Compositions and methods for treating Fabry disease

Abstract

Polynucleotides comprising nucleic acids encoding alpha-galactosidase A (GLA) are described. Also described are expression cassettes, vectors, cells, and cell lines comprising the polynucleotides, and methods of using the polynucleotides to treat lysosomal storage disorders, such as Fabry disease.

Description

[Technical field]

[0001] The present invention relates to the field of gene therapy. In particular, the present invention relates to an optimized cassette for the expression of human α-galactosidase A and its use for treating lysosomal storage diseases, in particular Fabry disease.

[0002] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 137,235, filed January 14, 2021, and U.S. Provisional Patent Application No. 63 / 264,356, filed November 19, 2021. The entire contents of the foregoing applications are incorporated herein by reference, including all text, tables, sequence listings and figures.

[0003] Reference to Electronically Submitted Sequence Listing This application contains a sequence listing that has been submitted electronically via EFS-Web as an ASCII formatted sequence listing under the file name "SequenceListing4WO", with a creation date of January 13, 2022, and having a size of 281 KB. The sequence listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Fabry disease is an X-linked lysosomal storage disorder with an estimated prevalence of approximately 1 in 40,000. Fabry disease is caused by a deficiency of the lysosomal enzyme α-galactosidase A (GLA; α-gal A). The enzyme deficiency results in the accumulation of the glycosphingolipids globotriaosylceramide (GL3 or GL-3 or Gb3) and globotriaosylsphingosine (lyso-GL3 or lyso-GL-3 or lyso-Gb3), leading to progressive renal disease, peripheral neuropathy, early-onset cerebrovascular disease, gastrointestinal symptoms, hypertrophic cardiomyopathy, arrhythmias, corneal verticillate opacities, and angiokeratoma. Patients with Fabry disease not treated with enzyme replacement therapy have a life expectancy from renal, cardiac, and / or cerebral complications due to vascular disease of 50 years in men and 70 years in women (Lidove et al., Int. J. Clin. Pract. 2007;61:293-302).

[0005] Enzyme replacement therapy (ERT) is available for Fabry disease, but does not represent a cure and requires weekly intravenous administration for the patient's lifetime. Furthermore, a significant proportion of patients render ERT ineffective by developing neutralizing antibodies (NAbs) against α-galactosidase (Linthorst et al., Kidney Int. 2004;66(4):1589-1595). Summary of the Invention

[0006] Disclosed herein is an optimized cassette for liver-directed expression of secretory form of human α-galactosidase A (GLA).These optimizations of cassette result in increased GLA secretion from liver, and allow hepatic gene transfer to achieve sufficient circulating levels of GLA in subjects to cross-correct GLA deficiency systemically.These cassettes are useful as gene therapy treatments for subjects with Fabry disease and other diseases and disorders that can be treated with GLA.

[0007] In one general aspect, the invention relates to a polynucleotide comprising a nucleic acid encoding alpha-galactosidase A (GLA), the nucleic acid being selected from the group consisting of: (1) a polynucleotide having at least 75% sequence identity to the sequence of SEQ ID NO:15; (2) a polynucleotide having at least 84% sequence identity to the sequence of SEQ ID NO:16; (3) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO:17; (4) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO:18; and (5) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO:19, and optionally, GLA comprises the amino acid sequence of SEQ ID NO:100.

[0008] In certain embodiments, the nucleic acid comprises fewer than 14 CpG dinucleotides, and optionally no CpG dinucleotides.

[0009] In one embodiment, the nucleic acid encoding GLA has the sequence of any of SEQ ID NOs:15-19.

[0010] In some embodiments, the nucleic acid encoding GLA further comprises one or more introns at any position within the nucleic acid encoding GLA, hi some embodiments, the intron is located between nucleotides 78 and 79 of the nucleic acid encoding GLA, the nucleotide positions of which are given with respect to the coding sequence for GLA having the sequence of SEQ ID NO:14.

[0011] In some embodiments, the intron is selected from the group consisting of the vitronectin 1 (VTN1) gene, the retinol binding protein 4 (RBP4) gene, the mouse IgG heavy chain A (IgHA) gene, and the mouse IgG heavy chain μ (IgHμ gene). In some embodiments, the one or more introns are selected from the sequences of SEQ ID NOs: 49-52.

[0012] In one embodiment, the nucleic acid encoding GLA has the sequence of any of SEQ ID NOs:43-46.

[0013] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100 with one amino acid substitution selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0014] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100 having any two amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0015] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100 having any three amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0016] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100 having any four amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0017] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100 having any five amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0018] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100 having any six amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0019] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100, with seven amino acid substitutions: Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0020] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:48.

[0021] In one embodiment, the nucleic acid encoding GLA comprises the coding sequence of SEQ ID NO:47.

[0022] In certain embodiments, the polynucleotide further comprises a second nucleic acid encoding a signal peptide sequence positioned 5' to the nucleic acid encoding GLA.

[0023] In certain embodiments, the signal peptide sequence is a heterologous signal peptide sequence.

[0024] In one embodiment, the signal peptide sequence is the endogenous or native GLA signal peptide sequence.

[0025] In certain embodiments, the signal peptide is selected from the group consisting of human chymotrypsinogen B2 signal peptide, AHSG signal peptide, CD300 signal peptide, LAMP1 signal peptide, Notch 2 signal peptide, ORM1 signal peptide, TF signal peptide, and native GLA signal peptide, or variants thereof.

[0026] In certain embodiments, the signal peptide is a human chymotrypsinogen B2 signal peptide, optionally having the amino acid sequence of SEQ ID NO:41, or a variant thereof.

[0027] In certain embodiments, the signal peptide is a human chymotrypsinogen B2 signal peptide, optionally having a coding sequence of any one of SEQ ID NOs: 1-5.

[0028] In one embodiment, the polynucleotide encodes a precursor GLA having the sequence of any of SEQ ID NOs:101-109.

[0029] In one embodiment, the polynucleotide comprises any of the sequences set forth in SEQ ID NOs:64-81.

[0030] In one embodiment, the invention relates to an expression cassette comprising a polynucleotide comprising a nucleic acid encoding GLA operably linked to an expression control element.

[0031] In one embodiment, the present invention relates to an expression cassette comprising a polynucleotide comprising a nucleic acid encoding human GLA operably linked to an expression control element.

[0032] In certain embodiments, the expression control element is a liver-specific expression control element.

[0033] In certain embodiments, the expression control elements of the expression cassette are located 5' to the polynucleotide, and the expression control elements optionally include an ApoE / hAAT enhancer / promoter sequence.

[0034] In certain embodiments, the expression cassette further comprises a polyadenylation sequence located 3' to the polynucleotide, which optionally comprises the bovine growth hormone (bGH) polyadenylation sequence.

[0035] In certain embodiments, the expression control element or polyadenylation sequence of the expression cassette has reduced CpGs compared to the wild-type expression control element or polyadenylation sequence.

[0036] In certain embodiments, the expression cassette further comprises an intron located between the 3' end of the expression control element and the 5' end of the polynucleotide, which optionally comprises the hBB2m1 intron.

[0037] In certain embodiments, the AAV ITRs flank the 5' and / or 3' ends of the polynucleotide or expression cassette.

[0038] In certain embodiments, the present invention relates to an adeno-associated virus (AAV) vector comprising the polynucleotide or expression cassette.

[0039] In one embodiment, an AAV vector comprises (a) one or more AAV capsids and (b) one or more AAV inverted terminal repeats (ITRs), where the AAV ITRs flank the 5' or 3' end of the polynucleotide or expression cassette.

[0040] In one embodiment, at least one of the ITRs of the AAV vector is modified to reduce CpGs.

[0041] In certain embodiments, the AAV vector has a capsid serotype that includes modified or mutated AAV VP1, VP2 and / or VP3 capsids with 90% or more, 95% or more, or 100% sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, AAV-2i8, SEQ ID NO:110, SEQ ID NO:36, SEQ ID NO:37, and / or LK03 (SEQ ID NO:42).

[0042] In one embodiment, the AAV vector comprises one or more ITRs of any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B, AAV serotypes, or combinations thereof.

[0043] In one embodiment, the AAV vector comprises a polynucleotide sequence of one of SEQ ID NOs:21-34, 53-56, and 91-99.

[0044] In certain embodiments, the invention relates to a non-viral vector comprising the polynucleotide or expression cassette.

[0045] In one embodiment, the present invention relates to a pharmaceutical composition comprising multiple AAV vectors or non-viral vectors in a biologically compatible carrier or excipient.Preferably, the multiple AAV vectors are provided in an amount sufficient to achieve a therapeutic effect.However, multiple compositions may be administered to achieve a therapeutic effect.

[0046] In certain embodiments, the pharmaceutical composition further comprises an empty AAV capsid.

[0047] In certain embodiments, the pharmaceutical composition further comprises a surfactant.

[0048] In one embodiment, the present invention relates to a method of treating a subject in need of GLA, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, AAV vector, non-viral vector, or pharmaceutical composition, wherein GLA is expressed in the subject.

[0049] In certain embodiments, the subject is a human.

[0050] In certain embodiments, the subject has Fabry disease.

[0051] In certain embodiments, the polynucleotide, expression cassette, AAV vector, non-viral vector, or pharmaceutical composition is administered to a subject intravenously, intraarterially, intracavitary, intramucosally, or via a catheter.

[0052] In one embodiment, the AAV vector is about 1 x 10 per kilogram of subject body weight. 8 ~Approx. 1×1014 The vector is administered to the subject in the range of vector genome (vg / kg).

[0053] In certain embodiments, the method reduces, reduces, or inhibits one or more symptoms of GLA or Fabry disease in a subject; or prevents or reduces the progression or worsening of the need for GLA or one or more symptoms of Fabry disease; or stabilizes the need for GLA or one or more symptoms of Fabry disease; or ameliorates the need for GLA or one or more symptoms of Fabry disease.

[0054] In certain embodiments, the invention relates to a cell comprising the polynucleotide or expression cassette.

[0055] In one embodiment, the present invention relates to a cell that produces an AAV vector.

[0056] In one embodiment, the invention relates to a method of producing an AAV vector, comprising: (a) introducing an AAV vector genome comprising a polynucleotide or expression cassette into a packaging helper cell; and (b) culturing the helper cell under conditions to produce the AAV vector. [Brief description of the drawings]

[0057] The foregoing Summary of the Invention, as well as the following Detailed Description of the Invention, are better understood when read in conjunction with the accompanying drawings. It is understood that the invention is not limited to the precise embodiments shown in the drawings. In the drawings: [Figure 1] FIG. 1 shows a schematic diagram of the expression vectors described herein. [Diagram 2]FIG. 2A shows a bar graph depicting serum GLA enzyme activity in male (left panel) and female (right panel) C57Bl / 6 mice administered AAV-encapsidated GLA expression cassettes containing different signal peptides (GLA, SP7, CD300, NOTCH2, ORM1 and TF, indicated along the x-axis) as determined by GLA enzyme activity assay 4 weeks after AAV transduction. FIG. 2B shows a bar graph depicting serum GLA protein levels in female C57Bl / 6 mice administered AAV-encapsidated GLA expression cassettes with wild-type signal peptide (GLA) or sp7 signal peptide (SP7), measured 6 weeks after AAV transduction; control represents the level of GLA protein in untreated mice; bar height represents the mean of 5 mice per group; error bars represent standard deviation from the mean; t-tests were performed comparing serum GLA protein levels in transduced mice to controls (*** p<0.001). [Diagram 3] FIG. 3 is a bar graph showing serum GLA enzyme activity in C57Bl / 6 and B6;129-GLA mice administered AAV-encapsidated sp7-GLA; bar heights represent the mean of 10-11 mice per group; error bars represent one standard deviation from the mean. [Figure 4]Figure 4A is a bar graph showing serum GLA enzyme activity as a function of AAV dose escalation in Fabry male mice administered AAV-encapsidated sp7-GLA, performed across two separate studies (study 1 and study 2) and measured 4 weeks after AAV administration; bar height represents the mean serum GLA activity of five mice per group; error bars indicate standard deviation from the mean; horizontal line indicates baseline (non-specific activity) observed in GLA knockout animals, defined as the maximum activity observed across a group of five GLA- / null mice that did not receive AAV-encapsidated sp7-GLA. Figure 4B is a graph showing the linearity of the dose-response observed for sp7-GLA AAV vector; data from GLA- / null male mice treated with AAV-encapsidated sp7-GLA shown in Figure 4A were replotted as a function of vector genome dose per mouse; data was fitted with a simple linear regression; 95% confidence intervals are indicated by dotted lines. [Diagram 5] Figure 5A is a bar graph showing GLA activity in the liver of male mice administered AAV-encapsidated sp7-GLA; baseline levels of activity were determined in samples from five GLA- / null untreated mice (control) and four age-matched GLA+ / null (WT) male mice; bar height represents the average GLA activity in liver lysates; error bars indicate standard deviation from the mean. Figure 5B is a bar graph showing GLA activity in the kidney of GLA- / null male mice administered AAV-encapsidated sp7-GLA (control) or four age-matched GLA+ / null (WT) male mice; bar height represents the average tissue GLA activity; error bars indicate standard deviation from the mean. [Figure 6] FIG. 6 is a bar graph showing serum GLA activity in C57Bl / 6 mice administered the AAV-encapsidated CpG-free codon-optimized GLA cassette described herein; GLA activity in serum was assayed 4 weeks after AAV transduction; bar height represents the average tissue GLA activity of 5 mice per group; error bars indicate standard deviation from the mean. [Figure 7]FIG. 7 is a bar graph showing serum GLA activity in C57Bl / 6 mice 42 days after AAV administration of codon-optimized GLA mutants (sp7-GLA-co4; sp7-GLA-var45), a GLA sequence with seven amino acid substitutions (GLA 7 mut; SPKL0031), or intron-containing mutants (intron IgHA; intron VTN1; intron RBP4; intron IgHμ) compared to sp7-GLA (lots A and B). [Figure 8] Figure 8A is a line graph showing a dose-dependent decrease in serum lyso-GL3 levels in B6;129-GLA - / - mice administered 4.4E11 vg / kg, 1.4E12 vg / kg, and 4.4E12 vg / kg AAV-sp7-GLA-co4. Lyso-GL3 levels were analyzed by mass spectrometry over a 28-day period. Figure 8B is a graph showing the linear relationship between α-gal A activity and lyso-GL-3 levels in serum, as measured using an in vitro 4-methylumbelliferyl β-D-galactopyranoside (4-MU-Gal) assay. [Figure 9] Figure 9A is a bar graph showing the reduction in lyso-GL3 levels in serum of B6;129-GLA - / - mice in response to intravenously administered doses of 4.4E11 vg / kg, 1.4E12 vg / kg, and 4.4E12 vg / kg AAV-sp7-GLA-co4, demonstrating a dose-dependent increase in the reduction of lyso-GL3. Figure 9B is a bar graph showing the reduction in levels of lyso-GL3 in kidney tissue of B6;129-GLA - / - mice one month after administration of AAV-sp7-GLA-co4 at doses of 4.4E11 vg / kg, 1.4E12 vg / kg, and 4.4E12 vg / kg, demonstrating a dose-dependent decrease in lyso-GL3. FIG. 9C is a bar graph showing reduced levels of lyso-GL3 in cardiac tissue of B6;129-GLA− / − mice one month after administration of AAV-sp7-GLA-co4 at doses of 4.4E11 vg / kg, 1.4E12 vg / kg, and 4.4E12 vg / kg, demonstrating a dose-dependent reduction in lyso-GL3. [Figure 10]Figure 10A is a graph of serum GLA activity over time after injection of AAV-sp7-GLA-co4 in a non-human primate (cynomolgus monkey). Figure 10B is a graph of serum GLA antigen levels over time after injection of AAV-sp7-GLA-co4 in a non-human primate (cynomolgus monkey). [Figure 11] 11 is a graph of serum GLA antigen levels measured over a 12-week time course in GLA knockout (B6;129-GLA − / − ) mice following IV injection of AAV-sp7-GLA (labeled "AAV-sp7.GLA") at doses of 2E11 vg / kg (upper triangles), 4E11 vg / kg (lower triangles), or 2E12 vg / kg (diamonds). Controls were wild-type (circles) or GLA knockout (squares) mice injected with vehicle only (labeled "WT+vehicle" and "GLAko," respectively). BQL indicates below limit of quantification. [Figure 12]FIG. 12A is a bar graph of GL-3 levels in the kidneys of GLA knockout (B6;129-GLA − / − ) mice measured 1 and 3 months after IV injection of AAV-sp7-GLA (denoted as “AAV-sp7.GLA”) at doses of 2E11 vg / kg, 4E11 vg / kg, or 2E12 vg / kg. Controls were wild-type mice (denoted as “GLA WT”) and GLA knockout mice (denoted as “GLA KO”). For each condition, the bars at 3 months and 1 month are presented from left to right, respectively. FIG. 12B is a bar graph of lyso-GL-3 levels in the kidneys of GLA knockout (B6;129-GLA − / − ) mice measured 1 and 3 months after IV injection of AAV-sp7-GLA (denoted as “AAV-sp7.GLA”) at doses of 2E11 vg / kg, 4E11 vg / kg, or 2E12 vg / kg. Controls were wild-type mice (denoted as “GLA WT”) and GLA knockout mice (denoted as “GLA KO”). For each condition, the bars at 3 months and 1 month are presented from left to right, respectively. FIG. 12C is a bar graph of GL-3 levels in the hearts of GLA knockout (B6;129-GLA − / −) mice measured 1 and 3 months after IV injection of AAV-sp7-GLA (denoted as “AAV-sp7.GLA”) at doses of 2E11 vg / kg, 4E11 vg / kg, or 2E12 vg / kg. Controls were wild-type mice (denoted as “GLA WT”) and GLA knockout mice (denoted as “GLA KO”). For each condition, the bars at 3 months and 1 month are presented from left to right, respectively. FIG 12D is a bar graph of lyso-GL-3 in the hearts of GLA knockout (B6;129-GLA- / -) mice measured 1 and 3 months after IV injection of AAV-sp7-GLA (denoted "AAV-sp7.GLA") at doses of 2E11 vg / kg, 4E11 vg / kg, or 2E12 vg / kg. Controls were wild type mice (denoted "GLA WT") and GLA knockout mice (denoted "GLA KO").In each condition, the 3-month and 1-month bars were presented from left to right, respectively. Detailed Description of the Invention

[0058] Various publications, articles and patents are cited or described in the Background and throughout this specification, and each of these references is incorporated herein by reference in its entirety. Discussion of documents, acts, materials, devices, articles and the like which is included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items form part of the prior art with respect to the invention disclosed or claimed.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, certain terms cited herein have the meanings set forth herein.

[0060] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0061] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise", as well as variations such as "comprises" and "comprising", are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including", or, in some cases, with the term "having".

[0062] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element, which element, step, or ingredient is relevant to the claimed invention. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of aspects or embodiments of the present invention, any of the above-mentioned words "containing," "including," and "having" may be replaced with the words "consisting of" or "consisting essentially of" to modify the scope of the disclosure.

[0063] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning and thus meets the requirements of the word "and / or" as used herein. The simultaneous applicability of two or more options is also understood to be within the meaning and thus meets the requirements of the term "and / or".

[0064] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

[0065] The term "about" as used herein refers to a value within 10% (i.e., ±10%) of the underlying parameter. For example, "about 1:10" means 1.1:10.1 or 0.9:9.9, about 5 hours means 4.5 hours or 5.5 hours, etc. The word "about" at the beginning of a value string modifies each of the values ​​by up to 10%.

[0066] All numerical values ​​or numerical ranges include integers within such ranges and fractions of integers or values ​​within such ranges unless the context clearly indicates otherwise. Thus, by way of example, a reference to a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. Thus, also by way of example, a reference to a numerical range such as "1 to 4" includes 2, 3, as well as 1.1, 1.2, 1.3, 1.4, etc. For example, "1 to 4 weeks" includes 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0067] Furthermore, a numerical range such as "0.01 to 10" includes 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dosage of "about 0.01 to about 10 mg / kg" per subject's body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

[0068] Reference to more (greater) or smaller integers includes any number greater or less than the referenced numerical value, respectively. Thus, for example, greater than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. For example, "two or more" administrations of a non-viral vector and / or immune cell modulator include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more administrations.

[0069] Additionally, references to numerical ranges such as "1 to 90" include 1.1, 1.2, 1.3, 1.4, 1.5, etc., as well as 81, 82, 83, 84, 85, etc. For example, "between about 1 minute and about 90 days" includes 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as 1 day, 2 days, 3 days, 4 days, 5 days, ..., 81 days, 82 days, 83 days, 84 days, 85 days, etc.

[0070] In an attempt to aid the reader of this application, the description is separated into various paragraphs or sections or directed to certain embodiments of the present invention. These separations should not be considered as separating the substance of a paragraph or section or embodiment from the substance of another paragraph or section or embodiment. On the contrary, those skilled in the art will understand that the description has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that may be contemplated. The discussion of any embodiment is merely illustrative and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples.

[0071] The description provided herein includes modified nucleic acids encoding GLA, expression cassettes comprising modified nucleic acids encoding GLA, viral vectors comprising modified nucleic acids encoding GLA, and non-viral vectors comprising modified nucleic acids encoding GLA. The invention also includes recombinant AAV particles comprising modified nucleic acids encoding GLA, non-viral particles comprising modified nucleic acids encoding GLA, pharmaceutical compositions comprising modified nucleic acids encoding GLA, methods of treating Fabry disease and other lysosomal storage diseases characterized by a GLA deficiency, and various constructs provided herein for use in treating Fabry disease and other lysosomal storage diseases characterized by a GLA deficiency.

[0072] nucleic acid The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In describing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0073] In certain embodiments, nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA, (polynucleotides and oligonucleotides), chromosomal DNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA inhibitor DNA or RNA (RNAi, e.g., small or small hairpin (sh)RNA, microRNA (miRNA), small or small interfering (si)RNA, trans-splicing RNA, or antisense RNA), locked nucleic acid analogs (LNA), oligonucleotide DNA (ODN) single and double stranded, immune stimulatory sequences (ISS), riboswitches, and ribozymes.

[0074] In certain embodiments, nucleic acids include naturally occurring polynucleotides, synthetic polynucleotides, and polynucleotides that have been intentionally modified or altered. Nucleic acids can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length.

[0075] According to some embodiments, the polynucleotide is a single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule. According to some embodiments, the polynucleotide is for therapeutic use, for example, ssDNA or dsDNA encoding a therapeutic transgene. According to some embodiments, the dsDNA molecule is a minicircle, nanoplasmid, open linear double-stranded DNA or closed-end linear double-stranded DNA (CELiD / ceDNA / doggybone DNA). According to some embodiments, the ssDNA molecule is a closed circular DNA or an open linear DNA.

[0076] "Transgene" is used herein to conveniently refer to a nucleic acid to be introduced into a cell or organism, or a nucleic acid that has been introduced.Transgene includes any nucleic acid, such as a heteropolynucleotide sequence, such as a modified nucleic acid encoding GLA, or a heterogeneous nucleic acid encoding a protein or peptide, or a nucleic acid (such as miRNA).The terms transgene and heterogeneous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0077] As used herein, "α-galactosidase A" or "GLA" or "α-gal A" refers to any nucleic acid or protein of GLA. In an embodiment, the nucleic acid encoding GLA encodes the human GLA protein. The complete DNA sequence of GLA, including introns and exons, is available at GenBank Accession No. X14448.1. The human GLA enzyme consists of 429 amino acids and is available at GenBank Accession Nos. X14448.1 and U78027. The full-length 429 amino acid human GLA enzyme is a precursor protein that includes a 31-residue signal peptide that is cleaved to yield a mature 398 amino acid subunit that contains four N-glycosylation consensus sequences. Unless otherwise indicated by the context, references to GLA include full-length precursor and mature α-galactosidase A. Examples of GLA include any naturally occurring GLA, its mature form and variants. An example of a full-length precursor GLA enzyme has the amino acid sequence of SEQ ID NO:12. An example of a mature GLA enzyme has the amino acid sequence of SEQ ID NO: 100. As used herein, a "nucleic acid encoding GLA" refers to a recombinant nucleic acid molecule that encodes a protein having at least a portion of the function or activity of the wild-type GLA protein. Examples of such nucleic acids include modified nucleic acid sequences that encode GLA.

[0078] The term "mutated protein" includes proteins that have a mutation in the gene encoding the protein, resulting in the protein being unable to achieve a stable conformation under conditions normally present in the endoplasmic reticulum (ER). The inability to achieve a stable conformation results in a substantial amount of the enzyme being degraded rather than being transported to the lysosome. Such mutations are sometimes referred to as "conformational mutations." Such mutations include, but are not limited to, missense mutations, and small in-frame deletions and insertions.

[0079] In one embodiment, the term "mutant GLA" includes GLA having a mutation in the gene encoding GLA that results in the enzyme being unable to achieve a stable conformation under conditions normally present in the ER. The inability to achieve a stable conformation results in a substantial amount of the enzyme being degraded rather than being transported to lysosomes.

[0080] As used herein, the term "modify" and grammatical variations thereof mean that a nucleic acid or protein deviates from a reference or parent sequence. A modified nucleic acid encoding GLA is altered compared to a reference (e.g., wild-type) or parent nucleic acid. Thus, a modified nucleic acid may have substantially the same, greater, or less activity or function as a reference or parent nucleic acid, but retains at least partial activity, function, and / or sequence identity with the reference or parent nucleic acid. A modified nucleic acid may be genetically modified to encode a variant or mutant GLA.

[0081] "Modified nucleic acid encoding GLA" means that the GLA nucleic acid has a modification compared to the parent unmodified nucleic acid encoding GLA. A specific example of a modification is a nucleotide substitution. The nucleotide substitution can be a silent mutation that encodes the same amino acid, or a missense mutation that encodes a different amino acid. The missense mutation can be a conservative or non-conservative mutation. Other examples of modifications include, for example, truncations and insertions. Modified nucleic acids can also include codon-optimized nucleic acids that encode the same protein as a wild-type protein or a nucleic acid that is not codon-optimized. Codon optimization can be used in a broader sense, including, for example, removing CpG dinucleotides.

[0082] As used herein, the term "modification" need not appear in every instance of reference to a nucleic acid encoding GLA.

[0083] In some embodiments, for modified nucleic acids encoding GLA, the GLA protein retains at least some of the functions or activities of wild-type GLA protein. The functions or activities of GLA protein include α-galactosidase A activity, a glycoside hydrolase enzyme that hydrolyzes terminal α-galactosyl moieties from glycolipids and glycoproteins. Thus, modified nucleic acids encoding GLA include modified forms so long as the encoded GLA retains a certain degree or aspect of the glycoside hydrolase activity of GLA.

[0084] As described herein, modified nucleic acids encoding GLA may exhibit different characteristics or properties compared to the reference nucleic acid or parent nucleic acid. For example, modified nucleic acids include sequences that have 100% identity to the reference nucleic acid encoding GLA described herein, as well as sequences that have less than 100% identity to the reference nucleic acid encoding GLA.

[0085] The terms "identity", "homology" and grammatical variations thereof mean that two or more referenced entities are the same when they are "aligned" sequences. Thus, for example, when two nucleic acids are identical, they have the same sequence, at least within the referenced region or portion. Identity can be over a defined range (region or domain) of sequences.

[0086] A "range" or "region" of identity refers to a portion of two or more referenced entities that are the same. Thus, if two protein or nucleic acid sequences are identical over one or more sequence ranges or regions, they share identity within that region. An "aligned" sequence refers to multiple protein (amino acid) or nucleic acid sequences, often including corrections for deleted or added bases or amino acids (gaps) compared to a reference sequence.

[0087] The identity can span the entire length or a portion of the sequence. In some embodiments, the length of the sequences that share percent identity is 2, 3, 4, 5 or more consecutive amino acids or nucleic acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. consecutive nucleic acids or amino acids. In some embodiments, the length of the sequences that share identity is 21 or more consecutive amino acids or nucleic acids, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. consecutive amino acids or nucleic acids. In a further embodiment, the length of the sequence sharing identity is 41 or more consecutive amino acids or nucleic acids, for example, 42, 43, 44, 45, 45, 47, 48, 49, 50, etc. In yet a further embodiment, the length of the sequence sharing identity is 50 or more consecutive amino acids or nucleic acids, for example, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 500, 500 to 1,000, etc.

[0088] As described herein, modified nucleic acids encoding GLA can be distinct from or exhibit 100% identity or less than 100% identity to a reference nucleic acid encoding GLA.

[0089] According to one embodiment, a nucleic acid encoding GLA has (1) 75% or more sequence identity, 76% or more sequence identity, 77% or more sequence identity, 78% or more sequence identity, 79% or more sequence identity, 80% or more sequence identity, 81% or more sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, or 89% or more sequence identity to the sequence of SEQ ID NO:15. (1) a polynucleotide having at least 75% sequence identity to the sequence of SEQ ID NO: 15, such as a sequence identity of 90% or more, a sequence identity of 91% or more, a sequence identity of 92% or more, a sequence identity of 93% or more, a sequence identity of 94% or more, a sequence identity of 95% or more, a sequence identity of 96% or more, a sequence identity of 97% or more, a sequence identity of 98% or more, a sequence identity of 99% or more, or a sequence identity of 99.5% or more; (2) a polynucleotide having at least 83% sequence identity or 84% sequence identity to the sequence of SEQ ID NO: 16. , 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.5% or more sequence identity, etc., at least 83% to the sequence of SEQ ID NO: 16. (3) a polynucleotide having a sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.(4) a polynucleotide having at least 85% sequence identity to the sequence of SEQ ID NO: 18, such as 5% or more sequence identity; (5) a polynucleotide having at least 85% sequence identity to the sequence of SEQ ID NO: 17, such as 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity. and (5) polynucleotides having at least 82% sequence identity to the sequence of SEQ ID NO: 16, such as 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 16. According to one embodiment, the nucleic acid encoding GLA has 100% sequence identity to any of the sequences of SEQ ID NOs: 15-19.

[0090] In some embodiments, the nucleic acid encoding GLA further comprises one or more introns located anywhere within the nucleic acid encoding GLA, hi some embodiments, the intron is located between nucleotides 78-79 of the nucleic acid encoding GLA, which nucleotide positions are given relative to the coding sequence for GLA having the sequence of SEQ ID NO:14.

[0091] In some embodiments, the intron is selected from the group consisting of the vitronectin 1 (VTN1) gene, the retinol binding protein 4 (RBP4) gene, the mouse IgG heavy chain A (IgHA) gene, and the mouse IgG heavy chain μ (IgHμ gene). In some embodiments, the one or more introns are selected from the sequences of SEQ ID NOs: 49-52.

[0092] In one embodiment, the nucleic acid encoding GLA has the sequence of any of SEQ ID NOs:43-46.

[0093] According to one embodiment, the nucleic acid of the invention encodes GLA having the amino acid sequence of SEQ ID NO:100.

[0094] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 100 with one amino acid substitution selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 100 with any two amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 100 with any three amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 100 with any four amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 100 with any five amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 100 with any six amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 100 with seven amino acid substitutions: Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO: 48.

[0095] In one embodiment, the nucleic acid encoding GLA comprises the coding sequence of SEQ ID NO:47.

[0096] Modified nucleic acids encoding GLA that exhibit different characteristics or properties compared to a reference or parent nucleic acid include nucleotide substitutions. For example, modified nucleic acids encoding GLA include nucleic acids that have a reduced number of CpG dinucleotides compared to a reference nucleic acid encoding GLA, referred to as CpG-reduced nucleic acids.

[0097] As used herein, the phrase "CpG reduced" or "CpG depleted" refers to a nucleic acid sequence that is generated synthetically or by mutation of a nucleic acid sequence such that one or more CpG dinucleotides (or motifs) are removed from the nucleic acid sequence. In an embodiment, all CpG motifs are removed to provide what is referred to herein as a modified CpG-free sequence. CpG motifs are suitably reduced or removed not only in coding sequences (e.g., transgenes) but also in non-coding sequences, including, for example, 5' untranslated regions (UTRs) and 3' UTRs, promoters, enhancers, signal peptides, polyA, ITRs, introns, and any other sequences present in a polynucleotide molecule.

[0098] According to certain embodiments, the nucleic acid encoding GLA contains fewer than 14 CpG dinucleotides, such as 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 CpG dinucleotides.

[0099] The phrase "consisting essentially of," when referring to a particular nucleotide or amino acid sequence, means a sequence that has the characteristics of the sequence of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself, as well as molecular modifications that do not affect the basic and novel characteristics of the sequence.

[0100] Nucleic acids, expression vectors, AAV vector genomes, non-viral vectors, plasmids containing modified nucleic acids encoding the GLA of the present invention can be prepared by using recombinant DNA technology techniques. The availability of nucleotide sequence information allows for the preparation of isolated nucleic acid molecules of the present invention by various means. Nucleic acids encoding GLA can be produced using a variety of standard cloning, recombinant DNA techniques, through cell expression or in vitro translation and chemical synthesis techniques. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to, (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides with shared structural features, for example, using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA with primers capable of annealing to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of subtracted nucleic acid libraries.

[0101] The nucleic acid may be maintained as DNA in any convenient cloning vector. In certain embodiments, the clone is maintained in a plasmid cloning / expression vector, such as pBluescript (Stratagene, La Jolla, Calif.), propagated in a suitable E. coli host cell. Alternatively, the nucleic acid may be maintained in a vector suitable for expression in mammalian cells, such as an AAV vector. In cases where post-translational modifications affect protein function, the nucleic acid molecule may be expressed in mammalian cells.

[0102] Expression cassette The present invention also provides an expression cassette comprising a polynucleotide comprising a nucleic acid encoding GLA described herein, operably linked to an expression control element. In one embodiment, the expression cassette comprises a nucleic acid encoding GLA, the nucleic acid being selected from the group consisting of: (1) a polynucleotide having at least 75% sequence identity (e.g., 75% to 100% identity) to the sequence of SEQ ID NO: 15; (2) a polynucleotide having at least 84% sequence identity (e.g., 84% to 100% identity) to the sequence of SEQ ID NO: 16; (3) a polynucleotide having at least 86% sequence identity (e.g., 86% to 100% identity) to the sequence of SEQ ID NO: 17; (4) a polynucleotide having at least 86% sequence identity (e.g., 86% to 100% identity) to the sequence of SEQ ID NO: 18; and (5) a polynucleotide having at least 83% sequence identity (e.g., 83% to 100% identity) to the sequence of SEQ ID NO: 19.

[0103] In one embodiment, GLA comprises the amino acid sequence of SEQ ID NO:100.

[0104] In an embodiment, the expression cassette comprises an appropriate secretory signal sequence or signal peptide that allows for the secretion of the polypeptide encoded by the polynucleotide molecule of the present invention. As used herein, the term "secretory signal sequence" or "signal peptide" or variations thereof are intended to refer to an amino acid sequence that functions to enhance the secretion of an operably linked polypeptide from a cell compared to the secretion level seen with the native polypeptide. A signal peptide is a short amino acid sequence, typically less than 20 amino acids in length, that directs a protein to or through the endoplasmic reticulum secretory pathway. "Enhanced" secretion means that the relative proportion of polypeptides synthesized by a cell that are secreted from the cell is increased, and does not require that the absolute amount of protein secreted is also increased. In an embodiment, essentially all of the polypeptide (i.e., at least 95%, 97%, 98%, 99% or more) is secreted. However, it is not essential that essentially all or most of the polypeptide is secreted, so long as the secretion level is enhanced compared to the native polypeptide. Generally, the secretory signal sequence is cleaved in the endoplasmic reticulum, and in an embodiment, the secretory signal sequence is cleaved prior to secretion. However, it is not essential that the secretory signal sequence be cleaved, so long as secretion of the polypeptide from the cell is enhanced and the polypeptide is functional. Thus, in certain embodiments, the secretory signal sequence is partially or completely retained. The secretory signal sequence may be derived in whole or in part from the secretory signal of the secreted polypeptide (i.e., from a precursor) and / or may be synthetic in whole or in part. The length of the secretory signal sequence is not critical, and generally, known secretory signal sequences are from about 10-15 amino acids in length to 50-60 amino acids in length. Furthermore, known secretory signals from secreted polypeptides may be altered or modified (e.g., by amino acid substitution, deletion, truncation or insertion), so long as the resulting secretory signal sequence functions to enhance secretion of the operably linked polypeptide. The secretory signal sequence of the present invention may comprise, consist essentially of, or consist of a naturally occurring secretory signal sequence or a variant thereof.Numerous secretory proteins and sequences that direct secretion from cells are known in the art. The secretory signal sequence of the present invention can also be wholly or partially synthetic or artificial. Synthetic or artificial secretory signal peptides are known in the art, see, for example, Barash et al., Biochem. Biophys. Res. Comm. 294:835-42 (2002).

[0105] In light of the present disclosure, any suitable signal peptide known to those of skill in the art can be used in the present invention. Examples of signal peptides include, but are not limited to, those found in the Signal Peptide Database (www.signalpeptide.de / ). Examples of signal peptides suitable for the present invention include, but are not limited to, wild-type GLA signal peptide, human chymotrypsinogen B2 signal peptide ("sp7"; 18 amino acid signal peptide of NCBI reference sequence NP_001020371), alpha 2-HS-glycoprotein (AHSG) signal peptide, CD300 signal peptide, lysosomal-associated membrane glycoprotein 1 (LAMP1) signal peptide, Notch 2 signal peptide, orosomucoid 1 (ORM1) signal peptide, transferrin (TF) signal peptide, secreton (artificial signal sequence described in Barash et al., Biochem Biophys Res Commun. 2002;294: 835-842), mouse IgKVIII, human IgKVIII, CD33, tPA, a-1 antitrypsin signal peptide, and naturally occurring secreted alkaline phosphatase (SEAP). Any conventional signal sequence that directs proteins through the endoplasmic reticulum secretory pathway can be used in the present invention, including variants of the signal peptides described above.

[0106] In certain embodiments, the signal peptide is the endogenous or native GLA signal peptide or a variant thereof.

[0107] In certain embodiments, the signal peptide is a heterologous signal peptide or a variant thereof.

[0108] In one embodiment, the signal peptide has a coding sequence of any of SEQ ID NOs:1-11 and 13.

[0109] In one embodiment, the signal peptide has an amino acid sequence of any one of SEQ ID NOs: 41 and 57-63.

[0110] In one embodiment, the expression cassette comprises a nucleic acid encoding a signal peptide sequence located at the 5' end of the nucleic acid encoding GLA. In one embodiment, the signal peptide is human chymotrypsinogen B2 signal peptide. In one embodiment, the signal peptide is human chymotrypsinogen B2 signal peptide having the amino acid sequence of SEQ ID NO: 41. In one embodiment, the signal peptide is human chymotrypsinogen B2 signal peptide having the coding sequence of any of SEQ ID NOs: 1-5.

[0111] In one embodiment, the polynucleotide encodes a GLA having the sequence of any of SEQ ID NOs:101-109.

[0112] In one embodiment, the polynucleotide comprises any of the sequences set forth in SEQ ID NOs:64-81.

[0113] In one embodiment, the expression control element is located 5' to a nucleic acid encoding GLA.

[0114] The term "expression cassette" as used herein refers to a nucleic acid construct that contains sufficient nucleic acid elements for expression of a polynucleotide molecule of the invention. Typically, an expression cassette contains a polynucleotide molecule of the invention operably linked to a promoter sequence.

[0115] "Expression control element" refers to a nucleic acid sequence that affects the expression of an operably linked nucleic acid. Expression control elements described herein include promoters and enhancers. Vector sequences, including AAV vectors and non-viral vectors, may include one or more "expression control elements". Typically, such elements are included to facilitate proper heteropolynucleotide transcription and proper translation (e.g., promoters, enhancers, splicing signals of introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, and stop codons, etc.). Such elements are typically referred to as "cis-acting" elements, acting in cis, but may also act in trans.

[0116] Expression control can be affected at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., within an intron). Expression control elements can be located adjacent to the transcribed sequence or at a distance away from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide), even at a significant distance. Nevertheless, due to the length limitations of the AAV vector, expression control elements in the AAV vector are typically within 1-1000 nucleotides from the transcription start site of the heterologous nucleic acid.

[0117] Functionally, expression of an operably linked nucleic acid can be at least partially controlled by an element (e.g., a promoter) that regulates transcription of the nucleic acid and, optionally, translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed nucleic acid sequence. A promoter typically increases the amount of expression from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0118] The term "operably linked" means that the regulatory sequence necessary for the expression of a nucleic acid sequence is arranged in a suitable position relative to the sequence so as to mediate the expression of the nucleic acid sequence. This same definition may be applied to the arrangement of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in expression vectors, such as rAAV vectors or non-viral vectors. The coding sequence may be operably linked to the regulatory sequence in sense or antisense orientation. In an embodiment, the promoter is a heterologous promoter.

[0119] The term "heterologous promoter" as used herein refers to a promoter that is not found operably linked to a given coding sequence in nature. In certain embodiments, the expression cassette may contain additional elements, such as introns, enhancers, polyadenylation sites, woodchuck response elements (WREs), and / or other elements known to affect the expression levels of coding sequences.

[0120] As used herein, the term "promoter" refers to a nucleotide sequence that can control the expression of a coding sequence or functional RNA. In general, the nucleic acid molecule of the present invention is located 3' of the promoter sequence. In an embodiment, the promoter sequence is composed of proximal and more distal upstream elements and can include enhancer elements.

[0121] As used herein, "enhancer" may refer to a sequence located adjacent to a heterologous nucleic acid. Enhancer elements are typically located upstream of a promoter element, but may also function and be located downstream or within the sequence. Thus, enhancer elements may be located 10-50 base pairs, 50-100 base pairs, 100-200 base pairs, or 200-300 base pairs, or more, upstream or downstream of a heterologous nucleic acid sequence. Enhancer elements typically increase expression of an operably linked nucleic acid provided by a promoter element.

[0122] An expression construct may include control elements that serve to drive expression in a particular cell or tissue type. Expression control elements (e.g., promoters) include those that are active in a particular tissue or cell type, referred to herein as "tissue-specific expression control elements / promoters." Tissue-specific expression control elements are typically active in a particular cell or tissue (e.g., liver). Expression control elements are typically active in a particular cell, tissue, or organ because they are recognized by a transcriptional activator protein, or other regulator of transcription, that is unique to that particular cell, tissue, or organ type. Such control elements are known to those of skill in the art (see, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al. (2010) Current protocols in molecular biology, John Wiley & Sons, New York).

[0123] The incorporation of tissue-specific regulatory elements in an expression construct provides at least partial tissue tropism for expression of a heterologous nucleic acid encoding a protein or inhibitory RNA. Examples of promoters active in the liver include the transthyretin (TTR) gene promoter; human alpha 1-antitrypsin (hAAT) promoter; apolipoprotein AI promoter; albumin, Miyatake, et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig, et al., Gene Ther. 3: 1002-9 (1996); alpha fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14(1996); human factor IX promoter; thyroxine-binding globulin (TBG) promoter; TTR minimal enhancer / promoter; alpha antitrypsin promoter; LSP (845 nt) (requires intronless scAAV); and LSP1 promoter. Examples of enhancers active in the liver are apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19(1997)).

[0124] Expression control elements also include ubiquitous or promiscuous promoters / enhancers that can drive expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in a variety of mammalian cell types, or synthetic elements that do not occur in nature (see, for example, Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic b-actin promoter, and the phosphoglycerate kinase (PGK) promoter.

[0125] Expression control elements can also confer expression in a regulatable manner, i.e., a signal or stimulus increases or decreases the expression of the operably linked heteropolynucleotide. Regulatable elements that increase the expression of the operably linked polynucleotide in response to a signal or stimulus are also called "inducible elements" (i.e., induced by a signal). Particular examples include, but are not limited to, hormone (e.g., steroid) inducible promoters. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present, with the greater the amount of signal or stimulus, the greater the increase or decrease in expression. Specific, non-limiting examples include the zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 98 / 10088); the tetracycline repression system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline inducible system (Gossen, et al., Science. 268: 1766-1769 (1995); see Harvey, et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); and the rapamycin inducible system (Magari, et al., J. Clin. Invest. 100:2865-2872 (1997); Rivera, et al., Nat. Medicine. 2:1028-1032 (1996)). Other regulatable control elements that may be useful in this context are those that are regulated by specific physiological conditions, e.g., temperature, acute phase, development.

[0126] Other examples of promoters include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (a mixture of CMV enhancer, chicken β-actin promoter (CBA) and rabbit β-globin intron) and other constitutive promoters, NSE (neuron-specific enolase), synapsin or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, SFFV promoter, Rous sarcoma virus (RSV) promoter, rat insulin promoter, TBG promoter and other liver-specific promoters, desmin promoter and similar muscle-specific promoters, EF1-α promoter, synthetic promoters, hybrid promoters, promoters with multiple tissue specificities, etc., all of which are well known and readily available to those skilled in the art. Other promoters may be of human origin or from other species, including mouse.

[0127] Expression control elements also include the native elements of heteropolynucleotides.Natural control elements (e.g., promoters) can be used when it is desired that the expression of heteropolynucleotides should mimic native expression.Natural elements can be used when the expression of heteropolynucleotides is regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli.Other native expression control elements, such as introns, polyadenylation sites, or Kozak consensus sequences, can also be used.

[0128] In the example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. More specifically, for example, two DNA sequences that are operably linked means that the two DNA sequences (in cis or trans) are arranged in a relationship such that at least one of the DNA sequences can exert a physiological effect on the other sequence.

[0129] Thus, additional elements for a vector include, but are not limited to, expression control (e.g., promoter / enhancer) elements, transcription termination signals or stop codons, 5' or 3' untranslated regions flanking sequences such as one or more copies of AAV ITR sequences (e.g., polyadenylation (polyA) sequences), or introns.

[0130] Additional elements include, for example, filler or stuffer polynucleotide sequences, e.g., to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically tolerate inserts of DNA, generally having a size range of about 4 kb to about 5.2 kb, or slightly larger. Thus, for shorter sequences, fillers or stuffers are included to adjust the length to be closer to or at the normal size of the viral genome sequence that is acceptable for packaging the AAV vector into a viral particle. In an embodiment, the filler / stuffer nucleic acid sequence is a non-translated (non-protein coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 kb, the filler or stuffer polynucleotide sequence has a length that, when combined with the sequence (e.g., inserted into a vector), has a total length of about 3.0 to 5.5 kb, or about 4.0 to 5.0 kb, or about 4.3 to 4.8 kb.

[0131] In one embodiment, the expression control element comprises an ApoE / hAAT enhancer / promoter sequence located 5' of the nucleic acid encoding GLA. In one embodiment, the ApoE / hAAT enhancer / promoter sequence has reduced CpGs compared to the wild-type ApoE / hAAT enhancer / promoter sequence. In one embodiment, the ApoE / hAAT enhancer / promoter sequence has the sequence of SEQ ID NO:38.

[0132] In some embodiments, the expression cassette comprises a polyadenylation (polyA) sequence located 3' to the nucleic acid encoding GLA. In some embodiments, the polyA sequence comprises a bovine growth hormone (bGH) polyadenylation sequence. In some embodiments, the bGH polyadenylation sequence has reduced CpGs compared to the wild-type bGH polyadenylation sequence. In some embodiments, the bGH polyadenylation sequence has the sequence of SEQ ID NO:20.

[0133] In some embodiments, the expression cassette further comprises an intron located between the 3' end of the expression control element and the 5' end of the nucleic acid encoding GLA. In some embodiments, the intron is an hBB2m1 intron. In some embodiments, the hBB2m1 intron sequence has reduced CpGs compared to the wild-type hBB2m1 intron sequence. In some embodiments, the hBB2m1 intron sequence has the sequence of SEQ ID NO:39.

[0134] In certain embodiments, the expression cassette further comprises one or more introns located anywhere within the nucleic acid encoding GLA. In certain embodiments, the intron is located at a site within the nucleic acid encoding GLA that matches the consensus nucleotide sequence of MAG / G, where M is adenine or cytosine, and " / " indicates the site of intron insertion. In certain embodiments, the intron is located between nucleotides 78-79 of the nucleic acid encoding GLA, the nucleotide positions being given relative to the coding sequence for GLA having the sequence of SEQ ID NO:14. In light of the present disclosure, any suitable intron known to those of skill in the art can be used in the present invention. Examples of suitable introns include, but are not limited to, introns from the vitronectin 1 (VTN1) gene, the retinol binding protein 4 (RBP4) gene, the mouse IgG heavy chain A (IgHA) gene, and the mouse IgG heavy chain μ (IgHμ gene). In certain embodiments, the one or more introns are selected from any of the sequences of SEQ ID NOs:49-52.

[0135] In one embodiment, the expression cassette has a sequence of any of SEQ ID NOs: 21 to 34, 53 to 56, and 91 to 99. In one embodiment, the expression cassette has a sequence that has at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.5% sequence identity, or 100% sequence identity to any of SEQ ID NOs: 21 to 34, 53 to 56, and 91 to 99.

[0136] Gene Delivery System Viral Vectors The present invention further provides a viral vector, such as an adeno-associated viral (AAV) vector, comprising a polynucleotide that includes a nucleic acid encoding a GLA as described herein.

[0137] The term "vector" or "gene transfer vector" as used herein refers to the nucleic acid molecule that contains a gene of interest.Examples of vectors include, but are not limited to, viral vectors delivered by retrovirus, adenovirus, adeno-associated virus, and lentivirus particle or virus-like particle (VLP), or VLPs similar to viral particles but non-infectious; and non-viral vectors delivered by non-viral gene transfer systems, such as microinjection, electroporation, liposome, high molecular weight natural polymer, high molecular weight synthetic polymer, and polymers composed of both natural and synthetic components.

[0138] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell, and optionally additional elements, such as heteropolynucleotide sequences, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), selection markers (e.g., antibiotic resistance), polyadenylation signals, etc.

[0139] As used herein, the term "delivery system" refers to any means of delivering a composition comprising nucleic acid sequence to cells or tissues.For example, gene transfer system can be a viral gene transfer system, such as intact virus, modified virus and VLP, to facilitate the delivery of viral vector to desired cells or tissues.Genetic transfer system can also be a non-viral delivery system that does not contain viral coat protein or does not form viral particles or VLPs, such as liposome-based system, polymer-based system, protein-based system, metal particle-based system, peptide cage system, etc.

[0140] Viral vectors are derived from or based on one or more nucleic acid elements that comprise a viral genome. Particular viral vectors include lentiviral vectors and adeno-associated viral (AAV) vectors.

[0141] The term "recombinant," as in variants of vectors, such as recombinant AAV (rAAV) vectors, and variants of sequences, such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e., modified) in a manner that is not generally found in nature. The term "recombinant" is not necessarily used herein in reference to AAV vectors, and sequences, such as polynucleotides, although recombinant forms that include polynucleotides are expressly included despite such omission.

[0142] A "recombinant AAV vector" or "rAAV" is derived from the wild-type genome of AAV by using molecular methods to remove the wild-type genome from the AAV genome and replace it with a non-natural nucleic acid sequence called a heterologous nucleic acid. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. rAAV is distinguished from the AAV genome because all or part of the AAV genome is replaced with a non-natural sequence with respect to the AAV genome nucleic acid. Thus, the incorporation of the non-natural sequence defines the AAV vector as a "recombinant" vector, which can be called a "rAAV vector".

[0143] The rAAV sequence can be packaged ex vivo, in vitro or in vivo for subsequent infection (transduction) of cells, and is referred to herein as a "particle". When a recombinant AAV vector sequence is encapsidated or packaged in an AAV particle, the particle can also be referred to as a "rAAV vector" or "rAAV particle". Such rAAV particles contain proteins that encapsidate or package the vector genome, and in the case of AAV, they are referred to as capsid proteins.

[0144] A vector "genome" refers to the portion of a recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., rAAV) particle. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid can be referred to as the "plasmid backbone," which is important for plasmid cloning and amplification, a process required for propagation and recombinant virus production. The plasmid backbone itself, with the exception of possible 3' ITR and / or 5' ITR cloning remnants, is not packaged or encapsidated into a viral (e.g., AAV) particle. Thus, a vector "genome" refers to the polynucleotide that is packaged or encapsidated by a virus (e.g., AAV).

[0145] Host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that may be or have been used as recipients of heterologous rAAV vectors. Cells derived from a stable human cell line, HEK293 (e.g., readily available from the American Type Culture Collection under accession number ATCC CRL1573), can be used. In one embodiment, a modified human embryonic kidney cell line (e.g., HEK293) transformed with adenovirus type 5 DNA fragments and expressing adenovirus E1a and E1b genes is used to generate recombinant AAV particles. The modified HEK293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV particles. Other host cell lines suitable for recombinant AAV production are described in International Application PCT / 2017 / 024951, the disclosure of which is incorporated herein in its entirety.

[0146] In certain embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct prior to or simultaneously with transfection of the AAV expression vector. A host cell with AAV helper functions can be referred to as a "helper cell" or a "packaging helper cell". Thus, AAV helper constructs may be used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions required for productive AAV transduction. AAV helper constructs often lack AAV ITRs and cannot replicate or package themselves. These constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Many AAV helper constructs are described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which code for both Rep and Cap expression constructs. Many other vectors are known that code for Rep and / or Cap expression products.

[0147] The method of producing recombinant AAV particles that can transduce mammalian cells is known in the art.For example, recombinant AAV particles can be produced as described in U.S. Patent No. 9,408,904; and International Application No. PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein in their entirety.

[0148] The present invention provides cells comprising nucleic acid encoding GLA, cells comprising expression cassettes comprising polynucleotides comprising nucleic acid encoding GLA, cells comprising viral vectors, such as AAV vectors, comprising nucleic acid encoding GLA, and cells comprising non-viral vectors comprising polynucleotides comprising nucleic acid encoding GLA.In some embodiments, cells produce viral vectors.In some embodiments, cells produce AAV vectors as described herein.

[0149] Also provided is a method for producing viral vectors, such as the AAV vectors described herein.In an embodiment, the method for producing AAV vectors comprises introducing the AAV vector genome comprising the nucleic acid encoding GLA described herein or the expression cassette comprising the nucleic acid encoding GLA described herein into packaging helper cells; and culturing the helper cells under conditions for producing AAV vectors.In an embodiment, the method for producing AAV vectors comprises introducing the nucleic acid encoding GLA or the expression cassette comprising the nucleic acid encoding GLA described herein into packaging helper cells; and culturing the helper cells under conditions for producing AAV vectors.

[0150] In some embodiments, the cell is a mammalian cell.

[0151] In some embodiments, the cells for vector production provide helper functions, such as AAV helper functions that package vectors into viral particles. In some aspects, the helper functions are Rep and / or Cap proteins for AAV vector packaging. In some embodiments, the cells for vector production can be stably or transiently transfected with polynucleotides that code for Rep and / or Cap protein sequences. In some embodiments, the cells for vector production provide Rep78 and / or Rep68 proteins. In such cells, the cells can be stably or transiently transfected with polynucleotides that code for Rep78 and / or Rep68 protein sequences.

[0152] In one embodiment, the cells for vector production are human embryonic kidney cells. In one embodiment, the cells for vector production are HEK-293 cells.

[0153] The term "transduce" and its grammatical variants refer to the introduction of a molecule, such as a rAAV vector, into a cell or host organism. The heterologous nucleic acid / transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. The introduced heterologous nucleic acid may also be present in the recipient cell or host organism extrachromosomally or only transiently.

[0154] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell (e.g., in a mammal, such as a cell or tissue or organ cell) refers to, for example, a genetic change in a cell following incorporation of a nucleic acid (e.g., a transgene) into the cell. Thus, a "transduced" cell is a cell into which an exogenous nucleic acid has been introduced, or a progeny thereof. The cell may be propagated and express the introduced protein. For gene therapy uses and methods, the transduced cell may be present in a subject.

[0155] The term "isolated," when used as a modifier of a composition, means that the compositions are made by the hand of man or are completely or at least partially separated from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they are normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, or cell membranes.

[0156] The term "isolated" does not exclude combinations produced by the hand of man that package or encapsidate the AAV vector genome and pharmaceutical preparation, such as rAAV sequences, or rAAV particles. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0157] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may contain at least 75%, or at least 85%, or about 90-99% by weight of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0158] The recombinant AAV vector and its method and use include any virus strain or serotype.As a non-limiting example, the recombinant AAV vector can be based on any AAV genome, such as LK03, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, Rh10, Rh74, AAV3B or AAV-2i8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or different from each other.As a non-limiting example, the recombinant AAV vector based on a specific serotype genome can be the same as the serotype of the capsid protein that packages the vector.In addition, the recombinant AAV vector genome can be based on an AAV serotype genome that is different from the serotype of the AAV capsid protein that packages the vector. For example, the AAV vector genome can be based on AAV2, while at least one of the three capsid proteins can be LK03, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B or AAV-2i8, or a variant thereof.

[0159] In certain embodiments, the adeno-associated virus (AAV) vectors include LK03, AAV2, AAV4, AAV6, AAV8, AAV9, AAV10, AAV12, Rh10, Rh74, AAV3B, and AAV-2i8, as well as any of the other AAV vectors described in, for example, WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO This includes variants thereof as described in International Application No. PCT / US2014 / 047670, which discloses RHM4-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6, US2013 / 0059732 (U.S. Patent No. 9,169,299, which discloses LK01, LK02, LK03, etc.) and WO 2016 / 210170, the disclosures of which are incorporated herein in their entireties.

[0160] As used herein, the term "serotype" is a category used to refer to AAVs that have a capsid that is serologically different from other AAV serotypes. Serological characteristics are determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). AAV variants, including capsid variants, may not be serologically distinct from a reference AAV or other AAV serotypes, but they differ in at least one nucleotide or amino acid residue compared to a reference or other AAV serotypes.

[0161] In the conventional definition, serotype means that the virus of interest is tested against sera specific to all the serotypes that exist and have been characterized for neutralizing activity, and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, there may or may not be serological differences with any of the existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, this new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are separate serotypes according to the traditional definition of serotype. Therefore, for convenience and to avoid duplication, the term "serotype" refers broadly to both serologically distinct viruses (e.g., AAV) as well as serologically indistinguishable viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0162] As described herein, the AAV capsid protein exhibits less than 100% sequence identity to a reference or parent sequence, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, LK03 (SEQ ID NO:42), AAV-2i8, the sequence of SEQ ID NO:110, the sequence of SEQ ID NO:36, and / or SEQ ID NO:37, but is different and identical to known AAV genes or proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, LK03 (SEQ ID NO:42) or AAV-2i8. In certain embodiments, the variant / mutant AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, LK03 (SEQ ID NO:42), AAV-2i8, the sequence of SEQ ID NO:110, the sequence of SEQ ID NO:36, the sequence of SEQ ID NO:37, and / or comprises or consists of a sequence at least 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5% etc., up to 99.5% etc. identical to a reference AAV capsid protein or parent AAV capsid protein, such as the sequence of SEQ ID NO:42.

[0163] In certain embodiments, a viral vector, such as an adeno-associated viral (AAV) vector, comprises any of the polynucleotides comprising a nucleic acid encoding GLA described herein, operably linked to an expression control element.

[0164] In certain embodiments, a viral vector, such as an adeno-associated viral (AAV) vector, comprises any of the expression cassettes comprising a polynucleotide comprising a nucleic acid encoding GLA described herein.

[0165] In certain embodiments, an AAV vector comprises one or more AAV capsids; and one or more AAV inverted terminal repeats (ITRs), where the AAV ITRs flank the 5' or 3' end of the polynucleotide or expression cassette.

[0166] In one embodiment, the AAV vector further comprises an intron located 5' or 3' to one or more ITRs.

[0167] In one embodiment, the AAV vector comprising at least one or more ITRs or introns has one or more ITRs or introns modified to have reduced CpGs.

[0168] In one embodiment, the AAV vectors of the invention are delivered via non-viral delivery systems, including, for example, those encapsulated in lipid nanoparticles (LNPs).

[0169] Non-viral methods In certain embodiments, the polynucleotides and expression cassettes of the invention are delivered or administered with non-viral delivery systems, including, for example, non-viral vectors, or chemical methods such as extracellular vesicles, or physical methods such as gene guns, electroporation, particle bombardment, ultrasound application, and magnetic cleavage.

[0170] Recombinant cells capable of expressing the GLA sequences of the present invention can be used for delivery or administration.

[0171] Naked DNA such as minicircles and transposons can be used for administration or delivery or lentiviral vectors.Furthermore, gene editing techniques such as zinc finger nucleases, meganucleases, TALENs and CRISPRs can also be used to deliver the coding sequence of the present invention.

[0172] In certain embodiments, the polynucleotides and expression cassettes of the invention are delivered as naked DNA, minicircles, transposons, or end-closed linear double-stranded DNA.

[0173] In certain embodiments, the polynucleotides and expression cassettes of the invention are delivered or administered in AAV vector particles or other viral particles that are further encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0174] In certain embodiments, the polynucleotides and expression cassettes of the present invention are delivered or administered with non-viral vectors.

[0175] As used herein, "non-viral vector" refers to a vector that is not delivered by a virus particle or a virus-like particle (VLP).According to an embodiment, the non-viral vector is a vector that is not delivered by a capsid.The vector can be encapsulated, mixed, or otherwise associated with the non-viral delivery nanoparticle.

[0176] In light of the present disclosure, any suitable non-viral delivery system known to those of skill in the art can be used in the present invention. Non-viral delivery nanoparticles can be, for example, lipid-based nanoparticles, polymer-based nanoparticles, protein-based nanoparticles, microparticles, microcapsules, metal particle-based nanoparticles, peptide cage nanoparticles, etc.

[0177] The non-viral delivery nanoparticles of the invention can be constructed by any method known in the art, and the non-viral vectors of the invention comprising nucleic acid molecules that contain therapeutic transgenes can be constructed by any method known in the art.

[0178] Lipid-Based Delivery Systems Lipid-based delivery systems are well known in the art, and any suitable lipid-based delivery system known to one of skill in the art in light of the present disclosure can be used in the present invention. Examples of lipid-based delivery systems include, for example, liposomes, lipid nanoparticles, micelles, or extracellular vesicles.

[0179] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles having nanoscale dimensions, i.e., in the range of about 10 to about 1000 nm, or about 50 to about 500 nm, or about 75 to about 127 nm, useful for delivery of AAV and non-viral vectors. Without being bound by theory, it is believed that LNPs provide polynucleotides, expression cassettes, AAV vectors, or non-viral vectors with partial or complete shielding from the immune system. Shielding allows delivery of the polynucleotide, expression cassette, AAV vector, or non-viral vector to tissues or cells while avoiding inducing a substantial immune response to the polynucleotide, expression cassette, AAV vector, or non-viral vector in vivo. Shielding may also allow repeated administration without inducing a substantial immune response to the polynucleotide, expression vector, AAV vector, or non-viral vector in vivo (e.g., in a subject such as a human). Shielding may also improve or increase polynucleotide, expression cassette, AAV vector, or non-viral vector delivery efficiency in vivo.

[0180] The pI (isoelectric point) of AAV is in the pH range of about 6 to about 6.5. Thus, the AAV surface has a slight negative charge. Therefore, it may be beneficial for the LNP to include a cationic lipid, such as, for example, an amino lipid. Exemplary amino lipids are described in U.S. Pat. Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,651, and U.S. Pat. Nos. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125, the disclosures of which are incorporated herein in their entireties.

[0181] The terms "cationic lipid" and "amino lipid" are used interchangeably herein and include lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino group (e.g., an alkylamino group or a dialkylamino group). Cationic lipids are typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and are substantially neutral at a pH above its pKa. The cationic lipid may be a titratable cationic lipid. In certain embodiments, the cationic lipid comprises a protonatable tertiary amino group (e.g., pH-titratable); a C18 alkyl chain (wherein each alkyl chain independently has 0-3 (e.g., 0, 1, 2, or 3) double bonds); and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0182] Cationic lipids include l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-i-linolenyloxy-N,N-dimethylaminopropane (g-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K)), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (DLin-K-DMA M-C2-DMA (also known as MC2)), (6Z,9Z,28Z,3l Z)-heptatriaconta-6,9,28,3l-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA (also known as MC3)), salts thereof, and mixtures thereof. Other cationic lipids also include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, y-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0183] Further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 2-dilinoleyl-3-acetoxypropane (DLin-DAC), and 2-dilinoleyl-3-acetoxypropane (DLin-DAC). Cypropane (DLin-MA), 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-Linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-Dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-Dioleyl-N,N-dimethylammonium chloride (DODAC), N-(l-(2,3-Dioleyloxy)propane) N-(1-propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyoxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propane ammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-l-(cis,cis-9,l2-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3-dimethyl-l-(cis These include, but are not limited to, cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS) and disubstituted spermine (D2S) or mixtures thereof.

[0184] Many commercially available preparations of cationic lipids can be used, such as LIPOFECTIN® (containing DOTMA and DOPE available from GIBCO / BRL) and LIPOFECT AMINE® (containing DOSPA and DOPE available from GIBCO / BRL).

[0185] In certain embodiments, the cationic lipid may be present in an amount from about 10% by weight of the LNP to about 85% by weight of the lipid nanoparticle, or from about 50% by weight of the LNP to about 75% by weight of the LNP.

[0186] Sterols can provide fluidity to the LNP. As used herein, "sterol" refers to any naturally occurring sterol of plant (phytosterol) or animal (zoosterol) origin, as well as non-naturally occurring synthetic sterols, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. The sterol can be any sterol conventionally used in the field of liposome, lipid vesicle or lipid particle preparations, most commonly cholesterol. Phytosterols can include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011 / 0177156, the disclosure of which is incorporated herein in its entirety. In certain embodiments, the sterol can be present in an amount of about 5% by weight of the LNP to about 50% by weight of the lipid nanoparticle, or about 10% by weight of the LNP to about 25% by weight of the LNP.

[0187] LNPs may include neutral lipids. Neutral lipids may include any lipid species that exists in either uncharged or neutral zwitterionic form at physiological pH. Such lipids include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside. The selection of neutral lipids is generally guided by, among other things, considerations of particle size and required stability. In certain embodiments, the neutral lipid component may be a lipid with two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0188] Lipids with various acyl chain groups of various chain lengths and degrees of saturation are available or can be isolated or synthesized by well-known techniques. In one embodiment, lipids containing saturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. In another group of embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths ranging from C14 to C22 are used. Additionally, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include, but are not limited to, l,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or any related phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups such as serine and inositol.

[0189] In certain embodiments, the neutral lipid may be present in an amount from about 0.1% by weight of the lipid nanoparticle to about 75% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.

[0190] The LNP-encapsulated nucleic acid, expression cassette, AAV vector, and non-viral vector can be incorporated into a pharmaceutical composition, such as a pharma- ceutically acceptable carrier or excipient. Such pharmaceutical compositions are particularly useful for the administration and delivery of the LNP-encapsulated nucleic acid, expression cassette, AAV vector, and non-viral vector to a subject in vivo or ex vivo.

[0191] Preparations of LNPs can be combined with additional components, non-limiting examples of which include polyethylene glycol (PEG) and sterols.

[0192] The term "PEG" refers to polyethylene glycol, a linear water-soluble polymer of ethylene PEG repeat units with two terminal hydroxyl groups. PEGs are classified by their molecular weight, e.g., PEG 2,000 has an average molecular weight of about 2,000 daltons, and PEG 5,000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies, and include, for example, PEGs of the following functional groups: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol succinate (MePEG-S), monomethoxypolyethyleneglycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycolamine (MePEG-NH2), monomethoxypolyethyleneglycol tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol imidazolylcarbonyl (MePEG-IM).

[0193] In some embodiments, the PEG may be a polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons, optionally substituted with alkyl, alkoxy, acyl, or aryl. In some embodiments, the PEG may be substituted with methyl at the terminal hydroxyl position. In some embodiments, the PEG may have an average molecular weight of about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or about 2,000 daltons, or about 750 daltons. The PEG may be substituted with alkyl, alkoxy, acyl, or aryl. In some embodiments, the terminal hydroxyl group may be substituted with a methoxy group or a methyl group.

[0194] PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397, the disclosures of which are incorporated herein in their entirety. Useful PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have various "anchor" lipid moieties for fixing PEG moieties to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide complexes (e.g., PEG-CerCl4 or PEG-CerC20), which are described in U.S. Patent No. 5,820,873, the disclosures of which are incorporated herein in their entirety (PEG-modified dialkylamines and PEG-modified l,2-diacyloxypropan-3-amines). In some embodiments, the PEG-modified lipids can be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, PEG can be in an amount between about 0.5% by weight of the LNP and about 20% by weight of the LNP, or between about 5% by weight of the LNP and about 15% by weight of the LNP.

[0195] Furthermore, LNP can be PEG-modified LNP and sterol-modified LNP. LNP can be the same or different LNP combined with additional components. In other words, the same LNP can be PEG-modified and sterol-modified, or the first LNP can be PEG-modified and the second LNP can be sterol-modified. Optionally, the first and second modified LNP can be combined.

[0196] In certain embodiments, the LNP prior to encapsulation may have a size ranging from about 10 to 500 nm, or from about 50 to about 200 nm, or from 75 to about 125 nm. In certain embodiments, the LNP-encapsulated nucleic acid, expression vector, AAV vector, or non-viral vector may have a size ranging from about 10 to 500 nm.

[0197] Polymer-Based Systems

[0198] Polymer-based delivery systems are well known in the art, and any suitable polymer-based delivery system or polymer nanoparticles known to those skilled in the art in light of the present disclosure can be used in the present invention.DNA can be entrapped in the polymer matrix of polymer nanoparticles, or adsorbed or conjugated onto the surface of nanoparticles.Examples of polymers commonly used for gene delivery include, for example, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI), chitosan, dendrimers, polyanhydrides, polycaprolactones, and polymethacrylates.

[0199] The polymeric non-viral vectors may have different sizes ranging from about 1 to about 1000 nm, optionally from about 10 to about 500 nm, optionally from about 50 to about 200 nm, optionally from about 100 to about 150 nm, optionally up to about 150 nm.

[0200] Protein-based systems Protein-based delivery systems are well known in the art, and any suitable protein-based delivery system or cell membrane-penetrating peptide (CPP) known to the skilled artisan in light of the present disclosure can be used in the present invention.

[0201] CPPs are short peptides (6-30 amino acid residues) that are potentially capable of intracellular penetration to deliver therapeutic molecules. The majority of CPPs consist mainly of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphipathic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., Tat, HIV-1 protein) or obtained by synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996-2006). Examples of CPPs include, for example, cationic CPPs (highly positively charged) (e.g., Tat peptide, penetratin, protamine, poly-L-lysine, polyarginine, etc.); amphipathic CPPs (chimeric or fusion peptides constructed from various sources that contain both positively and negatively charged amino acid sequences) (e.g., transportan, VT5, bactenecin-7 (Bac7), proline-rich peptides (PPRs), SAP (VRLPP)3, TP10, pep-1, MPG, etc.); membrane-philic CPPs (exhibiting both hydrophobic and amphipathic properties simultaneously, containing both large aromatic and small residues) (e.g., gH625, SPION-PEG-CPP NP, etc.); and hydrophobic CPPs (containing only non-polar motifs or residues) (e.g., SG3, PFVYLI, pep-7, fibroblast growth factor (FGF), etc.).

[0202] Proteinaceous non-viral vectors may have a variety of sizes ranging from about 1 to about 1000 nm, optionally from about 10 to about 500 nm, optionally from about 50 to about 200 nm, optionally from about 100 to about 150 nm, and optionally less than about 150 nm.

[0203] Peptide Cage System Peptide cage-based delivery systems are well known in the art, and any suitable peptide cage-based delivery system known to one of skill in the art in light of the present disclosure can be used in the present invention. In general, any proteinaceous material that can be assembled into a cage-like structure that forms a confined internal environment can be used. Several different types of protein "shells" can be assembled and loaded with different types of materials. For example, protein cages including shells of viral coat proteins (e.g., from Cowpea Chlorotic Mottle Virus (CCMV) protein coat) that encapsulate non-viral materials, as well as protein cages formed from non-viral proteins have been described (see, e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Application No. 20040028694, and U.S. Patent Application No. 20090035389, the disclosures of which are incorporated herein in their entirety). The peptide cage may comprise a proteinaceous shell that self-associates to form a protein cage (e.g., a structure with an internal cavity that is naturally accessible to solvent or can be made so by altering the solvent concentration, pH, equilibrium ratios).

[0204] Examples of protein cages derived from non-viral proteins include, for example, ferritins and apoferritins from both eukaryotic and prokaryotic species, e.g., 12- and 24-subunit ferritins; and protein cages formed from heat shock proteins (HSPs), e.g., the class of 24-subunit heat shock proteins that form the inner core space, the small HSP of Methanococcus jannaschii, the dodecameric Dsp HSP of Escherichia coli, the MrgA protein, etc. As will be appreciated by those skilled in the art, the monomers of the protein cages can be mutants (e.g., fragments) containing amino acid substitutions, insertions and deletions that occur naturally or can be synthesized.

[0205] The protein cages may have a variety of core sizes ranging from about 1 to about 1000 nm, optionally from about 10 to about 500 nm, optionally from about 50 to about 200 nm, optionally from about 100 to about 150 nm, optionally less than about 150 nm.

[0206] Pharmaceutical Compositions The present invention further provides pharmaceutical compositions comprising any of the polynucleotides comprising a nucleic acid encoding GLA, an expression cassette comprising a polynucleotide comprising a nucleic acid encoding GLA, a viral vector such as an AAV vector comprising a polynucleotide comprising a nucleic acid encoding GLA, or a non-viral vector comprising a polynucleotide comprising a nucleic acid encoding GLA as described herein.

[0207] rAAV vectors and non-viral vectors can be administered to patients via injection into biologically compatible carriers, for example, via intravenous injection.rAAV vectors and non-viral vectors can be administered alone or in combination with other molecules.Thus, rAAV vectors and non-viral vectors and other compositions, agents, drugs, biologics (proteins) can be incorporated into pharmaceutical compositions.Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo.

[0208] In certain embodiments, the pharmaceutical composition also comprises a pharma- ceutically acceptable carrier or excipient, including any pharmaceutical agent that does not itself induce a harmful immune response in the individual receiving the composition and that may be administered without undue toxicity.

[0209] As used herein, the terms "pharmaceutical acceptable" and "physiologically acceptable" refer to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutical acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, in administering nucleic acids, vectors, viral particles, or proteins to a subject.

[0210] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugar, and ethanol. Pharmaceutically acceptable salts may also be included therein, such as mineral acid salts, for example, hydrochloride, hydrobromide, phosphate, sulfate, and organic acid salts, for example, acetate, propionate, malonate, benzoate, and the like. Excipients also include proteins, such as albumin.

[0211] Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0212] Pharmaceutical compositions can be provided as salts, which can be formed with a variety of acids, including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base forms. In other cases, the preparation can be a lyophilized powder, which can contain any or all of the following: 1-50 mM histidine, 0.1-2% sucrose, and 2-7% mannitol, at a pH range of 4.5-5.5, which is combined with a buffer prior to use.

[0213] Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption enhancing or retarding agents that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharma-ceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) can also be incorporated into the composition.

[0214] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or known to those of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0215] Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of active compounds, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.Non-limiting examples include water, buffered saline, Hank's solution, Ringer's solution, dextrose, fructose, ethanol, animal oil, vegetable oil or synthetic oil.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

[0216] Moreover, the suspension of active compound can be prepared as a suitable oily injection suspension.Suitable lipophilic solvent or vehicle includes fatty oil such as sesame oil, or synthetic fatty acid ester such as ethyl oleate or triglyceride, or liposome.In some cases, suspension can contain suitable stabilizer or agent that increases the solubility of compound, so that highly concentrated solution can be prepared.

[0217] Cosolvents and adjuvants can be added to the formulation.Non-limiting examples of cosolvents include alcohols with hydroxyl groups or other polar groups, such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.Adjuvants include surfactants such as soybean lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0218] In certain embodiments, a pharmaceutical composition comprising any of the AAV vectors described herein further comprises an empty AAV capsid. In certain embodiments, in a pharmaceutical composition comprising an AAV vector and an empty AAV capsid, the ratio of empty AAV capsid to AAV vector is within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. In certain aspects, in a pharmaceutical composition comprising an AAV vector and an empty AAV capsid, the ratio of empty AAV capsid to AAV vector is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0219] In certain embodiments, the pharmaceutical composition comprises a surfactant.

[0220] After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment. Such labeling can include the amount, frequency, and method of administration.

[0221] Pharmaceutical compositions and delivery systems suitable for the compositions, methods and uses of the present invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, pp. 253-315).

[0222] An "effective amount" or a "sufficient amount" refers to an amount, in single or multiple doses, that alone or in combination with one or more other compositions (therapeutic agents such as drugs or immunosuppressants), treatments, protocols, or treatment regimens, provides a detectable response of any duration (long-term or short-term), any measurable or detectable degree of expected or desired result in the subject, or a benefit to the subject of any duration (e.g., minutes, hours, days, months, years, or being cured).

[0223] Compositions, such as pharmaceutical compositions, can be delivered to a subject to allow for production of the encoded protein. In certain embodiments, the pharmaceutical composition contains sufficient genetic material to allow the recipient to produce a therapeutically effective amount of the protein in the subject.

[0224] "Therapeutically effective amount" refers to the amount of active ingredient or ingredients that induces a desired biological or medical response in a subject. Therapeutically effective amount can be determined empirically and in a routine manner in relation to the stated purpose. For example, in vitro assays can be used as necessary to help identify optimal dosage ranges. The selection of a specific effective dose can be determined (e.g., through clinical trials) by those skilled in the art, taking into account several factors, including the disease to be treated or prevented, the associated symptoms, the patient's weight, the patient's immune status, and other factors known to those skilled in the art. The exact dosage to be used in the formulation also depends on the route of administration, and the severity of the disease, and should be determined according to the physician's judgment and each patient's circumstances. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

[0225] The compositions may be formulated and / or administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be formulated and / or administered to a patient alone or in combination with other agents that affect hemostasis (e.g., cofactors).

[0226] Treatment method The invention further provides a method of treating a subject in need of GLA, comprising administering to the subject a therapeutically effective amount of a nucleic acid, expression cassette, AAV vector, non-viral vector, or pharmaceutical composition of the invention, wherein GLA is expressed in the subject.

[0227] The methods and uses of the present invention include the delivery (transduction) of nucleic acid (transgene) to host cells, including dividing and / or non-dividing cells. The polynucleotides, expression cassettes, rAAV vectors, non-viral vectors, methods, uses and pharmaceutical preparations of the present invention are further useful in a method of delivering, administering or providing a protein encoded by a heterogeneous nucleic acid to a subject in need thereof as a method of treatment. In this way, the polynucleotide comprising the nucleic acid is transcribed and the protein is produced in vivo in the subject. The subject may benefit from or require the protein because they have a deficiency of the protein or because the production of the protein in the subject may provide a certain therapeutic effect as a treatment or other method.

[0228] The present invention is useful in humans and animals, including veterinary applications. Suitable subjects therefore include mammals, such as humans, as well as non-human mammals. The term "subject" refers to animals, typically mammals, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), livestock (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, pediatric, and adult subjects. Subjects include animal disease models, such as mouse and other animal models of protein / enzyme deficiencies, such as Fabry disease, and lysosomal storage diseases, as well as others known to those skilled in the art.

[0229] Suitable subjects for treatment according to the present invention include subjects who have insufficient amounts of GLA or are at risk of producing insufficient amounts of GLA, or subjects who produce abnormal, partially functional, or incapable GLA. Subjects can be tested for GLA activity to determine whether they are suitable for treatment according to the method of the present invention. Suitable subjects for treatment according to the present invention also include subjects who would benefit from GLA. Such subjects who could benefit from GLA include subjects with lysosomal storage diseases. Treated subjects can be monitored periodically after treatment, for example, every 1-4 weeks, every 1-6 months, every 6-12 months, or every 1, 2, 3, 4, 5, or more years.

[0230] The subject can be tested for immune response, for example, antibody against AAV. Thus, candidate subjects can be screened before treatment with the method of the present invention. The subject can also be tested for antibody against AAV after treatment, and optionally monitored for a period after treatment. Subjects with existing or developing AAV antibodies can be treated with immunosuppressants or other regimens described herein.

[0231] The subject suitable for treatment according to the present invention also includes the subject who has antibody against AAV or is at risk of producing antibody against AAV.rAAV vector can be administered or delivered to such subject using several techniques.For example, AAV empty capsid (i.e., AAV that lacks modified nucleic acid that codes for GLA) can be delivered to bind to AAV antibody in subject, thereby allowing the rAAV vector that contains heterogeneous nucleic acid to transduce the cells of the subject.

[0232] The modified nucleic acid, expression cassette, rAAV vector and non-viral vector of the present invention can be used to treat GLA deficiency.Therefore, in some embodiments, the modified nucleic acid encoding GLA, the expression cassette comprising the modified nucleic acid encoding GLA, rAAV vector and non-viral vector of the present invention can be used as a therapeutic agent and / or a preventive agent.

[0233] In one embodiment, the modified nucleic acid encoding GLA, the expression cassette comprising the modified nucleic acid encoding GLA, the rAAV vector, and the non-viral vector of the present invention can be used to treat Fabry disease. Administration of the modified nucleic acid encoding GLA, the expression cassette comprising the modified nucleic acid encoding GLA, the rAAV vector, and the non-viral vector of the present invention to a patient with Fabry disease or another lysosomal storage disease results in expression of the GLA protein.

[0234] In certain embodiments, methods according to the present invention may result in expression or activity of GLA at a level that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of GLA protein found in a subject not in need of GLA.

[0235] In certain embodiments, methods according to the present invention may result in expression or activity of GLA in the kidney at a level that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of GLA protein found in the kidney of a subject not requiring GLA.

[0236] In certain embodiments, methods according to the present invention may result in expression or activity of GLA in the heart at a level that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of GLA protein found in the heart of a subject not requiring GLA.

[0237] In certain embodiments, methods according to the present invention may result in expression or activity of GLA in the liver at a level that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of GLA protein found in the liver of a subject not requiring GLA.

[0238] In certain embodiments, methods according to the present invention may result in expression or activity of GLA in the bloodstream at a level that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of GLA protein found in the bloodstream of a subject not requiring GLA.

[0239] Subjects, animals, or patients administered the modified nucleic acids encoding GLA, expression cassettes comprising modified nucleic acids encoding GLA, rAAV vectors, and non-viral vectors of the present invention can be evaluated by various tests, assays, and functional evaluations to demonstrate, measure, and / or evaluate the efficacy of the modified nucleic acids encoding GLA, expression cassettes comprising modified nucleic acids encoding GLA, rAAV vectors, and non-viral vectors of the present invention as therapeutic and / or prophylactic agents.

[0240] Such tests and assays include measuring GLA activity (e.g., by using standard GLA activity assays) and / or GLA amount (e.g., by using Western blots with anti-GLA antibodies, or ELISA quantification) in biological samples such as blood, plasma, or urine (see, e.g., Christensen, E. et al., J Am Soc Nephrol. 2007 Mar;18(3):698-706); analysis of peak and steady-state vector-derived GLA enzyme levels assessed by total GLA protein and activity in plasma; analysis of GLA enzyme levels assessed by total GLA protein and activity in tissues by immunofluorescence and immunohistochemistry with cross-correction (see, e.g., Christensen, E. et al., 2007, Id.); measurement of GLA substrate accumulation of globotriaosylceramide (Gb3 or GL3) and globotriaosylsphingosine (lysoGb3 or lysoGL3) in tissues and serum (e.g., by lipid analysis by quantitative liquid chromatography tandem mass spectrometry or thin layer chromatography (see, e.g., Shu et al., 2007, Id.). al., J Biol Chem. 2007 Jul 20;282(29):20960-7, and Shu et al., J Glycomics Lipidomics. 2012;Suppl 2:1-6); measuring expression levels of Gb3 by immunofluorescence staining or electron microscopy (see, e.g., Braun et al., Cell Physiol Biochem. 2019;52(5):1139-1150); testing for GLA mRNA by quantitative reverse transcriptase PCR in AAV-transduced tissues; testing the immune response to the AAV capsid; and testing the immune response to the GLA transgene protein product.

[0241] Furthermore, the modified nucleic acid encoding GLA of the present invention, the expression cassette comprising the modified nucleic acid encoding GLA, the rAAV vector, and the non-viral vector can be used to treat lysosomal storage diseases. Lysosomal storage diseases include any disorder characterized by reduced or absent lysosomal enzyme activity. According to an embodiment, the modified nucleic acid encoding GLA of the present invention, the expression cassette comprising the modified nucleic acid encoding GLA, the rAAV vector, and the non-viral vector can be used to treat patients who need GLA. According to an embodiment, the modified nucleic acid encoding GLA of the present invention, the expression cassette comprising the modified nucleic acid encoding GLA, the rAAV vector, and the non-viral vector can be used to treat Fabry disease. According to an embodiment, the modified nucleic acid encoding GLA of the present invention, the expression cassette comprising the modified nucleic acid encoding GLA, the rAAV vector, and the non-viral vector can be used to reduce the level of sphingoglycolipids in the tissue of a subject.

[0242] As described herein, rAAV can penetrate cells and introduce nucleic acid / genetic material into cells, making it useful as a gene therapy vector.Since AAV is not associated with pathogenic disease in humans, rAAV vector can deliver heteropolynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial AAV pathogenesis or disease.

[0243] rAAV vectors have many desirable characteristics for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors has also shown no persistent toxicity, and immune responses are typically minimal or undetectable. AAV is known to infect a wide variety of cell types in vivo by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting many tissues, such as retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0244] For example, it may be desirable to introduce rAAV vectors that can provide multiple copies of GLA and therefore more GLA protein.Improved rAAV vectors and the methods for producing these vectors are described in detail in many references, patents and patent applications, including Wright JF (Hum. Gene Ther., 20:698-706, 2009).

[0245] Dosage may vary and depends on the type, onset, progression, severity, frequency, duration, or probability of the disease for which treatment is directed, the desired clinical endpoint, previous or contemporaneous treatment, the general health, age, sex, race, or immunological competence of the subject, and other factors understood by those skilled in the art.Dosage, number, frequency, or duration may be increased or decreased proportionately as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy, and the condition of the subject.Those skilled in the art will understand the factors that may affect the dosage and timing required to provide an amount sufficient to provide therapeutic or prophylactic benefit.

[0246] Dosages to achieve a therapeutic effect, such as the dosage of rAAV vector genome / kilogram of body weight (vg / kg), or the dosage of non-viral vectors, may vary based on several factors, including, but not limited to, the route of administration, the level of heteropolynucleotide expression required to achieve a therapeutic effect, the particular disease being treated, any host immune response to the viral vector, the host immune response to the heteropolynucleotide or expression product (protein), and the stability of the expressed protein. Those skilled in the art can determine the rAAV / vector genome or non-viral vector dosage range for treating a patient with a particular disease or disorder based on the aforementioned factors as well as other factors.

[0247] In general, the dosage of rAAV should be at least 1×10 per kilogram of subject body weight to achieve a therapeutic effect. 8vector genome (vg / kg), or more, e.g., 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 Or 1×10 14 , or more, vector genomes per kilogram of subject body weight (vg / kg).

[0248] For example, about 5 × 10 11 Recombinant AAV vg / kg or approximately 5 × 10 11 Recombinant AAV vg / kg dose greater than 1 x 10 12 Recombinant AAV vg / kg or approximately 1 × 10 12 Recombinant AAV vg / kg dose greater than 2 x 10 12 Recombinant AAV vg / kg or approximately 2 × 10 12 Recombinant AAV vg / kg dose greater than 3 x 10 12 Recombinant AAV vg / kg or approximately 3 × 10 12 Recombinant AAV vg / kg dose greater than 4 x 10 12 Recombinant AAV vg / kg or approximately 4 × 10 12 Recombinant AAV vg / kg dose greater than 5×10 12 Recombinant AAV vg / kg or approximately 5 × 10 12 Recombinant AAV vg / kg dose greater than 1 x 10 13 Recombinant AAV vg / kg or approximately 1 × 10 13 Recombinant AAV vg / kg dose greater than 2 x 10 13 Recombinant AAV vg / kg or approximately 2 × 10 13 Recombinant AAV vg / kg dose greater than 3 x 10 13 Recombinant AAV vg / kg or approximately 3 × 10 13 Recombinant AAV vg / kg dose greater than 4 x 10 13 Recombinant AAV vg / kg or approximately 4 × 10 13 Recombinant AAV vg / kg dose greater than 5×10 13 Recombinant AAV vg / kg or approximately 5 × 10 13Recombinant AAV vg / kg dose greater than 6×10 13 Recombinant AAV vg / kg or approximately 6 × 10 13 Doses greater than recombinant AAV vg / kg.

[0249] An exemplary dosage range of recombinant AAV vg / kg to be administered is about 5x10 11 ~about 6×10 13 Dose range of recombinant AAV vg / kg; approximately 5 x 10 11 ~Approx. 5.5×10 11 Dose range of recombinant AAV vg / kg; approximately 5.5 x 10 11 ~about 6×10 11 Dose range of recombinant AAV vg / kg; approximately 6 x 10 11 ~Approx. 6.5×10 11 Dose range of recombinant AAV vg / kg; approximately 6.5 x 10 11 ~Approx. 7×10 11 Dose range of recombinant AAV vg / kg; approximately 7 x 10 11 ~Approx. 7.5×10 11 Dose range of recombinant AAV vg / kg; approximately 7.5 x 10 11 ~Approx. 8×10 11 Dose range of recombinant AAV vg / kg; approximately 8 x 10 11 ~Approx. 8.5×10 11 Dose range of recombinant AAV vg / kg; approximately 8.5 x 10 11 ~Approx. 9×10 11 Dose range of recombinant AAV vg / kg; approximately 9 x 10 11 ~Approx. 9.5×10 11 Dose range of recombinant AAV vg / kg; approximately 9.5 x 10 11 ~Approx. 1×10 12 Dose range of recombinant AAV vg / kg; approximately 1 x 10 12 ~Approx. 1.5×10 12 Dose range of recombinant AAV vg / kg; approximately 1.5 x 10 12 ~about 2×10 12 Dose range of recombinant AAV vg / kg; approximately 2 x 10 12 ~Approx. 2.5×10 12 Dose range of recombinant AAV vg / kg; approximately 2.5 x 10 12 ~Approx. 3×1012 Dose range of recombinant AAV vg / kg; approximately 3 x 10 12 ~Approx. 3.5×10 12 Dose range of recombinant AAV vg / kg; approximately 3.5 x 10 12 ~Approx. 4×10 12 Dose range of recombinant AAV vg / kg; approximately 4 x 10 12 ~Approx. 4.5×10 12 Dose range of recombinant AAV vg / kg; approximately 4.5 x 10 12 ~Approx. 5×10 12 Dose range of recombinant AAV vg / kg; approximately 5 x 10 12 ~Approx. 5.5×10 12 Dose range of recombinant AAV vg / kg; approximately 5.5 x 10 12 ~about 6×10 12 Dose range of recombinant AAV vg / kg; approximately 6 x 10 12 ~Approx. 6.5×10 12 Dose range of recombinant AAV vg / kg; approximately 6.5 x 10 12 ~Approx. 7×10 12 Dose range of recombinant AAV vg / kg; approximately 7 x 10 12 ~Approx. 7.5×10 12 Dose range of recombinant AAV vg / kg; approximately 7.5 x 10 12 ~Approx. 8×10 12 Dose range of recombinant AAV vg / kg; approximately 8 x 10 12 ~Approx. 8.5×10 12 Dose range of recombinant AAV vg / kg; approximately 8.5 x 10 12 ~Approx. 9×10 12 Dose range of recombinant AAV vg / kg; approximately 9 x 10 12 ~Approx. 9.5×10 12 Dose range of recombinant AAV vg / kg; approximately 9.5 x 10 12 ~Approx. 1×10 13 Recombinant AAV vg / kg Dose range: approximately 1 x 10 13 ~Approx. 1.5×10 13 Dose range of recombinant AAV vg / kg; approximately 1.5 x 10 13 ~about 2×10 13 Dose range of recombinant AAV vg / kg; approximately 2 x 10 13 ~Approx. 2.5×10 13Dose range of recombinant AAV vg / kg; approximately 2.5 x 10 13 ~Approx. 3×10 13 Dose range of recombinant AAV vg / kg; approximately 3 x 10 13 ~Approx. 3.5×10 13 Dose range of recombinant AAV vg / kg; approximately 3.5 x 10 13 ~Approx. 4×10 13 Dose range of recombinant AAV vg / kg; approximately 4 x 10 13 ~Approx. 4.5×10 13 Dose range of recombinant AAV vg / kg; approximately 4.5 x 10 13 ~Approx. 5×10 13 Dose range of recombinant AAV vg / kg; approximately 5 x 10 13 ~Approx. 5.5×10 13 Dose range of recombinant AAV vg / kg; approximately 5.5 x 10 13 ~Approx. 6×10 13 Dose range of recombinant AAV vg / kg; approximately 6 x 10 13 ~Approx. 1×10 14 Dose ranges of recombinant AAV vg / kg.

[0250] In one embodiment, the AAV vg / kg is about 5×10 11 vg / kg, approximately 6 × 10 11 vg / kg, approximately 7 × 10 11 vg / kg, approximately 8 × 10 11 vg / kg, approximately 9 × 10 11 vg / kg, approximately 1 × 10 12 vg / kg, approximately 2 × 10 12 vg / kg, approximately 3 × 10 12 vg / kg, approximately 4 × 10 12 vg / kg, approximately 5 × 10 12 vg / kg, approximately 6 × 10 12 vg / kg, approximately 7 × 10 12 vg / kg, approximately 8 × 10 12 vg / kg, approximately 9 × 10 12vg / kg, approximately 1 × 10 13 vg / kg, approximately 2 × 10 13 vg / kg, approximately 3 × 10 13 vg / kg, approximately 4 × 10 13 vg / kg, approximately 5 × 10 13 vg / kg, approximately 6 × 10 13 It is administered at a dose of vg / kg.

[0251] As used herein, a "unit dosage form" refers to a physically discrete unit suitable as a unitary dose for a subject to be treated; when administered in one or more doses, each unit contains a predetermined amount, optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filler), calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect). Unit dosage forms can be present, for example, in ampoules and vials, which can contain liquid compositions, or compositions in lyophilized or lyophilized form; for example, a sterile liquid carrier can be added prior to administration or in vivo delivery. Individual unit dosage forms can be included in multi-dose kits or containers. rAAV particles, non-viral vectors, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0252] An "effective amount" or "sufficient amount" administered for treatment (e.g., to improve or provide a therapeutic benefit or improvement) is typically effective to provide a measurable response to one, more than one, or all adverse symptoms, consequences, or complications of a disease, e.g., one or more adverse symptoms, disorders, diseases, conditions, or complications caused by or associated with a disease, while a satisfactory result would be to reduce, decrease, inhibit, suppress, limit, or control the progression or worsening of the disease.

[0253] In one embodiment, a method according to the invention reduces, reduces or inhibits one or more symptoms of GLA need or Fabry disease; or prevents or reduces the progression or worsening of one or more symptoms of GLA need or Fabry disease; or stabilizes one or more symptoms of GLA need or Fabry disease; or ameliorates one or more symptoms of GLA need or Fabry disease.

[0254] An effective or sufficient amount does not have to be provided in a single dose, but can be multiple doses, and can be administered alone or in combination with another composition (e.g., drug), treatment, protocol or treatment regimen, but these are not required.For example, the amount can be increased proportionately as indicated by the needs of the subject, the type, condition and severity of the disease being treated, or the side effects of treatment (if any).In addition, an effective or sufficient amount does not have to be effective or sufficient when given in a single or multiple doses without a second composition (e.g., another drug or drug), because additional doses, amounts or periods beyond such doses, or additional compositions (e.g., drugs or drugs), treatments, protocols or treatment regimens may be included to be considered effective or sufficient in a given subject.An amount considered to be effective also includes an amount that results in a reduction in the use of another treatment, treatment regimen or protocol, such as the administration of a modified nucleic acid encoding GLA for the treatment of GLA deficiency (e.g., Fabry disease) or another lysosomal storage disease that can be treated with GLA.

[0255] Therefore, the method and use of the present invention also include, among others, the method and use that reduces the need or use of another compound, drug, medicine, treatment regimen, treatment protocol, process or therapy.For example, for GLA deficiency, if a given subject needs less frequent or reduced dosage or elimination of recombinant GLA administration to make up for the deficiency or missing GLA in the subject, the method or use of the present invention has therapeutic benefit.Therefore, according to the present invention, the method and use are provided that reduce the need or use of another treatment or treatment.

[0256] Effective amount or sufficient amount does not have to be effective in each and every subject that is treated, nor does it have to be effective in the majority of subjects that are treated in a given group or population.Effective amount or sufficient amount means effectiveness or sufficiency in a particular subject, not in a group or general population.As is typical for such methods, there will be subjects that show greater response or little or no response to a given treatment method or use.

[0257] Administration or in vivo delivery to a subject may be performed prior to the occurrence of adverse symptoms, conditions, complications, etc., caused by or associated with a disease. For example, screening (e.g., genetic) may be used to identify such subjects as candidates for the compositions, methods and uses of the present invention. Thus, such subjects include those that are screened positive for insufficient amounts or defects of functional gene products (e.g., GLA, or protein defects that lead to lysosomal storage diseases that can be treated with GLA), or that produce abnormal, partially functional or non-functional gene products (e.g., GLA, or proteins involved in lysosomal storage diseases that can be treated with GLA).

[0258] Administration to a subject or in vivo delivery according to the methods and uses of the invention as disclosed herein may be performed within 1-2 hours, 2-4 hours, 4-12 hours, 12-24 hours, or 24-72 hours after the subject has been identified as having a disease targeted for treatment or having one or more symptoms of the disease, or after the subject has been screened and identified as positive as described herein even though the subject does not have one or more symptoms of the disease. Of course, the methods and uses of the invention may be performed 1-7 days, 7-14 days, 14-24 days, 24-48 days, 48-64 days, or more days, months, or years after the subject has been identified as having a disease targeted for treatment or having one or more symptoms of the disease, or after the subject has been screened and identified as positive as described herein.

[0259] The term "ameliorate" refers to a detectable or measurable improvement in a subject's disease or symptoms thereof, or an underlying cellular response. Detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limitation or control in the occurrence, frequency, severity, progression or duration of a disease, or a complication caused by or associated with a disease, or an improvement in the symptoms or underlying causes or consequences of a disease, or reversal of a disease.

[0260] For Fabry disease, an effective amount is an amount that improves markers of Fabry disease, such as globotriaosylsphingosine (lyso-GB3) and those disclosed in U.S. Patent Application Publication No. 2010-0113517, the disclosure of which is incorporated herein in its entirety. Non-limiting examples of improvements in surrogate markers of Fabry disease disclosed in U.S. Patent Application Publication No. 2010 / 0113517 include increased levels or activity of α-Gal A in cells (e.g., fibroblasts) and tissues; decreased GL-3 accumulation; decreased plasma concentrations of homocysteine ​​and vascular cell adhesion molecule-1 (VCAM-1); decreased GL-3 accumulation in cardiomyocytes and valvular fibrocytes; reduced cardiac hypertrophy (particularly the left ventricle), amelioration of valvular insufficiency, and arrhythmias; amelioration of proteinuria; decreased urinary concentrations of lipids such as CTH, lactosylceramide, ceramide, and increased urinary concentrations of glucosylceramide and sphingomyelin; absence of laminated inclusions (zebra bodies) in glomerular epithelial cells; improved renal function; reduced hyperhidrosis; absence of angiokeratoma; and improvement in hearing abnormalities such as high-frequency sensorineural hearing loss, progressive hearing loss, sudden hearing loss, or tinnitus. Improvement of neurological symptoms includes prevention of transient ischemic attacks (TIA) or stroke; and improvement of neuropathic pain manifested as acroparesthesia (burning or tingling in the extremities). Another type of clinical marker that can be assessed for Fabry disease is the prevalence of adverse cardiovascular symptoms. Common cardiac-related signs and symptoms of Fabry disease include left ventricular hypertrophy, valvular disease (especially mitral valve prolapse and / or mitral regurgitation), premature coronary artery disease, angina, myocardial infarction, conduction abnormalities, arrhythmias, and congestive heart failure.

[0261] The therapeutic dose depends on, among other factors, the age and general condition of the subject, the severity of the disease or disorder. Thus, the therapeutically effective amount in humans falls within a relatively broad range that can be determined by a physician based on the response of each individual patient.

[0262] In further embodiments, the effective amount administered to a human subject is an increase in plasma GLA to the range of greater than 1 ng / ml, greater than 2 ng / ml, greater than 3 ng / ml, greater than 4 ng / ml, about 1 ng / ml, about 2 ng / ml, about 2.5 ng / ml, about 3 ng / ml, or about 3.5 ng / ml; greater than 1 nmol / h / mL, greater than 1.5 nmol / h / mL, greater than 2 nmol / h / mL, greater than 2.5 nmol / h / mL, greater than 3 nmol / h / mL, greater than 4 nmol / h / mL, greater than 5 nmol / h / mL, greater than 6 nmol / h / mL, greater than 7 nmol / h / mL, about 1 nmol / h / mL, about 1.5 nmol / h / mL, about 2 nmol / h / mL, about 2.5 nmol / h / mL, about 3 nmol / h / mL, about 4 nmol / h / mL, about 5 nmol / h / mL, about 6 nmol / h / mL, or about 7 nmol / h / mL. and / or a decrease in plasma lyso-Gb3 to less than 40 nmol / L, less than 30 nmol / L, less than 10 nmol / L, less than 5 nmol / L, or less than 2 nmol / L. Plasma GLA, plasma GLA activity, and plasma lyso-Gb3 can be measured using standard techniques, such as those provided or referenced in Tsukimura et al., Molecular Genetics and Metabolism Reports 1 (2014) 288-298.

[0263] The methods and uses of the present invention include delivery and administration systemically, locally or locally, or by any route, such as injection or infusion. Delivery of pharmaceutical compositions in vivo can generally be achieved by injection using a conventional syringe, but other delivery methods such as convection-enhanced delivery are also envisioned (see, e.g., U.S. Pat. No. 5,720,720, the disclosure of which is incorporated herein in its entirety). For example, the compositions can be delivered subcutaneously, intraepidermally, intradermally, intrathecally, intraorbitally, intramucosally, intranasally, intraperitoneally, intravenously, intrapleurally, intraarterially, intracavity, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application. Clinicians who specialize in the treatment of patients with Fabry disease or other lysosomal storage diseases can determine the optimal route for administration of AAV vectors and non-viral vectors based on a number of criteria, including but not limited to the patient's condition and the goals of treatment.

[0264] The composition may be administered alone. In some embodiments, the rAAV particles or non-viral vectors provide a therapeutic effect without an immunosuppressant. The therapeutic effect may be sustained, for example, for 2-4 days, 4-6 days, 6-8 days, 8-10 days, 10-14 days, 14-20 days, 20-25 days, 25-30 days, or 30-50 days or more, for example, 50-75 days, 75-100 days, 100-150 days, 150-200 days or more, in some cases, without the administration of an immunosuppressant. Thus, the therapeutic effect is provided for a period of time.

[0265] The rAAV vectors, non-viral vectors, methods, and uses of the present invention can be combined with any compound, agent, drug, treatment, or other treatment regimen or protocol that has a desired therapeutic, beneficial, additive, synergistic, or complementary activity or effect. Exemplary combination compositions and treatments include a second active ingredient, such as a biologic (protein), a drug (e.g., an immunosuppressant), and a drug. Such biologics (proteins), agents, drugs, treatments, and therapies can be administered or performed before, substantially simultaneously, or after any other method or use of the present invention.

[0266] The compound, agent, drug, treatment or other treatment regimen or protocol can be administered as a combination composition, or can be administered separately, such as simultaneously, or sequentially or consecutively (before or after) the delivery or administration of the nucleic acid, expression cassette, rAAV particle, or non-viral vector. Thus, the present invention provides a combination in which the method or use of the present invention is combined with any compound, agent, drug, treatment regimen, treatment protocol, process, therapy, or composition described herein or known to those skilled in the art. The compound, agent, drug, treatment regimen, treatment protocol, process, therapy, or composition can be administered or performed before, substantially simultaneously, or after the administration of the nucleic acid, expression cassette, non-viral vector, or rAAV particle of the present invention to a subject.

[0267] In certain embodiments, a nucleic acid, expression vector, non-viral vector, or rAAV particle of the invention is administered to a patient in combination with an immunosuppressant agent or regimen in which the patient has or is at risk of having an immune response to the rAAV particle and / or GLA protein. Such an immunosuppressant agent or regimen can be administered prior to, substantially simultaneously with, or after administration of the nucleic acid, expression cassette, non-viral vector, or rAAV vector of the invention.

[0268] In some embodiments, a subject or patient, such as a human patient with Fabry disease, has developed inhibitors against GLA protein (including anti-GLA antibodies and / or anti-GLA T cells), which may occur after treatment with conventional enzyme replacement therapy (e.g., after administration of recombinantly produced GLA protein). Such development of GLA inhibitors may occur in patients undergoing enzyme replacement therapy, particularly when the patient has undetectable GLA levels (as may be the case in pediatric Fabry disease), leading the patient's immune system to consider it "foreign". In some embodiments, a Fabry patient with GLA inhibitors is administered one or more regimens aimed at achieving immune tolerance or mitigating the immune response to GLA protein in the patient before, substantially simultaneously with, or after administration of the rAAV vector or non-viral vector of the invention. Such regimens to achieve immune tolerance or mitigate the immune response to the GLA protein may include administration of one or more immunosuppressive agents, including, but not limited to, methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, and synthetic vaccine particle (SVP™)-rapamycin (rapamycin encapsulated in a biodegradable nanoparticle), and / or administration of one or more immunosuppressive protocols or procedures, such as B cell depletion, immunoadsorption, and plasma exchange.

[0269] In some embodiments, the rAAV vector or non-viral vector is administered in conjunction with one or more immunosuppressive agents prior to, substantially simultaneously with, or after administration of the rAAV vector or non-viral vector, hi some embodiments, the one or more immunosuppressive agents are administered, e.g., 1-12 hours, 12-24 hours, or 24-48 hours, or 2-4 days, 4-6 days, 6-8 days, 8-10 days, 10-14 days, 14-20 days, 20-25 days, 25-30 days, 30-50 days, or longer, after administration of the rAAV vector or non-viral vector. Such administration of an immunosuppressant after a period of time after administration of the rAAV vector or non-viral vector can occur if there is a decrease in the encoded protein or inhibitor nucleic acid some time after the initial expression level, e.g., 20-25 days, 25-30 days, 30-50 days, 50-75 days, 75-100 days, 100-150 days, 150-200 days or more than 200 days after administration of the rAAV vector or non-viral vector.

[0270] In certain embodiments, the immunosuppressant is an anti-inflammatory agent.

[0271] In some embodiments, the immunosuppressant is a steroid, e.g., a corticosteroid. In some embodiments, the immunosuppressant is prednisone, prednisolone, a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g., CellCept®, Myfortic®), a CD52 inhibitor (e.g., alemtuzumab), CTLA4-Ig (abatacept, belatacept), an anti-CD3 monoclonal antibody, an anti-LFA-1 monoclonal antibody (e.g., efalizumab), an anti-CD40 monoclonal antibody (e.g., epratuzumab), an anti-CD20 monoclonal antibody (e.g., rituximab, olelizumab, ofatumumab, veltuzumab), a proteasome inhibitor (e.g., bortezomib), a TACI-Ig (e.g., atacicept), an anti-C5 mAb (e.g., eculizumab), mycophenolic acid, azathioprine, sirolimus everolimus, TNFR-Ig, anti-TNF monoclonal antibodies, tofacitinib, anti-IL-2R (e.g., basiliximab), anti-IL-17 monoclonal antibodies (e.g., secukinumab), anti-IL-6 monoclonal antibodies (e.g., anti-IL-6 antibody sirukumab, anti-IL-6 receptor antibody tocilizumab (Actemra®)), IL-10 inhibitors, TGFβ inhibitors, B cell targeting antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particles (SVP™)-rapamycin (biodegradable nanoparticle-encapsulated rapamycin), intravenous gamma globulin (IVIG), omalizumab, methotrexate, tyrosine kinase inhibitors administration of one or more immunosuppressive protocols or procedures, such as B-cell depletion, immunoadsorption, and plasma exchange, or combinations and / or derivatives thereof, and / or administration of one or more immunosuppressive protocols or procedures, such as B-cell depletion, immunoadsorption, and plasma exchange, or administration of other immunosuppressive agents ...

[0272] Immunosuppressive protocols, including the use of rapamycin, can be used alone or in combination with IL-10 to reduce, reduce, inhibit, prevent or block humoral and cellular immune responses to GLA protein. Liver gene transfer using the AAV vector of the present invention can be used to induce immune tolerance to GLA protein through the induction of regulatory T cells (Treg) and other mechanisms. Strategies to reduce (overcome) or avoid humoral immunity to AAV in systemic gene transfer include administering high vector doses, using AAV empty capsids as decoys to adsorb anti-AAV antibodies, administering immunosuppressants to reduce, reduce, inhibit, prevent or eradicate humoral immune responses to AAV, changing the AAV capsid serotype or modifying the AAV capsid to be less susceptible to neutralizing antibodies, using plasma exchange cycles to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers, and using delivery techniques such as balloon catheters followed by saline lavage. Such a strategy is described in Mingozzi et al., 2013, Blood, 122:23-36. Further strategies include using an AAV-specific plasmapheresis column to selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma, as described in Bertin et al., 2020, Sci. Rep. 10:864. See https: / / doi.org / 10.1038 / s41598-020-57893-z.

[0273] The ratio of AAV empty capsid to rAAV vector can be, for example, within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. The ratio can also be about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0274] The amount of AAV empty capsid administered can be adjusted based on the amount (titer) of AAV antibodies produced in a particular subject. The AAV empty capsid can be of any serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, LK03 (SEQ ID NO:42) AAV-2i8, sequence of SEQ ID NO:110, sequence of SEQ ID NO:36, and / or sequence of SEQ ID NO:37.

[0275] Alternatively, or in addition, rAAV vectors or non-viral vectors can be delivered by direct intramuscular injection (e.g., one or more slow-twitch fibers of muscle). In another alternative, a catheter introduced into the femoral artery can be used to deliver rAAV vectors or non-viral vectors to the liver via the hepatic artery. Non-surgical means, such as endoscopic retrograde cholangiopancreatography (ERCP), can also be performed to deliver rAAV vectors or non-viral vectors directly to the liver, thereby bypassing the bloodstream and AAV antibodies. Other ductal systems, such as the ducts of the submandibular gland, can also be used as portal veins to deliver rAAV vectors or non-viral vectors to subjects who develop or have pre-existing anti-AAV antibodies.

[0276] Further strategies to reduce humoral immunity to AAV include methods to remove, deplete, capture, and / or inactivate AAV antibodies, commonly referred to as apheresis, and more specifically plasmapheresis involving blood products. Apheresis or plasmapheresis is a process in which a human subject's plasma is circulated ex vivo (outside the body) through a device that modifies the plasma by adding, removing, and / or replacing components before returning to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). This procedure depletes, captures, inactivates, reduces, or removes immunoglobulins (antibodies) that bind to AAV, thereby reducing the titer of AAV antibodies in the treated subject that may contribute to AAV vector neutralization. One example is a device composed of an AAV capsid affinity matrix column. Passing a blood product (eg, plasma) through an AAV capsid affinity matrix will result in binding of only AAV antibodies, and all isotypes (including IgG, IgM, etc.).

[0277] A sufficient amount of plasmapheresis using an AAV capsid affinity matrix is ​​expected to substantially remove AAV capsid antibodies and reduce AAV capsid antibody titers (load) in humans. In an embodiment, the titers in treated subjects are substantially reduced to low levels (<1:5 or less, e.g., <1:4, or <1:3, or <1:2, or <1:1). The reduction in antibody titers is expected to be temporary, since B lymphocytes producing AAV capsid antibodies are expected to gradually restore AAV capsid antibody titers to the steady-state levels prior to plasmapheresis.

[0278] When pre-existing AAV antibody titers are reduced from 1:100 to 1:1, AAV antibody titer rebounds of about 0.15% (corresponding to a titer of 1:1.2), 0.43% (1:1.4), 0.9% (1:1.9), 1.7% (1:2.7), and 3.4% (1:4.4) occur 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours, respectively, after completion of the plasmapheresis procedure. Such temporary clearance of AAV antibodies from a subject corresponds to a time window (e.g., about 24 hours or less, e.g., 12 hours or less, or 6 hours or less, or 3 hours or less, or 2 hours or less, or 1 hour or less) during which an AAV vector may be administered to a subject and be expected to efficiently transduce a target tissue without substantial neutralization of the AAV vector by AAV antibodies.

[0279] When pre-existing AAV antibody titers are reduced from 1:1000 to 1:1, approximately 0.15% (corresponding to a titer of 1:2.5), 0.4% (1:5.3), 0.9% (1:9.7), 1.7% (1:18), and 3.4% (1:35) of AAV antibody titer rebound occurs 1 h, 3 h, 6 h, 12 h, and 24 h, respectively, after completion of the plasmapheresis procedure, thus providing a relatively short window for administration of AAV vectors.

[0280] AAV antibodies can be pre-existing and can be present at a level that reduces or blocks the therapeutic GLA gene transfer vector transduction of target cells.Alternatively, AAV antibodies can be developed after exposure to AAV or administration of AAV vector.When such antibodies develop after administration of AAV vector, these subjects can also be treated through apheresis, more specifically, plasmapheresis.

[0281] In some embodiments, the polynucleotides, expression cassettes, AAV vectors, and non-viral vectors of the present invention may be used in combination with methods for reducing antibody (e.g., IgG) levels in human plasma. In some embodiments, the polynucleotides, expression cassettes, AAV vectors, and non-viral vectors of the present invention may be used in combination with agents that block, inhibit, or reduce the interaction of IgG with neonatal Fc receptor (FcRn), such as anti-FcRn antibodies, to reduce IgG recycling, enhance IgG clearance in vivo, and / or to reduce circulating antibodies that bind to viral vectors, such as recombinant viral vectors, or bind to nucleic acids or polypeptides, proteins, or peptides encoded by therapeutic heteropolynucleotides packaged by recombinant viral vectors, or bind to therapeutic heteropolynucleotides.

[0282] In certain embodiments, antibody binding to the viral vector is reduced or inhibited by agents that reduce the interaction of IgG with FcRn, proteases, or glycosidases.

[0283] In certain embodiments, the polypeptides, expression cassettes, AAV vectors, or non-viral vectors of the invention may be used in combination with an endopeptidase (e.g., IdeS from Streptococcus pyogenes) or modified variants thereof, or an endoglycosidase (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof. In certain embodiments, the polypeptides, expression cassettes, AAV vectors, or non-viral vectors of the invention are administered to a subject in combination with an endopeptidase (e.g., IdeS from Streptococcus pyogenes) or modified variants thereof, or an endoglycosidase (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof to reduce or eliminate neutralizing antibodies against the AAV capsid, allowing the treatment of patients already considered ineligible for gene therapy or who develop AAV antibodies following AAV gene therapy. Such a strategy is described in Leborgne et al., 2020, Nat. Med., 26:1096-1101 (2020).

[0284] In certain embodiments, the nucleic acids, expression cassettes, AAV vectors, and non-viral vectors of the invention can be used in combination with, for example, bronchodilators; hearing aids; topical skin moisturizers; typical cardiac therapies such as diuretics, ACE inhibitors, cardiac devices, etc.; therapeutic agents for pain relief or renal protection; stroke prevention with antithrombotic and antiarrhythmic therapies; antiproteinuric agents in case of end-stage renal failure, renal dialysis and / or kidney transplantation; and metoclopramide, H2 blockers, and dietary therapy to ensure proper nutrition and gastrointestinal symptom management (see, e.g., Germain, Orphanet J Rare Dis. 2010 Nov 22;5:30; Ortiz et al., Mol Genet Metab. 2018 Apr;123(4):416-427; and Mehta et al., QJM: Inter. Jour. Med. 2010 Sept;103(9):641-659).

[0285] In certain embodiments, the polynucleotides, expression cassettes and AAV vectors of the invention can be used in combination with pharmaceutical chaperone therapy (also known as enzyme enhancement therapy), wherein one or more pharmaceutical chaperones can be administered prior to, simultaneously with, or following administration of the polynucleotides, expression cassettes, AAV vectors, or non-viral vectors of the invention for the treatment of lysosomal storage diseases, such as Fabry disease.

[0286] In certain embodiments, the polynucleotides, expression cassettes, AAV vectors, and non-viral vectors of the present invention may be used in combination with one or more pharmaceutical chaperones capable of stabilizing the GLA protein. Pharmaceutical chaperones that can be used in combination with the polynucleotides, expression cassettes and AAV vectors of the invention include, for example, 1-deoxygalactonojirimycin (DGJ), Migalastat hydrochloride, alpha-3,4-di-epihomonojirimycin, 4-epihomonojirimycin, 4-epifagomine, alpha allohomonojirimycin, N-methyldeoxygalactonojirimycin, beta-1-C-butyldeoxygalactonojirimycin, alpha galactohomonojirimycin, calystegine A3, calystegine B2, N-methylcalystegine B2 and N-methylcalystegine B3, and others described in U.S. Pat. Nos. 6,274,597, 6,774,135, and 6,599,919, the disclosures of which are incorporated herein in their entireties.

[0287] In some embodiments, the polynucleotides and expression cassettes of the invention are delivered or administered via AAV vector particles. In some embodiments, the polynucleotides and expression cassettes of the invention may be delivered or administered via other types of viral particles, including retroviruses, adenoviruses, helper-dependent adenoviruses, hybrid adenoviruses, herpes simplex viruses, lentiviruses, poxviruses, Epstein-Barr viruses, vaccinia viruses, and human cytomegalovirus particles. In some embodiments, the polynucleotides and expression cassettes of the invention may be delivered or administered via non-viral vectors.

[0288] kit The present invention provides a kit having packaging material and one or more components therein.The kit typically includes a label or package insert that includes a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein.The kit can include a collection of such components, such as rAAV particles or non-viral vectors, and optionally a second active ingredient, such as another compound, agent, drug, or composition.

[0289] A kit refers to the physical structure that contains one or more components of the kit.

[0290] The packaging material is capable of maintaining the components sterile and may be made from materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0291] The label or insert may include identification information of one or more components therein, dosage, mechanism of action, clinical pharmacology of the active ingredients, including pharmacokinetics and pharmacodynamics. The label or insert may include information identifying manufacturer information, lot number, manufacturing location and date, expiration date. The label or insert may include information regarding diseases for which the kit components may be used. The label or insert may include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or treatment regimen. The instructions may include dosage, use or duration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0292] The label or insert may include information regarding any benefit that the component may provide, such as a prophylactic or therapeutic benefit. The label or insert may include information regarding potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician regarding situations in which it is not appropriate to use a particular composition. Adverse side effects or complications may also occur if the subject has, may take, or is taking one or more other drugs that may be incompatible with the composition, or if the subject has, may take, or is taking another treatment protocol or treatment regimen that may be incompatible with the composition, and thus the instructions may include information regarding such incompatibilities.

[0293] Labels or inserts include "printed matter", such as "printed matter" separately or attached to paper or cardboard, or to a component, kit or packaging material (e.g., a box), or attached to an ampoule, tube or vial containing a kit component. Labels or inserts may also include computer readable media, such as a printed label with a bar code, optical disks, such as a CD- or DVD ROM / RAM, DVD, MP3, magnetic tape, or electronic storage media, such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, FLASH media, or memory type cards.

[0294] [Table 1] JPEG2024504611000002.jpg209170JPEG2024504611000003.jpg218170JPEG2024504611000004.jpg21 7170JPEG2024504611000005.jpg214170JPEG2024504611000006.jpg215170JPEG2024504611000007.j pg219170JPEG2024504611000008.jpg221170JPEG2024504611000009.jpg221170JPEG20245046110000 10.jpg214170JPEG2024504611000011.jpg220170JPEG2024504611000012.jpg221170JPEG2024504611 000013.jpg216170JPEG2024504611000014.jpg217170JPEG2024504611000015.jpg213170JPEG202450 4611000016.jpg222170JPEG2024504611000017.jpg221170JPEG2024504611000018.jpg222170JPEG20 24504611000019.jpg220170JPEG2024504611000020.jpg218170JPEG2024504611000021.jpg222170JP EG2024504611000022.jpg219170JPEG2024504611000023.jpg209170JPEG2024504611000024.jpg69170

[0295] In Table 1, the stop codons of SEQ ID NOs: 15 to 19 are shown in bold.

[0296] Further Aspects and Embodiments A first set of further aspects and embodiments are provided below:

[0297] 1. A polynucleotide comprising a nucleic acid encoding alpha-galactosidase A (GLA), wherein the nucleic acid is selected from the group consisting of: (1) a polynucleotide having at least 75% sequence identity to the sequence of SEQ ID NO:15; (2) a polynucleotide having at least 84% sequence identity to the sequence of SEQ ID NO:16; (3) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO:17; (4) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO:18; and (5) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO:19, and optionally, wherein GLA comprises the amino acid sequence of SEQ ID NO:100.

[0298] 2. The polynucleotide of 1., wherein the nucleic acid comprises fewer than 14 CpG dinucleotides, and optionally comprises 0 CpG dinucleotides.

[0299] 3. The polynucleotide according to 1. or 2, wherein the nucleic acid has any one of the sequences set forth in SEQ ID NOs: 15 to 19.

[0300] 4. A polynucleotide comprising a nucleic acid encoding an alpha-galactosidase A (GLA) protein, wherein the GLA protein has the amino acid sequence of SEQ ID NO:100 with one or more amino acid substitutions selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn, and optionally, wherein GLA comprises the amino acid sequence of SEQ ID NO:48.

[0301] 5. The polynucleotide according to 4., wherein the nucleic acid has the sequence of SEQ ID NO:47.

[0302] 6. A polynucleotide according to any one of 1. to 5., further comprising an intron located within the nucleic acid encoding GLA.

[0303] 7. The polynucleotide according to 6., wherein an intron is located between nucleotides 78 and 79 of the nucleic acid encoding GLA, the nucleotide positions being given with respect to the coding sequence for GLA having the sequence of SEQ ID NO:14.

[0304] 8. The polynucleotide according to 7., wherein the intron is selected from the group consisting of introns from the vitronectin 1 (VTN1) gene, the retinol binding protein 4 (RBP4) gene, the mouse IgG heavy chain A (IgHA) gene, and the mouse IgG heavy chain μ (IgHμ) gene, and optionally, the intron comprises one of the sequences set forth in SEQ ID NOs: 49-52.

[0305] 9. The polynucleotide according to any one of 6. to 8., wherein the nucleic acid has a sequence as set forth in any one of SEQ ID NOs: 43 to 46.

[0306] 10. The polynucleotide according to any one of 1. to 9., further comprising a second nucleic acid encoding a signal peptide sequence located at the 5' end of the nucleic acid encoding GLA.

[0307] 11. The polynucleotide according to 10, wherein the signal peptide sequence is a heterologous or endogenous or native signal peptide sequence.

[0308] 12. The polynucleotide according to 10. or 11., wherein the signal peptide is selected from the group consisting of human chymotrypsinogen B2 signal peptide, AHSG signal peptide, CD300 signal peptide, LAMP1 signal peptide, Notch 2 signal peptide, ORM1 signal peptide, TF signal peptide, and wild-type GLA signal peptide, or mutants thereof.

[0309] 13. The polynucleotide according to 12, wherein the signal peptide is human chymotrypsinogen B2 signal peptide.

[0310] 14. The polynucleotide according to 13, wherein the nucleic acid encoding the signal peptide has any one of the sequences set forth in SEQ ID NOs: 1 to 5.

[0311] 15. An expression cassette comprising the polynucleotide according to any one of 1. to 14., operably linked to an expression control element.

[0312] 16. The expression cassette according to 15, wherein the expression control element is a liver-specific expression control element.

[0313] 17. The expression cassette according to 15. or 16., wherein the expression control element is located 5' to the nucleic acid encoding GLA.

[0314] 18. An expression cassette described in any one of 15. to 17., further comprising a polyadenylation sequence located 3' of the nucleic acid encoding GLA.

[0315] 19. An expression cassette described in any one of 15. to 18., wherein the expression control element or polyadenylation sequence has reduced CpGs compared to the wild-type expression control element or polyadenylation sequence.

[0316] 20. An expression cassette described in any one of 15. to 19., wherein the expression control element comprises an ApoE / hAAT enhancer / promoter sequence.

[0317] 21. An expression cassette described in any of 15. to 20., wherein the polyadenylation sequence includes the bovine growth hormone (bGH) polyadenylation sequence.

[0318] 22. An expression cassette according to claim 20 or 21, wherein the ApoE / hAAT enhancer / promoter sequence or the bGH polyadenylation sequence has reduced CpGs compared to the wild-type ApoE / hAAT enhancer / promoter sequence or the bGH polyadenylation sequence.

[0319] 23. The expression cassette described in 20., wherein the ApoE / hAAT enhancer / promoter sequence comprises the sequence of SEQ ID NO:38.

[0320] 24. The expression cassette according to claim 21, wherein the bGH polyadenylation sequence comprises the sequence of SEQ ID NO:20.

[0321] 25. An expression cassette according to any one of 15. to 24., further comprising an intron located between the 3' end of the expression control element and the 5' end of the nucleic acid.

[0322] 26. The expression cassette according to 25., wherein the intron comprises the hBB2m1 intron.

[0323] 27. The expression cassette described in 26., wherein the intron sequence comprises the sequence of SEQ ID NO:39.

[0324] 28. An adeno-associated virus (AAV) vector comprising a polynucleotide or expression cassette according to any one of 1. to 27.

[0325] 29. The AAV vector described in 28., wherein the AAV vector comprises (a) one or more AAV capsids, and (b) one or more AAV inverted terminal repeats (ITRs), wherein the AAV ITRs flank the 5' or 3' end of the nucleic acid or expression cassette.

[0326] 30. The AAV vector described in 29., wherein at least one or more ITRs are modified to have reduced CpGs.

[0327] 31. The AAV vector is an altered, modified or mutated AAV having 90% or more sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO: 35), AAV3B, LK03 (SEQ ID NO: 42), AAV-2i8, SEQ ID NO: 110, SEQ ID NO: 36, and / or SEQ ID NO: 37. VP1, VP2 and / or VP3 capsids; or a capsid having 95% or greater sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35, AAV3B, LK03 (SEQ ID NO:42), AAV-2i8, SEQ ID NO:110, SEQ ID NO:36, and / or SEQ ID NO:37; or an AAV 1. The AAV vector according to any one of 28. to 30., having a capsid serotype including a capsid having 100% sequence identity to AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO: 35), AAV3B, LK03 (SEQ ID NO: 42), AAV-2i8, SEQ ID NO: 110, SEQ ID NO: 36, and / or SEQ ID NO: 37.

[0328] 32. An AAV vector described in any of 28. to 31., wherein the ITRs include one or more ITRs of any of the AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, and AAV3B, or a combination thereof.

[0329] 33. An AAV vector comprising a polynucleotide sequence of any one of SEQ ID NOs: 21 to 34 and 53 to 56.

[0330] 34. A pharmaceutical composition comprising multiple AAV vectors according to any of 28. to 33. in a biologically compatible carrier or excipient.

[0331] 35. The pharmaceutical composition described in 34., further comprising an empty AAV capsid.

[0332] 36. The pharmaceutical composition according to claim 35, wherein the ratio of empty AAV capsid to AAV vector is about 100:1 to about 50:1, about 50:1 to about 25:1, about 25:1 to about 10:1, about 10:1 to about 1:1, about 1:1 to about 1:10, about 1:10 to about 1:25, about 1:25 to about 1:50, or about 1:50 to about 1:100.

[0333] 37. The pharmaceutical composition described in any one of 34. to 36., further comprising a surfactant.

[0334] 38. A method for treating a subject in need of alpha-galactosidase A (GLA), comprising administering to the subject a therapeutically effective amount of a polynucleotide or expression cassette described in any of 1. to 24., or an AAV vector described in any of 25. to 30., or a pharmaceutical composition described in any of 31. to 34., wherein GLA is expressed in the subject.

[0335] 39. The method according to claim 38, wherein the subject has Fabry disease.

[0336] 40. The method according to 38. or 39., wherein the polynucleotide, expression cassette, AAV vector, or pharmaceutical composition is administered to the subject intravenously, intraarterially, intracavitarily, intramucosally, or via a catheter.

[0337] 41. The AAV vector delivers approximately 1 x 10 8 ~Approx. 1×10 14 The method according to any one of 38. to 40., wherein the vector is administered to the subject in the range of vector genome (vg / kg).

[0338] 42. A method according to any of 38. to 41., wherein the method reduces, reduces or inhibits the need for GLA or one or more symptoms of Fabry disease; or prevents or reduces the progression or worsening of the need for GLA or one or more symptoms of Fabry disease; or stabilizes the need for GLA or one or more symptoms of Fabry disease; or ameliorates the need for GLA or one or more symptoms of Fabry disease.

[0339] 43. A cell comprising a polynucleotide or expression cassette according to any one of 1. to 27.

[0340] 44. A cell that produces an AAV vector according to any one of 28. to 33.

[0341] 45. A method for producing an AAV vector described in any of 28. to 33., comprising: (A) introducing an AAV vector genome containing any of the polynucleotides or expression cassettes of 1. to 22. into a packaging helper cell; and (b) culturing the helper cell under conditions for producing the AAV vector.

[0342] A second set of further aspects and embodiments include:

[0343] Embodiment 1 is directed to a polynucleotide comprising a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding alpha-galactosidase A (GLA), wherein the nucleic acid sequence has at least about 85% sequence identity to the sequence of SEQ ID NO:15, and GLA has at least 95% sequence identity to the sequence of SEQ ID NO:100; (b) a nucleic acid sequence encoding alpha-galactosidase A (GLA) containing an inserted intron, wherein GLA containing the inserted intron has at least 95% sequence identity to the sequence of SEQ ID NO: 100 in the absence of the inserted intron; (c) a nucleic acid sequence encoding a precursor α-galactosidase A (GLA) comprising a signal peptide linked to the amino terminus of the precursor α-galactosidase A, wherein the signal peptide has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63; and GLA has at least 95% sequence identity to the sequence of SEQ ID NO:100; (d) A nucleic acid encoding α-galactosidase A (GLA) having an amino acid sequence that differs from SEQ ID NO: 100 by 1 to 7 amino acids, wherein at least one of the 1 to 7 amino acids is a substitution selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0344] Reference to "precursor" α-galactosidase A indicates the presence of a signal peptide. The signal peptide may be the naturally occurring peptide associated with GLA, or a different or heterologous signal peptide.

[0345] In different embodiments of aspect 1(a), the nucleic acid has at least 90%, at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO: 15, 16, 17, or 18, or at least 90%, at least 95%, at least 98%, or 100% sequence identity to bases 1 to 1194 of the sequence of SEQ ID NO: 15, 16, 17, or 18 (bases 1 to 1194 provide the amino acid coding region); independently, GLA has at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO: 100. When used independently, it is indicated that any of the provided sequence identities to the sequence of SEQ ID NO: 5 can be combined with any of the provided sequence identities of the sequence of SEQ ID NO: 100. For example, a nucleic acid having at least 90% sequence identity to the sequence of SEQ ID NO:15 may encode a GLA having at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:100; a nucleic acid having at least 95% sequence identity to the sequence of SEQ ID NO:15 may encode a GLA having at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:100; a nucleic acid of the sequence of SEQ ID NO:15 may encode a GLA having at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:100.

[0346] In different embodiments of aspect 1(b), the sequence identity of GLA to the sequence of SEQ ID NO:100 is at least 98% or 100% in the absence of an inserted intron.

[0347] In different embodiments of aspect 1(c), the signal peptide has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:41; has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:57; has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:58; has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:59; has at least 85% to the sequence of SEQ ID NO:60. , at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO: 61; at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO: 62; or at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO: 63; and with respect to GLA, each independently, at least 95%, at least 98% or 100% sequence identity to the sequence of SEQ ID NO: 100. Each independently indicates that each of the provided signal sequences along with the sequence identification can be combined with GLA having any of the provided sequence identities to the sequence of SEQ ID NO: 100.For example, a signal peptide having at least 85% sequence identity to the sequence of SEQ ID NO:41 may be combined with a GLA having at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:100; a signal peptide having at least 90% sequence identity to the sequence of SEQ ID NO:41 may be combined with a GLA having at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:100; a signal peptide having at least 95% sequence identity to the sequence of SEQ ID NO:41 may be combined with a GLA having at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:100; and a signal peptide of the sequence of SEQ ID NO:41 may be combined with a GLA having at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:100.

[0348] In a further embodiment of aspect 1(c), the precursor α-galactosidase A including a signal peptide has an amino acid sequence at least 95%, 97%, 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs:101, 102, 103, 104, 105, 106, 107, 108 and 109; and the precursor α-galactosidase A including a signal peptide has an amino acid sequence at least 95%, 97%, 99% or 100% identity to SEQ ID NO:109.

[0349] In a different embodiment of aspect 1(d), the nucleic acid encoding GLA differs from the sequence of SEQ ID NO:100 by one to seven amino acid substitutions, each of which is selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn, and GLA comprises the amino acid sequence of SEQ ID NO:48.

[0350] Embodiment 1 further describes aspect 1(d) and a different embodiment of aspect 1(d) by providing a nucleic acid that comprises at least 90%, at least 95%, at least 98% sequence identity to SEQ ID NO:47, or bases 1 to 1194 of SEQ ID NO:47, or is provided by the sequence of SEQ ID NO:47 or bases 1 to 1194 of SEQ ID NO:47.

[0351] When describing further aspects or embodiments, it is provided that the further explanation applies to each of the explanations provided in the referenced aspects or embodiments. For example, embodiment 1, which further describes aspect 1(d) and different embodiments of aspect 1(d), provides that the different embodiments of embodiment 1 can be independently applied to any explanations provided in different embodiments of aspect 1(d) and aspect 1(d).

[0352] Embodiment 2 further describes aspect 1(b) by providing that the GLA provided in the absence of an intron comprises any of aspect 1(a) and a different embodiment of aspect 1(a), aspect 1(b) and a different embodiment of aspect 1(b), aspect 1(d) and a different embodiment of aspect 1(d), or a GLA sequence as provided in embodiment 1, wherein the intron is located between nucleotides 78 and 79 of GLA, the nucleotide positions being given with reference to the coding sequence of GLA in SEQ ID NO:14.

[0353] Embodiment 3 further describes embodiment 1(d) and a different embodiment of embodiment 1(d), as well as embodiment 2, by providing that the intron has at least 90%, at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:49, 50, 51, or 52.

[0354] Embodiment 4 further describes aspect 1(d) by providing that the intron-containing GLA has at least 90%, at least 95%, at least 98% or 100% sequence identity to the sequence of SEQ ID NO:43, 44, 45 or 46.

[0355] Embodiment 5 further describes embodiment 1(a) and a different embodiment of embodiment 1(a), embodiment 1(b) and a different embodiment of embodiment 1(b), embodiment 1(d) and a different embodiment of embodiment 1(d), and embodiments 1, 2, 3, and 4 by providing a polynucleotide, wherein the second sequence encodes a signal peptide sequence located at the 5' end of the GLA nucleic acid sequence. The signal peptide sequence can be a heterologous, endogenous or naturally occurring signal peptide sequence, or a derivative thereof.

[0356]

[0023] Embodiment 6 further describes embodiment 5 by providing in different embodiments, wherein the peptide signal has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:41; has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:57; has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:58; has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:59. having at least 95% or 100% sequence identity; having at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:60; having at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:61; having at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:62; or having at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:63.

[0357] Embodiment 7 further describes aspect 1(a) and different embodiments of aspect 1(a), aspect (1b) and different embodiments of aspect 1(b), aspect 1(c) and different embodiments of aspect 1(c), aspect 1(d) and different embodiments of aspect 1(d), and a GLA coding sequence as provided in any of embodiments 1-6, wherein the GLA coding sequence comprises fewer than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 CpG dinucleotides.

[0358] Aspect 2 is directed to an expression cassette comprising a polynucleotide as provided in aspect 1(a) and a different embodiment of aspect 1(a), aspect 1(b) and a different embodiment of aspect 1(b), aspect 1(c) and a different embodiment of aspect 1(c), aspect 1(d) and a different embodiment of aspect 1(d), and a different embodiment of embodiments 1 to 7, wherein the polynucleotide is operably linked to an expression control element.

[0359] Embodiment 8 further describes embodiment 2, wherein the description of the polypeptide is as provided in embodiment 2 and the different embodiments of embodiment 2, and wherein the expression control element is a liver-specific expression control element, and wherein the expression control element comprises an ApoE / hAAT enhancer / promoter sequence, or comprises a sequence of SEQ ID NO:38 or a sequence having at least 98% sequence identity to SEQ ID NO:38.

[0360] References to a description in a referenced aspect or embodiment provide incorporation of the referenced aspect (including related embodiments) and the referenced embodiment (including different described embodiments). For example, references to a description of a polypeptide provided in aspect 2, in different embodiments, references are made to aspect 1(a) and different embodiments of aspect 1(a); aspect 1(b) and different embodiments of aspect 1(b); aspect 1(c) and different embodiments of aspect 1(c); aspect 1(d) and different embodiments of aspect 1(d), and polynucleotides provided in embodiments 1-7.

[0361] Embodiment 9 further describes the expression control element of embodiment 8 by providing that the expression control is located 5' to the polynucleotide.

[0362] Embodiment 10 further describes the expression cassettes of embodiment 2, embodiment 8 and embodiment 9, wherein the expression cassette further comprises a polyadenylation sequence located 3' of the polynucleotide. In a further embodiment, the polyadenylation sequence comprises a bovine growth hormone (bGH) polyadenylation sequence, comprising a sequence having at least 95% or 100% sequence identity to SEQ ID NO:20.

[0363]

[0036] Embodiment 11 further describes the expression cassette of aspect 2 and embodiments 9-10, wherein an intron is located between the 3' end of the expression control element and the 5' end of the polynucleotide. In a further embodiment, the intron comprises a sequence having at least 95% or 100% sequence identity to the sequence of SEQ ID NO:39.

[0364] Embodiment 12 further describes the expression cassette of aspect 2 and embodiments 9-11, wherein the expression control elements and / or polyadenylation sequences have reduced CpGs compared to wild-type expression control elements or polyadenylation sequences.

[0365] Embodiment 13 further describes the polypeptides of aspect 1 (including 1(a), 1(b), 1(c) and 1(d) and related embodiments) and embodiments 1-8, and the expression vectors of aspect 2 and embodiments 9-12, wherein the polypeptide or expression cassette further comprises an AAV inverted repeat (ITR) adjacent to its 5' end and / or an AAV ITR adjacent to its 3' end. Preferably, the 3' and 5' ends are adjacent to the ITR.

[0366] Aspect 3 is directed to an AAV plasmid genome comprising the polynucleotide or expression cassette of embodiment 13, wherein an origin of replication is present. In a further embodiment, a selectable marker is present.

[0367] Aspect 4 is directed to an adeno-associated virus (AAV) vector comprising a capsid and a polynucleotide or expression cassette as provided in embodiment 13. Reference to an AAV capsid includes naturally occurring AAV capsids as well as modified and mutated AAV capsids. The AAV capsid facilitates intracellular delivery of the polynucleotide or expression cassette, preferably the expression cassette.

[0368]

[0033] Embodiment 14 further describes the AAV vector of aspect 4, and the polypeptide and expression cassette of embodiment 13, wherein the ITRs comprise one or more ITRs of any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV3B serotypes, or combinations thereof.

[0369] Embodiment 15 further describes aspect 4, embodiment 13, and embodiment 14, in which, in different embodiments, the one adjacent to the 5' ITR at the 5' end is a 5' cloning remnant and / or the one adjacent to the 3' ITR at the 3' end is a 3' cloning remnant.

[0370] Embodiment 16 further describes aspect 4 and embodiments 13-15, wherein the 5' ITR and / or the 3' ITR are modified to have reduced CpGs.

[0371] Embodiment 17 further describes the AAV vector of aspect 4 and embodiments 13-16, wherein the expression cassette comprises a sequence having at least 95%, at least 98%, or 100% sequence identity to any of SEQ ID NOs:21-34, 53-56, and 91-99.

[0372] Embodiment 18 further describes the AAV vector of aspect 4 and embodiments 13-17, wherein the expression cassette comprises, or consists of, a sequence having at least 95%, at least 98%, or 100% sequence identity to any of SEQ ID NOs:91-95.

[0373] Embodiment 19 is a method for preparing a VP1, VP2 and / or VP3 vector comprising the steps of: a) preparing a VP1, VP2 and / or VP3 vector having at least 90%, at least 95%, at least 98%, or 100% sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, LK03 (SEQ ID NO:42) or AAV-2i8; or b) preparing a VP1, VP2 and / or VP3 vector having at least 90%, at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:110 or 42. or a VP1 having at least 90%, at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:110 or SEQ ID NO:42, and / or a VP2 having at least 90%, at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:36, and / or a VP3 having at least 90%, at least 95%, at least 98%, or 100% sequence identity to the sequence of SEQ ID NO:37.

[0374] Aspect 5 is directed to a pharmaceutical composition comprising an AAV vector as provided in aspect 4 and embodiments 13-19, and a biologically compatible carrier or excipient.

[0375] Embodiment 20 further describes the pharmaceutical composition of aspect 5, wherein the AAV vector is provided in an amount effective to increase GLA activity, preferably decrease globotriaosylsphingosine, in a human subject.

[0376] Embodiment 21 further describes the pharmaceutical composition of aspect 5, further comprising an empty AAV capsid. Reference to an empty AAV capsid refers to the same capsid used in the administered AAV vector, but the capsid lacks an AAV vector. In further aspects, the ratio of empty AAV capsid to AAV vector is about 100:1 to 1:100; about 100:1 to about 50:1; about 50:1 to about 25:1; about 25:1 to about 10:1; about 10:1 to about 1:1; about 1:1 to about 1:10; about 1:10 to about 1:25; about 1:25 to about 1:50; or about 1:50 to about 1:100.

[0377] Embodiment 22 further describes the pharmaceutical composition of aspect 5 and embodiments 20 and 21, wherein the composition further comprises a surfactant.

[0378] Aspect 6 is directed to a polypeptide selected from the group consisting of: (a) a nucleic acid sequence encoding a precursor α-galactosidase A comprising a signal peptide linked to the amino terminus of α-galactosidase A, wherein the signal peptide has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63; and said GLA has at least 95% sequence identity to the sequence of SEQ ID NO:100; and (b) α-galactosidase A (GLA) having an amino acid sequence that differs by 1 to 7 amino acids from the sequence of SEQ ID NO: 100, wherein at least one of the 1 to 7 amino acids is a substitution selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn.

[0379] In different embodiments of aspect 6(a), the signal peptide has at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:41; at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:57; at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:58; at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:60. having at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:61; having at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:62; or having at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO:63; each independently having at least 95%, at least 98%, or 100% sequence identity with respect to GLA to the sequence of SEQ ID NO:100.

[0380] In a different embodiment of aspect 6(b), GLA has an amino acid sequence that differs from SEQ ID NO:100 by 1 to 7 amino acid substitutions, wherein the 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions are each independently selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn; and GLA comprises the amino acid sequence of SEQ ID NO:48.

[0381] Aspect 7 is directed to a method of treating a subject in need of GLA, comprising administering to the subject a therapeutically effective amount of a polynucleotide, an expression cassette, an AAV vector, a pharmaceutical composition, or a polypeptide, or any of the aspects and embodiments described above in the second set of further aspects and embodiments.Preferably, the subject is a human.

[0382] Embodiment 23 further describes aspect 7, wherein the subject has Fabry disease; the method reduces, reduces or inhibits the need for GLA or one or more symptoms of Fabry disease; the method prevents or reduces the progression or worsening of the need for GLA or one or more symptoms of Fabry disease; the method stabilizes the need for GLA or one or more symptoms of Fabry disease; or the method improves one or more symptoms of the need for GLA or Fabry disease.

[0383] In embodiment 24, the AAV vector is administered in an amount of about 1×10 per kilogram of the subject's body weight. 8 ~Approx. 1×10 14 Embodiment 7 and embodiment 23 are further described in which the vector is administered to the subject in the range of vector genome (vg / kg).

[0384] Aspect 8 is directed to (i) a method according to aspect 7, embodiment 22 and embodiment 23; or (ii) a polynucleotide, an expression cassette, an AAV vector, a pharmaceutical composition, or a polypeptide in a second set of further aspects and embodiments, or any of the above aspects and embodiments, for use in the preparation of a medicament.

[0385] All of the features disclosed herein can be combined in any combination.Each feature disclosed herein can be replaced by an alternative feature that performs the same, equivalent, or similar purpose.Thus, unless otherwise specified, the features disclosed (e.g., modified nucleic acid encoding GLA, expression cassette comprising modified nucleic acid encoding GLA, rAAV particle comprising modified nucleic acid encoding GLA, and non-viral vector comprising modified nucleic acid encoding GLA) are examples of the same or similar feature type.

[0386] Several embodiments of the present invention have been described. Nevertheless, those skilled in the art can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Thus, the following examples are intended to illustrate, but in no way limit the scope of the claimed invention. EXAMPLES

[0387] Example 1 - Overview of GLA Expression Cassette The GLA expression cassette was designed as shown in Figure 1 and Table 2. Table 2 references the sequences of the precursor α-galactosidase A (signal peptide + GLA portion) and the expression cassette. All sequences provided in Table 2 included the 5'- and 3'-flanking AAV inverted terminal repeats (ITRs), the liver-specific ApoE / hAAT enhancer / promoter sequence, the human hemoglobin subunit beta (HBB2) intron, the signal peptide, the human GLA coding sequence, and the bovine growth hormone (bGH) polyadenylation (polyA) sequence.

[0388] [Table 2] JPEG2024504611000026.jpg108170

[0389] The expression cassette is packaged into an AAV viral particle by encapsidation into an AAV capsid, such as the AAV-4-1 capsid mutant described in International Patent Application Publication WO 2016 / 210170, the contents of which are incorporated herein in their entirety, or the LK03 capsid mutant described in U.S. Patent No. 9,169,299, the contents of which are incorporated herein in their entirety. Viral particles are generally produced using triple transfection protocols well known in the art.

[0390] Example 2 - Evaluation of signal peptides To evaluate the efficacy of GLA expression cassettes with various signal peptides in place of the native GLA signal peptide, expression cassettes with sequences of SEQ ID NO:21 (sp7.GLA), SEQ ID NO:23 (spCD300.GLA), SEQ ID NO:24 (spGLA.GLA), SEQ ID NO:26 (spNotch2.GLA), SEQ ID NO:27 (spORM1.GLA) and SEQ ID NO:29 (spTF.GLA) were packaged into AAV vectors containing capsids of SEQ ID NO:110, 36 and 37. Five to six male or female C57Bl / 6 mice per group were injected with 1.25 × 10 10 vg / mouse or 5×10 10 rAAV was injected intravenously via the tail vein at 1000 mg / mouse. An in vitro enzyme activity assay was used to measure the levels of circulating GLA enzyme activity in mouse serum. A standard curve was generated from serial dilutions of fluorescent 4-methylumbelliferyl (4-MU). GLA enzyme activity was expressed as the concentration of fluorescent 4-MU released per hour of co-incubation of serum and the synthetic enzyme substrate 4-methylumbelliferyl β-D-galactopyranoside (4-MU-Gal), expressed in nmol × mL. -1 ×hr -1 The activity of GLA enzyme was defined in units of 0.01 mg / mL. For all measurements of GLA enzyme activity, incubations of individual tissue samples with GLA and 4-MU-Gal were performed in duplicate. As shown in Figure 2A, the expression cassette containing the signal peptide sp7 from chymotrypsin B2 resulted in higher levels of serum GLA activity compared to the expression cassette containing the wild-type GLA signal peptide.

[0391] To evaluate the human GLA protein expression levels of the GLA cassettes relative to the endogenous levels of mouse GLA in serum, expression cassettes having the sequence of SEQ ID NO:21 (sp7.GLA) and the sequence of SEQ ID NO:24 (spGLA.GLA) were packaged into AAV vectors containing capsids of SEQ ID NO:110, 36, and 37, respectively. Five female C57Bl / 6 mice per group were injected with 5x10 10rAAV was injected intravenously via the tail vein at 1000 mg / mouse. Serum GLA protein was measured using capillary electrophoresis. Levels of circulating GLA in mouse serum collected 6 weeks after transduction were quantified against a standard curve of recombinant human GLA (ProteinSimple WesTM, Bio-Techne) and plotted in units of μg / mL (Figure 2B). GLA protein levels were also measured in untreated control C57Bl / 6 mice. Bar heights represent the mean serum GLA activity of five mice per group, error bars indicate one standard deviation from the mean, and the limit of quantification of the standard curve is indicated by a horizontal line (lower limit of quantification, indicated as "LOD" in the figure). Levels of GLA protein were significantly higher in mice treated with rAAV (AAVsp7.GLA or AAVspGLA.GLA) than in untreated mice (t test, *** p<0.001). Both rAAV vectors expressing GLA with its native signal peptide (GLA) and rAAV vectors expressing GLA with the chymotrypsinogen B2 signal peptide (SP7) induced serum GLA expression above baseline endogenous levels of mouse GLA in C57Bl / 6 mice (Figure 2B).

[0392] Example 3 - Evaluation of GLA cassettes in the Fabry mouse model The efficacy of the expression cassettes described herein was evaluated using a male GLA- / null knockout mouse model, which has a mixed B6;129 background rather than a C57Bl / 6 background.

[0393] 5×10 containing an expression cassette having the sequence of SEQ ID NO:21 (sp7.GLA) 10rAAV (SEQ ID NOs: 110, 36 and 37 capsids) (referred to herein as AAV-sp7-GLA) was injected intravenously via the tail vein into 10 C57Bl / 6, 11 B6;129-GLA+ / null and 11 B6;129-GLA- / null male mice. GLA activity in mouse serum collected 3, 4 and 6 weeks after rAAV administration was expressed as the concentration of fluorescent 4-MU released per hour of co-incubation of serum with the synthetic substrate 4-MU-Gal, expressed in nmol x mL -1 ×hr -1 The AAV-sp7-GLA rAAV showed higher potency in C57Bl / 6 mice compared to B6;129 mice. Serum GLA activity after transduction was lower in both B6;129-GLA+ / null and B6;129- / null mice, and therefore the lower potency was not a specific result of the GLA genotype or Fabry phenotype. As a result, to achieve serum GLA activity above 1000 nmol / mL-hr in Fabry model mice (B6;129 GLA null line), 5 × 10 9 vg / mouse to 5 × 10 11 A dose escalation study was performed to investigate GLA expression at a range of AAV doses up to 1000 ng / mouse.

[0394] Example 4 - Dose-finding study 5×10 9 vg / mouse, 8.9×10 9 vg / mouse, 1.6×10 10 vg / mouse, 2.8×10 10 vg / mouse, 5×10 10 vg / mouse, 1.58×10 11 vg / mouse, or 5×10 11vg / mouse of AAV-sp7-GLA was injected intravenously into B6;129 GLA- / null male mice (n=5 per group). Dose escalation was performed in two separate studies, Study 1 and Study 2. GLA activity in mouse serum collected 4 weeks after transduction was expressed as the concentration of fluorescent 4-MU released per hour of coincubation of serum with the synthetic substrate 4-MU-Gal, expressed in nmol × mL -1 ×hr -1 The serum GLA activity was measured in units of 100 μg / mL and plotted in Figures 4A and 4B. Bar heights represent the mean serum GLA activity of five mice per group, and error bars indicate one standard deviation from the mean. The maximum level of background serum GLA activity detected by the assay at week 4 in five untransduced control GLA- / null mice is shown as a horizontal line in Figure 4A. As shown, GLA expression showed a linear dose response to the amount of AAV administered (Figure 4B).

[0395] Example 5 - Renal uptake of GLA Tissue uptake of GLA, particularly in the kidney, is thought to be essential for the clinical efficacy of Fabry therapeutics. 9 vg / mouse, 8.9×10 9 vg / mouse, 1.6×10 10 vg / mouse, 2.8×10 10 vg / mouse, or 5×10 10 vg / mouse of AAV-sp7-GLA was injected intravenously via the tail vein into groups of five B6;129 GLA- / null male mice. Livers and kidneys were harvested 12 weeks after transduction and nmol × mg total lysate protein was determined. -1 ×hr -1GLA activity in tissue lysates was measured as the concentration of fluorescent 4-MU released per hour of co-incubation of tissue lysate and the synthetic substrate 4-MU-Gal, in units of 1000 and plotted in Figure 5A (liver) and Figure 5B (kidney). GLA activity was also assessed in tissue lysates of liver and kidney in five untreated B6;129 GLA- / null male control mice and four age-matched untreated B6;129 GLA+ / null (WT) male mice. Bar heights represent mean tissue GLA activity, error bars indicate one standard deviation from the mean, and horizontal lines indicate the limit of quantification of the calibration curve normalized to the protein concentration of the respective tissue lysate. As shown, increasing doses of AAV led to increased GLA activity observed in the liver and kidney of knockout male mice. Furthermore, GLA activity in the liver and kidney of GLA knockout animals was increased by 2.8 × 10 10 Doses of 100 vg / mouse or higher restored concentrations observed in normal animals.

[0396] In particular, 5×10 10 A vg / mouse dose of an AAV-sp7-GLA vector containing a liver-specific transgene promoter achieved GLA activity in the kidneys of Fabry mice approaching concentrations of GLA enzyme activity associated with wild-type mice (B6;129GLA+ / null, WT).

[0397] Example 6 - Evaluation of the GLA coding sequence The GLA portion of the expression cassette, having the sequence of SEQ ID NO:21 (sp7.GLA), was codon-optimized to remove CpG motifs and support maximal expression. Codon-optimized GLA mutant cassettes (SEQ ID NO:91 (GLAco4), SEQ ID NO:92 (GLAcoBC0), SEQ ID NO:93 (GLAcoH0), SEQ ID NO:94 (GLAcoH6), and SEQ ID NO:95 (GLAv45)) were encapsidated in AAV capsids and transfected into male C57Bl / 6 mice, five per group, at 5.0x10 10vg / mouse. Serum GLA activity was measured at week 4 as a proxy for relative transgene activity and plotted in FIG. 6. Bar heights represent mean tissue GLA activity and error bars indicate one standard deviation from the mean. As shown, CpG-free or CpG-reduced codon-optimized variants showed serum GLA activity comparable to that of the cassette having the sequence of SEQ ID NO:21.

[0398] Example 7 - Further optimization of the GLA expression cassette Further modifications were made to the GLA portion of the expression cassette provided in the sequence of SEQ ID NO:21 (sp7.GLA). Structurally guided mutagenesis was used to generate a GLA mutant (Q57K; Q111E; K213E; K237Q; F248T; G334E; G346N) ("GLA 7 mutant") containing seven amino acid substitutions (SEQ ID NO:47 (SPKL0031)). Additionally, a heterologous intron was introduced into the sp7-GLA coding sequence between nucleotides 78 / 79 of the GLA coding sequence of SEQ ID NO:14, resulting in SEQ ID NO:96 (IgHA, SEQ ID NO:97 (IgHμ), SEQ ID NO:98 (RBP4), and SEQ ID NO:99 (VTN1)). The expression cassette SEQ ID NO:95 (referred to as sp7-GLA-var45 in FIG. 7) was also included. All cassette sequences were encapsidated into AAV capsids and expressed at 2×10 9 vg / mouse with AAV-CAG-Gaussia (an AAV-encapsidated expression cassette carrying Gaussia luciferase under the control of the CAG promoter to normalize transduction efficiency) 10 Five male C57Bl / 6 mice per group were transduced at a dose of 10000000000000000 vg / mouse. As a proxy for relative transgene activity, serum GLA activity at 6 weeks was measured and plotted in Figure 7. Bar heights represent mean tissue GLA activity and error bars indicate one standard deviation from the mean. All modifications except for the IgHμ and RBP4 intron insertions resulted in a statistically significant increase in GLA activity in mouse serum.

[0399] Example 8 - Dose escalation study with codon-optimized AAV-sp7-GLA in a mouse model of Fabry disease Three doses (4.4E11 vg / kg, 1.4E12 vg / kg and 4.4E12 vg / kg) of AAV-encapsidated sp7-GLA-co4 (AAV-sp7-GLA-co4) were administered intravenously via the tail vein to GLA knockout mice (group of 5 male B6; GLA- / -). Serum was collected weekly; lyso-GL3 levels were analyzed by mass spectrometry, and GLA activity levels were measured using an in vitro 4-MU-Gal assay. As shown in Figure 8A, a dose-dependent response was observed in the levels of the Fabry disease biomarker (lyso-GL3) over the 28 days of the study. In addition, a linear relationship was observed between serum α-Gal A activity and lyso-GL3 levels (Figure 8B). A dose-dependent decrease in lyso-GL3 was observed in serum (Figure 9A) and in clinically relevant kidney (Figure 9B) and heart (Figure 9C) tissues.

[0400] Example 9 - Non-human primate (NHP) testing of AAV-sp7-GLA-co4 Four male and four female cynomolgus monkeys were administered 1e13 vg / kg of AAV-sp7-GLA-co4. Serum was collected weekly for 28 days to assess GLA activity levels by 4-MU-Gal assay (FIG. 10A) and GLA antigen levels measured using an ELISA specific for human α-Gal A (FIG. 10B). As observed, circulating GLA antigen and activity levels in monkeys administered AAV-sp7-GLA-CO4 were significantly above control levels.

[0401] Example 10 - Dose Escalation NHP Study of AAV-sp7-GLA-co4 A GLP-compliant 60-day dose-ranging study will be conducted using cynomolgus monkeys. The study period is intended to provide a sufficient window to determine peak expression and detect potential safety signals. AAV-sp7-GLA-co4 will be administered to NHPs in the group by a single intravenous (IV) infusion at the doses shown in Table 3 below.

[0402] [Table 3]

[0403] Serum samples will be collected at intervals for 60 days post-dose to measure levels of α-Gal A antigen, α-Gal A activity, and anti-α-Gal A IgG. Standard clinical pathology and anatomic pathology panel analyses will be performed. Biodistribution and germ cell transmission will be assessed.

[0404] Example 11 - Minimum Effective Dose Study A dose ranging study was performed in mice to determine the minimal effective dose that significantly reduced the Fabry biomarkers GL-3 and lyso-GL-3. GLA knockout mice (B6; 129-GLA - / -; also called GLAko or GLA KO) (n=20 per group) were intravenously (IV) injected with three doses of AAV-sp7-GLA (SEQ ID NO:21) ranging from 2E11 vg / kg to 2E12 vg / kg. Serum was collected from 10 mice every week for 6 weeks and at 9 and 12 weeks after AAV injection. Five mice were randomly selected from each group for 1, 3, 6, and 10 month time-takedown and analyzed for GLA antigen, GLA activity, and the Fabry biomarkers GL-3 and lyso-GL3. The levels of GLA activity were measured using an in vitro (4-MU-GAL) assay (data not shown). GLA antigen levels were measured using an α-Gal A specific ELISA.

[0405] A dose-dependent increase in circulating serum GLA was observed, with a linear relationship between GLA activity and GLA antigen expression levels (Figure 11). At week 2, serum GLA stabilized at ~70 ng / ml with 2E11 vg / kg, ~190 ng / ml with 4E11 vg / kg, and ~4 μg / ml with 2E12 vg / kg (Figure 11).

[0406] Levels of the Fabry biomarkers GL-3 and lyso-GL-3 in the heart and kidney were measured by LC / MS and analyzed using two-way ANOVA with multiple comparisons. At both 1 and 3 months, the levels of GL3 and lyso-GL-3 in the heart and kidney of GLAko mice administered AAV-sp7-GLA were significantly reduced compared to the levels of GL-3 and lyso-GL-3 in GLAko mice administered vehicle alone (p<0.05) (Figures 12A-12D).

[0407] The present invention is generally disclosed herein using positive language to describe a number of embodiments of the present invention. The present invention also specifically includes embodiments in which certain subjects are completely or partially excluded, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in some embodiments of the present invention, materials and / or method steps are excluded. Thus, the present invention is generally not expressed herein as to what the present invention does not include, but nevertheless, aspects that are not expressly excluded in the present invention are disclosed herein.

Claims

1. a) a nucleic acid sequence encoding α-galactosidase A (GLA), said nucleic acid sequence being at least 85% identical to the sequence of SEQ ID NO:15, and said GLA having a sequence at least 95% identical to the sequence of SEQ ID NO:100; b) a nucleic acid sequence encoding α-galactosidase A (GLA), said nucleic acid sequence including an intron in the coding sequence of said GLA, said GLA having at least 95% sequence identity to the sequence of SEQ ID NO: 100; c) a nucleic acid sequence encoding a precursor α-galactosidase A (GLA) containing an amino-terminal signal peptide, said signal peptide having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63; and said GLA having at least 95% sequence identity to the sequence of SEQ ID NO:100; and d) a nucleic acid sequence encoding an α-galactosidase A (GLA) having an amino acid sequence that differs from SEQ ID NO:100 by 1 to 7 amino acids, wherein at least one of the 1 to 7 amino acids is a substitution selected from the group consisting of Gln57Lys, Gln111Glu, Lys213Glu, Lys237Gln, Phe248Thr, Gly334Glu, and Gly346Asn. A polynucleotide comprising a nucleic acid sequence selected from the group consisting of:

2. The polynucleotide of claim 1, comprising a nucleic acid sequence having at least 95% identity to the sequence of SEQ ID NO:

15.

3. The polynucleotide of claim 2, wherein the nucleic acid sequence encodes GLA comprising the sequence of SEQ ID NO:

100.

4. The polynucleotide of any one of claims 1 to 3, wherein the nucleic acid sequence comprises less than 14 CpG dinucleotides.

5. The polynucleotide of claim 1, wherein the nucleic acid sequence has the sequence of any one of SEQ ID NOs: 15-19, or bases 1 to 1194 of any one of SEQ ID NOs: 15-19.

6. The polynucleotide of claim 1, wherein the nucleic acid sequence has a sequence at least 95% identical, at least 98% identical or 100% identical to the sequence of SEQ ID NO: 15, and the GLA has a sequence at least 98% identical or 100% identical to the sequence of SEQ ID NO:

100.

7. 10. The polynucleotide of claim 1, wherein the nucleic acid sequence encodes the GLA including the inserted intron, the GLA in the absence of the intron comprising the GLA sequence of any one of claims 1 to 6, the intron being located between nucleotides 78 and 79 of the nucleic acid encoding the GLA, the nucleotide positions being given with respect to the coding sequence for GLA having the sequence of SEQ ID NO:

14.

8. The polynucleotide of claim 7, wherein the intron comprises a sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, preferably the nucleic acid has the sequence of any of SEQ ID NOs:43-46.

9. The polynucleotide further comprises a second nucleic acid sequence, the second sequence encoding a signal peptide sequence located at the 5' end of the first nucleic acid sequence, preferably the signal peptide sequence is a heterologous signal peptide sequence, more preferably the signal peptide sequence is selected from the group consisting of human chymotrypsinogen B2 signal peptide (SEQ ID NO:41), AHSG signal peptide (SEQ ID NO:57), CD300 signal peptide (SEQ ID NO:58), GLA signal peptide (SEQ ID NO:59), LAMP1 signal peptide (SEQ ID NO:60), Notch signal peptide (SEQ ID NO:61), 9. The polynucleotide according to claim 1, wherein the signal peptide is selected from the group consisting of human chymotrypsinogen B2 signal peptide (SEQ ID NO: 61), ORM1 signal peptide (SEQ ID NO: 62), TF signal peptide (SEQ ID NO: 63), and wild-type GLA signal peptide (SEQ ID NO: 59), or a variant thereof having a sequence at least 90% identical to SEQ ID NO: 41 and any of SEQ ID NOs: 57 to 63, more preferably wherein the signal peptide is human chymotrypsinogen B2 signal peptide (SEQ ID NO: 41), and more preferably wherein the nucleic acid encoding the signal peptide has the sequence of any of SEQ ID NOs: 1 to 5.

10. 10. The polynucleotide of any one of claims 1 to 9, wherein the polynucleotide further comprises an AAV inverted repeat (ITR) adjacent to the 5' end of the polynucleotide and / or an AAV ITR adjacent to the 3' end of the polynucleotide.

11. 11. The expression cassette of any one of claims 1 to 10, wherein the GLA coding sequence is operably linked to an expression control element, preferably the expression control element is a liver-specific expression control element; or comprises an ApoE / hAAT enhancer / promoter sequence, preferably the ApoE / hAAT enhancer / promoter sequence has reduced CpG compared to a wild-type ApoE / hAAT enhancer / promoter sequence, more preferably the ApoE / hAAT enhancer / promoter sequence comprises the sequence of SEQ ID NO:

38.

12. The expression cassette of claim 11 , wherein the expression control element is located 5' to the nucleic acid encoding GLA.

13. 13. An expression cassette according to any one of claims 11 to 12, further comprising a polyadenylation sequence located 3' to the nucleic acid encoding GLA, preferably the polyadenylation sequence comprises the bovine growth hormone (bGH) polyadenylation sequence, more preferably the bGH polyadenylation sequence comprises the sequence of SEQ ID NO:

20.

14. 14. An expression cassette according to any one of claims 11 to 13, wherein the expression control elements and / or polyadenylation sequences have reduced CpGs compared to the wild-type expression control elements or wild-type polyadenylation sequences.

15. 15. An expression cassette according to any one of claims 11 to 14, further comprising an intron located between the 3' end of the expression control element and the 5' end of the polynucleotide, preferably the intron comprises the hBB2m1 intron (SEQ ID NO: 39).

16. 16. The expression cassette of claim 11, further comprising an ITR adjacent to the 5' end of the expression cassette and / or an ITR adjacent to the 3' end of the expression cassette.

17. 17. An AAV plasmid genome comprising the polynucleotide of claim 10 or the expression cassette of claim 16, and an origin of replication.

18. 17. An adeno-associated virus (AAV) vector comprising the polynucleotide of claim 10 or the expression cassette of claim 16 and a capsid.

19. The AAV vector of claim 18, wherein the AAV comprises the expression cassette, and the expression cassette further comprises the ITR adjacent to the 5' end of the expression cassette and the ITR adjacent to the 3' end of the expression cassette, preferably the 5' ITR and the 3' ITR are independently selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV3B ITRs, and more preferably at least one of the 5' ITR and 3' ITR has been modified to have reduced CpGs.

20. The AAV vector of claim 19, wherein the vector comprises a sequence having at least 95%, at least 99% or 100% sequence identity to any of SEQ ID NOs: 21-34, 53-56 and 91-99, or the vector consists of a sequence having at least 95%, at least 98% or 100% sequence identity to any of SEQ ID NOs: 91-95.

21. the capsid has at least 90%, at least 95%, at least 98%, or 100% sequence identity to the VP1, VP2, or VP3 capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, LK03 (SEQ ID NO:42), AAV-2i8; or an AAV having at least 90%, at least 95%, at least 98%, or 100% sequence identity to SEQ ID NO:110, SEQ ID NO:36, or SEQ ID NO:

37.

21. The AAV vector of any one of claims 18 to 20, comprising a VP1, VP2 or VP3 capsid, preferably wherein the capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:35), AAV3B, LK03 (SEQ ID NO:42) or AAV-2i8 capsid; or a capsid comprising SEQ ID NO:110, SEQ ID NO:36, and / or SEQ ID NO:

37.

22. A pharmaceutical composition according to any one of claims 18 to 21, comprising a plurality of AAV vectors according to any one of claims 34 to 41 in a biologically compatible carrier or excipient, preferably the plurality of AAV vectors being in an amount effective to increase GLA activity in a human subject, preferably comprising empty AAV capsids.

23. 23. The polynucleotide, expression cassette, AAV vector or pharmaceutical composition of any one of claims 1 to 22 for use in a method of treating a subject in need of alpha-galactose A (GLA), wherein GLA is expressed in the subject, preferably the method is for treating Fabry disease in a subject, more preferably the method reduces, diminishes or inhibits one or more symptoms of Fabry disease, more preferably the subject is a human.