement" in the context of sequence optimization refers to replacing a codon present in a reference nucleic acid sequence with another codon. A codon can be replaced in a reference nucleic acid sequence, for example, via chemical peptide synthesis or via recombinant methods known in the art. Thus, reference to "substitution" or "replacement" at a particular position in a nucleic acid sequence (e.g., mRNA) or within a particular region or subsequence of a nucleic acid sequence (e.g., mRNA) refers to the replacement of a codon with an alternative codon at such position or region.

[0077] Codon-optimized: The term "codon-optimized" or "codon optimization" refers to the alteration of codons in a polynucleotide encoding a protein (e.g., a GLA gene) such that the encoded protein is more efficiently expressed, for example, in a cell or organism. In some embodiments, a polynucleotide encoding an α-GAL enzyme may be codon-optimized for optimal production from the host organism(s) and / or cell type(s) selected for expression, taking into account GC content, cryptic splice sites, transcription termination signals, motifs that may affect RNA stability, and nucleic acid secondary structure, as well as any other factors of interest.

[0078] Engineered variant: The term "engineered α-GAL variant" or "engineered variant" refers to a GAL protein in which one or more amino acid residues have been modified by substitution, deletion, or insertion when compared to wild-type α-GAL. In some embodiments, engineered variants are characterized by improved efficacy and pharmacokinetic profiles, for example, due to modified structural attributes of the protein. In some embodiments, engineered α-GAL variants enhance clearance of substrates from tissues such as serum, kidney, heart, and / or liver. Engineered variants can be synthetic or recombinantly produced.

[0079] Gb3: As used herein, the term "Gb3" or "globotriaosylceramide" or "GB3" or "gb3" or "CD77" or "GL-3" refers to a type of glycosphingolipid that accumulates in lysosomes in Fabry disease and is considered the main culprit metabolite. GB3 is formed by alpha-linkage of galactose to lactosylceramide, catalyzed by A4GALT. GB3 is hydrolyzed at the terminal alpha linkage by GLA. Fabry disease is exemplified by the accumulation of GB3 in all organs (especially the heart and kidney), as well as in many cells and urine. Such accumulation is accompanied by a marked increase in the risk of stroke, heart disease (hypertrophic cardiomyopathy, rhythm and conduction system disorders, coronary artery disease, valvular abnormalities, etc.) and chronic proteinuric renal failure. In some embodiments, deacylated GB3 or lysoGb3 is also a useful biomarker for Fabry disease.

[0080] Gene: The term "gene" as used herein refers to a DNA region that encodes a protein or polypeptide (e.g., an alpha-galactosidase enzyme as described herein) as well as all DNA regions that regulate the production of such a protein or polypeptide, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequence. 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.

[0081] GLA gene: As used herein, the term "GLA gene" or "galactosidase gene" or "alpha-galactosidase gene" or "α-galactosidase gene" refers to a gene that encodes the enzyme alpha-galactosidase, which breaks down globotriaosylceramide. Genetic mutations in the GLA gene cause defects in the enzymatic function of alpha-galactosidase. In humans, the GLA gene is located on Xq22.1, the long (q) arm of the X chromosome at position 22.1. Some other names that may be referred to for the GLA gene include AGAL HUMAN, agalsidase alpha, alpha-D-galactosidase A, alpha-D-galactosidase galactohydrolase, alpha-galactosidase, alpha-galactosidase A, ceramide trihexosidase, GALA, galactosidase, or melibiase.

[0082] Galactosidase: The term "galactosidase" or "alpha galactosidase A" or "α-galactosidase A" or "α-GAL" as used herein refers to the enzyme encoded by the GLA gene. Human alpha galactosidase (EC 3.2.1.22) is a lysosomal enzyme that hydrolyzes terminal alpha galactosyl moieties from glycolipids and glycoproteins. As used herein, the term α-GAL may refer to the wild-type enzyme or a variant thereof. Deficiency of alpha galactosidase A causes Fabry disease (also known as diffuse truncal angiokeratoma, Anderson-Fabry disease, hereditary ectopic lipidosis, alpha galactosidase A deficiency, α-GAL deficiency, and ceramide trihexosidase deficiency), an X-linked congenital disorder of glycosphingolipid catabolism. In various embodiments described herein, a gene therapy platform for the treatment of Fabry disease is provided.

[0083] "Improved enzymatic properties": The term "improved enzymatic properties" refers to any property or attribute of an engineered α-GAL polypeptide that is improved compared to the same property or attribute of a reference α-GAL polypeptide (e.g., compared to a wild-type α-GAL polypeptide or another engineered α-GAL polypeptide). Improved properties include, but are not limited to, properties such as increased gene expression, increased protein production, increased thermoactivity, improved thermostability, increased activity at various pH values, improved stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate and / or product inhibition, improved chemical stability, improved functional group selectivity, improved solvent stability, increased resistance to acidic, neutral, or basic pH, resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, improved solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation), altered temperature profile, increased cellular uptake, improved lysosomal stability, improved ability to reduce GB3 cells, increased secretion from α-GAL producing cells, etc. In various embodiments, gene therapy vectors encompassed by the present disclosure comprise a nucleic acid sequence encoding an α-GAL polypeptide having one or more improved enzymatic properties compared to an α-GAL polypeptide, hi some embodiments, the nucleic acid sequence encoding an α-GAL polypeptide exhibiting one or more improved enzymatic properties is codon optimized.

[0084] In various embodiments, the codon-optimized and / or engineered α-GAL variants exhibit one or more of the above-mentioned improved properties. In certain embodiments, the α-GAL variants having the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 50 have improved serum and lysosomal stability, and the α-GAL variants having the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 55 have increased specific catalytic activity compared to the wild-type α-GAL polypeptide.

[0085] "Increased enzyme activity": The term "increased enzyme activity" refers to an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the percent conversion of substrate to product (e.g., the percent conversion of starting substrate to product in a particular time period) using a particular amount of engineered α-GAL enzyme compared to a reference α-GAL enzyme (e.g., a wild-type α-GAL enzyme or another engineered variant). Any suitable method known in the art and / or described herein can be used to determine enzyme activity. m , V max or k cat Any property related to enzyme activity may be affected, including classical enzyme properties such as, and changes therein may result in increased enzyme activity. The improved enzyme activity may range from about 1.1-fold higher than the enzyme activity of the corresponding wild-type enzyme, to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or even higher enzyme activity than the reference α-GAL enzyme.

[0086] Endogenous expression: The term "endogenous expression" and its grammatical equivalents refer to the expression of a gene within one or more cells into which a transgene is introduced. Endogenous expression uses the cell's own or existing transcription or translation machinery and resources to express the transgene. For example, in some embodiments, when the term is used to refer to an "endogenous α-GAL expression system," it means that α-GAL is expressed from within the cells of a tissue.

[0087] Nucleic acid: As used herein, the terms "nucleic acid", "polynucleotide", and "oligonucleotide" are used interchangeably to refer to a polymer of deoxyribonucleotides or ribonucleotides in a linear or cyclic conformation, in 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. The term can include known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar and / or phosphate moieties (e.g., thiophosphate backbone). In general, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of A will base-pair with T.

[0088] Functionally linked: As used herein, the terms "functionally linked" and "operatively linked" (or "operably linked") are used interchangeably with respect to the juxtaposition of two or more components (such as sequence elements) where the components are arranged in a manner that allows both components to function normally and allows the possibility that at least one of the components can mediate a function exerted on at least one of the other components. By way of example, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence, even if it is not adjacent to the coding sequence.

[0089] Physiological pH: As used herein, "physiological pH" generally refers to the pH range found in a subject's (e.g., human) blood.

[0090] Basic pH: The term "basic pH" (as used, for example, with respect to improved stability at or increased resistance to basic pH conditions) refers to a pH range of about 7-11.

[0091] Acidic pH: The term "acidic pH" (e.g., as used in reference to improved stability to or increased resistance to acidic pH conditions) refers to a pH range of about 1.5 to 4.5. Polypeptide: As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

[0092] Promoter: As used herein, the term "promoter" encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and the expression of the corresponding proteins or polypeptides may be ubiquitous, i.e., strongly active in a wide range of cells, tissues, and species, or may be cell type-specific, tissue-specific, or species-specific. In some embodiments, liver-specific promoters include, for example, the transthyretin promoter (TTR), the thyroxine-binding globulin (TBG) promoter, the hybrid liver-specific promoter (HLP), and the alpha-1-antitrypsin (AAT) promoter. Promoters may be "constitutive," i.e., constantly active, or "inducible," i.e., the promoter may be active or inactive depending on the presence or absence of biotic or abiotic factors. The nucleic acid construct or vector of the present invention also includes enhancer sequences, which may or may not be adjacent to the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0093] Sequence optimization: As used herein, the term "sequence optimization" refers to a process or series of processes in which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence having improved properties, e.g., improved protein expression or increased activity.

[0094] Tropism: As used herein, the term "tropism" or "tropicity" in the context of AAV refers to AAV capsid serotypes that have different transduction profiles for different tissue types. In some embodiments, "systemic tropism" and "systemic transduction" (and equivalent terms) indicate that a viral capsid or viral vector of the invention exhibits tropism for or transduces multiple tissues throughout the body, or multiple tissues or organs (e.g., more than one of the brain, lung, skeletal muscle, heart, liver, kidney, and / or pancreas), respectively.

[0095] Vector: As used herein, the term "vector" is capable of introducing a genetic sequence into a target cell. Typically, "vector construct", "expression vector", and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest, which can introduce a genetic sequence into a target cell. Thus, the term includes cloning and expression vehicles, as well as integrating vectors. In some embodiments, the vector is a virus, including, for example, vector nucleic acid in encapsulated form and viral particles in which the vector nucleic acid is packaged. In some embodiments, the vector is not a wild-type strain of virus, as it contains artificial mutations or modifications. In some embodiments, the vector is derived from a wild-type virus strain by genetic engineering (i.e., by deletion) to include a conditionally replicating virus, as detailed herein. In some embodiments, the vector is delivered by non-viral means. In some embodiments, the vectors described herein are gene therapy vectors and are used as carriers for the delivery of polynucleotide sequences (e.g., alpha-galactosidase enzyme) to cells. In certain embodiments, the gene therapy vectors described herein are recombinant AAV vectors (e.g., AAV8 or AAV9).

[0096] Wild-type: As used herein, the terms "wild-type" and "naturally-occurring" refer to a form of a nucleic acid or protein found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that is isolatable from a natural source and has not been intentionally modified by human manipulation.

[0097] The recitations herein of numerical ranges by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0098] Various aspects of the present invention are detailed in the following paragraphs. The use of the paragraphs is not intended to limit the present invention. Each paragraph is applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise specified. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] Fabry disease is an X-linked genetic disorder that results in abnormal production of the lysosomal hydrolase α-galactosidase A (α-GAL) due to mutations in the GLA gene. Because α-GAL is required for the catabolism of glycolipids such as sphingolipids, deficiency or dysfunction of α-GAL leads to the accumulation of sphingolipids in tissues. Fabry disease affects 1 in 40,000 males and results in a multisystemic disease that begins in childhood or adolescence. Clinical symptoms of Fabry disease include, but are not limited to, a burning, tingling, or prickling sensation or numbness in the extremities, heat intolerance, skin lesions called angiokeratomas, corneal opacities, arrhythmias, left ventricular hypertrophy, proteinuria, renal dysfunction, and cerebrovascular disorders such as strokes and / or seizures. Females who are heterozygous for the GLA gene can transmit the disease to their sons, but are usually asymptomatic. However, some females develop corneal clouding or more severe symptoms due to uneven X-chromosome inactivation.

[0100] Current treatment options for Fabry disease include recombinant enzyme replacement therapy (ERT). ERT slows the progression of Fabry disease but does not completely halt or reverse the disease. Current treatments for Fabry disease achieve a slowing of disease progression that is primarily limited to the kidneys and heart, with insufficient or no improvement in other organs / tissues. Fabry disease patients also require continuous protein-based infusions, which may result in infusion reactions and enhanced immunogenicity. The need for such ongoing disease management also increases the "treatment burden" or additional and ongoing work burden (i.e., what is needed and required) for patients to adhere to the recommendations made by clinicians to manage their morbidity and health status. In male patients with severe classical Fabry disease, the annual loss of renal function is -1mL / min / 1.73m 2 / year in healthy subjects, the maximum annual renal function loss was -6.82mL / min / 1.73m despite treatment. 2 / year (Germain et al, J Med Genet. 2015 May;52(5):353-8.2015). These needs may be addressed by vector delivery of GLA as described herein.

[0101] One advantage of gene therapy for the treatment of Fabry disease is the continuous α-GAL exposure rather than the intermittent α-GAL exposure afforded by ERT infusion. Gene therapy could potentially enable uptake by certain tissues and cell types (e.g., cardiomyocytes, peripheral neurons, and kidney podocytes) that are not easily achieved with infused ERT. The sustained availability of α-GAL in lysosomes could prevent reaccumulation of glycosphingolipids between doses. The greatly enhanced enzyme distribution to target cells could provide a transformative therapy that could achieve superior clinical benefit over current therapies. Furthermore, gene therapy with hepatocyte transduction could take advantage of the tolerogenic properties of the liver, inducing systemic immune tolerance to the transgene product and eliminating the risk of therapeutic compromise due to anti-drug antibodies. Without wishing to be bound by theory, it is believed that these advantages, combined with a single, long-term sustained dose, could address the need for highly therapeutically effective therapies and also reduce the treatment burden on patients and caregivers.

[0102] The present disclosure provides, inter alia, (1) an endogenous GLA expression system in tissues affected by Fabry disease, (2) methods for achieving sustained high expression of α-GAL to reduce the disease burden and treatment burden associated with the progression of Fabry disease, and (3) the use of vectors encoding GLA to achieve a reduction in Fabry disease-associated phenotypes.

[0103] In some embodiments, the endogenous GLA expression system provided herein comprises a viral vector controlled by a ubiquitous promoter that contains a sequence encoding α-GAL. In some embodiments, the promoter is a mammalian ubiquitous promoter. In some embodiments, the ubiquitous promoter is believed to achieve broad distribution of the encoded α-GAL in mammals. Current gene therapy methods for treating Fabry disease rely primarily on the use of liver-specific promoters, unlike the delivery vehicles described herein. Gene therapy systems for Fabry disease using liver-specific promoters rely on α-GAL production from the liver and "cross-correction" of other tissues. The present disclosure provides a system for endogenous expression of α-GAL in multiple target tissues, resulting in broader exposure of α-GAL and better treatment of Fabry disease and related conditions, using gene therapy systems using ubiquitous promoters. As described in more detail below, the ubiquitous promoter used in the present disclosure may be selected from one or more of the following: EF-1α promoter, UBC promoter, LSE beta-glucuronidase (GUSB) promoter, ubiquitous chromatin opening element (UCOE) promoter, GAPDH promoter, chicken beta actin (CBA) promoter, PGK promoter, and mini-EF1 promoter. In some embodiments, the ubiquitous promoter can be engineered based on one of the more known ubiquitous promoters.

[0104] The present disclosure provides a system for broader exposure of α-GAL and more effective treatment of Fabry disease and related conditions using a gene therapy system with a ubiquitous promoter. As detailed below, the ubiquitous promoter used in the present disclosure can be selected from one or more of the following: EF-1α promoter, UBC promoter, LSE beta-glucuronidase (GUSB) promoter, ubiquitous chromatin opening element (UCOE) promoter, GAPDH promoter, chicken beta actin (CBA) promoter, PGK promoter, and mini-EF1 promoter. In some embodiments, the ubiquitous promoter can be engineered based on one of the more known ubiquitous promoters.

[0105] In some embodiments, the endogenous GLA expression system provided herein comprises a viral vector that can improve the exposure or distribution of α-GAL in various tissues in a mammal. In some embodiments, the improved exposure or distribution of α-GAL in various tissues improves symptoms associated with Fabry disease. In some embodiments, the use of a viral vector complements the use of a ubiquitous promoter in providing broader tropism of GLA. In general, broader tropism of GLA is expected to improve symptoms of Fabry disease. In some embodiments, the viral vector is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 with a ubiquitous promoter. In some embodiments, a suitable viral vector with broad tropism can be engineered using combination elements of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 with a ubiquitous promoter.

[0106] In some embodiments, a viral vector encompassed by the present disclosure comprises a tissue-specific promoter, for example a liver-specific promoter, upstream of the nucleic acid sequence encoding an α-GAL polypeptide.

[0107] In some embodiments, the endogenous GLA expression system provided herein comprises a viral vector that can improve the exposure or distribution of α-GAL in various tissues in a mammal. In some embodiments, the improved exposure or distribution of α-GAL in various tissues improves symptoms associated with Fabry disease. In some embodiments, the use of a viral vector complements the use of a ubiquitous promoter in providing robust tissue distribution of α-GAL. In general, improved biodistribution of α-GAL is expected to improve symptoms of Fabry disease. In some embodiments, the viral vector is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 with a ubiquitous promoter. In some embodiments, a suitable viral vector with broad tropism can be engineered using combination elements of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 with a ubiquitous promoter.

[0108] In some embodiments, the endogenous GLA expression system provided herein is a viral vector expressing wild-type or variant α-GAL (e.g., an engineered variant). In some embodiments, the present disclosure provides a viral vector system comprising a ubiquitous promoter for broad tissue-wide distribution of wild-type α-GAL. In some embodiments, the present disclosure provides a viral vector system comprising a ubiquitous promoter for broad tissue-wide distribution of α-GAL variants.

[0109] In some embodiments, the GLA transgene encodes an enzyme that has improved serum or tissue stability of α-GAL compared to wild-type α-GAL. In some embodiments, the GLA transgene encodes an enzyme that has increased α-GAL activity compared to the wild-type enzyme. In some embodiments, the GLA transgene described herein encodes an α-GAL enzyme that includes one or more amino acid modifications at positions 1-100 of wild-type α-GAL. In some embodiments, the α-GAL enzyme encoded by the GLA transgene includes one or more amino acid modifications at positions 101-200 of wild-type α-GAL. In some embodiments, the α-GAL enzyme encoded by the GLA transgene includes one or more amino acid modifications at positions 201-300 of wild-type α-GAL. In some embodiments, the α-GAL enzyme encoded by the GLA transgene includes one or more amino acid modifications at positions 301-400 of wild-type α-GAL. In some embodiments, the α-GAL enzyme encoded by the GLA transgene comprises one or more amino acid modifications at positions 401-429 of wild-type α-GAL. In some embodiments, the modification can be an amino acid substitution. In some embodiments, the modification can be an amino acid deletion. In some embodiments, the modification can be an amino acid insertion. In some embodiments, the amino acid substitution can be a conservative substitution. In some embodiments, the amino acid substitution can be a non-conservative substitution. In some embodiments, the α-GAL encoded by the GLA transgene comprises an amino acid sequence selected from SEQ ID NOs: 7-17, 33, or 34.

[0110] Gene Therapy Vectors The vectors described herein comprise a GLA sequence. In some embodiments, the GLA sequence can be a naturally occurring (wild type) sequence (SEQ ID NO: 30). In some embodiments, the GLA sequence can be a modified sequence, such as a codon-optimized GLA sequence or an engineered or modified GLA sequence. Exemplary GLA sequences contemplated for use in the vectors of the present disclosure are listed in Table 1.

[0111] In some embodiments, the α-GAL encoded by the GLA transgene comprises a signal peptide sequence MQLRNPELHLGCALALRFLALVSWDIPGARA (SEQ ID NO: 76). In some embodiments, the α-GAL encoded by the GLA transgene comprises a signal peptide sequence at the N-terminus. In some embodiments, the α-GAL encoded by the GLA transgene comprises a signal peptide sequence at the C-terminus. In some embodiments, the α-GAL encoded by the GLA transgene comprises SEQ ID NO: 76 at the N-terminus. In some embodiments, the α-GAL encoded by the GLA transgene comprises SEQ ID NO: 76 at the N-terminus.

[0112] In some embodiments, the GLA sequence comprises the signal peptide sequence atgcagctgaggaacccagaactacatctgggctgcgcgcttgcgcttcgcttcctggccctcgtttcctgggacatccctggggctagagca (SEQ ID NO: 77). In some embodiments, the GLA sequence comprises a signal peptide sequence at the 5' end. In some embodiments, the GLA sequence comprises a signal peptide sequence at the 3' end. In some embodiments, the GLA sequence comprises SEQ ID NO: 77 at the 5' end. In some embodiments, the GLA sequence comprises SEQ ID NO: 77 at the 3' end.

[0113] [Table 1-1]

[0114] [Table 1-2]

[0115] [Table 1-3]

[0116] [Table 1-4]

[0117]

Table 1-5

[0118]

Table 1-6

[0119]

Table 1-7

[0120]

Table 1-8

[0121]

Table 1-9

[0122]

Table 1-10

[0123]

Table 1-11

[0124]

Table 1-12

[0125]

Table 1-13

[0126]

Table 1-14

[0127]

Table 1-15

[0128]

Table 1-16

[0129]

Table 1-17

[0130]

Table 1-18

[0131]

Table 1-19

[0132]

Table 1-20

[0133]

Table 1-21

[0134]

Table 1-22

[0135]

Table 1-23

[0136]

Table 1-24

[0137]

Table 1-25

[0138]

Table 1-26

[0139]

Table 1-27

[0140]

Table 1-28

[0141]

Table 1-29

[0142]

Table 1-30

[0143]

Table 1-31

[0144]

Table 1-32

[0145]

Table 1-33

[0146]

Table 1-34

[0147]

Table 1-35

[0148]

Table 1-36

[0149]

Table 1-37

[0150]

Table 1-38

[0151]

Table 1-39

[0152]

Table 1-40

[0153]

Table 1-41

[0154]

Table 1-42

[0155]

Table 1-43

[0156]

Table 1-44

[0157]

Table 1-45

[0158]

Table 1-46

[0159]

Table 1-47

[0160]

Table 1-48

[0161]

Table 1-49

[0162]

Table 1-50

[0163]

Table 1-51

[0164] In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to any one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is 70%-100% identical to SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is 75%-100% identical to SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is 80%-100% identical to SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is 85% to 100% identical to SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is 90% to 100% identical to SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is 95% to 100% identical to SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 70% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 75% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 80% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 85% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 90% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74.In some embodiments, the vector comprises a GLA sequence that is at least 95% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 96% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 97% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 98% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is at least 99% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74. In some embodiments, the vector comprises a GLA sequence that is 100% identical to one of SEQ ID NOs: 18-29, 31, 32, 35-45, and 61-74.

[0165] In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 35. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 36. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 37. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 38. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 39. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 40. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 41. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 42. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 43.In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 44. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 45. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 61. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 62. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 63. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 64. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 65. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 66. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 67.In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 68. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 69. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 70. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 71. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 72. In some embodiments, the disclosure encompasses gene therapy vectors that include a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO: 73. In some embodiments, the disclosure encompasses gene therapy vectors comprising a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical to SEQ ID NO:74.

[0166] In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 7. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 8. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 9. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 10. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 11. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 12. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nuclear sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO:13.In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 14. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 15. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 16. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 17. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 30. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 33. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nuclear sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO:34.In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 46. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 47. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 48. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 49. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 50. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 51. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nuclear sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO:52.In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 53. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 54. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 56. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 57. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 58. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nucleic sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO: 59. In some embodiments, the disclosure encompasses gene therapy vectors comprising a nuclear sequence encoding an α-GAL enzyme that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to SEQ ID NO:60.

[0167] In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 18. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 19. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 20. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 21. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 22. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 23. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 24. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 25. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 26. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 27. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 28. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:29. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:31. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:32. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:35. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:36. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:37. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:38. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:39. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO:40.In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 41. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 42. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 43. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 44. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 45. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 61. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 62. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 63. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 64. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 65. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 66. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 67. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 68. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 69. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 70. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 71. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 72. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 73. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence comprising SEQ ID NO: 74.

[0168] In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:7. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:8. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:9. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:10. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:11. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:12. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:13. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 14. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 15. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 16. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 17. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 30.In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:33. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:34. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:46. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:47. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:48. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:49. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:50. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:51. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:52. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:53. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:54. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO:55.In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 56. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 57. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 58. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 59. In some embodiments, the disclosure encompasses a gene therapy vector comprising a nucleic sequence encoding an α-GAL enzyme comprising SEQ ID NO: 60.

[0169] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such vector comprises: a) a 5' inverted repeat (ITR); b) a ubiquitous promoter; c) a nucleotide sequence encoding an α-GAL enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-17, 30, 33, 34, and 46-60; d) polyA; and e) a 3' ITR.

[0170] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:7, d) polyA, and e) a 3' ITR.

[0171] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:8, d) polyA, and e) a 3' ITR.

[0172] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:9, d) polyA, and e) a 3' ITR.

[0173] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:10, d) polyA, and e) a 3' ITR.

[0174] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:11, d) polyA, and e) a 3' ITR.

[0175] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:12, d) polyA, and e) a 3' ITR.

[0176] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:13, d) polyA, and e) a 3' ITR.

[0177] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:14, d) polyA, and e) a 3' ITR.

[0178] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:15, d) polyA, and e) a 3' ITR.

[0179] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 16, d) polyA, and e) a 3' ITR.

[0180] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:17, d) polyA, and e) a 3' ITR.

[0181] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:30, d) polyA, and e) a 3' ITR.

[0182] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:33, d) polyA, and e) a 3' ITR.

[0183] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:34, d) polyA, and e) a 3' ITR.

[0184] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:46, d) polyA, and e) a 3' ITR.

[0185] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:47, d) polyA, and e) a 3' ITR.

[0186] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:48, d) polyA, and e) a 3' ITR.

[0187] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:49, d) polyA, and e) a 3' ITR.

[0188] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:50, d) polyA, and e) a 3' ITR.

[0189] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:51, d) polyA, and e) a 3' ITR.

[0190] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:52, d) polyA, and e) a 3' ITR.

[0191] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:53, d) polyA, and e) a 3' ITR.

[0192] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:54, d) polyA, and e) a 3' ITR.

[0193] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:55, d) polyA, and e) a 3' ITR.

[0194] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:56, d) polyA, and e) a 3' ITR.

[0195] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:57, d) polyA, and e) a 3' ITR.

[0196] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:58, d) polyA, and e) a 3' ITR.

[0197] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:59, d) polyA, and e) a 3' ITR.

[0198] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:60, d) polyA, and e) a 3' ITR.

[0199] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:7, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0200] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:8, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0201] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:9, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0202] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 10, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0203] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:11, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0204] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 12, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0205] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 13, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0206] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 14, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0207] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 15, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0208] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 16, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0209] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 17, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0210] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:30, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0211] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:33, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0212] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:34, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0213] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:46, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0214] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:47, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0215] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:48, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0216] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:49, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0217] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:50, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0218] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:51, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0219] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:52, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0220] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:53, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0221] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:54, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0222] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:55, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0223] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:56, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0224] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:57, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0225] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:58, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0226] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:59, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0227] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:60, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0228] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:7, d) polyA, and e) a 3' ITR.

[0229] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:8, d) polyA, and e) a 3' ITR.

[0230] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:9, d) polyA, and e) a 3' ITR.

[0231] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:1, d) polyA, and e) a 3' ITR.

[0232] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:11, d) polyA, and e) a 3' ITR.

[0233] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:12, d) polyA, and e) a 3' ITR.

[0234] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 13, d) polyA, and e) a 3' ITR.

[0235] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:14, d) polyA, and e) a 3' ITR.

[0236] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:14, d) polyA, and e) a 3' ITR.

[0237] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 15, d) polyA, and e) a 3' ITR.

[0238] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 16, d) polyA, and e) a 3' ITR.

[0239] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 17, d) polyA, and e) a 3' ITR.

[0240] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:30, d) polyA, and e) a 3' ITR.

[0241] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:33, d) polyA, and e) a 3' ITR.

[0242] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:34, d) polyA, and e) a 3' ITR.

[0243] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:46, d) polyA, and e) a 3' ITR.

[0244] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:47, d) polyA, and e) a 3' ITR.

[0245] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:48, d) polyA, and e) a 3' ITR.

[0246] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:49, d) polyA, and e) a 3' ITR.

[0247] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:50, d) polyA, and e) a 3' ITR.

[0248] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:51, d) polyA, and e) a 3' ITR.

[0249] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:52, d) polyA, and e) a 3' ITR.

[0250] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:53, d) polyA, and e) a 3' ITR.

[0251] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:54, d) polyA, and e) a 3' ITR.

[0252] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:55, d) polyA, and e) a 3' ITR.

[0253] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:56, d) polyA, and e) a 3' ITR.

[0254] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:57, d) polyA, and e) a 3' ITR.

[0255] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:58, d) polyA, and e) a 3' ITR.

[0256] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:59, d) polyA, and e) a 3' ITR.

[0257] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:60, d) polyA, and e) a 3' ITR.

[0258] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:7, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0259] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:8, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0260] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:9, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0261] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 10, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0262] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:11, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0263] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 12, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0264] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 13, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0265] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 14, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0266] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 15, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0267] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 16, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0268] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO: 17, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0269] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:30, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0270] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:33, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0271] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:34, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0272] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:46, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0273] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:47, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0274] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:48, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0275] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:49, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0276] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:50, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0277] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:51, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0278] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:52, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0279] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:53, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0280] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:54, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0281] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:55, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0282] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:56, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0283] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:57, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0284] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:58, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0285] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:59, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0286] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding an α-GAL enzyme comprising SEQ ID NO:60, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0287] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:35, d) polyA, and e) a 3' ITR.

[0288] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:36, d) polyA, and e) a 3' ITR.

[0289] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:37, d) polyA, and e) a 3' ITR.

[0290] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:38, d) polyA, and e) a 3' ITR.

[0291] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:39, d) polyA, and e) a 3' ITR.

[0292] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:40, d) polyA, and e) a 3' ITR.

[0293] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:41, d) polyA, and e) a 3' ITR.

[0294] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:42, d) polyA, and e) a 3' ITR.

[0295] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:43, d) polyA, and e) a 3' ITR.

[0296] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:44, d) polyA, and e) a 3' ITR.

[0297] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:45, d) polyA, and e) a 3' ITR.

[0298] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:35, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0299] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:36, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0300] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:37, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0301] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:38, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0302] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:39, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0303] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:40, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0304] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:41, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0305] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:42, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0306] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:43, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0307] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:44, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0308] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:45, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0309] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:35, d) polyA, and e) a 3' ITR.

[0310] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:36, d) polyA, and e) a 3' ITR.

[0311] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:37, d) polyA, and e) a 3' ITR.

[0312] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:38, d) polyA, and e) a 3' ITR.

[0313] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:39, d) polyA, and e) a 3' ITR.

[0314] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:40, d) polyA, and e) a 3' ITR.

[0315] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:41, d) polyA, and e) a 3' ITR.

[0316] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:42, d) polyA, and e) a 3' ITR.

[0317] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:43, d) polyA, and e) a 3' ITR.

[0318] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:44, d) polyA, and e) a 3' ITR.

[0319] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:45, d) polyA, and e) a 3' ITR.

[0320] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:35, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0321] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:36, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0322] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:37, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0323] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:38, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0324] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:39, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0325] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:40, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0326] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:41, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0327] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:42, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0328] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:43, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0329] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:44, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0330] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:45, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0331] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:14, d) polyA, and e) a 3' ITR.

[0332] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:10, d) polyA, and e) a 3' ITR.

[0333] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:64, d) polyA, and e) a 3' ITR.

[0334] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:69, d) polyA, and e) a 3' ITR.

[0335] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme encoding SEQ ID NO:14, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0336] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme encoding SEQ ID NO:10, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0337] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme encoding SEQ ID NO:64, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0338] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence encoding an α-GAL enzyme encoding SEQ ID NO:69, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0339] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:14, d) polyA, and e) a 3' ITR.

[0340] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism to muscle or liver), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO: 10, d) polyA, and e) a 3' ITR.

[0341] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:64, d) polyA, and e) a 3' ITR.

[0342] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:69, d) polyA, and e) a 3' ITR.

[0343] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism for liver or muscle), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO: 14, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0344] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism for liver or muscle), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO: 10, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0345] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism for liver or muscle), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:64, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0346] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., with tropism for liver or muscle), c) a nucleotide sequence encoding the sequence set forth in SEQ ID NO:69, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0347] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:35, d) polyA, and e) a 3' ITR.

[0348] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:36, d) polyA, and e) a 3' ITR.

[0349] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:37, d) polyA, and e) a 3' ITR.

[0350] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:38, d) polyA, and e) a 3' ITR.

[0351] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:39, d) polyA, and e) a 3' ITR.

[0352] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:40, d) polyA, and e) a 3' ITR.

[0353] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:41, d) polyA, and e) a 3' ITR.

[0354] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:42, d) polyA, and e) a 3' ITR.

[0355] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:43, d) polyA, and e) a 3' ITR.

[0356] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:44, d) polyA, and e) a 3' ITR.

[0357] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, where such a vector comprises a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:45, d) polyA, and e) a 3' ITR.

[0358] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:35, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0359] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:36, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0360] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:37, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0361] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:38, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0362] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:39, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0363] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:40, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0364] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:41, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0365] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:42, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0366] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:43, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0367] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:44, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0368] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid with broad tissue tropism, the vector comprising a) a 5' inverted repeat (ITR), b) a ubiquitous promoter, c) a nucleotide sequence comprising SEQ ID NO:45, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0369] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:35, d) polyA, and e) a 3' ITR.

[0370] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:36, d) polyA, and e) a 3' ITR.

[0371] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:37, d) polyA, and e) a 3' ITR.

[0372] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:37, d) polyA, and e) a 3' ITR.

[0373] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:38, d) polyA, and e) a 3' ITR.

[0374] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:39, d) polyA, and e) a 3' ITR.

[0375] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:40, d) polyA, and e) a 3' ITR.

[0376] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:41, d) polyA, and e) a 3' ITR.

[0377] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:42, d) polyA, and e) a 3' ITR.

[0378] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:43, d) polyA, and e) a 3' ITR.

[0379] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:44, d) polyA, and e) a 3' ITR.

[0380] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, where such vector comprises a) a 5' inverted repeat (ITR), b) a tissue-specific promoter (e.g., one with tropism to muscle or liver), c) a nucleotide sequence comprising SEQ ID NO:45, d) polyA, and e) a 3' ITR.

[0381] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:35, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0382] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:36, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0383] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:37, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0384] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:38, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0385] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:39, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0386] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:40, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0387] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:41, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0388] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:42, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0389] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:43, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0390] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:44, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0391] In some embodiments, the disclosure encompasses a recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid, the vector comprising a) a 5' inverted repeat (ITR), b) a tissue-specific promoter, c) a nucleotide sequence comprising SEQ ID NO:45, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR.

[0392] The transgene delivered by the vector can be introduced into the cells of interest using a wide variety of methods. For example, either viral or non-viral vectors can be used to deliver the transgene of interest. Both viral and non-viral methods of vector delivery are contemplated by the provided methods. Thus, in some embodiments, the vectors described herein are delivered as viral vectors. In some embodiments, the vectors described herein are delivered as non-viral vectors.

[0393] The vectors described herein can be introduced into a cell as part of a viral vector or a non-viral vector molecule having additional sequences, such as an origin of replication, a promoter, and one or more genes. In some embodiments, the vector can be introduced as naked nucleic acid, such as a nucleic acid complexed with a liposome or an agent such as a poloxamer, or can be delivered by a virus (e.g., adenovirus, adeno-associated virus (AAV), herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV). In some embodiments, the vector is introduced using a viral vector.

[0394] Various viral vectors are known in the art, and include, for example, integrating vectors and non-integrating vectors.In some embodiments, the viral vector is a non-integrating viral vector.Non-integrating viral vectors include, for example, non-integrating lentivirus vectors or AAV vectors.Thus, in some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0395] In some embodiments, the AAV vector is modified in one or more regions, such as the AAV capsid, hi some embodiments, the rAAV vector is a rAAV9 vector.

[0396] In some embodiments, the rAAV vectors described herein comprise one or more of: (a) a 5' inverted repeat (ITR); (b) a ubiquitous promoter sequence; (c) a nucleotide sequence encoding wild-type α-GAL or a variant thereof; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); (e) polyA; and (f) a 3' ITR sequence.

[0397] In various embodiments, the rAAV vectors described herein for delivering a transgene (e.g., a gene encoding an alpha-galactosidase (α-GAL) protein) can be packaged using techniques known in the art and described herein. For example, in some embodiments, rAAV packaging utilizes packaging cells to form viral particles capable of infecting host cells. Such cells include, for example, HEK293, HeLa, HEK293T, Sf9 cells, or A549 cells, which are used to package adenovirus. Viral vectors used in gene therapy are usually made by producer cell lines that package nucleic acid vectors into viral particles. The vector typically contains minimal viral sequences required for packaging and subsequent integration into the host, other viral sequences being replaced by an expression cassette encoding the protein to be expressed. In this case, the protein to be expressed is α-GAL, either wild-type or modified α-GAL. Missing viral functions can be supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have terminal inverted repeat (ITR) sequences from the AAV genome, which are required for packaging and integration into the host genome. Viral DNA is packaged in cell lines that contain helper plasmids encoding other AAV genes, i.e., rep and cap, but lack ITR sequences. The cell lines are also infected with adenovirus as a helper. The helper virus promotes the replication of the AAV vector and the expression of AAV genes from the helper plasmid. Because it lacks ITR sequences, the helper plasmid is not packaged in significant amounts. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0398] In many gene therapy applications, it is desirable for gene therapy vectors to be delivered to specific tissue types with a certain specificity. Conventional gene therapy methods for the treatment of Fabry disease have limited success due to the poor tissue tropism of previously used gene therapy vectors. Unlike previously used vector designs, the vector designs provided herein have a wide distribution in tissues and cell types after being administered to subjects in need thereof. The rAAV vectors described herein have a wide tissue distribution, including, for example, the heart, liver, kidney and gastrointestinal tract.

[0399] In some embodiments, a rAAV vector capable of achieving broad α-GAL enzyme expression upon administration to a subject in need thereof comprises (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme, (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) with a mut6delATG mutation, (e) a bovine growth hormone (BGH) polyA, and (f) a 3' ITR.

[0400] A rAAV vector capable of achieving broad α-GAL enzyme expression upon administration to a subject in need thereof contains (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta-actin promoter, and a rabbit beta-globin intron, (c) a nucleotide sequence encoding α-GAL enzyme, (d) a bovine growth hormone (BGH) polyA, and (e) a 3' ITR.

[0401] In some embodiments, a rAAV vector capable of achieving broad α-GAL enzyme expression upon administration to a subject in need thereof is packaged within an AAV9 capsid, and the rAAV vector comprises (a) a 5' inverted repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron, (c) a nucleotide sequence encoding α-GAL enzyme, (d) a bovine growth hormone (BGH) polyA, and (e) a 3' ITR.

[0402] In some embodiments, the rAAV9 vector optionally includes a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (e.g., one with a mut6delATG mutation) between the nucleotide sequence encoding the α-GAL enzyme and the polyA sequence.

[0403] Exemplary sequences of rAAV are shown below in Table 2. In some embodiments, the rAAV vector comprises a rAAV vector element comprising a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the vector element sequences shown in the table below. In some embodiments, the rAAV vector comprises a vector element nucleotide sequence that is identical to the vector element nucleotide sequence shown in the table below.

[0404] [Table 2-1]

[0405] [Table 2-2]

[0406] [Table 2-3]

[0407] In some embodiments, the present disclosure encompasses gene therapy vectors comprising modified GLA gene sequences. Such modifications may be made to improve expression characteristics. Such modifications may include, but are not limited to, the insertion of a translation initiation site (e.g., methionine), the addition of a Kozak sequence, the insertion of a signal peptide, and / or codon optimization. Thus, in some embodiments, the GLA gene is modified to include the insertion of a translation initiation site. In some embodiments, the GLA gene is modified to include the addition of a Kozak sequence. In some embodiments, the GLA gene is modified to include a signal peptide. In some embodiments, the GLA gene is codon optimized. In other embodiments, the GLA gene is engineered. In yet other embodiments, the GLA gene is codon optimized and engineered.

[0408] In some embodiments, the vector comprises an ID tag, e.g., a stuffer sequence. The purpose of the ID tag includes, for example, allowing a technician to identify the vector. In certain embodiments, the vector comprises a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE) element. In some embodiments, the vector comprises a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE). Various optimized or variant forms of WPRE are known in the art, including WPRE3, WPREmut6delATG, among others. Other variant WPRE forms include, for example, WPRE2, WPRE_wt (GenBank Accession No. J04514), WPRE_wt (GenBank Accession No. J02442), and WPREmut6. The WPRE element can comprise a wild-type sequence or a modified WPRE element sequence. Various mutant forms of WPRE are known, including, for example, mut6delATG (SEQ ID NO: 4). In some embodiments, the vector comprises mut6delATG (SEQ ID NO: 4).

[0409] The vectors described herein include one or more promoter sequences. In some embodiments, the promoter sequence is a ubiquitous promoter sequence. Any suitable promoter region or promoter sequence can be used, so long as the promoter region promotes the expression of a coding sequence in a mammalian cell. In certain embodiments, the promoter region promotes the expression of a coding sequence, such as GLA, in a mammalian cell. In some embodiments, the promoter controlling the expression of the GLA transgene is a ubiquitous promoter. In some embodiments, the ubiquitous promoter is selected from one or more of the following: GAPDH promoter, mini-EF1 promoter, CMV promoter EF-1α promoter, PGK promoter, UBC promoter, LSE beta-glucuronidase (GUSB) promoter, or ubiquitous chromatin opening element (UCOE) and / or chicken beta actin promoter. In some embodiments, the ubiquitous promoter includes a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a ubiquitous promoter including a rabbit beta globin intron.

[0410] In some embodiments, the vectors described herein comprise one or more polyA sequences, hi some embodiments, the polyA is selected from human growth hormone polyA (hGHpA), synthetic polyA (SPA), Simian Virus 40 late polyA (SV40pA), and bovine growth hormone (BGH) polyA.

[0411] In some embodiments, the present disclosure provides an expression cassette comprising a polynucleotide sequence comprising: (a) a 5' inverted repeat (ITR); (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron; (c) a nucleotide sequence encoding an α-GAL enzyme; (d) optionally a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising a mut6delATG mutation; (e) a bovine growth hormone (BGH) polyA; and (f) a 3' ITR. In some embodiments, the elements of the expression cassette are in 5' to 3' order. In various embodiments, one or more of (a) to (f) are operably linked in 5' to 3' order.

[0412] In some embodiments, the vector is introduced into a cell. Thus, in some embodiments, a cell is provided, said cell comprising a vector described herein. In some embodiments, the cell is in vitro, in situ, or in vivo. Thus, in some embodiments, the cell comprising a vector described herein is in vitro. In some embodiments, the cell comprising a vector described herein is in situ. In some embodiments, the cell comprising a vector described herein is in vivo.

[0413] Pharmaceutical Compositions Exemplary pharmaceutical compositions containing the vectors described herein are detailed below.

[0414] Pharmaceutically acceptable carriers are determined, in part, by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions available.

[0415] Formulations for both ex vivo and in vivo administration include suspensions or emulsions. Active ingredients are often mixed with excipients that are pharma- ceutically acceptable and compatible with active ingredients. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, or other agents that enhance the effectiveness of pharmaceutical compositions.

[0416] Treatment methods The vector of the present disclosure can be used to treat subjects with Fabry disease. Thus, the vector of the present disclosure can be used to treat subjects with Fabry disease, thereby alleviating one or more symptoms associated with the disease. In some embodiments, the vector of the present disclosure can be used to treat subjects with low or no expression of α-GAL.

[0417] Non-limiting examples of symptoms of Fabry disease include neuropathic pain, hypohidrosis or anhidrosis, exercise intolerance, abdominal cramps, diarrhea, angiokeratoma, verticillata, tinnitus, proteinuria, chronic kidney disease, hypertension, coronary insufficiency, atrioventricular conduction disorders, arrhythmias and valvular dysfunction, left ventricular hypertrophy, seizures, and stroke.

[0418] In some embodiments, the vectors provided herein are used as prophylactic treatments in subjects with Fabry disease. Prophylactic treatments can be administered, for example, to subjects who have not yet developed the disease but are susceptible to or otherwise at risk for a particular biological condition, including Fabry disease (e.g., subjects may have a mutation that causes Fabry disease, but are asymptomatic or have an unknown status of the mutation that causes Fabry disease). In some embodiments, therapeutic treatments can be administered, for example, to subjects already suffering from Fabry disease, to improve or stabilize the subject's condition (e.g., patients who are already experiencing symptoms of Fabry disease).

[0419] In some embodiments, the rAAV vector remains episomal after administration to a subject in need thereof. In some embodiments, the rAAV vector does not remain episomal after administration to a subject in need thereof. For example, in some embodiments, the rAAV vector is integrated into the genome of the subject. Such integration can be achieved by using various gene editing techniques, such as, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), ARCUS genome editing, and / or CRISPR-Cas systems.

[0420] In some embodiments, pharmaceutical compositions comprising the rAAV vectors described herein are used to treat subjects in need thereof. Pharmaceutical compositions containing the rAAV vectors or particles of the present invention contain pharma- ceutical acceptable excipients, diluents or carriers. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to subjects in therapeutically effective amounts.

[0421] The rAAV vector is administered to a subject in need thereof by a suitable route. In some embodiments, the rAAV vector is administered intravenously, intraperitoneally, subcutaneously, or intradermally. In some embodiments, the rAAV vector is administered intravenously. In some embodiments, intradermal administration includes administration by using a "gene gun" or gene gun particle delivery system. In some embodiments, the rAAV vector is administered by a non-viral lipid nanoparticle. For example, a composition comprising a rAAV vector may include one or more diluents, buffers, liposomes, lipids, lipid complexes. In some embodiments, the rAAV vector is contained within a microsphere or nanoparticle, such as a lipid nanoparticle.

[0422] In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 2-15 weeks after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 2 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 3 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 4 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 5 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 6 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 7 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 8 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 9 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about 10 weeks. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about week 11. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about week 12. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about week 13. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about week 14. In some embodiments, functional α-GAL is detectable in the subject's plasma or serum at about week 15. In some embodiments, functional α-GAL is detectable in the subject's hepatocytes at about 2-15 weeks after administration of the rAAV vector.

[0423] In some embodiments, functional α-GAL is detectable in the plasma of a subject for at least 3 months, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, or 20 years after administration of the rAAV vector. Thus, in some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 3 months after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 6 months after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 12 months after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 2 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 3 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 4 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 5 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 6 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 7 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 8 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 9 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for at least 10 years after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the plasma or serum of a subject for the remainder of the subject's life after administration of the rAAV vector.

[0424] In some embodiments, administered rAAV containing GLA results in the production of active α-GAL comparable to that seen after administration of purified GLA protein delivered intravenously. In some embodiments, administered rAAV containing GLA results in the production of greater amounts of active α-GAL compared to administration of purified α-GAL protein delivered intravenously.

[0425] In some embodiments, the administered rAAV containing GLA results in a reduction in globotriaosylceramide (GB3) in the subject. In some embodiments, the administered rAAV containing GLA reduces the subject's GB3 by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or about 10% compared to the subject's baseline GB3 amount before administering the rAAV containing GLA. Thus, in some embodiments, the administered rAAV containing GLA reduces the subject's GB3 by about 95%. In some embodiments, the administered rAAV containing GLA reduces the subject's GB3 by about 90%. In some embodiments, the administered rAAV containing GLA reduces the subject's GB3 by about 85%. In some embodiments, the administered rAAV containing GLA reduces the subject's GB3 by about 80%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 75%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 70%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 65%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 60%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 55%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 50%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 45%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 40%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 35%. In some embodiments, an administered rAAV comprising GLA reduces the subject's GB3 by about 30%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 25%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 20%.In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 15%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 10%.

[0426] In some embodiments, the administered rAAV containing GLA reduces GB3 in a subject for at least about 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years.

[0427] In some embodiments, after administration of an AAV vector to a subject, the amount of functional α-GAL detectable in the circulating blood is about 2 to 100 times, or more than 10 times, more than 20 times, more than 30 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, more than 95 times, or more than 100 times or more greater than the amount of functional α-GAL detectable in the subject before administration of an rAAV containing a GLA transgene.

[0428] In some embodiments, after administration of the AAV vector to a subject, the amount of detectable active α-GAL meets or exceeds the human therapeutic concentration, i.e., the concentration of α-GAL considered to be the normal circulating concentration in humans (e.g., 5-9 nmol / hr / ml). In some embodiments, the concentration of active α-GAL after administration of the rAAV vector is about 2-35 times or more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 55 times, more than 60 times, more than 65 times, more than 70 times, more than 75 times, more than 80 times, more than 85 times, more than 90 times, more than 95 times, or more than 100 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 2 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 3 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 4 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 5 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 6 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 7 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 8 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 9 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 10 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 11 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 15 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 20 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 25 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 30-fold higher than the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 35-fold higher than the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 40-fold higher than the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 45-fold higher than the human therapeutic concentration.In some embodiments, the concentration of active α-GAL after administration is about 50 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 55 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 60 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 65 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 70 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 75 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 80 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 85 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 90 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 95 times the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 100-fold higher than the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 200-fold higher than the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 300-fold higher than the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about 400-fold higher than the human therapeutic concentration. In some embodiments, the concentration of active α-GAL after administration is about or more than 500-fold higher than the human therapeutic concentration.

[0429] Thus, administration of a rAAV vector containing the GLA transgene results in robust and sustained expression compared to a single administration of purified α-GAL to a subject in need.

[0430] In some embodiments, the rAAV vector containing GLA transgene is delivered as a single dose per subject.In some embodiments, the subject is delivered with the minimum effective dose (MED).As used herein, MED refers to the dose of rAAV GLA vector that is required to achieve the α-GAL activity that leads to GB3 reduction in subject.

[0431] Vector titers are determined based on the DNA content of the vector preparation. In some embodiments, quantitative PCR or optimized quantitative PCR is used to determine the DNA content of the rAAV GLA vector preparation. In one embodiment, the dosage is about 1×10, inclusive. 11 Genome copies (GC) / kg (body weight) ~ approx. 1 x 10 13 It is GC / kg.

[0432] The dosage required to achieve therapeutic benefit in a subject in need thereof is lower than that achieved using other types of rAAV capsids, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and / or AAV8, which contain a liver-specific promoter. In some embodiments, the rAAV gene therapy vectors expressing alpha-galactosidase protein described herein are administered to a subject at a lower or equivalent dosage than that used for gene therapy vectors containing a liver-specific promoter, yet surprisingly exhibit higher serum and tissue exposure.

[0433] In some embodiments, the rAAV GLA vector composition is administered at about 1.0×10 9 GC~approx. 1.0×10 15 It can be formulated in a dosage unit to contain a replication-deficient virus amount in the range of GC. As used herein, the term "dosage" can refer to the total dosage delivered to a subject during the course of treatment, or the amount delivered in a single (multiple) administration.

[0434] In some embodiments, the dosage is sufficient to reduce plasma GB3 levels in the patient by 25% or more. In some embodiments, the rAAV expressing α-GAL is administered in combination with one or more therapeutic agents for the treatment of Fabry disease.

[0435] Combination therapy The compositions and methods of the invention can be used in combination with other therapeutic agents known in the art to be used to treat Fabry disease or its complications, including, but not limited to, ERT (e.g., agalsidase beta), pain relief medications (e.g., lidocaine, diphenylhydantoin, carbamazepine, gabapentin, phenytoin, neurotropin, opioids), indigestion medications (e.g., metoclopramide, H2 blockers), vitamin D supplementation, etc., beta blockers (metoprolol, acebutolol, bisoprolol, atenolol, propranolol, etc.), anticoagulant medications (heparin, warfarin, apixaban, rivaroxaban).

[0436] The compositions and methods of the present invention can be used in conjunction with other forms of treatment, including, but not limited to, physical exercise (e.g., dialysis, kidney transplantation), salt restriction in the diet, intake of dietary fiber, placement of a pacemaker, and heart transplantation.

[0437] Production of rAAV viral vectors Methods for making and isolating AAV viral vectors suitable for delivery to subjects are known in the art.See, for example, US Patent No. 7,790,449, US Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7,588,772B2.In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct(s) encoding rep and cap.In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs.In each of these systems, AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring separation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to recover AAV (i.e., El, E2a, VA, and E4 of adenovirus or UL5, UL8, UL52, and UL29 of herpesvirus, and herpesvirus polymerase are also supplied in trans by the system). In these new systems, helper functions can be supplied by transient transfection of cells with constructs encoding the necessary helper functions, or cells can be engineered to stably contain genes encoding helper functions whose expression can be controlled at the transcriptional or post-transcriptional level.

[0438] In some embodiments, the expression cassette and rep / cap genes flanked by ITRs are introduced into a desired cell or cell line by infection with a baculovirus-based vector.

[0439] In some embodiments, the expression cassette and rep / cap genes flanked by ITRs are introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems, see, e.g., the summary in Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of which are incorporated herein by reference in their entirety. Methods of making and using these and other AAV production systems are also described in U.S. Patent Nos. 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065, the contents of each of which are incorporated herein by reference in their entirety. For general overviews, see, e.g., Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al, 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med 10:717-733, each of which is incorporated herein by reference in its entirety, and the references cited below.

[0440] The methods used to construct the vectors described herein are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known and the selection of a suitable method is not a limitation of the present invention. See, for example, K. Fisher et al, (1993) J. Virol, 70:520-532 and U.S. Patent No. 5,478,745.

[0441] Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill in the art can readily construct any number of other plasmids suitable for use in the present invention. The properties, construction and use of such plasmids, and other vectors, in the present invention will be readily apparent to those of skill in the art from the present disclosure.

[0442] In one embodiment, the production plasmid is one described herein or in WO2012 / 158757, which is incorporated herein by reference. A variety of plasmids are known in the art for use in producing rAAV vectors and are useful herein. The production plasmid is cultured in a host cell that expresses the cap and / or rep proteins of AAV. In the host cell, each rAAV genome is rescued and packaged into a capsid or envelope protein to form an infectious viral particle.

[0443] In certain embodiments, the rAAV expression cassette, vector (such as an rAAV vector), virus (such as an rAAV), or production plasmid comprises an AAV terminal inverted repeat sequence, a codon-optimized nucleic acid sequence encoding an α-GAL polypeptide, and an expression control sequence directing expression of the encoded protein, and is present in a host cell. In other embodiments, the rAAV expression cassette, virus, vector (such as an rAAV vector), or production plasmid further comprises one or more of an intron, a Kozak sequence, a polyA, a post-transcriptional regulatory element, and others. In one embodiment, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus (WHP) Post-transcriptional Regulatory Element (WPRE). In various embodiments, the nucleic acid sequence comprises a signal peptide upstream of the transgene encoding the α-GAL polypeptide. In some embodiments, the signal peptide is at the N-terminus of the α-GAL polypeptide. In some embodiments, the signal peptide is at the C-terminus of the α-GAL polypeptide.

[0444] Various methods are known in the art related to AAV vector production and purification. See, for example, Mizukami, Hiroaki, et al. A Protocol for AAV vector production and purification, US Patent Publication Nos. US20070015238 and US20120322861. For example, a plasmid containing a gene of interest is combined with one or more helper plasmids, such as a helper plasmid containing a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2 and VP3, including the modified VP2 region described herein), and transfected into a recombinant cell so that the rAAV can be packaged and subsequently purified.

[0445] In some embodiments, packaging is performed in a helper or producer cell, such as a mammalian cell or an insect cell. Exemplary mammalian cells include, but are not limited to, HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, e.g., ATCC® CRL-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-10™, or ATCC® CCL-61™). Exemplary insect cells include, but are not limited to, Sf9 cells (see, e.g., ATCC® CRL-1711™). Helper cells may contain rep and / or cap genes encoding Rep and / or Cap proteins for use in the methods described herein. In some embodiments, packaging is performed in vitro.

[0446] In some embodiments, the plasmid containing or comprising the gene of interest is combined with one or more helper plasmids, e.g., a helper plasmid containing the rep gene of a first serotype and a cap gene of the same serotype or a different serotype, and transfected into a helper cell such that the rAAV is packaged.

[0447] In some embodiments, the one or more helper plasmids include a first helper plasmid that includes the rep and cap genes, and a second helper plasmid that includes one or more of the helper genes Ela, Elb, E4, E2a, and VA. For clarity, the helper genes are genes that encode the helper proteins Ela, Elb, E4, E2a, and VA. In some embodiments, the cap gene is modified such that one or more of the proteins VP1, VP2, and VP3 are not expressed. In some embodiments, the cap gene is modified such that VP2 is not expressed. Methods for making such modifications are known in the art (Lux et al. (2005), J Virology, 79:11776-87).

[0448] Helper plasmids and methods for making such plasmids are generally known in the art and are generally commercially available (e.g., pDF6, pRep, pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA, Cellbiolabs, San Diego, CA, Agilent Technologies, Santa Clara, Ca, and Addgene, Cambridge, MA; pxx6, Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kem, A. et al. al.(2003),Identification of a Heparin-Binding Motif on Adeno- Associated Virus Type 2 Capsids,Journal of Virology,Vol.77,11072-11081.,Grimm et al.(2003),Helper Virus-Free,Optically Controllable,and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850, see Kronenberg et al. (2005). EXAMPLES

[0449] Exemplary features, objects, and advantages of the present invention will become apparent in the following examples. It should be understood, however, that the examples, while illustrating embodiments of the present invention, are provided by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0450] The data in the following examples was generated using a GAL transgene containing the signal peptide sequence of SEQ ID NO:77, encoding an α-GAL enzyme containing the signal peptide sequence of SEQ ID NO:76.

[0451] Example 1. Design and purification of a viral vector for expression of α-GAL enzyme This example summarizes exemplary viral vector designs encompassed by this disclosure.

[0452] Recombinant adeno-associated virus 9 (rAAV9) was generated to express wild-type human α-GAL or α-GAL variants (e.g., amino acid sequences shown in Table 1) under ubiquitous promoter control in a viral vector. A WPRE element was linked to the 3' end of the wild-type GLA transgene to increase transgene expression and improve mRNA stability. A bovine growth hormone polyA tail was added to the 3' end of the WPRE element. A DNA construct of promoter-GLA-WPRE-BGHpA was incorporated between the terminal inverted repeats of a circular plasmid vector. Figure 1 shows an exemplary rAAV9 vector construct.

[0453] Using methods known in the art, the terminal inverted repeats and rep sequences of AAV2 were used to package the rAAV vector. rAAV9 stocks were produced using the adenovirus-free triple-plasmid co-transfection method using HEK-293T cells and purified using ultracentrifugation over cesium chloride. The titer of vg particle number was determined by quantitative PCR.

[0454] The purified rAAV9 virus suspension was diluted in a formulation buffer consisting of 1.5 mM KH2PO4 (potassium dihydrogen phosphate), 2.7 mM KCl (potassium chloride), 8.1 mM Na2HPO4 (disodium hydrogen phosphate), 136.9 mM NaCl (sodium chloride) and 0.001% Pluronic F-68. A null vector carrying the rAAV9 capsid (rAAV9-null) was used as a control.

[0455] Example 2. Serum stability of α-GAL after rAAV9 administration In this example, we show that the GLA transgene delivered by rAAV9 resulted in serum α-GAL protein expression for at least 12 weeks after administration of the vector to mice.

[0456] First, rAAV9 vectors were produced and purified as described above in Example 1. Then, 6- to 9-month-old male GLAko mice were injected with 2.5 × 10 purified rAAV9 vectors encoding α-GAL. 11 vg / kg, 6.25×10 12 As a negative control, a group of GLAko mice were administered 6.25 × 10 12 The animals were dosed at 1 mg / kg / day. Serum was collected at multiple time points during the study and at the end of the 12 week period. A portion of the animals received a 1 mg / kg dose of α-GAL ERT 24 hours prior to sacrifice and collection of tissues to be used as positive controls. Serum was collected 1 hour after administration of α-GAL ERT and circulating enzyme C was assayed. max concentration was obtained.

[0457] The amount of α-GAL in serum was measured using ELISA. Briefly, high-binding MSD black plates (MSD, Cat. No. L15XB) were coated overnight at 4°C with polyclonal sheep anti-human α-GLA capture antibody (R&D systems, Cat. No. AF6146) in 0.2M sodium carbonate-bicarbonate buffer (Thermoscientific, Cat. No. 28382). The coated plates were then washed three times with D'PBS and wash buffer containing 0.05% Tween-20. The plates were blocked for 1 hour with blocking buffer (PBS containing 3% BSA) before adding samples or purified α-GLA protein standards in dilution buffer (PBS containing 1% BSA). Binding was allowed for 1 hour with slow shaking and washed three times with wash buffer. Polyclonal rabbit anti-human α-GLA (Novus Biologics, Cat. No. H00002717-D01P) in dilution buffer was then added and incubated for 1 h, followed by washing 3 times with wash buffer, and detection antibody, sulfo-tag goat anti-rabbit antibody (MSD, Cat. No. R32AB-1), was added and left at room temperature for 1 h. Plates were read on an MSD Sector Imager S600 system with 1× reading buffer (MSD, Cat. No. R92TC-1). A standard curve was used to calculate the final α-GAL concentration. Tissue α-GAL protein concentrations were normalized by total protein concentration determined by BCA assay. None of the antibodies used in this assay recognized mouse α-GAL protein. A rapid increase in α-GAL was observed in serum 2 weeks after vector administration, reaching more than 4500-fold the normal value in a similar high-dose group of rAAV9-WT at 12 weeks. Even at the lowest dose, rAAV9-WT showed no significant difference in C of the positive control α-GAL ERT. max It was observed that the results of the study yielded a higher constant concentration of serum α-GAL enzyme than that of the control (Figure 2).

[0458] At 12 weeks post-treatment, multiple organs were harvested for further evaluation, including liver, kidney, heart, and gastrointestinal tract. For histological evaluation, fixation in 10% NBF was performed. For physiological evaluation, tissues were snap frozen and stored at -80°C.

[0459] Dose-dependent sustained supraphysiological exposure of α-GAL was observed in peripheral organs such as liver (Figure 3A), kidney (Figure 3B), heart (Figure 3C) and gastrointestinal tract (Figures 3D and 3E), which was accompanied by reduced accumulation of GB3 and lysoGb3 in these animals (Figures 3F-3M). It was further observed that higher doses of rAAV9-WT reduced GB3 and lysoGb3 concentrations by more than 95% in serum and kidney (Figures 3F, 3H, 3J, 3L) and more than 99% in liver and heart (Figures 3G, 3I, 3K, 3M). Similarly, lysoGb3 concentrations in all tissues tested were reduced by more than 99% after high-dose rAAV9-WT injection. In contrast, as can be seen in Figures 3F-3M, mice administered α-GAL protein had a moderate reduction in substrate concentrations in serum and liver, but not in kidney and heart.

[0460] Overall, we observed that treatment with rAAV9-WT encoding α-GAL improved the clearance of GB3 and lysoGb3 in various tissues, and expression of α-GAL was sustained for at least 12 weeks.

[0461] Example 3. Effect of rAAV9-WT treatment of G3Stg / GLAko mice. This example shows the in vivo effect of rAAV9-WT encoding α-GAL expression in various tissues of a mouse model of severe Fabry disease, in which the substrate concentration in various tissues is significantly higher than in GLAko mice. The data from this example demonstrate that the GLA transgene delivered by rAAV9-WT reduces the accumulation of GB3 associated with Fabry disease.

[0462] First, the rAAV9-WT vector was produced and purified as described in Example 1 above.

[0463] G3Stg / GLA knockout mice were then generated by crossing GLA knockout mice with a C57BL / 6 background with GB3 synthase transgenic mice. Established mice were purchased from Jackson lab and breeding colonies were maintained at Taconic Biosciences. G3Stg / GLA knockout mice have significant deposition of GB3 substrates in the viscera, resulting in severe renal, GI and neurological phenotypes such as albuminuria, reduced renal osmolality, delayed colonic propulsive motility and loss of thermosensitivity, reflecting some symptoms of Fabry disease (Taguchi et.al.,2013). C57BL / 6 mice (Charles River Laboratories) were used as wild type controls in this experiment. Animals were maintained in a controlled environment at Melior Discoveries with no more than four mice per cage in a ventilated cage rack system on a 12-hour dark / 12-hour light cycle (lights on at 7:00 am) and fed standard rodent chow and water ad libitum.

[0464] Treatment with rAAV9-WT caused a sustained dose-dependent increase in both serum α-GAL concentration and α-GAL activity (Figure 4A). At the highest dose of 6.24e12vg / kg, serum α-GAL activity was 10,000-fold higher than WT serum α-GAL activity. Dose-dependent increases in tissue α-GAL concentration and activity were observed in all tissues examined, including liver (Figure 4B), kidney (Figure 4C), heart (Figure 4D), gastrointestinal tract (duodenum Figure 4E and colon Figure 4F), and brain (Figure 4G).

[0465] We next analyzed the amount of GB3 in severe Fabry (G3Stg / GLAko) mice transfected with rAAV9-WT encoding α-GAL using mass spectrometry. Briefly, substrate samples were first extracted using chloroform:methanol (v / v 2:1) and formic acid before performing HPLC and LC-MS / MS (Applied Biosystem API5000, Turbo Ion Spray Ionization, positive ion mode). The concentrations of GB3 and lyso-Gb3 in various tissues from these animals were observed to show a dose-dependent decrease in accumulated substrate concentration in each tissue (Figure 5A-5L).

[0466] We observed that rAAV9-WT expressed α-GAL in Fabry disease mice and reduced the accumulation of GB3 associated with Fabry disease.

[0467] Example 4. Phenotypic effect of rAAV9 on a mouse model of Fabry disease (G3Stg / GLAko mice) This example shows the phenotypic effect of rAAV9-WT encoding α-GAL on G3Stg / GLAko mice. Fabry disease mice treated with rAAV9-WT showed improved overall body weight over time.

[0468] First, the rAAV9-WT vector was produced and purified as described in Example 1 above.

[0469] G3Stg / GLAko mice were then treated with rAAV9 encoding the α-GAL enzyme as in the mice described in Example 3. Body weight was monitored throughout the study.

[0470] G3Stg / GLAko mice showed a significant weight loss over the study period, with mice treated with rAAV9-WT encoding the α-GAL enzyme weighing more compared to mice administered the null AAV vector (Figure 6). WT mice showed a constant weight gain over the study period, but G3Stg / GLAko mice treated with null vector and low doses of rAAV9-WT began to lose weight from 16-18 weeks of age onwards, with animals in the null vector group finishing the study with lower weights than at the start of the study (22.2 ± 0.5 g / mouse at 28 weeks vs. 27.0 ± 0.6 g / mouse at start, p<0.0001). Treatment with the highest dose of rAAV9-WT showed significantly less weight loss at the end (28.5 ± 0.8 g / mouse vs. 22.2 ± 0.5 g / null AAV mouse, p<0.01). Treatment with high doses of rAAV9-WT thus prevented weight loss, demonstrating the benefit of this gene therapy candidate on overall health.

[0471] The results of this example demonstrated that rAAV9-WT encoding the α-GAL enzyme can be used to induce significant improvement in treating the symptoms of Fabry disease.

[0472] Example 5. Restoration of renal function defects by rAAV9 in a Fabry disease model (G3Stg / GLAko mice) In this example, the effect of rAAV9-WT encoding α-GAL enzyme on renal function in G3Stg / GLAko mice was examined.

[0473] First, the rAAV9-WT vector was produced and purified as described in Example 1 above.

[0474] G3Stg / GLAko mice were then treated with rAAV9 as in the mice described in Example 3. Renal function was assessed by measuring the concentrations of markers such as blood urea nitrogen and urinary albumin.

[0475] rAAV9-WT encoding the α-GAL enzyme caused a significant decrease in serum BUN in mice treated with the highest dose of the gene therapy drug (Figure 7A). It was observed that serum albumin concentrations in Fabry mice treated with the highest dose of the gene therapy drug (GT) were similar to those in WT mice (Figure 7B).

[0476] This example demonstrates that rAAV9-WT encoding the α-GAL enzyme rescues the renal phenotype associated with Fabry disease, specifically, restoring BUN and urinary albumin concentrations.

[0477] Example 6. Reversal of neuropathy associated with Fabry disease in a mouse model (G3Stg / GLAko mice) In this example, the effect of rAAV9-WT encoding α-GAL enzyme on neuropathy markers in a mouse model of Fabry disease was examined.

[0478] First, the rAAV9-WT vector was produced and purified as described in Example 1 above.

[0479] G3Stg / GLAko mice were then treated with rAAV9 as in the mice described in Example 3. G3Stg / GLAko mice show some signs of neuropathy as observed in histology of peripheral neurons after sacrifice. Footpads from the hind limbs and dorsal root ganglia from these animals were harvested for analysis by immunohistochemistry to assess small fiber neuron density to monitor overall neuropathology in these animals. In animals treated with the highest dose of GT, there was a marked reduction in dorsal root nerve vacuolation, restored to WT levels (Figure 8A). Finally, there was also a dose-dependent increase in PGP9.5 (a neuronal marker) and MPZ (a marker of myelinated nerves) in the paws of treated animals (Figures 8B and 8C).

[0480] This example demonstrates that α-GLA enzyme produced by rAAV9-WT is associated with improved neuropathy as assessed by marker expression in a mouse model of Fabry disease.

[0481] Example 7. Normalization of autophagy dysregulation in a mouse model of Fabry disease (G3Stg / GLAko mice) In this example, we investigated the effect of rAAV9-WT encoding the α-GAL enzyme on autophagy dysregulation in a mouse model of Fabry disease.

[0482] Dysregulation of autophagy has been reported in Fabry patients (Chevrier et al,Autophagy.2010 Jul;6(5):589-99.) and may play an important role in Fabry neuropathy. The protein p62 is a classical receptor for autophagy and accumulates in kidneys and fibroblasts of Fabry patients, and similar accumulation was observed in kidney, heart, and smooth muscle of G3Stg / GLAko mice. Treatment with rAAV9-WT completely eliminated p62 accumulation from kidney and heart when administered at a dose of 6.25e12vg / kg (Figure 9A-F).

[0483] Furthermore, chronic inflammation in Fabry patients contributes to organ damage (Pinto et al, High Blood Press Cardiovasc Prev. 2020) and is likely responsible for the increased DRG mass. Treatment of G3Stg / GLAko mice with rAAV9-WT abolishes inflammation in the DRG, as demonstrated by a reduction in the macrophage marker CD68 by histological evaluation (Figure 9G-I).

[0484] Example 8. A ubiquitous transduction approach using rAAV9 administration results in high circulating α-GAL concentrations, high exposure of α-GAL in target tissues, and significantly reduced substrates compared to a liver-directed rAAV8 approach In this example, we show that the GLA transgene delivered by the rAAV9-ubiquitous promoter resulted in higher α-GAL protein expression in serum for at least 12 weeks than GLA delivered by the rAAV8-liver-specific promoter.

[0485] First, rAAV9-WT and rAAV8-WT encoding α-GAL were produced and purified as described in Example 1 above. Then, 2.5 × 10 purified rAAV9 or rAAV8 were inoculated into 6- to 9-month-old male GLAko mice. 11 Mice were followed for 12 weeks after treatment with α-GAL ERT administered intravenously (IV) at a dose of 1 mg / kg. Serum was collected at multiple time points during the study and at the end of the 12 weeks. A subset of animals received a 1 mg / kg dose of α-GAL ERT 24 hours prior to sacrifice and collection of tissues to be used as positive controls. Serum was collected 1 hour after administration of α-GAL ERT and circulating enzyme C was assayed. max concentration was obtained.

[0486] The amount of α-GAL in serum was measured using ELISA as described in Example 2. The C of rAAV9-WT was significantly higher than that of rAAV8-WT and the positive control α-GAL ERT. max It was observed that the α-GAL enzyme concentration was higher than that of the control (FIG. 11A).

[0487] Twelve weeks after dosing, kidneys were harvested for further evaluation.

[0488] Significantly higher levels of α-GAL exposure compared to rAAV8-WT or α-GAL were observed in the kidneys of animals treated with rAAV9-WT (FIG. 11B), which was accompanied by reduced accumulation of GB3 in these animals (FIG. 11C). Furthermore, it was observed that treatment with rAAV9 reduced GB3 by more than 86%, whereas treatment with rAAV8 reduced GB3 by 78%, and treatment with a single dose of α-GAL at 1 mg / kg did not result in any reduction in GB3 in the kidneys.

[0489] Thus, overall, we observed that treatment with rAAV9, which has a ubiquitous promoter driving GLA expression, reduced GB3 in the kidney more efficiently than liver-targeted rAAV8 and resulted in higher sustained α-GAL activity in serum for at least 12 weeks.

[0490] Without wishing to be bound by theory, it is contemplated that a liver-specific promoter may also be used in conjunction with an otherwise modified / codon-optimized GLA transgene to express the enzyme at higher levels.

[0491] Example 9. Administration of rAAV9 with a ubiquitous promoter to drive GLA expression resulted in dose-dependent restoration of renal function, whereas liver-targeted rAAV8 to drive GLA expression improved renal function only at high doses In this example, the effects of rAAV9 and rAAV8 on renal function in G3Stg / GLAko mice were examined.

[0492] First, rAAV8 and rAAV9 were produced and purified as described in Example 1 above.

[0493] G3Stg / GLAko mice were then treated with rAAV8 and rAAV9 as in mice described in Example 3. Wild-type animals were treated with vehicle as a control. Renal function was assessed by measuring blood urea nitrogen.

[0494] We observed that rAAV9-WT caused a dose-dependent decrease in serum BUN, restoring it to levels close to those of healthy wild-type animals at the highest dose of 6.25e12vg / kg (Figure 12A). Treatment of animals with liver-targeted rAAV8-WT did not result in improvement in serum BUN at a dose of 2.5e11vg / kg, and only responded at a 25-fold higher dose of 6.25e12vg / kg (Figure 12B).

[0495] This example demonstrates that rAAV9, which drives GLA expression through a ubiquitous promoter, rescues the renal phenotype associated with Fabry disease more efficiently than liver-targeted rAAV8.

[0496] Example 10. High exposure of α-GAL variants compared to wild-type α-GAL in various tissues following administration of plasmids expressing these variants via tail vein hydrodynamic injection. This example shows the serum stability and tissue biodistribution of various α-GAL variants in vivo.

[0497] Plasmids expressing either wild-type or engineered human alpha-galactosidase (α-α-GAL) under the control of a ubiquitous promoter were tested in a mouse model of Fabry disease. In the first study, the following plasmids were tested: WT expressing wild-type α-GAL, and A, B, C, D, E, F expressing engineered α-GAL proteins. In the second study, the WT plasmid expressing the WT α-GAL protein, and the 002, 003, 004, 005, 006, and 007 plasmids expressing engineered α-GAL variants were tested.

[0498] In the first study, 12-14 week old male GLAko mice were each administered 50ug of plasmid DNA by hydrodynamic gene delivery via tail vein injection. One group was included in the study where GLAko mice were injected with buffer only as a negative control. Another group was included in the study where WT animals were treated with buffer. Animals were sacrificed 2 days after injection. Serum was collected by cardiac puncture at the terminal endpoint and tissues such as heart and kidney were collected after perfusion with PBS. Samples were snap frozen and stored at -80°C. Serum and tissue samples were analyzed for α-GAL activity.

[0499] The second study used male mice aged 10-12 weeks. The same study design was followed as in the first study. An additional group was included in the study: GLAko mice were injected with recombinant human α-GAL protein at a dose of 1 mg / kg as a positive control.

[0500] Tissues were homogenized in lysis buffer containing 10 mM HEPES and EDTA-free 1.5x Halt protease inhibitor cocktail supplemented with 0.5% Triton-X 100, centrifuged, and the supernatant was collected for analytical assays. Alpha-galactosidase activity in the supernatant or serum was measured using a fluorescent substrate. Briefly, 2 ul of biological sample was incubated with 15 uL of 4-MU-a-GLA substrate solution (Research Products International Company, Catalog No. M65400) containing a-galactosidase B inhibitor (N-acetyl-D-galactosamine, Sigma Catalog No. A-2795) for 60 min at 37°C. The enzymatic reaction is stopped by the addition of 200 uL of glycine carbonate stop solution (pH 10.7). 4-MU product was measured by a fluorescence microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The concentration of 4-MU in the test samples was calculated from a 4-MU standard curve on the same plate. Tissue activity was normalized based on total protein concentration determined by BCA assay.

[0501] In the first study, we observed that mice injected with plasmids A-F had significantly higher levels of α-GAL activity in the circulation as well as in the heart and kidney compared to WT (Figure 10A-C). Plasmid A expresses wild-type human α-GAL protein, whereas the other plasmids express α-GAL variants engineered for improved serum stability and tissue uptake, which was reflected in these results. Plasmid D produced the highest α-GAL activity in both serum and tissue.

[0502] In a second study, mice injected with plasmids 002-007 (expressing engineered α-α-GAL) had significantly higher levels of α-GAL activity in the circulation as well as in the heart and kidney compared with 001 (expressing WT α-GAL) (Figures S10D-S10F). In this study, variant 004 produced the highest serum and tissue α-GAL activity.

[0503] This example demonstrates that plasmids containing various variant α-GAL transgenes express the enzyme with significantly increased serum stability and tissue biodistribution compared to plasmids containing wild-type GLA.

[0504] Example 11. Comparison of vectors expressing engineered α-α-GAL with vectors expressing WT α-GAL In this example, we examined enzyme activity in serum and tissues following administration of viral vectors encoding engineered α-α-GAL and WT α-α-GAL.

[0505] First, four variants of rAAV9 vectors (referred to herein as rAAV9-WT, rAAV9-A, rAAV9-005, and rAAV9-D) were produced and purified as described in Example 1 above.

[0506] Research 1 In Study 1, 12-13 week-old G3Stg / GLAko male mice were administered rAAV9-A, rAAV9-005, rAAV9-D, and rAAV9-WT at two different doses of 5.0 × 10 10 and 2.5 x 10 11 vg / kg or null control at 2.5 × 10 11 vg / kg and monitored for 4 weeks post-dose. Table 3 shows the specific α-GAL variant used in each rAAV9. WT:WT sibling mice of the same genetic background were used as controls and received vehicle only. Mice were assigned to each test article group in a quasi-randomized manner based on pre-dose body weight to ensure balance between groups. Serum was collected at multiple time points during the study. Blood was collected by retro-orbital or tail vein bleeding during the study and by cardiac puncture at termination and processed to obtain serum. At the end of the study, terminal serum was collected and mice were perfused to harvest organs including liver, kidney and heart, which were then flash frozen on dry ice and stored at -80°C. Analytical evaluation included measurement of α-GAL enzyme activity and analysis of substrate concentrations in serum and various tissues.

[0507] [Table 3]

[0508] Alpha-galactosidase activity: Tissues were homogenized in lysis buffer containing 10 mM HEPES and EDTA-free 1.5xHalt protease inhibitor cocktail supplemented with 0.5% Triton-X 100, centrifuged, and the supernatant was collected for analytical assays. Alpha-galactosidase activity in the supernatant or serum was measured using a fluorescent substrate. Briefly, 2 ul of biological sample was incubated with 15 uL of 4-MU-a-gal substrate solution (Research Products International Company, Catalog No. M65400) containing a-galactosidase B inhibitor (N-acetyl-D-galactosamine, Sigma Catalog No. A-2795) for 60 min at 37°C. The enzymatic reaction is stopped by the addition of 200 uL of glycine carbonate stop solution (pH 10.7). The 4-MU product is measured by a fluorescence microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The concentration of 4-MU in the test samples is calculated from a 4-MU standard curve on the same plate. Tissue activity is normalized to total protein concentration as determined by BCA assay (Thermo Scientific, Cat. No. 23225).

[0509] Mus musculus, G3Stg / GLA knockout mice were generated by crossing GLA knockout mice with a C57BL / 6 background with GB3 synthase transgenic mice. Established mice were purchased from Jackson lab and breeding colonies were maintained at Taconic Biosciences. Animals were maintained in a controlled environment at Takeda or Melior Discoveries with no more than four mice per cage in a ventilated cage rack system on a 12-hour dark / 12-hour light cycle (lights on at 7:00 AM) and fed standard rodent chow and water ad libitum.

[0510] 5.0 × 10 of rAAV9-A, rAAV9-005, and rAAV9-D to male G3Stg / GLAko mice 10 vg / kg or 2.5 × 10 11 A single intravenous administration of either rAAV9-WT or rAAV9-WT resulted in sustained higher α-GAL activity in serum over the 4-week study period compared with administration of rAAV9-WT.

[0511] Research 2 In study 2, 14- to 16-week-old G3Stg / GLAko male mice were injected with 2.5 × 10 11 rAAV9-40, rAAV9-41, and rAAV9-42 were administered once at a dose of 2.5 × 10 vg / kg and monitored for 4 weeks after administration. Table 3 shows the specific α-GAL variant used with each rAAV9. WT:WT sibling mice were used as controls and received vehicle only. Following the same study design as the previous study, aged G3Stg / GLAko mice (14-16 weeks old) were used and administered 2.5 × 10 11 A single dose at vg / kg also produced sustained α-GAL activity in serum at levels higher than those of rAAV9-WT-treated animals through 4 weeks.

[0512] Figures 13A and 13B show the α-galactosidase activity of each variant in serum. rAAV9-A, rAAV9-005, and rAAV9-D were incubated at 2.5 × 10 11 The serum α-GAL activity of animals administered rAAV9-WT at a dose of 1000 vg / kg was observed to be more than 16,000-fold, 16,000-fold, and 46,000-fold higher than their normal α-GAL activity in WT mice measured in vehicle-treated WT:WT animals, respectively. In contrast, the serum activity of α-GAL after administration of rAAV9-WT reached slightly more than 1,000-fold higher than normal. Furthermore, elevated α-GAL activity in tissues (kidney, heart, and liver) was also observed in animals treated with rAAV9-A, rAAV9-005, and rAAV9-D at two different doses compared to animals treated with the rAAV9-WT group. Figures 13C-E summarize the α-galactosidase activity of each variant in the kidney, heart, and liver.

[0513] Figure 14A shows serum α-galactosidase activity of various variants in G3Stg / GLAko mice (14-16 weeks old). The increase in serum α-GAL activity in the rAAV9-40 and rAAV9-42 groups was 11,000-fold compared to the WT:WT vehicle control, and in the rAAV9-41 group was over 6,600-fold, whereas serum α-GAL activity in rAAV9-WT-treated animals was only 780-fold higher than the WT:WT control group. All tissues evaluated in the rAAV9-40, rAAV9-42, and rAAV9-41 groups also showed higher α-GAL activity than in the rAAV9-WT-treated animals, with the exception of the liver of the rAAV9-41 animals, which was lower than that of the animals treated with rAAV9-WT (Figures 14B-D).

[0514] Table 4 summarizes the α-α-GAL activity in serum and tissues for each variant.

[0515] [Table 4]

[0516] Example 12. Comparison of reduction of GB3 and lysoGb3 substrates in variants In this example, the effects of engineered α-GAL and WT α-GAL variants on the reduction of GB3 and lysoGb3 were examined.

[0517] Quantification of GB3 and lysoGb3 substrates Substrates from serum and tissue samples were analyzed using an LC-MS methodology. Samples were first extracted using chloroform:methanol (v / v 2:1) and formic acid, followed by HPLC and LC-MS / MS (Applied Biosystem API5000, Turbo Ion Spray Ionization, positive ion mode).

[0518] Both substrates, GB3 and lysoGb3, were observed to be substantially decreased in mice treated with viral vectors expressing α-GAL compared to rAAV9-null controls in both studies, due to persistently high α-GAL activity in serum and tissues. Figures 15A-15D summarize serum and tissue GB3 at 4 weeks after IV administration of rAAV9-A, rAAV9-005, rAAV9-D, rAAV9-WT, and null control at two different doses from Study 1 (described in Example 10). Figures 16A-16D summarize serum and tissue lysoGb3 at 4 weeks after IV administration of rAAV9-A, rAAV9-005, rAAV9-D, rAAV9-WT, and null control at two different doses from Study 1. Figures 17A-17D summarize serum and tissue Gb3 at 4 weeks after IV administration of rAAV9-A, rAAV9-005, rAAV9-D, rAAV9-WT, and a null control at two different doses from Study 2 (described in Example 10). Figures 18A-18D summarize serum and tissue lysoGb3 at 4 weeks after IV administration of rAAV9-A, rAAV9-005, rAAV9-D, rAAV9-WT, and a null control at two different doses from Study 2. In the liver, where α-GAL activity was highest after administration of the viral vectors, all viral vectors showed α-GAL activity of 2.5×10 11vg / kg. In the heart and kidney, rAAV9-A, rAAV9-005, rAAV9-D, rAAV9-41, and rAAV9-42 were more efficient than rAAV9-WT in reducing GB3. The one exception to this was the kidney in animals treated with rAAV9-41. Table 5 shows the results of the 2.5×10 11 vg / kg and 5.0×10 10 Table 6 shows serum and tissue GB3 and lysoGb3 substrates at 4 weeks after IV administration of rAAV9-A, rAAV9-005, rAAV9-D, rAAV9-WT, and rAAV9 null control at a dose of 2.5×10 11 Shown are GB3 and lysoGb3 substrates in serum and tissues 4 weeks after IV administration of rAAV9-40, rAAV9-41, rAAV9-42, rAAV9-WT, and rAAV9-null control at vg / kg.

[0519] Table 7 shows a comparison of tissue GB3 matrix reduction as a percentage of the G3Stg / GLAko null control group (rAAV9-null) at 4 weeks following administration of rAAV9 test articles from Study 1 and Study 2.

[0520] Cardiac GB3 reduction was 86% or more in animals treated with viral vectors expressing engineered α-GAL (as high as 95% in animals treated with rAAV9-42) compared to animals treated with null vector. In contrast, cardiac GB3 reduction was only 70% and 58% in animals treated with rAAV9-WT in Study 1 and Study 2, respectively (Table 7). In this mouse model, substrate accumulation occurs gradually with age. Because older mice were used in Study 2, the percentage of substrate clearance by rAAV9-WT was lower in this study than in Study 1. Compared to a 74% reduction in kidney GB3 in animals treated with rAAV9-WT in Study 1, kidney GB3 reduction was 96%, 95%, and 93% after treatment with rAAV9-A, rAAV9-005, and rAAV9-D, respectively (Table 7). In study 2, treatment with rAAV9-40, rAAV9-41, and rAAV9-42 reduced renal GB3 substrate by 86%, 51%, and 86%, respectively, compared with animals treated with null vector, with a 79% reduction in animals treated with rAAV9-WT (Table 7). In mice with Fabry disease symptoms, a dose of 2.5e11vg / kg of viral vector expressing an engineered α-GAL variant nearly normalized lysoGb3 substrate concentrations to those of vehicle-treated WT:WT mice (Tables 5 and 6).

[0521] [Table 5]

[0522] [Table 6]

[0523] [Table 7]

[0524] Example 13 - In vitro evaluation of plasmids expressing codon-optimized engineered α-GAL variants. In this example, the α-GAL activity of various codon-optimized α-GAL variants in two different cell lines was examined.

[0525] The DNA sequences of engineered α-GAL variants D and 004 were further codon-optimized to improve expression from human liver, kidney and heart tissues. Up to six separate DNA sequences were generated for engineered α-GAL variants D and 004, respectively. These were then incorporated into plasmids and used for transfection.

[0526] Huh7 (human hepatoma) or HEK293 (human embryonic kidney) cells were transfected with plasmids expressing different α-GAL variants using the lipofectamine 3000 reagent kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, cells were seeded at 125,000 cells per well in a 12-well plate format with 1 mL of growth medium per well and maintained overnight at 37 °C and 5% CO2. The next day, fresh medium was added (1 mL per well) before transfection. For each plasmid, 1 μg of plasmid DNA was added to 2 ul of P3000 reagent, 1.5 μl of lipofectamine 3000, and enough OptiMEM medium was added to make 100 ul and incubated at room temperature for 10-15 min. This mixture was then added to the cells and incubated overnight at 37 °C and 5% CO2. The next day, the medium was refreshed and the cells were incubated for another day before the supernatant was collected for analysis of α-GAL activity.

[0527] The supernatant from the transfected cells was collected and assayed for α-GAL enzyme activity. All of the codon-optimized variants of D resulted in significantly higher α-GAL activity in Huh7 cells compared to WT α-GAL or non-optimized enzyme D (Figure 19A). In contrast, in HEK293 cells, only three of the optimized variants showed good activity in the supernatant (Figure 19C). In the case of engineered variant 004, only one of the codon-optimized variants showed relatively good activity in both Huh7 and HEK293 cells (Figures 19B, 19D). The plasmids expressing the two of the codon-optimized engineered variants were then packaged into rAAV9 viruses and tested in vivo, as described in Example 13.

[0528] Example 14 - In vivo evaluation of plasmids expressing codon-optimized engineered α-GAL variants. In this example, the α-GAL activity of various codon-optimized α-GAL variants was examined in Fabry disease model mice (G3Stg / GLAko).

[0529] 10–12 week-old G3Stg / GLAko male mice were injected with rAAV9-D3 and rAAV9-004-3 at four different doses of 2.5 × 10 8 , 2.5×10 9 , 2.5×10 10 and 2.5 x 10 11 vg / kg, or null control rAAV9-null at 2.5 × 10 11Mice were dosed once at 100 mg / kg and monitored for 4 weeks after dosing. WT:WT and WT:CAR sibling mice of the same genetic background served as controls and received vehicle only. Mice were assigned to each test article group in a quasi-randomized manner based on pre-dosing body weight to ensure balance between groups. Serum was collected at multiple time points during the study. Blood was collected by retro-orbital or tail vein bleeding during the study and by cardiac puncture at termination and processed to obtain serum. At the end of the study, terminal serum was collected and mice were perfused to harvest organs including liver, kidney and heart, which were then flash frozen on dry ice and stored at -80°C. Analytical evaluations included measurement of α-GAL enzyme activity and analysis of substrate concentrations in serum and various tissues.

[0530] Tissues were homogenized in lysis buffer containing 10 mM HEPES and EDTA-free 1.5xHalt protease inhibitor cocktail supplemented with 0.5% Triton-X 100, centrifuged, and the supernatant was collected for analytical assay. Alpha-galactosidase activity in the supernatant or serum was measured using a fluorescent substrate. Briefly, 2 μl of biological sample was incubated with 15 uL of 4-MU-a-gal substrate solution (Research Products International Company, Catalog No. M65400) containing a-galactosidase B inhibitor (N-acetyl-D-galactosamine, Sigma Catalog No. A-2795) at 37°C for 60 min. The enzymatic reaction is stopped by the addition of 200 μL of glycine carbonate stop solution (pH 10.7). The 4-MU product is measured by a fluorescence microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The concentration of 4-MU in the test samples is calculated from a 4-MU standard curve on the same plate. Tissue activity is normalized to total protein concentration as determined by BCA assay (Thermo Scientific, Cat. No. 23225).

[0531] In G3Stg / GLAko mice, dose-dependent expression of α-GAL was observed following intravenous administration of rAAV9-D3 and rAAV9-004-3 at doses ranging from 2.5e8vg / kg to 2.5e11vg / kg to male G3Stg / GLAko mice, resulting in sustained high α-GAL activity in serum over the 4-week study period (Figure 20A). Serum α-GAL activity in animals administered rAAV9-D3 and 004-3 at a dose of 2.5e11vg / kg was more than two logs higher than normal α-GAL activity in WT mice as measured in vehicle-treated WT animals. Circulating α-GAL concentrations were undetectable in G3Stg / GLAko mice treated with rAAV9-null. A dose-dependent increase in α-GAL activity was observed in tissues (kidney, heart, and liver) of animals treated with rAAV9-D3 and 004-3 (Figures 20B-20D). Significant α-GAL activity above WT normal values ​​was observed at doses of 2.5e10vg / kg and above for rAAV9-D3 and rAAV9-004-3.

[0532] Example 15 - In vivo evaluation of GB3 and lysoGb3 substrates using plasmids expressing codon-optimized engineered α-GAL variants. In this example, we investigated the reduction of GB3 substrates and lysoGb3 substrates in various codon-optimized α-GAL variants in Fabry disease model mice (G3Stg / GLAko).

[0533] G3Stg / GLAko male mice were administered rAAV9-D3 and rAAV9-004-3 at four different doses as described in Example 14. Furthermore, tissues were homogenized as previously mentioned in Example 14. Substrates from serum and tissue samples were analyzed using LC-MS methodology. Samples were first extracted using chloroform:methanol (v / v 2:1) and formic acid before running HPLC and LC-MS / MS (Applied Biosystem API5000, Turbo Ion Spray Ionization, positive ion mode).

[0534] A dose-dependent decrease in both GB3 and lysoGb3 substrates was observed in mice treated with rAAV9-D3 and rAAV9-004-3 compared to rAAV9-null controls (Figures 21A-21H). At the maximum dose of 2.5e11vg / kg, treatment of animals with either rAAV9-D3 or rAAV9-044-3 resulted in decreased concentrations of GB3 and lysoGb3 substrates in key target tissues, including kidney and heart, to WT normal values. Similar trends were observed in liver and serum.

[0535] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is not intended that the scope of the invention be limited to the above Description, but rather, it is as set forth in the following claims.

Claims

1. A recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid having broad tissue tropism, a. a 5' inverted repeat (ITR); b. a ubiquitous promoter; c. a nucleotide sequence encoding the α-GAL enzyme; d. PolyA; e. a 3′ ITR.

2. A recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid having broad tissue tropism, a. a 5' inverted repeat (ITR); b. a ubiquitous promoter; c. a nucleotide sequence encoding the α-GAL enzyme; d. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); e. PolyA, f. 3′ ITR.

3. 3. The recombinant rAAV vector of claim 1 or 2, wherein the AAV capsid is a broad tropism AAV capsid selected from an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, or an AAV9 capsid.

4. The recombinant rAAV vector of claim 3, wherein the broad tropism AAV capsid is AAV9.

5. 2. The rAAV vector of claim 1, wherein the ubiquitous promoter is selected from a chicken beta actin (CBA) promoter, an EF-1α promoter, a PGK promoter, a UBC promoter, a LSE beta-glucuronidase (GUSB) promoter, or a ubiquitous chromatin opening element (UCOE) promoter.

6. 2. The rAAV vector of claim 1, wherein the ubiquitous promoter comprises a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron.

7. The rAAV vector of claim 1, wherein the ubiquitous promoter comprises a truncated EF-1α promoter and one or more introns.

8. 8. The rAAV vector of claim 7, wherein the one or more introns are from a chicken β-actin gene and / or a rabbit β-globin gene.

9. The rAAV vector of claim 4, wherein the AAV9 capsid is naturally occurring or modified.

10. The rAAV vector of claim 2 , wherein the WPRE sequence is modified.

11. The rAAV vector of claim 10, wherein the WPRE sequence is WPRE mut6delATG.

12. 3. The rAAV vector of claim 1 or 2, wherein the polyA is bovine growth hormone (BGH) polyA.

13. 4. The rAAV vector of any one of the preceding claims, wherein the nucleotide sequence encoding the α-GAL enzyme is codon-optimized.

14. The rAAV vector of claim 13, wherein the nucleotide sequence encoding the α-GAL enzyme is codon-optimized for human cells.

15. The rAAV vector of claim 1 or 2, wherein the α-GAL enzyme has an unmodified sequence.

16. 3. Use of the recombinant adeno-associated virus vector (rAAV) of claim 1 or 2 in the manufacture of a medicament for the treatment of Fabry disease.

17. A pharmaceutical composition comprising the rAAV vector of any one of claims 1 to 15.

18. A cell comprising the rAAV vector of any one of claims 1 to 15.

19. 1. Use of a recombinant adeno-associated virus vector (rAAV) in the manufacture of a medicament for the treatment of Fabry disease, the rAAV being packaged within a capsid having broad tissue tropism, the rAAV comprising: a. a 5' inverted repeat (ITR); b. Ubiquitous promoters, including the cytomegalovirus (CMV) enhancer, the chicken beta actin promoter, and the rabbit beta globin intron; c. a nucleotide sequence encoding the α-GAL enzyme; d. PolyA; e. a 3′ ITR.

20. 1. Use of a recombinant adeno-associated virus vector (rAAV) in the manufacture of a medicament for the treatment of Fabry disease, the rAAV being packaged within a capsid having broad tissue tropism, the rAAV comprising: a. a 5' inverted repeat (ITR); b. Ubiquitous promoters, including the cytomegalovirus (CMV) enhancer, the chicken beta actin promoter, and the rabbit beta globin intron; c. a nucleotide sequence encoding the α-GAL enzyme; d. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); e. PolyA, f. a 3' ITR.

21. 21. The use of claim 19 or 20, wherein the AAV capsid is a broad tropism AAV capsid selected from an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, or an AAV9 capsid.

22. The use of claim 21 , wherein the broad tropism AAV capsid is AAV9.

23. The use according to claim 19 or 20, wherein the nucleotide sequence encoding the α-GAL enzyme is codon-optimized.

24. The use according to claim 19 or 20, wherein the nucleotide sequence encoding the α-GAL enzyme is engineered.

25. The use according to claim 24, wherein the nucleotide sequence encoding the α-GAL enzyme has been engineered and codon-optimized.

26. The use according to claim 19 or 20, wherein the α-GAL enzyme has an unmodified sequence.

27. The use according to claim 20, wherein the WPRE is WPRE mut6delATG.

28. 21. The use according to claim 20, wherein the polyA is bovine growth hormone (BGH) polyA.

29. The use according to claim 19 or 20, wherein the rAAV vector is administered intravenously, subcutaneously, or transdermally.

30. The use according to claim 29, wherein the transdermal administration is performed by a gene gun.

31. The use of claim 19 or 20, wherein the rAAV vector exists as an episome after administration.

32. The use of claim 19 or 20, wherein the rAAV vector achieves a therapeutic effect at a lower dose than a rAAV vector comprising an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, or an AAV8 capsid that is specifically directed to the liver using a liver-specific promoter.

33. The use of claim 19 or 20, wherein the subject has detectable α-GAL in serum for at least 5 weeks, 10 weeks, 15 weeks, 26 weeks, 1 year, 5 years, 10 years or 15 years after administration of the rAAV vector.

34. The use of claim 33, wherein the subject has detectable α-GAL in serum for more than 15 weeks.

35. 21. The use of claim 19 or 20, wherein administration results in alpha-GAL enzyme expression in one or more of the liver, kidney, heart, and gastrointestinal tract of the subject.

36. The use of claim 19 or 20, wherein administration of the rAAV vector results in a reduction in the amount of globotriaosylceramide (gb3) in one or more of the subject's liver, heart, kidneys and gastrointestinal tract.

37. 1. A method for expressing an α-GAL enzyme in a cell, comprising administering a rAAV vector packaged within an AAV9 capsid, said vector comprising: a. a 5' inverted repeat (ITR); b. Ubiquitous promoters, including the cytomegalovirus (CMV) enhancer, the chicken beta actin promoter, and the rabbit beta globin intron; c. a nucleotide sequence encoding the α-GAL enzyme; d. bovine growth hormone (BGH) poly A; e. a 3′ ITR.

38. 1. A method for expressing an α-GAL enzyme in a cell, comprising administering a rAAV vector packaged within an AAV9 capsid, said vector comprising: a. a 5' inverted repeat (ITR); b. Ubiquitous promoters, including the cytomegalovirus (CMV) enhancer, the chicken beta actin promoter, and the rabbit beta globin intron; c. a nucleotide sequence encoding the α-GAL enzyme; d. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) carrying the mut6delATG mutation; e. bovine growth hormone (BGH) poly A; f. a 3' ITR.

39. A recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid having broad tissue tropism, a. a 5' inverted repeat (ITR); b. a liver-specific promoter; c. a nucleotide sequence encoding the α-GAL enzyme; d. PolyA; e. a 3′ ITR.

40. A recombinant adeno-associated virus (rAAV) vector packaged within an AAV capsid having broad tissue tropism, a. a 5' inverted repeat (ITR); b. a liver-specific promoter; c. a nucleotide sequence encoding the α-GAL enzyme; d. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); e. PolyA, f. 3′ ITR.

41. The vector of claim 39 or 40, wherein the nucleotide sequence encoding the α-GAL enzyme is codon-optimized.

42. 41. The vector of claim 39 or 40, wherein the nucleotide sequence encoding the α-GAL enzyme is engineered.

43. 41. The vector of claim 39 or 40, wherein the nucleotide sequence encoding the α-GAL enzyme is both codon optimized and engineered.

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