Polynucleotides

Abstract

The present invention relates to polynucleotides comprising a GBA nucleotide sequence encoding a GCase protein or fragment thereof, and wherein a portion of the encoding sequence is not the wild type. The invention also relates to viral particles comprising a recombinant genome comprising the polynucleotides of the invention, compositions comprising the polynucleotides or viral particles, and methods and uses of the polynucleotides, viral particles or compositions.

Description

[0001] This application is a divisional application of CN202080017551.2. Field of the Invention

[0002] The present invention relates to polynucleotides comprising a GBA nucleotide sequence encoding beta-glucocerebrosidase (GCase), viral particles comprising the polynucleotides, and therapies utilizing the polynucleotides. Background of the Invention

[0003] Gaucher disease (GD) is an autosomal recessive lipid storage disorder characterized by the accumulation of glucocerebroside in cells of the macrophage-monocyte system. GD is caused by mutations in the housekeeping GBA gene, which impair the activity and / or production of the enzyme β-glucocerebrosidase (GCase).

[0004] There are three main types of GD, characterized by specific identified mutations, and each type can present with different clinical symptoms. Type 1 GD has minimal or no involvement of the central nervous system but is primarily characterized by visceral manifestations such as an enlarged spleen and liver, low blood cell counts, bleeding problems, and bone disease. Over the past 20 years, enzyme replacement therapy has become the standard treatment for type 1 GD. In addition to the high cost (approximately US$200,000 or approximately £150,000 per patient per year), enzyme replacement therapy treatment in GD typically requires lifelong injections one or more times every other week. This results in a high proportion of GD patients experiencing a high level of treatment burden.

[0005] Therefore, there is a need to provide an effective therapeutic vector for the treatment of GD, ie, a vector that allows high-level GCase expression.

[0006] This application relates to a gene therapy method for treating GD, comprising administering viral particles containing a GBA polynucleotide encoding GCase. The polynucleotides and viral particles described herein can provide higher GCase expression compared to polynucleotides containing a polynucleotide encoding wild-type GCase. This gene therapy method can avoid the need for frequent and lifelong intravenous injections of GCase. SUMMARY OF THE INVENTION

[0007] This application demonstrates that specific modifications to GBA nucleotide sequences encoding GCase can help increase the expression level and activity of GCase polypeptides expressed in vitro and / or in vivo. For example, this application demonstrates that the use of codon-optimized GBA nucleotide sequences can increase the expression and / or activity of the encoded GCase protein. Such modified (i.e., non-wild-type) and / or codon-optimized GBA nucleotide sequences can be further modified to provide further improvements in the expression and / or activity of the encoded GCase protein. Further modifications can include providing further modifications within the GBA nucleotide sequence, such as removing CpG motifs and / or using specific gene regulatory elements comprising specific promoter and / or enhancer sequences. It is believed that such modifications to GBA nucleotide sequences can enhance the efficacy of such nucleotide sequences in treating GD.

[0008] These modifications provide GBA nucleotide sequences that are highly expressed (eg, in the liver) and encode GCase polypeptides or fragments thereof. As demonstrated in the Examples, the polynucleotides of the present invention express GCase activity at higher levels than wild-type GBA.

[0009] Thus, in a first aspect of the present invention, a polynucleotide comprising a GBA nucleotide sequence is provided, wherein the GBA nucleotide sequence encodes a β-glucocerebrosidase (GCase) protein or a fragment thereof, and wherein at least a portion of the GBA nucleotide sequence is not wild type.

[0010] In a second aspect of the present invention, a polynucleotide comprising a GBA nucleotide sequence is provided, wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, 1000 to 1494, 1000 to 1611, 1300 to 1494, 1300 to 1611 or about 1494 nucleotides of SEQ ID NOs: 1-8.

[0011] In a third aspect of the present invention, there is provided a viral particle comprising a recombinant genome comprising a polynucleotide of the present invention.

[0012] In a fourth aspect of the present invention, there is provided a composition comprising the polynucleotide or viral particle of the present invention and a pharmaceutically acceptable excipient.

[0013] In a fifth aspect of the present invention, a method of treatment is provided, comprising administering to a patient an effective amount of the polynucleotide or viral particle of the present invention.

[0014] In a sixth aspect of the present invention, there is provided use of the polynucleotide, viral particle or composition of the present invention in the preparation of a medicament for a therapeutic method.

[0015] In a seventh aspect of the present invention, there is provided use of the polynucleotide, viral particle or composition of the present invention in the preparation of a medicament for achieving stable GCase activity in a subject.

[0016] In an eighth aspect of the invention, there is provided the use of a polynucleotide, viral particle or composition of the invention in the preparation of a medicament for providing a higher bioavailability of GCase in a subject than the bioavailability from GCase enzyme replacement therapy, wherein the bioavailability is measured over a period of 2 weeks after administration.

[0017] In a ninth aspect of the present invention, a method is provided for achieving stable GCase activity in a subject by administering to the subject a polynucleotide, viral particle or composition of the present invention.

[0018] In a tenth aspect of the invention, a method is provided for providing a bioavailability of GCase in a subject that is greater than the bioavailability from GCase enzyme replacement therapy by administering to the subject a polynucleotide, viral particle or composition of the invention, wherein the bioavailability is measured over a period of 2 weeks after administration.

[0019] In an eleventh aspect of the present invention, a polynucleotide, a viral particle or a composition of the present invention is provided for use in a method for expressing a GBA nucleotide sequence in a subject and achieving stable GCase activity.

[0020] In a twelfth aspect of the invention, a polynucleotide, viral particle or composition of the invention is provided for use in a method of expressing a GBA nucleotide sequence and providing increased GCase bioavailability in a subject compared to the bioavailability from GCase enzyme replacement therapy, wherein the bioavailability is measured over a period of 2 weeks after administration.

[0021] In a thirteenth aspect of the present invention, there is provided use of a polynucleotide, viral particle or composition of the present invention in the preparation of a medicament for reducing the level of hexosylceramide and / or hexosylsphingosine in a subject suffering from a disease or condition associated with GCase deficiency.

[0022] In a fourteenth aspect of the invention, methods are provided for reducing hexosylceramide and / or hexosylsphingosine levels in a subject suffering from a disease or disorder associated with GCase deficiency by administering to the subject a polynucleotide, viral particle or composition of the invention.

[0023] In a fifteenth aspect of the invention, a polynucleotide, viral particle or composition of the invention is provided for use in a method of reducing the level of hexosylceramide and / or hexosylsphingosine in a subject suffering from a disease or condition associated with GCase deficiency, optionally wherein reducing the level of hexosylceramide and / or hexosylsphingosine results in treating the disease or condition associated with GCase deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 - Schematic representation of the GBA cassettes from constructs FLF-PL01, FLF-PL28, and FLF-PL64. LSP-S and LSP-L: liver-specific promoters; GBAwt: wild-type human GBA nucleotide sequence; GBAco: codon-optimized human GBA nucleotide sequence (except for the segment encoding the signal peptide, the termini of which are indicated by dashed lines).

[0025] Figure 2 Dose-dependent hepatic expression and secretion of human GCase into the murine bloodstream following injection of AAV2 / 8-FLF-PL28. (A) Representative images of mouse livers stained for GCase 12 weeks after injection of AAV2 / 8-PL28. DAB (3,3'-diaminobenzidine) was used to visualize GCase, and hematoxylin was used as a counterstain. (B) GCase levels measured by activity assay in the serum of mice treated with increasing doses of AAV2 / 8-PL28. n = 5 C57BL / 6 mice per treatment group. Error bars indicate mean ± SD.

[0026] Figure 3 Relative GCase levels were observed for each tested GBA codon-optimized construct (FLF-PL16 to FLF-PL36; '16' to '36') following transfection into Huh-7 cells. Each construct was independently tested in 3 to 5 experiments. Data shown here represent GCase activity relative to the wild-type GBA construct FLF-PL01 ('01'). Error bars represent mean ± SD.

[0027] Figure 4 GCase activity was measured in the bloodstream of mice following injection of the vectors AAV2 / 8-FLF-PL-01, 21, 28, 30, 36, and 37 (see Example 5 for construct descriptions). (A) GCase activity levels found in mouse serum 8 weeks after injection of the tested GBA constructs. (B) GCase activity levels observed in mouse serum 4, 8, 12, and 36 weeks after injection of the constructs FLF-PL01 and FLF-PL28. Error bars represent mean ± SD, n = 5-8 animals per experimental group. *p ≤ 0.05; **p ≤ 0.001 (one-way ANOVA).

[0028] Figure 5 Figure 3. GCase uptake levels in spleen and bone marrow tissues after AAV2 / 8-FLF-PL28 injection in wild-type mice. Representative images of GBA-stained spleen and bone marrow tissues from naive or AAV2 / 8-PL28-treated mice 4 weeks after injection are shown. DAB (3,3'-diaminobenzidine) was used to visualize GBA, and hematoxylin was used as a counterstain.

[0029] Figure 6 Following injection of AAV2 / 8-FLF-PL28 into wild-type mice, colocalization of human GCase with the classic murine macrophage marker F4 / 80 was observed in the spleen. Representative immunofluorescence images of spleen tissue stained with GBA and F4 / 80 antibodies. DAPI (blue) was used to visualize cell nuclei.

[0030] Figure 7 —GCase activity levels found in the bloodstream of mice 4 weeks after injection of AAV2 / 8-FLF-PL28 and FLF-PL64. 12 GCase activity in mouse sera collected 4 weeks after vg / kg dose injection. Error bars represent mean ± SD. N = 5 C57BL / 6 mice per treatment group.

[0031] Figure 8 - Uptake levels observed in the spleen, bone marrow, and lung following AAV2 / 8-FLF-PL28 and FLF-PL64 injection were observed in mice 5 weeks after treatment.

[0032] Figure 9 — sequence listing.

[0033] Figure 10 ——GCase secretion level of human cell lines after transduction with AAV-FLF-PL64. 5 Cells were transduced at an MOI of 100 vg / cell. (A) Active GBA levels were determined using 4MU-Glc as a substrate. (B) Transduction levels for each cell line were determined by qPCR using primers specific for the polyadenylation sequence. Blank values ​​for each cell line were subtracted to obtain values ​​for active GCase levels. Error bars represent the mean ± SD of duplicate wells.

[0034] Figure 11 :(A) Enzyme replacement therapy ( Pharmacokinetics and half-life calculations of VPRIV (60 U / kg) in wild-type mice. One-phase decay model equation: Y0 is the value of Y when X (time) is zero. Plateau is the value of Y at infinite time. K is the rate constant. Tau is the time constant. Half-life is expressed in time units on the X-axis. Span is the difference between Y0 and Plateau. (B) Comparison of serum pharmacokinetic profiles of GCase activity in wild-type mice following a single injection of enzyme replacement therapy (VPRIV (60 U / kg), solid black) and FLF-PL64 gene therapy.

[0035] Figure 12 Immunostaining of GCase in the liver, spleen, and bones of mice following administration of VPRIV or FLF-PL64. DAB (3,3'-diaminobenzidine) was used to visualize GCase, and hematoxylin was used as a counterstain. FLF-PL64 samples were obtained five weeks after injection, while VPRIV-treated samples were collected at the indicated times. Each image represents n = 5 C57BL / 6 mice per treatment group. All images are at the same magnification.

[0036] Figure 13 : When administering velaglucerase alfa (labeled as ERT) or AAV-GBA (AAV-FLF-PL64), after gba 9v / null Increased GCase activity was observed in the liver (a), leukocytes (b), spleen (c), and bone marrow (d) of mice. ERT samples were collected 1-2 hours after the last injection, corresponding to the peak of tissue uptake. AAV-GBA (AAV-FLF-PL64) samples were collected 12 weeks after injection and corresponded to steady-state uptake levels. GCase activity is expressed as a percentage of the activity measured in wild-type healthy mice (20 weeks of age). All mice were treated at 8 weeks of age, pre-overt symptomatology. The ERT dose was 60 U / kg, administered by injection every two weeks; AAV-FLF-PL64 was administered at 2×10 12 vg / kg injection. n = 10. ****P ≤ 0.0001

[0037] Figure 14 :AAV-GBA (AAV-FLF-PL64) gene therapy can reduce gba 9v / nullActivated macrophages and inflammation in mouse liver. Upper panel: H&E-stained liver sections showing representative images of each group. Storage cells are identified by circles. Lower left panel: Graph showing the comparison between storage cells counted in the AAV-FLF-PL64 and ERT-treated groups and the vehicle control group. Lower right panel: Graph showing CD68-positive cells counted in the AAV-FLF-PL64 and ERT-treated groups compared with the vehicle control group after staining with anti-CD68 antibody. AAV-GBA (AAV-FLF-PL64) was injected at 2×10 12 vg / kg injection; samples collected 12 weeks after infection, ERT dose of 60 U / kg, administered every two weeks. ERT samples were collected 1-2 hours after the last injection. Mean ± SEM, (n = 10), **P ≤ 0.005, ****P ≤ 0.0005

[0038] Figure 15 :AAV-GBA (AAV-FLF-PL64) gene therapy in gba 9v / null In mice, velaglucerase alfa ( LC / MS analysis of hexosylceramide and hexosylsphingosine levels in the liver, spleen, and bone marrow of AAV-FLF-PL64 and ERT-treated groups. Levels were normalized to those measured in the vehicle control group. AAV-GBA (AAV-FLF-PL64) was injected at 2×10 12 vg / kg injection; samples collected 12 weeks after infection; ERT dose was 60 U / kg, administered every two weeks. ERT samples were collected 1-2 hours after the last injection. Mean ± SEM, (n = 10), **P ≤ 0.005, ****P ≤ 0.0005

[0039] Details

[0040] General Definition

[0041] Unless defined otherwise, 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.

[0042] In general, the term "comprising" is intended to mean including but not limited to. For example, the phrase "a polynucleotide comprising a GBA nucleotide sequence" should be interpreted to mean that the polynucleotide has a GBA nucleotide sequence, but the polynucleotide may contain additional nucleotides.

[0043] In some embodiments of the present invention, the word "comprising" is replaced by the phrase "consisting of". The term "consisting of" is intended to be limiting. For example, the phrase "a polynucleotide consisting of a GBA nucleotide sequence" should be understood to mean that the polynucleotide has a GBA nucleotide sequence and does not contain additional nucleotides.

[0044] As used herein, when referring to two endpoints to define a range of values, "to" should be understood as "up to and including." Thus, a range defined as "5 to 10" includes all values ​​greater than 5 and less than 10, as well as the discrete values ​​5 and 10 themselves.

[0045] The terms "protein" and "polypeptide" are used interchangeably herein and are intended to refer to polymeric chains of amino acids of any length.

[0046] For the purposes of the present invention, in order to determine the percent identity of two sequences (e.g., two polynucleotides or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for optimal alignment with the second sequence). The nucleotides or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid residue as the corresponding position in the second sequence, the nucleotides or amino acids at that position are identical. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions in the reference sequence × 100).

[0047] Sequence comparisons are typically performed over the length of a reference sequence. For example, if a user wishes to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. For example, to assess whether a sequence is at least 80% identical to SEQ ID NO: 1 (an example of a reference sequence), one skilled in the art would compare over the length of SEQ ID NO: 1 and identify how many positions in the test sequence are identical to positions in SEQ ID NO: 1. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1, gaps or missing positions should be considered as different positions.

[0048] Those skilled in the art are aware of different computer programs that can be used to determine the homology or identity between two sequences. For example, comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm, which has been incorporated into the GAP program of the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using a Blosum 62 matrix or a PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0049] For the purposes of the present invention, the term "fragment" refers to a contiguous portion of a sequence. For example, a 50-nucleotide fragment of SEQ ID NO: 1 refers to 50 contiguous nucleotides of SEQ ID NO: 1.

[0050] polynucleotides

[0051] In one aspect, the present invention provides a polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-glucocerebrosidase (GCase) protein or a fragment thereof, and wherein at least a portion of the GBA nucleotide sequence is not wild type.

[0052] The polynucleotide may further comprise one or more of the following features: The GBA nucleotide sequence or the portion of the GBA nucleotide sequence that is not wild-type may be codon-optimized. The polynucleotide may (additionally) comprise a portion that is not codon-optimized. The polynucleotide may comprise an intron or a fragment of an intron.

[0053] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, deoxyribonucleotides, ribonucleotides, or their analogs. For example, a polynucleotide may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). A polynucleotide may be composed of DNA. A polynucleotide may be mRNA. Because a polynucleotide may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).

[0054] GBA nucleotide sequence encoding GCase

[0055] In one aspect, the polynucleotides provided herein comprise a GBA nucleotide sequence. The GBA nucleotide sequence typically encodes a β-glucocerebrosidase (GCase) protein or a fragment thereof.

[0056] The term "coding sequence" refers to a nucleotide sequence comprising an open reading frame comprising codons encoding the encoded polypeptide. For example, a nucleotide sequence encoding a GCase protein or a fragment thereof comprises codons encoding the amino acid sequence of the GCase protein or a fragment thereof. An example of a GBA nucleotide sequence encoding a wild-type GCase protein is provided in SEQ ID NO:9.

[0057] A GBA nucleotide sequence may be interrupted by non-coding nucleotides (e.g., introns), but only nucleotides encoding a polypeptide should be considered part of the GBA nucleotide sequence. For example, a GBA nucleotide sequence encoding a GCase protein will include any codons that encode amino acids that are components of the GCase protein expressed from the coding sequence, regardless of whether these codons are consecutive in sequence or separated by one or more non-coding nucleotides. In other words, a GBA polynucleotide comprising coding nucleotide segments interrupted by non-coding nucleotide segments will be considered to comprise a "GBA nucleotide sequence" consisting of directly juxtaposed non-contiguous coding segments (i.e., minus the non-coding segments). However, for purposes of this disclosure, stop codons will be considered part of the full-length coding sequence.

[0058] The GBA nucleotide sequence encoding GCase and / or the GCase coding sequence as described herein may also include codons for a signal peptide. It is well known that some proteins, particularly those exported to various tissues, are expressed with signal peptides. The signal peptide can be located at the N-terminus of the protein sequence (in this case, at the 5' end of the coding sequence), and many signal peptides are cleaved after cellular processing. Therefore, herein, a mature protein or polypeptide (e.g., a mature GCase protein or polypeptide) will be considered to be a protein or polypeptide produced after the signal peptide has been processed and removed / cleaved (and therefore no longer forms part of the polypeptide sequence).

[0059] The following table describes the codons that encode each amino acid:

[0060]

[0061] The corresponding RNA codon will contain a U in place of a T in the table above.

[0062] One aspect of the application provides a polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase protein or fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, 1000 to 1494, 1000 to 1611, 1300 to 1494, 1300 to 1611, or about 1494 nucleotides of any one of SEQ ID NOs: 1-8. Optionally, all or part of the GBA nucleotide sequence is codon optimized. In one embodiment, the GBA nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1300 nucleotides of SEQ ID NOs: 1-8. In one embodiment, the GBA nucleotide sequence comprises a sequence that is at least 99% identical to a fragment of at least 1300 nucleotides of SEQ ID NOs: 1-8.

[0063] In one example, the GBA nucleotide sequence can comprise a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1. In one embodiment, the GBA nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1. In one embodiment, the GBA nucleotide sequence comprises a sequence that is at least 99% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1. The GBA nucleotide sequence can comprise a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 5. The GBA nucleotide sequence can comprise a sequence that is at least 98% identical to SEQ ID NO: 1. The GBA nucleotide sequence can comprise a sequence that is at least 99% identical to SEQ ID NO: 1. The GBA nucleotide sequence can comprise a sequence that is at least 98% identical to SEQ ID NO: 5. The GBA nucleotide sequence can comprise a sequence that is at least 99% identical to SEQ ID NO: 5. In one embodiment, the GBA nucleotide sequence can comprise SEQ ID NO: 1. In another embodiment, the GBA nucleotide sequence can comprise SEQ ID NO: 5.

[0064] The GBA nucleotide sequence can comprise the sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 that encodes a GCase protein having GCase activity. In these examples, the variant of SEQ ID NO: 1 is identical to SEQ ID NO: 1 except that the variant comprises nucleotide substitutions such that the GCase protein has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. In these examples, the variant of SEQ ID NO: 1 can have 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 20, or at most 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 1. The variant of SEQ ID NO: 1 can have 1, at most 2, at most 3, at most 4, at most 5, or at most 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 1. In one example, the variant of SEQ ID NO: 1 has up to 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 and / or encodes a GCase protein having up to 3 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. In one example, the variant of SEQ ID NO: 1 has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 and / or encodes a GCase protein having up to 2 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. In one example, the variant of SEQ ID NO: 1 has 1 nucleotide substitution relative to the sequence of SEQ ID NO: 1 and / or encodes a GCase protein having up to 1 amino acid substitution relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0065] The GBA nucleotide sequence can comprise the sequence of SEQ ID NO: 5 or a variant of SEQ ID NO: 5 encoding a GCase protein having GCase activity. In these examples, the variant of SEQ ID NO: 5 is identical to SEQ ID NO: 5 except that the variant comprises nucleotide substitutions such that the GCase protein has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. In these examples, the variant of SEQ ID NO: 5 can have 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 20, or at most 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 5. The variant of SEQ ID NO: 5 can have 1, at most 2, at most 3, at most 4, at most 5, or at most 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 5. In one example, the variant of SEQ ID NO: 5 has up to 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and / or encodes a GCase protein having up to 3 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. In one example, the variant of SEQ ID NO: 5 has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and / or encodes a GCase protein having up to 2 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. In one example, the variant of SEQ ID NO: 5 has 1 nucleotide substitution relative to the sequence of SEQ ID NO: 5 and / or encodes a GCase protein having up to 1 amino acid substitution relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0066] GCase protein or its fragments

[0067] The polynucleotide comprises a GBA nucleotide sequence encoding a GCase protein or a fragment thereof.

[0068] β-Glucocerebrosidase (GCase) is an enzyme with glucosylceramidase activity (EC 3.2.1.45) that hydrolyzes the β-glucosidic bond of the chemical glucocerebroside, an intermediate in glycolipid metabolism and abundant in cell membranes. Mutations in the GBA gene (encoding GCase) lead to the accumulation of glucocerebroside in macrophages, which infiltrate numerous vital organs and manifest as Gaucher disease (GD). The representative wild-type GCase polypeptide is encoded by SEQ ID NO:9.

[0069] GCase (e.g., the GCase of SEQ ID NO:25 encoded by SEQ ID NO:9) is initially expressed as a precursor "immature" form comprising a signal peptide (amino acid residues 1 to 39 of SEQ ID NO:25 and codons 1 to 39 of SEQ ID NO:9), and a mature GCase polypeptide region. After processing, the "mature" form of GCase lacks the signal peptide. The term "mature GCase" or "mature GCase polypeptide" refers to a GCase polypeptide that does not contain a signal peptide, such as the GCase encoded by SEQ ID NOs:1-4. A typical GCase signal peptide can be encoded by the nucleotide sequence of SEQ ID NO:17 and have a polypeptide sequence of SEQ ID NO:18.

[0070] The GCase or fragment thereof may be a variant GCase or fragment thereof, i.e., a GCase that does not have a sequence identical to SEQ ID NO: 25. In one embodiment, the GCase or fragment thereof is encoded by a polypeptide of the present invention and / or a GBA nucleotide sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 25; or at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of SEQ ID NO: 25 that is at least 300, at least 350, at least 400, less than or equal to 536, less than or equal to 497, 300 to 536, or 300 to 497 amino acids in length. In one embodiment, the GCase protein or fragment thereof is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO:25; or at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of SEQ ID NO:25 that is approximately 497 amino acids in length. The GCase protein or fragment thereof may have the sequence of SEQ ID NO:25. Preferably, the GCase protein or fragment thereof does not contain the signal peptide of SEQ ID NO:18. Preferably, the GCase protein or fragment thereof is functional. A functional GCase protein or fragment is a protein or fragment that hydrolyzes glucocerebroside.

[0071] The GBA nucleotide sequence can encode a GCase protein having 1, up to 2, up to 3, up to 4, or up to 5 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. In such an example, the GBA nucleotide sequence can encode a GCase protein having up to 3 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. The GBA nucleotide sequence can encode a GCase protein having up to 2 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25. The GBA nucleotide sequence can encode a variant GCase protein having up to 1 amino acid substitution relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0072] Determining whether a GCase protein or fragment encoded by a GBA nucleotide sequence is functional is within the capabilities of those skilled in the art. One skilled in the art need only express the GCase nucleotide sequence and test whether the expressed protein is active. For example, one skilled in the art can prepare viral particles of the present invention comprising a GBA nucleotide sequence linked to an operable promoter and transduce cells with the viral particles under conditions suitable for expressing the GCase protein or fragment thereof. Fluorescence assays, such as the "Serum GBA Activity Assay" described in Example 1, can be used to analyze the activity (amount) of the expressed GCase protein or fragment.

[0073] For example, a suitable fluorescence assay is as follows. β-glucocerebrosidase (acid β-glucosidase; GCase) activity can be measured fluorescently using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate. Briefly, serum samples (0.5 μL, 1:50 dilution) can be measured at 37°C for 30 minutes in 50 mM sodium citrate, 25 mM taurocholate, pH approximately 5.75, 6 mM 4MU-Glc. Relative fluorescence levels (RFU) can then be assessed using excitation and emission wavelengths of 365 nm and 445 nm, respectively. GCase is expressed as nanomoles / h / mL serum based on a 4-methylumbelliferyl (4-MU) standard curve.

[0074] A portion of the GBA nucleotide sequence is not wild type

[0075] A portion of the GBA nucleotide sequence (e.g., a coding sequence encoding a GCase protein or a fragment thereof) may not be wild-type. The GBA nucleotide sequence encoding a wild-type GCase is represented by SEQ ID NO: 9, and a GBA nucleotide sequence comprising a portion different from the sequence of SEQ ID NO: 9 comprises a portion that is not wild-type.

[0076] In one embodiment, the portion of the GBA nucleotide sequence that is not wild type is codon-optimized. Codon-optimization can improve expression of a nucleotide sequence (e.g., a GBA nucleotide sequence) in a particular tissue and / or in a particular organism. For example, if a nucleotide sequence is codon-optimized for expression in human liver, the nucleotide sequence is modified to increase the number of codons that can be favored in human liver (in a sense, such codons correspond to a higher abundance of tRNA species than other tRNA species that are specific for the same amino acid). One of skill in the art will appreciate that codon-optimization of a sequence can not require changing every codon, especially since "favored codons" can already be present in certain positions.

[0077] Such codon-optimization can be influenced by other factors. For example, one can see that the presence of CpG has a deleterious effect on expression, so a user can decide not to use favored codons in positions where doing so would introduce a CpG into the sequence; this would still be considered codon-optimization. In one embodiment, a favored codon ending in a C nucleotide would not be included in the codon-optimized coding sequence portion of the sequence where the next codon in the sequence begins with a G. For example, the codon CTC encodes leucine. In a scheme where CTC is a favored codon, it should not be used for leucine in the coding sequence where the next codon begins with a G, e.g., the codon GTT (alternatively, the next codon can be chosen - where possible - to avoid a G in the first position).

[0078] Determining the frequency of each codon used in a portion of a nucleotide sequence is simple. One of skill in the art need only enter the sequence of the portion into one of the readily available algorithms that looks at codon usage and view the results. Alternatively, a user can simply count them.

[0079] In one embodiment, the polynucleotide of the application comprises a GBA nucleotide sequence in which 67% of the codons encoding histidine are CAC and 33% of the codons encoding histidine are CAT.

[0080] Optionally, the portion of the codon-optimized GBA nucleotide sequence is codon-optimized for expression in human liver. Optionally, the GBA nucleotide sequence is codon-optimized for expression in human liver. Optionally, the portion of the codon-optimized GBA nucleotide sequence is a contiguous portion.

[0081] The codon-optimized portion can correspond to a sequence encoding a portion of the GCase protein or the entire GCase protein. For example, the coding sequence can be full-length (e.g., SEQ ID NO: 9), including a signal peptide that is not part of the mature GCase protein, and the entire coding sequence can be codon-optimized. Therefore, reference herein to "a portion of a GBA sequence is codon-optimized" should be understood to mean "at least a portion of the GBA sequence is codon-optimized." Optionally, the portion of the codon-optimized GBA nucleotide sequence is at least 1000, at least 1200, at least 1300, less than 1600, less than 1500, 1000 to 1600, 1000 to 1500, 1300 to 1500, or about 1494 nucleotides in length. Optionally, the codon-optimized GBA nucleotide sequence portion encodes (corresponds to) the mature GCase protein. For example, the GBA nucleotide sequence may encode a precursor GCase protein (ie, include a signal peptide), and if the portion of the codon-optimized GBA nucleotide sequence corresponds to the mature GCase protein, the signal peptide is not codon-optimized.

[0082] Thus, in some embodiments, a portion of the GBA nucleotide sequence may not be codon-optimized, e.g., a portion of the coding sequence is not codon-optimized for expression in the liver. In some embodiments, the portion that is not codon-optimized is at least 80, at least 90, at least 100, at least 110, less than 200, less than 170, less than 140, or about 117 nucleotides. In some embodiments, the portion of the GBA nucleotide sequence that is not codon-optimized is a portion that encodes a signal peptide.

[0083] As described above, providing a polynucleotide sequence comprising a partially or fully codon-optimized GBA nucleotide sequence can ensure high-level expression of the encoded polypeptide (i.e., a GCase polypeptide). Those skilled in the art will appreciate that expression of GCase from a polynucleotide sequence (e.g., a GBA nucleotide sequence of the present invention) or from a viral particle of the present invention generally requires the presence of a promoter sequence or region upstream of and / or operably linked to the polynucleotide sequence. Therefore, in one embodiment, the present invention provides a polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase polypeptide that, when operably linked to the promoter sequence, results in high-level expression of the GCase polypeptide in human hepatocytes. In some embodiments, the promoter sequence can be part of a transcriptional regulatory element. In some embodiments, the promoter sequence can be a liver-specific promoter sequence. In one embodiment, the promoter sequence is a promoter having SEQ ID NO: 12. In another embodiment, the promoter sequence is a promoter having SEQ ID NO: 15.

[0084] It will also be understood by those skilled in the art that when comparing a polynucleotide or vector of the present invention to a reference (comparison) polynucleotide or vector, such as a reference polynucleotide or viral particle comprising the GBA nucleotide sequence of SEQ ID NO: 9, the reference polynucleotide or vector can be identical to the polynucleotide or vector of the present invention, except that the GBA nucleotide sequence is different. In other words, the different GBA nucleotide sequences being compared can be operably linked to the same promoter sequence. In some embodiments, the different GBA nucleotide sequences being tested can be operably linked to different (designated) promoter sequences.

[0085] Thus, in one embodiment, the GCase polypeptide encoded by the GBA nucleotide sequence is expressed at a higher level in human hepatocytes compared to a reference wild-type GBA sequence. The reference wild-type GBA nucleotide sequence may be SEQ ID NO:9. In one embodiment, the polypeptide encoded by the GBA nucleotide sequence is expressed at a higher level in human hepatocytes compared to a polypeptide encoded by a nucleotide sequence comprising the GBA nucleotide sequence of SEQ ID NO:9 and a promoter element of SEQ ID NO:13 (wherein the GBA nucleotide sequence of SEQ ID NO:9 and the promoter element of SEQ ID NO:13 are preferably operably linked). In one embodiment, the polypeptide encoded by the GBA nucleotide sequence is expressed at a higher level in human hepatocytes compared to a polypeptide encoded by a nucleotide sequence comprising the GBA nucleotide sequence of SEQ ID NO:9 and a transcriptional regulatory element of SEQ ID NO:10 (wherein the GBA nucleotide sequence of SEQ ID NO:9 and the promoter element of SEQ ID NO:10 are preferably operably linked). In such embodiments, the GCase encoded by the GBA nucleotide sequence can be expressed at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, or at least 1.5-fold in human hepatocytes. In one embodiment, the GCase polypeptide encoded by the GBA nucleotide sequence is expressed at a higher level or at a non-statistically significantly different level in human hepatocytes compared to a polypeptide encoded by an otherwise identical reference polynucleotide comprising the GBA nucleotide sequence of SEQ ID NO: 9 and operably linked to the promoter of SEQ ID NO: 13, wherein the two polynucleotides are delivered to the cells in the same manner and in the same amount.

[0086] In one embodiment, when a polynucleotide sequence comprising a GBA nucleotide sequence is administered to a subject or a non-human mammal, such as a mouse, GCase is present in the serum of the subject or non-human animal at higher levels (for example, 4 or 8 or 12 weeks after administration) compared to GCase encoded by an otherwise identical nucleotide sequence comprising the GBA nucleotide sequence of SEQ ID NO: 9 operably linked to a promoter element of SEQ ID NO: 12, 13 or 15, wherein the polynucleotide comprising the GBA nucleotide sequence is administered in the same manner and in the same amount.

[0087] One skilled in the art can determine whether GCase is expressed at a higher level from a given GBA nucleotide sequence (e.g., a codon-optimized GBA nucleotide sequence) compared to a reference sequence (e.g., a wild-type GBA nucleotide sequence, such as SEQ ID NO: 9) by transducing some cells with viral particles comprising the GBA nucleotide sequence and some cells with particles comprising the reference sequence. The cells can be cultured under conditions suitable for expressing the GCase protein or fragments thereof encoded by the GBA nucleotide sequence, and the levels of the expressed GCase protein can be compared. The level of expressed GCase protein can be assessed using a fluorescence assay as described in the section entitled "GCase Protein or Fragments thereof" or an ELISA using a GCase-specific antibody. Suitable cells include cultured human hepatocytes, such as Huh-7 cells.

[0088] As mentioned above, the presence of CpG (i.e., CG dinucleotides) may reduce expression efficiency. This is because CpG may be methylated, and their methylation may lead to gene silencing and thus reduce expression. In addition, high CpG content may trigger a TLR response, increasing the risk of anti-AAV immune response. For this reason, it is preferred that the codon-optimized coding sequence portion comprises a reduced number of CpGs compared to the corresponding portion of the reference wild-type GBA nucleotide sequence (e.g., SEQ ID NO: 9). In one embodiment, the codon-optimized GBA nucleotide sequence portion (which may be all GBA nucleotide sequences) comprises less than 40, less than 20, less than 10, or less than 5 CpGs. In one embodiment, the codon-optimized GBA nucleotide sequence portion (which may be all GBA nucleotide sequences) comprises less than 5, less than 4, less than 3, or less than 2 CpGs / 100nt. In some embodiments, the codon-optimized coding sequence portion does not contain CpG, i.e., does not contain (0) CG dinucleotides.

[0089] In one embodiment, the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, 1000 to 1494, 1300 to 1494, or about 1494 nucleotides of SEQ ID NOs: 1-4. In one embodiment, the codon-optimized portion of the coding sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NOs: 1-4. In one embodiment, the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, 1000 to 1494, 1300 to 1494, or about 1494 nucleotides of SEQ ID NO: 1. In one embodiment, the codon-optimized portion of the GBA nucleotide sequence is at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1. In one embodiment, the codon-optimized coding sequence portion is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1.

[0090] The present invention provides a polynucleotide comprising a GBA nucleotide sequence encoding a GCase protein or a fragment thereof, wherein the GBA sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 1. Optionally, the sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to SEQ ID NO: 1 is codon-optimized.

[0091] Non-codon-optimized coding sequence

[0092] In one embodiment, the GBA nucleotide sequence comprises a portion that is not codon-optimized. The portion that is not codon-optimized may be a continuous portion.

[0093] Thus, as is understood in the art, a non-codon optimized portion has not been modified to include a greater number of preferred codons compared to the wild-type sequence. The contiguous non-codon optimized polynucleotide sequence is the wild-type sequence.

[0094] Optionally, the non-codon optimized portion is at least 80, at least 90, at least 100, at least 110, less than 200, less than 170, less than 140, or about 117 nucleotides. In some embodiments, the non-codon optimized portion of the GBA nucleotide sequence encodes (corresponds to) all or a portion of the signal peptide. Optionally, the non-codon optimized portion encodes all or a portion of the GCase signal peptide. In some embodiments, the non-codon optimized portion of the GBA nucleotide sequence is a portion having a sequence of SEQ ID NO: 17.

[0095] The polynucleotide may further comprise a transcriptional regulatory element

[0096] The polynucleotide may comprise transcriptional regulatory elements.

[0097] In one embodiment, the transcriptional regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 10. In one embodiment, the polynucleotide comprises a transcriptional regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 10. Optionally, the polynucleotide comprises a transcriptional regulatory element that is at least 98% identical to SEQ ID NO: 10. Optionally, the polynucleotide comprises the transcriptional regulatory element of SEQ ID NO: 10. Optionally, the polynucleotide comprises a transcriptional regulatory element consisting of SEQ ID NO: 10.

[0098] In another embodiment, the transcriptional regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14. In one embodiment, the polynucleotide comprises a transcriptional regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14. Optionally, the polynucleotide comprises a transcriptional regulatory element that is at least 98% identical to SEQ ID NO: 14. Optionally, the polynucleotide comprises the transcriptional regulatory element of SEQ ID NO: 14. Optionally, the polynucleotide comprises a transcriptional regulatory element consisting of SEQ ID NO: 14.

[0099] Any suitable transcriptional regulatory element may be used, such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, and LSP, which are all liver-specific transcriptional regulatory elements. These transcriptional regulatory elements are described in more detail in the following references: HLP1: McIntosh J. et al., Blood 2013 Apr 25, 121(17): 3335-44; LP1: Nathwani et al., Blood. 2006 April 1, 107(7): 2653-2661; HCR-hAAT: Miao et al., Mol Ther. 2000; 1: 522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637-645(1996); and LSP: Wang et al., Proc Natl Acad Sci US A. 1999 March 30, 96(7): 3906-3910.

[0100] The transcriptional regulatory element may comprise a promoter and / or enhancer, such as promoter elements and / or enhancer elements from HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, and LSP. Each of these transcriptional regulatory elements comprises a promoter, an enhancer, and optionally other nucleotides.

[0101] In one embodiment, the transcriptional regulatory element includes an enhancer, and the enhancer is human apolipoprotein E (ApoE) liver locus control region (HCR; Miao et al (2000), Molecular Therapy 1 (6): 522) or its fragment. In one embodiment, the transcriptional regulatory element includes the fragment of the HCR enhancer, and the fragment is a length of at least 80, at least 90, at least 100, less than 192, 80 to 192, 90 to 192, 100 to 250 or 117 to 192 nucleotide. Optionally, the fragment length of the HCR enhancer is 100 to 250 nucleotide. In another embodiment, the fragment of the HCR enhancer is a length of at least 150, at least 190, at least 230, less than 400, 150 to 400, 190 to 370, 230 to 340, 250 to 340 or about 321 nucleotide. Optionally, the fragment of the HCR enhancer is 250 to 340 nucleotides in length.

[0102] Suitable HCR enhancer element fragments are described in SEQ ID NOs: 11 and 16. Optionally, the transcriptional regulatory element comprises an enhancer that is at least 80, at least 90, at least 100, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides in length, and the enhancer comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 11. Optionally, the transcriptional regulatory element comprises an enhancer that is 117 to 192 nucleotides in length, and the enhancer comprises a polynucleotide sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO. 11. Optionally, the transcriptional regulatory element comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 90, at least 100, or at least 110 nucleotides of SEQ ID NO: 11. Optionally, the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 11. Optionally, the polynucleotide comprises an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 11. Optionally, the polynucleotide comprises the enhancer of SEQ ID NO: 11. Optionally, the transcriptional regulatory element comprises an HCR enhancer fragment that is equal to or less than 321 nucleotides, equal to or less than 192 nucleotides, or equal to or less than 117 nucleotides in length and comprises SEQ ID NO: 11.

[0103] In another embodiment, the transcriptional regulatory element comprises an enhancer that is at least 150, at least 190, at least 230, less than 400, 150 to 400, 190 to 370, 230 to 340, 250 to 340, or about 318 nucleotides in length, and the enhancer comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 16. Optionally, the transcriptional regulatory element comprises an enhancer that is 250 to 340 nucleotides in length, and the enhancer comprises a polynucleotide sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 16. Optionally, the transcriptional regulatory element comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 250 nucleotides of SEQ ID NO: 16. Optionally, the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 16. Optionally, the polynucleotide comprises an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 16. Optionally, the polynucleotide comprises the enhancer of SEQ ID NO: 16.

[0104] In one embodiment, the transcriptional regulatory element comprises a promoter that is a human alpha-1 antitrypsin promoter (A1AT; Miao et al (2000), Molecular Therapy 1(6):522) or a fragment thereof. Optionally, the fragment of the A1AT promoter is at least 100, at least 120, at least 150, at least 180, less than 255, 100 to 255, 150 to 225, 150 to 300, or 180 to 255 nucleotides in length. Optionally, the fragment of the A1AT promoter is 150 to 300 nucleotides in length. In another embodiment, the fragment of the A1AT promoter is at least 200, at least 250, at least 300, less than 500, 200 to 500, 250 to 500, or 350 to 450 nucleotides in length. Optionally, the fragment of the A1AT promoter is 350 to 450 nucleotides in length.

[0105] Suitable A1AT promoter fragments are described in SEQ ID NOs: 12 and 15. Optionally, the transcriptional regulatory element comprises a promoter that is at least 100, at least 120, at least 150, at least 180, less than 255, 100 to 255, 150 to 300, or 180 to 255 nucleotides in length, and the promoter comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 12. Optionally, the transcriptional regulatory element comprises a promoter that is 180 to 255 nucleotides in length, and the promoter comprises a polynucleotide sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 12. Optionally, the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 100, at least 120, or at least 150 nucleotides of SEQ ID NO: 12. Optionally, the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 12. Optionally, the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 12. Optionally, the polynucleotide comprises the promoter of SEQ ID NO: 12. Optionally, the transcriptional regulatory element comprises an A1AT promoter having a length of equal to or less than 418 nucleotides, equal to or less than 255 nucleotides, or equal to or less than 185 nucleotides and comprises SEQ ID NO:12.

[0106] In another embodiment, the transcriptional regulatory element comprises a promoter of at least 200, at least 250, at least 300, less than 500, 200 to 500, 250 to 500, 350 to 450, or about 418 nucleotides in length, and the promoter comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15. Optionally, the transcriptional regulatory element comprises a promoter of 350 to 450 nucleotides in length, and the promoter comprises a polynucleotide sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15. Optionally, the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 350 nucleotides of SEQ ID NO: 15. Optionally, the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15. Optionally, the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 15. Optionally, the polynucleotide comprises the promoter of SEQ ID NO: 15.

[0107] If it is intended that polynucleotide be expressed in the liver, the promoter can be a liver-specific promoter. Optionally, the promoter is a human liver-specific promoter. A "liver-specific promoter" is a promoter that provides expression at a higher level in hepatocytes than in other general cells. For example, those skilled in the art can determine whether a promoter is a liver-specific promoter by comparing the expression of the polynucleotide in hepatocytes (such as Huh-7 cells) with the expression of the polynucleotide in cells from other tissues. If the expression level in hepatocytes is higher than in cells from other tissues, the promoter is a liver-specific promoter. Optionally, if a transcriptional regulatory element or promoter promotes higher levels of protein expression in hepatocytes compared to cells from at least one other organ or tissue and the level at which the transcriptional regulatory element or promoter promotes protein expression in cells from at least one other organ or tissue is less than 40%, less than 30%, less than 25%, less than 15%, less than 10% or less than 5% of the level at which the transcriptional regulatory element or promoter promotes protein expression in hepatocytes, the transcriptional regulatory element or promoter is liver-specific. Optionally, the cell from at least one other organ or tissue is at least one of nephrocytes, pancreatic cells, mammary cells, neuroblastoma cells, pneumonocytes and early B cells. Optionally, the cell from at least one other organ or tissue is at least one of nephrocytes, pancreatic cells, mammary cells, neuroblastoma cells, pneumonocytes and early B cells. Optionally, the cell from at least one other organ or tissue is at least one of HEK293T cells, PANC1 cells, BxPC-3 cells, MCF7 cells, 1643 cells, MRC-9 cells and 697 cells. Optionally, the cell from at least one other organ or tissue is at least one of HEK293T cells, PANC1 cells, BxPC-3 cells, MCF7 cells, 1643 cells, MRC-9 cells and 697 cells.

[0108] In one embodiment, the polynucleotides of the present invention can provide GCase that is specifically expressed in the liver. In such instances, the polynucleotides can promote significantly greater GCase expression in hepatocytes compared to at least one other tissue type or organ. In one example, the polynucleotides of the present invention that provide GCase that is specifically expressed in the liver comprise a liver-specific promoter.

[0109] Optionally, the polynucleotides of the invention can provide GCase expressed at a higher level in hepatocytes compared to cells from at least one other organ or tissue, and such that GCase is expressed in the one other organ or tissue at a level that is less than 40%, less than 30%, less than 25%, less than 15%, less than 10% or less than 5% of the level of GCase expression in hepatocytes when measured in the same assay.

[0110] Optionally, the cell from at least one other organ or tissue is at least one of nephrocytes, pancreatic cells, mammary cells, neuroblastoma cells, pneumonocytes and early B cells. Optionally, the cell from at least one other organ or tissue is at least one of nephrocytes, pancreatic cells, mammary cells, neuroblastoma cells, pneumonocytes and early B cells. Optionally, the cell from at least one other organ or tissue is at least one of HEK293T cells, PANC1 cells, BxPC-3 cells, MCF7 cells, 1643 cells, MRC-9 cells and 697 cells. Optionally, the cell from at least one other organ or tissue is at least one of HEK293T cells, PANC1 cells, BxPC-3 cells, MCF7 cells, 1643 cells, MRC-9 cells and 697 cells.

[0111] Viral particles containing polynucleotides

[0112] The present invention further provides a viral particle comprising a recombinant genome comprising a polynucleotide of the present invention. For the purposes of the present invention, the term "viral particle" refers to all or part of a viral particle. For example, a viral particle comprises a recombinant genome and may further comprise a capsid. The viral particle can be a gene therapy vector. As used herein, the terms "viral particle" and "vector" are used interchangeably. For the purposes of this application, a "gene therapy" vector is a viral particle that can be used for gene therapy, i.e., a viral particle that contains all the functional elements required for expression of a transgene (e.g., a GBA nucleotide sequence) upon administration to a host cell.

[0113] Suitable viral particles include parvovirus, retrovirus, lentivirus or herpes simplex virus. Parvovirus can be adeno-associated virus (AAV). Viral particles are preferably recombinant adeno-associated virus (AAV) vectors or lentiviral vectors. More preferably, the viral particles are AAV viral particles. Unless the context indicates otherwise, the terms AAV and rAAV are used interchangeably herein.

[0114] The genomic structure of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides in length. Inverted terminal repeat (ITR) sequences flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, VP2, and VP3) form the capsid. The terminal ~145 nt (ITR) are self-complementary and are organized to form an energetically stable intramolecular duplex that forms a T-shaped hairpin. These hairpin structures act as origins of viral DNA replication, serving as primers for the cellular DNA polymerase complex. Upon wild type (wt) AAV infection in mammalian cells, the Rep genes (i.e., encoding Rep78 and Rep52 proteins) are expressed from the P5 promoter and the P19 promoter, respectively, and both of these Rep proteins play a role in the replication of the viral genome. Splicing events in the Rep ORF result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that the Rep78 and Rep52 proteins encoded by the unspliced mRNA in mammalian cells are sufficient to produce AAV vectors. Likewise, in insect cells, the Rep78 and Rep52 proteins are sufficient to produce AAV vectors.

[0115] The recombinant viral genome of the present application can comprise an ITR. An AAV vector of the present application can function with only one ITR. Thus, the viral genome comprises at least one ITR, but more typically two ITRs (one at either end of the viral genome, i.e., one at the 5' end and one at the 3' end). There can be intervening sequences between the polynucleotide of the present application and the one or more ITRs. The polynucleotide can be incorporated into a viral particle between two conventional ITRs or on either side of an engineered ITR with two D regions.

[0116] The AAV sequences that can be used to generate AAV vectors in the present invention can be derived from the genome of any AAV serotype. In general, AAV serotypes have genomic sequences that share significant homology at the amino acid and nucleic acid levels, provide a set of identical genetic functions, produce physically and functionally substantially equivalent virions, and replicate and assemble via nearly identical mechanisms. For an overview of the genomic sequences and genomic similarities of various AAV serotypes, see, for example, GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716; Chiorini et al, 1997; Srivastava et al, 1983; Chiorini et al, 1999; Rutledge et al, 1998; and Wu et al, 2000. AAV serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, or 12 can be used in the present invention. Sequences from AAV serotypes may be mutated or engineered when used to produce gene therapy vectors.

[0117] Optionally, the AAV vector comprises an ITR sequence derived from AAV1, AAV2, AAV4 and / or AAV6. Preferably, the ITR sequence is an AAV2 ITR sequence. In this article, the term AAVx / y refers to a viral particle comprising a genomic component, such as at least an ITR from AAVx (wherein x is the AAV serotype number) and having a capsid from AAVy (wherein y is the number of the same or different serotype). For example, an AAV2 / 8 vector can comprise a portion of the viral genome (including ITRs) from an AAV2 strain and a capsid from an AAV8 strain.

[0118] In one embodiment, the viral particle is an AAV viral particle comprising a capsid. AAV capsids are generally formed from three proteins, VP1, VP2, and VP3. The amino acid sequence of VP1 comprises the sequence of VP2. The portion of VP1 that is not part of VP2 is referred to as VP1 unique or VP1U. The amino acid sequence of VP2 comprises the sequence of VP3. The portion of VP2 that is not part of VP3 is referred to as VP2 unique or VP2U. Optionally, the viral particle comprises a hepatotrophic capsid or a CNS-tropic capsid. Whether a viral particle (capsid) is tropic for a particular tissue can be assessed, for example, by administering such particles expressing a marker gene such as luciferase and performing in vivo imaging at multiple time points (e.g., as described in Zincarelli et al (2008), Molecular Therapy, 16: 1073-1080). Particles that drive strong marker expression in liver or CNS tissue, respectively, particularly if in contrast to less expression in other tissues, will be considered hepatotrophic or CNS-tropic.

[0119] In some embodiments, the hepatotrophic capsid can be an AAV3-derived or AAV3B-derived capsid. Optionally, the hepatotrophic capsid comprises a sequence that is at least 98%, at least 99%, or at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736, or 700 to 736 amino acids of SEQ ID NO: 19, 20, or 24. Optionally, the hepatotrophic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 19. Optionally, the hepatotrophic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 20. Optionally, the hepatotrophic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 24. Optionally, the CNS-tropic capsid comprises a sequence that is at least 98%, at least 99%, at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736, or 700 to 736 amino acids of SEQ ID NO: 21. Optionally, the CNS-tropic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 21. Viral particles of the application can be “hybrid” particles, in which the viral ITR and viral capsid are from different parvoviruses, e.g., different AAV serotypes. Preferably, the viral ITR and capsid are from different AAV serotypes, in which case such viral particles are referred to as transcapsidated or pseudotyped. Likewise, parvoviruses can have “chimeric” capsids (e.g., comprising sequences from different parvoviruses, preferably different AAV serotypes) or “targeted” capsids (e.g., directed tropism).

[0120] In some embodiments, the recombinant AAV genome comprises complete ITRs, comprising functional terminal decomposition sites (TRS). This AAV genome may comprise one or two decomposable ITRs, i.e., the ITR comprises a functional TRS, on which site-specific incisions can occur to produce a free 3' hydroxyl group, which can serve as a substrate for DNA polymerase to unwind and replicate the ITR. Preferably, the recombinant genome is single-stranded (i.e., it is packaged into viral particles in a single-stranded form). Optionally, the recombinant genome is not packaged in a self-complementary configuration, i.e., the genome does not contain a single covalently linked polynucleotide chain with a large number of self-complementary parts annealed in the viral particles. Alternatively, the recombinant genome can be packaged in a "monomer duplex" form. "Monomer duplex" is described in WO 2011 / 122950. The genome can be packaged into two substantially complementary but non-covalently linked polynucleotides that anneal in the viral particles.

[0121] The viral particle may also contain a polyadenylic acid sequence. The polyadenylic acid sequence may be located downstream of the nucleotide sequence encoding the functional GCase protein. The polyadenylic acid sequence may be a bovine growth hormone polyadenylic acid sequence (bGHpA—SEQ ID NO: 23). The polyadenylic acid sequence may be 250 to 270 nucleotides in length.

[0122] The viral particle may further comprise an intron sequence, such as a viral intron sequence, optionally the SV40 intron sequence (SEQ ID NO: 22).

[0123] In one embodiment, the viral particle comprises a polynucleotide sequence comprising a promoter element, an intron sequence (e.g., an SV40 intron sequence), a GBA nucleotide sequence, and a polyadenylation sequence (e.g., a bGHpA sequence). In such an embodiment, the intron sequence (e.g., an SV40 intron sequence) may be located between the promoter element and the GBA nucleotide sequence. In such an embodiment, the polyadenylation sequence (e.g., a bGHpA sequence) may be located downstream of the GBA nucleotide sequence.

[0124] The viral particles of the present invention optionally overexpress GCase in the host cell. For example, the viral particles of the present invention express GCase protein or a fragment thereof at a higher level in Huh-7 cells after transduction compared to an otherwise identical viral particle comprising the GBA nucleotide sequence of SEQ ID NO: 9 transduced into a comparable Huh-7 cell population in comparable amounts. Optionally, after transduction into a Huh-7 cell population, the viral particles of the present invention express GCase protein at a higher level than a viral particle comprising the GBA nucleotide sequence of SEQ ID NO: 9 and the transcriptional regulatory element of SEQ ID NO: 10 or the promoter of SEQ ID NO: 12. Optionally, after transduction into a Huh-7 cell population, the viral particles of the present invention express GCase protein or a fragment thereof at a higher level than a comparable viral particle comprising the GBA nucleotide sequence of SEQ ID NO: 9 and the transcriptional regulatory element of SEQ ID NO: 10 or the promoter of SEQ ID NO: 12 transduced into a comparable Huh-7 cell population in comparable amounts. Optionally, after transduction into a Huh-7 cell population, the viral particles express GCase protein at a level comparable to (i.e., a level that is not statistically significantly different from) a viral particle comprising a GBA nucleotide sequence of SEQ ID NO: 9 and a promoter element of SEQ ID NO: 13 that is transduced into a comparable Huh-7 cell population in comparable amounts. In such embodiments, the term "comparable viral particles" refers to viral particles that are identical to the AAV viral particles of the present invention, except that the comparable viral particles comprise different GBA nucleotide sequences and different transcriptional regulatory elements. Optionally, the comparable viral particles comprise the same transcriptional regulatory elements as the AAV viral particles of the present invention. Optionally, activity is assessed using a chromogenic assay, such as the fluorometric assay discussed above.

[0125] In one embodiment, a viral particle is provided comprising a polynucleotide sequence comprising:

[0126] a) a GBA nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 5, operably linked to:

[0127] b) a transcriptional regulatory sequence having at least 98% sequence identity to SEQ ID NO: 14;

[0128] The viral particle further comprises a capsid that is at least 98% identical to SEQ ID NO: 20.

[0129] In one embodiment, a viral particle is provided comprising a polynucleotide sequence comprising:

[0130] a) a GBA nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 5 operably linked to:

[0131] b) a transcriptional regulatory sequence having at least 98% sequence identity to SEQ ID NO: 10;

[0132] wherein the viral particle further comprises a capsid having at least 98% identity to SEQ ID NO: 20.

[0133] Compositions, methods and uses

[0134] In another aspect of the application, there is provided a composition comprising a polynucleotide or viral particle of the application and a pharmaceutically acceptable excipient.

[0135] The pharmaceutically acceptable excipient can include a carrier, diluent and / or other pharmaceutical agent, pharmaceutical agent or adjuvant, etc. Optionally, the pharmaceutically acceptable excipient includes a saline solution. Optionally, the pharmaceutically acceptable excipient includes human serum albumin.

[0136] The application further provides a method of expressing a GBA nucleotide sequence and achieving stable GCase activity in a subject and / or providing higher bioavailability of GCase in a subject compared to bioavailability from GCase enzyme replacement therapy, wherein the bioavailability is measured over a period of 2 weeks post administration, wherein the method comprises administering to the subject a polynucleotide, viral particle or composition of the application.

[0137] The application further provides a polynucleotide, viral particle or composition of the application for use in a method of treatment. Optionally, the method of treatment comprises administering to a patient an effective amount of a polynucleotide or viral particle of the application.

[0138] The application further provides a method of treatment comprising administering to a patient an effective amount of a polynucleotide or viral particle of the application.

[0139] The application further provides use of a polynucleotide, viral particle or composition of the application in the manufacture of a medicament for a method of treatment. Optionally, the method of treatment comprises administering to a patient an effective amount of a polynucleotide or viral particle of the application. Optionally, the method of treatment is gene therapy. "Gene therapy" involves administration of a viral particle of the application which is capable of expressing a transgene (e.g. a GBA nucleotide sequence) in the host to which it is administered.

[0140] Optionally, the method of treatment is a method of treating a disease associated with GCase deficiency. As discussed above, GCase deficiency may lead to the accumulation of glucocerebroside in macrophages, which infiltrate many vital organs, leading to a variety of diseases, including synucleopathy (as discussed in WO08 / 144591) or Parkinson's disease. Optionally, the method of treatment is a method of treating Parkinson's disease or synucleopathy.

[0141] Optionally, the method of treatment is a method of treating a lysosomal storage disease such as Gaucher disease (GD), e.g., type I, type II, or type III GD. Preferably, the lysosomal storage disease is characterized by bruising, fatigue, anemia, low platelet count, and hepatosplenomegaly. Optionally, the method of treatment is a method of treating GD, e.g., type I GD. In some embodiments, the patient is a patient with GD, e.g., type I GD. Optionally, the patient has an antibody or inhibitor to recombinant GCase (e.g., imiglucerase, velaglucerase alfa, or taliglucerase alfa) that the patient has previously been treated with as part of enzyme replacement therapy. Optionally, the polynucleotide and / or vector / viral particle is administered intravenously. Optionally, the polynucleotide and / or vector / viral particle is administered to the patient only once (i.e., a single dose).

[0142] When GD is "treated" in the above-described methods, it means that one or more symptoms of GD Type I are improved. This does not mean that the symptoms of GD Type I are completely cured so that they are no longer present in the patient, but in certain methods, this may be the case. Therefore, in all cases, the term "treating" can be replaced with the term "improving". The treatment method may result in one or more symptoms of GD Type I being less severe than before treatment. Optionally, the treatment method results in an increase in the amount / concentration of circulating GCase in the patient's blood, and / or an increase in the overall level of GCase activity detectable in a given volume of blood and / or macrophages, relative to the situation before administration. In one embodiment, the treatment method results in one or more of the following, relative to the situation before administration: an increase in hemoglobin concentration; an increase in platelet count; a decrease in spleen size; a decrease in liver size.

[0143] Furthermore, the methods of the present invention can "prevent" diseases such as Gaucher disease. Gaucher disease is often associated with the accumulation of glucocerebrosidase in various tissues, and if the methods of the present invention are performed on young subjects (e.g., adolescents, young adults, children, or infants), it should be possible to prevent the development of Gaucher disease. Therefore, in all cases, the term "treatment" can be replaced with the term "prevention."

[0144] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., increasing the level of functional GCase in a subject (thereby resulting in a level of functional GCase sufficient to ameliorate the symptoms of GD (e.g., type I GD)).

[0145] Optionally, less than 1×10 11 less than 1×10 12 less than 5×10 12 less than 2×10 12 less than 1.5×10 12 less than 3×10 12 less than 1×10 13 less than 2×10 13 or less than 3×10 13 The vector / viral particles are administered at a dose of 100 vector genomes. Optionally, the dose of the vector / viral particles administered is selected so that the subject expresses GCase at a level of 10%-90%, 20%-80%, 30%-70%, 25%-50%, 20%-150%, 30%-140%, 40%-130%, 50%-120%, 60%-110%, or 70%-100% of the level of a healthy subject not suffering from GD.

[0146] Optionally, the patient administered the polynucleotide, viral particle, or composition may have a GCase activity level of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 μmol / h / ml. Optionally, GCase activity is measured using a fluorescent substrate specific for GCase. Optionally, GCase activity is measured fluorescently using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate. Optionally, GCase activity is measured in serum, plasma, macrophages, spleen, liver, and / or bone marrow of the subject.

[0147] In one embodiment, GCase activity can be measured fluorometrically using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate as follows: (1) serum samples or tissues (liver, spleen, bone marrow) are collected, snap-frozen, and lysed; (2) tissue lysates or serum / plasma samples are mixed in 50 mM sodium citrate, 25 mM taurocholate, pH=5.75, and 6 mM 4MU-Glc and incubated at 37°C for 30 minutes; (3) one volume (100 μl) of stop solution (0.5 M glycine, 0.3 M NaOH, pH 10.0) is added to terminate the reaction; (4) relative fluorescence levels (RFUs) are assessed using a Spectramax I3X (Molecular devices) using excitation and emission wavelengths of 365 nm and 445 nm, respectively, and then the fluorescence levels are converted to nanomoles / h / mL based on a 4-methylumbelliferyl (4-MU, Sigma-Aldrich) standard curve.

[0148] Optionally, a patient administered the polynucleotide, viral particle, or composition may have a higher level of GCase activity at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks, or at least 35 weeks after administration, as compared to the activity measured in a subject administered an effective dose of GCase enzyme replacement therapy, when measured in the same assay at the same time point after administration. Optionally, a patient administered the polynucleotide, viral particle, or composition may have a 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold higher level of GCase activity at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks, or at least 35 weeks after administration, as compared to the activity measured in a subject administered an effective dose of GCase enzyme replacement therapy, when measured in the same assay at the same time point after administration.

[0149] Optionally, the dose of the vector / viral particle administered is selected so that the subject has a higher GCase bioavailability compared to the bioavailability from GCase enzyme replacement therapy. Bioavailability can be measured (e.g., estimated or calculated) by any method known in the art. The bioavailability of GCase in the serum, macrophages, spleen, liver, and / or bone marrow of the subject can be measured. In one example, the area under the curve ("AUC") method according to Example 8 can be used to estimate bioavailability. In one example, bioavailability can be estimated by estimating the total GCase activity available in the serum, plasma, macrophages, spleen, liver, and / or bone marrow of the subject. Optionally, it is calculated over a defined time period and refers to the total activity or concentration of GCase within that time period. Optionally, GCase activity is measured using a fluorescent substrate specific for GCase. Optionally, GCase activity is measured fluorescently using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate. Optionally, measure the GCase activity in the serum, plasma, macrophage, spleen, liver and / or bone marrow of the experimenter. Optionally, measure the GCase activity in the leukocyte of the experimenter. Optionally, the bioavailability is measured in the time period of 2 weeks after administration. Optionally, the bioavailability is measured in the time period of 5 weeks after administration. Optionally, measure the bioavailability in the serum. In one example, when measured in the same assay at the same time point after administration, compared with the bioavailability measured in the experimenter of the GCase enzyme replacement therapy using an effective dose, higher GCase bioavailability is achieved in the experimenter in the time period of at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks or at least 35 weeks after administration.

[0150] Optionally, a patient (e.g., a patient suffering from a disease or condition associated with GCase deficiency) administered a polynucleotide, viral particle, or composition of the invention may have reduced hexosylceramide and / or hexosylsphingosine levels after administration, preferably when hexosylceramide and / or hexosylsphingosine levels are measured 6 weeks, 8 weeks, 10 weeks, or 12 weeks after administration. Hexosylceramide and / or hexosylsphingosine levels may be reduced by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 2 to 3-fold, 2 to 4-fold, 2 to 5-fold, 2 to 6-fold, or 3 to 5-fold compared to the (starting) hexosylceramide and / or hexosylsphingosine levels at the time of administration of the polynucleotide, viral particle, or composition of the invention. For example, after administration of the polynucleotide viral particles or compositions of the invention (e.g., 6 weeks, 8 weeks, 10 weeks, or 12 weeks after administration), the patient's hexosylceramide and / or hexosylsphingosine levels may be 50% or less, 40% or less, 30% or less, 25% or less, or 20% or less compared to the (initial) hexosylceramide and / or hexosylsphingosine levels at the time of administration of the polynucleotide, viral particles, or compositions of the invention. Optionally, the patient's hexosylceramide and / or hexosylsphingosine levels may be increased when compared to healthy subjects or subjects without a disease or condition associated with GCase deficiency. For example, the hexosylceramide and / or hexosylsphingosine levels are measured in the spleen, liver, and / or bone marrow of the patient / subject. The hexosylceramide and / or hexosylsphingosine levels can be measured in the patient / subject's serum and / or leukocytes (e.g., macrophages). Methods for measuring hexosylceramide and / or hexosylsphingosine levels are known in the art, and preferably hexosylceramide and / or hexosylsphingosine levels are measured using mass spectrometry (LC / MS analysis), for example, by the methods described in Example 9. Optionally, the reduction in hexosylceramide and / or hexosylsphingosine levels (e.g., in the patient's / subject's serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) is greater than the reduction achieved from GCase enzyme replacement therapy, preferably when the hexosylceramide and / or hexosylsphingosine levels are measured after at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after the start of treatment. For example, the levels of a polynucleotide, viral particle or composition of the invention at least 6 weeks (e.g., at 6 weeks), at least 8 weeks (e.g., at 8 weeks), at least 10 weeks (e.g., at 10 weeks) or at least 12 weeks (e.g., at 12 weeks) after administration can be compared to the levels at least 6 weeks (e.g., at 6 weeks), at least 8 weeks (e.g., at 8 weeks), at least 10 weeks (e.g., at 10 weeks) or at least 12 weeks (e.g., at 12 weeks) from the first administration of GCase enzyme replacement therapy, respectively.As a specific example, the level of hexosylceramide and / or hexosylsphingosine can be measured at least 12 weeks (e.g., at 12 weeks) after the administration of the polynucleotides, viral particles or compositions of the present invention, and compared to the level measured at least 12 weeks (e.g., at 12 weeks) after the first administration of GCase enzyme replacement therapy. Preferably, the level of hexosylceramide and / or hexosylsphingosine is measured in the same assay at the same time point after administration. Optionally, GCase enzyme replacement therapy can be administered every two weeks. Optionally, the reduction in the level of hexosylceramide in the subject (or patient) after the administration of the polynucleotides, viral particles or compositions of the present invention is such that the level of hexosylceramide (e.g., in serum, leukocytes (e.g., macrophages), liver and / or spleen) does not exceed 200%, 150% or 125% of the level of hexosylceramide measured in healthy subjects or subjects not suffering from a disease or condition associated with GCase deficiency. In one example, the reduction in the level of hexosylceramide and / or hexosylsphingosine can represent a reduction in the level of glucosylceramide and / or glucosylsphingosine, respectively. For example, a decrease in hexosylceramide can represent a decrease in glucosylceramide. As a further example, a decrease in hexosylsphingosine levels can represent a decrease in glucosylsphingosine levels.

[0151] In one example, the reduction in hexosylceramide and / or hexosylsphingosine levels is a reduction in glucosylceramide and / or glucosylsphingosine levels, respectively. In other words, a patient (e.g., a patient suffering from a disease or condition associated with GCase deficiency) administering a polynucleotide, viral particle, or composition of the present invention may have reduced glucosylceramide and / or glucosylsphingosine levels after administration, preferably measuring glucosylceramide and / or glucosylsphingosine levels 6 weeks, 8 weeks, 10 weeks, or 12 weeks after administration. Glucosylceramide and / or glucosylsphingosine levels may be reduced by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 2 to 3-fold, 2 to 4-fold, 2 to 5-fold, 2 to 6-fold, or 3 to 5-fold compared to the (initial) glucosylceramide and / or glucosylsphingosine levels when administering a polynucleotide, viral particle, or composition of the present invention. For example, compared to the (initial) glucosylceramide and / or glucosylsphingosine levels at the time of administration of the polynucleotide, viral particle or composition of the present invention, after administration of the polynucleotide viral particle or composition of the present invention (e.g., 6 weeks, 8 weeks, 10 weeks or 12 weeks after administration), the patient's glucosylceramide and / or glucosylsphingosine levels may be 50% or less, 40% or less, 30% or less, 25% or less, 20% or less. Optionally, when compared to healthy subjects or subjects who do not suffer from a disease or condition associated with GCase deficiency, the patient's glucosylceramide and / or glucosylsphingosine levels may be increased. For example, glucosylceramide and / or glucosylsphingosine levels are measured in the spleen, liver and / or bone marrow of the patient / subject. Glucosylceramide and / or glucosylsphingosine levels can be measured in the serum and / or leukocytes (e.g., macrophages) of the patient / subject. Methods for measuring glucosylceramide and / or glucosylsphingosine levels are known in the art, and glucosylceramide and / or glucosylsphingosine levels are preferably measured using mass spectrometry (LC / MS analysis), for example, by the method described in Example 9. Optionally, the reduction in glucosylceramide and / or glucosylsphingosine levels (e.g., in the patient's / subject's serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) is greater than the reduction achieved by GCase enzyme replacement therapy, preferably when glucosylceramide and / or glucosylsphingosine levels are measured after at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after the start of treatment. For example, the level at least 6 weeks (e.g., at 6 weeks), at least 8 weeks (e.g., at 8 weeks), at least 10 weeks (e.g., at 10 weeks), or at least 12 weeks (e.g., at 12 weeks) after administration of the polynucleotide, viral particle or composition of the invention can be compared with the level at least 6 weeks (e.g., at 6 weeks), at least 8 weeks (e.g., at 8 weeks), at least 10 weeks (e.g., at 10 weeks), or at least 12 weeks (e.g., at 12 weeks), respectively, from the first administration of GCase enzyme replacement therapy.As a specific example, glucosylceramide and / or glucosylceramide levels can be measured at least 12 weeks after administration of the polynucleotides, viral particles or compositions of the present invention (e.g., at 12 weeks) and compared to the levels measured at least 12 weeks after the first administration of GCase enzyme replacement therapy (e.g., at 12 weeks). Preferably, glucosylceramide and / or glucosylceramide levels are measured in the same assay at the same time point after administration. Optionally, GCase enzyme replacement therapy can be administered every two weeks. Optionally, the reduction in glucosylceramide levels in the subject (or patient) after administration of the polynucleotides, viral particles or compositions of the present invention is such that the glucosylceramide levels (e.g., in serum, leukocytes (e.g., macrophages), liver and / or spleen) do not exceed 200%, 150% or 125% of the glucosylceramide levels measured in healthy subjects or subjects not suffering from a disease or condition associated with GCase deficiency.

[0152] Optionally, a patient (e.g., a patient suffering from a disease or condition associated with GCase deficiency) administered a polynucleotide, viral particle, or composition of the invention may show a decrease in the number of storage cells and / or activated macrophages in the liver following administration, preferably when the cells are counted at least 6 weeks (e.g., at 6 weeks), at least 8 weeks (e.g., at 8 weeks), at least 10 weeks (e.g., at 10 weeks), or at least 12 weeks (e.g., at 12 weeks) after administration. A decrease in the number of storage cells and / or activated macrophages in the liver may indicate a decrease in inflammation and therefore a therapeutic benefit. The number of activated macrophages can be measured by measuring CD68 阳性 The number of cells can be indicated or estimated. Storage cells and CD68 can be identified by methods known in the art, such as the method described in Example 9. 阳性 cell.

[0153] "GCase enzyme replacement therapy" may refer to any therapy comprising administering a GCase polypeptide to a subject. The GCase polypeptide may be wild-type, such as a GCase polypeptide having the amino acid sequence of SEQ ID NO:25. The GCase polypeptide may be administered at any suitable dose, optionally at a dose of 40 to 100 U / kg BW, 50 to 80 U / kg BW, 60 to 70 U / kg BW, or about 60 U / kg BW. The GCase polypeptide may be administered by any suitable route, optionally by intravenous or subcutaneous injection.

[0154] A GCase activity level of at least X% (e.g., at least 20%) refers to a GCase activity level that is at least X% (e.g., 20%) of the range of normal GCase levels measured from a sample such as spleen or bone marrow. Those skilled in the art will readily understand the meaning of reference to a percentage of normal GCase activity levels, which is determined in routine clinical practice, for example, by comparison with a control sample from a healthy subject.

[0155] The term "stable GCase activity" or "stable GCase activity level" refers to a GCase activity level that remains at or above a certain level for a continuous period of at least 5 weeks. In other words, the activity may fluctuate above the activity level, but as long as it remains above the minimum threshold, it is still considered stable. In some embodiments, the GCase activity level remains at or above a certain level for a continuous period of at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks. For example, if the activity level remains at least 20% for a continuous period of at least 5 weeks, then the patient has a stable GCase activity level of at least 20%. In such an example, the GCase activity level may continue to be at least 20% after at least 5 weeks and thus remain at at least 20% for a cumulative continuous period of at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks. If the GCase activity level remains at or above a certain level for a continuous period of at least 5 weeks, then the patient has a stable GCase activity level. Optionally, a patient administered a polynucleotide, viral particle, or composition may have a stable GCase activity level of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50% relative to the GCase activity of a healthy subject. Optionally, a patient administered a polynucleotide, viral particle, or composition may have a stable GCase activity level of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 μmol / h / ml. Optionally, GCase activity is measured using a fluorescent substrate specific for GCase. Optionally, GCase activity is measured fluorescently using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate. Optionally, GCase activity is measured in the serum, macrophages, spleen, liver, and / or bone marrow of the subject.

[0156] Optionally, the GCase activity level is stable after at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks after administration of the polynucleotide, viral particle, or composition. For example, if the patient has a stable GCase activity level of at least 20% after at least 5 weeks of administration of the polynucleotide, viral particle, or composition, then there is a GCase activity level of at least 20%, wherein the GCase activity level is maintained at at least 20% for a continuous period of at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks after the initial at least 5 weeks of administration.

[0157] Optionally, the level of GCase activity is at or above a certain level (e.g., 20%, 25%, 30%, 35% or 40%; and / or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 μmol / h / ml) at least 5 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks or at least 50 weeks after administration of the polynucleotide, viral particle or composition. For example, about 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 61 weeks, 62 weeks, 63 weeks, 64 weeks, 65 weeks, 66 weeks, 67 weeks, 68 weeks, 69 weeks, 70 weeks, 71 weeks, 72 weeks, 73 weeks, 74 weeks, 75 weeks, 76 weeks, 77 weeks, 78 weeks, 79 weeks, 80 weeks At the time point of 8 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks or 52 weeks, the GCase activity level is at or above a certain level (e.g., 20%, 25%, 30%, 35% or 40%; and / or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 μmol / h / ml).

[0158] The present invention will now be described with reference to the following examples, which are illustrative only and should not be construed in any way as limiting the scope of the invention.

[0159] Sequence Listing

[0160]

[0161] Aspects of the Invention

[0162] The present invention is further described in the following aspects.

[0163] 1. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-glucocerebrosidase (GCase) protein or a fragment thereof, and wherein at least a portion of the GBA nucleotide sequence is not wild type.

[0164] 2. The polynucleotide of aspect 1, wherein the portion of the GBA nucleotide sequence that is not wild-type is codon-optimized.

[0165] 3. The polynucleotide of aspect 1 or 2, wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-8.

[0166] 4. The polynucleotide of any one of aspects 1 to 3, wherein the GBA nucleotide sequence comprises the sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 encoding a GCase protein having GCase activity.

[0167] 5. The polynucleotide of aspect 4, wherein the variant of SEQ ID NO: 1 is identical to SEQ ID NO: 1 except that the variant of SEQ ID NO: 1 comprises nucleotide substitutions such that the GCase protein has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9 or at most 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0168] 6. The polynucleotide of aspect 4 or 5, wherein the variant of SEQ ID NO: 1 has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 20 or at most 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 1.

[0169] 7. The polynucleotide of any one of aspects 4 to 6, wherein the variant of SEQ ID NO: 1 has 1, at most 2, at most 3, at most 4, at most 5 or at most 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 1.

[0170] 8. The polynucleotide of any one of aspects 4 to 7, wherein the variant of SEQ ID NO: 1 has a maximum of 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 and / or encodes a GCase protein having a maximum of 3 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0171] 9. The polynucleotide of any one of aspects 4 to 8, wherein the variant of SEQ ID NO: 1 has a maximum of 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 and / or encodes a GCase protein having a maximum of 2 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0172] 10. The polynucleotide of any one of aspects 4 to 9, wherein the variant of SEQ ID NO: 1 has 1 nucleotide substitution relative to the sequence of SEQ ID NO: 1 and / or encodes a GCase protein having at most 1 amino acid substitution relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0173] 11. The polynucleotide of any one of aspects 1 to 3, wherein the GBA nucleotide sequence comprises the sequence of SEQ ID NO: 5 or a variant of SEQ ID NO: 5 encoding a GCase protein having GCase activity.

[0174] 12. The polynucleotide of aspect 11, wherein the variant of SEQ ID NO: 5 is identical to SEQ ID NO: 5 except that the variant of SEQ ID NO: 5 comprises nucleotide substitutions such that the GCase protein has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9 or at most 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0175] 13. The polynucleotide of aspect 11 or 12, wherein the variant of SEQ ID NO: 5 has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 20 or at most 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 5.

[0176] 14. The polynucleotide of any one of aspects 11 to 13, wherein the variant of SEQ ID NO: 5 has 1, at most 2, at most 3, at most 4, at most 5 or at most 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 5.

[0177] 15. The polynucleotide of any one of aspects 11 to 14, wherein the variant of SEQ ID NO: 5 has a maximum of 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and / or encodes a GCase protein having a maximum of 3 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0178] 16. The polynucleotide of any one of aspects 11 to 15, wherein the variant of SEQ ID NO: 5 has a maximum of 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and / or encodes a GCase protein having a maximum of 2 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0179] 17. The polynucleotide of any one of aspects 11 to 16, wherein the variant of SEQ ID NO: 5 has 1 nucleotide substitution relative to the sequence of SEQ ID NO: 5 and / or encodes a GCase protein having at most 1 amino acid substitution relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0180] 18. The polynucleotide of any of the preceding aspects, wherein the GBA nucleotide sequence encodes a GCase protein having 1, up to 2, up to 3, up to 4 or up to 5 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0181] 19. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence encodes a GCase protein having up to 3 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0182] 20. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence encodes a GCase protein having a maximum of 2 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0183] 21. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence encodes a GCase protein having at most one amino acid substitution relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25.

[0184] 22. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, 1000 to 1494, 1000 to 1611, 1300 to 1494, 1300 to 1611, about 1494, or about 1611 nucleotides of any one of SEQ ID NOs: 1-8.

[0185] 23. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1300 nucleotides of any one of SEQ ID NOs: 1-8.

[0186] 24. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 99% identical to a fragment of at least 1300 nucleotides of any one of SEQ ID NOs: 1-8.

[0187] 25. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 98% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-8.

[0188] 26. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-8.

[0189] 27. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1.

[0190] 28. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 99% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1.

[0191] 29. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 1.

[0192] 30. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 1.

[0193] 31. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 5.

[0194] 32. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 5.

[0195] 33. The polynucleotide of any one of the preceding aspects, wherein at least a portion of the GBA nucleotide sequence is codon-optimized.

[0196] 34. The polynucleotide of aspect 33, wherein the codon-optimized portion of the GBA nucleotide sequence is codon-optimized for expression in human hepatocytes.

[0197] 35. The polynucleotide of aspect 33, wherein the GBA nucleotide sequence is codon-optimized for expression in human hepatocytes.

[0198] 36. The polynucleotide of any one of aspects 2 to 35, wherein the codon-optimized portion of the GBA nucleotide sequence is a contiguous portion.

[0199] 37. The polynucleotide of any one of aspects 2 to 36, wherein the codon-optimized portion of the GBA nucleotide sequence is at least 1000, at least 1200, at least 1300, less than 1494, 1000 to 1494, 1300 to 1494, or about 1494 nucleotides in length.

[0200] 38. The polynucleotide of any one of aspects 2 to 37, wherein the codon-optimized portion of the GBA nucleotide sequence corresponds to a mature GCase protein.

[0201] 39. The polynucleotide of any one of aspects 2 to 38, wherein the codon-optimized portion of the GBA nucleotide sequence does not encode all or part of the signal peptide.

[0202] 40. The polynucleotide of any one of aspects 2 to 39, wherein the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence comprises a reduced number of CpGs compared to the corresponding portion of the wild-type GBA nucleotide sequence.

[0203] 41. The polynucleotide of aspect 40, wherein the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence comprises less than 40, less than 20, less than 18, less than 10 or less than 5 CpGs.

[0204] 42. The polynucleotide of aspect 41, wherein the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence comprises less than 5, less than 4, less than 3 or less than 2 CpGs per 100 nucleotides.

[0205] 43. The polynucleotide of aspect 41 or 42, wherein the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence does not contain CpG.

[0206] 44. The polynucleotide of any one of aspects 40 to 43, wherein the wild-type GBA nucleotide sequence is SEQ ID NO: 9.

[0207] 45. The polynucleotide of any one of aspects 2 to 44, wherein the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, 1000 to 1494, 1300 to 1494 or about 1494 nucleotides of any one of SEQ ID NOs: 1-4.

[0208] 46. ​​The polynucleotide of aspect 45, wherein the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to any one of SEQ ID NOs: 1-4.

[0209] 47. The polynucleotide of aspects 2 to 46, wherein the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, 1000 to 1494, 1300 to 1494 or about 1494 nucleotides of SEQ ID NO: 1.

[0210] 48. The polynucleotide of aspect 47, wherein the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1.

[0211] 49. The polynucleotide of aspects 2 to 48, wherein the codon-optimized portion of the GBA nucleotide sequence is at least 99.5%, at least 99.8% or 100% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1.

[0212] 50. The polynucleotide of aspect 49, wherein the codon-optimized portion of the GBA nucleotide sequence is at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1.

[0213] 51. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a portion that is not codon-optimized.

[0214] 52. The polynucleotide of aspect 51, wherein the non-codon-optimized portion encodes all or a portion of the GCase signal peptide.

[0215] 53. The polynucleotide of aspect 51 or 52, wherein the non-codon optimized portion is at least 80, at least 90, at least 100, at least 110, less than 200, less than 170, less than 140 or about 117 nucleotides.

[0216] 54. The polynucleotide of any one of aspects 51 to 53, wherein the non-codon-optimized portion comprises 1 or more CpGs.

[0217] 55. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide further comprises a transcriptional regulatory element.

[0218] 56. The polynucleotide of aspect 55, wherein the transcriptional regulatory element comprises a liver-specific promoter.

[0219] 57. The polynucleotide of aspect 55 or 56, wherein the transcriptional regulatory element comprises an A1AT promoter or a fragment of an A1AT promoter.

[0220] 58. The polynucleotide of aspect 57, wherein the fragment of the A1AT promoter is at least 100, at least 120, at least 150, at least 180, less than 255, 100 to 255, 150 to 225, 150 to 300, or 180 to 255 nucleotides in length.

[0221] 59. The polynucleotide of aspect 58, wherein the fragment of the A1AT promoter is 180 to 255 nucleotides in length.

[0222] 60. The polynucleotide of any of the preceding aspects, wherein the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 15.

[0223] 61. The polynucleotide of aspect 60, wherein the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 15.

[0224] 62. The polynucleotide of aspect 61, wherein the polynucleotide comprises the promoter of SEQ ID NO: 12 or SEQ ID NO: 15.

[0225] 63. The polynucleotide of any one of aspects 55 to 62, wherein the transcriptional regulatory element comprises a fragment of the A1AT promoter having a length equal to or less than 418 nucleotides, equal to or less than 255 nucleotides, or equal to or less than 185 nucleotides and comprises SEQ ID NO: 12.

[0226] 64. The polynucleotide of any one of aspects 55 to 63, wherein the transcriptional regulatory element comprises an enhancer.

[0227] 65. The polynucleotide of aspect 64, wherein the enhancer is an HCR enhancer or a fragment of an HCR enhancer.

[0228] 66. The polynucleotide of aspect 65, wherein the fragment of the HCR enhancer is a fragment of at least 80, at least 90, at least 100, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides in length.

[0229] 67. The polynucleotide of aspect 66, wherein the fragment length of the HCR enhancer is 117 to 192 nucleotides.

[0230] 68. The polynucleotide of any of the preceding aspects, wherein the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 16.

[0231] 69. The polynucleotide of aspect 68, wherein the polynucleotide comprises an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 16.

[0232] 70. The polynucleotide of aspect 69, wherein the polynucleotide comprises the enhancer of SEQ ID NO: 11 or SEQ ID NO: 16.

[0233] 71. The polynucleotide of any one of aspects 55 to 70, wherein the transcriptional regulatory element comprises a fragment of an HCR enhancer having a length equal to or less than 321 nucleotides, equal to or less than 192 nucleotides, or equal to or less than 117 nucleotides and comprises SEQ ID NO: 11.

[0234] 72. The polynucleotide of any one of aspects 55 to 71, wherein the transcriptional regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 10.

[0235] 73. The polynucleotide of aspect 72, wherein the transcriptional regulatory element has the sequence of SEQ ID NO: 10.

[0236] 74. The polynucleotide of any one of the preceding aspects, wherein:

[0237] (i) the GBA nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1 or 5; and

[0238] (ii) the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 12 and / or an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 11.

[0239] 75. The polynucleotide of any one of the preceding aspects, wherein:

[0240] (i) the GBA nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1 or 5; and

[0241] (ii) the polynucleotide comprises a transcriptional regulatory element that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 10.

[0242] 76. The polynucleotide of aspect 57, wherein the A1AT promoter or fragment of the A1AT promoter is at least 200, at least 250, at least 300, less than 500, 200 to 500, 250 to 500, 350 to 450, or about 418 nucleotides in length.

[0243] 77. The polynucleotide of aspect 76, wherein the A1AT promoter or the fragment of the A1AT promoter has a length of 350 to 450 nucleotides.

[0244] 78. The polynucleotide of aspect 65, wherein the HCR enhancer or fragment of the HCR enhancer is a fragment of at least 150, at least 190, at least 230, less than 400, 150 to 400, 190 to 370, 230 to 340, 250 to 340 or about 321 nucleotides in length.

[0245] 79. The polynucleotide of aspect 78, wherein the HCR enhancer or the fragment of the HCR enhancer is 250 to 340 nucleotides in length.

[0246] 80. The polynucleotide of aspects 55 to 79, wherein the transcriptional regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 14.

[0247] 81. The polynucleotide of aspect 80, wherein the transcriptional regulatory element has the sequence of SEQ ID NO: 14.

[0248] 82. The polynucleotide of aspects 1 to 56 or 76 to 79, wherein:

[0249] (i) the GBA nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1 or 5; and

[0250] (ii) the polynucleotide comprises a transcriptional regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO:14.

[0251] 83. The polynucleotide of aspects 1 to 56 or 76 to 79, wherein:

[0252] (i) the GBA nucleotide sequence comprises a sequence that is at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 1 or 5; and

[0253] (ii) the polynucleotide comprises a promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 15 and / or an enhancer that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 16.

[0254] 84. The polynucleotide of any preceding aspect, wherein the GCase encoded by the GBA nucleotide sequence is expressed at a higher level in human hepatocytes than the GCase encoded by the wild-type GBA nucleotide sequence in an otherwise identical reference polynucleotide.

[0255] 85. The polynucleotide of any of the preceding aspects, wherein the expression level of GCase encoded by the GBA nucleotide sequence in human hepatocytes is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, or at least 1.5-fold compared to the GCase encoded by the wild-type GBA nucleotide sequence in the reference polynucleotide.

[0256] 86. The polynucleotide of aspect 84 or 85, wherein the reference polynucleotide comprises the wild-type GBA nucleotide sequence of SEQ ID NO: 9.

[0257] 87. The polynucleotide of aspect 86, wherein the reference polynucleotide comprises the promoter of SEQ ID NO: 13.

[0258] 88. The polynucleotide of any of the preceding aspects, wherein the GCase encoded by the GBA nucleotide sequence is expressed at a higher level or at a non-statistically significantly different level in human hepatocytes compared to the GCase encoded by an otherwise identical reference polynucleotide comprising the GBA nucleotide sequence of SEQ ID NO: 9 and operably linked to the promoter of SEQ ID NO: 13.

[0259] 89. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide comprises DNA or RNA.

[0260] 90. A viral particle comprising a recombinant genome comprising the polynucleotide of any one of the preceding aspects.

[0261] 91. The viral particle of aspect 90, which is an AAV viral particle, an adenoviral viral particle or a lentiviral viral particle.

[0262] 92. The viral particle of aspect 91, which is an AAV viral particle.

[0263] 93. The viral particle of any one of aspects 90 to 92, wherein the viral particle comprises a hepatotropic capsid or a CNS-tropic capsid.

[0264] 94. The viral particle of aspect 93, wherein the hepatotropic capsid comprises a sequence that is at least 98%, at least 99% or at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736 or 700 to 736 amino acids of SEQ ID NO: 19 or 20.

[0265] 95. The viral particle of aspect 94, wherein the hepatotropic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 19.

[0266] 96. The viral particle of aspect 94, wherein the hepatotropic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 20.

[0267] 97. The viral particle of aspect 93, wherein the CNS-tropic capsid comprises a sequence that is at least 98%, at least 99% or at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736 or 700 to 736 amino acids of SEQ ID NO: 21.

[0268] 98. The viral particle of aspect 97, wherein the CNS-tropic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 21.

[0269] 99. The viral particle of any one of aspects 90 to 98, wherein the recombinant genome further comprises:

[0270] a) AAV2 ITR;

[0271] b) a polyadenylation sequence; and / or

[0272] c) Introns.

[0273] 100. The viral particle of aspect 99, wherein the recombinant genome is single-stranded.

[0274] 101. The viral particle of any one of aspects 90 to 100, wherein upon transduction into Huh-7 cells, the viral particle expresses GCase or a fragment thereof such that the GCase activity in the transduced cells is greater than the activity of GCase or a fragment thereof in cells transduced with otherwise identical viral particles comprising the GBA nucleotide sequence of SEQ ID NO: 9.

[0275] 102. The viral particle of any one of aspects 90 to 101, wherein upon transduction into Huh-7 cells, the viral particle expresses GCase or a fragment thereof such that the GCase activity in the transduced cells is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 20 times the activity of GCase or a fragment thereof in cells transduced with otherwise identical viral particles comprising the GBA nucleotide sequence of SEQ ID NO: 9.

[0276] 103. The viral particle of aspect 101 or 102, wherein the activity is measured using a fluorescent substrate specific for GCase.

[0277] 104. A composition comprising the polynucleotide or viral particle of any one of the preceding aspects and a pharmaceutically acceptable excipient.

[0278] 105. The polynucleotide, viral particle or composition of any preceding aspect for use in a method of treatment.

[0279] 106. The polynucleotide, viral particle or composition for use according to aspect 105, wherein the method of treatment comprises administering to the patient an effective amount of the polynucleotide, composition or viral particle of any one of aspects 1 to 104.

[0280] 107. A method of treatment comprising administering to a patient an effective amount of the polynucleotide, composition or viral particle of any one of aspects 1 to 104.

[0281] 108. Use of the polynucleotide, viral particle or composition of any one of aspects 1 to 104 in the preparation of a medicament for use in a method of treatment.

[0282] 109. The use of aspect 108, wherein the method of treatment comprises administering to the patient an effective amount of the polynucleotide or viral particle of any one of aspects 1 to 104.

[0283] 110. The polynucleotide, viral particle, composition, use or method of any one of aspects 105 to 109, wherein the method of treatment is a method of treating a disease associated with GCase deficiency.

[0284] 111. The polynucleotide, viral particle, composition, use or method of any one of aspects 105 to 109, wherein the method of treatment is a method of treating Parkinson's disease.

[0285] 112. The polynucleotide, viral particle, composition, use or method of any one of aspects 105 to 109, wherein the method of treatment is a method of treating Gaucher disease.

[0286] 113. The polynucleotide, viral particle, composition, use or method of aspect 112, wherein the Gaucher disease is type I Gaucher disease.

[0287] 114. The polynucleotide, viral particle, composition, use or method of aspect 112, wherein the Gaucher disease is type II Gaucher disease.

[0288] 115. The polynucleotide, viral particle, composition, use or method of aspect 112, wherein the Gaucher disease is type III Gaucher disease.

[0289] 116. The polynucleotide, viral particle, composition, use or method of any one of aspects 112 to 115, wherein the patient has antibodies or inhibitors against recombinant GCase, and the patient has previously been treated with recombinant GCase as part of enzyme replacement therapy.

[0290] 117. Use of the polynucleotide, viral particle or composition of any one of aspects 1 to 104 in the preparation of a medicament for achieving stable GCase activity in a subject.

[0291] 118. Use of the polynucleotide, viral particle, or composition of any one of aspects 1 to 104 in the manufacture of a medicament for providing a higher bioavailability of GCase than from GCase enzyme replacement therapy in a subject, wherein bioavailability is measured over a period of 2 weeks after administration.

[0292] 119. A method of achieving stable GCase activity in a subject by administering to the subject the polynucleotide, viral particle, or composition of any one of aspects 1 to 104.

[0293] 120. A method of providing a higher bioavailability of GCase than from GCase enzyme replacement therapy in a subject by administering to the subject the polynucleotide, viral particle, or composition of any one of aspects 1 to 104, wherein bioavailability is measured over a period of 2 weeks after administration.

[0294] 121. The method or use of any one of aspects 117 to 120, wherein achieving stable GCase activity in the subject or providing a higher bioavailability of GCase in the subject treats a disease in the subject.

[0295] 122. The polynucleotide, viral particle, or composition of any one of aspects 1 to 104 for use in a method of expressing a GBA nucleotide sequence and achieving stable GCase activity in a subject.

[0296] 123. The polynucleotide, viral particle, or composition of any one of aspects 1 to 104 for use in a method of expressing a GBA nucleotide sequence and providing a higher bioavailability of GCase than from GCase enzyme replacement therapy in a subject, wherein bioavailability is measured over a period of 2 weeks after administration.

[0297] 124. The polynucleotide, viral particle, or composition for use of aspect 122 or 123, wherein achieving stable GCase activity and / or providing a higher bioavailability of GCase results in treating a disease in the subject.

[0298] 125. The polynucleotide, viral particle, or composition, use, or method of any one of aspects 117 to 124, wherein GCase activity and / or bioavailability is measured using a fluorescent substrate specific for GCase.

[0299] 126. The polynucleotide, viral particle, or composition, use, or method of any one of aspects 117 to 125, wherein GCase activity is measured in serum or plasma of the subject.

[0300] 127. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 126, wherein GCase activity is measured in macrophages of the subject.

[0301] 128. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 127, wherein the GCase activity of the subject is stabilized at a level of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 μmol / h / ml.

[0302] 129. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 128, wherein the GCase activity in the subject is stabilized at a level of at least 3 μmol / h / ml.

[0303] 130. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 129, wherein the GCase activity in the subject is stabilized at a level of at least 5 μmol / h / ml.

[0304] 131. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 130, wherein the GCase activity in the subject is stabilized at a level of at least 9 μmol / h / ml.

[0305] 132. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 131, wherein the method comprises administering to a subject an effective dose of the polynucleotide, viral particle or composition.

[0306] 133. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 132, wherein the stabilized GCase activity is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% of the GCase activity relative to the GCase activity of a healthy subject.

[0307] 134. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 133, wherein the stabilized GCase activity is 10% to 100%, 20% to 90%, 30% to 70%, 40% to 70% or 50% to 70% of the GCase activity relative to the GCase activity of a healthy subject.

[0308] 135. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 134, wherein the stabilized GCase activity is stable for at least 5 weeks after administration.

[0309] 136. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 135, wherein the stabilized GCase activity is stable for at least 10 weeks after administration.

[0310] 137. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 136, wherein the stabilized GCase activity is stable for at least 15 weeks after administration.

[0311] 138. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 137, wherein the stabilized GCase activity is stable for at least 20 weeks after administration.

[0312] 139. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 138, wherein the stabilized GCase activity is stable for at least 25 weeks after administration.

[0313] 140. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 139, wherein the stabilized GCase activity is stable for at least 30 weeks after administration.

[0314] 141. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 140, wherein the stabilized GCase activity is stable for at least 35 weeks after administration.

[0315] 142. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 141, wherein the stabilized GCase activity is stable for at least 40 weeks after administration.

[0316] 143. The polynucleotide, viral particle or composition for use, method or use of any one of aspects 117 to 142, wherein the method achieves higher GCase activity in the liver, spleen and / or bone marrow of the subject at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks or at least 35 weeks after administration when compared to the activity measured in a subject administered an effective dose of a GCase enzyme replacement therapy when measured in the same assay at the same time point after administration.

[0317] 144. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 143, wherein the method achieves higher GCase bioavailability in the liver, spleen and / or bone marrow of the subject over a period of at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks or at least 35 weeks after administration when compared to the bioavailability measured in a subject administered an effective dose of GCase enzyme replacement therapy when measured in the same assay at the same time point after administration.

[0318] 145. The polynucleotide, viral particle or composition, use or method of any one of aspects 118, 120 or 123 to 144, wherein the GCase enzyme replacement therapy comprises administering a GCase polypeptide having the sequence of SEQ ID NO: 25.

[0319] 146. The polynucleotide, viral particle or composition, use or method of aspect 145, wherein the GCase enzyme replacement therapy comprises administering the GCase polypeptide at a dose of 40 to 100, 50 to 80, 60 to 70 or about 60 U / kg BW.

[0320] 147. The polynucleotide, viral particle or composition, use or method of any one of aspects 121 or 124 to 146, wherein the disease is Gaucher disease.

[0321] 148. The polynucleotide, viral particle or composition, use or method of aspect 147, wherein the Gaucher disease is type I Gaucher disease.

[0322] 149. The polynucleotide, viral particle or composition, use or method of aspect 147, wherein the Gaucher disease is type II Gaucher disease.

[0323] 150. The polynucleotide, viral particle or composition, use or method of aspect 147, wherein the Gaucher disease is type III Gaucher disease.

[0324] 151. The polynucleotide, viral particle or composition, use or method of any one of aspects 117 to 150, wherein the patient has antibodies or inhibitors against recombinant GCase, the patient having previously been treated with recombinant GCase as part of enzyme replacement therapy.

[0325] 152. Use of the polynucleotide, viral particle or composition of any one of aspects 1 to 104 in the preparation of a medicament for reducing the level of hexosylceramide and / or hexosylsphingosine in a subject suffering from a disease or condition associated with GCase deficiency.

[0326] 153. A method of reducing hexosylceramide and / or hexosylsphingosine levels in a subject suffering from a disease or disorder associated with GCase deficiency by administering to the subject the polynucleotide, viral particle, or composition of any one of aspects 1 to 104.

[0327] 154. The use or method of aspect 152 or 153, wherein reducing the level of hexosylceramide and / or hexosylsphingosine in the subject treats a disease or condition associated with GCase deficiency.

[0328] 155. The polynucleotide, viral particle or composition of any one of aspects 1 to 104 for use in a method of reducing the level of hexosylceramide and / or hexosylsphingosine in a subject suffering from a disease or condition associated with GCase deficiency, optionally wherein reducing the level of hexosylceramide and / or hexosylsphingosine results in treating the disease or condition associated with GCase deficiency.

[0329] 156. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 155, wherein the level of hexosylceramide and / or hexosylsphingosine is reduced by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 2 to 3-fold, 2 to 4-fold, 2 to 5-fold, 2 to 6-fold or 3 to 5-fold compared to the level of hexosylceramide and / or hexosylsphingosine when the polynucleotide, viral particle or composition of any one of aspects 1 to 104 is administered.

[0330] 157. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 156, wherein the reduction in hexosylceramide and / or hexosylsphingosine levels is greater than the reduction achieved in a subject administered an effective dose of GCase enzyme replacement therapy, optionally when the hexosylceramide and / or hexosylsphingosine levels are measured at least 6 weeks, at least 8 weeks, at least 10 weeks or at least 12 weeks after administration.

[0331] 158. The polynucleotide, viral particle or composition, use or method of aspect 157, wherein the GCase enzyme replacement therapy comprises administering a GCase polypeptide having the sequence of SEQ ID NO: 25.

[0332] 159. The polynucleotide, viral particle or composition, use or method of aspect 158, wherein the GCase enzyme replacement therapy comprises administering the GCase polypeptide at a dose of 40 to 100, 50 to 80, 60 to 70 or about 60 U / kg BW.

[0333] 160. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 159, wherein the level of hexosylceramide and / or hexosylsphingosine is measured in macrophages of the subject.

[0334] 161. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 160, wherein the level of hexosylceramide and / or hexosylsphingosine is measured in the spleen of the subject.

[0335] 162. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 161, wherein the level of hexosylceramide and / or hexosylsphingosine is measured in the liver of the subject.

[0336] 163. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 162, wherein the hexosylceramide and / or hexosylsphingosine levels are measured in the serum of the subject.

[0337] 164. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 163, wherein the hexosylceramide and / or hexosylsphingosine levels are measured by mass spectrometry.

[0338] 165. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 164, wherein the disease is Gaucher disease.

[0339] 166. The polynucleotide, viral particle or composition, use or method of aspect 165, wherein the Gaucher disease is type I Gaucher disease.

[0340] 167. The polynucleotide, viral particle or composition, use or method of aspect 165, wherein the Gaucher disease is type II Gaucher disease.

[0341] 168. The polynucleotide, viral particle or composition, use or method of aspect 165, wherein the Gaucher disease is type III Gaucher disease.

[0342] 169. The polynucleotide, viral particle or composition, use or method of any one of aspects 152 to 168, wherein the patient has antibodies or inhibitors against recombinant GCase, and the patient has previously been treated with recombinant GCase as part of enzyme replacement therapy.

[0343] Further aspects of the invention

[0344] The present invention is also described in the following aspects.

[0345] 1. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-glucocerebrosidase (GCase) protein or a fragment thereof, and wherein at least a portion of the GBA nucleotide sequence is not wild-type, optionally wherein the portion of the GBA nucleotide sequence that is not wild-type is codon-optimized, more optionally wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-8.

[0346] 2. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, 1000 to 1494, 1000 to 1600, 1300 to 1494, 1300 to 1611, about 1494, or about 1611 nucleotides of any one of SEQ ID NOs: 1-8.

[0347] 3. The polynucleotide of any one of aspects 1 to 2, wherein at least a portion of the GBA nucleotide sequence is codon-optimized.

[0348] 4. The polynucleotide of aspect 3, wherein:

[0349] (a) at least a portion of the codon-optimized GBA nucleotide sequence is codon-optimized for expression in human hepatocytes;

[0350] (b) wherein the codon-optimized portion of the GBA nucleotide sequence is a continuous portion;

[0351] (c) the codon-optimized portion of the GBA nucleotide sequence is at least 1000, at least 1200, at least 1300, less than 1494, between 1000 and 1494, between 1300 and 1494, or about 1494 nucleotides in length;

[0352] (d) The codon-optimized portion of the GBA nucleotide sequence corresponds to the mature GCase protein;

[0353] (e) the codon-optimized portion of the GBA nucleotide sequence does not encode all or part of the signal peptide;

[0354] (f) the GBA nucleotide sequence, or the codon-optimized portion of the GBA nucleotide sequence, comprises a reduced number of CpGs compared to the corresponding portion of the wild-type GBA nucleotide sequence; optionally wherein the GBA nucleotide sequence, or the codon-optimized portion of the GBA nucleotide sequence, comprises less than 40, less than 20, less than 18, less than 10 or less than 5 CpGs, more optionally wherein the GBA nucleotide sequence, or the codon-optimized portion of the GBA nucleotide sequence, comprises less than 5, less than 4, less than 3 or less than 2 CpGs per 100 nucleotides, more optionally wherein the GBA nucleotide sequence, or the codon-optimized portion of the GBA nucleotide sequence, contains no CpGs, preferably wherein the wild-type GBA nucleotide sequence is SEQ ID NO: 9; and / or

[0355] (g) the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, between 1000 and 1494, between 1300 and 1494, or about 1494 nucleotides of any one of SEQ ID NOs: 1-4.

[0356] 5. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide sequence comprises a non-codon optimized portion, optionally wherein:

[0357] (a) the non-codon-optimized portion encodes the entire GCase signal peptide or a portion of the GCase signal peptide;

[0358] (b) a non-codon-optimized portion of at least 80, at least 90, at least 100, at least 110, less than 200, less than 170, less than 140, or about 117 nucleotides; and / or

[0359] (c) The non-codon-optimized portion contains one or more CpGs.

[0360] 6. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide further comprises a transcriptional regulatory element, optionally wherein the transcriptional regulatory element comprises a liver-specific promoter and / or enhancer.

[0361] 7. The polynucleotide of aspect 6, wherein the transcriptional regulatory element comprises an A1AT promoter or a fragment of an A1AT promoter, optionally wherein

[0362] (a) the A1AT promoter or a fragment of the A1AT promoter is at least 100, at least 120, at least 150, at least 180, less than 255, 100 to 255, 150 to 225, 150 to 300, or 180 to 255 nucleotides in length, more optionally wherein the fragment of the A1AT promoter is 180 to 255 nucleotides in length;

[0363] (b) the A1AT promoter or a fragment of the A1AT promoter is at least 200, at least 250, at least 300, less than 500, 200 to 500, 250 to 500, 350 to 450, or about 418 nucleotides in length, more optionally wherein the fragment of the A1AT promoter is 350 to 450 nucleotides in length;

[0364] (c) the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 15.

[0365] 8. The polynucleotide of aspect 6 or 7, wherein the enhancer is an HCR enhancer or a fragment of an HCR enhancer, optionally wherein:

[0366] (a) the HCR enhancer or fragment of the HCR enhancer is a fragment of at least 80, at least 90, at least 100, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides in length, more optionally wherein the fragment of the HCR enhancer is 117 to 192 nucleotides in length;

[0367] (b) the HCR enhancer or fragment of the HCR enhancer is at least 150, at least 190, at least 230, less than 400, 150 to 400, 190 to 370, 230 to 340, 250 to 340, or about 321 nucleotides in length, more optionally wherein the fragment of the HCR enhancer is 250 to 340 nucleotides in length

[0368] (c) the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 16.

[0369] 9. The polynucleotide of aspect 6, wherein the transcriptional regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 10 or 14.

[0370] 10. The polynucleotide of any preceding aspect, wherein the GCase encoded by the GBA nucleotide sequence is expressed at a higher level in human hepatocytes than the GCase encoded by the wild-type GBA nucleotide sequence in an otherwise identical reference polynucleotide, optionally wherein the GCase encoded by the GBA nucleotide sequence is expressed at a level in human hepatocytes that is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold or at least 1.5-fold greater than the GCase encoded by the wild-type GBA nucleotide sequence in the reference polynucleotide, more optionally wherein the reference polynucleotide comprises the wild-type GBA nucleotide sequence of SEQ ID NO: 9, optionally wherein the reference polynucleotide comprises the promoter of SEQ ID NO: 13.

[0371] 11. A viral particle comprising a recombinant genome comprising the polynucleotide of any one of the preceding aspects.

[0372] 12. The viral particle of aspect 11, which is an AAV viral particle, an adenoviral viral particle or a lentiviral viral particle, optionally an AAV viral particle.

[0373] 13. The viral particle according to any one of aspects 11-12, wherein the viral particle comprises a hepatotropic capsid or a CNS-tropic capsid.

[0374] 14. The viral particle of aspect 13, wherein the hepatotropic capsid comprises a sequence that is at least 98%, at least 99%, or at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736, or 700 to 736 amino acids of SEQ ID NO: 19, 20, or 24.

[0375] 15. The viral particle of aspect 13, wherein the CNS-tropic capsid comprises a sequence that is at least 98%, at least 99%, or at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736, or 700 to 736 amino acids of SEQ ID NO: 21.

[0376] 16. The viral particle of any one of aspects 11 to 15, wherein the recombinant genome further comprises:

[0377] a) AAV2 ITR;

[0378] b) a polyadenylation sequence; and / or

[0379] c) introns;

[0380] Optionally wherein the recombinant genome is single-stranded.

[0381] 17. The viral particle of any one of aspects 11 to 16, wherein upon transduction into Huh-7 cells, the viral particles express GCase or a fragment thereof such that the GCase activity in the transduced cells is greater than the activity of GCase or a fragment thereof in cells transduced with otherwise identical viral particles comprising the GBA nucleotide sequence of SEQ ID NO: 9, optionally wherein upon transduction into Huh-7 cells, the viral particles express GCase or a fragment thereof such that the GCase activity in the transduced cells is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold or at least 20-fold greater than the activity of GCase or a fragment thereof in cells transduced with otherwise identical viral particles comprising the GBA nucleotide sequence of SEQ ID NO: 9, more optionally wherein the activity is measured using a fluorescent substrate specific for GCase.

[0382] 18. A composition comprising the polynucleotide or viral particle of any one of the preceding aspects and a pharmaceutically acceptable excipient.

[0383] 19. The polynucleotide, viral particle or composition of any preceding aspect for use in a method of treatment.

[0384] 20. The polynucleotide, viral particle or composition for use according to aspect 19, wherein the method of treatment comprises administering to the patient an effective amount of the polynucleotide, composition or viral particle according to any one of aspects 1 to 17.

[0385] 21. The polynucleotide, viral particle or composition for use according to any one of aspects 19 to 20, wherein the method of treatment is a method of treating a disease associated with GCase deficiency.

[0386] 22. The polynucleotide, viral particle or composition for use according to any one of aspects 19 to 20, wherein the method of treatment is a method of treating Parkinson's disease.

[0387] 23. The polynucleotide, viral particle or composition for use according to any one of aspects 19 to 20, wherein the method of treatment is a method of treating Gaucher disease.

[0388] 24. The polynucleotide, viral particle or composition for use according to aspect 23, wherein the Gaucher disease is type I, type II or type III Gaucher disease.

[0389] 25. The polynucleotide, viral particle or composition for use according to any one of aspects 23 to 24, wherein the patient has antibodies or inhibitors against recombinant GCase, the patient having previously been treated with recombinant GCase as part of enzyme replacement therapy. Example

[0390] Example 1 - Method

[0391] Unless otherwise stated, the following general procedures were followed in the examples described below.

[0392] Production of rAAV

[0393] AAV2 / 8 particles were produced by transiently transfecting HEK293T cells with plasmids encoding the AAV Rep and Cap, adenoviral helper functions, and a recombinant genome containing a GBA construct. AAV2 / 8 particles were purified using an aPOROS CaptureSelect affinity column, titrated by qPCR, and characterized by alkaline gel analysis.

[0394] Mouse study design

[0395] AAV viral particles carrying the GBA transgene under the transcriptional control of a hepatocyte-specific promoter were administered into the tail vein of 6-8 week-old wild-type (C57BL / 6) male mice. As described for each study, AAV doses ranged from 6 × 10 11 vg / kg to 6×10 12 vg / kg. For each experiment, an additional group of animals received no treatment and served as a control for treatment efficacy. To assess the kinetics and persistence of transgene expression, serum GCase levels were measured at various time intervals (4, 8, and 12 weeks) after injection. Mice were followed up to 12 weeks after AAV treatment and sacrificed for biochemical and pathological analysis.

[0396] Serum and tissue GBA activity assay

[0397] β-glucocerebrosidase (acid β-glucosidase; GCase) activity was measured fluorometrically using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate. Serum samples were obtained from mouse blood and stored at -80°C. Tissues (liver, spleen, bone marrow) were harvested, snap-frozen, and lysed. β-glucocerebrosidase (acid β-glucosidase, GCase) activity was measured fluorometrically using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate. On the day of the assay, serum was diluted (0.5 μL, 1:50) and assayed in 50 mM sodium citrate, 25 mM taurocholate, pH = 5.75, 6 mM 4MU-Glc at 37°C for 30 minutes. For tissue samples, tissue protein lysates were assayed directly. The reaction was terminated by adding a volume (100 μl) of stop solution (0.5 M glycine, 0.3 M NaOH, pH 10.0). Spectramax I3X (Molecular devices) was used to assess relative fluorescence levels (RFU) using excitation and emission wavelengths of 365 nm and 445 nm, respectively. Fluorescence levels were then converted to nanomoles / h / mL (serum) or nmol / h / mg total protein (tissue) based on a 4-methylumbelliferone (4-MU, Sigma-Aldrich) standard curve.

[0398] Vector genome copy number

[0399] To determine the number of vector genomes per hepatocyte after rAAV injection, DNA was isolated from frozen liver samples using the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's instructions. After DNA isolation, qPCR was performed using a primer set that binds to a region common to the LSP-S and LSP-L promoters, allowing estimation of AAV copy number.

[0400] Immunohistochemistry

[0401] Rabbit anti-human GCase (Abcam ab125065; 1:100) was used to visualize GCase in mouse tissues. Rat anti-F4 / 80 (Abcam ab6640; 1:100) was used to visualize mouse macrophages. Formalin-fixed mouse tissues were deparaffinized with xylene and ethanol washes, followed by antigen retrieval according to the Ventana CC1 product recommendations. Immunohistochemical staining was performed using a Ventana Discovery XT instrument and the Ventana DAB Map Detection Kit (760-124). Sections were counterstained with hematoxylin. FITC-conjugated and Texas Red-conjugated secondary antibodies were used for immunofluorescence staining. DAPI was used to visualize cell nuclei. Signals were visualized using a confocal fluorescence microscope (Zeiss).

[0402] Huh-7 transfection and potency assay

[0403] One day before transfection, the hepatocyte cell line Huh-7 was cultured at 3×10 5 The cell density of 10 cells was plated in 12-well plates. For transfection, FuGENE was used at a ratio of 4 μl per microgram of plasmid and added to Huh-7 cells overnight in the presence of 10% serum (fetal bovine serum, FBS). The transfection medium was replaced and the cells were incubated for 24 hours with a medium supplemented with insulin-transferrin-selenium (ITS, ThermoFisher Scientific) and 25mM Hepes buffer. Huh-7 cell transduction was performed for 24 hours in the presence of serum at a defined multiplicity of infection (MOI), and then the medium was replaced and incubated in fresh medium for 24 hours. As described above, GCase activity was measured using 20 μl of culture medium using 4MU-Glc as a substrate.

[0404] Statistical analysis

[0405] Statistical analysis was performed using Prism 7 (Graph Pad) software. Column analysis was performed by one-way analysis of variance. P values ​​and sample sizes are indicated in the figure legends.

[0406] To roughly estimate bioavailability (AUC), a one-stage decay model equation was used in GraphPad Prism: Y = (Y0-Plateau)*exp(-K*X)+Plateau. Y0 is the value of Y when X (time) is zero, expressed in the same units as Y. Plateau is the value of Y at infinite time, expressed in the same units as Y. K is the rate constant, expressed as the reciprocal of the time units on the X axis (i.e., if X is in minutes, then K is in minutes). -1Tau is the time constant, expressed in the same units as the X-axis and calculated as the inverse of K. Half-life is expressed in the time units of the X-axis, calculated as ln(2) / K. Span is the difference between Y0 and Plateau, expressed in the same units as the Y-values. AUC calculations employ the linear trapezoidal method. AUC is expressed as U*h / L, where one unit is defined as the amount of enzyme required to hydrolyze 1 pmol / h of 4-methylumbelliferyl-beta-D-glucopyranoside substrate at 37°C.

[0407] Example 2 - GBA constructs

[0408] To assess whether a liver-directed gene therapy approach could be used to treat Gaucher disease (GD), the human full-length GBA coding sequence (found at GenBank Accession No. NM_000157.3; SEQ ID NO: 9) was cloned into a liver-specific promoter-driven adeno-associated virus (AAV) vector. In the FLF-PL01 AAV construct Figure 1 A), the GBA wild-type sequence (GBAwt, not codon-optimized) was driven by a liver-specific promoter referred to herein as "LSP-S" (SEQ ID NO: 10). To determine the sequence that would be best for expression, sequences were designed using a variety of different codon-optimization strategies. In one example AAV construct (FLF-PL28), the GBA codon sequence was optimized and driven by the same liver-specific promoter, LSP-S Figure 1 B). The FLF-PL64 construct contains the same GBA codon-optimized sequence as FLF-PL28, but differs in that it contains a longer transcriptional regulatory element referred to herein as "LSP-L" (SEQ ID NO: 14) instead of LSP-S Figure 1 C).

[0409] Example 3 - Wild-type GBA transgene expression analysis

[0410] To assess whether the (wild-type) GBA construct FLF-PL01 would result in liver expression of beta-glucocerebrosidase (GCase) and subsequent secretion into the bloodstream, FLF-PL01 was pseudotyped into AAV2 / 8. rAAV particles were prepared and titrated as described above and characterized by alkaline gel analysis prior to use in mice. Eight-week-old wild-type (C57BL / 6) mice were treated with a single injection of AAV2 / 8-FLF-PL01 at doses of 6 x 1010 11 vg / kg to 6 x 1011 12 vg / kg. Control (naive) mice were untreated. Serum samples were collected at 4, 8, and 12 weeks post-AAV injection for assessment of levels of circulating active GCase. GCase activity was determined as described above and immunohistochemical staining was performed. Sections were counterstained with hematoxylin.

[0411] Injection of wild-type mice with AAV2 / 8-FLF-PL01 resulted in increased expression of human GCase in the liver of treated animals ( Figure 2 A). With the increase of vector dose, the expression level of GCase in liver increased. At the vector dose of 6×10 11 An approximately 12-fold increase was observed in the vg / kg group, and at 2×10 12 A 43-fold increase was observed in the vg / kg group and at 6×10 12 A 57-fold increase was observed in the vg / kg dose group ( Figure 2 B) These data demonstrate that AAV2 / 8-FLF-PL01 drives GCase expression to levels that result in significant GCase release into the blood and potentially entry into macrophages in GD-affected tissues.

[0412] Example 4 - In vitro GCase expression analysis of codon-optimized constructs

[0413] Codon usage tables of various liver-expressed sequences were used to generate GBA sequences that were codon-optimized throughout the entire segment corresponding to the mature GCase protein (but not the signal peptide coding region). With the exception of one such codon-optimized GBA sequence ('FLF-PL36'), the resulting sequences were then further manually modified to remove CpGs, cryptic splice sites, premature stop codons, and unwanted amino acid substitutions. 21 codon-optimized GBA sequences were created and tested for GCase expression levels after transfection of the human hepatocyte cell line Huh-7. Huh-7 cells were plated at 3 × 10 cells per well. 5 The cells were plated onto 12-well plates at a cell density of 100 cells and transfected as described above. GCase activity was measured using 20 microliters of culture medium using 4MU-Glc as a substrate. The results of this analysis allowed the identification of GBA codon optimizations (FLF-PL21, FLF-PL28, FLF-PL30, and FLF-PL36) that demonstrated increased GCase expression relative to the wild-type GBA sequence, FLF-PL01 ( Figure 3 ).

[0414] Example 5 - In vivo GCase expression analysis of codon-optimized constructs

[0415] The four constructs identified in Example 4 (FLF-PL21, FLF-PL28, FLF-PL30, and FLF-PL36) were pseudotyped with AAV2 / 8 and expressed at 2×10 12Wild-type mice were injected with a dose of 1000 vg / kg. The experiment also included the non-codon-optimized construct FLF-PL01 and a construct (FLF-PL37) containing the same wild-type GBA sequence as FLF-PL01, driven by the strong synthetic CAG promoter. Control (naive) mice were untreated. Animals were sacrificed at multiple time points up to 36 weeks after injection, and serum and tissue samples were collected.

[0416] Figure 4 A shows the results of GCase activity 8 weeks after injection in mice injected with a non-codon-optimized GBA sequence driven by the LSP-S promoter (FLF-PL01), codon-optimized GBA constructs also driven by the LSP-S promoter (FLF-PL21, FLF-PL28, FLF-PL30, and FLF-PL36), and a non-codon-optimized GBA sequence driven by the CAG promoter (FLF-PL37). All four GBA codon-optimized constructs showed increased levels of GCase activity in the bloodstream when injected into mice compared to FLF-PL01 ( Figure 4 A). The FLF-PL28 construct exhibited the greatest increase in GCase release into the bloodstream (approximately 6-fold) compared to a non-codon-optimized construct (FLF-PL01) driven by the same LSP-S promoter. Elevated GCase levels driven by FLF-PL28 relative to FLF-PL01 were observed throughout the 36-week study period ( Figure 4 B).

[0417] Of particular note, the GCase levels observed in mice injected with FLF-PL28, which contains a liver-specific promoter, were as high as those driven by the FLF-PL37 construct, in which the wild-type GBA sequence was expressed from the ubiquitous and strong CAG promoter ( Figure 4 A).

[0418] At the end of the period, spleens and bone marrow were collected and fixed in formalin before being embedded in paraffin. GBA immunostaining analysis on paraffin sections showed that, consistent with circulating GCase levels, tissue uptake of GCase was increased in mice treated with the FLF-PL28GBA codon-optimized construct compared to the non-codon-optimized construct FLF-PL01 ( Figure 5 ).

[0419] To assess the level of macrophage uptake of FLF-PL28 in the spleen after liver-directed GBA expression, immunofluorescence analysis was performed using antibodies against the mouse pan-macrophage marker F4 / 80 and GBA. The majority of F4 / 80-positive cells showed expression of human-specific GBA, indicating that the majority of GCase uptake in the spleen occurred in macrophages ( Figure 6 ).

[0420] Example 6 - Analysis of the effect of promoters on GCase activity in vivo

[0421] To test whether promoter engineering could further increase expression of the GBA codon-optimized sequence, the GBA construct from FLF-PL28 was placed under a liver-specific promoter (referred to herein as "LSP-L"; SEQ ID NO: 14) to generate construct FLF-PL64 (Example 2, Figure 1 C).

[0422] This new construct was used to prepare AAV2 / 8 vectors and was expressed at 2 × 10 12 vg / kg was injected into wild-type mice. Control (naive) mice were left untreated. After 5 weeks, the animals were sacrificed and serum and tissues were collected.

[0423] Analysis of GCase activity in serum showed that AAV2 / 8-FLF-PL64 resulted in increased GCase expression in the bloodstream of mice compared with mice treated with AAV2 / 8-FLF-PL28 (approximately 2.5-fold, P = 0.0001, one-way ANOVA) ( Figure 7 ).

[0424] Like construct FLF-PL28, FLF-PL64 allows robust uptake of GCase into GD target tissues such as spleen, bone marrow, and lung ( Figure 8 ).

[0425] Example 7 - Liver Expression Selectivity of AAV Vectors with GBA Constructs

[0426] To analyze the selectivity of the LSP-L promoter for hepatic cell lines, eight human cell lines derived from various tissues were selected. The table below summarizes the details of each cell line and its origin.

[0427] Table 1. Human cell lines evaluated in this example.

[0428] cell lines source Growth Source species HUH-7 Hepatocellular carcinoma (liver) Adherence people HEK293T kidney Adherence people PANC-1 Pancreatic (epithelioid carcinoma) Adherence people BxPC-3 Pancreas (adenocarcinoma) Adherence people MCF7 Breast (epithelial; adenocarcinoma) Adherence people 1643 neuroblastoma Adherence people MRC-9 Normal lung fibroblasts (embryonic) Adherence people 697 B-cell leukemia (early B-cell) Suspension people

[0429] The eight human cell lines described in Table 1 above were grown in DMEM, IMDM, or RPMI medium supplemented with 10% FBS. For each cell line, AAV-FLF-PL64 (AAV with hepatotropic capsid = SEQ ID NO: 20) was used at 1×10 5 The multiplicity of infection (MOI) of Vg / cell was 2×10 4 cells / well. All experiments were performed in duplicate. Suspension cells were counted and transduced into 48-well plates in serum-free medium (300 μl / well). For adherent cell lines, the medium was aspirated, then washed with PBS (1X) and treated with 5 ml TripLE for 5 minutes at 37°C, 5% CO2 to dissociate the cells. The reaction was terminated by adding 5 ml of complete medium. Countess TM The dissociated cells were counted using a II automated cell counter (ThermoFisher) and centrifuged (250 × g for 5 min) and then counted at 2 × 10 5 The cells were resuspended in complete medium at a density of 10 cells / ml. These cells were plated into 96-well plates (2×10 4 Cells were grown in 4% paraformaldehyde (50 cells / well) to allow attachment for 5 hours prior to transduction. The transduction mix was prepared in X-VIVO medium (50 μl / well) and added to the cells. Three hours later, 100 μl / well of complete medium was added. One day after transduction, the medium for each cell line was replaced with complete medium (+25 mM HEPES for secretion analysis).

[0430] GCase activity was determined by fluorescence using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc) as a substrate.

[0431] GCase activity was measured from the culture supernatant of each cell line to determine the level of GCase secreted after transduction with AAV-FLF-PL64 ( Figure 10 When the LSP-L promoter drove the GBA transgene, GCase secretion was detected only in the HUH-7 cell line. The observed level of active GCase in HUH-7 cells was approximately 5.0 nmol / h / ml [5.1 ± 0.1 nmol / h / ml]. No detectable levels of active GCase were observed in any of the other cell lines analyzed.

[0432] Example 8 - Comparison with ERT therapy

[0433] The purpose of this embodiment is to combine FLF-PL64 with (60 U / kg BW) were compared when mice were administered a single injection. comprises the same amino acid sequence and similar glycosylation pattern as the native enzyme GCase (i.e. SEQ ID NO: 25) and thus provides a suitable comparison. Patients receiving enzyme replacement therapy (ERT) are typically treated with biweekly intravenous infusions of ERT (infusion duration of 1-2 hours, at a clinical dose of 60 U / kg.

[0434] powder (400 units, Shire) for the preparation of infusion solutions was obtained and kept under refrigeration and protected from light until reconstitution. One vial (400 U) was reconstituted with 4.3 ml of sterile water according to the manufacturer's recommendations to reach a solution of 100 U / ml. After reconstitution, the VPRIV solution was immediately snap-frozen into single-use aliquots and stored at (-80°C) for later use.

[0435] (60 U / kg BW) or FLF-PL64 (formulated as AAV2 / 8 particles, 2 x 10 12 vg / kg) was administered as a single intravenous injection to wild-type mice. The levels of active GCase in serum and tissues were determined at different time points up to 1 week, and at 3 and 5 weeks after injection. The levels of active GCase were determined using 4-methylumbelliferyl-beta-D-glucopyranoside (4MU-Glc) fluorescence.

[0436] As shown in Figure Figure 11 (A), VPRIV was rapidly cleared from the mouse blood. VPRIV reached a Cmax of 12.7 μmol / h / ml at two minutes post-injection; the estimated half-life was approximately 5.6 minutes. Residual levels of active GCase were only detectable in serum at approximately 20 minutes post-injection. These levels remained close to the untreated control group for the remainder of the study. A comparison of VPRIV with FLF-PL64 was performed by analysing mice with stable expression of GCase Figure 11 (B). Treatment with FLF-PL64 also resulted in an increase in the levels of active GCase in the mouse blood (Cmax 9.4 μmol / h / ml) Figure 11 (B). However, although the levels of active GCase were not as high as those observed with post-injection, these levels remained constant for the duration of the study. Table 2 below shows the predicted bioavailability of the mice during the 2-week interval following injection of ERT or FLF-PL64.

[0437] Table 2. Predicted bioavailability (AUC) of C57BL / 6 mice during the 2-week interval following a single injection of ERT (60 U / kg BW) or AAV-FLF-PL64 (2 x 10 12 vg / kg).​​

[0438]

[0439] Figure 12 Shows the application GCase immunostaining in the liver, spleen, and bone marrow of mice treated with AAV2 / 8-FLF-PL64 or AAV2 / 8-FLF-PL64. Representative images of each animal group are shown. DAB (3,3'-diaminobenzidine) was used to visualize GCase, and hematoxylin was used as a counterstain. The FLF-PL64-treated samples were obtained five weeks after injection, while The processed samples were collected as marked. The semi-quantitative analysis of these images is shown in Table 3 below:

[0440] Table 3: Administration of ERT Relative levels of GCase immunoreactivity observed in mouse liver, spleen, and bone marrow after treatment with FLF-PL64 or FLF-PL64. "-" indicates GCase-negative staining; "+" indicates GCase-positive staining.

[0441]

[0442] Example 9 - In vivo studies of therapeutic potential

[0443] 1. Methods

[0444] Mouse methods

[0445] 9V / null mice, carrying the Gba1 mutation D409V / D409V (9V / 9V), were used as a model of Gaucher disease in this study. 9V / null mice have a nearly normal lifespan but exhibit visceral abnormalities (inflammation and storage cells) and substrate accumulation (Xu et al. Am J Pathol. 2003 Nov; 163(5): 2093-101; Xu et al. PLoS One. 2010 May 20; 5(5): e10750). 9V / null mice were generated by crossing mice carrying the Gba1 mutation D409V / D409V (9V / 9V) with Gba1 null / WT mice. Approximately two 9V / null mice were generated per litter. The backgrounds of the 9V / null and WT mice were C57BL / 6, 129SvEvBrd, and FVB. 9V / null mice from multiple litters were randomly assigned to each treatment group on a rolling basis. Both male and female mice were included in each group, attempting to balance sex within each group. All mice were housed under pathogen-free conditions, monitored daily, and weighed weekly. All AAV-treated mice exhibited normal growth and weight gain.

[0446] At the end of the study, mice were euthanized with pentobarbital (100 mg / kg) and perfused transcardially with saline. The liver, spleen, and lungs were then dissected.

[0447] AAV / VPRIV preparation and administration

[0448] Aliquots of AAV8-FLF-PL64 were stored at -80°C. Prior to injection, the aliquots were thawed on ice and diluted with X-VIVO10 (Lonza, pH 7.4, 4°C) and gently mixed by briefly vortexing at low speed. The diluted AAV was kept on ice prior to injection and used within 2 hours.

[0449] Resuspend and aliquot (25, 50, 100 μl) and store at -80 ° C. Before injection, the aliquots were thawed on ice and diluted to the specified dose with acidified X-VIVO 10 (Lonza, pH 5.5, 4 ° C) and gently mixed by briefly vortexing at low speed. The diluted enzyme was kept on ice before injection and used within 2 hours.

[0450] AAV (2 × 10 12 8-week-old 9V / null mice were administered once with the indicated doses of 5 μL / g body weight (BW) of AAV (vg / kg) and vehicle (X-vivo). WT mice were administered with vehicle. AAV and vehicle were administered to mice simultaneously and briefly under isoflurane via the tail vein. Administered via tail vein bolus injection to 9V / null mice, anesthetized with a mixture of isoflurane and oxygen at 60 U / kg and 2.5 μL / g BW in a biobubble chamber, every two weeks for 7 injections starting at 8 weeks of age.

[0451] Tissue collection

[0452] At 12, 16, and 20 weeks of age, blood (approximately 100 μL) was collected from the tail vein into tubes containing 0.5 M EDTA (5 μL). Freshly collected blood samples were kept on ice and separated into plasma within 2 hours for determination of GCase activity. Each plasma collection and activity assay for each treatment group was performed within 2 hours of the scheduled enzyme injection. A separate portion of blood (approximately 400 μL) was processed to isolate white blood cells (WBCs) for GCase activity assay. The collected WBCs were stored at -80°C.

[0453] Tissues (liver, lung, spleen, bone marrow) were collected at the end point of the experiment (20 weeks of age). Tissue collection from each group was performed within 2 hours of the last scheduled enzyme injection. Liver, lung, and spleen samples were divided into four aliquots, three of which were frozen in separate tubes and stored at -80°C prior to GCase activity assays, protein, and substrate analysis. The remaining aliquot was fixed in 10% formalin for histological analysis. Bone marrow cells were collected from the femur and tibia of both legs of the mice, frozen in two tubes, and stored at -80°C for GCase activity and substrate assays.

[0454] GCase activity assay

[0455] Use Precellys Evolution tissue homogenizer in 1% sodium taurocholate and 1% Triton X-100 (Tc / Tx) to homogenize tissue, carry out two cycles (each 20 seconds, 30 seconds interval) at 4 ℃.Cells (bone marrow (BM) and leukocytes (WBC)) are homogenized with ultrasonic treatment at 4 ℃ in 1% Tc / Tx.Tissue and cell lysates (2 μ L) are diluted (5 times) with the reaction buffer (0.025M citrate-phosphate buffer, pH 5.6) in the assay mixture.The diluted lysates (10 μ L) (each sample in triplicate) are loaded onto the reaction plate.With 4-methylumbelliferyl-β-D-pyranoglucoside (4MU-glucose, 4mM) (Biosynth AG, Switzerland) in the presence and absence of 2mM Conduritol B epoxide (Millipore.CA) at 37 ℃, hatch 1 hour fluorescence determination GCase activity. Protein concentration was determined using BCA protein assay reagent (Pierce, Rockford, IL).

[0456] Plasma was diluted in 0.025 M citrate-phosphate buffer, pH 5.6. GCase activity was determined fluorometrically as above using 4-methylumbelliferyl-β-D-glucopyranoside (4MU-glucose, 4 mM) (Biosynth AG, Switzerland).

[0457] Substrate analysis

[0458] Frozen tissue was weighed and homogenized in 3.6 mL of methanol / chloroform / H2O (2:1:0.6 v / v / v). Aliquots (500 μL) of the lysate were subjected to LC / MS analysis. Quantified hexosylceramide and hexosylsphingosine were normalized by tissue weight.

[0459] Plasma was diluted in water (40 μL plasma + 60 μL water) and subjected to LC / MS analysis. Substrate levels were normalized by plasma volume.

[0460] Bone marrow cells were suspended in 200 μL of water, sonicated, and vortexed to prepare a cell lysate. 160 μL of the lysate was subjected to LC / MS analysis. The protein concentration of the remaining lysate was determined. Substrate levels were normalized to mg protein.

[0461] LC / MS analysis was performed to analyze the concentrations of hexosylceramide and hexosylsphingosine. Since galactosylceramide and galactosylsphingosine levels are very low in this mouse model, the measured hexosylceramide and hexosylsphingosine concentrations represent the levels of glucosylceramide and glucosylsphingosine, respectively.

[0462] Histological analysis

[0463] Liver, lung, spleen and bone marrow were dissected from saline-perfused mice, fixed in formalin (10%) and embedded in paraffin.Fixed tissues were cut into 4 μm sections and mounted on slides.

[0464] Storage cell count

[0465] Tissue sections were stained with hematoxylin and eosin (H&E) using an Autostainner (Leica Autostainner XL). The stained tissue was scanned using an Aperio AT2 (Leica, 40x). Tissue images were processed using Aperio ImageScope (V12.4.0.0543). Ten 20x size photographs (500 μm × 800 μm images) were selected from the liver and lungs of each mouse for analysis. Storage cells were counted from each image. The cell count average of the 10 images in the data graph was calculated. The definition of "storage cells" was based on the size of the cells (macrophages), for example, storage cells in the liver were >10 μm in size and storage cells in the lungs were >15 μm in size.

[0466] CD68 staining and quantification

[0467] Tissue sections were stained with rabbit anti-mouse CD68 antibody (1:25. Abcam Ab53444) in a Discover Ultra automated IHC / ISH slide stainer. Tissues were counterstained with hematoxylin for nuclei. Stained tissues were scanned with Aperio AT2 (Leica, 40 times) and images were acquired using Aperio ImageScope (V12.4.0.0543). Liver and lung images of 20 times the size (500 μm × 800 μm) were used for quantitative analysis. IHC signals from 5 liver or lung images of each mouse were analyzed using Image J (Fiji, v5.1). The average CD68 signal for each mouse was calculated for the data graph.

[0468] Statistical analysis

[0469] Data were analyzed by Student's t test or one-way ANOVA. Graphs and statistical analyses were generated using PRISM 8 software (PRISM version 8.0.1).

[0470] 2. Results

[0471] GCase activity

[0472] AAV-FLF-PL64 treatment restored the active GCase level in 9V / null mice by measuring GCase activity in cells and tissues. White blood cell (WBC), bone marrow, and tissue samples were collected at the time of administration when the mice were 20 weeks old.

[0473] Shown to increase the viability of all tested cells and tissues ( Figure 13 ). As mentioned above, Tissues from the treated group were collected within 2 hours after the last injection, and this is consistent with previous data showing that this was During the time period when it is at its C-max in the tissue.

[0474] AAV-FLF-PL64 was also shown to significantly increase GCase activity in all tissues after only a single administration ( Figure 13 Compared to vehicle-9V / null, GCase activity in the liver increased 4.7-fold and in the spleen increased 2.5-fold. In white blood cells, GCase activity in the AAV-FLF-PL64-treated group significantly increased 7-9-fold. Specifically, GCase activity in white blood cells reached approximately 82% of WT activity.

[0475] Histology of tissues

[0476] Visceral pathology in 9V / null mice was determined by counting foamy macrophages as storage cells and quantifying CD68 staining signals on activated macrophages. Storage cells were counted in H&E-stained liver sections. CD68 signal (brown) intensity was quantified in liver and lung sections stained with anti-CD68 antibodies.

[0477] Storage cells ≥10 μm in the liver were counted from 10 images per tissue per mouse. The number of storage cells was not detected in the group ( Figure 14 ).

[0478] In the AAV-FLF-PL64-treated group, CD68 signal in the liver was also significantly reduced. AAV-FLF-PL64 treatment reduced CD68 signal to approximately 25% of vehicle-9V / null levels. In contrast, CD68 signal in the VPRIV group was approximately 37% of vehicle-9V / null levels ( Figure 14 ).

[0479] Substrate accumulation

[0480] 9V / null mice are known to accumulate glycolipid substrates in the liver, lungs, and spleen (Xu et al. PLoS One. 2010 May 20;5(5):e10750). For example, this study showed that hexosylceramide in the control vehicle-9V / null group was 7.97-fold higher in the liver and 3.57-fold higher in the spleen than in the WT group (data not shown).

[0481] Compared with vehicle-9V / null, the AAV-FLF-PL64 treated group showed a significant decrease in hexosylceramide and hexosylsphingosine in the liver and spleen ( Figure 15 Specifically, hexosylceramide levels in the AAV-FLF-PL64-treated group were reduced to 1.20-fold wild-type levels in the liver and 1.03-fold wild-type levels in the spleen (data not shown). A similar reduction to near-WT levels was observed when bone marrow was analyzed (data not shown).

[0482] on the other hand, Treatment showed a significant decrease in hexosylceramide only in the liver, while the hexosylceramide levels in other tested tissues did not change significantly. There did not appear to be any significant effects on hexosylsphingosine levels in any of the tissues tested.

[0483] It should be understood that although the present invention has been described by way of example, these examples are by no means limiting and may be modified within the scope of the appended claims. The preferred features of each embodiment of the present invention are the same as those of each of the other embodiments. All publications cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference as if each individual publication was specifically and individually indicated to be incorporated herein by reference.

Claims

1. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-glucocerebrosidase (GCase) protein or a fragment thereof, and wherein at least a portion of the GBA nucleotide sequence is not wild-type, optionally wherein the portion of the GBA nucleotide sequence that is not wild-type is codon-optimized, more optionally wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises the following sequence: (i) 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-8; (ii) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-8; and / or (iii) a variant of any one of SEQ ID NOs: 1-8 encoding a GCase protein having GCase activity, wherein the variant is identical to SEQ ID NOs: 1-8, respectively, except that the variant comprises nucleotide substitutions such that the GCase protein has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9 or at most 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO:

25.

2. The polynucleotide according to claim 1, wherein the GBA nucleotide sequence comprises the following sequence: (i) 100% identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5; (ii) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.8% identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5; and / or (iii) a variant of SEQ ID NO: 1 or SEQ ID NO: 5 encoding a GCase protein having GCase activity, wherein the variant is identical to SEQ ID NO: 1 or SEQ ID NO: 5, respectively, except that the variant comprises nucleotide substitutions such that the GCase protein has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9 or at most 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO:

25.

3. The polynucleotide according to claim 1 or 2, wherein the variant is a variant of SEQ ID NO: 1, and the variant of SEQ ID NO: 1: (i) identical to SEQ ID NO: 1 except that the variant comprises nucleotide substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25; (ii) has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 20 or at most 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 1; (iii) has 1, at most 2, at most 3, at most 4, at most 5 or at most 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 1; (iv) having up to 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 and / or encoding a GCase protein having up to 3 amino acid substitutions relative to the wild-type amino acid GCase sequence of SEQ ID NO: 25; (v) has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 and / or encodes a GCase protein having up to 2 amino acid substitutions relative to the wild-type amino acid GCase sequence of SEQ ID NO: 25; and / or (vi) having one nucleotide substitution relative to the sequence of SEQ ID NO: 1 and / or encoding a GCase protein having at most one amino acid substitution relative to the wild-type amino acid GCase sequence of SEQ ID NO:

25.

4. The polynucleotide according to claim 1 or 2, wherein the variant is a variant of SEQ ID NO: 5, and the variant of SEQ ID NO: 5: (i) identical to SEQ ID NO: 5 except that the variant comprises nucleotide substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25; (ii) has 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 20 or at most 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 5; (iii) has 1, at most 2, at most 3, at most 4, at most 5, or at most 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 5; (iv) having up to 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and / or encoding a GCase protein having up to 3 amino acid substitutions relative to the wild-type amino acid GCase sequence of SEQ ID NO: 25; (v) has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and / or encodes a GCase protein having up to 2 amino acid substitutions relative to the wild-type amino acid GCase sequence of SEQ ID NO: 25; and / or (vi) having one nucleotide substitution relative to the sequence of SEQ ID NO: 5 and / or encoding a GCase protein having at most one amino acid substitution relative to the wild-type amino acid GCase sequence of SEQ ID NO:

25.

5. The polynucleotide according to any one of the preceding claims, wherein the GBA nucleotide sequence encodes a GCase protein having: (i) up to 3 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25; (ii) up to 2 amino acid substitutions relative to the wild-type GCase amino acid sequence of SEQ ID NO: 25; and / or (iii) a maximum of one amino acid substitution relative to the wild-type GCase amino acid sequence of SEQ ID NO:

25.

6. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, 1000 to 1494, 1000 to 1611, 1300 to 1494, 1300 to 1611, about 1494, or about 1611 nucleotides of any one of SEQ ID NOs: 1-8.

7. The polynucleotide of any one of the preceding claims, wherein at least a portion of the GBA nucleotide sequence is codon-optimized.

8. The polynucleotide according to claim 7, wherein: (a) at least a portion of the codon-optimized GBA nucleotide sequence is codon-optimized for expression in human hepatocytes; (b) the codon-optimized portion of the GBA nucleotide sequence is a continuous portion; (c) the GBA nucleotide sequence is codon-optimized for expression in human hepatocytes; (d) the codon-optimized portion of the GBA nucleotide sequence is at least 1000, at least 1200, at least 1300, less than 1494, between 1000 and 1494, between 1300 and 1494, or about 1494 nucleotides in length; (e) the codon-optimized portion of the GBA nucleotide sequence corresponds to the mature GCase protein; (f) the codon-optimized portion of the GBA nucleotide sequence does not encode all or part of the signal peptide; (g) the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence comprises a reduced number of CpGs compared to a corresponding portion of a wild-type GBA nucleotide sequence; optionally wherein the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence comprises less than 40, less than 20, less than 18, less than 10 or less than 5 CpGs, more optionally wherein the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence comprises less than 5, less than 4, less than 3 or less than 2 CpGs per 100 nucleotides, more optionally wherein the GBA nucleotide sequence or the codon-optimized portion of the GBA nucleotide sequence contains no CpGs, preferably wherein the wild-type GBA nucleotide sequence is SEQ ID NO: 9; (h) the codon-optimized portion of the GBA nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, between 1000 and 1494, between 1300 and 1494, or about 1494 nucleotides of any one of SEQ ID NOs: 1-4; and / or (i) the codon-optimized portion of the GBA nucleotide sequence is at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO:

1.

9. The polynucleotide of any one of the preceding claims, wherein the GBA nucleotide sequence comprises a non-codon-optimized portion, optionally wherein: (a) the non-codon-optimized portion encodes the entire GCase signal peptide or a portion of the GCase signal peptide; (b) the non-codon-optimized portion is at least 80, at least 90, at least 100, at least 110, less than 200, less than 170, less than 140, or about 117 nucleotides; and / or (c) The non-codon-optimized portion comprises one or more CpGs.

10. The polynucleotide according to any one of the preceding claims, wherein the polynucleotide further comprises a transcriptional regulatory element, optionally wherein the transcriptional regulatory element comprises an enhancer and / or a liver-specific promoter.

11. The polynucleotide of claim 10, wherein the transcriptional regulatory element comprises an A1AT promoter or a fragment of an A1AT promoter, optionally wherein: (a) the A1AT promoter or a fragment of the A1AT promoter is at least 100, at least 120, at least 150, at least 180, less than 255, 100 to 255, 150 to 225, 150 to 300, or 180 to 255 nucleotides in length, more optionally wherein the fragment of the A1AT promoter is 180 to 255 nucleotides in length; (b) the A1AT promoter or a fragment of the A1AT promoter is at least 200, at least 250, at least 300, less than 500, 200 to 500, 250 to 500, 350 to 450, or about 418 nucleotides in length, more optionally wherein the fragment of the A1AT promoter is 350 to 450 nucleotides in length; and / or (c) the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 15 or SEQ ID NO:

12.

12. The polynucleotide of claim 10 or 11, wherein the enhancer is a HCR enhancer or a fragment of a HCR enhancer, optionally wherein: (a) the HCR enhancer or the fragment of the HCR enhancer is a fragment of at least 80, at least 90, at least 100, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides in length, more optionally wherein the fragment of the HCR enhancer is 117 to 192 nucleotides in length; (b) the HCR enhancer or the fragment of the HCR enhancer is a fragment of at least 150, at least 190, at least 230, less than 400, 150 to 400, 190 to 370, 230 to 340, 250 to 340, or about 321 nucleotides in length, more optionally wherein the fragment of the HCR enhancer is 250 to 340 nucleotides in length; (c) the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 16 or SEQ ID NO:

11.

13. The polynucleotide of claim 10, wherein the transcriptional regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8% or 100% identical to SEQ ID NO: 14 or 10.

14. The polynucleotide of any preceding claim, wherein the GCase encoded by the GBA nucleotide sequence is expressed at a higher level in human hepatocytes than the GCase encoded by the wild-type GBA nucleotide sequence in an otherwise identical reference polynucleotide, optionally wherein the GCase encoded by the GBA nucleotide sequence is expressed at a level in human hepatocytes that is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, or at least 1.5-fold higher than the GCase encoded by the wild-type GBA nucleotide sequence in the reference polynucleotide, more optionally wherein the reference polynucleotide comprises the wild-type GBA nucleotide sequence of SEQ ID NO: 9, optionally wherein the reference polynucleotide comprises the promoter of SEQ ID NO:

13.

15. A viral particle comprising a recombinant genome comprising the polynucleotide of any one of the preceding claims.

16. The viral particle according to claim 15, which is an AAV viral particle, an adenoviral viral particle or a lentiviral viral particle, optionally an AAV viral particle.

17. The viral particle according to claim 15 or 16, wherein the viral particle comprises a hepatotropic capsid or a CNS-tropic capsid.

18. The viral particle of claim 17, wherein the hepatotropic capsid comprises a sequence that is at least 98%, at least 99%, at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736, or 700 to 736 amino acids of SEQ ID NO: 19, 20, or 24, optionally wherein the hepatotropic capsid comprises a sequence that is at least 99% identical to SEQ ID NO: 19 or 20.

19. The viral particle of claim 17, wherein the CNS-tropic capsid comprises a sequence that is at least 98%, at least 99%, at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, 600 to 736, 650 to 736, or 700 to 736 amino acids of SEQ ID NO: 21, optionally wherein the CNS-tropic capsid comprises a sequence that is at least 99% identical to SEQ ID NO:

21.

20. The viral particle of any one of claims 15 to 19, wherein the recombinant genome further comprises: a) AAV2 ITR; b) a polyadenylation sequence; and / or c) introns; Optionally wherein the recombinant genome is single stranded.

21. The viral particle of any one of claims 15 to 20, wherein upon transduction into Huh-7 cells, the viral particle expresses GCase or a fragment thereof such that the GCase activity in the transduced cells is greater than the activity of GCase or a fragment thereof in cells transduced with an otherwise identical viral particle comprising the GBA nucleotide sequence of SEQ ID NO: 9, optionally wherein upon transduction into Huh-7 cells, the viral particle expresses GCase or a fragment thereof such that the GCase activity in the transduced cells is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold greater than the activity of GCase or a fragment thereof in cells transduced with an otherwise identical viral particle comprising the GBA nucleotide sequence of SEQ ID NO: 9, more optionally wherein the activity is measured using a fluorogenic substrate specific for GCase.

22. A composition comprising a polynucleotide or viral particle according to any one of the preceding claims and a pharmaceutically acceptable excipient.

23. A polynucleotide, viral particle or composition according to any preceding claim for use in a method of treatment.

24. The polynucleotide, viral particle or composition for use according to claim 23, wherein the method of treatment comprises administering to a patient an effective amount of a polynucleotide, composition or viral particle according to any one of claims 1 to 22.

25. The polynucleotide, viral particle or composition for use according to any one of claims 23 to 24, wherein the method of treatment is a method of treating a disease associated with GCase deficiency.

26. The polynucleotide, viral particle or composition for use according to any one of claims 23 to 24, wherein the method of treatment is a method of treating Parkinson's disease.

27. The polynucleotide, viral particle or composition for use according to any one of claims 23 to 24, wherein the method of treatment is a method of treating Gaucher disease, optionally wherein: (i) the Gaucher disease is type I, type II or type III Gaucher disease; and / or (ii) The patient has antibodies or inhibitors against recombinant GCase and the patient has previously been treated with recombinant GCase as part of enzyme replacement therapy.

28. The polynucleotide, viral particle or composition of any one of claims 1 to 22, for use in a method of expressing the GBA nucleotide sequence and achieving stable GCase activity in a subject.

29. The polynucleotide, viral particle or composition of any one of claims 1 to 22 for use in a method of expressing the GBA nucleotide sequence in a subject and providing greater GCase bioavailability than from GCase enzyme replacement therapy, wherein the bioavailability is measured over a period of 2 weeks following administration.

30. The polynucleotide, viral particle or composition for use according to claim 28 or 29, wherein achieving stable GCase activity and / or providing higher GCase bioavailability results in treating a disease in the subject.

31. A polynucleotide, viral particle or composition for use according to any one of claims 28 to 30, wherein: (i) measuring the activity and / or bioavailability of the GCase using a fluorescent substrate specific for GCase; (ii) measuring GCase activity in the serum or plasma of the subject; (iii) measuring GCase activity in macrophages of the subject; (iv) the subject's GCase activity is stabilized at a level of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 μmol / h / ml; (v) the subject's GCase activity is stabilized at a level of at least 3 μmol / h / ml; (vi) the subject's GCase activity is stabilized at a level of at least 5 μmol / h / ml; (vii) the subject's GCase activity is stabilized at a level of at least 9 μmol / h / ml; (viii) the method comprises administering to the subject an effective dose of the polynucleotide, viral particle or composition; (ix) the stable GCase activity is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the GCase activity of a healthy subject; (x) the stable GCase activity is 10% to 100%, 20% to 90%, 30% to 70%, 40% to 70%, or 50% to 70% of the GCase activity of a healthy subject; (xi) the stabilized GCase activity is stable for at least 5 weeks after administration; (xii) the stabilized GCase activity is stable for at least 10 weeks after administration; (xiii) the stabilized GCase activity is stable for at least 15 weeks after administration; (xiv) the stabilized GCase activity is stable for at least 20 weeks after administration; (xv) the stabilized GCase activity is stable for at least 25 weeks after administration; (xvi) the stabilized GCase activity is stable for at least 30 weeks after administration; (xvii) the stabilized GCase activity is stable for at least 35 weeks after administration; (xviii) the stabilized GCase activity is stable for at least 40 weeks after administration; (xix) the method achieves higher GCase activity in the liver, spleen, and / or bone marrow of the subject at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks, or at least 35 weeks after administration, when compared to the activity measured in a subject administered an effective dose of GCase enzyme replacement therapy, when measured in the same assay at the same time point after administration; and / or (xx) the method achieves greater GCase bioavailability in the liver, spleen, and / or bone marrow of a subject for a period of at least 5 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks, or at least 35 weeks after administration, when compared to the bioavailability measured in a subject administered an effective dose of GCase enzyme replacement therapy, when measured in the same assay at the same time point after administration.

32. The polynucleotide, viral particle or composition for use according to any one of claims 30 to 31, wherein the disease is Gaucher disease, optionally wherein the Gaucher disease is Type I, Type II or Type III Gaucher disease.

33. The polynucleotide, viral particle or composition of any one of claims 1 to 22 for use in a method of reducing hexosylceramide and / or hexosylsphingosine levels in a subject suffering from a disease or condition associated with GCase deficiency, optionally wherein reducing hexosylceramide and / or hexosylsphingosine levels results in treating the disease or condition associated with GCase deficiency.

34. The polynucleotide, viral particle or composition for use according to claim 33, wherein: (i) the hexosylceramide and / or hexosylsphingosine levels are reduced by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 2 to 3-fold, 2 to 4-fold, 2 to 5-fold, 2 to 6-fold, or 3 to 5-fold when compared to the levels of hexosylceramide and / or hexosylsphingosine when the polynucleotide, viral particle, or composition of any one of claims 1 to 22 is administered; (ii) the reduction in hexosylceramide and / or hexosylsphingosine levels is greater than the reduction achieved in a subject administered an effective dose of GCase enzyme replacement therapy, optionally when the hexosylceramide and / or hexosylsphingosine levels are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration; (iii) measuring the level of hexosylceramide and / or hexosylsphingosine in macrophages of the subject; (iv) measuring the level of the hexosylceramide and / or hexosylsphingosine in the spleen of the subject; (v) measuring the level of the hexosylceramide and / or hexosylsphingosine in the liver of the subject; (vi) measuring the level of the hexosylceramide and / or hexosylsphingosine in the serum of the subject; (vii) measuring the hexosylceramide and / or hexosylsphingosine levels by mass spectrometry; and / or (viii) the disease is Gaucher disease, optionally wherein the Gaucher disease is Type I, Type II or Type III Gaucher disease.

35. The polynucleotide, viral particle or composition for use according to any one of claims 23 to 34, wherein the patient has antibodies or inhibitors against recombinant GCase, and the patient has previously been treated with recombinant GCase as part of enzyme replacement therapy.

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