Compositions for use in treating Fabry disease

Abstract

The present disclosure provides, inter alia, a gene therapy approach for treating Fabry disease in a subject, particularly for alleviating peripheral neuropathy associated with Fabry disease, comprising administering to a subject in need thereof a recombinant adeno-associated viral vector (rAAV) expressing a GLA transgene with broad tissue tropism, improved stability, and reduced immunogenicity, packaged in an AAV capsid.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 401,089, filed August 25, 2022, U.S. Provisional Patent Application No. 63 / 482,948, filed February 2, 2023, and U.S. Provisional Patent Application No. 63 / 500,742, filed May 8, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Reference to sequence listing

[0001] This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The Sequence Listing file entitled MIL-022WO1.SL.XML was created on Aug. 21, 2023, and is 28,159 bytes in size. [Background technology]

[0003] Fabry disease is a rare, progressive, congenital metabolic disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-GAL) as a result of mutations in the GLA gene. Fabry disease affects approximately 1 in 40,000 males and typically manifests as a multisystemic disorder with onset during childhood or adolescence. Fabry disease can also affect females and can present with a variety of symptoms. The lack of α-GAL enzyme activity leads to the progressive, systematic accumulation of its primary substrate, globotriaosylceramide (GB3), and its deacetylated, soluble form, globotriaosylsphingosine (lysoGb3). If left untreated, patients with Fabry disease have a short life expectancy, often resulting in death around age 40 or 50 due to vascular disease affecting the kidneys, heart, and / or central nervous system.

[0004] Neurological symptoms of Fabry disease involve the peripheral nervous system, where accumulation of globotriaosylceramide is observed in Schwann cells and dorsal root ganglia. Some patients with Fabry disease experience pain, which may be due to lipid deposition in the dorsal root ganglia and sympathetic ganglia or to small fiber neuropathy. Peripheral neuropathy affects more than one-quarter of patients with Fabry disease and is characterized by loss of small myelinated and unmyelinated fibers, with large fibers largely spared (Ohnishi and Dyck. Loss of small peripheral sensory neurons in Fabry disease. Arch Neurol 1974;31:120).

[0005] Enzyme replacement therapy (ERT) by introducing a functional enzyme is currently an approved treatment for Fabry disease. While ERT is effective in many cases, this treatment requires lifelong intravenous administration of α-GAL every two weeks. While ERT eliminates symptoms associated with Fabry disease, it is not curative and does not halt disease progression. The poor pharmacological response is largely due to the enzyme's short circulating half-life and suboptimal intracellular delivery. Therefore, there remains a need for therapies to treat Fabry disease that can halt disease progression and potentially be curative. Gene therapy is a promising treatment option. Summary of the Invention

[0006] This application relates to a gene therapy approach using a recombinant adeno-associated viral vector (rAAV) to mediate the transfer and expression of the alpha-galactosidase A (GLA) gene. In particular, this application discloses gene therapy methods and compositions for alleviating, treating, and / or preventing peripheral neuropathy in Fabry disease. rAAV vectors expressing a GLA transgene result in sustained exposure of high levels of functional alpha-GAL enzyme in multiple tissues of Fabry disease patients, particularly in the nervous system (e.g., PNS), that are equivalent to or greater than normal enzyme function in healthy individuals. Restored alpha-GAL reduces the accumulation of substrates Gb3 and lysoGb3 in neural tissues, particularly in the dorsal root ganglion, and improves neural abnormalities, such as maintaining the density of small-diameter nerve fibers (e.g., myelinated nerve fibers).

[0007] In some embodiments, the rAAV vectors used to treat and / or prevent peripheral neuropathy in patients with Fabry disease according to the present disclosure have broad tissue tropism and utilize a ubiquitous promoter to drive widespread gene expression, resulting in sustained high levels of protein expression and robust protein exposure in a wide range of tissues, and / or reduced Gb3 or lysoGb3 levels. Furthermore, the GLA transgene used in the present application is codon-optimized and / or expresses an engineered variant of α-GAL, resulting in increased in vivo α-GAL activity and / or reduced in vivo lysoGb3 or Gb3 levels. Furthermore, this genetic approach, which promotes expression of a GLA variant encoding an α-GAL protein with extended half-life and improved cellular uptake, further increases α-GAL exposure in key target tissues, such as nervous tissue, resulting in improved treatment outcomes for Fabry disease, including, for example, improved peripheral neuropathy outcomes.

[0008] In some embodiments described herein, promoters specific to the liver or nervous system may be used.

[0009] As detailed below, the rAAV-based gene therapy approach described herein results in overall improvement in 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.

[0010] In particular, this gene therapy approach is suitable for alleviating peripheral neuropathy in Fabry disease. Accordingly, one aspect of the present invention provides a method for treating, alleviating, and / or preventing peripheral neuropathy in a subject diagnosed with Fabry disease, the method comprising administering to the subject a composition comprising a recombinant adeno-associated viral vector (rAAV) comprising a polynucleotide encoding the α-GAL enzyme or a variant thereof. Symptoms of peripheral neuropathy may manifest as neuropathic pain, thermal hypoesthesia, hearing loss, other sensory disorders, and / or gastrointestinal disorders.

[0011] In some embodiments, provided are methods for ameliorating peripheral abnormalities of nerve fibers in a subject with Fabry disease, the methods comprising administering to the subject a composition comprising a recombinant adeno-associated viral vector (rAAV) comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof. In some embodiments, small diameter nerve fibers, including small diameter myelinated nerve fibers and unmyelinated nerve fibers, are preserved. In some embodiments, the density of small sensory nerves is maintained.

[0012] In some embodiments, the rAAV vector expressing the GLA transgene described herein is used to reduce the accumulation of globotriaosylceramide (Gb3) in the peripheral nervous system caused by α-galactosidase A deficiency, including administering it to a subject in need thereof. In some embodiments, the accumulation of Gb3 in the dorsal root ganglion (DRG) is reduced. In other embodiments, the expression of LAMP1 in the DRG is reduced.

[0013] In some embodiments, the rAAV vector expressing the GLA transgene has broad tissue tropism, and the vector comprises (a) a 5' inverted terminal repeat (ITR), (b) a ubiquitous promoter, (c) a polynucleotide encoding an α-GAL enzyme or a variant thereof, (d) polyA, and (e) a 3' ITR.

[0014] In some embodiments, rAAV vectors that can be used to treat and / or prevent peripheral neuropathy in patients with Fabry disease exhibit broad tissue tropism and include (a) a 5' inverted terminal repeat (ITR), (b) a ubiquitous promoter, (c) a polynucleotide encoding an α-GAL enzyme or a variant thereof, (d) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), (e) polyA, and (f) a 3' ITR.

[0015] In some embodiments, an rAAV vector that can be used to treat and / or prevent peripheral neuropathy in patients with Fabry disease comprises (a) a 5' inverted terminal repeat (ITR), (b) a liver-specific promoter, (c) a polynucleotide encoding an α-GAL enzyme or a variant thereof, (d) polyA, and (e) a 3' ITR. In other embodiments, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The liver-specific promoter drives expression of the α-GAL enzyme in the liver, which then crosses the blood-brain barrier (BBB) ​​and reaches the nervous system, e.g., the PNS.

[0016] In some embodiments, the rAAV vectors described herein are packaged with AAV capsids that have broad tissue tropism.

[0017] Various types of AAV capsids with broad tissue tropism 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.

[0018] Thus, in some embodiments, an AAV capsid with broad tropism comprises an AAV1 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV2 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV3 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV4 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV5 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV6 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV7 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV8 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV9 capsid.

[0019] In one embodiment, the AAV capsid comprises an AAV9 capsid. The AAV9 capsid is native or modified.

[0020] In some embodiments, the ubiquitous promoter is selected from a chicken beta-actin (CBA) promoter, a CAG 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. 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 a chicken beta-actin (CBA) promoter. In some embodiments, the ubiquitous promoter is an EF-1α promoter. In some embodiments, the EF-1α promoter is combined with a chimeric intron derived from chicken beta-actin and rabbit beta-globin genes. 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.

[0021] In some embodiments, the ubiquitous promoter comprises a cytomegalovirus (CMV) enhancer, a chicken beta-actin promoter, and a rabbit beta-globin intron.

[0022] In some embodiments, the ubiquitous promoter comprises a truncated EF-1α promoter and one or more introns.

[0023] In some embodiments, the one or more introns are derived from chicken beta actin and / or rabbit beta globin genes.

[0024] In some embodiments, the rAAV vector with broad tissue tropism comprises a liver-specific promoter, such as, but 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.

[0025] In some embodiments, the WPRE sequence is optional or modified. In one embodiment, the WPRE sequence is WPRE mut6delATG.

[0026] Exemplary poly A sequences that can be included in gene therapy vectors encompassed by the present disclosure include human growth hormone poly A (hGHpA), synthetic poly A (SPA), simian virus 40 late poly A (SV 40pA), and bovine growth hormone (BGH) poly A. In certain embodiments, the poly A is bovine growth hormone (BGH) poly A.

[0027] In some embodiments, the GLA transgene expressing the α-GAL enzyme comprises a GLA gene having a wild-type sequence (SEQ ID NOs: 1 and 5) or a modified sequence as described herein, including, for example, codon-optimized GLA and / or engineered variants of GLA.

[0028] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is codon-optimized. In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is codon-optimized for human cells.

[0029] In some embodiments, the α-GAL enzyme has an unmodified sequence. In other embodiments, the α-GAL enzyme has a modified sequence.

[0030] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is modified. In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is modified and codon-optimized. In some embodiments, the modified sequence comprises one or more amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified α-GAL enzyme variant has improved stability (e.g., serum stability), intracellular activity (e.g., lysosomal activity), and / or specific catalytic activity compared to the wild-type α-GAL enzyme.

[0031] As a non-limiting example, an α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, an α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, an α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, an α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 7.

[0032] As non-limiting examples, the GLA transgene comprises a nucleotide sequence selected from SEQ ID NOs: 8-10 and 12-13. In some embodiments, the GLA transgene comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the GLA transgene comprises the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the GLA transgene comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the GLA transgene comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the GLA transgene comprises the nucleotide sequence of SEQ ID NO: 13.

[0033] As a non-limiting example, the method includes administering to a subject in need thereof an rAAV vector packaged in a broad tissue tropism rAAV9 capsid, the vector including (a) a 5' inverted terminal repeat (ITR), (b) a ubiquitous promoter including a cytomegalovirus (CMV) enhancer, a chicken β-actin promoter, and a rabbit β-globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme or a variant thereof, (d) polyA, and (e) a 3' ITR.

[0034] In another example, the method includes administering to a Fabry disease patient exhibiting symptoms of peripheral neuropathy an rAAV vector packaged in an rAAV9 capsid with broad tissue tropism, the vector including (a) a 5' inverted terminal 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 posttranscriptional regulatory element (WPRE), (e) polyA, and (f) a 3' ITR.

[0035] 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.

[0036] In some embodiments, transdermal administration is by gene gun.

[0037] In some embodiments, the rAAV vectors of the disclosure are administered in a volume of 1.0 x 10 10 vg / kg (viral genome / kilogram of body weight) ~ 1.0 × 10 14 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1.0 x 10 vg / kg. 10vg / kg~5.0×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1.0 x 10 vg / kg. 10 vg / kg~1.0×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1.0 x 10 vg / kg. 10 vg / kg~5.0×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1.0 x 10 vg / kg. 10 vg / kg~1.0×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1.0 x 10 vg / kg. 10 vg / kg~5.0×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1.0 x 10 vg / kg. 10 vg / kg~2.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5.0 x 10 vg / kg. 10 vg / kg~1.0×10 14 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5.0 x 10 vg / kg. 10 vg / kg~5.0×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5.0 x 10 vg / kg. 10 vg / kg~1.0×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5.0 x 10 vg / kg. 10 vg / kg~5.0×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5.0 x 10 vg / kg. 10 vg / kg~1.0×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5.0 x 10 vg / kg. 10 vg / kg~5.0×10 11In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5.0 x 10 vg / kg. 10 vg / kg~2.5×10 11 Administer at doses ranging from 0.1 mg / kg to 0.2 mg / kg.

[0038] 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, 18 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 duration of the subject's life.

[0039] In some embodiments, expression of the modified α-GAL enzyme results in 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1000-fold, 3000-fold, 10,000-fold, 15,000-fold, 20,000-fold, 25,000-fold, or 30,000-fold higher serum α-GAL concentrations compared to expression of WT α-GAL. In some embodiments, expression of the modified α-GAL enzyme results in 3-fold, 10-fold, 30-fold, 100-fold, 1000-fold, 3000-fold, 10,000-fold, 15,000-fold, 20,000-fold, or 30,000-fold higher intracellular enzyme amounts compared to expression of WT α-GAL.

[0040] 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.

[0041] In another aspect, the present invention provides methods for reducing or ameliorating gastrointestinal symptoms in a subject diagnosed with Fabry disease, the methods comprising administering to the subject a composition comprising a recombinant adeno-associated viral vector (rAAV) comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof, wherein the subject diagnosed with Fabry disease has or is developing one or more gastrointestinal symptoms. Gastrointestinal symptoms include intestinal motility disorders, autonomic dysfunction, vascular disorders, and myopathy. In some embodiments, the treatment reverses vacuolization in the gastrointestinal tract. [Brief explanation of the drawings]

[0042] [Figure 1] 1 shows an exemplary vector diagram of rAAV9 (generally referred to herein as "rAAV9") containing wild-type α-GAL under the control of a ubiquitous promoter, as described herein. [Figure 2A] 1 is a histogram of α-Gal activity in serum 18 weeks after administration of two different doses (5×10 10 and 2.5×10 11 vg / kg) of rAAV vector expressing a GLA transgene, as well as the controls shown in Table 4. [Figure 2B] α-Gal activity in the kidney 18 weeks after administration of two different doses (5×10 10 and 2.5×10 11 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4, is shown. [Figure 2C] α-Gal activity in the heart after 18 weeks of administration of two different doses (5×10 10 and 2.5×10 11 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4, is shown. [Figure 2D] 1 shows α-Gal activity in the liver 18 weeks after administration of two different doses (5×10 10 and 2.5×10 11 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4. [Figure 2E] 1 is a graph showing alpha-galactosidase activity in the kidney and heart of Fabry disease mice treated with variant 1 or wild-type human alpha-galactosidase. [Figure 2F] 1 is a graph showing alpha-galactosidase activity in vehicle control mice, Fabry mice transfected with wild-type alpha-galactosidase, and Fabry mice transfected with Variant 1. [Figure 2G] This is a graph showing serum alpha-galactosidase concentrations in primates transduced with different concentrations of AAV variant 1 (6.25e12 or 3e13 vg / kg). [Figure 3A] Serum Gb3 substrate concentrations after 18 weeks of administration of two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4, are shown. [Figure 3B] 1 shows kidney Gb3 substrate concentrations 18 weeks after administration of two different doses (5×10 10 and 2.5×10 11 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4. [Figure 3C] Cardiac Gb3 substrate levels after 18 weeks of administration of two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4, are shown. [Figure 3D] Hepatic Gb3 substrate concentrations after 18 weeks of administration of two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4, are shown. [Figure 3E] Graph showing percent reduction in kidney or cardiac Gb3 volume upon treatment with variant 1 or vehicle control. [Figure 4A] Serum lysoGb3 substrate concentrations are shown 18 weeks after administration of two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4. [Figure 4B] The lysoGb3 substrate concentrations in the kidney are shown 18 weeks after administration of two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4. [Figure 4C] The lysoGb3 substrate concentration in the heart after 18 weeks of administration of two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4, is shown. [Figure 4D] The lysoGb3 substrate concentration in the liver after 18 weeks of administration of two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4, is shown. [Figure 5]Hot plate latency after 18 weeks is shown for mice administered two different doses (5x1010 and 2.5x1011 vg / kg) of rAAV vector expressing the GLA transgene, as well as the controls shown in Table 4. [Figure 6] Representative examples of MPZ IHC staining of DRG from WT mice, Gb3Stg / GLAko mice treated with rAAV9 variant 1 at a dose of 2.5×10 11 vg / kg, and Gb3Stg / GLAko mice administered with rAAV9 null vector are shown. [Figure 7A] Immunohistochemical staining of kidney tissue is shown. The results demonstrate supraphysiological α-Gal A enzyme exposure in kidney target cells (podocytes) of Gb3Stg / GLAko mice treated with the rAAV9-α-Gal gene therapy vector at a dose of 2.5 x 10 vg / kg. The rAAV9-null vector was used as a control. [Figure 7B] Immunohistochemical staining of cardiac tissue is shown. The results demonstrate supraphysiological α-Gal A enzyme exposure in cardiac target cells (cardiomyocytes) of Gb3Stg / GLAko mice treated with the rAAV9-α-Gal gene therapy vector at a dose of 2.5 x 10 vg / kg. The rAAV9-null vector was used as a control. [Figure 8A] Figure 1 shows normalization of vacuolization and lysosomal burden in the dorsal nerve root (DNR) of a Gb3Stg / GLAko mouse treated with a 2.5 x 10 vg / kg dose of the rAAV9-α-Gal gene therapy construct. The rAAV9-null vector was used as a control. The upper panel shows HE (hematoxylin and eosin) staining of the dorsal nerve root. The lower panel shows immunohistochemical staining for lysosome-associated membrane protein 1 (LAMP1). [Figure 8B] Graph showing the percentage of LAMP1-positive areas in Fabry disease mice (baseline, control, or variant 1 gene therapy-treated) and normal mice. [Figure 8C] Graph showing hot plate latency in control or variant 1 gene-treated Fabry disease mice and normal mice. [Figure 8D] 1 is a graph showing the correlation between DRG and latency. [Figure 8E] MRI images showing DRG volume in normal mice and WT aGAL-expressing Fabry disease mice transduced with vehicle control or gene therapy (GT). [Figure 8F] Graph showing DRG volume in vehicle control or gene-treated WT aGAL-expressing Fabry disease mice, or normal mice. [Figure 9] This figure shows the recovery of gastrointestinal pathological vacuolation and lysosomal burden in Gb3Stg / GLAko mice treated with rAAV9-α-Gal gene therapy vector at a dose of 2.5 × 10 vg / kg. The rAAV9-null vector was used as a control. The upper panel shows hematoxylin and eosin (HE) staining of transverse smooth muscle. Vacuoles in the transverse smooth muscle of G3Stg / GlaKO duodenum are restored after treatment with rAAV9-α-Gal gene therapy vector. The middle panel shows α-Gal A enzyme exposure. α-Gal A positive staining is observed in mice treated with rAAV9-α-Gal gene therapy vector. The lower panel shows immunohistochemical staining of LAMP1, a lysosome-associated membrane protein 1. LAMP1-positive staining in smooth muscle and myenteric ganglion cells is observed only in untreated and rAAV9-null vector-treated mice. [Figure 10] A shows IHC of hepatocytes treated with rAAV8-hα-GAL, which has a ubiquitous promoter, and B shows IHC of hepatocytes treated with rAAV9-hα-GAL, which has a liver-specific promoter. [Figure 11-1] A shows the predicted pharmacokinetics of α-galactosidase concentrations in the heart, kidney, liver, and plasma at 3e11vg / kg in humans. B shows the predicted pharmacokinetics of α-galactosidase concentrations in the heart, kidney, liver, and plasma at 1e11vg / kg in humans. C shows the predicted pharmacokinetics of α-galactosidase concentrations in the heart, kidney, liver, and plasma in future humans treated with ERT. [Figure 11-2]D shows the predicted pharmacokinetics of α-galactosidase concentrations in the heart, kidney, liver, and plasma when a future human is treated with 3e11vg / kg. E shows the predicted pharmacokinetics of Gb3 reduction in the heart, kidney, liver, and plasma when a future human is treated with 1e11vg / kg. F shows the predicted pharmacokinetics of Gb3 reduction in the heart, kidney, liver, and plasma when a future human is treated with ERT. [Figure 12A] Graph showing alpha-galactosidase activity over a 4-week period in mice injected with two different doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2. [Figure 12B] 1 is a bar graph showing alpha-galactosidase activity in terminal serum at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12C] 1 is a bar graph showing alpha-galactosidase activity in the kidney at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12D] 1 is a bar graph showing alpha-galactosidase activity in the heart at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12E] 1 is a bar graph showing alpha-galactosidase activity in the liver at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12F] 1 is a bar graph showing terminal serum Gb3 substrate levels at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12G] 1 is a bar graph showing Gb3 substrate levels in the kidney at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12H]1 is a bar graph showing Gb3 substrate levels in the kidney at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12I] 1 is a bar graph showing Gb3 substrate levels in the liver at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12J] 1 is a bar graph showing terminal serum LysoGb3 substrate levels at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12K] 1 is a bar graph showing LysoGb3 substrate levels in the kidney at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12L] 1 is a bar graph showing LysoGb3 substrate levels in the heart at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12M] 1 is a bar graph showing LysoGb3 substrate levels in the liver at two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12N] Graph showing vector genome copy numbers in various tissues when treated with two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 12O] Graph showing mRNA copy numbers in various tissues when treated with two doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL-variant 2 compared to the control group. [Figure 13A] 1 is a graph showing serum α-galactosidase activity over 32 weeks in mice after intravenous injection of two different doses of rAAV9-hα-GAL variant 1 compared to a control group. [Figure 13B] 1 is a graph showing α-galactosidase activity in serum of non-human primates after intravenous injection of rAAV9-hα-GAL variant 1 over a 28-day period. [Figure 13C] 1 is a graph showing α-galactosidase activity in serum of non-human primates after intravenous injection of rAAV9-hα-GAL variant 1 over a 90-day period. DETAILED DESCRIPTION OF THE INVENTION

[0043] definition Approximately or about: As used herein, the term "approximately," when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, unless otherwise specified or otherwise clear from the context, 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 less) from the stated reference value (except where 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 near the stated reference value on either side of the stated reference value.

[0044] 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).

[0045] 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 simultaneously or within a period of time such that there may be overlap in the effects of each agent on the patient. In some embodiments, the administration of the agents is spaced sufficiently close together so that a combined (e.g., synergistic) effect is achieved.

[0046] 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 substituted in a parent or reference sequence (e.g., a wild-type GLA polypeptide sequence) via, for example, chemical peptide synthesis or via recombinant methods known in the art. Thus, a 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 schema AnY, where A is the single-letter code corresponding to the amino acid naturally occurring or originally present at position n, and Y is the alternative amino acid residue. In other embodiments, the substitution pattern can be described according to the schema An(YZ), where A is the single-letter code corresponding to the amino acid residue that is substituted for the amino acid naturally occurring or originally present at position X, and Y and Z are the substituted alternative amino acid residues.

[0047] The abbreviations used for 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, an amino acid is referred to as having an α-carbon (C α ) can be in either the L- or D-configuration. In various embodiments described herein, one or more amino acids in the wild-type GLA sequence can be substituted with a different amino acid, thereby resulting in a variant of the α-GAL protein.

[0048] Substitutions in the amino acid sequence of a protein or polypeptide can be conservative or non-conservative in 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 can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid having a hydroxyl side chain can be substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine), an amino acid having an aromatic side chain can be substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid having a basic side chain can be substituted with another amino acid having a basic side chain (e.g., lysine and arginine), an amino acid having an acidic side chain can be substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid), and / or a hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid, respectively. Non-conservative substitutions refer to the substitution of an amino acid in a polypeptide (e.g., the 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 can be an acidic amino acid substituted with a basic or aliphatic amino acid, an aromatic amino acid substituted with a small amino acid, and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0049] 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 substitution of an amino acid residue with an alternative amino acid residue is made by substituting a codon encoding a first amino acid with a codon encoding a second amino acid.

[0050] AVV vector: As used herein, the term "AAV vector" includes capsid proteins and a viral genome, the viral genome including at least one transgene region and at least one inverted terminal repeat (ITR). An AAV vector and / or its constituent capsid and viral genome may be modified to alter its tropism for a particular cell type, tissue, organ, or organism. In the context of the present invention, the viral genome includes a nucleic acid sequence encoding a GLA transgene, e.g., α-GAL or a variant thereof.

[0051] 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.

[0052] 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 result in 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 thought to contribute, at least in part, to the rate or severity of proteinuria or kidney damage in Fabry disease. Blood urea nitrogen measures the efficiency of the kidneys in removing urea from the blood. High BUN levels indicate poor kidney function.

[0053] 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 comprising a GLA polypeptide and a second part (e.g., fused to the first part by genetic engineering) comprising 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).

[0054] 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 a "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.

[0055] 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) so 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 can 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.

[0056] Dosage: As used herein, the terms "dose" and "administration" are used interchangeably. Dose refers to the amount of active ingredient given to an individual per administration. Dosage varies depending on various factors, including frequency of administration, the individual's size and tolerance, severity of symptoms, risk of side effects, and route of administration. One skilled in the art will recognize that dosage may vary depending on the above factors or based on the progress of treatment. The term "dosage form" refers to a specific format of a pharmaceutical product and varies depending on the route of administration.

[0057] Modified Variant: The term "modified α-GAL variant" or "modified 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, modified variants are characterized by improved efficacy and pharmacokinetic properties, e.g., due to modified structural attributes of the protein. In some embodiments, modified α-GAL variants enhance substrate clearance from tissues such as serum, kidney, heart, and / or liver. Modified variants can be synthetic or recombinantly produced.

[0058] 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 thought to be the primary causative metabolite. GB3 is formed by the α-linkage of galactose to lactosylceramide, catalyzed by A4GALT. GB3 is hydrolyzed at the terminal α-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 associated with a significantly increased 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.

[0059] Gene: As used herein, the term "gene" refers to a DNA region that encodes a protein or polypeptide (e.g., an α-galactosidase enzyme as described herein) and 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, a gene includes, but is 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.

[0060] GLA gene: As used herein, the term "GLA gene" or "galactosidase gene" or "α-galactosidase gene" or "α-galactosidase gene" refers to a gene encoding the enzyme α-galactosidase, which breaks down globotriaosylceramide. Genetic mutations in the GLA gene result in defects in the enzymatic function of α-galactosidase. In humans, the GLA gene is located on the long (q) arm of the X chromosome at position 22.1, Xq22.1. Some other names that may refer to the GLA gene include AGAL HUMAN, agalsidase α, α-D-galactosidase A, α-D-galactosidase galactohydrolase, α-galactosidase, α-galactosidase A, ceramide trihexosidase, GALA, galactosidase, or melibiase.

[0061] Galactosidase: The term "galactosidase" or "alpha-galactosidase A" or "alpha-galactosidase A" or "alpha-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 alpha-GAL can 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, alpha-GAL deficiency, and ceramide trihexosidase deficiency), an X-linked congenital disorder of glycosphingolipid catabolism. Various embodiments described herein provide gene therapy platforms for the treatment of Fabry disease.

[0062] "Hypoalgesia or Hypoalgesia": As used herein, the terms "hypoalgesia" or "hypoalgesia" refer to a decreased sensitivity to painful stimuli. Hypoalgesia occurs when nociceptive (painful) stimuli are interrupted or diminished anywhere along the pathway between the input (nociceptors) and where the stimuli are processed and perceived in consciousness as pain. Pain relief can be mild, such as massaging a stubbed toe to relieve pain or taking aspirin to relieve a headache, or severe, such as under strong anesthesia.

[0063] "Improved enzymatic property": The term "improved enzymatic property" refers to any property or characteristic of a modified α-GAL polypeptide that is improved compared to the same property or characteristic of a reference α-GAL polypeptide (e.g., compared to a wild-type α-GAL polypeptide or another modified 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 tolerance to acidic, neutral, or basic pH, resistance to proteolytic activity (i.e., decreased susceptibility to proteolysis), reduced aggregation, improved solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation), altered temperature properties, 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 that has one or more improved enzymatic properties compared to a reference α-GAL polypeptide. In some embodiments, the nucleic acid sequence encoding the α-GAL polypeptide exhibiting one or more improved enzymatic properties is codon-optimized.

[0064] In various embodiments, the codon-optimized and / or engineered α-GAL variants exhibit one or more of the aforementioned improved properties: in certain embodiments, the α-GAL variants have improved serum and lysosomal stability, and in other embodiments, the α-GAL variants have improved specific catalytic activity relative to the wild-type α-GAL polypeptide.

[0065] "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 rate of substrate-to-product conversion (e.g., the rate of conversion of starting substrate to product in a specific time period) using a particular amount of modified α-GAL enzyme compared to a reference α-GAL enzyme (e.g., a wild-type α-GAL enzyme or another modified 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 associated with enzyme activity can be affected, including classical enzyme properties such as, and changes in these properties can result in increased enzyme activity. Improved enzyme activity can range from about 1.1-fold 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 greater enzyme activity than the reference α-GAL enzyme.

[0066] 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 its 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.

[0067] Nucleic Acid: As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a polymer of deoxyribonucleotides or ribonucleotides in 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 terms can encompass known analogs of natural nucleotides as well as nucleotides that are modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). In general, an analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T.

[0068] Neuropathy: As used herein, the term "neuropathy" refers to damage and / or dysfunction of one or more nerves. "Peripheral neuropathy" refers to any damage and / or dysfunction affecting the peripheral nervous system (PNS). "Peripheral neuropathy" may manifest as one or a combination of motor, sensory, sensorimotor, or autonomic dysfunction. Peripheral neuropathy has been shown to be associated with systemic diseases, including Fabry disease. For the purposes of this disclosure, peripheral neuropathy associated with Fabry disease primarily manifests as sensory dysfunction (e.g., decreased heat sensitivity). One type of peripheral neuropathy is "demyelinating peripheral neuropathy," which is a broad class of peripheral neuropathy associated with the destruction or removal from nerves of myelin, the lipid-rich sheath that surrounds and insulates nerve fibers.

[0069] Operable Linkage: As used herein, the terms "operably linked" and "operably linked" (or "operably linked") are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) where the components are positioned in a manner that permits both components to function normally and allows for the potential for at least one of the components to mediate a function exerted by 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 usually 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.

[0070] Physiological pH: As used herein, "physiological pH" generally refers to the pH range found in a subject's (e.g., human) blood, i.e., pH 7.4.

[0071] Basic pH: The term "basic pH" (as used, for example, in reference to improved stability at or increased tolerance to basic pH conditions) refers to a pH range of about 7-11.

[0072] Acidic pH: The term "acidic pH" (e.g., as used in reference to improved stability to or increased tolerance to acidic pH conditions) refers to a pH range of about 1.5 to 6. 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 analogs or modified derivatives of the corresponding naturally occurring amino acids.

[0073] 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 corresponding protein or polypeptide expression can be ubiquitous, i.e., strongly active in a wide range of cells, tissues, and species, or cell-type-, tissue-, 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 can be "constitutive," i.e., constantly active, or "inducible," i.e., the promoter can be active or inactive depending on the presence or absence of biotic or abiotic factors. The nucleic acid constructs or vectors of the present invention also include enhancer sequences, which may or may not be adjacent to the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of the native gene.

[0074] 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 with improved properties, e.g., improved protein expression or increased activity.

[0075] The terms "individual," "subject," "subject in need thereof," and "patient," used interchangeably herein, refer to mammals, including, but not limited to, mice, monkeys, humans, mammalian farm animals, mammalian sport animals, and mammalian pets. In preferred embodiments, the individual is a human. In various embodiments, the subject or a subject in need thereof is a Fabry disease patient exhibiting one or more symptoms associated with peripheral neuropathy. Therapeutically effective: A "therapeutically effective" amount or dose, or a "sufficient / effective" amount or dose, is a dose that produces an effect when administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0076] Treatment: As used herein, the term "treatment" or "therapy" generally refers to achieving a desired physiological effect. This effect may be prophylactic, meaning that a disease or condition or its symptoms are completely or partially prevented, and / or therapeutic, meaning that an injury, disease, or condition is partially or completely cured and / or a harmful effect resulting from an injury, disease, or condition is alleviated, including arresting or reversing the progression of a disease or condition. Treatment also includes prophylactic use to reduce the effects of injury once it has occurred. For example, in one embodiment, the present invention includes pre-administration to reduce damage before surgery involving the peripheral nervous system. Treatment can also refer to delaying onset, ameliorating symptoms, improving patient survival, increasing survival time or rate, and the like. The effect of treatment may be compared to an individual or population of individuals not receiving treatment.

[0077] 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, respectively, multiple tissues throughout the body, or multiple tissues or organs (e.g., two or more of the brain, lung, skeletal muscle, heart, liver, kidney, and / or pancreas).

[0078] Thermohypoesthesia: As used herein, the term "thermoesthesia" or "thermoparesthesia" or "thermohypesthesia" refers to insensibility or hyposensitivity to thermal stimuli.

[0079] Vector: As used herein, the term "vector" refers to a gene capable of introducing a gene 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 gene 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, encapsulated forms of vector nucleic acid and viral particles in which the vector nucleic acid is packaged. In some embodiments, the vector is not a wild-type strain of virus because 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).

[0080] Wild-type: As used herein, the terms "wild-type" and "naturally-occurring" refer to a form of a nucleic acid or protein that is found in nature. For example, a wild-type polypeptide or polynucleotide sequence is one that is present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0081] 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 deemed to be modified by the term "about."

[0082] Various aspects of the present invention are detailed in the following sections. The use of sections is not intended to limit the present invention. Each section may be applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0083] Detailed Description This disclosure relates to rAAV-based gene therapy for the treatment and / or prevention of Fabry disease, particularly for the amelioration and / or prevention or treatment of neuropathy in Fabry disease. The disclosure provides, inter alia, (1) an endogenous GLA expression system in tissues affected by Fabry disease, (2) methods for achieving sustained high expression of alpha-galactosidase (alpha-GAL) to reduce the disease burden and treatment burden associated with the progression of Fabry disease, and (3) the use of rAAV vectors encoding GLA to achieve amelioration of Fabry disease-associated phenotypes, particularly peripheral neuropathy in Fabry disease.

[0084] In some embodiments, the gene therapy treatments described herein utilize rAAV vectors with broad tissue tropism, including ubiquitous promoters to drive widespread gene expression, resulting in sustained high levels of protein expression and robust protein exposure in a wide range of tissues, and / or reduced Gb3 or lysoGb3 levels. Furthermore, the GLA transgene used in this application is codon-optimized and / or expresses an engineered variant of α-GAL, resulting in increased in vivo α-GAL activity and / or reduced in vivo lysoGb3 or Gb3 levels. Furthermore, this genetic approach, which promotes expression of a GLA variant encoding an α-GAL protein with extended half-life and improved cellular uptake, further increases α-GAL exposure in key target tissues, such as nervous tissue, improving treatment outcomes for Fabry disease, including, for example, improved peripheral neuropathy outcomes.

[0085] 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, such as peripheral neuropathy.

[0086] Alpha-galactosidase and Fabry disease Fabry disease is an X-linked genetic disorder caused by abnormal 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.

[0087] Fabry disease is a systemic metabolic disorder that affects many tissues and organs, including the kidneys, heart, lungs, and nervous system. Neurological symptoms of Fabry disease include peripheral nervous system (PNS) dysfunction, with accumulation of globotriaosylceramide in Schwann cells and dorsal root ganglia. PNS dysfunction primarily affects small A-delta and C-fibers and is likely responsible for the altered autonomic function and neuropathic pain seen in Fabry disease. Other associated neurological problems include hypohidrosis and other abnormalities related to nervous system dysfunction.

[0088] Peripheral neuropathy in Fabry disease manifests as neuropathic pain, decreased temperature and cold sensation (i.e., thermohypoesthesia), hearing loss, other sensory disturbances, and in some cases gastrointestinal problems.

[0089] Patients with Fabry disease begin experiencing pain in the second half of life or during adolescence (Ries et al. Pediatric Fabry disease. Pediatrics. 2005;115:e344-355). Neuropathic pain in Fabry disease can generally be persistent (i.e., chronic) or occur in intermittent episodes triggered by changes in body or ambient temperature or other stressful situations (MacDermot and MacDermot, Neuropathic pain in Anderson-Fabry disease: pathology and therapeutic options. Eur J Pharmacol. 2001;429:121-125). The intermittent pain of Fabry disease, called "Fabry crises," typically begins in the extremities and radiates proximally and can be triggered by movement, illness, temperature changes, or other physical and emotional stressors. This neuropathic pain is also associated with a lack of temperature sensation.

[0090] Neuropathy in Fabry disease is associated with significantly elevated thresholds for sensing cold and warmth (i.e., thermal hypoesthesia or decreased thermal sensitivity) in the hands and feet (Luciano et al. Physiological characterization of neuropathy in Fabry's disease. Muscle Nerve. 2002;26:622-629; and Dutsch et al. Small fiber dysfunction predominates in Fabry neuropathy. J Clin Neurophysiol. 2002;19:575-586). Well-established biophysical sensory testing techniques can be used to measure these thresholds (Dyck et al., A 4, 2, and 1 stepping algorithm for quick and accurate estimation of cutaneous sensation threshold. Neurology. 1993;43:1508-1512).

[0091] Increased thresholds for hot and cold sensations (hypoesthesia) in men and carrier women have been shown to begin with burning pain and acute discomfort (Hilz, et al., Lower limb cold exposure induces pain and prolonged small fiber dysfunction in Fabry patients. Pain, 2000;84:361-365). A reduction in the number of small myelinated nerve fibers has been reported in human patients (Onishi and Dyck, Loss of small peripheral sensory neurons in Fabry disease. Histologic and morphometric evaluation of cutaneous nerves, spinal ganglia, and posterior columns. Arch. Neurol. 1974;31:120-127). A study of pain-related nerve damage based on the characterization of postmortem tissue from several patients with Fabry disease showed a significant reduction in thin, small-diameter myelinated and unmyelinated nerve fibers (Hilz, et al., Enzyme replacement therapy improves function of C-, Adelta-, and Abeta-nerve fibers in Fabry neuropathy. Neurology; 2004; 62: 1066-1072).

[0092] Several studies have shown that Fabry disease is associated with a peripheral neuropathy that primarily affects small-diameter myelinated (Aδ) and unmyelinated (C) fibers. Likely causative mechanisms include nerve ischemia caused by glycolipid accumulation within the vasa nervorum or intrinsic nerve dysfunction (Luciano et al. Physiological characterization of neuropathy in Fabry's disease. Muscle Nerve. 2002;26:622-629; Hilz et al. Lower limb cold exposure induces pain and prolonged small fiber dysfunction in Fabry patients. Pain. 2000;84:361-365; and Gadoth and Sandbank. Involvement of dorsal root ganglia in Fabry's disease. J Med Genet. 1983;20:309-312).

[0093] Sensory fiber neuropathy (SFSN) in patients with Fabry disease has been found to be associated with sensorineural hearing loss (Ries et al., Neuropathic and cerebrovascular correlates of hearing loss in Fabry disease. Brain 2007, 130:143-150).

[0094] Other neurological deficits in Fabry disease include decreased vibration thresholds and nerve conduction abnormalities in some patients. Pathological examination of peripheral nerves (e.g., the sural nerve) usually reveals a normal number of large myelinated fibers but a significant reduction in small unmyelinated fibers. Glycolipid deposition in sensory ganglia is associated with peripheral neuropathy. Furthermore, this neuropathy in Fabry disease is associated with a severe loss of intraepidermal innervation.

[0095] In Fabry disease knockout mice, abnormal accumulation of Gb3 due to defective lysosomal α-galactosidase activity has been shown to be associated with abnormal sciatic nerve morphology, a reduction in the number of unmyelinated and small myelinated axons, preservation of large myelinated axons, and behavioral and thermal sensitivity, characteristics similar to those described in human patients (Rodrigues et al., Neurophysiological, behavioral and morphological abnormalities in the Fabry knockout mice; Neurobiol. Dis., 2009, 33(1):48-56). This may explain the changes in heat sensitivity (hypoalgesia) observed in Fabry knockout mice.

[0096] In summary, it is well established that GB3 inclusions are present in DRG neurons in both patients and preclinical rodent models, and that these inclusions cause cell swelling, including increases in the diameter of DRG neuron cell bodies and total DRG volume. GB3 content can be over 10-fold higher in DRG than in other brain regions (e.g., Kaye et al., Nervous system involvement in Fabry's disease: clinical pathological and biochemical correlation. Ann Neurol 1988;23:505-509); these high levels of GB3 can lead to DRG neuron stress and cell death. In addition to DRG neuron cell bodies, lipid inclusions are prominent in peripheral nerve axons, which correlate with axonal morphological abnormalities, such as irregularly shaped axons, enlarged axons, and uneven myelin. This dramatic loss of small-diameter fibers is most evident in long axons in the distal lower limbs. Although innervation of proximal regions, such as the thigh, may also be reduced, loss is most evident around the foot. Studies suggest that fiber loss is significantly greater in the skin than in the peripheral nerve trunks.

[0097] 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 primarily limited to the kidneys and heart, with inadequate or no improvement in other organs / tissues, particularly the nervous system. Patients with Fabry disease also require continuous protein-based infusions, which can lead to infusion reactions and enhanced immunogenicity. The need for such ongoing disease management also increases the "treatment burden" or additional and ongoing workload (i.e., the need and demands) for patients to adhere to the recommendations made by clinicians to manage their morbidity and health status. Male patients with severe classic Fabry disease experience an annual loss of renal function of -1 mL / min / 1.73 m 2 Despite treatment, the annual loss of renal function was up to -6.82 mL / min / 1.73 m compared with healthy controls. 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.

[0098] Gene therapy is a promising approach for the treatment and prevention of Fabry disease. One advantage of a gene therapy approach to the treatment of Fabry disease is continuous α-GAL exposure, rather than the intermittent α-GAL exposure afforded by ERT infusion. A gene therapy approach could potentially enable uptake by certain tissues and cell types (e.g., the peripheral nervous system) that are not easily achieved with infused ERT. Continuous availability of α-GAL in lysosomes could prevent reaccumulation of glycosphingolipids between doses. Significantly enhanced enzyme distribution to target cells could provide a transformative therapy with the potential to achieve superior clinical benefit over current therapies. Furthermore, gene therapy involving hepatocyte transduction could exploit 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, these advantages, combined with a single, long-term, sustained dose, could address the need for highly therapeutically effective therapies and reduce the treatment burden on patients and caregivers.

[0099] As discussed below, the present disclosure provides GLA gene therapy for treating and / or ameliorating one or more neuropathic symptoms in Fabry disease.

[0100] Gene Therapy Vectors One aspect of the present disclosure provides a GLA expression system (i.e., a GLA transgene) comprising a viral vector containing a polynucleotide encoding α-GAL or a variant thereof, controlled by a ubiquitous promoter. In some aspects, the viral vector is a recombinant AAV vector. In particular, recombinant AAV (rAAV) vectors have broad tissue tropism. The rAAV vectors discussed in this application have broad tissue tropism and utilize a ubiquitous promoter to promote widespread gene expression, resulting in sustained high levels of protein expression, robust protein exposure in a wide range of tissues, and / or reduced GB3 or lysoGb3 levels. Furthermore, the GLA transgene is codon-optimized and / or expresses an engineered variant of α-GAL, resulting in increased in vivo α-GAL activity and / or reduced in vivo lysoGb3 or GB3 levels.

[0101] α-GAL enzyme and variants The viral vectors described herein comprise a polynucleotide encoding an α-GAL enzyme or a variant thereof. In some embodiments, the α-GAL enzyme is a native (wild-type) enzyme having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5. In some embodiments, the α-GAL enzyme is a functional variant, such as a modified α-GAL enzyme.

[0102] Exemplary amino acid sequences of α-GAL enzymes and variants thereof contemplated for use in the vectors of the present disclosure are shown in Table 1 below.

[0103] [Table 1-1]

[0104] [Table 1-2]

[0105] In some embodiments, the α-GAL enzyme encoded by the GLA transgene comprises the signal peptide sequence MQLRNPELHLGCALALRFLALVSWDIPGARA (SEQ ID NO: 4). 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 enzyme encoded by the GLA transgene comprises a signal peptide sequence at the C-terminus. In some embodiments, the α-GAL enzyme encoded by the GLA transgene comprises SEQ ID NO: 4 at the N-terminus. As a non-limiting example, an α-GAL enzyme comprising a signal peptide comprises an amino acid sequence selected from SEQ ID NOs: 5-7 (Table 1).

[0106] In some embodiments, the modified sequence comprises 1 to 25, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, or 15 to 25 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5).

[0107] In some embodiments, the modified sequence comprises one to ten amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). For example, in some embodiments, the modified sequence comprises one to nine amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises one to eight amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises one to seven amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises one to six amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises one to five amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises one to four amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 1). In some embodiments, the modified sequence comprises one to three amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5).

[0108] In some embodiments, the modified sequence comprises 10 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5).

[0109] In some embodiments, the GLA transgene expresses recombinant α-GAL. The recombinant α-GAL comprises a polypeptide sequence having 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:5, or a functional fragment thereof. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 86% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 87% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 88% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 89% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 91% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 92% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 93% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 94% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 96% sequence identity to SEQ ID NO:5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 97% sequence identity to SEQ ID NO:5.In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 98% sequence identity to SEQ ID NO: 5. In some embodiments, the recombinant α-GAL comprises a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 5.

[0110] In some embodiments, the recombinant α-GAL comprises at least one substitution or substitutions in SEQ ID NO: 5 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.

[0111] Additional exemplary GLA transgene 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.

[0112] In some embodiments, the modified α-GAL enzyme has improved stability (e.g., serum stability), intracellular stability (e.g., lysosomal activity), and / or specific catalytic activity compared to the wild-type α-GAL enzyme (SEQ ID NO: 5).

[0113] In some embodiments, the present 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 any one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is 70% to 100% identical to SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is 75% to 100% identical to SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is 80% to 100% identical to SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is 85% to 100% identical to SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is 90% to 100% identical to SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is 95% to 100% identical to SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 70% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 75% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 80% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 85% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 90% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 91% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 92% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 93% identical to one of SEQ ID NOs: 1-3 and 5-7.In some embodiments, the vector comprises a GLA sequence that is at least 94% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 95% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 96% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 97% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 98% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is at least 99% identical to one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence that is 100% identical to one of SEQ ID NOs: 1-3 and 5-7.

[0114] 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: 2. 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: 3. 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: 6. In some embodiments, the present 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:7.

[0115] In some embodiments, a GLA gene therapy vector comprises a polynucleotide encoding an α-GAL enzyme or a variant thereof. In some embodiments, the disclosure encompasses gene therapy vectors comprising a polynucleotide 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: 1. In some embodiments, the disclosure encompasses gene therapy vectors comprising a polynucleotide 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: 2. In some embodiments, the disclosure encompasses gene therapy vectors comprising a polynucleotide 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: 3. In some embodiments, the disclosure encompasses gene therapy vectors comprising a polynucleotide 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: 5. In some embodiments, the disclosure encompasses gene therapy vectors comprising a polynucleotide 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: 6. In some embodiments, the disclosure encompasses gene therapy vectors comprising a polynucleotide 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.

[0116] In other embodiments, the GLA transgene of the present disclosure may comprise α-GAL or a variant thereof (e.g., the sequence in Table 1 of PCT / US22 / 17998) disclosed in Applicant's previous PCT application PCT / US22 / 17998 (the contents of which are incorporated herein by reference in their entirety).

[0117] In some embodiments, the GLA transgene comprises a signal peptide sequence The GLA sequence comprises a nucleic acid sequence encoding 5' atgcagctgaggaacccagaactacatctgggctgcgcgcttgcgcttcgcttcctggccctcgtttcctgggacatccctggggctagagca 3' (SEQ ID NO: 11). 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: 11 at the 5' end. In some embodiments, the GLA sequence comprises SEQ ID NO: 11 at the 3' end.

[0118] In some embodiments, the polynucleotide encoding the α-GAL enzyme or variant thereof comprises at least one chemical modification. In some embodiments, the polynucleotide encoding the α-GAL enzyme or variant thereof is codon-optimized.

[0119] As a non-limiting example, the polynucleotide comprises the nucleotide sequence of Table 2. In some embodiments, the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequence of SEQ ID NO: 13.

[0120] [Table 2-1]

[0121] [Table 2-2]

[0122] [Table 2-3]

[0123] [Table 2-4]

[0124] [Table 2-5]

[0125] In some embodiments, the polynucleotide is codon-optimized. In some embodiments, the polynucleotide of the GLA gene therapy vector encoding the α-GAL enzyme or variant thereof comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to any one of SEQ ID NOs: 8-10 and 12-13.

[0126] In some embodiments, a polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% identical to SEQ ID NO: 8. In some embodiments, a polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% identical to SEQ ID NO: 9. In some embodiments, a polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to SEQ ID NO: 12. In some embodiments, a polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to SEQ ID NO: 13.

[0127] In other embodiments, the polynucleotide encoding α-GAL or a variant thereof may comprise a nucleic acid sequence disclosed in Applicant's previous PCT application No. PCT / US22 / 17998 (the contents of which are incorporated herein by reference in their entirety) (e.g., the sequences in Table 1 of PCT / US22 / 17998).

[0128] Recombinant AAV (rAAV) vectors A transgene delivered by a vector can be introduced into a target cell using a variety of methods. For example, either viral or non-viral vectors can be used to deliver a transgene of interest. Both viral and non-viral vector delivery methods are contemplated in the methods provided herein. 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. In some embodiments, the vectors can be introduced as naked nucleic acid or as nucleic acid complexed with an agent such as a liposome or poloxamer. Various viral vectors for delivering transgenes are known in the art, including, for example, either integrating or non-integrating vectors. In some embodiments, the viral vector is a non-integrating viral vector. Non-integrating viral vectors include, for example, non-integrating lentiviral or AAV vectors. In other embodiments, the GLA transgene of the present invention can be delivered by a virus (e.g., adenovirus, adeno-associated virus (AAV), herpesvirus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)). A vector may contain additional sequences, such as, for example, an origin of replication, a promoter, and one or more genes.

[0129] As a non-limiting example, the GLA transgene described herein is introduced using a viral vector, which is an adeno-associated virus (AAV)-derived vector. In accordance with the present disclosure, a recombinant adeno-associated virus (rAAV) vector is used for the GLA transgene.

[0130] Generally, rAAV vectors comprise a capsid and a viral genome modified to include a transgene, for example, a transgene for expressing α-GAL enzyme (i.e., a polynucleotide encoding α-GAL enzyme or a variant thereof). The AAV vectors described herein can comprise or be derived from any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction, and immunogenic properties. Without wishing to be bound by theory, it is believed that AAV capsid proteins often drive the tropism of AAV particles to specific tissues.

[0131] In many gene therapy applications, it is desirable to deliver gene therapy vectors with specificity to specific tissue types. Conventional gene therapy approaches for the treatment of Fabry disease have had 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 administration to a subject in need thereof. The rAAV vectors described herein have a wide tissue distribution, including, for example, the heart, liver, kidney, gastrointestinal tract, and nervous tissue.

[0132] 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. 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. "Broad tissue tropism" means that the capsid can enable gene transfer into two, 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 enables gene transfer into one or more of the following tissues: the liver, kidney, heart, gastrointestinal tract, and / or peripheral neurons of a subject.

[0133] 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.

[0134] Thus, in some embodiments, an AAV capsid with broad tropism comprises an AAV1 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV2 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV3 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV4 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV5 sequence. In some embodiments, an AAV capsid with broad tropism comprises an AAV6 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV7 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV8 capsid. In some embodiments, an AAV capsid with broad tropism comprises an AAV9 capsid.

[0135] Without wishing to be bound by theory, it is understood that the AAV capsid sequence comprises the VP1 region. In some embodiments, the parent AAV capsid sequence comprises the VP1, VP2, and / or VP3 regions, or any combination thereof. The parent VP1 sequence may be considered synonymous with the parent AAV capsid sequence.

[0136] In some embodiments, the rAAV capsid sequence may comprise an amino acid sequence having 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the above.

[0137] Recombinant or modified AAV vectors may contain modified capsids with enhanced tropism to target a wide range of tissues or to target neural tissues (e.g., DRG). Capsid modification methods have been used to identify capsids with enhanced delivery to a wide range of tissues (e.g., kidney, liver, lung, heart, brain, spinal cord, DRG). Various methods have been used, including mutagenesis, DNA barcoding, directed evolution, random peptide insertion, and capsid shuffling and / or chimeras.

[0138] In a preferred embodiment, the rAAV vectors described herein comprise an AAV9 capsid sequence. In some examples, the rAAV vectors described herein comprise a capsid sequence that is 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the AAV9 capsid sequence.

[0139] AAV vectors contain one or more inverted terminal repeat (ITR) sequences. Inverted terminal repeats (ITRs) traditionally cap both the 5' and 3' ends of the viral genome and provide origins of replication for the viral genome. Without wishing to be bound by theory, AAV viral vectors typically contain two ITR sequences at the 5' and 3' ends of the ssDNA, forming energetically stable double-stranded regions. In some embodiments, the AAV vector is a recombinant AAV viral vector that is replication-deficient and lacks sequences encoding functional Rep and Cap proteins within the viral genome. These defective AAV vectors may lack most or all of the parent coding sequence and essentially retain only one or two AAV ITR sequences and a GLA transgene for delivery to cells, tissues, organs, or organisms.

[0140] In some embodiments, the rAAV vectors described herein contain at least one ITR and a GLA transgene. In some embodiments, the rAAV vectors have two ITRs. These two ITRs flank the 5' and 3' ends of the transgene region. The ITRs function as origins of replication containing replication recognition sites. The ITRs contain sequence regions that can be complementary and symmetrically arranged. The ITRs incorporated into the viral genomes described herein can consist of natural or recombinantly obtained polynucleotide sequences. The ITRs can be derived from the same serotype as the AAV capsid or its derivatives, selected from any of the known AAV serotypes. The ITRs can also be of a different serotype from the capsid.

[0141] Independently, the length of each ITR may be about 100 to about 150 nucleotides. The length of an ITR may be about 100 to 105 nucleotides, 106 to 110 nucleotides, 111 to 115 nucleotides, 116 to 120 nucleotides, 121 to 125 nucleotides, 126 to 130 nucleotides, 131 to 135 nucleotides, 136 to 140 nucleotides, 141 to 145 nucleotides, or 146 to 150 nucleotides. In some embodiments, the length of an ITR is 140 to 142 nucleotides. Non-limiting examples of ITR lengths are 102, 105, 130, 140, 141, 142, and 145 nucleotides. ITRs encompassed by this disclosure include ITRs that have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to known AAV serotype ITR sequences.

[0142] In some embodiments, the rAAV vector comprises at least one element to enhance transgene specificity and expression (see, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015). Non-limiting examples of elements that enhance transgene specificity and expression include promoters, endogenous miRNAs, posttranscriptional regulatory elements (PREs), polyadenylation (polyA) signal sequences and upstream enhancers (USEs), CMV enhancers, and introns.

[0143] Those skilled in the art will recognize that expression of a transgene in a target cell may require a specific promoter, including, but not limited to, a species-specific, inducible, tissue-specific, or cell cycle-specific promoter (Parr et al., Nat. Med. 1997, 3:1145-1149). In some embodiments, the promoter is believed to be efficient at driving expression of a transgene (e.g., a GLA transgene) in an rAAV vector. In some embodiments, the promoter is a promoter believed to be efficient at driving expression in a targeted cell. In some embodiments, the promoter is a promoter that has tropism for a targeted cell, such as a neuron.

[0144] By way of non-limiting example, a promoter is selected to provide persistent expression of the transgene in tissues and / or cells of the central or peripheral nervous system.

[0145] The promoter may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include those derived from viruses, plants, mammals, or humans. In some embodiments, the promoter may be derived from a human cell or system. In some embodiments, the promoter may be truncated or mutated.

[0146] Promoters that drive or enhance expression in most tissues include, but are not limited to, the human elongation factor 1I subunit (EF1I) promoter, the cytomegalovirus (CMV) immediate-early enhancer and / or promoter, the chicken u-actin (CBA) promoter and its derivative CAG, the u-glucuronidase (GUSB) promoter, or the ubiquitin C (UBC) promoter. In some embodiments, the viral vector comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include EF-1I, PGK, UBC, GUSB (hGBp), and UCOE (the promoter of HNRPA2B1-CBX3).

[0147] In some embodiments, the promoter sequence is a ubiquitous promoter sequence. In some embodiments, the promoter is a mammalian ubiquitous promoter that drives expression of a coding sequence (e.g., a GLA transgene) in mammalian cells. In some embodiments, an rAAV vector expressing a GLA transgene with a ubiquitous promoter allows the encoded α-GAL to be widely distributed to multiple target tissues in a mammal, such as the kidney, liver, lung, heart, and nervous system, thereby achieving broader exposure of α-GAL and better treatment of Fabry disease and its associated symptoms. As non-limiting examples, the ubiquitous promoter used in the present disclosure can be selected from one or more of the following: EF-1α promoter, UBC promoter, LSE β-glucuronidase (GUSB) promoter, ubiquitous chromatin opening element (UCOE) promoter, GAPDH promoter, chicken β-actin (CBA) promoter, PGK promoter, CMV promoter, and mini-EF1 promoter. In some embodiments, the ubiquitous promoter can be modified from one of the more well-known ubiquitous promoters. In some embodiments, the ubiquitous promoter comprises a cytomegalovirus (CMV) enhancer, a chicken beta-actin promoter, and a rabbit beta-globin intron.

[0148] In some embodiments, cell type-specific promoters may be used to increase exposure of the GLA transgene to excitatory neurons (e.g., glutamatergic), inhibitory neurons (e.g., GABAergic), neurons of the sympathetic or parasympathetic nervous system, sensory neurons, neurons of the dorsal root ganglion, dorsal root nerves, motor neurons, or supporting cells of the nervous system, such as microglia, astrocytes, oligodendrocytes, and / or Schwann cells.

[0149] In some embodiments, the promoter may be a combination of two or more components of the same or different starting promoters or parent promoters.

[0150] In some embodiments, rAAV vector components can be selected and / or modified to further tailor the specificity and efficiency of expression of the GLA transgene in the nervous system (e.g., DRG).

[0151] In some embodiments, the rAAV vector optionally comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (e.g., having a mut6delATG mutation). In some examples, the WPRE element is located between the nucleic acid sequence encoding the α-GAL enzyme or variant thereof and the polyA sequence.

[0152] In some embodiments, while using a capsid sequence with a broad tropism and ubiquitous promoter, the rAAV vector may optionally further include a nervous system targeting peptide to further increase expression of the GLA transgene in the nervous system to alleviate neurological damage in Fabry disease (e.g., DRG and other cells of the CNS and PNS). In some embodiments, the targeting peptide may direct the AAV particles to cells or tissues of the PNS, such as, but not limited to, the dorsal root ganglion (DRG). The length of the targeting peptide may vary. In some embodiments, the length of the targeting peptide is 3 to 20 amino acids. As non-limiting examples, the length of a targeting peptide can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids.

[0153] In certain aspects, the rAAV vector further comprises a post-transcriptional regulatory element (PRE). 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 sequence. Various mutant forms of WPRE are known, including, for example, mut6delATG (SEQ ID NO: 17). In some embodiments, the vector comprises mut6delATG (SEQ ID NO: 17).

[0154] 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 start site (e.g., methionine), the addition of a Kozak sequence (gccacca), 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 start 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 modified. In yet other embodiments, the GLA gene is codon-optimized and modified.

[0155] 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.

[0156] In some embodiments, the vector comprises an ID tag, such as a stuffer sequence. The purpose of the ID tag includes, for example, allowing a technician to identify the vector. An example of a DNA tag sequence is shown in Table 3 (SEQ ID NO: 20).

[0157] In some embodiments, the AAV vector is modified in one or more regions, such as the AAV capsid. In some embodiments, the rAAV vector is an rAAV9 vector.

[0158] Exemplary sequences of rAAV vectors are shown below in Table 2. In some embodiments, the rAAV vector comprises an rAAV vector element comprising a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the vector element sequences shown below in Table 3. In some embodiments, the rAAV vector comprises a vector element nucleotide sequence that is identical to the vector element nucleotide sequence shown below in Table 3.

[0159] [Table 3-1]

[0160] [Table 3-2]

[0161] In some embodiments, the disclosure encompasses an rAAV vector packaged within an AAV capsid with broad tissue tropism, where the rAAV vector comprises a) a 5' inverted terminal repeat (ITR), b) a ubiquitous promoter, c) a polynucleotide encoding an α-GAL enzyme or a variant thereof, d) polyA, and e) a 3' ITR sequence.

[0162] In some embodiments, the rAAV vector further comprises a post-transcriptional regulatory element (PRE). In some embodiments, the rAAV vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (e.g., one having a mut6delATG mutation) between the nucleotide sequence encoding the α-GAL enzyme and the polyA sequence. As a non-limiting example, the rAAV vector comprises a) a 5' inverted terminal repeat (ITR), b) a ubiquitous promoter, c) a polynucleotide encoding the α-GAL enzyme or a variant thereof, d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e) polyA, and f) a 3' ITR sequence.

[0163] In other embodiments, an rAAV vector capable of achieving broad α-GAL enzyme expression upon administration to a subject in need thereof comprises (a) a 5' inverted terminal repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken β-actin promoter, and a rabbit β-globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme or a variant thereof, (d) a bovine growth hormone (BGH) polyA, and (e) a 3' ITR.

[0164] In some embodiments, an rAAV vector capable of achieving broad α-GAL enzyme expression upon administration to a subject in need thereof comprises (a) a 5' inverted terminal repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken β-actin promoter, and a rabbit β-globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme or a variant thereof, (d) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) with a mut6delATG mutation, (e) a bovine growth hormone (BGH) polyA, and (f) a 3' ITR.

[0165] 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 terminal repeat (ITR), (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken β-actin promoter, and a rabbit β-globin intron, (c) a nucleotide sequence encoding an α-GAL enzyme or a variant thereof, (d) a bovine growth hormone (BGH) polyA, and (e) a 3' ITR sequence.

[0166] 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 terminal repeat (ITR); (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken β-actin promoter, and a rabbit β-globin intron; (c) a nucleotide sequence encoding an α-GAL enzyme or a variant thereof; (d) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) with a mut6delATG mutation; (e) a bovine growth hormone (BGH) polyA; and (f) a 3' ITR sequence.

[0167] In some embodiments, the present disclosure provides an expression cassette comprising a polynucleotide sequence comprising: (a) a 5' inverted terminal repeat (ITR); (b) a ubiquitous promoter comprising a cytomegalovirus (CMV) enhancer, a chicken β-actin promoter, and a rabbit β-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 above expression cassette elements are present in 5' to 3' order. In various embodiments, one or more of (a) through (f) are operably linked in 5' to 3' order.

[0168] In one example, the disclosure provides an rAAV vector packaged in an AAV capsid, the rAAV vector comprising: (a) a 5' inverted terminal repeat (ITR) comprising SEQ ID NO: 14; (b) a promoter comprising SEQ ID NO: 15; (c) a nucleotide sequence encoding an α-GAL enzyme comprising the amino acid sequence of any one of SEQ ID NOs: 1-3 and 5-7; (d) a bovine growth hormone (BGH) polyA comprising SEQ ID NO: 17; and (e) a 3' ITR sequence comprising SEQ ID NO: 18. In another example, the disclosure provides an rAAV vector packaged in an AAV capsid, the rAAV vector comprising: (a) a 5' inverted terminal repeat (ITR) comprising SEQ ID NO: 14; (b) a promoter comprising SEQ ID NO: 15; (c) a nucleotide sequence encoding an α-GAL enzyme comprising the amino acid sequence of any one of SEQ ID NOs: 1-3 and 5-7; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising SEQ ID NO: 17; (e) a bovine growth hormone (BGH) polyA comprising SEQ ID NO: 18; and (f) a 3' ITR sequence comprising SEQ ID NO: 18.

[0169] As a non-limiting example, the present disclosure provides an rAAV vector comprising (a) a 5' inverted terminal repeat (ITR) comprising SEQ ID NO: 14, (b) a promoter comprising SEQ ID NO: 15, (c) a nucleotide sequence encoding an α-GAL enzyme or a variant thereof comprising any one of SEQ ID NOs: 8-10 and 12-13, (d) a bovine growth hormone (BGH) polyA comprising SEQ ID NO: 17, and (e) a 3' ITR sequence comprising SEQ ID NO: 18. In some embodiments, the present disclosure provides an rAAV vector comprising (a) a 5' inverted terminal repeat (ITR) comprising SEQ ID NO: 14, (b) a promoter comprising SEQ ID NO: 15, (c) a nucleotide sequence encoding an α-GAL enzyme or a variant thereof comprising any one of SEQ ID NOs: 8-10 and 12-13, (d) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising SEQ ID NO: 16, (e) a bovine growth hormone (BGH) polyA comprising SEQ ID NO: 17, and (f) a 3' ITR sequence comprising SEQ ID NO: 18.

[0170] rAAV vector production In various embodiments, the rAAV vectors described herein for delivering a transgene (e.g., a gene encoding an alpha-galactosidase (alpha-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 as HEK293, HeLa, HEK293T, Sf9 cells, or A549 cells.

[0171] Viral vectors used in gene therapy are typically generated 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, with other viral sequences replaced by an expression cassette encoding the protein to be expressed. In this case, the protein to be expressed is the α-GAL enzyme or a variant thereof. Missing viral functions can be supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the inverted terminal repeat (ITR) sequences from the AAV genome, which are required for packaging and integration into the host genome.

[0172] Methods for producing and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. Patent Nos. 7,790,449, 7,282,199, and 7,588,772, PCT Publication Nos. WO 2003 / 042397, WO 2005 / 033321, and WO 2006 / 110689. In one system, a packaging cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct(s) encoding the AAV rep and cap proteins. 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 viral particles are produced in response to infection with a helper adenovirus or herpesvirus (e.g., adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase), which helps separate the rAAV vector from contaminating viruses.

[0173] Other systems may be used that do not require helper virus infection to recover AAV. In these newer systems, helper functions may be supplied by transient transfection of cells with constructs encoding the necessary helper functions, or cells may be engineered to stably contain genes encoding helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level.

[0174] In some embodiments, the expression cassette flanked by the ITRs and rep / cap genes is introduced into packaging cells by infection with a baculovirus-based vector (e.g., Zhang et al., Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production; Human Gene Therapy, 2009;20:922-929, the contents of which are incorporated herein by reference in their entirety). Other AAV production systems and methods are also described in, for example, 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.

[0175] Methods and techniques for generating transgene expression cassettes, rAAV vectors, helper plasmids, and constructs, such as genetic engineering, recombinant engineering, and synthetic techniques, are well known in the art (see, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)). Similarly, methods for generating rAAV virions are well known, and the selection of a suitable method is not a limitation of the present invention.

[0176] Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known to those of skill in the art and are readily available. 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 of the present invention will be readily apparent to those of skill in the art from the present disclosure.

[0177] In certain embodiments, an rAAV expression cassette, vector (e.g., an rAAV vector), virus (e.g., an rAAV), or production plasmid comprises an AAV inverted terminal repeat, a codon-optimized nucleic acid sequence encoding an α-GAL polypeptide, and expression control sequences directing expression of the encoded protein, and is present in a host cell. In other embodiments, the rAAV expression cassette, virus, vector (e.g., 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.

[0178] In some embodiments, the helper plasmids include a first helper plasmid containing the rep gene and the cap gene, and a second helper plasmid containing one or more of the helper genes Ela, Elb, E4, E2a, and VA. For clarity, helper genes are genes encoding the helper proteins Ela, Elb, E4, E2a, and VA. In some embodiments, the cap gene is modified to prevent expression of one or more of the proteins VP1, VP2, and VP3. In some embodiments, the cap gene is modified to prevent expression of VP2. Methods for making such modifications are known in the art (Lux et al. (2005), J. Virology, 79:11776-87). Helper plasmids, and methods for producing such plasmids, are generally known in the art and are generally commercially available (see, e.g., pDF6, pRep, pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E and pDP8.ape).

[0179] In some embodiments, the plasmid containing the transgene 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 or a different serotype, and transfected into helper cells such that the rAAV is packaged.

[0180] In some embodiments, packaging is performed in helper or producer cells, such as mammalian or insect cells. 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.

[0181] Various methods related to AAV vector production and purification are known in the art. See, for example, Mizukami, Hiroaki, et al., A Protocol for AAV vector production and purification, U.S. Patent Publication Nos. US20070015238 and US20120322861. For example, a plasmid containing a gene of interest can be combined with one or more helper plasmids containing, for example, the rep gene (encoding, for example, Rep78, Rep68, Rep52, and Rep40) and the cap gene (encoding VP1, VP2, and VP3, including the modified VP2 region described herein) and transfected into recombinant cells, thereby packaging the rAAV and subsequently purifying it.

[0182] Pharmaceutical Composition Exemplary pharmaceutical compositions containing the vectors described herein are detailed below.

[0183] 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 is a wide variety of suitable formulations of pharmaceutical compositions available.

[0184] Formulations for both ex vivo and in vivo administration include suspensions or emulsions. The active ingredient is often mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. 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 (e.g., wetting or emulsifying agents, pH buffering agents, stabilizers, or other agents that enhance the effectiveness of the pharmaceutical composition).

[0185] In some embodiments, pharmaceutical compositions containing the rAAV vectors described herein are provided. Pharmaceutical compositions containing the rAAV vectors or particles of the present invention contain a pharmaceutically acceptable excipient, diluent, or carrier. 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.

[0186] Therapeutic Applications and Methods of Use The transgenes and rAAV vectors of the present disclosure can be used to treat subjects with Fabry disease. Thus, the vectors 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 vectors of the present disclosure can be used to treat subjects with low or no expression of α-GAL and / or deficient activity of the α-GAL enzyme.

[0187] Non-limiting examples of symptoms of Fabry disease include neuropathic pain, hypohidrosis or anhidrosis, exercise intolerance, abdominal cramps, diarrhea, angiokeratoma, cornea verticillata, tinnitus, proteinuria, chronic kidney disease, hypertension, coronary insufficiency, atrioventricular conduction disorders, arrhythmias and valvular dysfunction, left ventricular hypertrophy, seizures and stroke.

[0188] Neuropathic symptoms of Fabry disease include neuropathic pain, decreased temperature and cold sensation (i.e., thermohypesthesia), hearing loss, and, in some cases, gastrointestinal problems. Neuropathy can be caused by peripheral abnormalities of peripheral nerve fibers, particularly small nerve fibers (myelinated and unmyelinated). The damage is caused, in part, by the accumulation of GB3 in the peripheral nervous system, including the DRG.

[0189] In various embodiments described herein, rAAV vectors according to the present disclosure are used to treat peripheral neuropathy and / or one or more symptoms associated with peripheral neuropathy in patients diagnosed with Fabry disease. The patient is experiencing or developing one or more symptoms associated with peripheral neuropathy, such as neuropathic pain. Peripheral neuropathic pain in patients with Fabry disease can manifest as chronic, burning pain and overlapping attacks of acute, severe pain, paresthesia, thermal and cold sensations (primarily cold sensations), and paresthesia (e.g., painless tingling). Symptoms associated with autonomic nervous system dysfunction include decreased sweating, impaired pupil constriction and impaired salivary and tear secretion, gastrointestinal motility disorders (abdominal cramps, bloating, diarrhea, nausea), and sensory loss.

[0190] The rAAV vector expressing the GLA transgene described herein is particularly suitable for alleviating symptoms associated with Fabry disease. In some embodiments, the rAAV vector expressing the GLA transgene can be used to alleviate peripheral neuropathy in a subject diagnosed with Fabry disease. This method includes administering to the subject a composition comprising an rAAV expressing the α-GAL enzyme or its variant, as described herein. This method alleviates symptoms of neuropathy, such as neuropathic pain, decreased temperature sensation (i.e., thermosensitivity), hearing loss, other sensory disorders, and, in some cases, gastrointestinal disorders.

[0191] In some embodiments, the rAAV vectors described herein are used to ameliorate peripheral nerve fiber abnormalities in Fabry disease. Peripheral nerves are small-diameter nerve fibers, including myelinated and unmyelinated nerve fibers. The nerve fibers are cutaneous sensory nerves. In some embodiments, treatment with the vector can maintain the density of nerve fibers in the peripheral nervous system.

[0192] In some embodiments, rAAV vectors are used to reduce GB3 and lysoGb3 accumulation in the peripheral nervous system. In some embodiments, administration of the rAAV vectors described herein improves morphological defects in DRGs, including reduced expression of the surrogate biomarker LAMP1. LAMP1 (lysosomal-associated membrane protein 1) is a biomarker for neurodegeneration. Patients with Fabry disease exhibit severe dorsal root ganglion hypertrophy with dysfunctional perfusion, which is thought to result from glycolipid accumulation in the dorsal root ganglion, mediating a direct neurotoxic effect and reducing blood supply to neurons. Increased exposure of the α-GAL enzyme can reduce LAMP1 in DRGs.

[0193] In some embodiments, an rAAV vector for a GLA transgene reduces globotriaosylceramide (GB3) in a subject. In some embodiments, the rAAV vector reduces GB3 in a subject 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 level before administering the rAAV containing GLA. Thus, in some embodiments, an administered rAAV containing GLA reduces GB3 in a subject by about 95%. In some embodiments, an administered rAAV containing GLA reduces GB3 in a subject by about 90%. In some embodiments, an administered rAAV containing GLA reduces GB3 in a subject by about 85%. In some embodiments, an administered rAAV containing GLA reduces GB3 in a subject by about 80%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 75%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 70%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 65%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 60%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 55%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 50%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 45%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 40%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 35%. In some embodiments, an administered rAAV comprising GLA reduces a subject's GB3 by about 30%. In some embodiments, an administered rAAV comprising GLA reduces GB3 in a subject by about 25%. In some embodiments, an administered rAAV comprising GLA reduces GB3 in a subject by about 20%. In some embodiments, an administered rAAV comprising GLA reduces GB3 in a subject by about 15%.In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 10%.

[0194] In some embodiments, serum GB3 is reduced 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 level before administration of the rAAV expressing a GLA transgene. In some embodiments, kidney, heart, and / or liver GB3 is reduced 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 level before administration of the rAAV expressing a GLA transgene.

[0195] In some embodiments, rAAV vectors are used to reduce GB3 and lysoGb3 accumulation in the kidney. In some embodiments, rAAV vectors are used to reduce GB3 and lysoGb3 accumulation in the liver. In some embodiments, rAAV vectors are used to reduce GB3 and lysoGb3 accumulation in the heart. In some embodiments, rAAV vectors are used to reduce GB3 and lysoGb3 accumulation in serum.

[0196] In some embodiments, an administered rAAV containing GLA produces α-GAL enzyme in the kidney of a subject. In some embodiments, an administered rAAV containing GLA produces α-GAL enzyme in the heart of a subject. In some embodiments, an administered rAAV containing GLA produces α-GAL enzyme in the liver of a subject. In some embodiments, an administered rAAV containing GLA produces α-GAL enzyme in the serum of a subject.

[0197] In some embodiments, LAMP1 levels in the peripheral nervous system (e.g., DRG) are reduced 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 LAMP1 expression before administration of the rAAV expressing the GLA transgene. In some embodiments, rAAV gene therapy reduces lysosomal autophagy organelles in the DRG.

[0198] In some embodiments, the administered rAAV containing a GLA transgene 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.

[0199] In some embodiments, vector-mediated gene therapy provides consistent expression and high α-GAL activity in serum and other tissues (e.g., kidney, heart, and liver, and the nervous system), hi some embodiments, vector-mediated gene therapy provides consistent expression and high α-GAL activity in the peripheral nervous system (e.g., DRG).

[0200] In some examples, functional α-GAL is detectable in the serum and tissues of a subject about 2 to 18 weeks after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the serum and tissues of a subject 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 weeks after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the serum and tissues of a subject 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 or more after administration of the rAAV vector.

[0201] In some embodiments, functional α-GAL is detectable in the subject's nervous system (e.g., DRG) about 2-18 weeks after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the subject's serum and tissues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 weeks or more after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the subject's nervous system (e.g., DRG) 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.

[0202] In some embodiments, after administration of an AAV vector to a subject, the amount of functional α-GAL detectable in the circulation is about 2 to 1000 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 greater than the amount of functional α-GAL detectable in the subject before administration of an rAAV containing a GLA transgene.

[0203] In some embodiments, after administration of an AAV vector to a subject, the amount of functional α-GAL detectable in the PNS 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 greater than the amount of functional α-GAL detectable in the subject before administration of an rAAV containing a GLA transgene.

[0204] In some embodiments, after administration of an AAV vector to a subject, the detectable concentration of 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 level of active α-GAL after administration of an 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, more than 100 times, more than 200 times, more than 300 times, more than 400 times, more than 500 times, more than 600 times, more than 700 times, more than 800 times, more than 900 times, or more than 1000 times the human therapeutic concentration.

[0205] Thus, administration of an 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.

[0206] In some embodiments, after administering the AAV vector to a subject, a 10% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 15% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 20% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 25% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 30% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 35% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 40% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 45% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 50% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 55% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 60% reduction in Gb3 is achieved in ERT-resistant cell types. In some embodiments, after administering the AAV vector to a subject, a 65% reduction in Gb3 is achieved in ERT-resistant cell types.

[0207] In some embodiments, the improvement in Fabry disease symptoms lasts for at least 1 week. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 2 weeks. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 3 weeks. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 4 weeks. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 1 month. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 2 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 3 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 4 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 5 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 6 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 7 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 8 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 9 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 10 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 11 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least 12 months. In some embodiments, the improvement in Fabry disease symptoms lasts for at least one year. In some embodiments, the improvement in Fabry disease symptoms lasts for more than one year.

[0208] In some embodiments, a 10% reduction in Gb3 is achieved in ERT-resistant cell types at 6 months after administering the AAV vector to a subject. In some embodiments, a 15% reduction in Gb3 is achieved in ERT-resistant cell types at 6 months after administering the AAV vector to a subject. In some embodiments, a 20% reduction in Gb3 is achieved in ERT-resistant cell types at 6 months after administering the AAV vector to a subject. In some embodiments, a 25% reduction in Gb3 is achieved in ERT-resistant cell types at 6 months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 is achieved in ERT-resistant cell types at 6 months after administering the AAV vector to a subject. In some embodiments, a 35% reduction in Gb3 is achieved in ERT-resistant cell types at 6 months after administering the AAV vector to a subject. In some embodiments, a 40% reduction in Gb3 is achieved in ERT-resistant cell types at 6 months after administering the AAV vector to a subject. In some embodiments, a 45% reduction in Gb3 in ERT-resistant cell types is achieved 6 months after administration of the AAV vector to a subject. In some embodiments, a 50% reduction in Gb3 in ERT-resistant cell types is achieved 6 months after administration of the AAV vector to a subject.

[0209] In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved one month after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved two months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved three months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved four months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved five months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved six months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved seven months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved 8 months after administering the AAV vector to a subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved 9 months after administering the AAV vector to a subject.

[0210] The rAAV vector is administered to a subject diagnosed with Fabry disease 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 using a "gene gun" or gene gun particle delivery system. In some embodiments, the rAAV vector is administered via a non-viral lipid nanoparticle. For example, a composition comprising an rAAV vector may include one or more diluents, buffers, liposomes, lipids, or lipid complexes. In some embodiments, the rAAV vector is contained within a nanoparticle, such as a microsphere or lipid nanoparticle.

[0211] In some embodiments, the rAAV vector is administered spinally, e.g., by spinal injection. 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 subject's genome. 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.

[0212] In some embodiments, the rAAV vector that comprises GLA transgene is administered to the subject in need as a single dose.In some embodiments, the rAAV vector is administered to the subject at 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 causes GB3 amount reduction in the subject.

[0213] In some embodiments, the dose is 1×10 10 vg / kg (viral genome / kilogram of body weight) ~ 1 x 10 14 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1 x 10 10 vg / kg~5×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1 x 10 vg / kg. 10 vg / kg~1×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1 x 10 vg / kg. 10 vg / kg~5×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1 x 10 vg / kg. 10 vg / kg~1×10 12In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1 x 10 vg / kg. 10 vg / kg~5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 1 x 10 vg / kg. 10 vg / kg~2.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5×10 10 vg / kg~1×10 14 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5×10 10 vg / kg~5×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5×10 10 vg / kg~1×10 13 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5×10 10 vg / kg~5×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5×10 10 vg / kg~1×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5×10 10 vg / kg~5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at doses ranging from 5×10 10 vg / kg~2.5×10 11 Administer at doses ranging from 0.1 mg / kg to 0.2 mg / kg.

[0214] In some embodiments, the rAAV vectors of the disclosure are administered in a volume of 1.5 x 10 10 ~2.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 1 x 10 vg / kg. 10 ~2×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2.5 x 10 vg / kg.10 ~3.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2.5 x 10 vg / kg. 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 3.5 x 10 vg / kg. 10 ~4.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 4.5 x 10 vg / kg. 10 ~5.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 5.5 x 10 vg / kg. 10 ~6.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 6.5 x 10 vg / kg. 10 ~7.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 7.5 x 10 vg / kg. 10 ~8.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 8.5 x 10 vg / kg. 10 ~9.5×10 10 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 9.5 x 10 vg / kg. 10 ~1×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 1 x 10 vg / kg. 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 1 x 10 vg / kg. 11 ~1.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 1.5 x 10 vg / kg. 11 ~2.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2.5 x 10 vg / kg. 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2 x 10 vg / kg. 11 ~2.5×10 11In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2.5E11 to 3.5 x 10 vg / kg. 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 3.5 x 10 vg / kg. 11 ~4.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 4.5 x 10 to 5.5 x 10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 5.5 x 10 vg / kg. 11 ~6.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 6.5 x 10 vg / kg. 11 ~7.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 7.5 x 10 vg / kg. 11 ~8.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 8.5 x 10 vg / kg. 11 ~9.5×10 11 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 9.5 x 10 vg / kg. 11 ~1×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 1 x 10 vg / kg. 12 vg / kg~2×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 1.5 x 10 12 vg / kg~2.5×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2.5 x 10 12 vg / kg~3.5×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 3.5 x 10 12 vg / kg~4.5×10 12 In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 4.5 x 10 12 vg / kg~5.5×10 12Administer at a dose of

[0215] In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2.5×10 vg / kg to 1×10 vg / kg. In some embodiments, the rAAV vectors of the disclosure are administered at a dose of 2.5×10 vg / kg to 2.5×10 vg / kg.

[0216] In some embodiments, rAAV gene therapy vectors expressing the alpha-galactosidase proteins described herein are administered to a subject at doses lower than or equivalent to those used with other gene therapy vectors (e.g., AAVs with liver-specific promoters), yet still exhibit higher serum and tissue exposure, e.g., higher and sustained exposure in the PNS (e.g., DRG).

[0217] In some embodiments, rAAV vectors expressing a GLA transgene provided herein are used as prophylactic treatments in subjects with Fabry disease to prevent the onset and / or progression of one or more symptoms of Fabry disease. In one embodiment, rAAV vectors expressing a GLA transgene provided herein are used as prophylactic treatments in subjects with Fabry disease to prevent the onset and / or progression of one or more peripheral neuropathy manifestations.

[0218] A prophylactic treatment may be administered, for example, to a subject who is not yet ill but who is susceptible to or otherwise at risk for a particular biological condition, including Fabry disease (e.g., a subject may have a mutation that causes Fabry disease but is asymptomatic or whose Fabry disease-causing mutation status is unknown). In some embodiments, therapeutic treatment may be administered to a subject who already has Fabry disease, for example, to improve or stabilize the subject's condition (e.g., a patient who is already experiencing symptoms of Fabry disease).

[0219] peripheral neuropathy In some embodiments, the methods further include evaluation methods and tests to assess improvement in peripheral neuropathy in Fabry disease patients treated with rAAV-mediated gene therapy as contemplated in the present disclosure.

[0220] As discussed in this disclosure, hot plate latency tests in a mouse model of Fabry disease show that G3Stg / GLAko mice treated with an rAAV vector containing the GLA transgene significantly responded to heat stimuli with sensitivity similar to that of wild-type mice (e.g., Figure 5). The results observed in mice suggest that rAAV-mediated GLA gene therapy may achieve similar improvements in heat sensitivity in patients with Fabry disease. Therefore, peripheral neuropathy in patients with Fabry disease can be measured using commonly used clinical methods to evaluate the efficacy of rAAV-mediated GLA gene therapy described herein.

[0221] Patients with Fabry disease may be diagnosed at an early age with the onset of neuropathy-related symptoms. In some cases, patients may be diagnosed with the onset of neuropathy-related symptoms in adulthood. Physicians (e.g., neurologists) generally recognize a set of distinct early symptoms of Fabry disease, some of which are associated with small fiber neuropathy. Symptoms include chronic burning pain, attacks of severe pain, paresthesias / paresthesias, sensory loss, hypohidrosis / anhidrosis, abdominal cramps, diarrhea (after meals), bloating, nausea, and tinnitus and hearing loss.

[0222] Neuropathic pain scales may be used during initial assessment and follow-up after treatment. Neuropathic symptom and change questionnaires assess the number, severity, and change of symptoms, as well as the function of motor, autonomic, large-diameter, and small-diameter sensory nerve fibers (Dyck et al., Longitudinal assessment of diabetic polyneuropathy using a composite score in the Rochester Diabetic Neuropathy Study cohort. Neurology. 1997, 49:229-239). Other available neuropathic pain assessment tools include the Leeds Assessment of Neuropathic Symptoms and Signs, the Neuropathic Pain Questionnaire, the Neuropathic Pain Symptom Inventory, the Douleur Neuropathique en 4 questions, pain DETECT, and the ID-pain. Composite symptom scores have been used to grade neuropathic pain in clinical studies of patients with diabetes and Fabry disease with peripheral neuropathy.

[0223] Neurological testing of peripheral small-fiber-mediated sensation may be performed before and after treatment, using, for example, a fairly crude thermal sensation test in which tubes filled with cold and hot water, cold and hot tuning forks, a reflex hammer handle, or a thermal disk with a polyvinyl surface on one side and a metal surface on the other (similar to a "Minnesota Thermal Disc") are placed on the patient's foot.

[0224] A patient's pain perception and hyperalgesia can be tested before and after treatment by applying indentation pressure to the skin with a sharp pin. An inability to distinguish between pinprick and blunt pressure suggests small fiber neuropathy.

[0225] In some embodiments, Fabry disease patients who have or are developing one or more neuropathy-related symptoms are treated with the rAAV vector expressing the GLA transgene described herein.The same evaluation is followed after treatment to assess the improvement of neuropathy-related symptoms.In some examples, evaluation is performed 2 weeks, 5 weeks, 10 weeks, 18 weeks, 6 months, or more after treatment.

[0226] In some embodiments, peripheral neuropathy is quantified by a computer-based sensory assessment device, as described by Dyck and O'Brien (Quantitative sensation testing in epidemiological and therapeutic studies of peripheral neuropathy. Muscle Nerve. 1999;22:659-662) and Schiffmann, et al. (Enzyme replacement therapy improves peripheral nerve and sweat function in Fabry disease. Muscle Nerve. 2003;28:703-710), the contents of which are incorporated herein by reference in their entireties.

[0227] In some embodiments, the patient being treated with the method measures thermal sensation. The perception of cold in the feet is a primary indicator of Fabry disease neuropathy and can be quantified on a scale of 1 to 25 in units of just noticeable difference (JND) to assess treatment outcome (see Ries et al., 2007). With respect to experimental measurements focused on sensation and perception, the term "just noticeable difference" or "JND" refers to the amount that must be changed for the difference to be perceptible and detectable at least most of the time.

[0228] In some embodiments, quantitative sensory testing (QST) may be used to examine improvement in a patient's neurological deficits, and QST may be performed according to standard procedures (Rolke et al., Pain. 2006;123:231-243, the contents of which are incorporated herein by reference in their entirety). In some embodiments, calibrated thermal electrodes may be used for QST before and after rAAV treatment.

[0229] In some embodiments, peripheral nerve fiber morphology is imaged to measure treatment outcome.

[0230] In some embodiments, a skin biopsy is taken from the patient to assess cutaneous nerve fiber density, for example, a skin specimen is taken from the lower leg and subjected to standard immunostaining procedures to assess nerve fiber morphology.

[0231] In some embodiments, nerve conduction activity may be measured before and after rAAV treatment. As a non-limiting example, microneurography may be used to record action potentials in human nerve fibers (e.g., skin fibers).

[0232] Combination therapy The compositions and methods of the present invention can also be used in combination with other therapeutic agents known in the art used to treat Fabry disease or its complications, including, but not limited to, SRT (substrate synthesis inhibition therapy), ERT (e.g., agalsidase beta), pain relief medications (e.g., lidocaine, diphenylhydantoin, carbamazepine, gabapentin, phenytoin, neurotropin, opioids), dyspepsia medications (e.g., metoclopramide, H2 blockers), vitamin D supplementation, etc., beta blockers (metoprolol, acebutolol, bisoprolol, atenolol, propranolol, etc.), and anticoagulant medications (heparin, warfarin, apixaban, rivaroxaban).

[0233] The compositions and methods of the present invention can also be used in conjunction with other forms of treatment, including, but not limited to, physical exercise (e.g., dialysis, kidney transplantation), dietary salt restriction, dietary fiber intake, pacemaker placement, and heart transplantation. [Example]

[0234] Illustrative features, objects, and advantages of the present invention will be apparent from the following examples. It should be understood, however, that the examples, while illustrating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0235] Example 1. Production and purification of a viral vector expressing α-GAL enzyme This example summarizes the viral vectors encompassed by the present disclosure.

[0236] Recombinant adeno-associated virus 9 (rAAV9) was constructed to express wild-type human α-GAL or α-GAL variants (e.g., the amino acid sequences shown in Table 1) under the control of a ubiquitous promoter in a viral vector. A WPRE element was linked to the 3' end of the 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. The promoter-GLA-WPRE-BGHpA DNA construct was integrated between the inverted terminal repeats of a circular plasmid vector. Figure 1 shows an exemplary rAAV9 vector construct.

[0237] Using methods known in the art, rAAV vectors were packaged using the AAV2 inverted terminal repeat and rep sequences. rAAV9 stocks were produced using an adenovirus-free triple-plasmid co-transfection method in HEK-293T cells and purified using cesium chloride ultracentrifugation. vg particle count titers were determined by quantitative PCR.

[0238] The purified rAAV9 virus suspension was diluted with 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.

[0239] Two rAAV vectors, Variant 1 and Variant 2, were prepared and used in the following studies. A diagram of the transgene expression cassette is shown in Figure 1. Variant 1 contains the codon-optimized nucleic acid sequence of SEQ ID NO: 12. Variant 2 contains the codon-optimized nucleic acid sequence of SEQ ID NO: 13.

[0240] Example 2. α-GAL activity in serum after administration In this example, we demonstrate that variant 1 and variant 2 resulted in expression of α-GAL protein in serum for at least 18 weeks after administration of the vector to mice.

[0241] For this study, we used an exacerbated mouse model of Fabry disease (G3Stg / GlaKO) generated by crossing Gla knockout (GlaKO) mice with transgenic mice expressing the human Gb3 synthase (G3Stg) to increase Gb3 accumulation. These mice had up to 10-fold higher levels of the substrate GB3 in various tissues compared with age-matched GlaKO mice, reflecting the symptoms observed in Fabry disease patients.

[0242] Male G3Stg / GLAko mice aged 8–12 weeks were divided into groups (n = 6–12 / group) and injected with purified rAAV9 vectors expressing variant 1 and variant 2 at two different doses (2.5 × 10 11 ,5.0×10 10 rAAV9-null vector (control) was administered intravenously (IV) at 2.5 × 1011 Groups of G3Stg / GLAko mice were administered 200 mg / kg of ribozyme. A vehicle-treated wild-type (WT:WT) group was also used as a control. The study design is summarized in Table 4 below.

[0243] G3Stg / GLAko mice showed a significant loss of body weight over the study period, and mice treated with rAAV9 vectors expressing variant 1 or variant 2 weighed more compared with mice receiving null AAV vector or vehicle alone (data not shown).

[0244] Serum was collected at multiple time points during the study and at the end of the 18-week period.

[0245] [Table 4]

[0246] The serum α-GAL levels were measured 18 weeks after administration.

[0247] Example 3. α-GAL activity in serum and various tissues after vector administration This example demonstrates sustained activity of α-GAL in the kidney, heart, and liver 18 weeks after intravenous administration of the vector in the same experiment described in Example 2, as shown in Table 4. A sustained increase in serum α-GAL activity was also observed throughout the study after vector administration at both doses (Figure 2A). Serum α-galactosidase activity was increased by at least 6,700-fold compared to vehicle.

[0248] Mouse tissues were homogenized in lysis buffer containing 10 mM HEPES and EDTA-free 1.5x Halt protease inhibitor cocktail supplemented with 0.5% Triton-X100, centrifuged, and the supernatant was collected for analytical assays. α-Galactosidase (α-GAL) activity in the supernatant or serum was measured using a fluorescent substrate. Briefly, 2 μL of biological sample was incubated with 15 μL of 4-MU-α-gal substrate solution (Research Products International Company, catalog number M65400) containing α-galactosidase B inhibitor (N-acetyl-D-galactosamine, Sigma catalog number A-2795) at 37°C for 60 minutes. The enzymatic reaction was stopped by the addition of 200 μL of glycine carbonate stop solution (pH 10.7). 4-MU product was measured using 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 to total protein concentration as determined by the BCA assay (Thermo Scientific, Cat. No. 23225).

[0249] The data from this example showed that both variant 1 and variant 2 induced persistent expression of α-GAL in the kidney, heart, and liver (Figures 2B, 2C, and 2D). Higher exposure in the liver meant that more enzyme crossed the BBB and reached the nervous system (e.g., PNS). α-galactosidase activity in the kidney was increased by at least 150-fold compared to vehicle. α-galactosidase activity in the heart was increased by at least 950-fold compared to vehicle.

[0250] Furthermore, Figure 2E compares the α-galactosidase activity in the kidney and heart of Fabry mice transfected with 5e10 vg / kg of human wild-type α-galactosidase and variant 1, respectively. Table 5 shows the fold increase in serum activity following transfection with wild-type human α-galactosidase and variant 1.

[0251] [Table 5]

[0252] Figure 2F shows that elevated α-GAL activity was also observed in the serum of mice transfected with variant 1 and human α-galactosidase compared with the control group. However, the α-galactosidase activity was higher in mice transfected with variant 1 compared with wild-type human α-galactosidase.

[0253] Figure 2G shows serum α-galactosidase concentrations in non-human primates (NHPs) after 21 days of single intravenous administration of two different doses (6.25e12vg / kg and 3e13vg / kg) of Variant 1. In NHPs, serum α-GAL levels were observed to reach more than 50-fold the predicted effective therapeutic level, even at the lowest dose tested, 6.25e12vg / kg.

[0254] High levels of enzyme exposure in the kidney and heart were confirmed by immunohistochemistry (Figures 7A and 7B). Furthermore, α-GAL A enzyme exposure was detected in difficult-to-access cell types, such as cardiac cardiomyocytes and renal podocytes (Figures 7A and 7B).

[0255] Example 4. Substrate levels after vector administration In this example, we investigated the effect of variant 1 and variant 2 on the reduction of GB3 and lysoGb3, which shows GB3 and lysoGb3 substrate levels 18 weeks after vector administration, as described in Example 2.

[0256] G3Stg / GLAko mice have significantly elevated substrate levels in various tissues. Levels of GB3 and lysoGb3 were analyzed by mass spectrometry in severe Fabry disease (G3Stg / GLAko) mice treated with the constructs listed in Table 4. Substrates from serum and tissue samples were analyzed using LC-MS. Briefly, substrate 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).

[0257] We observed that both variant 1 and variant 2 were able to reduce Fabry disease-associated Gb3 accumulation in terminal serum, kidney, heart, and liver (Figures 3A-3D). A greater than 90% reduction in Gb3 was observed in kidney and heart compared to vehicle-treated Fabry disease mice. Furthermore, Figure 3E compares the substrate reduction in kidney and heart of Fabry mice when wild-type human α-galactosidase or variant 1 was introduced. Table 6 shows the fold reduction in kidney and heart of Fabry mice when wild-type human α-galactosidase or variant 1 was introduced.

[0258] [Table 6]

[0259] Similarly, after 18 weeks of treatment, we observed a decrease in lysoGb3 accumulation in serum, kidney, heart, and liver (Figures 4A-4D). The reduction in substrate in multiple tissues demonstrates a systemic effect of rAAV-directed gene therapy. Treatment with variant 1 or variant 2 resulted in complete or near-complete normalization of substrate in Fabry disease mice.

[0260] Example 5. Restoration of thermosensitivity by variant 1 and variant 2 in a mouse model of Fabry disease (G3Stg / GLAko mice) This example demonstrates the effect of rAAV vectors expressing variant 1 and variant 2 on the thermosensitivity of G3Stg / GLAko mice.

[0261] In this study, we used the hot plate latency test to test the response of mice to thermal stimuli. The hot plate test (Eddy and Leimbach, 1953) is a simple behavioral screen used to estimate the effect of NCEs on pain detection thresholds. It is based on the principle that when rodents are placed on a hot surface, they will initially demonstrate the aversive effects of the thermal stimulus by licking or flicking their paws, and ultimately by overt attempts to escape the environment (jumping). Substances that alter the nociceptive threshold either prolong (analgesic effect) or shorten (hyperalgesic effect) the latency to lick / jump. The hot plate test is a rapid and relatively inexpensive method for assessing acute thermal pain, and its advantage over tail flick / tail withdrawal is the opportunity to test thermal sensitivity. This simple sensitivity test in mice is often used to examine neuropathic pain (e.g., heat-induced) in human patients.

[0262] For the study, mice from each group (Table 4) were tested on a hot plate before administration and 2, 4, 8, 12, 16, and 18 weeks after administration. The hot plate (Columbus Instruments) was preheated to 55°C. To prevent escape, an open, cylindrical, transparent Plexiglas tube with a diameter of 30 cm was placed on the hot plate, leaving the animal's paw exposed to the hot plate. A stopwatch was used to measure the time from placing the mouse on the hot plate to the first paw lick. The latency to respond by licking or shaking / flapping the hind paw, whichever occurred first, was measured to the nearest 0.1 second. If no pain response was measured, the mouse was automatically removed after 1 minute to prevent tissue damage.

[0263] Mice receiving variant 1 and variant 2 at a dose of 2.5e11vg / kg restored thermal sensitivity to WT thresholds (Figure 5).

[0264] Example 6. Amelioration of Fabry disease-associated neurological disorders in a mouse model (G3Stg / GLAko mice) In this example, the expression of neuropathic markers was examined in a mouse model of Fabry disease 18 weeks after vector administration.

[0265] G3Stg / GLAko mice exhibit some signs of neuropathy, as observed in histology of peripheral neurons after sacrifice. Footpads from the hind paws of these animals were collected for immunohistochemical analysis to assess small-fiber nerves and monitor any neuropathology in these animals. MPZ (myelin protein zero, the most abundant protein in nerve myelin) staining (Figure 6) showed that dermal nerve bundle morphology improved and density maintained in G3Stg / GLAko mice treated with rAAV9-variant 1, comparable to that of WT mice. As shown in Figure 6, intraepidermal nerve fiber density was maintained in G3Stg / GLAko mice treated with the rAAV9-α-GAL gene therapy vector, but not in G3Stg / GLAko mice treated with the control vector (rAAV-null).

[0266] Example 7. Neuronal structure and lysosomal burden in a mouse model of Fabry disease In this example, we investigated the neural structure and lysosomal burden in a mouse model of Fabry disease 18 weeks after administration of the rAAV9-α-GAL gene therapy vector.

[0267] Dorsal nerve root (DNR) samples were collected from G3Stg / GLAko mice treated with the rAAV9-α-GAL gene therapy vector. Samples were also collected from wild-type and control vector (rAAV-null)-treated G3Stg / GLAko mice. Tissues were fixed and stained. Histological evaluation of fixed tissues was performed by immunohistochemistry or hematoxylin and eosin (HE) staining. Anti-LAMP1 antibody was used for immunohistochemistry.

[0268] As shown in Figure 8A, vacuoles were observed only in G3Stg / GLAko mice treated with the control vector. Treatment with the rAAV9-α-Gal gene therapy vector demonstrated reversal of vacuolation in G3Stg / GLAko mice. LAMP1-positive staining was observed only in G3Stg / GLAko mice treated with the control vector, indicating a reduction in lysosomal load. Substrate clearance resulted in various structural and functional improvements in treated mice, including normalization of lysosomal load and prevention of dorsal root nerve vacuolation.

[0269] Furthermore, Figures 8B, 8C, and 8D demonstrate that reversal of structural pathology in the peripheral nervous system (PNS) led to the restoration and maintenance of sensory impairment. Figure 8B shows the percentage of LAMP1-positive areas in Fabry disease mice compared with Fabry disease mice treated with variant 1 or vehicle and normal mice. Variant 1-treated Fabry disease mice showed a decrease in LAMP1-positive areas compared with vehicle-treated Fabry disease mice. Furthermore, hot plate latencies were observed on days 2 and 12 in Fabry disease mice treated with variant 1 and control (vehicle) compared with normal control (vehicle-treated) mice (Figure 8C). Hot plate latencies, indicating sensory impairment in Fabry disease mice, were observed to be restored by partial clearance of DRG substrate. Furthermore, DRG substrate load, assessed using the logarithm of LAMP1 density, correlated with hot plate latencies, whereas small fiber density did not correlate with hot plate latencies (Figure 8D). This finding appears to be consistent with reports of the mechanisms of PNS pathology in patients.

[0270] Figure 8E shows mouse DRG-MRI images to assess the effect of gene therapy (GT) treatment on DRG volume in Fabry disease mice treated with vehicle and a tool gene therapy construct expressing wild-type human α-galactosidase (GT) compared with normal mice. Figure 8F shows histograms of DRG area in gene-treated mice versus control (vehicle-treated) or normal mice. A sustained reduction in DRG volume was observed in gene-treated mice up to 24 weeks. Gene therapy is expected to substantially improve neurophysiological function in humans with Fabry disease. Fabry disease patients exhibited a significant increase in DRG volume, which was unaffected by ERT treatment. Therefore, DRG volume is a potential surrogate marker for monitoring the efficacy of treatment on PNS pathology in Fabry disease.

[0271] Example 8. Gastrointestinal vacuolization and lysosomal burden in a mouse model of Fabry disease In this example, vacuolation and lysosomal burden in the gastrointestinal tract of a mouse model of Fabry disease were examined 18 weeks after administration of the rAAV9-α-GAL gene therapy vector.

[0272] Gastrointestinal smooth muscle tissue samples were collected from G3Stg / GLAko mice treated with the rAAV9-α-GAL gene therapy vector. Samples were also collected from wild-type mice, untreated G3Stg / GLAko mice, and G3Stg / GLAko mice treated with a control vector (MY011). Tissues were fixed and stained. Histological evaluation of fixed tissues was performed by immunohistochemistry or hematoxylin and eosin (HE) staining. An anti-LAMP1 antibody was used for immunohistochemistry.

[0273] As shown in Figure 9, treatment with the rAAV-α-Gal gene therapy vector almost completely reversed pathological vacuolation and significantly reduced lysosomal burden in myenteric ganglion cells and transverse smooth muscle of the gastrointestinal tract. Exposure of the α-Gal A enzyme was observed in mice treated with the rAAV9-α-Gal gene therapy vector. Vacuoles in the transverse smooth muscle of the G3Stg / GlaKO duodenum were reversed after treatment with the rAAV9-α-Gal gene therapy vector. LAMP1-positive staining in smooth muscle and myenteric ganglion cells was observed only in untreated and rAAV-null-treated mice, but not in mice treated with the rAAV9-α-Gal gene therapy vector, suggesting a reversal of lysosomal burden in the gastrointestinal tract.

[0274] These immunohistochemistry results confirm high levels of enzyme exposure in various tissues and cells, including cardiomyocytes, podocytes, peripheral neurons, and DRG. Taken together, these studies suggest that the rAAV-α-Gal gene therapy vector mediates modified α-Gal expression, preventing further disease progression and even reversing certain symptoms in a mouse model of severe Fabry disease.

[0275] Example 9. Comparison of gene therapy using rAAV8-hα-GAL with a ubiquitous promoter and a liver-specific promoter in GlaKO mice. In this example, the effects of gene therapy were evaluated using rAAV8-hα-GAL (containing a liver-specific promoter) or rAAV9-hα-GAL (containing a ubiquitous promoter) vectors encoding wild-type α-galactosidase.

[0276] Figures 10A and 10B show the immunohistochemistry results of GlaKO mice administered a single intravenous dose of rAAV8-hα-GAL or rAAV9-hα-GAL, encoding wild-type α-galactosidase and carrying a liver-specific or ubiquitous promoter, at 2.5e11vg / kg for 12 weeks.

[0277] Table 7 summarizes the comparison of ERT and rAAV treatment in GlaKO mice.

[0278] [Table 7]

[0279] rAAV administration increased serum and tissue exposure of hα-GAL and improved substrate clearance in Fabry disease mice. Fabry disease mice treated with AAV-hαGAL containing both ubiquitous and liver-specific promoters (GlaKO mice) showed favorable mRNA expression in the liver, potentially improving safety.

[0280] Example 10. PK of α-galactosidase production and substrate reduction in humans using translational modeling Target α-GAL levels predicted to provide superior efficacy over ERT can be achieved at significantly lower doses in human Fabry disease patients. This example demonstrates a translational modeling approach to demonstrate α-galactosidase production or Gb3 reduction in humans compared with ERT over a 13-month period.

[0281] Figures 11A-11B show predicted α-galactosidase production in various tissues over 13 months at different doses. Figure 11C shows predicted α-galactosidase production in various tissues with ERT. Figures 11D-11E show predicted Gb3 reduction in various tissues over 13 months at different doses. Figure 11F shows predicted Gb3 reduction in various tissues with ERT. A reduction of at least 30% in Gb3 at 6 months in ERT-resistant cell types may represent a meaningful change leading to improved function.

[0282] Example 11. Minimum effective dose of adeno-associated viral vector expressing human alpha-galactosidase (alpha-GAL) variants The objective of this example was to evaluate the minimally effective dose of two recombinant adeno-associated viral vectors expressing human alpha-galactosidase (alpha-GAL) variants in symptomatic mouse models of Fabry disease (G3Stg / GLAko or HEMI;CAR) administered as a single intravenous (IV) dose at four different doses and monitored over a four-week period.

[0283] rAAV9-hα-GAL-variant 1 and rAAV9-hα-GAL-variant 2 were added to 2.5 × 10 8 , 2.5×10 9 , 2.5×10 10 , and 2.5 × 10 11 The null vector (rAAV9-null) was administered at 2.5 × 10 11 vg / kg was used as a control, but did not produce circulating α-GAL activity and no human protein was detected.

[0284] Mice treated with rAAV9-hα-GAL-variant 1 and rAAV9-hα-GAL-variant 2 showed a dose-response increase in serum α-GAL activity 1 week after injection, which persisted throughout the 4-week study (Figure 12A). Tables 8A and 8B show α-GAL activity in terminal serum and key tissues, including kidney, heart, and liver (also shown in Figures 12B-E).

[0285] [Table 8A]

[0286] [Table 8B]

[0287] High α-GAL activity is 2.5 × 10 10 and 2.5 x 10 11 was detected only at the top two doses of 2.5×10 vg / kg. 11 At rAAV9-hα-GAL-variant 1 and rAAV9-hα-GAL-variant 2, high α-GAL activity was achieved in a dose-response manner in terminal serum (>6,900-fold and >10,000-fold higher than WT) and major target tissues (e.g., kidney >150-fold and >100-fold, heart >900-fold and >500-fold, and liver >750-fold and approximately 1,000-fold for rAAV9-hα-GAL-variant 1 and rAAV9-hα-GAL-variant 2, respectively; Table 8B).

[0288] As expected, human α-GAL protein levels were not detected in any of the control samples. Protein levels in serum and evaluated tissues increased in a dose-dependent manner, reflecting α-GAL activity in each sample type and dose (Table 9). The two lower doses (2.5 × 10 8 , 2.5×10 9 At 2.5 × 10 vg / kg, none of the variant test articles produced detectable levels of protein in serum or tissues. 11Mice receiving rAAV9-hα-GAL-variant 1 at 1000 mg / kg had higher α-GAL protein and α-GAL activity in serum and liver than mice receiving the same dose of rAAV9-hα-GAL-variant 1, but had lower exposure to kidney and heart, likely due to differences in tissue uptake rates between the two variants.

[0289] [Table 9]

[0290] Gb3 and lysoGb3 2.5 x 10 null vector rAAV9 11 In disease controls injected at 100 mg / kg, both Gb3 and lysoGb3 substrate accumulated in terminal serum and major tissues compared with WT:WT controls. WT:CAR mice carrying a knock-in copy of the G3S gene exhibited more Gb3 substrate than WT:WT but less than G3Stg / GLAko, particularly in the heart and serum (Table 10, Figures 12F-12M).

[0291] When mice were treated with either rAAV9-variant 1 or rAAV9-variant 2, both Gb3 substrates were dose-dependently reduced in terminal serum and target tissues, with the highest dose of 2.5 × 10 11 vg / kg, which corresponds to a >85% reduction in tissue Gb3 from G3Stg / GLAko null controls. 10 Mice treated with 2.5×10 vg / kg had near normalization of Gb3 substrate in the liver (>75%) and terminal serum (>), but reduced availability in the kidney and heart. 10 At the vg / kg dose, rAAV9-variant 2 reduced Gb3 in the kidney and heart slightly better (53% and 61%, respectively) than rAAV9-variant 1 (46% and 51%, respectively).

[0292] [Table 10]

[0293] A similar dose-dependent trend was observed for the reduction of lysoGb3 substrate (Table 11 and Figures 12J-12M).

[0294] [Table 11]

[0295] Vector genome and mRNA copy number Vector genome (vg) and mRNA copy numbers were determined from kidney, heart, and liver tissues. As expected, no vector genome copies were detected in the WT:WT and WT:CAR controls. 2.5 × 10 copies were detected for all tissues. 11 At the highest dose of 2.5 × 10 vg / kg, the vg copy number was detectable, and similar results were obtained for the mRNA copy number (Figures 12N-12O). 10 At low doses of vg / kg, only liver vg and mRNA are detectable.

[0296] The top two doses (2.5 × 10 10 and 2.5 x 10 11 It was observed that only 100 mg / kg of riboflavin (vg / kg) showed significant exposure and substrate reduction effects in serum and evaluated tissues in a dose-dependent manner.

[0297] Example 12. Minimum effective dose of adeno-associated virus vector expressing human alpha-galactosidase (alpha-GAL) variants The objective of this example was to evaluate the durability of rAAV9-variant 1 in GlaKO male mice at two different doses (1e11 and 2.5e10 vg / kg) for 8 months with interim sacrifice 3 months after injection. rAAV9-null was used as a negative control only at the highest dose of 1e11 vg / kg.

[0298] GlaKO and WT control animals were injected with two different doses of variant 1 (1e11 and 2.5e10 vg / kg) or the highest dose (1e11 vg / kg) of rAAV9 null control via the intravenous route of administration and evaluated for serum and tissue exposure, substrate depletion, urine and blood chemistries, histopathological evaluation, and echocardiograms.

[0299] Data generated from this study are serum and tissue α-GAL activity in the 8-month cohort (Table 12). Serum α-GAL activity over the 8-month period is plotted in Figure 13A.

[0300] [Table 12-1]

[0301] [Table 12-2]

[0302] [Table 12-3]

[0303] At both concentrations tested, sustained α-GAL activity was observed in the liver, kidney, and heart throughout the study period.

[0304] Recombinant adenovirus rAAV9-hα-GAL variant 1 was administered intravenously to non-human primates. Obtaining AAV9-seronegative animals was difficult. Despite high pre-existing NAbs, high transgene expression was observed in most treated animals (Figures 13B and 13C). Figures 13B and 13C show serum α-galactosidase concentrations over 28 to 90 days at different concentrations.

[0305] The results show that although α-Gal expression levels in NHPs were lower than in mice, they reached >3000-fold normal levels and >50-fold the target clinical level at a dose as low as 6.25e12vg / kg. The NHP results support a low therapeutic dose of rAAV9-hα-GAL variant 1 in the e12vg range.

[0306] 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. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A method for reducing or ameliorating peripheral neuropathy in a subject diagnosed with Fabry disease, the method comprising administering to the subject a composition comprising a recombinant adeno-associated viral vector (rAAV) comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof, wherein the subject diagnosed with Fabry disease has or is developing one or more symptoms associated with neuropathy.

2. 10. The method of claim 1, wherein the peripheral neuropathy manifests as neuropathic pain, heat hypoesthesia, hearing loss, other sensory disturbances, and / or gastrointestinal disturbances.

3. The method of claim 2, wherein the neuropathic pain is alleviated.

4. The method of claim 2, wherein said thermal hypoesthesia is reduced.

5. 1. A method for ameliorating peripheral abnormalities of nerve fibers in a subject with Fabry disease, said method comprising administering to said subject a composition comprising a recombinant adeno-associated viral vector (rAAV) comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof.

6. The method of claim 5, wherein the nerve fibers are small diameter nerve fibers.

7. 7. The method of claim 6, wherein the improvement comprises preservation of small diameter myelinated nerve fibers.

8. 7. The method of claim 6, wherein the improvement comprises preservation of small diameter unmyelinated nerve fibers.

9. The method of any one of claims 5 to 8, wherein the density of sensory nerve fibers is maintained.

10. 10. The method of any one of the preceding claims, wherein the administration of the rAAV reduces the accumulation of globotriaosylceramide (GB3) in the peripheral nervous system, as indicated by a decrease in LAMP1 levels in DRG.

11. A method for reducing accumulation of globotriaosylceramide (GB3) in the peripheral nervous system caused by α-galactosidase A deficiency, comprising administering to a subject in need thereof a composition comprising a recombinant adeno-associated viral vector (rAAV) containing a polynucleotide encoding the α-GAL enzyme or a variant thereof.

12. 12. The method of claim 11, wherein the accumulation in the dorsal root ganglia (DRG) is reduced as indicated by a decrease in LAMP1 levels in the DRG.

13. 12. The method of claim 11, wherein accumulation in the peripheral nerve is reduced as indicated by a decrease in LAMP1 levels within the nerve.

14. 1. A method for treating a neurological disorder in a subject suffering from Fabry disease, comprising: (a) determining that the subject is suffering from or is developing one or more neuropathy-related symptoms; (b) administering to the subject a therapeutically effective amount of a composition comprising a recombinant adeno-associated viral vector (rAAV) comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof; (c) assessing the one or more neuropathy-related symptoms in the subject after the treatment. The method of treatment comprises:

15. 15. The method of claim 14, wherein the one or more neuropathy-associated symptoms comprise chronic burning pain, attacks of severe pain, paresthesias / paresthesias, loss of sensation, hypohidrosis / anhidrosis, abdominal cramps, diarrhea (after meals), bloating, nausea, and tinnitus and hearing loss.

16. 16. The method of claim 14 or 15, wherein the assessment is performed 2 weeks, 5 weeks, 10 weeks, 18 weeks, or 6 months after the administration of the composition.

17. The rAAV vector is an rAAV vector packaged in a capsid having broad tissue tropism, and the vector is a. 5' inverted terminal repeat (ITR), b. ubiquitous promoters, c. a polynucleotide encoding the α-GAL enzyme; d. Poly A, and e. 3' ITR 10. A method according to any one of the preceding claims, comprising:

18. The rAAV vector is an rAAV vector packaged in a capsid having broad tissue tropism, and the vector is a. 5' inverted terminal repeat (ITR), b. liver-specific promoters; c. a polynucleotide encoding the α-GAL enzyme; d. Poly A, and e. 3' ITR The method according to any one of claims 1 to 16, comprising:

19. The rAAV vector is an rAAV vector packaged in a capsid having specific tropism for the nervous system, and the vector comprises: a. 5' inverted terminal repeat (ITR), b. ubiquitous promoters, c. a polynucleotide encoding the α-GAL enzyme; d. Poly A, and e. 3' ITR The method according to any one of claims 1 to 16, comprising:

20. 20. The method of any one of claims 17 to 19, wherein the vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

21. The method of any one of claims 17 to 20, wherein the polynucleotide encodes an α-GAL enzyme comprising an amino acid sequence selected from SEQ ID NOs: 2, 3, 6, and 7.

22. The method of any one of claims 17 to 21, wherein the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequence of SEQ ID NOs: 9, 10, 12, and 13.

23. The method of any one of claims 17 to 22, wherein the AAV capsid is an omnitropic 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.

24. 24. The method of claim 23, wherein the broad tropism AAV capsid is an AAV9 capsid.

25. 25. The method of claim 24, wherein the AAV9 capsid is native or modified.

26. 26. The method of any one of claims 17 to 25, wherein the ubiquitous promoter is a chicken beta actin (CBA) promoter, an EF-1α promoter, a PGK promoter, a UBC promoter, a LSE beta glucuronidase (GUSB) promoter, a ubiquitous chromatin opening element (UCOE) promoter, a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, or comprises one or more introns.

27. 27. The method of claim 26, wherein the ubiquitous promoter comprises a cytomegalovirus (CMV) enhancer, a chicken beta-actin promoter, and a rabbit beta-globin intron.

28. 27. The method of claim 26, wherein the ubiquitous promoter comprises a truncated EF-1α promoter and one or more introns.

29. The method of any one of claims 20 to 28, wherein the WPRE sequence is modified.

30. 30. The method of claim 29, wherein the WPRE sequence is WPRE mut6delATG.

31. The method of any one of claims 17 to 30, wherein the polyA is bovine growth hormone (BGH) polyA.

32. 10. The method of any one of the preceding claims, wherein the rAAV vector is administered by intravenous, subcutaneous, or transdermal administration.

33. 33. The method of claim 32, wherein the rAAV is administered by intravenous administration.

34. 10. The method of any one of the preceding claims, wherein the subject has detectable α-GAL in the serum for at least 5 weeks, 10 weeks, 15 weeks, 18 weeks, 20 weeks, 24 weeks, 26 weeks, 30 weeks, 40 weeks, 1 year, 5 years, 10 years, or 15 years after administration of the rAAV vector.

35. 35. The method of claim 34, wherein the subject has detectable α-GAL in serum for more than 18 weeks.

36. The rAAV was added to approximately 1 x 10 １０ vg / kg (vector genome / body weight) to approximately 1 x 10 １４ 10. The method of any one of the preceding claims, wherein the subject is administered a dose in the range of 0.15 vg / kg (vector genome / body weight).

37. The rAAV was diluted to about 1.0 x 10 １０ vg / kg~5.0×10 １３ 37. The method of claim 36, wherein the subject is administered a dose in the range of 0.15 mg / kg.

38. 1. A method for reducing or ameliorating gastrointestinal symptoms in a subject diagnosed with Fabry disease, the method comprising administering to the subject a composition comprising a recombinant adeno-associated viral vector (rAAV) comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof, wherein the subject diagnosed with Fabry disease has or is developing one or more gastrointestinal symptoms.

39. 39. The method of claim 38, wherein the gastrointestinal symptoms include intestinal motility disorders, autonomic dysfunction, vascular disorders, and myopathy.

40. 40. The method of claim 39, wherein administering the rAAV to the subject reverses vacuolation in the gastrointestinal tract.

41. 10. The method of claim 9, wherein the density of the intraepidermal nerve fibers is maintained.

42. 7. The method of claim 6, wherein the nerve fiber is a dorsal nerve root and vacuolation within the dorsal nerve root is maintained.

43. The rAAV vector is an rAAV vector packaged in a capsid having broad tissue tropism, and the vector is a. 5' inverted terminal repeat (ITR), b. ubiquitous promoters, c. a polynucleotide encoding the α-GAL enzyme; d. Poly A, and e. 3' ITR The method of any one of claims 38 to 40, comprising:

44. The rAAV vector is an rAAV vector packaged in a capsid having broad tissue tropism, and the vector is a. 5' inverted terminal repeat (ITR), b. liver-specific promoters; c. a polynucleotide encoding the α-GAL enzyme; d. Poly A, and e. 3' ITR The method of any one of claims 38 to 40, comprising:

45. The rAAV vector is an rAAV vector packaged in a capsid having specific tropism for the nervous system, and the vector comprises: a. 5' inverted terminal repeat (ITR), b. ubiquitous promoters, c. a polynucleotide encoding the α-GAL enzyme; d. Poly A, and e. 3' ITR The method of any one of claims 38 to 40, comprising:

46. 46. ​​The method of any one of claims 43 to 45, wherein the vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

47. 46. ​​The method of any one of claims 43 to 45, wherein the polynucleotide encodes an α-GAL enzyme comprising an amino acid sequence selected from SEQ ID NOs: 2, 3, 6, and 7.

48. 48. The method of any one of claims 43 to 47, wherein the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequence of SEQ ID NOs: 9, 10, 12, and 13.

49. The method of any one of claims 43 to 48, wherein the AAV capsid is an omnitropic 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.

50. 50. The method of claim 49, wherein the broad tropism AAV capsid is an AAV9 capsid.

51. 51. The method of claim 50, wherein the AAV9 capsid is native or modified.

52. 52. The method of any one of claims 43 to 51, wherein the ubiquitous promoter is a chicken beta actin (CBA) promoter, an EF-1α promoter, a PGK promoter, a UBC promoter, a LSE beta glucuronidase (GUSB) promoter, a ubiquitous chromatin opening element (UCOE) promoter, a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, or comprises one or more introns.

53. 53. The method of claim 52, wherein the ubiquitous promoter comprises a cytomegalovirus (CMV) enhancer, a chicken beta-actin promoter, and a rabbit beta-globin intron.

54. 53. The method of claim 52, wherein the ubiquitous promoter comprises a truncated EF-1α promoter and one or more introns.

55. 55. The method of any one of claims 46 to 54, wherein the WPRE sequence is modified.

56. 56. The method of claim 55, wherein the WPRE sequence is WPRE mut6delATG.

57. 57. The method of any one of claims 43 to 56, wherein the polyA is bovine growth hormone (BGH) polyA.

58. 58. The method of any one of claims 43 to 57, wherein the rAAV vector is administered intravenously, subcutaneously, or transdermally.

59. 59. The method of claim 58, wherein the rAAV is administered by intravenous administration.

60. The rAAV was added to approximately 1 x 10 １０ vg / kg (vector genome / body weight) to approximately 1 x 10 １４ 60. The method of any one of claims 43 to 59, wherein the subject is administered a dose in the range of vg / kg (vector genome / body weight).

61. The rAAV was diluted to about 1.0 x 10 １０ vg / kg~5.0×10 １３ 61. The method of claim 60, wherein the subject is administered a dose in the range of 0.15 mg / kg.

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