Composition for treating Fabry disease

Abstract

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

Description

[0001] Cross-referenced applications

[0002] This application claims priority and the benefits of: U.S. Provisional Patent Application Serial No. 63 / 401,089 filed on August 25, 2022; U.S. Provisional Patent Application Serial No. 63 / 482,948 filed on February 2, 2023; and U.S. Provisional Patent Application Serial No. 63 / 500,742 filed on May 8, 2023; the contents of each of which are incorporated herein by reference in their entirety.

[0003] Reference to a sequence listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The sequence listing file is named MIL-022WO1.SL.XML, created on August 21, 2023, and is 28,159 bytes in size. Background Art

[0005] Fabry disease is a rare, progressive inborn error of metabolism caused by a mutation in the GLA gene that results in a deficiency of the lysosomal enzyme alpha-galactosidase A (α-GAL). Fabry disease affects 1 in 40,000 males who present with multisystem disease, usually during childhood or adolescence. Fabry disease can also affect females and present with a variety of symptoms. The lack of α-GAL enzyme activity results in a gradual systemic accumulation of its major substrate, ceramide trihexosylceramide (GB3) and its deacetylated, soluble form, trihexosylceramide (lysoGb3). Without treatment, patients with Fabry disease have a shortened life expectancy, usually ending in death in their fourth or fifth decade due to vascular disease affecting the kidneys, heart, and / or central nervous system.

[0006] Neurological manifestations of Fabry disease involve the peripheral nervous system, where accumulation of ceramide trihexosides is found in Schwann cells and dorsal root ganglia. Fabry disease in some patients is associated with the development of pain, which may be caused by lipid deposition or small fiber neuropathy in the dorsal root ganglia and sympathetic ganglia. Peripheral neuropathy affects more than a quarter of patients with Fabry disease and is characterized by loss of small myelinated and unmyelinated fibers, while larger fibers are largely spared (Ohnishi and Dyck. Loss of small peripheral sensory neurons in Fabry disease. Arch Neurol 1974;31:120)

[0007] Enzyme replacement therapy ("ERT"), which introduces a functional enzyme, is the currently approved treatment for Fabry disease. Although ERT is effective in many cases, the treatment requires lifelong intravenous administration of α-GAL every two weeks. ERT resolves the symptoms associated with Fabry disease, but it is not a cure and does not prevent the progression of the disease. The lack of pharmacological response is mainly due to the short circulation half-life of the enzyme and suboptimal cellular delivery. Therefore, the industry is in urgent need of therapies for the treatment of Fabry disease that can prevent disease progression and may be curative. Gene therapy is a promising treatment. Summary of the invention

[0008] The present application relates to a gene therapy method for transferring and expressing the alpha-galactosidase A (GLA) gene using a recombinant adeno-associated virus vector (rAAV). Specifically, the present application discloses, among other things, a gene therapy method and composition for alleviating, treating and / or preventing peripheral neuropathy in Fabry disease. The rAAV vector expressing the GLA transgene causes a sustained high level of exposure of the functional alpha-GAL enzyme in multiple tissues of Fabry disease patients, which is comparable to or higher than the normal enzyme function of healthy people, particularly in the nervous system (e.g., PNS). The restored alpha-GAL reduces the accumulation of substrates Gb3 and lysoGb3 in neural tissues, particularly in dorsal root ganglia, and improves neural abnormalities, including retaining the density of small nerve fibers (e.g., myelinated nerve fibers).

[0009] In some embodiments, the rAAV vector for treating and / or preventing peripheral neuropathy in patients with Fabry disease according to the present disclosure has a broad tissue tropism and utilizes a ubiquitous promoter to drive broad gene expression, thereby causing sustained high levels of protein expression and stable exposure of the protein to multiple tissues and / or reduced levels of Gb3 or lysoGb3. In addition, the GLA transgene used in the present application is codon optimized and / or expresses an engineered variant of α-GAL, causing increased α-GAL activity in vivo, and / or reduced levels of lysoGb3 or Gb3 in vivo. In addition, the present genetic method of driving the expression of GLA variants encoding α-GAL proteins with increased half-life and improved cellular uptake further increases α-GAL exposure in key target tissues (e.g., neural tissues), thereby allowing better treatment outcomes for Fabry disease, such as improved outcomes for peripheral neuropathy.

[0010] In some embodiments described herein, liver- or nervous system-specific promoters may be used.

[0011] As described in more detail below, the rAAV-based gene therapy approach described herein results in overall improvement in health status in a mouse model of Fabry disease, as evidenced by weight gain, improved renal function, and neurological symptoms, and is further expected to be true in humans as well.

[0012] Specifically, the gene therapy method of the present invention is suitable for alleviating peripheral neuropathy in Fabry disease. Therefore, in one aspect of the present invention, a method for treating, alleviating and / or preventing peripheral neuropathy in a subject diagnosed with Fabry disease is provided; the method comprises administering to the subject a composition comprising a recombinant adeno-associated viral vector (rAAV), the recombinant adeno-associated viral vector comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof. Symptoms of peripheral neuropathy may manifest as neuropathic pain, decreased thermal sensation, hearing loss, other sensory deficits and / or gastrointestinal disturbances.

[0013] In some embodiments, a method for improving abnormalities around nerve fibers in a subject with Fabry disease is provided; the method comprising administering to the subject a composition comprising a recombinant adeno-associated viral vector (rAAV), the recombinant adeno-associated viral vector comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof. In some embodiments, small nerve fibers are retained, including small myelinated and unmyelinated nerve fibers. In some embodiments, the density of small sensory nerves is retained.

[0014] In some embodiments, the rAAV vector expressing the GLA transgene as described herein is used to reduce the accumulation of ceramide trihexosyl (Gb3) in the peripheral nervous system caused by α-galactosidase A deficiency, which includes administering to a subject in need thereof. In some embodiments, the accumulation of Gb3 in dorsal root ganglia (DRG) is reduced. In other embodiments, the expression of LAMP1 in DRG is reduced.

[0015] 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) polyadenylation; and (e) a 3' ITR.

[0016] In some embodiments, rAAV vectors useful for treating and / or preventing peripheral neuropathy in patients with Fabry disease exhibit broad tissue tropism and comprise: (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) polyadenylation; and (f) a 3' ITR.

[0017] In some embodiments, the rAAV vector that can be used to treat and / or prevent peripheral neuropathy in patients with Fabry disease comprises: (a) a 5' terminal inverted repeat (ITR); (b) a liver-specific promoter; (c) a polynucleotide encoding an α-GAL enzyme or a variant thereof; (d) polyadenylation; 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 induces expression of the α-GAL enzyme in the liver, which can cross the blood-brain barrier (BBB) ​​to the nervous system, for example, to the PNS.

[0018] In some embodiments, a rAAV vector as described herein is packaged with an AAV capsid that has broad tissue tropism.

[0019] Various 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 broadly tropistic 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.

[0020] Thus, in some embodiments, the AAV capsid having wide tropism comprises an AAV1 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV2 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV3 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV4 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV5 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV6 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV7 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV8 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV9 capsid.

[0021] In one embodiment, the AAV capsid comprises an AAV9 capsid. The AAV9 capsid is naturally occurring or modified.

[0022] 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 β-glucuronidase (GUSB) promoter, or a ubiquitous chromatin opening element (UCOE) promoter. In some embodiments, the ubiquitous promoter comprises a CBh (CMV enhancer, a chicken beta actin promoter, a chicken-beta actin-MVM hybrid intron). Therefore, 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 from a chicken beta actin and a rabbit beta globulin gene. In some embodiments, the ubiquitous promoter is a UBC promoter. In some embodiments, the ubiquitous promoter is a LSE β-glucuronidase (GUSB) promoter. In some embodiments, the ubiquitous promoter is a ubiquitous chromatin opening element (UCOE) promoter.

[0023] In some embodiments, the ubiquitous promoter comprises a cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a rabbit beta globin intron.

[0024] In some embodiments, the ubiquitous promoter comprises a shortened EF-1α promoter and one or more introns.

[0025] In some embodiments, the one or more introns are from the chicken β-actin and / or rabbit β-globin genes.

[0026] In some embodiments, the rAAV vector with broad tissue tropism comprises a liver-specific promoter. Exemplary liver-specific promoters include, but are not limited to, 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.

[0027] In some embodiments, the WPRE sequence is optional or modified. In one embodiment, the WPRE sequence is WPRE mut6delATG.

[0028] Exemplary polyadenylation sequences that can be included in gene therapy vectors encompassed by the present disclosure include human growth hormone polyadenylation (hGHpA), synthetic polyadenylation (SPA), simian virus 40 late polyadenylation (SV 40pA), and bovine growth hormone (BGH) polyadenylation. In a specific embodiment, the polyadenylation is bovine growth hormone (BGH) polyadenylation.

[0029] In some embodiments, the GLA transgene expressing the α-GAL enzyme comprises a GLA gene having a wild-type sequence (SEQ ID No: 1 and 5) or a GLA gene having a modified sequence as described herein. Such modified GLA sequences include, for example, codon-optimized GLA and / or engineered variants of GLA.

[0030] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is codon optimized. In some examples, the nucleotide sequence encoding the α-GAL enzyme is codon optimized for human cells.

[0031] In some embodiments, the α-GAL enzyme has an unmodified sequence. In other embodiments, the α-GAL enzyme has a modified sequence.

[0032] In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is engineered. In some embodiments, the nucleotide sequence encoding the α-GAL enzyme is engineered 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 increased stability (e.g., serum stability), intracellular activity (e.g., lysosomal activity) and / or specific catalytic activity compared to the wild-type α-GAL enzyme.

[0033] As a non-limiting example, the α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the α-GAL enzyme variant comprises the amino acid sequence of SEQ ID NO: 7.

[0034] As a non-limiting example, 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.

[0035] As a non-limiting example, the method of the present invention comprises administering to a subject in need thereof a rAAV vector packaged in a rAAV9 capsid having broad tissue tropism, the vector comprising: (a) a 5' terminal inverted 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) polyadenylation; and (e) a 3' ITR.

[0036] In another example, the method of the present invention comprises administering to a Fabry disease patient exhibiting symptoms of peripheral neuropathy a rAAV vector packaged in a rAAV9 capsid with broad tissue tropism, the vector comprising: (a) a 5' terminal inverted 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); (e) polyadenylation; and (f) a 3' ITR.

[0037] In some embodiments, the rAAV vector is administered by intravenous, subcutaneous, or transdermal administration. Thus, in some embodiments, the rAAV vector is administered intravenously to a subject in need thereof. In some embodiments, the rAAV vector is administered subcutaneously to a subject in need thereof. In some embodiments, the rAAV vector is administered transdermally to a subject in need thereof.

[0038] In some embodiments, transdermal administration is performed by gene gun.

[0039] In some embodiments, the rAAV vectors of the present disclosure are expressed at 1.0×10 10 vg / kg (viral genome / kg body weight) to 1.0×10 14 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.0×10 10 vg / kg to 5.0×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.0×10 10 vg / kg to 1.0×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.0×10 10 vg / kg to 5.0×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.0×10 10 vg / kg to 1.0×1012 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.0×10 10 vg / kg to 5.0×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.0×10 10 vg / kg to 2.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.0×10 10 vg / kg to 1.0×10 14 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.0×10 10 vg / kg to 5.0×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.0×10 10 vg / kg to 1.0×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.0×10 10 vg / kg to 5.0×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.0×10 10 vg / kg to 1.0×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.0×10 10 vg / kg to 5.0×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.0×10 10 vg / kg to 2.5×10 11 The dose was administered in the range of vg / kg.

[0040] 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, the subject has detectable α-GAL in the serum for at least 10 years after administration of the rAAV vector. In some embodiments, the subject has detectable α-GAL in the serum for at least 15 years after administration of the rAAV vector. In some embodiments, the subject has detectable α-GAL in the serum for at least 20 years after administration of the rAAV vector. In some embodiments, the subject has detectable α-GAL in the serum for the lifetime of the subject after administration of the rAAV vector.

[0041] In some embodiments, expression of the modified α-GAL enzyme provides 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, 30,000-fold higher serum α-GAL levels than expression of WT α-GAL. In some embodiments, expression of the modified α-GAL enzyme provides 3-fold, 10-fold, 30-fold, 100-fold, 1000-fold, 3000-fold, 10,000-fold, 15,000-fold, 20,000-fold, 30,000-fold higher intracellular enzyme levels than expression of WT α-GAL.

[0042] In some embodiments, administration causes exposure of the α-GAL enzyme in one or more of the liver, kidney, heart, gastrointestinal tract, brain, and / or peripheral neurons of the subject. Thus, in some embodiments, administration causes exposure of the α-GAL enzyme in the liver. In some embodiments, administration causes exposure of the α-GAL enzyme in the kidney. In some embodiments, administration causes exposure of the α-GAL enzyme in the heart. In some embodiments, administration causes exposure of the α-GAL enzyme in the gastrointestinal tract and cells associated with the gastrointestinal tract. In some embodiments, administration causes exposure of the α-GAL enzyme in the brain. In some embodiments, administration causes exposure of the α-GAL enzyme in peripheral neurons.

[0043] In another aspect, the present invention provides a method for alleviating or improving 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), the recombinant adeno-associated viral vector comprising a polynucleotide encoding an α-GAL enzyme or a variant thereof, wherein the subject diagnosed with Fabry disease has or is showing one or more gastrointestinal symptoms. Gastrointestinal symptoms include intestinal motility disorders, impaired autonomic function, vasculopathy, and myopathy. In some embodiments, treatment reverses cavitation in the gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 An exemplary vector map of rAAV9 (generally referred to herein as "rAAV9") comprising wild-type α-GAL under the control of a ubiquitous promoter as described herein is shown.

[0045] Figure 2A As shown in Table 4, two different doses (5×10 10 and 2.5×10 11 Histogram of α-Gal activity in serum 18 weeks after administration of rAAV vector expressing GLA transgene (vg / kg) and control.

[0046] Figure 2B-2D The results are shown in Table 4 at two different doses (5 × 10 10 and 2.5×10 11 α-Gal activity in different tissues: kidney (2B), heart (2C), and liver (2D) after 18 weeks of administration of rAAV vectors expressing the GLA transgene and controls (vg / kg). Figure 2E is a graph showing α-galactosidase activity in the kidney and heart of Fabry disease mice treated with variant 1 or wild-type human α-galactosidase. Figure 2F is a graph showing α-galactosidase activity in vehicle control mice, wild-type α-galactosidase-transduced Fabry mice, and variant 1-transduced Fabry mice. Figure 2Gis a graph showing serum alpha-galactosidase concentrations in primates transduced with different concentrations (6.25el2 or 3el3 vg / kg) of AAV variant 1.

[0047] Figures 3A-3E The results are shown in Table 4 at two different doses (5 × 10 10 and 2.5×10 11 vg / kg) administration of rAAV vectors expressing the GLA transgene and controls for 18 weeks before and after administration of Gb3 substrate concentrations in serum (3A), kidney (3B), heart (3C), and liver (3D). Figure 3E is a graph showing the percent reduction in Gb3 volume in the kidney or heart when treated with variant 1 or vehicle control.

[0048] Figures 4A-4D The results are shown in Table 4 at two different doses (5 × 10 10 and 2.5×10 11 vg / kg) administration of rAAV vectors expressing the GLA transgene and controls for 18 weeks before and after lysoGb3 substrate concentrations in serum (4A), kidney (4B), heart (4C), and liver (4D).

[0049] Figure 5 The results are shown in Table 4 at two different doses (5×10 10 and 2.5×10 11 Figure 3 Hot plate latency of mice 18 weeks after administration of rAAV vector expressing GLA transgene (vg / kg) and control.

[0050] Figure 6 WT mice, rAAV9-variant 1 at 2.5×10 11 Representative of MPZ IHC staining of DRG in Gb3Stg / GLAko mice treated with vg / kg dose and Gb3Stg / GLAko mice administered with rAAV9 null vector.

[0051] Fig. 7A Immunohistochemical staining of renal tissue is shown. The results showed that the rAAV9-α-Gal gene therapy vector was used at 2.5×10 11 Supraphysiological α-GalA enzyme exposure was present in renal target cells (podocytes) of Gb3Stg / GLAko mice treated with vg / kg doses. rAAV9 null vector was used as a control.

[0052] Figure 7B Immunohistochemical staining of cardiac tissue is shown. The results showed that the rAAV9-α-Gal gene therapy vector was used at 2.5×10 11Supraphysiological α-GalA enzyme exposure was present in cardiac target cells (cardiomyocytes) in Gb3Stg / GLAko mice treated with vg / kg doses. rAAV9 null vector was used as a control.

[0053] Fig. 8A demonstrated the use of rAAV9-α-Gal gene therapy constructs at 2.5 × 10 11 Normalization of vacuolation and lysosomal load in dorsal nerve roots (DNR) in vg / kg dose-treated Gb3Stg / GLAko mice. rAAV9 null vector was used as a control. The upper figure is HE (hematoxylin and eosin) staining of dorsal nerve roots. The lower figure is immunohistochemical staining of LAMP1 (lysosomal associated membrane protein 1). Figure 8B is a graph showing the percentage of LAMP1 positive area at baseline, in Fabry disease mice treated with control or variant 1 gene therapy, and in normal mice. Figure 8C is a graph showing hot plate latencies in Fabry disease mice and normal mice treated with control or variant 1 gene therapy. Fig.8D is a graph showing the correlation between DRG and latency. Fig. 8E are MRI images showing DRG volume in normal mice and Fabry disease mice transduced with vehicle control or gene therapy (GT) expressing WT aGAL. Fig.8F is a graph showing DRG volume in Fabry disease mice or normal mice treated with vehicle control or gene therapy expressing WT aGAL.

[0054] Fig. 9 demonstrated the use of rAAV9-α-Gal gene therapy vector at 2.5×10 11 Reversal of pathological vacuolation and lysosomal load in the gastrointestinal tract in Gb3Stg / GLAko mice treated with a vg / kg dose. The rAAV9 null vector was used as a control. The upper figure shows HE (hematoxylin and eosin) staining of transverse smooth muscle. Vacuoles in the transverse smooth muscle of the G3Stg / GlaKO duodenum were reversed after treatment with the rAAV9-α-Gal gene therapy vector. The middle figure shows the exposure of the α-Gal A enzyme. α-Gal A positive staining was observed in mice treated with the rAAV9-α-Gal gene therapy vector. The lower figure shows immunohistochemical staining for LAMP1 (lysosomal associated membrane protein 1). LAMP1 positive staining in smooth muscle and enteric myoganglion cells was observed only in untreated and rAAV9 null vector-treated mice.

[0055] Fig. 10A Shown are IHC of hepatocytes treated with rAAV8-hα-GAL with a ubiquitous promoter. Fig. 10BIHC of hepatocytes treated with rAAV9-hα-GAL with a liver-specific promoter is shown.

[0056] Fig.11A Predicted pharmacokinetics in human heart, kidney, liver and plasma at 3e11 vg / kg based on α-galactosidase concentrations are presented. Fig. 11B Predicted pharmacokinetics in human heart, kidney, liver and plasma at 1e11 vg / kg based on α-galactosidase concentration are presented. Fig. 11C The predicted pharmacokinetics of future ERT treatment in humans based on α-galactosidase concentrations in the heart, kidney, liver, and plasma are presented. Fig.11D Projected pharmacokinetics in humans based on α-galactosidase concentrations in the heart, kidney, liver, and plasma for future treatment at 3e11 vg / kg are presented. Fig.11E Projected pharmacokinetics based on Gb3 reduction in heart, kidney, liver, and plasma for future treatment in humans at 1e11 vg / kg are presented. Fig.11F The projected pharmacokinetics based on Gb3 reduction in the heart, kidney, liver, and plasma during future treatment with ERT in humans are presented.

[0057] Fig. 12A is a graph showing α-galactosidase activity in mice over 4 weeks when rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 were injected at 2 different doses. Fig. 12B is a bar graph showing alpha-galactosidase activity in terminal serum at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig. 12C is a bar graph showing alpha-galactosidase activity in kidney at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig.12D is a bar graph showing alpha-galactosidase activity in the heart at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig.12E is a bar graph showing alpha-galactosidase activity in the liver at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig.12F is a bar graph showing Gb3 substrate levels in terminal serum at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Figure 12G is a bar graph showing Gb3 substrate levels in kidney at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig.12H is a bar graph showing Gb3 substrate levels in kidney at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig.12I is a bar graph showing Gb3 substrate levels in the liver at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig.12J is a bar graph showing the LysoGb3 substrate levels in terminal serum at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Figure 12K is a bar graph showing the LysoGb3 substrate levels in kidney at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Figure 12L is a bar graph showing the LysoGb3 substrate levels in the heart at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Figure 12M is a bar graph showing the LysoGb3 substrate levels in the liver at 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control. Fig.12N is a graph showing vector genome copies in various tissues when treated with 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to controls. Fig.12O is a graph showing mRNA copies in various tissues when treated with 2 doses of rAAV9-hα-GAL variant 1 and rAAV9-hα-GAL variant 2 compared to control.

[0058] Fig.13A is a graph showing α-galactosidase activity in mouse serum during 32 weeks following intravenous injection of rAAV9-hα-GAL variant 1 at two different doses compared to control. Fig. 13B is a graph showing α-galactosidase activity in the serum of non-human primates when rAAV9-hα-GAL variant 1 was injected intravenously over 28 days. Fig. 13C is a graph showing α-galactosidase activity in the serum of non-human primates when rAAV9-hα-GAL variant 1 was injected intravenously over 90 days.

[0059] definition

[0060] About or approximately: As used herein, the term "about" refers to a value similar to the stated reference value when applied to one or more values ​​of interest. In certain embodiments, unless otherwise specified or apparent from the context (except that such number exceeds 100% of possible values), the term "about" refers to a series of values ​​in either direction (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value. It should be understood that when the term "about" or "approximately" is used to modify the stated reference value, the stated reference value itself is covered together with the value of the reference value approaching the stated reference value on both sides of the stated reference value.

[0061] Administration: The terms "administer," "administration," and "administering" refer to providing a composition of the invention (eg, a recombinant gene therapy vector expressing alpha-galactosidase) to a subject in need thereof (eg, to a human affected by Fabry disease).

[0062] Administration in combination: As used herein, the term "administered in combination" or "combined administration" means administering two or more agents to a subject simultaneously or within an interval so that the effects of each agent on the patient may overlap. In some embodiments, the administration of the agents is sufficiently close to achieve a combined (e.g., synergistic) effect.

[0063] 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. Amino acids in a parent or reference sequence (e.g., a wild-type GLA polypeptide sequence) may be substituted, for example, via chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to "substitution at position X" refers to the replacement of the amino acid present at position X with an alternative amino acid residue. In some aspects, the substitution pattern may be described according to the formula AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the amino acid residue to be substituted. In other aspects, the substitution pattern may be described according to the formula An(YZ), wherein A is the single letter code corresponding to the amino acid residue that replaces the amino acid naturally or originally present at position X, and Y and Z are the alternative amino acid residues to be substituted.

[0064] Abbreviations 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), glutamic acid (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 three-letter abbreviations are used, the amino acid may be surrounded by the α-carbon (C or S) unless explicitly preceded by "L" or "D" or clear from the context in which the abbreviation is used. α ) is in L- or D-configuration. In various embodiments described herein, one or more amino acids in the wild-type GLA sequence can be substituted with different amino acids to generate variants of α-GAL proteins.

[0065] Substitutions in a protein or polypeptide amino acid sequence can be conservative or non-conservative in nature. Conservative amino acid substitutions refer to the replacement of a residue with a different residue having a similar side chain, and thus generally involve replacing an amino acid in a polypeptide (e.g., an α-GAL amino acid sequence) with an amino acid within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be substituted with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid may be replaced with another hydrophobic or hydrophilic amino acid, respectively. Non-conservative substitutions refer to substitutions of amino acids in a polypeptide (e.g., α-GAL amino acid sequence) with amino acids having significantly different side chain properties. As examples and not limitations, exemplary non-conservative substitutions may be acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids.

[0066] In the context of the present disclosure, substitutions (even when they are referred to as amino acid substitutions) are made at the nucleic acid level, i.e., replacement of an amino acid residue with an alternative amino acid residue is performed by replacing the codon encoding the first amino acid residue with the codon encoding the second amino acid.

[0067] AVV vector: As used herein, the term "AAV vector" comprises a capsid protein and a viral genome, wherein the viral genome comprises at least one transgenic region and at least one terminal inverted repeat sequence (ITR). AAV vectors and / or their component capsids and viral genomes can be engineered to change the tropism for specific cell types, tissues, organs or organisms. In the context of the present invention, the viral genome comprises a GLA transgene, such as a nucleic acid sequence encoding α-GAL or a variant thereof.

[0068] 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, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, animals are transgenic animals, genetically engineered animals, and / or clones.

[0069] Blood urea nitrogen: As used herein, the term "blood urea nitrogen" or "BUN" refers to the amount of urea in the blood. Blood urea nitrogen is elevated in kidney-related pathologies. Chronic kidney disease is one of the cardinal features of Fabry disease, leading to end-stage renal failure. Gb-3 deposition in glomerular podocytes is thought to be responsible, at least in part, for the rate of progression or severity of proteinuria or renal involvement in Fabry disease. Blood urea nitrogen measures how efficiently the kidneys remove urea from the blood. High BUN levels indicate poor kidney function.

[0070] Chimera: As used herein, a "chimera" is an entity having two or more inconsistent or heterogeneous parts or regions. For example, a chimeric molecule may comprise a first part comprising a GLA polypeptide and a second part (e.g., genetically fused to the first part) comprising a second therapeutic protein (e.g., a protein with a different enzymatic activity, an antigen binding portion, or a portion capable of extending the plasma half-life of α-GAL, such as the Fc region of an antibody).

[0071] Codon substitution: As used herein, the term "codon substitution" or "codon replacement" in the context of sequence optimization refers to the replacement of a codon present in a reference nucleic acid sequence with another codon. Codons in a reference nucleic acid sequence can be replaced, for example, via chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to a "replacement" or "substitution" at a certain position of a nucleic acid sequence (e.g., mRNA) or within a certain region or subsequence of a nucleic acid sequence (e.g., mRNA) refers to the replacement of a codon at that position or region by an alternative codon.

[0072] Codon optimization: The term "codon optimized" or "codon optimization" refers to changes in the codons of a polynucleotide encoding a protein (e.g., a GLA gene) such that the encoded protein is more efficiently expressed, for example, in a cell or organism. In some embodiments, the polynucleotide encoding the α-GAL enzyme may be codon optimized for optimal production in a host organism and / or cell type selected for expression for GC content, cryptic splice sites, transcription termination signals, motifs and nucleic acid secondary structures that may affect RNA stability, and any other factors of interest.

[0073] Dose: As used herein, the terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual per administration. The dose will vary depending on a variety of factors, including the frequency of administration; the size and tolerance of the individual; the severity of the condition; the risk of side effects; and the route of administration. Those skilled in the art will recognize that the dose may be modified based on the above factors or based on the progress of the treatment. The term "dosage form" refers to the specific format of the drug and depends on the route of administration.

[0074] Engineered variants: The term "engineered α-GAL variant" or "engineered variant" refers to a GAL protein in which one or more amino acid residues have been modified by substitution, deletion, or insertion compared to wild-type α-GAL. In some embodiments, the engineered variants are characterized by improved efficacy and pharmacokinetic profiles due to, for example, altered structural attributes of the protein. In some embodiments, the engineered α-GAL variants enhance substrate clearance from tissues, such as serum, kidney, heart, and / or liver. Engineered variants can be produced synthetically or recombinantly.

[0075] Gb3: As used herein, the term "Gb3" or "ceramide trihexosyl" or "GB3" or "gb3" or "CD77" or "GL-3" refers to a class of glycosphingolipids that accumulate in lysosomes in Fabry disease and are considered to be the main pathogenic metabolite. GB3 is formed by the alpha linkage of galactose to lactosylceramide catalyzed by A4GALT. GB3 is hydrolyzed by GLA at the terminal alpha linkage. Fabry disease is characterized by the accumulation of GB3 in all organs (especially the heart and kidneys), as well as in many cells and urine. Such accumulation is accompanied by a significantly increased risk of stroke, heart disease (hypertrophic cardiomyopathy, rhythm and conduction system disorders, coronary artery disease, valvular abnormalities, etc.), and chronic proteinuria renal failure. In some embodiments, deacylated GB3 or lysoGb3 is also a valuable biomarker for Fabry disease.

[0076] Gene: As used herein, the term "gene" refers to a DNA region encoding a protein or polypeptide (e.g., α-galactosidase as described herein), as well as all DNA regions that regulate the production of a protein or polypeptide, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. 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, replication origins, matrix attachment sites, and locus control regions.

[0077] GLA gene: As used herein, the term "GLA gene" or "galactosidase gene" or "alpha-galactosidase gene" or "alpha-galactosidase gene" refers to a gene encoding an enzyme alpha-galactosidase that breaks down ceramide trihexosyl. Genetic mutations in the GLA gene result in defective enzymatic function of alpha-galactosidase. In humans, the GLA gene is located at Xq22.1, i.e., position 22.1 on the long (q) arm of the X chromosome. Some other names that the GLA gene may be called include AGAL HUMAN, Agalsidase α, α-D-galactosidase A, α-D-galactosidase galactose hydrolase, α-galactosidase, α-galactosidase A, ceramide trihexosylase, GALA, galactosidase, or melibiase.

[0078] Galactosidase: As used herein, the term "galactosidase" or "alpha galactosidase A" or "alpha-galactosidase A" or "alpha-GAL" refers to the enzyme encoded by the GLA gene. Human alpha galactosidase (EC 3.2.1.22) is a lysosomal enzyme that hydrolyzes the terminal alpha galactosyl moiety of glycolipids and glycoproteins. As used herein, the term alpha-GAL may refer to the wild-type enzyme or a variant thereof. Alpha galactosidase A deficiency causes Fabry disease (also known as diffuse angiokeratoma corporis, Anderson-Fabry disease, hereditary ectopic lipidosis, alpha-galactosidase A deficiency, alpha-GAL deficiency, and ceramide trihexosidase deficiency), which is an X-linked inborn error of glycosphingolipid catabolism. In various embodiments described herein, a gene therapy platform for treating Fabry disease is provided.

[0079] "Hypoalgesia or hypoalgesia": As used herein, the term "hypoalgesia" or "hypoalgesia" refers to a decrease in sensitivity to painful stimuli. Harmful (Pain) stimulation in the input ( Nociceptors ) and processed and recognized in consciousness painHypoalgesia occurs when the path between the body and the lungs is interrupted or reduced somewhere. The hypoalgesic effect may be mild, such as massaging a stubbed toe to relieve pain or taking aspirin to relieve a headache, or it may be severe, such as being under strong anesthesia.

[0080] "Improved enzyme property": The term "improved enzyme property" refers to any property or attribute of an engineered α-GAL polypeptide that is improved relative to the same property or attribute of a reference α-GAL polypeptide (e.g., compared to a wild-type α-GAL polypeptide or another engineered α-GAL polypeptide). Improved properties include, but are not limited to, properties such as increased gene expression, increased protein production, increased thermal activity, increased thermal stability, increased activity at different pH levels, increased stability, increased enzyme activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate and / or product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic, neutral or alkaline pH, increased resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation), altered temperature profile, increased cellular uptake, increased lysosomal stability, increased ability to deplete GB3 cells, increased secretion from α-GAL producing cells, etc. In various embodiments, the gene therapy vectors encompassed by the present disclosure comprise a nucleic acid sequence encoding an α-GAL polypeptide comprising one or more improved enzymatic properties relative to a reference α-GAL polypeptide. In some embodiments, a nucleic acid sequence encoding an α-GAL polypeptide that exhibits one or more improved enzymatic properties is codon optimized.

[0081] In various embodiments, the codon-optimized and / or engineered α-GAL variants exhibit one or more of the aforementioned improved properties. In a specific embodiment, the α-GAL variants have improved serum and lysosomal stability, and in other embodiments, the α-GAL variants have increased specific catalytic activity relative to the wild-type α-GAL polypeptide.

[0082] "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 percent conversion of substrate to product (e.g., percent conversion of a starting amount of substrate to product over a specified period of time) using a specified amount of an engineered α-GAL enzyme compared to a reference α-GAL enzyme (e.g., a wild-type α-GAL enzyme or another engineered variant). Any suitable method known in the art and / or those described herein can be used to determine enzyme activity. Any property associated with enzyme activity may be affected, including K m 、Vmax or k cat The improvement in enzyme activity can be from about 1.1 times the enzyme activity of the corresponding wild-type enzyme to up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times or more of the enzyme activity compared to the reference α-GAL enzyme.

[0083] Endogenous expression: The term "endogenous expression" and its grammatical equivalents refer to the expression of a gene within one or more cells into which the transgene is introduced. Endogenous expression uses the cell's own or pre-existing transcriptional or translational 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.

[0084] Nucleic acid: As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer in a linear or circular conformation and in single-stranded or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limiting the length of the polymer. The terms may encompass known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). In general, analogs of a particular nucleotide have the same base pairing specificity; i.e., an analog of A will base pair with T.

[0085] 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" can 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. In the context of the present disclosure, peripheral neuropathy associated with Fabry disease is primarily manifested as sensory dysfunction (e.g., low thermal sensitivity). One type of peripheral neuropathy is "demyelinating peripheral neuropathy," a widespread type of peripheral neuropathy associated with the destruction or removal of myelin (the lipid-rich sheath that surrounds and insulates nerve fibers) in the nerves.

[0086] Operably linked: As used herein, the terms "operably linked" and "operatively linked" (or "operably linked") are used interchangeably in reference to the juxtaposition of two or more components (e.g., sequence elements) wherein the components are arranged so that both components function normally and so that at least one component can regulate the function exerted on at least one of the other components. For example, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if the transcriptional regulatory sequence 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 typically operably linked to a coding sequence in cis, 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 they are not linked.

[0087] Physiological pH: As used herein, "physiological pH" means the pH range normally found in the blood of a subject (eg, a human), ie, pH 7.4.

[0088] Alkaline pH: The term "alkaline pH" (eg, as used in reference to improved stability under alkaline pH conditions or increased tolerance to alkaline pH) means a pH range of about 7 to 11.

[0089] Acidic pH: The term "acidic pH" (e.g., in reference to improved stability to acidic pH conditions or increased tolerance to acidic pH) means a pH range of about 1.5 to 6. Polypeptide: As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to 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 corresponding naturally occurring amino acids.

[0090] 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 may be ubiquitous, meaning strongly active in a wide range of cells, tissues, and species, or cell type-specific, tissue-specific, or species-specific. In some embodiments, liver-specific promoters include, for example, the transthyretin promoter (TTR); the thyroxine-binding globulin (TBG) promoter; the hybrid liver-specific promoter (HLP) and the alpha-1-antitrypsin (AAT) promoter. Promoters may be "constitutive," meaning continuously active, or "inducible," meaning that the promoter can be activated or inactivated by the presence or absence of biological or abiotic factors. The nucleic acid construct or vector of the present invention also includes an enhancer sequence, which may be connected or unconnected to the promoter sequence. The enhancer sequence affects promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0091] Sequence optimization: As used herein, the term "sequence optimization" refers to a process or series of processes by which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, thereby generating a nucleic acid sequence with improved properties, such as improved protein expression or increased activity.

[0092] The terms "individual", "subject", "subject in need thereof" and "patient" are used interchangeably herein and refer to mammals, including but not limited to rodents, apes, humans, mammalian farm animals, mammalian sports animals and mammalian pets. In preferred embodiments, the individual is a human. In various embodiments, the subject or subject in need thereof is a Fabry disease patient who exhibits 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 the effect of administration. The exact dose will depend on the purpose of the treatment and can be determined by a person skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Volumes 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).

[0093] Treatment: As used herein, the term "treatment" or "therapy" generally means obtaining a desired physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or disorder or its symptoms, and / or may be therapeutic in terms of partially or completely curing an injury, disease or disorder and / or ameliorating side effects attributed to the injury, disease or disorder, and includes inhibiting the development of a disease or disorder or causing the regression of a disease or disorder. Treatment may also include prophylactic use to mitigate the effects of an injury, if it occurs. For example, in one aspect, the invention includes pre-administration prior to surgery involving the peripheral nervous system to mitigate the injury. Treatment may also refer to any delay in onset, improvement in symptoms, improvement in patient survival, increase in survival time or survival rate, etc. The therapeutic effect may be compared to an individual or group of individuals who have not received treatment.

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

[0095] Thermal hypoesthesia: As used herein, the term "thermal hypoesthesia" or "thermal insensitivity" or "thermal hypoesthesia" refers to insensitivity or less sensitivity to thermal stimulation.

[0096] Vector: As used herein, the term "vector" is capable of transferring a gene sequence to a target cell. In general, "vector construct", "expression vector" and "gene transfer vector" mean any nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring a gene sequence to a target cell. Therefore, the term includes cloning and expression vehicles and integration vectors. In some embodiments, the vector is a virus, which includes, for example, an encapsulated form of a vector nucleic acid and a viral particle in which the vector nucleic acid has been packaged. In some embodiments, the vector is not a wild-type strain because it contains artificial mutations or modifications. In some embodiments, the vector is derived from a wild-type strain by genetic manipulation (i.e., by deletion) to contain a conditionally replicating virus, as further described herein. In some embodiments, the vector is delivered by a non-viral means. In some embodiments, the vector described herein is a gene therapy vector, which is used as a vehicle for delivering a polynucleotide sequence (e.g., α-galactosidase) to a cell. In a specific embodiment, the gene therapy vector described herein is a recombinant AAV vector (e.g., AAV8 or AAV9).

[0097] Wild-type: As used herein, the terms "wild-type" and "naturally occurring" refer to the form of a nucleic acid or protein found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation.

[0098] The recitation of numerical ranges by endpoints herein includes all numbers and fractions within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It should also be understood that all numbers and fractions thereof are assumed to be modified by the term "about."

[0099] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the present invention. Each section may apply to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or". As used herein, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" include single and plural references. DETAILED DESCRIPTION

[0100] The present disclosure relates to rAAV-based gene therapy for treating and / or preventing Fabry disease, in particular for improving and / or preventing or treating neuropathy in Fabry disease. The present disclosure provides, among other things, (1) an intrinsic GLA expression system in tissues affected by Fabry disease; (2) a method for achieving sustained and high expression of α-galactosidase (α-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 improve Fabry disease-related phenotypes, in particular peripheral neuropathy in Fabry disease.

[0101] In some embodiments, the gene therapy treatment described herein utilizes rAAV vectors with broad tissue tropism, and the rAAV vectors contain ubiquitous promoters to drive broad gene expression, resulting in sustained high levels of protein expression and robust protein exposure to a wide range of tissues and / or reduced levels of GB3 or lysoGb3. In addition, the GLA transgene used in the present application is codon optimized and / or expresses an engineered variant of α-GAL, resulting in increased α-GAL activity in vivo, and / or reduced levels of lysoGb3 or Gb3 in vivo. In addition, the present gene method of driving the expression of GLA variants encoding α-GAL proteins with increased half-life and improved cellular uptake further increases α-GAL exposure in key target tissues (e.g., neural tissues), thereby allowing better treatment outcomes for Fabry disease, such as improved outcomes for peripheral neuropathy.

[0102] The methods and compositions provided herein can be used to achieve sustained expression of GLA in a variety of tissues affected by Fabry disease. Thus, the present application provides compositions and methods that are highly effective in treating Fabry disease and alleviating associated symptoms such as peripheral neuropathy.

[0103] Alpha-galactosidase and Fabry disease

[0104] Fabry disease is an X-linked genetic disorder caused by mutations in the GLA gene that result in abnormal lysosomal hydrolase α-galactosidase A (α-GAL). Since α-GAL is essential for the catabolism of glycolipids (such as sphingolipids), the deficiency or dysfunction of α-GAL leads to the accumulation of sphingolipids in tissues.

[0105] Fabry disease is a systemic metabolic disorder that affects multiple tissues and organs including the kidneys, heart, lungs, and nervous system. Neurological manifestations of Fabry disease include peripheral nervous system (PNS) involvement with accumulation of ceramide trihexosylceramide in Schwann cells and dorsal root ganglia. Peripheral nervous system involvement primarily affects small Aδ and C fibers and may be causally related to the autonomic changes and neuropathic pain seen in Fabry disease. Other associated neurological problems include hypohidrosis and other abnormalities related to nervous system dysfunction.

[0106] Peripheral neuropathy in Fabry disease manifests as neuropathic pain, decreased cold and temperature sensation (ie, thermal hypoesthesia), hearing loss, other sensory deficits, and possibly gastrointestinal disturbances.

[0107] Fabry disease patients begin to experience pain at the end of the first decade of life or in adolescence (Ries et al. Pediatric Fabry disease. Pediatrics. 2005; 115: e344-355). In general, neuropathic pain in Fabry disease may be continuous (i.e., chronic), or composed of intermittent attacks caused by changes in body or environmental temperature and other stressful situations (MacDermot and MacDermot, Neuropathic pain in Anderson-Fabry disease: pathology and therapeutic options. Eur J Pharmacol. 2001; 429: 121-125). Paroxysmal pain in Fabry disease (called "Fabry disease crisis") usually starts from the limbs and radiates to the proximal end, and may be caused by movement, disease, temperature changes or other physical and emotional stress. This neuropathic pain is also associated with a lack of temperature perception.

[0108] The neuropathy of Fabry disease is associated with significantly increased thresholds for detecting cold and warmth in the hands and feet (i.e., decreased thermal sensation or hypothermia) (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). Established biophysical quantitative sensory testing techniques can be used to measure those 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).

[0109] Increased perception thresholds for warm and cold temperatures (hyposthesia) in male and female carriers have been shown to induce 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 diameter 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). Studies of nerve damage leading to pain, based on autopsy characterization of tissue from a small number of Fabry disease patients, have shown a significant reduction in small, thinly 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).

[0110] Several studies have shown that Fabry disease is associated with a peripheral neuropathy that primarily affects small myelinated (Aδ) and unmyelinated (C) fibers. The underlying causal mechanism may be due to neuronal ischemia or intrinsic neuronal dysfunction caused by glycolipid accumulation in the vasa nervorum (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).

[0111] It was found that sensory nerve fiber neuropathy (SFSN) in patients with Fabry disease is associated with sensorineural hearing loss (Ries et al., Neuropathic and cerebrovascular correlates of hearing loss inFabry disease. Brain 2007, 130:143-150).

[0112] Other neuropathies of Fabry disease include impaired vibration thresholds and abnormal nerve conduction in some patients. Pathological examination of peripheral nerves (e.g., sural nerve) usually shows a normal number of large myelinated fibers, but a significant loss of unmyelinated small fibers. Glycolipid deposition in sensory ganglia is associated with peripheral neuropathy. In addition, this neuropathy of Fabry disease is associated with a severe loss of intraepidermal innervation.

[0113] It has been shown that in Fabry knockout mice, abnormal accumulation of Gb3 due to lack of lysosomal α-galactosidase activity is associated with abnormal sciatic nerve morphology, reduced number of unmyelinated axons and small myelinated axons and preservation of large diameter myelinated axons, behavior and sensitivity to thermal stimulation, showing similar features 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). Fabry knockout mice may explain the observed changes in thermal sensitivity (hypopalgesia).

[0114] In summary, it is well known that GB3 inclusions are present in DRG neurons in patients and rodent preclinical models, and these inclusions cause cell swelling, including increased diameter of DRG neuron cell bodies and total DRG volume. The GB3 content in DRG may be more than 10 times higher than that in brain regions (e.g., Kaye et al., Nervous system involvement in Fabry's disease: clinicopathological and biochemical correlation. Ann Neurol 1988; 23: 505-509); these high levels of Gb3 can lead to DRG neuron stress and death. In addition to DRG neuron cell bodies, lipid inclusions are also prominent in the axons of peripheral nerves and are associated with axonal morphological abnormalities, including irregularly shaped axons, enlarged axons, and uneven myelin. The significant loss of such small fibers is most prominent in the distal long axons of the lower limbs. For example, the innervation of the proximal part of the thigh may also be reduced, but this type of loss is most obvious around the foot. Studies have shown that fiber loss in the skin is much higher than fiber loss in peripheral nerve trunks.

[0115] Current treatment options for Fabry disease include recombinant enzyme replacement therapy (ERT). ERT slows the progression of Fabry disease but does not completely stop or reverse the disease. Current treatments for Fabry disease primarily achieve a slowing of disease progression limited to the kidneys and heart, with insufficient or no improvement in other organs / tissues, particularly the nervous system. Fabry disease patients also require continuous protein-based infusions, which sometimes result in infusion reactions and increased immunogenicity. Such ongoing disease management requirements also increase the patient’s “treatment burden” or additional and ongoing workload (i.e., the necessity and requirement) for them to comply with the recommendations made by their clinicians to manage their morbidity and health. In severely affected male patients with classic Fabry disease, the rate of renal function loss associated with -1 mL / min / 1.73 m per year is 1.5 times that of placebo. 2 Compared with healthy subjects, the annual renal function loss was as high as -6.82 mL / min / 1.73 m2 / year despite treatment. 2 / year (Germain et al., J Med Genet. 2015 May;52(5):353-8.2015). These needs may be addressed by the GLA vector delivery described herein.

[0116] Gene therapy is a promising approach for the treatment and prevention of Fabry disease. One of the advantages of gene therapy approaches for the treatment of Fabry disease is continuous α-GAL exposure, rather than the intermittent α-GAL exposure provided by ERT infusions. Gene therapy approaches may allow for uptake by certain tissues and cell types (e.g., the peripheral nervous system), which is not easily achieved with infused ERT. The continued availability of α-GAL in lysosomes prevents the re-accumulation of glycosphingolipids between doses. The significant enhancement of the distribution of enzymes to target cells provides a transformative therapy that has the potential to achieve clinical benefits superior to existing therapies. In addition, gene therapy with hepatocyte transduction can take advantage of liver tolerance and induce systemic immune tolerance to transgenic products, eliminating the risk of reduced therapeutic efficacy due to anti-drug antibodies. Without wishing to be bound by theory, it is believed that these benefits, combined with a single, long-acting dose, can both meet the need for treatments with significantly higher therapeutic efficacy and reduce the burden of treatment on patients and caregivers.

[0117] As discussed below, the present disclosure provides GLA gene therapy for treating and / or ameliorating one or more neurological manifestations in Fabry disease.

[0118] Gene therapy vectors

[0119] In one aspect of the present disclosure, a GLA expression system (i.e., GLA transgenic) is provided, which comprises a viral vector containing a polynucleotide encoding α-GAL or its variant controlled by a ubiquitous promoter. In some aspects, the viral vector is a recombinant AAV vector. Specifically, the recombinant AAV (rAAV) vector has a wide range of tissue tropism. The rAAV vector considered in the present application has a wide range of tissue tropism, and utilizes a ubiquitous promoter to drive a wide range of gene expression, causing sustained high-level protein expression and robust protein exposure to a wide range of tissues and / or GB3 or lysoGb3 levels to decrease. In addition, the GLA transgenic carries out codon optimization and / or expresses an engineered variant of α-GAL, causing an increase in α-GAL activity in vivo, and / or a reduction in lysoGb3 or GB3 levels in vivo.

[0120] α-GAL enzyme and variants

[0121] 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 naturally occurring (wild-type) enzyme having an 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.

[0122] Exemplary amino acid sequences of α-GAL enzymes and variants thereof contemplated for use in the vectors of the present disclosure are provided in Table 1 below.

[0123] Table 1: Amino acid sequences of α-GAL enzymes and their variants

[0124]

[0125]

[0126]

[0127]

[0128] 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, the α-GAL enzyme comprising a signal peptide comprises an amino acid sequence selected from SEQ ID NO.: 5-7 (Table 1).

[0129] 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).

[0130] In some embodiments, the modified sequence comprises 1 to 10 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). For example, in some embodiments, the modified sequence comprises 1 to 9 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises 1 to 8 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises 1 to 7 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises 1 to 6 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises 1 to 5 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5). In some embodiments, the modified sequence comprises 1 to 4 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 1). In some embodiments, the modified sequence comprises 1 to 3 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5).

[0131] In some embodiments, the modified sequence comprises 10 amino acid substitutions compared to the wild-type α-GAL enzyme (SEQ ID NO: 5).

[0132] In some embodiments, the GLA transgene expresses a 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 greater 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.

[0133] In some embodiments, the recombinant α-GAL comprises at least one substitution at one or more positions in SEQ ID NO:5 selected from the group consisting of: 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.

[0134] Additional exemplary GLA transgenes and α-GAL enzyme sequences can be found in PCT Publication Nos: PCT / US2021 / 019811, PCT / US2019 / 067493, and PCT / US2015 / 063329, each of which is herein incorporated by reference in its entirety.

[0135] In some embodiments, the modified α-GAL enzyme has increased stability (eg, serum stability), intracellular stability (eg, lysosomal activity), and / or specific catalytic activity compared to the wild-type α-GAL enzyme (SEQ ID NO: 5).

[0136] In some embodiments, the present disclosure encompasses a gene therapy vector comprising a GLA sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to any one of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having 70% to 100% identity to SEQ ID NOs. 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having 75% to 100% identity to SEQ ID NOs. 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having 80% to 100% identity to SEQ ID NOs. 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having 85% to 100% identity to SEQ ID Nos: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having 90% to 100% identity to SEQ ID Nos: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having 95% to 100% identity with SEQ ID No: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having at least 70% identity with one of SEQ ID No: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having at least 75% identity with one of SEQ ID No: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having at least 80% identity with one of SEQ ID No: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having at least 85% identity with one of SEQ ID No: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having at least 90% identity with one of SEQ ID No: 1-3 and 5-7. In some embodiments, the vector comprises a GLA sequence having at least 91% identity with one of SEQ ID No: 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.

[0137] In some embodiments, the present disclosure encompasses a gene therapy vector comprising a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 3. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 6. In some embodiments, the disclosure encompasses a gene therapy vector comprising a GLA sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 7.

[0138] In some embodiments, the GLA gene therapy vector comprises a polynucleotide encoding an α-GAL enzyme or a variant thereof. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a polynucleotide encoding an α-GAL enzyme comprising 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to SEQ ID NO: 1. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a polynucleotide encoding an α-GAL enzyme comprising 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to SEQ ID NO: 2. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a polynucleotide encoding an α-GAL enzyme comprising 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to SEQ ID NO: 3. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a polynucleotide encoding an alpha-GAL enzyme comprising 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to SEQ ID NO: 5. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a polynucleotide encoding an alpha-GAL enzyme comprising 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to SEQ ID NO: 6. In some embodiments, the present disclosure encompasses a gene therapy vector comprising a polynucleotide encoding an alpha-GAL enzyme comprising 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to SEQ ID NO: 7.

[0139] In other embodiments, the GLA transgene of the present disclosure may comprise α-GAL or a variant thereof disclosed in the applicant's previous PCT application NO: PCT / US22 / 17998 (eg, the sequences in Table 1 of PCT / US22 / 17998); the contents of which are incorporated herein by reference in their entirety.

[0140] In some embodiments, the GLA transgene comprises a nucleic acid sequence encoding the signal peptide sequence 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.

[0141] In some embodiments, the polynucleotide encoding the α-GAL enzyme or a variant thereof comprises at least one chemical modification. In some embodiments, the polynucleotide encoding the α-GAL enzyme or a variant thereof is codon optimized.

[0142] As non-limiting examples, the polynucleotide comprises a nucleotide sequence in 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.

[0143] Table 2: Nucleotide sequences of α-GAL and its variants

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150] 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 that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% identical to any one of SEQ ID NOs: 8-10 and 12-13.

[0151] In some embodiments, the polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence that is 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, the polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence that is 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, the polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence that is 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, the polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% identical to SEQ ID NO: 12. In some embodiments, the polynucleotide encoding α-GAL or a variant thereof comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% identical to SEQ ID NO: 13.

[0152] In other embodiments, the polynucleotide encoding α-GAL or a variant thereof may comprise a nucleic acid sequence disclosed in the applicant's previous PCT application NO: PCT / US22 / 17998 (eg, a sequence in Table 1 of PCT / US22 / 17998); the contents of which are incorporated herein by reference in their entirety.

[0153] Recombinant AAV (rAAV) Vectors

[0154] Transgenes delivered by vectors can be introduced into cells of interest using a variety of methods. For example, viral or non-viral vectors can be used to deliver transgenes of interest. The methods provided herein consider both viral and non-viral vector delivery methods. Therefore, in some embodiments, the vectors described herein are delivered in viral vectors. In some embodiments, the vectors described herein are delivered in non-viral vectors. In some embodiments, the vectors can be introduced as naked nucleic acids, as nucleic acids complexed with agents such as liposomes or poloxamer. Various viral vectors are known in the art for delivering transgenes, and include, for example, integrated or non-integrated vectors. In some embodiments, the viral vector is a non-integrated viral vector. Non-integrated viral vectors include, for example, non-integrated lentiviral vectors or AAV vectors. In other embodiments, the GLA transgene of the present invention can be delivered by viruses (e.g., adenovirus, adeno-associated virus (AAV), herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)). The vector may have additional sequences, such as replication origins, promoters, and one or more genes.

[0155] As a non-limiting example, the GLA transgene described herein is introduced using a viral vector; the viral vector is an adeno-associated virus (AAV) derived vector. According to the present disclosure, a recombinant adeno-associated virus (rAAV) vector is used for the GLA transgene.

[0156] In general, rAAV vectors comprise a capsid and a viral genome that is modified to include a transgene, such as a transgene for expressing an α-GAL enzyme (i.e., a polynucleotide encoding an α-GAL enzyme or a variant thereof). The AAV vectors described herein may comprise or be derived from any natural or recombinant AAV serotype. AAV serotypes may differ in features such as, but not limited to, packaging, tropism, transduction, and immunogenicity profiles. Although not wishing to be bound by theory, it is generally believed that the AAV capsid protein is a driving factor in the tropism of AAV particles to specific tissues.

[0157] In many gene therapy applications, it is desirable that gene therapy vector delivery has specificity for a particular tissue type. Due to the reduced tissue tropism of previously used gene therapy vectors, previous gene therapy approaches have had limited success in treating Fabry disease. Unlike previously used vector designs, the vector designs provided here have broad tissue and cell type distribution once administered to a subject in need. The rAAV vectors described herein have a wide tissue distribution and include, for example, heart, liver, kidney, gastrointestinal tract, and neural tissue.

[0158] A variety of AAV capsids with broad tissue tropism (the terms "broad tissue tropism", "wide tropism" are used interchangeably herein) can be used in the rAAV vectors described herein. Various capsids and associated tropisms are described in Curr Opin Vir. 2016 Dec 21: 75-80 (the contents of which are incorporated herein by reference). "Broad tissue tropism" means that the capsid enables gene transfer to two or more than 2, 3, 4, 5, 6, 7, 8 or more tissue types. For example, in some embodiments, the capsid with broad tissue tropism enables gene transfer to one or more of the following tissues: liver, kidney, heart, gastrointestinal tract and / or peripheral neurons of a subject.

[0159] For example, in some embodiments, the AAV capsid is a broadly tropic 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.

[0160] Thus, in some embodiments, the AAV capsid having wide tropism comprises an AAV1 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV2 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV3 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV4 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV5 sequence. In some embodiments, the AAV capsid having wide tropism comprises an AAV6 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV7 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV8 capsid. In some embodiments, the AAV capsid having wide tropism comprises an AAV9 capsid.

[0161] Although not 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 can be considered synonymous with the parent AAV capsid sequence.

[0162] In some embodiments, the rAAV capsid sequence may comprise an amino acid sequence that is 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the amino acid sequences described above.

[0163] Recombinant or engineered AAV vectors may include capsids engineered to have enhanced tropism for targeting broad tissues or targeting neural tissues (e.g., DRG). Capsid engineering methods have been used to attempt to identify capsids with enhanced transduction of broad tissues (e.g., kidney, liver, lung, heart, brain, spinal cord, DRG). A variety of methods have been used, including mutation methods, DNA barcoding, directed evolution, random peptide insertion, and capsid shuffling and / or chimeras.

[0164] In a preferred embodiment, the rAAV vectors described herein comprise an AAV9 capsid sequence. In some instances, 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.

[0165] AAV vectors include one or more terminal inverted repeat (ITR) sequences. The terminal inverted repeat sequence (Inverted terminal repeat, ITR) traditionally caps the viral genome at the 5' end and the 3' end, providing a replication origin for the viral genome. Although it is not desired to be bound by theory, the AAV viral vector generally includes two ITR sequences at the 5' end and the 3' end of the ssDNA, which form an energy-stable double-stranded region. In some embodiments, the AAV vector is a recombinant AAV viral vector, which has a replication defect and lacks the sequence encoding the functional Rep and Cap proteins in its viral genome. These defective AAV vectors may lack most or all of the parental coding sequences, and essentially carry only one or two AAV ITR sequences and GLA transgenes to deliver to cells, tissues, organs or organisms.

[0166] In some embodiments, the rAAV vector described herein comprises at least one ITR and a GLA transgene. In some embodiments, the rAAV vector has two ITRs. The two ITRs are flanked by a transgenic region at the 5' end and the 3' end. The ITR acts as a replication origin and comprises a replication recognition site. The ITR comprises a sequence region that is complementary and symmetrically arranged. As described herein, the ITR incorporated into the viral genome may be composed of a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence. The ITR may be derived from the same serotype as the AAV capsid, selected from any known AAV serotype or a derivative thereof. The ITR may have a different serotype from the capsid.

[0167] In some embodiments, the ITR length is 140-142 nucleotides. The non-limiting examples of ITR length are 102, 105, 130, 140, 141, 142, 145 nucleotides. ITRs encompassed by the present disclosure include ITRs having 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 a known AAV serotype ITR sequence.

[0168] In some embodiments, the rAAV vector comprises at least one element that enhances 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, post-transcriptional regulatory elements (PREs), polyadenylation (poly-A) signal sequences and upstream enhancers (USEs), CMV enhancers, and introns.

[0169] Those skilled in the art will recognize that specific promoters may be required for expression of transgenes in target cells, including but not limited to species-specific, inducible, tissue-specific, or cell cycle-specific promoters (Parr et al., Nat. Med. 1997, 3: 1145-1149). In some embodiments, the promoter is considered to be effective when it drives expression of a transgene (e.g., a GLA transgene) of an rAAV vector. In some embodiments, the promoter is a promoter that is considered effective when it drives expression in a targeted cell. In some embodiments, the promoter is a promoter that is tropistic for targeted cells, such as neurons.

[0170] As a non-limiting example, a promoter is selected for sustained expression of a transgene in tissues and / or cells of the central or peripheral nervous system.

[0171] 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 those derived from human cells or systems. In some embodiments, the promoter may be truncated or mutated.

[0172] Promoters that drive or promote expression in most tissues include, but are not limited to, human elongation factor 1Į-subunit (EF1Į) promoter, cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken ȕ-actin (CBA) promoter and its derivatives CAG, ȕ glucuronidase (GUSB) promoter or ubiquitin C (UBC) promoter. In some embodiments, the viral vector comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include EF-1Į, PGK, UBC, GUSB (hGBp) and UCOE (promoter of HNRPA2B1-CBX3).

[0173] In some embodiments, the promoter sequence is a ubiquitous promoter sequence. In some embodiments, the promoter is a mammalian ubiquitous promoter that promotes the expression of a coding sequence (e.g., a GLA transgenic) in a mammalian cell. In some embodiments, the rAAV vector expressing the GLA transgenic under a ubiquitous promoter realizes the wide distribution of the encoded α-GAL on multiple target tissues such as kidney, liver, lung, heart, and nervous system in mammals, thereby causing α-GAL to be more widely exposed and better treat Fabry disease and related symptoms. As a non-limiting example, the ubiquitous promoter used in the present disclosure can be selected from one or more of 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 miniature EF1 promoter. In some embodiments, the ubiquitous promoter can be engineered 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.

[0174] In some embodiments, cell type specific promoters can 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 ganglia, dorsal root nerves, motor neurons, or supporting cells of the nervous system, such as microglia, astrocytes, oligodendrocytes, and / or Schwann cells.

[0175] In some embodiments, the promoter can be the same or different starting promoters or a combination of two or more components of a parent promoter.

[0176] In some embodiments, rAAV vector components can be selected and / or engineered to further tailor the specificity and efficiency of GLA transgene expression in the nervous system (eg, DRG).

[0177] 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 a variant thereof and the polyadenylation sequence.

[0178] In some embodiments, in order to further increase the expression of the GLA transgene in the nervous system to alleviate the neuropathy (e.g., DRG and other cells of the CNS and PNS) in Fabry disease when using a capsid sequence with a broad tropism and ubiquitous promoter, the rAAV vector may also optionally include a targeting peptide for the nervous system. In some embodiments, the targeting peptide can direct the AAV particles to the cells or tissues of the PNS. The cells or tissues of the PNS may be, but are not limited to, dorsal root ganglia (DRG). The length of the targeting peptide may vary. In some embodiments, the length of the targeting peptide is 3-20 amino acids. As non-limiting examples, the 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 in length.

[0179] In certain embodiments, the rAAV vector further comprises a post-transcriptional regulatory element (PRE). In some embodiments, the vector comprises a woodchuck hepatitis virus post-transcriptional control element (WPRE). Various optimized or variant forms of WPRE are known in the art, and include WPRE3, WPREmut6delATG, etc. Other variant WPRE forms include, for example, WPRE2, WPRE_wt (GenBank deposit number J04514); WPRE_wt (GenBank deposit number J02442) and WPREmut6. The WPRE element may comprise a wild-type sequence or a modified WPRE element sequence. Various mutant forms of WPRE are known, and include, for example, mut6delATG (SEQ ID NO: 17). In some embodiments, the vector comprises mut6delATG (SEQ ID NO: 17).

[0180] In some embodiments, the present disclosure encompasses a gene therapy vector comprising a modified GLA gene sequence. Such modifications may be performed to improve expression characteristics. Such modifications may include, but are not limited to, insertion of a translation start site (e.g., methionine), addition of a Kozak sequence (gccacca), insertion of a signal peptide, and / or codon optimization. Thus, in some embodiments, the GLA gene is modified to include insertion of a translation start site. In some embodiments, the GLA gene is modified to include addition of a Kozak sequence. In some embodiments, the GLA gene is modified to include a signal peptide. In some embodiments, the transgene is codon optimized. In other embodiments, the GLA gene is engineered. In other embodiments, the GLA gene is codon optimized and engineered.

[0181] In some embodiments, the vectors described herein comprise one or more polyadenylation sequences. In some embodiments, the polyadenylation is selected from human growth hormone polyadenylation (hGHpA), synthetic polyadenylation (SPA), simian virus 40 late polyadenylation (SV40pA), and bovine growth hormone (BGH) polyadenylation.

[0182] In some embodiments, the vector comprises an ID tag, such as a stuffer sequence. The purpose of the ID tag includes, for example, enabling a technician to identify the vector. An example of a DNA tag sequence is listed in Table 3 (SEQ ID NO: 20).

[0183] 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 a rAAV9 vector.

[0184] Exemplary sequences of rAAV vectors are shown below in Table 2. In some embodiments, the rAAV vector comprises a rAAV vector element comprising a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a vector element sequence shown below in Table 3. In some embodiments, the rAAV vector comprises a vector element nucleotide sequence that is identical to a vector element nucleotide sequence shown below in Table 3.

[0185] Table 3: Exemplary vector component sequences

[0186]

[0187]

[0188]

[0189]

[0190] In some embodiments, the present disclosure encompasses a rAAV vector packaged in an AAV capsid with broad tissue tropism, wherein the rAAV vector comprises: a) a 5′ terminal inverted repeat (ITR); b) a ubiquitous promoter; c) a polynucleotide encoding an α-GAL enzyme or a variant thereof; d) polyadenylation; and e) a 3′ ITR sequence.

[0191] 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., having a mut6delATG mutation) between the nucleotide sequence encoding the α-GAL enzyme and the polyadenylic acid sequence. As a non-limiting example, the rAAV vector comprises: a) a 5' terminal inverted repeat sequence (ITR); b) a ubiquitous promoter; c) a polynucleotide encoding an α-GAL enzyme or a variant thereof; d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); e) polyadenylic acid; and f) a 3' ITR sequence.

[0192] In other embodiments, the rAAV vector capable of achieving ubiquitous α-GAL enzyme expression upon administration to a subject in need thereof comprises: (a) a 5' terminal inverted 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) polyadenylation; and (e) a 3' ITR.

[0193] In some embodiments, a rAAV vector capable of achieving ubiquitous α-GAL enzyme expression after administration to a subject in need thereof comprises: (a) a 5' terminal inverted 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) having a mut6delATG mutation; (e) a bovine growth hormone (BGH) polyadenylation; and (f) a 3' ITR.

[0194] In some embodiments, a rAAV vector capable of achieving ubiquitous α-GAL enzyme expression after administration to a subject in need thereof is packaged in an AAV9 capsid, wherein the rAAV vector comprises: (a) a 5' terminal inverted repeat sequence (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) polyadenylation; and (e) a 3' ITR sequence.

[0195] In some embodiments, a rAAV vector capable of achieving ubiquitous α-GAL enzyme expression after administration to a subject in need thereof is packaged in an AAV9 capsid, wherein the rAAV vector comprises: (a) a 5' terminal inverted repeat sequence (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) having a mut6delATG mutation; (e) a bovine growth hormone (BGH) polyadenylation; and (f) a 3' ITR sequence.

[0196] In some embodiments, the present disclosure provides an expression cassette comprising a polynucleotide sequence including: (a) a 5' terminal inverted 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) an optional woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a mut6delATG mutation; (e) a bovine growth hormone (BGH) polyadenylation; and (f) a 3' ITR. In some embodiments, the elements in the above expression cassette are present in a 5' to 3' order. In various embodiments, one or more of (a) to (f) are operably linked in a 5' to 3' order.

[0197] In one example, the present disclosure provides an rAAV vector packaged in an AAV capsid, the rAAV vector comprising (a) a 5' terminal inverted repeat sequence (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) polyadenylation comprising SEQ ID NO: 17; and (e) a 3' ITR sequence comprising SEQ ID NO: 18.

[0198] In another example, the present disclosure provides an rAAV vector packaged in an AAV capsid, the rAAV vector comprising (a) a 5' terminal inverted repeat sequence (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) polyadenylation comprising SEQ ID NO: 18; and (f) a 3' ITR sequence comprising SEQ ID NO: 18.

[0199] As a non-limiting example, the present disclosure provides an rAAV vector comprising (a) a 5' terminal inverted repeat sequence (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) polyadenylation 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' terminal inverted repeat sequence (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 post-transcriptional regulatory element (WPRE) comprising SEQ ID NO: 16; (e) a bovine growth hormone (BGH) polyadenylation comprising SEQ ID NO: 17; and (f) a 3'ITR sequence comprising SEQ ID NO: 18.

[0200] Production of rAAV vectors

[0201] In various embodiments, the rAAV vectors described herein for delivery of transgenes (e.g., genes encoding α-galactosidase (α-GAL) proteins) can be packaged using techniques known in the art and as 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.

[0202] The viral vectors used in gene therapy are usually produced by a production cell line that packages nucleic acid vectors into viral particles. The vector usually contains the minimum viral sequence required for packaging and subsequent integration into the host, and other viral sequences are replaced by expression cassettes encoding proteins to be expressed. In this case, the protein to be expressed is an α-GAL enzyme or a variant thereof. The missing viral function can be provided in trans by a packaging cell line. For example, AAV vectors used in gene therapy usually only have terminal inverted repeat (ITR) sequences required for packaging and integration into the host genome from the AAV genome.

[0203] Methods for producing and separating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. Patent Nos. 7790449, 7282199, and 7588772; PCT publications WO 2003 / 042397, WO 2005 / 033321, and WO 2006 / 110689. In one system, the packaging cell line is transiently transfected with a construct encoding a transgenic flanking ITR and a construct encoding AAV rep and cap proteins. In a second system, a packaging cell line stably supplied with rep and cap is transiently transfected with a transgenic flanking ITR. In each of these systems, AAV virions 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 to separate rAAV vectors from contaminated viruses.

[0204] Other systems that do not require infection with a helper virus to recover AAV can also be used. In these newer systems, helper functions can be provided by transiently transfecting cells with a construct encoding the desired helper function, or cells can be engineered to stably contain genes encoding helper functions, whose expression can be controlled at the transcriptional or post-transcriptional level.

[0205] In some embodiments, an expression cassette flanked by 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-associatedvirus production; Human Gene Therapy, 2009; 20:922-929, the contents of which are incorporated herein by reference). Other AAV production systems and methods are also described in, e.g., 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.

[0206] Methods and techniques for producing transgenic expression cassettes, rAAV vectors and helper plasmids and constructs, such as genetic engineering, recombineering and synthetic techniques, are well known in the art (see Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)). Similarly, methods for producing rAAV virions are also well known, and the selection of suitable methods is not limiting to the present invention.

[0207] Many plasmids and other cloning and expression vectors that can be used according to the present invention are well known and readily available to those skilled in the art. In addition, those skilled in the art can easily construct many other plasmids suitable for use in the present invention. The properties, construction and use of such plasmids and other vectors in the present invention will be apparent to those skilled in the art in this disclosure.

[0208] In certain embodiments, the rAAV expression cassette, vector (e.g., rAAV vector), virus (e.g., rAAV), production plasmid comprises an AAV terminal inverted repeat sequence, a codon-optimized nucleic acid sequence encoding an α-GAL polypeptide, and an expression control sequence directing the expression of the encoded protein, and is present in a host cell. In other embodiments, the rAAV expression cassette, virus, vector (e.g., rAAV vector), production plasmid further comprises one or more of an intron, a Kozak sequence, polyadenylation, a post-transcriptional regulatory element, and the like. 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 a transgene encoding an α-GAL polypeptide. In some embodiments, the signal peptide is located at the N-terminus of the α-GAL polypeptide. In some embodiments, the signal peptide is located at the C-terminus of the α-GAL polypeptide.

[0209] In some embodiments, the helper plasmid includes a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising one or more of the following helper genes: Ela gene, Elb gene, E4 gene, E2a gene and VA gene. For the sake of clarity, helper genes are genes encoding helper proteins Ela, Elb, E4, E2a and VA. In some embodiments, the cap gene is modified so that one or more of proteins VP1, VP2 and VP3 are not expressed. In some embodiments, the cap gene is modified so that VP2 is not expressed. Methods for carrying out such modifications are known in the art (Lux et al. (2005), J Virology, 79: 11776-87). Helper plasmids and methods for making such plasmids are well known in the art and are generally commercially available (see, for example, pDF6, pRep, pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E, and pDP8.ape).

[0210] In some embodiments, a plasmid containing the transgene is combined with one or more helper plasmids, for example, containing the rep gene of a first serotype and the cap gene of the same serotype or a different serotype, and transfected into helper cells to package rAAV.

[0211] In some embodiments, packaging is performed in a helper cell or a production cell, such as a mammalian cell or an insect cell. Exemplary mammalian cells include, but are not limited to, HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, for example, 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, for example, ATCC® CRL-1711™). Helper cells may include rep and / or cap genes encoding Rep proteins and / or Cap proteins for use in the methods described herein. In some embodiments, packaging is performed in vitro.

[0212] Various methods related to the production and purification of AAV vectors 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 auxiliary plasmids, such as those containing rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encoding VP1, VP2, and VP3, including modified VP2 regions as described herein), and transfected into recombinant cells so that rAAV can be packaged and subsequently purified.

[0213] Pharmaceutical composition

[0214] Exemplary pharmaceutical compositions comprising the vectors described herein are described in detail below.

[0215] 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.Thus, a variety of suitable formulations of pharmaceutical compositions may be utilized.

[0216] Preparations for in vitro and in vivo administration include suspensions in liquids or emulsified liquids. The active ingredient is often mixed with a pharmaceutically acceptable and compatible excipient for the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, the composition may contain a small amount of auxiliary substances, such as a wetting agent or emulsifier, a pH buffer, a stabilizer, or other reagents that improve the effectiveness of the pharmaceutical composition.

[0217] In some embodiments, a pharmaceutical composition is provided, comprising an rAAV vector as described herein. The pharmaceutical composition containing rAAV vectors or particles of the present invention contains a pharmaceutically acceptable excipient, diluent or carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Such carriers can be prepared by conventional methods and administered to subjects in a therapeutically effective amount.

[0218] Therapeutic applications and methods of use

[0219] The transgenic 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 reducing one or more symptoms associated with the disease. In some embodiments, the vectors of the present disclosure can be used to treat subjects with reduced α-GAL expression or no α-GAL expression and / or impaired α-GAL enzyme activity.

[0220] Non-limiting examples of symptoms of Fabry disease include neuropathic pain, hypohidrosis or anhidrosis, exercise intolerance, abdominal cramps, diarrhea, angiokeratomas, keratovertebrae, tinnitus, proteinuria, chronic kidney disease, hypertension, coronary insufficiency, AV conduction disorders, arrhythmias and valvular dysfunction, left ventricular hypertrophy, seizures, and stroke.

[0221] Neuropathy in Fabry disease includes neuropathic pain, hypoesthesia of cold and heat (i.e., decreased heat sensation), hearing loss, and possible gastrointestinal disturbances. Peripheral abnormalities of peripheral nerve fibers, especially small nerve fibers (myelinated and unmyelinated), can cause neuropathy. Part of the reason for the damage is the accumulation of GB3 in the peripheral nervous system (e.g., DRG).

[0222] In various embodiments described herein, the 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 has or is showing one or more symptoms associated with peripheral neuropathy, such as neuropathic pain. Peripheral neuropathic pain in patients with Fabry disease may manifest as chronic burning pain, and superimposed attacks of acute severe pain, dysesthesia, thermal sensory disturbances (mainly feeling cold) and paresthesias (such as painless tingling). Symptoms associated with autonomic nervous system dysfunction may include hypohidrosis, impaired pupil constriction, and impaired saliva and tear production, gastrointestinal dysmotility (abdominal cramps, bloating, diarrhea, nausea) and loss of sensation.

[0223] The rAAV vector expressing the GLA transgene as 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. The method includes administering to the subject a composition comprising an rAAV expressing an α-GAL enzyme or a variant thereof as described herein. The method alleviates the manifestations of neuropathy, including neuropathic pain, decreased sensation of cold and heat (i.e., decreased sensation of heat), hearing loss, other sensory deficits, and possible gastrointestinal disturbances.

[0224] In some embodiments, the rAAV vectors described herein are used to modify peripheral abnormalities of nerve fibers in Fabry disease. Peripheral nerves are small nerve fibers, including myelinated and unmyelinated nerve fibers. The nerve fibers are cutaneous sensory nerves. In some embodiments, treatment with the vectors can maintain nerve fiber density in the peripheral nervous system.

[0225] In some embodiments, rAAV vectors are used to reduce the accumulation of GB3 and lysoGb3 in the peripheral nervous system. In some embodiments, after administering rAAV vectors as described herein, morphological defects in DRG are improved, including reducing the expression of alternative biomarker LAMP1. LAMP 1 (lysosomal associated membrane protein 1) is a biomarker for neurodegeneration. Fabry disease patients have severely enlarged dorsal root ganglia with perfusion dysfunction, which may be caused by direct neurotoxic effects mediated by glycolipid deposition in dorsal root ganglia and reduced blood supply to neurons. Increased exposure to α-GAL enzymes can reduce LAMP1 in DRG.

[0226] In some embodiments, the rAAV vector for GLA transgene causes ceramide trihexosides (GB3) in the subject to reduce. In some embodiments, compared with the baseline GB3 level of the subject before the rAAV comprising GLA, the GB3 in the subject reduces by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or about 10%. Therefore, in some embodiments, the rAAV comprising GLA used reduces the GB3 in the subject by about 95%. In some embodiments, the rAAV comprising GLA used reduces the GB3 in the subject by about 90%. In some embodiments, the rAAV comprising GLA used reduces the GB3 in the subject by about 85%. In some embodiments, the rAAV comprising GLA used reduces the GB3 in the subject by about 80%. In some embodiments, the rAAV comprising GLA used reduces the GB3 in the subject by about 75%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 70%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 65%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 60%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 55%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 50%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 45%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 40%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 35%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 30%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 25%. In some embodiments, the administered rAAV comprising GLA reduces GB3 in a subject by about 20%. In some embodiments, the 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%.

[0227] In some embodiments, GB3 in serum 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 prior to administration of the rAAV expressing the GLA transgene. In some embodiments, GB3 in kidney, heart, and / or liver 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 prior to administration of the rAAV expressing the GLA transgene.

[0228] In some embodiments, the rAAV vector is used to reduce the accumulation of GB3 and lysoGb3 in the kidney. In some embodiments, the rAAV vector is used to reduce the accumulation of GB3 and lysoGb3 in the liver. In some embodiments, the rAAV vector is used to reduce the accumulation of GB3 and lysoGb3 in the heart. In some embodiments, the rAAV vector is used to reduce the accumulation of GB3 and lysoGb3 in serum.

[0229] In some embodiments, the administered rAAV comprising GLA causes production of the α-GAL enzyme in the kidney of the subject. In some embodiments, the administered rAAV comprising GLA causes production of the α-GAL enzyme in the heart of the subject. In some embodiments, the administered rAAV comprising GLA causes production of the α-GAL enzyme in the liver of the subject. In some embodiments, the administered rAAV comprising GLA causes production of the α-GAL enzyme in the serum of the subject.

[0230] In some embodiments, LAMP1 levels in the peripheral nervous system (e.g., in 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 prior to administration of the rAAV expressing the GLA transgene. In some embodiments, the rAAV gene therapy reduces lysosomal autophagic organelles in the DRG.

[0231] In some embodiments, the administered rAAV transgene comprising GLA reduces GB3 in the 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.

[0232] In some embodiments, vector-mediated gene therapy provides consistent expression and high α-GAL activity in serum and other tissues (e.g., kidney, heart, liver, and nervous system). In some embodiments, vector-mediated gene therapy provides consistent expression and high α-GAL activity in the peripheral nervous system (e.g., DRG).

[0233] In some examples, functional α-GAL is detectable in the serum and tissues of the 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 the subject 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks or 18 weeks after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the serum and tissues of the 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.

[0234] In some embodiments, functional α-GAL is detectable in the nervous system (e.g., DRG) of the subject at 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 the subject at 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, or 18 weeks or more after administration of the rAAV vector. In some embodiments, functional α-GAL is detectable in the nervous system (e.g., DRG) of the 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 after administration of the rAAV vector.

[0235] In some embodiments, the level of functional α-GAL detectable in the circulation after administration of the AAV vector to a subject is between about 2-fold and 1000-fold higher, or 10-fold higher, 20-fold higher, 30-fold higher, 40-fold higher, 50-fold higher, 60-fold higher, 70-fold higher, 80-fold higher, 90-fold higher, 95-fold higher, or 100-fold higher than the amount of functional α-GAL detectable in the subject before administration of the rAAV comprising the GLA transgene.

[0236] In some embodiments, the level of functional α-GAL detectable in the PNS after administration of the AAV vector to the subject is between about 2-fold and 100-fold higher, or greater than 10-fold, greater than 20-fold, greater than 30-fold, greater than 40-fold, greater than 50-fold, greater than 60-fold, greater than 70-fold, greater than 80-fold, greater than 90-fold, greater than 95-fold, or 100-fold or more higher than the amount of functional α-GAL detectable in the subject before administration of the rAAV comprising the GLA transgene.

[0237] In some embodiments, the level of active α-GAL detectable after the AAV vector is administered to the subject reaches or exceeds the human therapeutic level, i.e., the level of α-GAL considered to be the normal circulating level of humans (e.g., 5-9 nmol / h / ml). In some embodiments, the level of active α-GAL after administration of the rAAV vector is between about 2 and 35 times the human therapeutic level, or greater than 35 times, greater than 40 times, greater than 45 times, greater than 50 times, greater than 55 times, greater than 60 times, greater than 65 times, greater than 70 times, greater than 75 times, greater than 80 times, greater than 85 times, greater than 90 times, greater than 95 times, greater than 100 times, greater than 200 times, greater than 300 times, greater than 400 times, greater than 500 times, greater than 600 times, greater than 700 times, greater than 800 times, greater than 900 times, or greater than 1000 times.

[0238] Thus, administration of a rAAV vector comprising a GLA transgene results in sustained robust expression compared to a single administration of purified α-GAL to a subject in need thereof.

[0239] In some embodiments, after the AAV vector is administered to the subject, a 10% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to the subject, a 15% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to the subject, a 20% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to the subject, a 25% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to the subject, a 35% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to the subject, a 40% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to the subject, a 45% Gb3 reduction in ERT-resistant cell types is achieved. In some embodiments, after the AAV vector is administered to a subject, a 50% reduction in Gb3 in an ERT-resistant cell type is achieved. In some embodiments, after the AAV vector is administered to a subject, a 55% reduction in Gb3 in an ERT-resistant cell type is achieved. In some embodiments, after the AAV vector is administered to a subject, a 60% reduction in Gb3 in an ERT-resistant cell type is achieved. In some embodiments, after the AAV vector is administered to a subject, a 65% reduction in Gb3 in an ERT-resistant cell type is achieved.

[0240] 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 persists for at least 1 year. In some embodiments, the improvement in Fabry disease symptoms persists for 1 year or longer.

[0241] In some embodiments, after the AAV vector is administered to the subject, a 10% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, after the AAV vector is administered to the subject, a 15% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, after the AAV vector is administered to the subject, a 20% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, after the AAV vector is administered to the subject, a 25% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, after the AAV vector is administered to the subject, a 35% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, after the AAV vector is administered to the subject, a 40% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, a 45% reduction in Gb3 in ERT-resistant cell types is achieved at 6 months following administration of the AAV vector to a subject. In some embodiments, a 50% reduction in Gb3 in ERT-resistant cell types is achieved at 6 months following administration of the AAV vector to a subject.

[0242] In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 1 month. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 2 months. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 3 months. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 4 months. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 5 months. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 6 months. In some embodiments, after the AAV vector is administered to the subject, a 30% Gb3 reduction in ERT-resistant cell types is achieved at 7 months. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved at 8 months following administration of the AAV vector to the subject. In some embodiments, a 30% reduction in Gb3 in ERT-resistant cell types is achieved at 9 months following administration of the AAV vector to the subject.

[0243] The rAAV vector is administered to a subject diagnosed with Fabry disease via a suitable route. In some embodiments, the rAAV vector is administered by intravenous, intraperitoneal, subcutaneous or intradermal administration. In some embodiments, the rAAV vector is administered intravenously. In some embodiments, intradermal administration includes administration by using a "gene gun" or a bioballistic particle delivery system. In some embodiments, the rAAV vector is administered via non-viral lipid nanoparticles. For example, a composition comprising an rAAV vector may include one or more diluents, buffers, liposomes, lipids, lipid complexes. In some embodiments, the rAAV vector is contained in a microsphere or nanoparticle (e.g., lipid nanoparticle).

[0244] In some embodiments, the rAAV vector is applied by spinal application, such as spinal injection. In some embodiments, the rAAV vector remains free after being applied to a subject in need. In some embodiments, the rAAV vector does not remain free after being applied to a subject in need. For example, in some embodiments, the rAAV vector is integrated into the genome of the subject. Such integration can be achieved, for example, by using various gene editing techniques, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), ARCUS genome editing and / or CRISPR-Cas systems.

[0245] In some embodiments, the rAAV vector comprising the GLA transgene is administered as a single dose to a subject in need thereof. In some embodiments, the rAAV vector is administered to a subject at a minimum effective dose (MED). As used herein, MED refers to the rAAV GLA vector dose required to achieve α-GAL activity in a subject, causing a decrease in GB3 levels.

[0246] In some embodiments, the dose is between 1×10 10 vg / kg (viral genome / kg body weight) to 1×10 14 In some embodiments, the rAAV vectors of the present disclosure are dosed at 1×10 10 vg / kg to 5×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 10 vg / kg to 1×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 10 vg / kg to 5×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 10 vg / kg to 1×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 10 vg / kg to 5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 10 vg / kg to 2.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5×10 10 vg / kg to 1×10 14 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5×10 10 vg / kg to 5×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5×10 10 vg / kg to 1×10 13 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5×10 10 vg / kg to 5×10 12In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5×10 10 vg / kg to 1×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5×10 10 vg / kg to 5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5×10 10 vg / kg to 2.5×10 11 The dose was administered in the range of vg / kg.

[0247] In some embodiments, the rAAV vectors of the present disclosure are expressed at 1.5×10 10 Up to 2.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 10 Up to 2×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2.5×10 10 Up to 3.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 3.5×10 10 to 4.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 4.5×10 10 Up to 5.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.5×10 10 Up to 6.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 6.5×10 10 to 7.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 7.5×10 10 Up to 8.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 8.5×10 10 to 9.5×10 10 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 9.5×10 10 Up to 1×1011 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 11 to 1.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.5×10 11 Up to 2.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2×10 11 Up to 2.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2.5E11 to 3.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 3.5×10 11 to 4.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 4.5×1011 to 5.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 5.5×10 11 Up to 6.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 6.5×10 11 to 7.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 7.5×10 11 Up to 8.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 8.5×10 11 to 9.5×10 11 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 9.5×10 11 Up to 1×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1×10 12 vg / kg to 2×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 1.5×10 12 vg / kg to 2.5×1012 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2.5×10 12 vg / kg to 3.5×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 3.5×10 12 vg / kg to 4.5×10 12 In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 4.5×10 12 vg / kg to 5.5×10 12 dosage administration.

[0248] In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2.5×1010 vg / kg to 1×1011 vg / kg. In some embodiments, the rAAV vectors of the present disclosure are administered at a dose of 2.5×1010 vg / kg to 2.5×1012 vg / kg.

[0249] In some embodiments, a rAAV gene therapy vector expressing an α-galactosidase protein as described herein is administered to a subject at a dose that is lower than or equal to the dose used with other gene therapy vectors (e.g., AAV with a liver-specific promoter); however, still exhibits higher serum and tissue exposure, such as high and sustained exposure in the PNS (e.g., DRG).

[0250] In some embodiments, the rAAV vectors expressing the GLA transgene provided herein are used as a prophylactic treatment in subjects with Fabry disease to prevent the onset and / or progression of one or more symptoms in Fabry disease. In one embodiment, the rAAV vectors expressing the GLA transgene provided herein are used as a prophylactic treatment in subjects with Fabry disease to prevent the onset and / or progression of one or more peripheral neuropathy manifestations.

[0251] Prophylactic treatments can be administered, for example, to subjects who are not yet ill, but who are susceptible to or at risk for a particular biological disorder, including Fabry disease (e.g., a subject may have a mutation that causes Fabry disease but is asymptomatic, or the status of the mutation that causes Fabry disease is unknown). In some embodiments, therapeutic treatments can be administered, for example, to subjects who already have Fabry disease, to improve or stabilize the subject's disorder (e.g., a patient who has developed symptoms of Fabry disease).

[0252] Peripheral neuropathy

[0253] In some embodiments, the methods of the invention further include assessment methods and tests for evaluating improvement of peripheral neuropathy in Fabry disease patients treated with rAAV-mediated gene therapy contemplated in the present disclosure.

[0254] As discussed in the present disclosure, hot plate latency tests in a mouse model of Fabry disease showed that G3Stg / GLAko mice treated with a rAAV vector containing a GLA transgene responded significantly to heat stimulation, with similar sensitivity to wild-type mice (e.g., Figure 5 ). The results observed in mice indicate that rAAV-mediated GLA gene therapy can similarly improve hypothermia in patients with Fabry disease. Therefore, peripheral neuropathy in patients with Fabry disease can be measured using clinical methods commonly used to evaluate the efficiency of rAAV-mediated GLA gene therapy as described herein.

[0255] Fabry disease patients can be diagnosed and show neuropathy related symptoms when they are young. In some cases, patients can be diagnosed and show neuropathy related symptoms in adulthood. Doctors (such as neurologists) can usually recognize a series of obvious early features of Fabry disease, some of which are related to small fiber neuropathy. Features include chronic burning pain, severe pain attacks, paresthesia / dysesthesia, loss of sensation, less sweat / no sweat, abdominal cramps, (postprandial) diarrhea, bloating, nausea and tinnitus, hearing loss.

[0256] Neuropathic pain assessment scale can be used for initial assessment and follow-up examination after treatment. Neuropathic symptoms and changes questionnaire assesses the number, severity and changes of symptoms, as well as motor nerve, autonomic nerve, large fiber and small fiber sensory nerve function (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 Leeds neuropathic symptoms and signs assessment (Leeds Assessment of Neuropathic Symptoms and Signs), neuropathic pain questionnaire, neuropathic pain symptom checklist, dual neuropathy 4 questions, pain DETECT and ID-pain. In clinical studies of diabetes and Fabry disease patients with peripheral neuropathy, total symptom scores have been used for the classification of neuropathic pain.

[0257] Neurological examination of peripheral small fiber-mediated sensation can be performed before and after treatment. For example, a relatively crude thermal perception test can be used, which consists of placing tubes filled with cold and warm water, cold and warm tuning forks, the handle of a reflex hammer, or a hot plate with a polyethylene surface on one side and a metal surface on the other side (similar to "Minnesota Thermal Discs") on the patient's foot.

[0258] The patient's pain perception and hyperalgesia can be tested before and after treatment by applying a sharp needle to the skin with just enough pressure. Loss of the ability to differentiate between the pinprick sensation and the sensation of applied dull pressure suggests a small fiber neuropathy.

[0259] In some embodiments, a Fabry disease patient who has or is experiencing one or more neuropathy-related symptoms is treated with a rAAV vector expressing a GLA transgene as described herein. Improvement of neuropathy-related symptoms is assessed based on the same post-treatment assessment. In some instances, the assessment is performed 2 weeks, 5 weeks, 10 weeks, 18 weeks, 6 months, or more after treatment.

[0260] In some embodiments, peripheral neuropathy is quantified by a computer-assessed sensory assessor 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 each of which are incorporated herein by reference in their entirety.

[0261] In some embodiments, the perception of cold and warmth is measured in patients treated with the methods of the invention. Cold sensation in the feet is a major marker of Fabry disease neuropathy and can be quantified in units of just-notice difference (JND) on a scale of 1 to 25 to assess the efficacy of treatment (see Ries et al., 2007). In the context of experimental measurements focusing on sensation and perception, the term "just-notice difference" or "JND" is the amount that must change in order for the difference to be noticeable, detectable, at least half the time.

[0262] In some embodiments, quantitative sensory testing (QST) can be used to investigate improvements in neuropathy in patients; QST can 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, a calibrated thermal pattern can be used for QST before and after rAAV treatment.

[0263] In some embodiments, the morphology of peripheral nerve fibers is imaged and the treatment results are measured.

[0264] In some embodiments, skin pathology sections are taken from patients for evaluation of skin nerve fiber density. For example, skin samples are obtained from the calf and subjected to standard immunostaining to evaluate nerve fiber morphology.

[0265] In some embodiments, neuronal conduction activity can be measured before and after rAAV treatment.As a non-limiting example, microneurographic recordings can be used to record action potentials of human nerve fibers (eg, skin fibers).

[0266] Combination therapy

[0267] The compositions and methods of the present invention can also be used in combination with other therapies known in the art for treating Fabry disease or its complications, including but not limited to: SRT (substrate reduction therapy), ERT (e.g., agalsidase beta), analgesics (e.g., lidocaine, diphenylhydantoin, carbamazepine, gabapentin, phenytoin, neurotrophins, opioids), dyspepsia treatments (e.g., metoclopramide, H-2 blockers), vitamin D replacement agents, etc., beta blockers (metoprolol, acebutolol, bisoprolol, atenolol, propranolol, etc.), anticoagulant therapy (heparin, warfarin, Apixaban, Rivaroxaban).

[0268] The compositions and methods of the present invention may also be used in conjunction with other forms of therapy, including but not limited to: physical exercise (eg, dialysis, kidney transplantation); dietary salt restriction, fiber intake, pacemaker implantation, and heart transplantation.

[0269] Example

[0270] The exemplary features, objects and advantages of the present invention will be apparent in the following examples. However, it should be understood that the examples, although indicating embodiments of the present invention, are given by way of illustration only and are not limiting. It will be apparent to those skilled in the art that various changes and modifications within the scope of the present invention will be apparent from the examples.

[0271] Example 1. Production and purification of viral vector expressing α-GAL enzyme

[0272] This example summarizes the viral vectors contemplated by the present disclosure.

[0273] A recombinant adeno-associated virus 9 (rAAV9) was developed to express wild-type human α-GAL or α-GAL variants (e.g., amino acid sequences shown in Table 1) under the control of a ubiquitous promoter in a viral vector. The WPRE element was linked to the 3' end of the GLA transgene to increase transgene expression, thereby improving mRNA stability. A bovine growth hormone polyadenylation tail was appended to the 3' end of the WPRE element. The DNA construct of promoter-GLA-WPRE-BGHpA was integrated between the terminal inverted repeats of a circular plasmid vector. Figure 1 Exemplary rAAV9 vector constructs are shown.

[0274] Using methods in the art, the rAAV vector was encapsulated using the AAV2 terminal inverted repeat sequence and the rep sequence. The rAAV9 stock solution was produced using HEK-293T cells by a three-plasmid co-transfection method without adenovirus and purified by cesium chloride ultracentrifugation. The titer of the number of viral genome particles was determined by quantitative PCR.

[0275] Purified rAAV9 virus suspension was diluted in a formulation buffer consisting of 1.5 mM KH2PO4 (monopotassium phosphate), 2.7 mM KCl (potassium chloride), 8.1 mM Na2HPO4 (disodium phosphate), 136.9 mM NaCl (sodium chloride) and 0.001% Pluronic F-68. A null vector with rAAV9 capsid (rAAV9 null) was used as a control.

[0276] Two rAAV vectors, variant 1 and variant 2, were prepared and used in the following studies. Figure 1 A diagram of the transgenic expression cassette is shown in . Variant 1 comprises the codon-optimized nucleic acid sequence of SEQ ID NO: 12. Variant 2 comprises the codon-optimized nucleic acid sequence of SEQ ID NO: 13.

[0277] Example 2. α-GAL Activity in Serum after Administration

[0278] This example demonstrates that variant 1 and variant 2 provide expression of α-GAL protein in serum for at least 18 weeks after administration of the vector to mice.

[0279] An aggravated Fabry disease mouse model (G3Stg / GlaKO), generated by mating Gla knockout (GlaKO) mice with transgenic mice expressing human Gb3 synthase (G3Stg) to increase Gb3 accumulation, was used for this study. These mice had up to 10-fold higher levels of the substrate GB3 in various tissues compared to age-matched GlaKO mice, reflecting the symptoms observed in Fabry disease patients.

[0280] Male G3Stg / GLAko mice aged 8-12 weeks were divided into groups (n=6-12 per group) and 2.5×10 11 , 5.0 × 10 10 Purified rAAV9 vectors expressing variant 1 and variant 2 were administered intravenously (IV) at two different doses of 1.5 × 10 vg / kg and mice were followed for 18 weeks. rAAV9 null vector (control) was used as a negative control at 2.5 × 10 11 vg / kg was administered to a group of G3Stg / GLAko mice. A vehicle-treated wild-type (WT:WT) group was also used as a control. The study design is summarized in Table 4 below.

[0281] G3Stg / GLAko mice lost significant weight during the study; mice treated with rAAV9 vectors expressing variant 1 or variant 2 gained more weight than mice given an ineffective AAV vector or vehicle alone (data not shown).

[0282] Serum was collected at various time points during the study and at the end of 18 weeks.

[0283] Table 4: Study design

[0284]

[0285] The amount of α-GAL in serum was measured 18 weeks after administration.

[0286] Example 3. α-GAL activity in serum and various tissues after vector administration

[0287] This example illustrates that in the same experiment discussed in Example 2, sustained α-GAL activity in kidney, heart and liver was observed 18 weeks after intravenous administration of vector as shown in Table 4. A sustained increase in serum α-GAL activity was also observed after both doses of vector throughout the study ( Figure 2A ). It was seen that the α-galactosidase activity in the serum was increased by at least 6700-fold compared to the vehicle.

[0288] Tissues from mice were homogenized in EDTA-free lysis buffer containing 10 mM HEPES with 0.5% Triton-X 100 and 1.5x Halt protease inhibitor cocktail, centrifuged, and supernatants were collected for analysis. Alpha-galactosidase (α-GAL) activity in supernatants or serum was measured using a fluorescent substrate. Briefly, 2 ul of biological sample was incubated with 15 µL of 4-MU-a-gal substrate solution (Research Products International Company, catalog number M65400) and 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 adding 200 µL of glycine carbonate stop solution, pH 10.7. The 4-MU product was measured using a fluorescence plate reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The concentration of 4-MU in the test sample was calculated based on the calibration curve of 4-MU on the same plate. Tissue activity was normalized to total protein concentration determined by BCA assay (ThermoScientific, Cat. No. 23225).

[0289] The data in this example show that both variant 1 and variant 2 continuously express α-GAL in the kidney, heart and liver ( Figure 2B , 2C and 2D). The higher exposure in the liver indicates a higher amount of enzyme crossing the BBB to the nervous system (e.g., PNS). The α-galactosidase activity in the kidney increased more than 150-fold compared to the vehicle. The α-galactosidase activity in the heart increased more than 950-fold compared to the vehicle.

[0290] in addition, Figure 2E , comparing α-galactosidase activity in kidney and heart of Fabry mice transduced with 5e10 vg / kg human wild-type α-galactosidase and variant 1, respectively. Table 5 shows the fold increase of wild-type human α-galactosidase and variant 1 transduced sera.

[0291] Table 5: Fold increase in serum, kidney and heart α-galactosidase activity in Fabry disease mice transduced with wild-type α-galactosidase and variant 1

[0292]

[0293] Figure 2F It was shown that increased α-GAL activity was also observed in the serum of mice transduced with variant-1 and human α-galactosidase compared to vehicle control. However, the α-galactosidase activity of mice transduced with variant 1 was higher compared to wild-type human α-galactosidase.

[0294] Figure 2G Serum α-galactosidase concentrations up to 21 days after a single intravenous administration of two different doses of variant 1, 6.25e12 vg / kg and 3e13 vg / kg, in non-human primates (NHPs) are shown. It was observed that in NHPs, even at the lowest tested dose of 6.25e12 vg / kg, α-GAL serum levels were >50-fold higher than the predicted effective therapeutic level.

[0295] Immunohistochemistry results in kidney and heart confirmed high levels of enzyme exposure ( Fig. 7A and 7B ). In addition, α-GAL A enzyme exposure has been detected in difficult-to-access cell types such as cardiomyocytes in the heart and podocytes in the kidney ( Fig. 7A and 7B ).

[0296] Example 4. Substrate Levels after Application of Vector

[0297] This example examines the effects of variant 1 and variant 2 on GB3 and lysoGb3 reduction. It shows GB3 and lysoGb3 substrate levels 18 weeks after administration of the vector as discussed in Example 2.

[0298] Substrate levels are significantly higher in various tissues of G3Stg / GLAko mice. The levels of GB3 and lyso-Gb3 in the severe Fabry disease (G3Stg / GLAko) mice treated with constructs as shown in Table 4 were analyzed by mass spectrometry. The substrate LC-MS method was used to analyze serum and tissue samples. Briefly, substrate samples were first extracted with chloroform: methanol (v / v 2: 1) and formic acid, and then HPLC and LC-MS / MS (Applied Biosystem API5000, Turbo Ion SprayIonization, positive ion mode) were performed.

[0299] It was observed that both variant 1 and variant 2 reduced Fabry disease-associated GB3 accumulation in terminal serum, kidney, heart, and liver ( Figures 3A-3D ). Gb3 was observed to be reduced by more than 90% in the kidney and heart of Fabry mice compared to vehicle control treated mice. Figure 3E Comparison of substrate reduction in kidney and heart of Fabry disease mice transduced with wild-type human α-galactosidase or variant 1. Table 6 shows the fold reduction in kidney and heart of Fabry disease mice transduced with wild-type human α-galactosidase or variant 1.

[0300] Table 6: Fold reduction in kidney and heart of Fabry disease mice transduced with wild-type human α-galactosidase or variant 1

[0301]

[0302] Similarly, decreased lysoGb3 accumulation was observed in serum, kidney, heart, and liver after 18 weeks of administration ( Figures 4A-4D ). The reduction of substrates in multiple tissues suggests that rAAV-induced gene therapy has a systemic effect. Treatment with variant 1 or variant 2 resulted in complete or near-complete normalization of substrates in Fabry mice.

[0303] Example 5. Restoration of thermal sensitivity caused by variants 1 and 2 in a mouse model of Fabry disease (G3Stg / GLAko mice)

[0304] This example demonstrates the effects of rAAV vectors expressing variant 1 and variant 2 on thermosensitivity in G3Stg / GLAko mice.

[0305] The hot plate latency test was used in this study to test the response of mice to thermal stimulation. The hot plate test of Eddy and Leimbach (1953) is a simple behavioral screen for evaluating the effect of NCE on the threshold for detecting pain. It is based on the following principle: when rodents are placed on a hot surface, they initially lick or tap their paws, and eventually demonstrate the aversive effect of thermal stimulation by openly trying to escape the environment (jumping). Substances that change the threshold of nociception can increase the licking / jump latency (analgesic effect) or reduce the latency (hyperalgesia). The hot plate test is also a fast and relatively inexpensive method for evaluating acute thermal pain, and the advantage over tail flicking / tail withdrawal is the opportunity to test thermal sensitivity. This simple sensitivity test in mice is often used to indicate neuropathic pain (e.g., by heat) in human patients.

[0306] In the test, mice from each group were tested on a hot plate before and after administration at 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 18 weeks (Table 4). The hot plate (Columbus Instruments) was preheated to 55°C. An open cylindrical transparent plexiglas with a diameter of 30 cm was placed on the top of the hot plate to prevent mice from escaping, but the paws of the animals were exposed to the hot plate. A stopwatch was used to measure the time from when the mouse was placed on the hot plate to the first licking of the paw. The latency of the reaction was measured to the nearest 0.1 second with the hind paw licking or shaking / swaying (whichever came first). If no pain response was measured, the mouse was automatically removed after 1 minute to prevent tissue damage.

[0307] Thermostats of mice dosed with variant 1 and variant 2 at 2.5e11 vg / kg were restored to WT threshold values ​​( Figure 5 ).

[0308] Example 6. Improvement of Fabry disease-associated neuropathy in a mouse model (G3Stg / GLAko mice)

[0309] This example examines the expression of neuropathy markers in a Fabry disease mouse model 18 weeks after vector administration.

[0310] G3Stg / GLAko mice exhibit several signs of neuropathy, as observed in the histology of peripheral neurons after sacrifice. The footpads of the hind paws from these animals were collected and analyzed by immunohistochemistry to assess small fiber nerves to monitor any neuronal pathology in these animals. MPZ (myelin protein zero, the most abundant protein in nerve myelin) staining ( Figure 6 ) revealed that the morphology of dermal nerve bundles in G3Stg / GLAko mice treated with rAAV9-variant 1 was improved and the density was preserved, comparable to that of WT mice. Figure 6 As shown, the intraepidermal nerve fiber density was preserved in G3Stg / GLAko mice treated with the rAAV9-α-GAL gene therapy vector, whereas the nerve density was not preserved in G3Stg / GLAko mice treated with the control vector (rAAV null).

[0311] Example 7. Neural structure and lysosomal burden in a mouse model of Fabry disease

[0312] This example examines neural structure and lysosomal burden in a mouse model of Fabry disease 18 weeks after administration of a rAAV9-α-GAL gene therapy vector.

[0313] Dorsal nerve root (DNR) samples were collected from G3Stg / GLAko mice treated with rAAV9-α-GAL gene therapy vectors. Samples were also collected from wild-type mice and G3Stg / GLAko mice treated with control vectors (rAAV ineffective). The tissues were fixed and processed for staining. Histological evaluation was performed on fixed tissues that were stained with immunohistochemistry or hematoxylin and eosin (HE). Anti-LAMP1 antibody was used for immunohistochemical staining.

[0314] like Fig. 8A As shown in Figure 2, vacuoles were observed only in G3Stg / GLAko mice treated with a control vector. Treatment with the rAAV9-α-Gal gene therapy vector was shown to reverse vacuolation in G3Stg / GLAko mice. LAMP1-positive staining was observed only in G3Stg / GLAko mice treated with a control vector, indicating a reduction in lysosomal load. Substrate depletion had multiple improvements in the structure and function of the treated mice, including normalization of lysosomal load and prevention of vacuolation in dorsal root nerves.

[0315] also, Figure 8B, Figure 8C and Fig.8D Demonstrated reversal of peripheral nervous system (PNS) structural pathology translated into restoration and preservation of sensory loss. Figure 8B The percentage of LAMP1 positive areas in Fabry mice compared to Fabry mice treated with variant 1 or vehicle and normal mice is shown. It was observed that the Lamp positive area was reduced in Fabry mice treated with variant 1 compared to Fabry mice treated with vehicle. In addition, the hot plate latency of Fabry mice treated with variant 1 and control (vehicle) on days 2 and 12 was observed and compared to normal control (vehicle-treated) mice ( Figure 8C ). It was observed that the hot plate latency depicting sensory loss in Fabry disease mice was restored with partial DRG substrate removal. Furthermore, DRG substrate load, as assessed using the logarithm of LAMP1 density, showed a correlation with hot plate latency, whereas small fiber density showed no correlation with hot plate latency ( Fig.8D ). We believe this finding is consistent with reports on the pathogenesis of PNS in patients.

[0316] Fig. 8E Mouse DRG-MRI evaluating 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 to normal mice. Fig.8F It is a histogram of DRG area in gene therapy treated mice versus control (vehicle treated) mice or normal mice. It was observed that the gene therapy treated mice showed a continuous decrease in DRG volume until the 24th week. It is expected that gene therapy will greatly improve the neurophysiological state of people with Fabry disease. The DRG volume of Fabry disease patients increases significantly and is not affected by ERT treatment. Therefore, DRG volume becomes a potential alternative marker for monitoring the efficacy of treatment of Fabry disease PNS pathology.

[0317] Example 8. Vacuolization and lysosomal loading in the gastrointestinal tract in a mouse model of Fabry disease

[0318] This example examines vacuolation and lysosomal loading in the gastrointestinal tract 18 weeks after administration of rAAV9-α-GAL gene therapy vectors in a mouse model of Fabry disease.

[0319] Smooth muscle tissue samples from the gastrointestinal tract were collected from G3Stg / GLAko mice treated with rAAV9-α-GAL gene therapy vectors. Samples were also collected from wild-type mice, untreated G3Stg / GLAko mice, and G3Stg / GLAko mice treated with a control vector (MY011). The tissues were fixed and processed for staining. Histological evaluation was performed on fixed tissues stained with immunohistochemistry or hematoxylin and eosin (HE). Anti-LAMP1 antibodies were used for immunohistochemical staining.

[0320] like Fig. 9 As shown, rAAV-α-Gal gene therapy vector treatment almost completely reversed pathological vacuolation and significantly reduced lysosomal load in myoenteric ganglion cells and transverse smooth muscle of the gastrointestinal tract. α-Gal A enzyme exposure was observed in mice treated with rAAV9-α-Gal gene therapy vector. Vacuoles in transverse smooth muscle of the duodenum of G3Stg / GlaKO were reversed after treatment with rAAV9-α-Gal gene therapy vector. LAMP1-positive staining in smooth muscle and myoenteric ganglion cells was only observed in untreated and rAAV-ineffectively treated mice, but not in mice treated with rAAV9-α-Gal gene therapy vector, indicating that lysosomal load in the gastrointestinal tract was reversed.

[0321] These immunohistochemical results confirmed high levels of enzyme exposure in different tissues and cells, including cardiomyocytes, podocytes, peripheral neurons, and DRGs. Collectively, these studies suggest that rAAV-α-Gal gene therapy vectors can mediate expression of engineered α-GAL to prevent further disease progression and even reverse some symptoms in a mouse model of severe Fabry disease.

[0322] Example 9. Comparison of gene therapy using rAAV8-hα-GAL with ubiquitous and liver-specific promoters in GlaKO mice.

[0323] This example evaluates the effect of gene therapy using rAAV8-hα-GAL (with a liver-specific promoter) or rAAV9-hα-GAL (with a ubiquitous promoter) vectors encoding wild-type α-galactosidase.

[0324] Fig. 10A and Fig. 10B Shown are the results of immunohistochemistry in GlaKO mice given a single intravenous dose of 2.5e11 vg / kg for 12 weeks with rAAV8-hα-GAL or rAAV9-hα-GAL encoding wild-type α-galactosidase with a liver-specific promoter or a ubiquitous promoter.

[0325] Table 7 summarizes the comparison between ERT and rAAV therapy in GlaKO mice.

[0326] Table 7: Comparison of ERT and rAAV therapy

[0327]

[0328] It was observed that rAAV administration in Fabry mice showed higher serum and tissue exposure of hα-GAL and better substrate clearance. It was observed that Fabry mice treated with AAV-hαGAL with ubiquitous and liver-specific promoters (GlaKO mice) showed better mRNA expression in the liver and will likely provide better safety.

[0329] Example 10. PK of Human α-Galactosidase Production and Substrate Reduction Using Translational Modeling

[0330] It is predicted that target α-GAL levels that are superior to ERT efficacy can be achieved at significantly lower doses in human Fabry disease patients.This example demonstrates a translational modeling approach to show the production of α-galactosidase or reduction in human Gb3 over 13 months compared to ERT.

[0331] Figure 11A-11B Expected α-galactosidase production in various tissues at different doses over 13 months is shown. Fig. 11C The predicted α-galactosidase production in various tissues using ERT is shown. Figure 11D-11E The predicted Gb3 reduction in various tissues at different doses over 13 months is shown. Fig.11F Expected Gb3 reductions in various tissues using ERT are demonstrated. A reduction of at least 30% in Gb3 in ERT-resistant cell types at 6 months may provide meaningful changes that may translate into functional improvements.

[0332] Example 11. Minimum Efficacy Dose of Adeno-Associated Virus Vectors Expressing Human α-Galactosidase (α-GAL) Variants

[0333] The goal of this example is to evaluate the minimal effective dose of two recombinant adeno-associated viral vectors expressing human α-galactosidase (α-GAL) variants in a mouse model with Fabry disease symptoms (G3Stg / GLAko or HEMI; CAR) when administered intravenously (IV) once at 4 different doses and monitored for a duration of 4 weeks.

[0334] rAAV9-hα-GAL-variant 1 and rAAV9-hα-GAL-variant 2 were quantified at 2.5 × 10 8 , 2.5×10 9 , 2.5×10 10 and 2.5×10 11vg / kg. An ineffective vector (rAAV9-ineffective) was used as a control and the expression of 2.5×10 11 No circulating α-GAL activity or human protein was detected at 4% vg / kg.

[0335] Mice treated with rAAV9-hα-GAL-variant 1 and rAAV9-hα-GAL-variant 2 showed a dose-responsive increase in α-GAL activity in serum 1 week after injection and persisted for 4 weeks of the study ( Fig. 12A Tables 8A and 8B show the final serum and key tissues such as kidney, heart and liver (also shown in Figure 12B-12E ) in the presence of α-GAL.

[0336] Table 8A: Activity of α-GAL in serum and tissues

[0337]

[0338] Table 8B: α-GAL activity folds in serum and tissues

[0339]

[0340] Only in 2.5×10 10 and 2.5×10 11 High α-GAL activity was detected at the first 2 doses of 2.5×10 11 At 544 vg / kg, high α-GAL activity was achieved in a dose-responsive manner in terminal serum (6,900-fold and 10,000-fold over WT activity) and in key target tissues (e.g., kidney >150-fold and >100-fold; heart >900-fold and >500-fold; liver >750-fold and approximately 1,000-fold for rAAV9-hα-GAL-variant 1 and rAAV9-hα-GAL-variant 2, respectively; Table 8B).

[0341] Human α-GAL protein levels were not detected in any of the control samples as expected. Protein levels increased in a dose-dependent manner in serum and the tissues evaluated, reflecting the α-GAL activity of each sample type and dose (Table 9). 8 , 2.5×10 9 vg / kg), none of the variant test articles were able to produce detectable protein levels in serum or tissues. 11Mice treated with 100 vg / kg showed higher α-GAL protein and activity in serum and liver than mice treated with rAAV9-hα-GAL-variant 1 at the same dose, but less exposure in kidney and heart, which may be the result of differences in tissue uptake kinetics between the 2 variants.

[0342] Table 9: α-GAL concentrations in serum and tissues

[0343]

[0344] α-GAL = α-galactosidase A; GLAKO = α-GAL deficiency. Values ​​are mean α-GAL concentrations (ng / mL serum or mgtt protein). BLQ = below limit of quantitation

[0345] Gb3 and lysoGb3

[0346] In the presence of an ineffective vector rAAV9, 2.5 × 10 11 In the disease controls injected with 100 vg / kg, Gb3 and lysoGb3 substrates accumulated in terminal serum and kidney tissues compared to WT:WT controls. WT:CAR mice with knock-in copies of the G3S gene showed higher Gb3 substrates, especially in the heart and serum, than WT:WT, but lower than G3Stg / GLAko (Table 10, Figure 12F-12M ).

[0347] When mice were treated with rAAV9-variant 1 or rAAV9-variant 2, Gb3 substrate was reduced in both the terminal serum and target tissues in a dose-dependent manner, with the highest dose of 2.5 × 10 11 vg / kg. This translates into a reduction of >85% in tissue Gb3 relative to the G3Stg / GLAko null control. 10 In mice treated with 2.5×10 10 At the vg / kg dose, rAAV9-variant 2 reduced Gb3 in kidney and heart (53% and 61%, respectively) slightly better than rAAV9-variant 1 (46% and 51%, respectively).

[0348] Table 10: Percent reduction of Gb3 substrate

[0349]

[0350] Gb3 = ceramide trihexosyl-3; GLAKO = α-GAL deficiency. Values ​​are mean % reduction compared to placebo (rAAV9).

[0351] A similar dose-dependent trend was observed for the reduction of lysoGb3 substrate (Tables 11 and Figure 12J-12M ).

[0352] Table 11: LysoGb3 substrate reduction percentage

[0353]

[0354]

[0355] Lyso Gb3 = lysosomal ceramide trihexosyl-3; GLAKO = α-GAL deficiency. Values ​​are mean % reduction compared to placebo (rAAV9-).

[0356] Vector genome and mRNA copy number

[0357] The vector genome (vg) and mRNA copy numbers were measured in kidney, heart, and liver tissues. No vector genome copies were detected in the WT:WT and WT:CAR controls as expected. The highest dose was 2.5×10 11 vg / kg treatment resulted in detectable vg copy numbers in all tissues, which translated into similar results in mRNA copy numbers ( Figure 12N-Figure 12O ). At a low dose of 2.5×10 10 vg / kg, only liver vg and mRNA could be detected.

[0358] It was observed that only the first two doses (2.5×10 10 and 2.5×10 11 vg / kg) showed significant exposure and substrate reduction efficacy in serum and evaluated tissues in a dose-dependent manner.

[0359] Example 12. Minimum Efficacy Dose of Adeno-Associated Virus Vectors Expressing Human α-Galactosidase (α-GAL) Variants

[0360] The objective of this example was to evaluate the durability of rAAV9-Variant 1 in GlaKO male mice over 8 months with provisional sacrifice 3 months after injection of 2 different doses (1e11 and 2.5e10 vg / kg). rAAV9 null was used as a negative control, with only the highest dose of 1e11 vg / kg.

[0361] GlaKO and WT control animals were injected with 2 different doses (1e11 and 2.5e10 vg / kg) of variant 1 or the highest dose (1e11 vg / kg) of rAAV9 placebo via the intravenous route of administration and evaluated for serum and tissue exposure, substrate reduction, urine and blood chemistries, histopathological evaluation, and echocardiography.

[0362] The data generated from this study will be serum and tissue α-GAL activity for the cohort over the 8-month period (Table 12). Fig.13A Drawn in.

[0363]

[0364]

[0365] Sustained α-GAL activity was observed in the liver, kidney, and heart throughout the test period at both concentrations tested.

[0366] The recombinant adenovirus rAAV9-hα-GAL variant 1 was administered intravenously to non-human primates. This is challenging for AAV9 seronegative source animals. Despite high pre-existing Nabs, high transgene expression was observed in most treated animals ( Fig. 13B , Fig. 13C ). Fig. 13B and Fig. 13C Serum α-galactosidase concentrations over 28-90 days at different concentrations are shown.

[0367] The results showed that the expression level of α-Gal in NHPs at a low dose of 6.25e12 vg / kg was lower than that in mice, but still exceeded normal levels by 3000-fold and exceeded clinical target levels by 50-fold. The results in NHPs support low rAAV9-hα-GAL variant 1 therapeutic doses in the e12 vg range.

[0368] Equivalents and scope

[0369] 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 invention is not intended to be limited to the above description, but rather is set forth in the following claims.

Claims

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 experiencing one or more neuropathy-related symptoms.

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

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

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

5. A method for ameliorating perineurofibrotic abnormalities in a subject 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. The method of claim 5 , wherein the nerve fibers are small nerve fibers.

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

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

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

10. The method of any of the preceding claims, wherein administration of the rAAV reduces accumulation of ceramide trihexosylceramide (GB3) in the peripheral nervous system as evidenced by a decrease in LAMP1 levels in DRGs.

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

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

13. The method of claim 11, wherein accumulation in peripheral nerves is reduced as evidenced by a decrease in LAMP1 levels in the nerves.

14. A method of treating neuropathy in a subject suffering from Fabry disease, the method comprising (a) determining that the subject has or is experiencing 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; and (c) evaluating said one or more neuropathy-related symptoms in said subject after said treatment.

15. The method of claim 14, wherein the one or more neuropathy-related symptoms include chronic burning pain, severe pain attacks, paresthesia / dysesthesia, sensory loss, hypohidrosis / anhidrosis, abdominal cramps, (postprandial) diarrhea, bloating, nausea, and tinnitus and hearing loss.

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

17. The method of any one of the preceding claims, wherein the rAAV vector is a rAAV vector packaged in a capsid with broad tissue tropism, the vector comprising: a. 5' terminal inverted repeat sequence (ITR); b. Ubiquitous promoter; c. the polynucleotide encoding the α-GAL enzyme; d. polyadenylation; and e. 3' ITR.

18. The method of any one of claims 1 to 16, wherein the rAAV vector is a rAAV vector packaged in a capsid with broad tissue tropism, the vector comprising: a. 5' terminal inverted repeat sequence (ITR); b. Liver-specific promoter; c. the polynucleotide encoding the α-GAL enzyme; d. polyadenylation; and e. 3' ITR.

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

20. The method of any one of claims 17-19, wherein the vector further comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

21. The method of any one of claims 17-20, 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 17-21, wherein the polynucleotide encoding the α-GAL enzyme comprises the nucleotide sequences of SEQ ID Nos: 9, 10, 12 and 13.

23. The method of any one of claims 17-22, wherein the AAV capsid is a broadly tropic 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. The method of claim 23, wherein the broadly tropic AAV capsid is an AAV9 capsid.

25. The method of claim 24, wherein the AAV9 capsid is naturally occurring or engineered.

26. The method of any one of claims 17-25, wherein the ubiquitous promoter comprises 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 one or more introns.

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

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

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

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

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

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

34. The method of any of the preceding claims, wherein the subject has detectable α-GAL in 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. The method of claim 34, wherein the subject has detectable α-GAL in serum for greater than 18 weeks.

36. The method of any of the preceding claims, wherein the rAAV is present in an amount of about 1×10 10 vg / kg (vector genome / body weight) to about 1×10 14 A dose in the range of vg / kg (vector genomes / body weight) is administered to the subject.

37. The method of claim 36, wherein the rAAV is present at about 1.0 × 10 10 vg / kg to 5.0 × 10 13 A dose within the range of vg / kg is administered to the subject.

38. 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 experiencing one or more gastrointestinal symptoms.

39. The method of claim 38, wherein the gastrointestinal symptoms include intestinal dysmotility, impaired autonomic function, vasculopathy, and myopathy.

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

41. The method of claim 9, wherein intraepidermal nerve fiber density is preserved.

42. The method of claim 6, wherein the nerve fiber is a dorsal nerve root and cavitation in the dorsal nerve root is preserved.

43. The method of any one of claims 38-40, wherein the rAAV vector is a rAAV vector packaged in a capsid with broad tissue tropism, the vector comprising: a. 5' terminal inverted repeat sequence (ITR); b. Ubiquitous promoter; c. the polynucleotide encoding the α-GAL enzyme; d. polyadenylation; and e. 3' ITR.

44. The method of any one of claims 38-40, wherein the rAAV vector is a rAAV vector packaged in a capsid with broad tissue tropism, the vector comprising: a. 5' terminal inverted repeat sequence (ITR); b. Liver-specific promoter; c. the polynucleotide encoding the α-GAL enzyme; d. polyadenylation; and e. 3' ITR.

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

46. ​​The method of any one of claims 43-45, wherein the vector further comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

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

48. The method of any one of claims 43-47, 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 43-48, wherein the AAV capsid is a broadly tropic 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. The method of claim 49, wherein the broadly tropic AAV capsid is an AAV9 capsid.

51. The method of claim 50, wherein the AAV9 capsid is naturally occurring or engineered.

52. The method of any one of claims 43-51, wherein the ubiquitous promoter comprises 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 one or more introns.

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. The method of claim 52, wherein the ubiquitous promoter comprises a shortened EF-1 alpha promoter and one or more introns.

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

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

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

58. The method of any one of claims 43-57, wherein the rAAV vector is administered by intravenous, subcutaneous, or transdermal administration.

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

60. The method of any one of claims 43-59, wherein the rAAV is present in an amount of about 1×10 10 vg / kg (vector genome / body weight) to about 1×10 14 A dose in the range of vg / kg (vector genomes / body weight) is administered to the subject.

61. The method of claim 60, wherein the rAAV is present at about 1.0×10 10 vg / kg to 5.0×10 13 A dose within the range of vg / kg is administered to the subject.

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