Improved stability variant beta-glucocerebrosidase

Abstract

The present invention relates to modified β-glucocerebrosidase (GCase) polypeptides and polynucleotides comprising modified glucocerebrosidase (GBA) nucleotide sequences. The present invention further relates to viral particles comprising recombinant genomes comprising polynucleotides of the invention, and compositions comprising modified GCase polypeptides, polynucleotides, or viral particles of the invention. The present invention also relates to methods and uses of the modified GCase polypeptides, polynucleotides, viral particles, and / or compositions of the invention. The present invention further relates to modified GCase polypeptides, polynucleotides, viral particles, or compositions of the invention for use in a method of treatment or for use in the manufacture of a medicament for use in a method of treatment.

Description

[Technical Field]

[0001] The present invention relates to a modified β-glucocerebrosidase (GCase) polypeptide and a polynucleotide comprising a modified glucocerebrosidase (GBA) nucleotide sequence. The present invention further relates to a viral particle comprising a recombinant genome comprising the polynucleotide of the present invention, and a composition comprising the modified GCase polypeptide, polynucleotide, or viral particle of the present invention. The present invention also relates to methods and uses of the modified GCase polypeptide, polynucleotide, viral particle, and / or composition of the present invention. The present invention further relates to the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention for use in therapeutic methods or for use in the manufacture of pharmaceuticals for use in therapeutic methods. [Background technology]

[0002] Gaucher disease (GD) is an autosomal recessive lipid storage disorder characterized by the deposition of glucocerebrosides in macrophage-monocyte cells. GD is caused by mutations in the housekeeping GBA gene that impair the activity and / or production of the enzyme β-glucocerebrosidase (GCase). GCase is an enzyme with glucosylceramidase activity (EC3.2.1.45) that hydrolyzes the β-glucosidic bond of glucocerebrosides, a chemical intermediate in glycolipid metabolism that is abundant in cell membranes. This mutation can lead to the production of a less active misfolded GCase, which can result in the accumulation of glucocerebrosides in macrophages that infiltrate many vital organs, manifesting as GD.

[0003] There are three main types of GD characterized by specific identified mutations, and each type can present different clinical symptoms. Type 1 GD hardly or does not affect the central nervous system, and is mainly characterized by symptoms of organs such as splenomegaly and hepatomegaly, reduction in blood cell count, bleeding problems, and bone diseases. In the past 20 years, enzyme replacement therapy has emerged as the standard treatment for type 1 GD. In addition to being expensive (about $200,000 or about £150,000 / patient / year), treatment with enzyme replacement therapy for GD generally requires injections more than once every other week throughout life. As a result, the proportion of GD patients with a large treatment burden increases.

[0004] Therefore, there is a need to provide an improved and effective therapy for treating GD, that is, to enhance the availability of functional GCase to the affected target organs and avoid the need for frequent and lifelong intravenous injections of GCase.

Summary of the Invention

[0005] The present invention relates to a modified β-glucosylceramidase (GCase) polypeptide comprising one or more amino acid substitution mutations.

[0006] Therefore, in a first aspect of the present invention, there is provided a modified β-glucosylceramidase (GCase) polypeptide comprising at least one mutation, wherein the at least one mutation (i) provides higher effective activity; and / or (ii) provides increased stability; and / or (iii) provides structural stabilization at physiological pH; and / or (iv) provides a longer half-life; and / or (v) provides increased thermal stability a polypeptide is provided.

[0007] A second aspect of the present invention provides a modified β-glucocerebrosidase (GCase) polypeptide having at least one mutation at a position selected from the group consisting of 351, 380, 272, 262, 313, 404, 407, 482, 484, 490, 494, 503, and 534 of SEQ ID NO: 1.

[0008] A third aspect of the present invention provides a polynucleotide comprising a modified glucocerebrosidase (GBA) nucleotide sequence encoding the modified GCase polypeptide of the present invention.

[0009] A fourth aspect of the present invention provides a viral particle comprising a recombinant genome containing the polynucleotide of the present invention.

[0010] A fifth aspect of the present invention provides a composition comprising a modified GCase polypeptide, polynucleotide, or viral particle of the present invention and a pharmaceutically acceptable excipient.

[0011] A sixth aspect of the present invention provides a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention for use in a therapeutic method.

[0012] A seventh aspect of the present invention provides a therapeutic method comprising administering an effective amount of a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention to a patient.

[0013] An eighth aspect of the present invention provides the use of a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention in the manufacture of a pharmaceutical for use in a therapeutic method.

[0014] In a ninth aspect of the present invention, a modified GCase polypeptide, polynucleotide, viral particle, or composition for use in the treatment of a disease is provided, wherein the modified GCase polypeptide or the encoded modified GCase polypeptide has increased stability at pH 7.4 compared to the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0015] A tenth aspect of the present invention provides a method for treating a disease by administering a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention, wherein the modified GCase polypeptide or the encoded modified GCase polypeptide has increased stability at pH 7.4 compared to the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0016] An eleventh aspect of the present invention provides the use of a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention in the treatment of a disease, wherein the modified GCase polypeptide or the encoded modified GCase polypeptide has increased stability at pH 7.4 compared to the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0017] A twelfth aspect of the present invention provides a modified GCase polypeptide, polynucleotide, viral particle, or composition for use in the treatment of a disease, wherein the treatment of the disease comprises administering a lower dose of the modified GCase polypeptide, polynucleotide, viral particle, or composition compared to the dose required for administering an equivalent polypeptide, polynucleotide, viral particle, or composition comprising or encoding the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0018] A thirteenth aspect of the present invention provides a method for treating a disease by administering a GCase polypeptide, polynucleotide, viral particle, or composition of the present invention, wherein the treatment of the disease is provided in a lower dose of a modified GCase polypeptide, polynucleotide, viral particle, or composition compared to the dose required for administering an equivalent polypeptide, polynucleotide, viral particle, or composition comprising or encoding the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0019] A fourteenth aspect of the present invention provides a use of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention in the treatment of a disease, wherein the treatment of the disease is performed by administering a lower dose of the modified GCase polypeptide, polynucleotide, viral particle, or composition compared to the dose required for administration of an equivalent polypeptide, polynucleotide, viral particle, or composition comprising or encoding the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0020] In a fifteenth aspect of the present invention, a modified GCase polypeptide comprising at least one mutation, (i) Having higher effective activity; and / or (ii) Having increased stability; and / or (iii) Having structural stability at physiological pH; and / or (iv) Having a half-life longer than; and / or (v) Provides increased thermal stability A modified GCase polypeptide is provided.

[0021] A sixteenth aspect of the present invention provides a modified GCase polypeptide comprising at least one mutation, wherein the at least one mutation reduces the number of human leukocyte antigen (HLA)-I and / or HLA-II binders.

[0022] A seventeenth aspect of the present invention provides a modified GCase polypeptide comprising at least one mutation and having a reduced number of HLA-I and / or HLA-II binders.

[0023] In the 18th aspect, in subjects suffering from a disease or condition related to GCase deficiency, (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. The use of a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention in the manufacture of pharmaceuticals is provided.

[0024] In the 19th aspect, by administering the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention to a subject suffering from a disease or condition related to GCase deficiency, the subject will, (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. A method is provided.

[0025] In the 20th aspect, in subjects suffering from a disease or condition related to GCase deficiency, (iv) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (v) Reduce the number of storage cells; and / or (vi) Reduce the level of activated macrophages. A modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention for use in the method, Optional (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. This provides a modified GCase polypeptide, polynucleotide, viral particle, or composition that leads to the treatment of diseases or conditions associated with the GCase deficiency.

[0026] GCase is a lysosomal protein that functions normally and is stable at acidic pH. It is typically unstable at physiological pH (e.g., pH 7.4) with a half-life of only a few minutes. See, for example, Example 2 and Figure 1, which show that the activity of VPRIV (veraglucerase alpha, a commercially available ERT that is a recombinant GCase with the same amino acid sequence as the naturally occurring human GCase polypeptide) dramatically decreases after incubation at pH 7.4. Sequence ID 3 contains the sequence of veraglucerase alpha. Existing enzyme replacement therapy (ERT) is the standard treatment approach for those with GD. ERT typically delivers GCase enzymes intravenously, which means that such treatments encounter problems with effective delivery of GCase enzymes to target organs due to the distance the GCase enzymes must travel and their instability in the blood. We have developed a modified GCase polypeptide with increased stability. We have identified amino acid substitutions in the GCase polypeptide that can increase its stability.

[0027] While we do not wish to be bound by theory, this amino acid mutation is thought to improve stability at physiological pH (e.g., pH 7.4), thereby extending the time that GCase remains stable and ultimately contributing to higher effective activity. The modified GCase polypeptide according to the present invention may have higher residual activity than the wild-type GCase polypeptide under various pH conditions (see, for example, Examples 3 and 4). The modified GCase polypeptide according to the present invention may have a longer half-life under various pH conditions than the wild-type GCase polypeptide (see, for example, Example 5). The modified GCase polypeptide according to the present invention may exhibit higher GCase effective activity than the wild-type GCase polypeptide (see, for example, Examples 8 and 9). The modified GCase polypeptide according to the present invention may have improved thermal stability (see, for example, Example 12). The modified GCase polypeptide according to the present invention may be provided as a protein for enzyme replacement therapy. The modified GCase polypeptide according to the present invention may also be provided by expression in the liver (e.g., in the form of gene therapy) and transport of the protein through the blood to target organs. When the modified GCase polypeptide according to the present invention is provided (for example, in the form of gene therapy), it may be taken up more readily in the relevant tissues than when the wild-type GCase polypeptide is provided (see, for example, Example 10). Furthermore, the inventors have found that the modified GCase polypeptide according to the present invention may have a reduced potential immunogenicity risk compared to the wild-type GCase polypeptide. The modified GCase polypeptide according to the present invention may have a lower predicted number of "potent" binders for HLA-I and a lower predicted number of potent binders for HLA-II (see, for example, Example 7).

[0028] By extending the stable time of GCase and consequently providing higher active efficacy, the modified GCase polypeptide of the present invention can achieve greater therapeutic efficacy when administered at the same or similar doses as less stable GCases (e.g., wild-type GCase). Furthermore, the modified GCase polypeptide of the present invention may achieve the same therapeutic efficacy as less stable GCases (e.g., wild-type GCase) when administered at lower doses. Therefore, treatment costs can be reduced and the safety profile can be improved. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 shows the enzyme activity of VPRIV in PBS (pH 7.4). The residual enzyme activity (residual GCase activity) was measured after incubation with PBS for 0 minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes. The residual enzyme activity at each time point was calculated as a percentage of the initial activity. [Figure 2] Figures 2A and 2B provide a comparison of the residual enzyme activity of various GCase variants (#21, #68, #69, #85, and #21+85; see Table 3), wild-type GCase, and VPRIV (velaglucerase alpha, a recombinant GCase with the same amino acid sequence as naturally occurring human GCase; VPRIV is a commercially available ERT) incubated at 37°C for 7 days under various conditions, namely in AB buffer (pH 5.6) (Figure 2A) and PBS buffer (pH 7.4) (Figure 2B). Residual enzyme activity (residual GCase activity) was measured after incubation for 0 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 3 days, 4 days, 5 days, 6 days, and 7 days. The residual enzyme activity at each time point was calculated as a percentage of the initial activity. [Figure 3]Figures 3A and 3B provide a comparison of the residual enzyme activity of various GCase variants (#21, #85, and #21+85; see Table 3), wild-type GCase, and VPRIV (veraglucerase alpha, a recombinant GCase with the same amino acid sequence as naturally occurring human GCase; VPRIV is a commercially available ERT) incubated at 37°C for 7 days under various conditions, i.e., in human plasma (Figure 3A) and human serum (Figure 3B). Residual enzyme activity (residual GCase activity) was measured after incubation for 0 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 3 days, 4 days, 5 days, 6 days, and 7 days. The residual enzyme activity at each time point was calculated as a percentage of the initial activity. [Figure 4] Figures 4A-4C provide a comparison of the half-lives of purified GCase variant #85 (Figure 4B) and veraglucerase alpha (VPRIV, a recombinant GCase with the same amino acid sequence as naturally occurring human GCase) (Figure 4A) in various physiological matrices, namely AB buffer (pH 5.6), PBS buffer (pH 7.4), mouse serum, mouse plasma, and human serum. Residual enzyme activity (residual GCase activity) was measured after incubation with the physiological matrix for 0 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 24 hours, 48 ​​hours, 120 hours, and 144 hours (not all time points are shown). The residual enzyme activity at each time point was calculated as a percentage of the initial activity. The half-lives (minutes) of purified GCase variant #85 and wild-type GCase (veraglucerase alpha) were determined (Figure 4C). The time calculated for "lysosomal pH" corresponds to the result using AB buffer (pH 5.6), while the time calculated for "physiological pH" corresponds to the result using PBS buffer (pH 7.4). [Figure 5]Figures 5A–C provide a comparison of the enzymatic efficiency (i.e., the ability of the GCase to process the substrate) of GCase variant #85 with the enzymatic efficiency reported by others for wild-type GCase and the enzyme replacement therapy (ERT) imiglucerase and veraglucerase alpha. Enzymatic efficiency was measured by treatment with 4-methylumbelliferyl-β-D-glucopyranosideuronic acid (4-MUG). In Figures 5A and 5B, 1 nM and 3.35 nM of GCase variant #85 were incubated with 2.5 × 10⁶ nM, 5 × 10⁶ nM, 7.5 × 10⁶ nM, or 1 × 10⁷ nM of 4-MUG, respectively. The 4-MU formation rate at each 4-MUG concentration was plotted against 4-MUG concentration and fitted using the Michaelis-Menten model. The rate is measured in nM / sec. Figure 5C provides Kcat(s-1) and Km(mM) values ​​for GCase variant #85, in addition to Km(mM) values ​​reported by others for wild-type GCase and enzyme replacement therapy (ERT) imiglucerase and veraglucerase alfa. [Figure 6] Figure 6 shows the results of an in silico assessment of the potential immunogenicity risk profile of GCase variant #85. Fragments of GCase variant #85 and wild-type GCase (GCaseWT) were evaluated for their binding ability to predicted HLA class I and HLA class II. MHC: Major Histocompatibility Complex, HLA: Human Leukocyte Antigen. [Figure 7]Figure 7 shows the effective GCase activity of numerous GCase variants and wild-type GCase after transduction of AAV2 / '37' into Huh-7 cells (human hepatocyte cell line). The effective GCase activity of GCase variant #21, GCase variant #85, and GCase variant #21+85 were tested. Effective GCase activity is expressed as nmol / hour / mL based on the 4-MU standard curve. The MOIs used for transduction were 5 × 10³ vg / cell, 1 × 10⁴ vg / cell, and 5 × 10⁴ vg / cell. MOI: Infection multiplicity. Untreated controls were also included. Results were normalized by vector genome copy number (determined according to the chapter titled "Vector Genome Copy Number" in Example 1). Normalization was performed across MOIs to account for transduction efficiency. When "GCase activity" is mentioned in the figure, it refers to "effective GCase activity". [Figure 8-1] Figures 8A and 8B show the levels of effective GCase activity in plasma (Figure 8A) and spleen (Figure 8B) after injection of AAV2 / 8 virus particles expressing a sequence encoding one of the following GCase variants: GCase variant #21, GCase variant #85, GCase variant #21+85, or wild-type GCase, into wild-type (C57BL / 6) male mice. Control (naive) mice were left untreated. AAV2 / 8 virus particles were injected into mice at a dose of 6 × 10¹⁰ vg / kg. Animals were sorted 4 weeks after treatment. The level of effective GCase activity is expressed as mU / mL (plasma) or mU / mg (protein) (spleen and bone marrow) according to the VPRIV standard curve. Results were normalized by vector genome copy number (determined according to the chapter titled "Vector Genome Copy Number" in Example 1). When "GCase activity" is mentioned in the figures, it refers to "effective GCase activity". [Figure 8-2]Figure 8C shows the level of effective GCase activity in the bone marrow (Figure 8C) after injection of AAV2 / 8 virus particles expressing a sequence encoding one of the GCase variants GCase variant #21, GCase variant #85, and GCase variant #21+85, or wild-type GCase, into wild-type (C57BL / 6) male mice. Control (naive) mice were left untreated. AAV2 / 8 virus particles were injected into mice at a dose of 6 × 10¹⁰ vg / kg. Animals were sorted 4 weeks after treatment. The level of effective GCase activity is expressed as mU / mL (plasma) or mU / mg (protein) (spleen and bone marrow) according to the VPRIV standard curve. Results were normalized by vector genome copy number (determined according to the chapter titled "Vector Genome Copy Number" in Example 1). When "GCase activity" is mentioned in the figure, it means "effective GCase activity". [Figure 9] Figure 9 shows the levels of GCase in the liver, spleen, lungs, and bone marrow of wild-type (C57BL / 6) male mice after injection with AAV2 / 8 virus particles expressing GCase variant #85 or a sequence encoding wild-type GCase, as determined by immunohistochemical staining. Animals were sorted 4 weeks after treatment. [Figure 10-1]Figures 10A and 10B show the GCase activity levels in plasma (Figure 10A) and spleen (Figure 10B) of wild-type (C57BL / 6) male mice after injection of AAV2 / 8 virus particles expressing a sequence encoding GCase variant #85. Control (naive) mice were left untreated. AAV2 / 8 virus particles were injected into mice at the following doses: 2 × 10⁹ vg / kg, 2 × 10¹⁰ vg / kg, 2 × 10¹¹ vg / kg, 6 × 10¹¹ vg / kg, and 2 × 10¹² vg / kg. For Figure 10A, plasma was obtained 14, 28, and 42 days after treatment. For Figure 10B, animals were selected 6 weeks after treatment. The effective GCase activity level in plasma is expressed in mU / mL (plasma), and the effective GCase activity level in tissues and WBCs is expressed in mU / mg (protein). All of these were determined using the VPRIV standard curve. The results were normalized by the vector genome copy number (determined according to the chapter titled "Vector Genome Copy Number" in Example 1). When "GCase activity" is mentioned in the figures, it refers to "effective GCase activity". [Figure 10-2] Figures 10C-D show the GCase activity levels in the bone marrow (Figure 10C) and lungs (Figure 10D) of wild-type (C57BL / 6) male mice after injection of AAV2 / 8 virus particles expressing the sequence encoding GCase variant #85. Control (naive) mice were left untreated. AAV2 / 8 virus particles were injected into mice at the following doses: 2 × 10⁹ vg / kg, 2 × 10¹⁰ vg / kg, 2 × 10¹¹ vg / kg, 6 × 10¹¹ vg / kg, and 2 × 10¹² vg / kg. For Figures 10C-10D, animals were selected 6 weeks after treatment. Effective GCase activity levels in plasma are expressed in mU / mL (plasma), and effective GCase activity levels in tissue and WBC are expressed in mU / mg (protein), all of which were determined using the VPRIV standard curve. The results were normalized by the vector genome copy number (determined according to the chapter titled "Vector Genome Copy Number" in Example 1). When "GCase activity" is mentioned in the figures, it refers to "effective GCase activity". [Figure 10-3]Figure 10E shows the GCase activity levels in leukocytes (WBCs) (Figure 10E) after injection of AAV2 / 8 virus particles expressing the sequence encoding GCase variant #85 into wild-type (C57BL / 6) male mice. Control (naive) mice were left untreated. AAV2 / 8 virus particles were injected into mice at the following doses: 2 × 10⁹ vg / kg, 2 × 10¹⁰ vg / kg, 2 × 10¹¹ vg / kg, 6 × 10¹¹ vg / kg, and 2 × 10¹² vg / kg. For Figure 10E, animals were selected 6 weeks after treatment. Effective GCase activity levels in plasma are expressed in mU / mL (plasma), and effective GCase activity levels in tissues and WBCs are expressed in mU / mg (protein), all of which were determined using the VPRIV standard curve. The results were normalized by the vector genome copy number (determined according to the chapter titled "Vector Genome Copy Number" in Example 1). When "GCase activity" is mentioned in the figures, it refers to "effective GCase activity". [Figure 11-1] Sequence List [Figure 11-2] Sequence list (continued) [Figure 11-3] Sequence list (continued) [Figure 11-4] Sequence list (continued) [Figure 11-5] Sequence list (continued) [Figure 11-6] Sequence list (continued) [Figure 11-7] Sequence list (continued) [Figure 11-8] Sequence list (continued) [Figure 11-9] Sequence list (continued) [Figure 11-10] Sequence list (continued) [Figure 11-11] Sequence list (continued) [Figure 11-12] Sequence list (continued) [Figure 11-13] Sequence list (continued) [Figure 11-14] Sequence list (continued) [Figure 11-15] Sequence list (continued) [Figure 11-16] Sequence list (continued) [Figure 11-17] Sequence list (continued) [Figure 11-18] Sequence list (continued) [Figure 11-19] Sequence list (continued) [Figure 11-20] Sequence list (continued) [Figure 11-21] Sequence list (continued) [Figure 11-22] Sequence list (continued) [Figure 11-23] Sequence list (continued) [Figure 11-24] Sequence list (continued) [Figure 11-25] Sequence list (continued) [Figure 11-26] Sequence list (continued) [Figure 11-27] Sequence list (continued) [Figure 11-28] Sequence list (continued) [Figure 11-29] Sequence list (continued) [Figure 12] Figure 12 shows a comparison of the thermal stability between GCase variant #85 and veraglycerase alpha (VPRIV) at concentrations of 1.5 μM, 3 μM, and 6 μM in solutions at pH 5.75 (Figure 12A) and pH 7 (Figure 12B). [Figure 13] Figure 13 shows a comparison of GCase activity levels in liver (Figure 13A), leukocytes (Figure 13B), bone marrow (Figure 13C), spleen (Figure 13D), and lungs (Figure 13E) tissues in Gba-deficient mice after a single injection of AAV encoding GCase variant #85 (at a dose of 2 × 10¹² vg / kg, "AAV") or during the course of veraglucerase alfa (VPRIV delivered bi-weekly at 60 U / kg, "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. The mean level of GCase activity is shown relative to the mean level of GCase activity observed in non-deficient (wild-type) mice, and results are calculated as a percentage (y-axis, "% of WT"). The dotted line represents the mean level of GCase activity in wild-type mice for comparison. "Untreated" = control. Data are expressed as mean ± SD. [Figure 14]Figure 14 shows a comparison of plasma hexosylsphingosine levels in Gba-deficient mice after a single injection of a specified dose of AAV or during the administration of veraglucerase alfa (VPRIV, delivered every two weeks at 60 U / kg as "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. **** = statistically significant difference compared to the untreated control "0" (p<0.0001). Analysis performed = one-way ANOVA. [Figure 15-1] Figure 15A shows a comparison of hexosylsphingosine levels in the liver (Figure 15A) of Gba-deficient mice after a single injection of AAV at specified doses (2 × 10¹¹ vg / kg and 2 × 10¹² vg / kg) or during the administration of veraglucerase alfa (VPRIV, delivered every two weeks at 60 U / kg as "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. * = difference from untreated control "0" (p < 0.1). **** = statistically significant difference from untreated control "0" (p < 0.0001). Analysis performed = one-way ANOVA. [Figure 15-2] Figure 15B shows a comparison of hexosylsphingosine levels in the spleen (Figure 15B) of Gba-deficient mice after a single injection of AAV at specified doses (2 × 10¹¹ vg / kg and 2 × 10¹² vg / kg) or during the administration of veraglucerase alfa (VPRIV, delivered every two weeks at 60 U / kg as "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. * = difference from untreated control "0" (p < 0.1). **** = statistically significant difference from untreated control "0" (p < 0.0001). Analysis performed = one-way ANOVA. [Figure 15-3]Figure 15C shows a comparison of hexosylsphingosine levels in the bone marrow (Figure 15C) of Gba-deficient mice after a single injection of AAV at specified doses (2 × 10¹¹ vg / kg and 2 × 10¹² vg / kg) or during the course of veraglucerase alfa (VPRIV, delivered every two weeks at 60 U / kg as "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. * = difference from untreated control "0" (p < 0.1). **** = statistically significant difference from untreated control "0" (p < 0.0001). Analysis performed = one-way ANOVA. [Figure 15-4] Figure 15D shows a comparison of hexosylsphingosine levels in the lungs (Figure 15D) of Gba-deficient mice after a single injection of AAV at specified doses (2 × 10¹¹ vg / kg and 2 × 10¹² vg / kg) or during the administration of veraglucerase alfa (VPRIV, delivered every two weeks at 60 U / kg as "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. * = difference from untreated control "0" (p < 0.1). **** = statistically significant difference from untreated control "0" (p < 0.0001). Analysis performed = one-way ANOVA. [Figure 16-1] Figure 16A shows a comparison of CD68 density levels in the lungs of Gba-deficient mice after a single injection of a specified dose of AAV or during the course of veraglucerase alfa (VPRIV, delivered every two weeks at 60 U / kg as "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. For comparison, CD68 density and storage cell count levels in non-deficient mice ("wild-type") are shown. 0 = untreated control. The p-value relative to the untreated control "0" is shown. Data are expressed as mean ± SD. [Figure 16-2]Figure 16B shows a comparison of lung storage cell count levels (Figure 16B) in Gba-deficient mice after a single injection of a specified dose of AAV or during the course of veraglucerase alfa (VPRIV, delivered every two weeks at 60 U / kg as "ERT"). Measurements were taken 12 weeks after AAV injection and within 2 hours of ERT delivery. For comparison, CD68 density and storage cell count levels in non-deficient mice ("wild-type") are shown. 0 = untreated control. The p-value relative to the untreated control "0" is shown. Data are expressed as mean ± SD. [Figure 17-1] Figure 17A shows the rapid and sustained increase in plasma GCase activity levels after administration of AAV (2 × 10¹² vg / kg single injection) encoding GCase variant #85 to rhesus monkeys, observed up to 57 days after AAV administration (Figure 17A). AAV demonstrated a favorable safety profile and was found to be well-tolerated. [Figure 17-2] Figure 17B shows the rapid and sustained increase in plasma GCase activity levels after administration of AAV (2 × 10¹² vg / kg single injection) encoding GCase variant #85 to rhesus monkeys, observed up to 170 days after AAV administration (Figure 17B). AAV demonstrated a favorable safety profile and was found to be well-tolerated. In Figure 17B, animal 17-020 was euthanized on day 83 for tissue uptake testing. [Figure 18]Figure 18 compares the levels of GCase activity in liver, leukocytes, bone marrow, spleen, and lung tissues after a single injection of AAV encoding GCase variant #85 ("AAV-#85", doses of 2 × 10¹¹ vg / kg and 2 × 10¹² vg / kg), during the course of veraglucerase alfa (VPRIV, "ERT", delivered every two weeks at 60 U / kg), or after a single injection of AAV encoding wild-type GCase ("AAV-WT", dose of 2 × 10¹² vg / kg) in Gba-deficient mice. Measurements were taken 12 weeks after AAV injection and within 2 hours of the last ERT delivery. The mean level of GCase activity is shown relative to the mean level of GCase activity observed in non-deficient (wild-type) mice, and the results are calculated as a percentage (y axis, "% relative to WT"). The dotted line represents the mean level of GCase activity in wild-type mice for comparison. "Untreated" = control. Data are expressed as mean ± SEM. n=9~16 / treatment group. *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001, one-way ANOVA. The p-values ​​relative to the untreated control "0" are shown. For the x-axis, 1=untreated control; 2=2×10¹¹vg / kg "AAV-#85"; 3=2×10¹²vg / kg "AAV-#85"; 4="ERT"; 5=2×10¹²vg / kg "AAV-WT". [Figure 19-1] Figure 19 shows a comparison of hexosylsphingosine levels in plasma (Figure 19A) and liver (Figure 19B) of Gba-deficient mice after a single injection of AAV encoding GCase variant #85 ("AAV-#85", doses of 2 × 10¹¹ vg / kg or 2 × 10¹² vg / kg), during the process of veraglucerase alfa (VPRIV, "ERT", delivered every two weeks at 60 U / kg), or after a single injection of AAV encoding wild-type GCase ("AAV-WT", doses of 2 × 10¹² vg / kg). Measurements were taken 12 weeks after AAV injection and within 2 hours of the last ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. n = 9-16 / group. *P≦0.05;****P≦0.0001, one-way ANOVA. Shows p-values ​​relative to the untreated control "0". [Figure 19-2] Figure 19 shows a comparison of hexosylsphingosine levels in the spleen (Figure 19C) and bone marrow (Figure 19D) of Gba-deficient mice after a single injection of AAV encoding GCase variant #85 ("AAV-#85", doses of 2 × 10¹¹ vg / kg or 2 × 10¹² vg / kg), during the course of veraglucerase alfa (VPRIV, "ERT", delivered every two weeks at 60 U / kg), or after a single injection of AAV encoding wild-type GCase ("AAV-WT", doses of 2 × 10¹² vg / kg). Measurements were taken 12 weeks after AAV injection and within 2 hours of the last ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. n = 9-16 / group. *P≦0.05;****P≦0.0001, one-way ANOVA. Shows p-values ​​relative to the untreated control "0". [Figure 19-3] Figure 19 shows a comparison of hexosylsphingosine levels in the lungs (Figure 19E) of Gba-deficient mice after a single injection of AAV encoding GCase variant #85 ("AAV-#85", doses of 2 × 10¹¹ vg / kg or 2 × 10¹² vg / kg), during the process of veraglucerase alfa (VPRIV, "ERT", delivered every two weeks at 60 U / kg), or after a single injection of AAV encoding wild-type GCase ("AAV-WT", doses of 2 × 10¹² vg / kg). Measurements were taken 12 weeks after AAV injection and within 2 hours of the last ERT delivery. For comparison, hexosylsphingosine levels in non-deficient mice (wild-type) are shown. 0 = untreated control. Data are expressed as mean ± SD. n = 9-16 / group. *P ≤ 0.05;****P ≤ 0.0001, one-way ANOVA. The p-value is shown relative to the untreated control "0". [Figure 20]Figure 20 shows a comparison of plasma GCase activity levels in Gba-deficient mice after injection of AAV encoding GCase variant #85 ("AAV-#85") or wild-type GCase ("AAV-WT") at different doses (2 × 10¹⁰ vg / kg, 2 × 10¹¹ vg / kg, or 2 × 10¹² vg / kg). Measurements were taken 12 weeks after AAV injection. Data are expressed as mean ± SD. n = 9 to 16 / treatment group. ***P ≤ 0.001, ***P ≤ 0.0001, Student's t-test.

[0030] Explanation of the sequence list

[0031] [Table 1-1]

[0032] [Table 1-2]

[0033] [Table 1-3] [Modes for carrying out the invention]

[0034] general definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0035] Generally, the term "contains" is intended to mean that it contains but is not limited to. For example, the expression "modified GCase polypeptide containing two mutations" should be interpreted as meaning that the modified GCase polypeptide has at least two mutations, but may contain further mutations. Similarly, the expression "polynucleotide containing a modified glucocerebrosidase (GBA) nucleotide sequence" refers to a polynucleotide having a modified GBA nucleotide sequence, but the polynucleotide may contain additional nucleotides.

[0036] In some embodiments of the present invention, the word “contains” is replaced with the expression “essentially consists of.” The term “essentially consists of” means that certain further components may be present, i.e., those that do not substantially affect the essential characteristics of the subject.

[0037] In some embodiments of the present invention, the word “contains” is replaced with the expression “consisting of.” The term “consisting of” is intended to be restrictive. For example, the expression “modified GCase polypeptide consisting of two mutations” should be interpreted as meaning that the modified GCase polypeptide has only two mutations and no additional mutations. Similarly, the expression “polynucleotide consisting of a modified glucocerebrosidase (GBA) nucleotide sequence” should be understood as meaning that the polynucleotide has a modified GBA nucleotide sequence and no additional nucleotides.

[0038] In some embodiments of the present invention, the word "possess" can be replaced with the word "include" or the expression "consist of".

[0039] When used herein, the "~" symbol, when referring to two endpoints to define a range of values, should be interpreted as meaning "between and including" (greater than or equal to and less than or equal to). Therefore, the range defined as "5~10" includes not only the discrete values ​​of 5 and 10 themselves, but also all values ​​greater than 5 and less than 10.

[0040] The terms "protein" and "polypeptide" are used interchangeably herein and are intended to refer to polymer chains of amino acids of any length.

[0041] The terms “mutation,” “substitutional mutation,” and “amino acid substitution” are used interchangeably herein and are intended to mean the substitution of one amino acid in an amino acid sequence with a different amino acid. For example, a modified GCase polypeptide containing a mutation at the position corresponding to position 272 of SEQ ID NO: 1 may be equivalent to a GCase polypeptide having the sequence of SEQ ID NO: 1, except that the amino acid at position 272 of the GCase polypeptide is different from the amino acid at position 272 of SEQ ID NO: 1. Alternatively, a modified GCase polypeptide containing a mutation at the position corresponding to position 272 of SEQ ID NO: 1 may be equivalent to a GCase polypeptide having the sequence of SEQ ID NO: 1, except that some amino acids, including the amino acid at position 272, are different from the corresponding amino acids in SEQ ID NO: 1 (for example, the contiguous portion of the GCase polypeptide including position 272 may contain amino acids different from the corresponding amino acids in SEQ ID NO: 1). In the expressions “the mutation at the position corresponding to position X of SEQ ID NO: Z is a substitution by amino acid Y” and “at least one mutation contains a mutation at the position corresponding to position X of SEQ ID NO: Z,” the residue being substituted is the amino acid at the position corresponding to position X of SEQ ID NO: Z. Amino acid Y is a different amino acid that replaces the original or native amino acid in the amino acid sequence at the position corresponding to position X of sequence number Z. In the expressions "substitution of amino acid X" or "amino acid X to be substituted," amino acid X is the original or native amino acid present in the amino acid sequence that is to be replaced. For example, a glutamate substitution means that the original or native glutamate amino acid is replaced by another amino acid. In the expressions "substitution by amino acid Y" or "mutation to amino acid Y," amino acid Y is a different amino acid that replaces the original or native amino acid in the amino acid sequence. For example, a glutamine substitution means that the original or native (non-glutamine) amino acid is replaced by glutamine. The standard abbreviation used to define substitutional mutations lists the original or native amino acid at the position in the amino acid sequence that is to be substituted, and the amino acid that will replace the original or native amino acid.For example, a modified GCase polypeptide containing the substitution mutation E272Q refers to a modified GCase amino acid sequence that includes a substitution of the glutamic acid residue at the position corresponding to position 272 with a glutamine residue (i.e., a glutamine residue is present at the position corresponding to position 272).

[0042] The amino acid "corresponding" to the specified position in a given sequence number may be the amino acid at the specified position in the particular sequence number mentioned. For example, the amino acid "corresponding to position 272 of sequence number 1" may be the amino acid at position 272 of sequence number 1. Alternatively, the amino acid "corresponding" to the specified position in a given sequence number may be an amino acid from another amino acid sequence that corresponds to the specified position in the given sequence number. For example, the amino acid "corresponding to position 272 of sequence number 1" may be an amino acid from another amino acid sequence that corresponds to position 272 of sequence number 1. Determining which amino acid in another amino acid sequence "corresponds" to the specified position in a given sequence number is within the capabilities of a person skilled in the art. For example, a person skilled in the art can perform a sequence alignment between another amino acid sequence and the given sequence number using a suitable alignment algorithm, such as the Needleman and Wunsch algorithm described herein, and determine which region of the other amino acid sequence aligns to the specified position in the given sequence number. For example, a person skilled in the art can align another amino acid sequence with sequence number 1 and determine which amino acid aligns, and thus corresponds, to, for example, position 272 of sequence number 1.

[0043] The term "conservative substitution" refers to a substitution mutation in which one amino acid is replaced by another amino acid that has similar biochemical properties such as size, charge, or hydrophobicity. Amino acids can be classified into groups based on the structure of their side chains: aliphatic (glycine, alanine, valine, leucine, isoleucine); hydroxyl / sulfur-containing (serine, threonine, cysteine, methionine); cyclic (proline); aromatic (phenylalanine, tyrosine, tryptophan); basic (histidine, lysine, arginine); acidic (aspartic acid, glutamic acid); and acid amine (asparagine, glutamine). Therefore, a "conservative substitution" refers to a substitution mutation in which one amino acid is replaced by another amino acid from the same group. Conversely, the term "non-conservative substitution" refers to a substitution in which one amino acid is replaced by another amino acid that has different biochemical properties, i.e., a substitution mutation in which one amino acid is replaced by an amino acid from a different group. For example, the substitution of aspartic acid with glutamic acid can be considered a "conservative substitution," while the substitution of glutamic acid with glutamine can be considered a "non-conservative" substitution.

[0044] The terms “wild-type” and “native” are used interchangeably herein and are intended to describe naturally occurring forms. For example, “wild-type GCase amino acid sequence” refers to a GCase amino acid sequence found in nature.

[0045] The term "physiological pH" refers to the pH that is normally present in the human body, typically pH 7.35–7.45. For the purposes of this invention, the term "physiological pH" specifically refers to the pH found in blood, approximately pH 7.4, typically pH 7.35–7.45. Therefore, if at least one mutation provides structural stabilization at physiological pH, this is understood to provide structural stabilization at a pH that can be found in blood, e.g., pH 7.4. As a further example, if the stability of a polypeptide is measured at physiological pH, the stability is measured at a pH level that can be found in blood, e.g., pH 7.4. Preferably, the physiological pH is pH 7.4.

[0046] The term "lysosome pH" refers to the pH level of a lysosome. In other words, lysosome pH is the pH level that can be found within a lysosome, for example, pH 5.6. For example, if the stability of a polypeptide is measured by lysosome pH, the stability is measured at the pH level that can be found in the lysosome, for example, pH 5.6. Lysosome pH may be between pH 5.4 and pH 5.8. Lysosome pH may be between pH 5.5 and pH 5.7. Lysosome pH may be between pH 5.55 and pH 5.65. Preferably, lysosome pH is pH 5.6.

[0047] The terms "AAV virus particle" and "AAV vector" are used interchangeably herein.

[0048] In the context of describing the length of a nucleotide or amino acid sequence, the term “around” indicates that the sequence may contain or consist of a given number of nucleotides or amino acids plus or minus 10%, more specifically plus or minus 5%, more specifically plus or minus 1%, or more specifically plus or minus a single integer. For example, a reference to a nucleotide sequence “around” 1494 nucleotides in length may refer to a nucleotide sequence of 1345–1643 nucleotides in length, more specifically 1420–1568 nucleotides in length, more specifically 1480–1508 nucleotides in length, or more specifically 1493–1495 nucleotides in length.

[0049] When used in the context of a length of time (e.g., 16 hours), the term "around" indicates that the length of time includes plus or minus 10%, more specifically plus or minus 5%, or more specifically plus or minus 1% of the specified length of time.

[0050] For the purposes of this invention, in order to determine the identity percentage of two sequences (such as sequences of two polynucleotides or two polypeptides), the sequences are aligned for optimal comparison purposes (for example, a gap may be introduced in the first sequence for optimal alignment with the second sequence). Then, the nucleotide or amino acid residues at each position are compared. If a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotide or amino acid at that position is identical. The identity percentage between the two sequences is a function of the number of identical positions shared by the sequences (i.e., identity % = number of identical positions / total number of positions in the reference sequence × 100).

[0051] Typically, sequence comparison is performed on the entire length of a reference sequence. For example, if a user wants to determine whether a given ("test") sequence is 95% identical to sequence number 1, sequence number 1 would be the reference sequence. To evaluate whether a sequence is at least 95% identical to sequence number 1 (an example of a reference sequence), a person skilled in the art would perform an alignment on the entire length of sequence number 1 and identify how many locations in the test sequence are identical to sequence number 1. If at least 95% of the locations are identical, the test sequence is at least 95% identical to sequence number 1. If the test sequence is shorter than sequence number 1, any gaps or missing locations should be considered non-identical.

[0052] Those skilled in the art are aware of the various computer programs available for determining homology or identity between two sequences. For example, sequence comparison and determination of the percentage of identity between two sequences can be performed using mathematical algorithms. In one embodiment, the percentage of identity between two amino acid or nucleic acid sequences is determined using either a Blosum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, using the Needleman and Wunsch (1970) algorithm incorporated into the GAP program of the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ).

[0053] The singular forms "a," "an," and "the" refer to multiple objects unless explicitly indicated otherwise in the context. Therefore, for example, a reference to "amino acids" includes two or more examples or versions of such amino acids.

[0054] For the purposes of this invention, the term "fragment" refers to a contiguous portion of a sequence. For example, the 50-amino acid fragment of SEQ ID NO: 1 refers to the 50 consecutive amino acids of SEQ ID NO: 1.

[0055] Whether above or below, all publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0056] Modified GCase polypeptide This invention provides a modified GCase polypeptide (also referred to as a GCase variant) containing at least one mutation. The term "modified" means that the polypeptide has at least one difference compared to the wild-type GCase polypeptide, for example, that a mutation has been introduced. It is thought that when transitioning from an acidic pH to a physiological pH, previously positively charged residues may become neutral, and neutral residues may become negatively charged, resulting in the disappearance of attractive forces or the formation of repulsive forces, which can destabilize the polypeptide. While we do not wish to be bound by theory, it is thought that this amino acid mutation may help improve stability at physiological pH (and therefore in blood), thereby extending the time that GCase is stable and, consequently, active. For example, substituting an amino acid that is a proton donor at acidic pH but not at physiological pH (e.g., glutamic acid) with an amino acid that is a proton donor at both acidic and physiological pH (e.g., glutamine) may help stabilize the GCase polypeptide.

[0057] Optionally, at least one mutation contains (or consists of) 20 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations. Optionally, at least one mutation contains (or consists of) 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations. Optionally, at least one mutation contains (or consists of) 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations. Optionally, at least one mutation contains (or consists of) 3 or fewer mutations. Optionally, at least one mutation contains (or consists of) 2 or fewer mutations. Optionally, at least one mutation contains (or consists of) 1 mutation. Optionally, at least one mutation contains (or consists of) 2 mutations.

[0058] The modified GCase polypeptide of the present invention is functional. Functional GCase polypeptides hydrolyze glucocerebrosides. Determining whether a GCase polypeptide is functional is within the capabilities of those skilled in the art. Those skilled in the art only need to test whether the GCase polypeptide is active. For example, those skilled in the art can determine the specific activity of a GCase polypeptide as discussed herein. A modified GCase polypeptide is functional if it has at least 20% (optionally, at least 30%, at least 40%, at least 50%, or at least 75%) of the specific activity of a wild-type GCase polypeptide (determined by the same method).

[0059] Preferably, the modified GCase polypeptide of the present invention is a modified human GCase polypeptide.

[0060] In this specification, the "activity" (also referred to as "GCase activity" or "enzyme activity") of a GCase polypeptide refers to the activity observed in a functional assay for determining the activity of the GCase polypeptide. The activity of a GCase polypeptide can be analyzed using a colorimetric assay or a fluorescence assay, such as those described herein. For example, a preferred fluorescence assay is as follows: The GCase polypeptide is incubated with 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG), a fluorescent substrate of GCase. When 4-MUG is hydrolyzed by GCase, the fluorescent product 4-methylumbelliferone (4-MU) is released. The levels of the generated fluorescent product can be measured using an emission wavelength of 365 nm and an excitation wavelength of 445 nm, respectively. Optionally, the fluorescence level is converted to nanomoles / time / mL based on a 4-MU (e.g., Sigma-Aldrich) standard curve. Optionally, the fluorescence level is converted to mU / mL (e.g., plasma) or mU / mg (e.g., protein) based on a VPRIV standard curve. Optionally, the GCase polypeptide is incubated with 4-MUG for 30 minutes. Optionally, the GCase polypeptide is incubated with 4-MUG at 37°C. Optionally, the GCase polypeptide is incubated with 4-MUG at 37°C for 30 minutes at pH 5.75. Optionally, the GCase polypeptide is incubated with 4-MUG at 37°C for 1 hour in the presence and absence of an irreversible GCase inhibitor, such as conzlitol B epoxide. For example, a portion of the sample is incubated in the presence of an irreversible GCase inhibitor, such as conzlitol B epoxide, and a portion of the sample is incubated in its absence, and the results are compared to obtain the GCase activity level. Optionally, the GCase polypeptide is incubated at pH 5.6 or pH 5.75. The activity of the GCase polypeptide can be measured according to the protocol described in the chapter titled “Determination of GCase Activity” of Example 1 or the protocol described in the paragraph beginning “GCase Activity Assay” in the chapter titled “Method” of Example 13.

[0061] In some embodiments, the GCase polypeptide is purified before measuring its activity. The protein can be purified using techniques known in the art, such as column chromatography (e.g., affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography), HPLC, or separation based on charge or hydrophobicity. For example, the protein can be purified using a Sepharose column (affinity chromatography resin). For example, the protein can be purified using the Sepharose column (affinity chromatography resin) described in Example 1. The GCase polypeptide activity can be measured by a fluorescence assay (such as those described herein) performed on the purified GCase polypeptide.

[0062] In this specification, "effective activity" (also referred to as "effective GCase activity") refers to the activity of the GCase polypeptide observed after the expression of the GBA nucleotide sequence encoding the GCase polypeptide in a biological system. "Effective activity" is a function of the enzymatic efficiency and stability of the GCase polypeptide. "Effective activity" may also be referred to as "total activity obtained." In some embodiments, increased effective activity is a result of increased stability of the modified GCase polypeptide. In some embodiments, increased effective activity is a result of increased enzymatic efficiency of the modified GCase polypeptide. In some embodiments, increased effective activity is a result of increased enzymatic efficiency and stability of the modified GCase polypeptide. In some embodiments, the effective activity of the GCase polypeptide is determined by generating AAV particles containing a polynucleotide including a GBA nucleotide sequence encoding the GCase polypeptide operably linked to a promoter, transducing the AAV particles into cells (e.g., human cells, optionally Huh-7 cells), collecting the GCase polypeptide from the cells and / or culture medium, and optionally measuring the activity of the GCase polypeptide using a fluorescence assay. Optionally, AAV particles containing polynucleotides with a GBA nucleotide sequence encoding a GCase polypeptide are incubated with Huh-7 cells for approximately 16 hours to induce transduction, and the GCase polypeptide is collected from the culture medium. Optionally, the AAV particles contain the capsid of SEQ ID NO: 37. Optionally, transduction is performed for 5 × 10⁻⁶ cells. 3 vg / cell, 1×10 4 vg / cell, or 5×10 4The assay is performed based on the infection multiplicity of vg / cell. The activity of the collected GCase polypeptides can be measured according to the fluorescence assay protocols described herein. For example, GCase polypeptides may be incubated with 4-MUG at 37°C for 30 minutes. Optionally, GCase polypeptides are incubated with 4-MUG at 37°C for 30 minutes at pH 5.75. Optionally, GCase polypeptides are incubated with 4-MUG at 37°C for 1 hour in the presence and absence of an irreversible GCase inhibitor, such as conzlitol B epoxide. For example, a portion of the sample is incubated in the presence of an irreversible GCase inhibitor, such as conzlitol B epoxide, and a portion of the sample is incubated in its absence, and the results are compared to obtain the GCase activity level. Optionally, GCase polypeptides are incubated at pH 5.6 or pH 5.75. The levels of the generated fluorescence product can be measured using emission wavelengths of 365 nm and excitation wavelengths of 445 nm, respectively. Fluorescence levels can be converted to nanomoles / time / mL based on a 4-methylumbelliferone (4-MU, e.g., Sigma-Aldrich) standard curve. Optionally, the activity of the collected GCase polypeptide is measured according to the fluorescence assay protocol described in the chapter titled “Determination of GCase Activity” of Example 1 or the paragraph beginning with “GCase Activity Assay” in the chapter titled “Methods” of Example 13. Optionally, the activity is normalized by the vector genome copy number. The number of vector genomes present in the transduced cells can be determined (e.g., by qPCR) and used to adjust the measured GCase polypeptide activity. Optionally, the number of vector genomes present in the transduced cells is determined by qPCR. Optionally, the number of vector genomes present in the transduced cells is determined by qPCR using a primer set that binds to the promoter (e.g., rather than to the LSP-L promoter). Optionally, the vector genome copy number is determined according to the first paragraph of the chapter titled “Vector Genome Copy Number” of Example 1. Optionally, the effective activity of the GCase polypeptide can be determined using the methodology of Example 8.

[0063] In some embodiments, the effective activity of the GCase polypeptide is determined by generating AAV particles comprising a polynucleotide comprising a GBA nucleotide sequence encoding a GCase polypeptide operably linked to a promoter, injecting the AAV particles into mice, and optionally measuring the activity levels in plasma, white blood cells, and / or tissues (such as liver, spleen, bone marrow, and / or lung, etc.) from the mice using a fluorescence assay. Optionally, the AAV particles comprising a polynucleotide comprising a GBA nucleotide sequence encoding a GCase polypeptide may be injected into mice via a single tail vein injection at a dose of, for example, 2×10 9 vg / kg to 2×10 12 vg / kg. Optionally, the dose may be selected from 2×10 9 vg / kg, 2×10 10 vg / kg, 2×10 11 vg / kg, 6×10 11 vg / kg, and 2×10 12 vg / kg. Optionally, the dose is 6×10 10The concentration is vg / kg. Optionally, AAV particles contain AAV8 capsid. Plasma samples may be obtained 14 days after treatment (by post-orbital blood collection) or 28 days after treatment (by post-orbital blood collection). Animals may be sorted 4 or 6 weeks after treatment to collect plasma, leukocytes, and / or other tissues (e.g., liver, spleen, bone marrow, and / or lung). Optionally, animals are sorted 4 weeks after treatment. The activity of GCase polypeptides in the collected plasma, leukocytes, and / or other tissues can be measured according to the fluorescence assay protocol described herein. For example, GCase polypeptides may be incubated with 4-MUG at 37°C for 30 minutes. Optionally, GCase polypeptides are incubated with 4-MUG at pH 5.75 at 37°C for 30 minutes. Optionally, GCase polypeptides are incubated with 4-MUG at 37°C for 1 hour in and without an irreversible GCase inhibitor, such as conzlitol B epoxide. For example, a portion of the sample is incubated in the presence of an irreversible GCase inhibitor, such as conzlitol B epoxide, and a portion is incubated in its absence, and the results are compared to obtain the GCase activity level. Optionally, GCase polypeptides are incubated at pH 5.6 or pH 5.75. The level of the resulting fluorescence product can be measured using an emission wavelength of 365 nm and an excitation wavelength of 445 nm, respectively. The activity of GCase polypeptides in plasma, tissue, and WBC can be expressed as mU / mL (plasma) (for plasma) or mU / mg (protein) (for tissue and WBC) using the VPRIV standard curve. Optionally, GCase activity can be calculated as nmol / hour / mL or nmol / hour / mg for plasma or tissue, respectively, using the 4-MU standard curve. Optionally, the activity of GCase polypeptides in collected plasma, leukocytes, and / or other tissues may be measured according to the fluorescence assay protocol described in the chapter titled “Determination of GCase Activity” of Example 1 or the paragraph beginning with “GCase Activity Assay” in the chapter titled “Methods” of Example 13. Optionally, the activity is normalized by the vector genome copy number.The number of vector genomes present in the liver after injection of AAV particles can be determined (e.g., by qPCR) and used to adjust the activity of the measured GCase polypeptide. Optionally, the number of vector genomes present in the liver after injection of AAV particles can be determined by qPCR. Optionally, the number of vector genomes present in the liver after injection of AAV particles can be determined by qPCR using a primer set that binds to a promoter (e.g., to the LSP-L promoter). Optionally, the vector genome copy number can be determined according to the second paragraph of the chapter titled "Vector Genome Copy Number" in Example 1. Optionally, the effective activity of the GCase polypeptide can be determined using the methodology of Example 9.

[0064] The present invention provides a modified GCase polypeptide comprising at least one mutation. (i) Provides higher effective activity; and / or (ii) to provide increased stability; and / or (iii) Provides structural stabilization at physiological pH; and / or (iv) Provides a longer half-life; and / or (v) Provides increased thermal stability It is possible.

[0065] In some embodiments, at least one mutation provides higher efficacy. Exemplary mutations providing higher efficacy are disclosed herein. “At least one mutation provides higher efficacy” means that a modified GCase polypeptide containing at least one mutation has higher efficacy than a reference GCase polypeptide that does not contain the at least one mutation but is otherwise identical to the modified GCase polypeptide containing the at least one mutation. For example, if at least one mutation provides higher efficacy, and the modified GCase polypeptide containing at least one mutation is otherwise identical to the wild-type GCase polypeptide except that it contains at least one mutation (e.g., mutations W351C and A380C), then the modified GCase polypeptide containing at least one mutation has higher efficacy than the wild-type GCase polypeptide. In some embodiments, if a “test” polypeptide identical to SEQ ID NO: 1 except for at least one mutation has higher efficacy compared to the polypeptide of SEQ ID NO: 1, and optionally, if the “test” polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, then at least one mutation “provides higher efficacy.” A modified GCase polypeptide containing at least one mutation does not necessarily have to have higher active efficacy than a wild-type GCase polypeptide. For example, a modified GCase polypeptide containing at least one mutation may contain one or more further modifications (such as deletions) that reduce the polypeptide's active efficacy. In such cases, the modified GCase polypeptide may not have the same active efficacy as a wild-type GCase polypeptide, but nevertheless, the at least one mutation disclosed herein may help provide higher active efficacy to the modified GCase polypeptide so that it has higher active efficacy than a reference GCase polypeptide that is otherwise identical to the modified GCase polypeptide except for the lack of at least one mutation. Alternatively, the at least one mutation may provide increased active efficacy that restores the active efficacy of the modified GCase polypeptide to the level of the wild-type GCase polypeptide.

[0066] In some embodiments, a modified GCase polypeptide containing at least one mutation has higher effective activity. In some embodiments, a modified GCase polypeptide containing at least one mutation has higher effective activity than the reference GCase polypeptide described herein. For example, the reference GCase polypeptide may be a wild-type GCase polypeptide. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 4 or 5. In some embodiments, the effective activity of the modified GCase is compared with the effective activity of the reference GCase polypeptide. Exemplary mutations that increase the effective activity of the modified GCase polypeptide are disclosed herein.

[0067] A "reference GCase polypeptide" is a GCase polypeptide that does not contain at least one mutation of the modified GCase polypeptide of the present invention. Optionally, the reference GCase polypeptide may not contain at least one mutation but may otherwise be identical to the modified GCase polypeptide of the present invention. The reference GCase polypeptide may be any one of the polypeptides of SEQ ID NOs: 1 to 5. The reference GCase polypeptide may be a wild-type GCase polypeptide. The reference GCase polypeptide may be any wild-type GCase polypeptide. The reference GCase polypeptide may be any one of the polypeptides of SEQ ID NOs: 1 to 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 1. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 3. Optionally, the reference GCase polypeptide is not a wild-type GCase polypeptide and does not contain at least one mutation. Optionally, the reference GCase polypeptide contains a leucine mutation at the position corresponding to position 184 of SEQ ID NO: 1, optionally H184L. Optionally, the reference GCase polypeptide includes a leucine mutation at the position corresponding to position 184 of SEQ ID NO: 1, optionally H184L, and the reference GCase polypeptide also includes an asparagine mutation at the position corresponding to position 360 of SEQ ID NO: 1, optionally K360N. Optionally, the reference GCase polypeptide includes both H184L and K360N. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 4 or 5. In some embodiments, the reference GCase polypeptide includes the polypeptide of SEQ ID NO: 41 or 44. In some embodiments, the reference GCase polypeptide includes the polypeptide of SEQ ID NO: 43 or 46. In some embodiments, the reference GCase polypeptide includes the polypeptide of SEQ ID NO: 42 or 45.

[0068] In some embodiments, the reference GCase polypeptide may be a wild-type GCase polypeptide such as VPRIV. Optionally, VPRIV is reconstituted with sterile water or nuclease-free water. In some embodiments, the reference GCase polypeptide may be imiglucerase. In some embodiments, the reference GCase polypeptide contains a histidine mutation, optionally R534H, at the position corresponding to position 534 of SEQ ID NO: 1. In some embodiments, the reference GCase polypeptide contains or is the sequence of SEQ ID NO: 1 or 2, except for the histidine mutation at the position corresponding to position 534 of SEQ ID NO: 1.

[0069] To determine whether a modified GCase polypeptide has higher effective activity than a reference GCase polypeptide (such as a wild-type GCase polypeptide as described herein), the effective activity of the modified GCase polypeptide and the reference GCase polypeptide is determined by the same method, and the results are compared. The method may be any of the methods described herein for determining effective activity.

[0070] The modified GCase polypeptide of the present invention may have an effective activity at least 1.2 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, and at least 50 times higher than the effective activity of a reference GCase polypeptide (such as a wild-type GCase polypeptide). Modified GCase polypeptides may have at least 1.2 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 10 times, at least 15 times, at least 20 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times higher effective activity than reference GCase polypeptides (wild-type GCase polypeptides, etc.). Modified GCase polypeptides may have at least 5 times, at least 10 times, at least 25 times, or at least 50 times higher effective activity than reference GCase polypeptides (wild-type GCase polypeptides, etc.). Modified GCase polypeptides may have an effective activity 1.2 to 3 times, 1.2 to 5 times, 3 to 6 times, 5 to 10 times, 1.5 to 8 times, 2 to 10 times, 3 to 15 times, 6 to 15 times, 10 to 15 times, 15 to 20 times, 10 to 30 times, 20 to 30 times, 25 to 30 times, 25 to 40 times, or 30 to 50 times higher than the effective activity of a reference GCase polypeptide (such as a wild-type GCase polypeptide). Modified GCase polypeptides may have an effective activity 2 to 100 times, 10 to 100 times, 10 to 80 times, 25 to 100 times, or 25 to 80 times higher than the effective activity of a reference GCase polypeptide (such as a wild-type GCase polypeptide). When referring to a multiplier change in activity, the term "~" includes the specified value.Therefore, for example, "1.5 times to 8 times" includes values ​​of 1.5 times and 8 times.

[0071] In some embodiments, at least one mutation provides increased stability (also referred to as higher stability). Exemplary mutations that provide increased stability are disclosed herein. "At least one mutation provides increased stability" means that a modified GCase polypeptide containing at least one mutation has higher stability than a reference GCase polypeptide that does not contain the at least one mutation but is otherwise identical to the modified GCase polypeptide containing the at least one mutation. For example, if at least one mutation provides increased stability and the modified GCase polypeptide containing at least one mutation is otherwise identical to the wild-type GCase polypeptide except that it contains at least one mutation (e.g., mutations W351C and A380C), then the modified GCase polypeptide containing at least one mutation has higher stability than the wild-type GCase polypeptide. In some embodiments, if a "test" polypeptide that is identical to SEQ ID NO: 1 except for at least one mutation has higher stability than the polypeptide of SEQ ID NO: 1, and optionally, if the "test" polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, then at least one mutation "provides increased stability". A modified GCase polypeptide containing at least one mutation does not necessarily have to be more stable than a wild-type GCase polypeptide. For example, a modified GCase polypeptide containing at least one mutation may contain one or more further modifications (such as deletions) that reduce the polypeptide's stability. In such cases, the modified GCase polypeptide may not have the same stability as a wild-type GCase polypeptide, but nevertheless, the at least one mutation disclosed herein may help provide higher stability to the modified GCase polypeptide so that it is more stable than a reference GCase polypeptide that is otherwise identical to the modified GCase polypeptide except for the lack of at least one mutation. Alternatively, the at least one mutation may provide increased stability that restores the stability of the modified GCase polypeptide to the level of stability of a wild-type GCase polypeptide.

[0072] In some embodiments, a modified GCase polypeptide containing at least one mutation exhibits increased stability. In some embodiments, a modified GCase polypeptide containing at least one mutation exhibits increased stability compared to a reference GCase polypeptide described herein. In some embodiments, the increased stability provided by at least one mutation or the increased stability of a modified GCase polypeptide containing at least one mutation is increased stability at pH 7.4. In some embodiments, the increased stability is measured after incubation at pH 7.4 and 37°C for 120 minutes. In some embodiments, the increased stability provided by at least one mutation or the increased stability of a modified GCase polypeptide containing at least one mutation is increased stability at pH 5.6. In some embodiments, the increased stability is measured after incubation at pH 5.6 and 37°C for 72 hours. In some embodiments, the increased stability is increased stability compared to a reference GCase polypeptide described herein. For example, the reference GCase polypeptide may be a wild-type GCase polypeptide. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 4 or 5. Optionally, a modified GCase polypeptide containing at least one mutation has increased stability compared to the reference GCase polypeptide. Optionally, a modified GCase polypeptide containing at least one mutation has increased stability compared to the reference GCase polypeptide, and this increased stability is at pH 7.4. In some embodiments, the stability of the modified GCase is compared to the stability of the reference GCase polypeptide. Exemplary mutations that increase the stability of the modified GCase polypeptide are disclosed herein. For example, a mutation that enables crosslinking of disulfide bonds.

[0073] Modified GCase polypeptides, which have increased stability compared to the reference GCase polypeptide, retain a higher percentage of GCase activity over time than the reference GCase polypeptide. In some embodiments, the stability of the GCase polypeptide is determined by measuring the residual enzyme activity of the GCase polypeptide. In this specification, references to “residual enzyme activity” (also referred to as “residual GCase activity” or “residual activity”) refer to the activity of the GCase polypeptide retained after a specified period. The residual enzyme activity of GCase may be measured in samples after one or more specified periods. Optionally, the sample is obtained by transfecting host cells (e.g., Expi293F cells) with an expression vector containing a nucleotide sequence encoding the GCase polypeptide and collecting the GCase polypeptide from the culture medium. Optionally, the GCase polypeptide is collected from the culture medium four days after transfection. Optionally, samples are obtained according to the methods described in the chapters of Example 1 titled “Amplification of Expi293F Cells,” “Day Before Transfection of Expi293F Cells,” “Transfection of Expi293F Cells,” and “Collection of Transfected Expi293F Cells.” Samples containing GCase polypeptides may be transferred to a matrix (e.g., AB buffer at pH 5.6, PBS at pH 7.4, serum, or plasma) and incubated. The pH of the matrix may be selected to determine the residual activity (or stability) at a particular pH. The incubation temperature may be selected to determine the residual activity (or stability) at a particular temperature. In addition to taking aliquots of the sample at the initial time point, aliquots of the incubated sample containing GCase polypeptides may be taken at a series of time points, e.g., two or more, three or more, four or more, five or more, or six or more time points, and the GCase activity in each aliquot may be determined. The initial time point (i.e., 0 minutes or 0 hours) is the time when the sample is transferred to the matrix for incubation. By comparing the GCase activity at a given time point with the GCase activity at an initial time point, it becomes possible to determine the remaining GCase activity at that time point.Aliquotes of the sample containing GCase polypeptide may be taken at 0 minutes, 30 minutes, 60 minutes, 120 minutes, 3 days, 4 days, 5 days, 6 days, and / or 7 days. Optionally, the sample is incubated in PBS at pH 7.4 for 120 minutes. Optionally, the sample is incubated in PBS at pH 7.4 for 72 hours. Optionally, the sample is incubated in PBS at pH 7.4 for 7 days. Optionally, the sample is incubated in AB buffer at pH 5.6 for 72 hours. Optionally, the residual enzyme activity in the sample is measured according to the chapter titled “Stability Assessment” in Example 1. The activity of GCase polypeptide in aliquots can be measured according to the fluorescence assay protocol described herein. For example, GCase polypeptide may be incubated with 4-MUG at 37°C for 30 minutes. Optionally, GCase polypeptide is incubated with 4-MUG at pH 5.75 at 37°C for 30 minutes. Optionally, GCase polypeptides are incubated with 4-MUG at 37°C for 1 hour in and without an irreversible GCase inhibitor, such as conzlitol B epoxide. For example, a portion of the sample is incubated in the presence of an irreversible GCase inhibitor, such as conzlitol B epoxide, and a portion of the sample is incubated in its absence, and the results are compared to obtain the GCase activity level. Optionally, GCase polypeptides are incubated at pH 5.6 or pH 5.75. The level of the resulting fluorescence product can be measured using an emission wavelength of 365 nm and an excitation wavelength of 445 nm, respectively. Optionally, the activity of GCase polypeptides in aliquots can be measured according to the fluorescence assay protocol described in the chapter titled “Determination of GCase Activity” in Example 1 or the paragraph beginning “GCase Activity Assay” in the chapter titled “Method” in Example 13. The GCase activity at a given time point may be compared with the initial GCase activity of the sample (i.e., the GCase activity at the initial time point is also determined), and the remaining GCase activity may be calculated as a percentage of the initial GCase activity.Therefore, a modified GCase polypeptide with increased stability compared to the reference GCase polypeptide may have higher residual activity than the reference GCase polypeptide (i.e., as a percentage of the initial GCase polypeptide activity). Optionally, the reference GCase polypeptide may be a wild-type GCase polypeptide such as VPRIV. Optionally, VPRIV may be reconstituted with nuclease-free water or sterile water. Optionally, the residual enzyme activity of the GCase polypeptide may be determined using the methodology of Example 3. Optionally, the residual enzyme activity of the GCase polypeptide may be determined using the methodology of Example 4.

[0074] Optionally, specific activity may be measured at each time point. "Specific activity" refers to the activity per unit of GCase polypeptide, thereby "normalizing" the activity to take into account the amount or concentration of GCase polypeptide in the sample. This can be done, for example, by measuring the concentration of GCase polypeptide in the sample using a standard ELISA assay and dividing the activity by the GCase concentration. "Specific activity" can also be measured using a fluorescence assay. The fluorescence assay may be any one of the fluorescence assays described herein.

[0075] In one example of an ELISA assay, an antibody that binds to a GCase polypeptide may be conjugated to a plate. A sample containing an unknown concentration of GCase polypeptide may be passed over the plate. A second detection antibody that binds to the GCase polypeptide may be applied to the plate, and any excess detection antibody may be washed off. The remaining (i.e., not washed off) detection antibody binds to the GCase polypeptide. The detection antibody may be ligated to an enzyme such as horseradish peroxidase. By measuring the amount of detection antibody, the level of detection antibody bound to the GCase polypeptide on the plate can be measured. For example, if the detection antibody is ligated to horseradish peroxidase, the horseradish peroxidase can catalyze the production of a blue reaction product from a substrate such as TMB (3,3',5,5'-tetramethylbenzidine), and the level of this blue product can be detected by its absorbance at 450 nm. The level of the blue product is proportional to the amount of detection antibody remaining after the washing step, which is proportional to the amount of GCase polypeptide in the sample. Alternatively, for example, when using purified protein, the amount or concentration of GCase polypeptide may be measured spectrophotometrically.

[0076] In some embodiments, the stability of a GCase polypeptide is determined by determining its half-life. The half-life can be determined as described herein.

[0077] To determine whether a modified GCase polypeptide has increased stability compared to a reference GCase polypeptide (such as a wild-type GCase polypeptide as described herein), the stability of the modified GCase polypeptide and the reference GCase polypeptide is determined by the same method, and the results are compared. The method may be any of the methods described herein for determining stability.

[0078] The stability or residual activity of the modified GCase polypeptide may be determined after incubation for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. Optionally, the increased stability or increased residual activity of the modified GCase polypeptide in the sample may be observed after incubation for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days.

[0079] The residual activity of a modified GCase polypeptide can be expressed as a percentage of the initial GCase activity. Where it is stated that a modified GCase polypeptide "retains" at least X% (e.g., X may be 30) of activity at a particular time point, this should be interpreted as meaning that the modified GCase polypeptide has at least X% residual activity at a particular time point (e.g., at least 30% residual activity). When measured as described herein (e.g., by measuring activity over time and comparing the activity at a given time point to the initial GCase activity of the sample), a modified GCase polypeptide may retain at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% activity. Activity may be measured after incubation at pH 5.6. Modified GCase polypeptides can retain at least 70%, at least 75%, at least 80%, or at least 85% activity after incubation at pH 5.6 and 37°C for 120 minutes. Modified GCase polypeptides can retain at least 80% or at least 85% activity after incubation at pH 5.6 and 37°C for 120 minutes. Modified GCase polypeptides can retain at least 90% or at least 95% activity after incubation at pH 5.6 and 37°C for 120 minutes. Modified GCase polypeptides can retain at least 60% activity after incubation at pH 5.6 and 37°C for 72 hours. Modified GCase polypeptides can retain at least 80% activity after incubation at pH 5.6 and 37°C for 72 hours. Modified GCase polypeptides can retain at least 85% activity after incubation at pH 5.6 and 37°C for 72 hours. The modified GCase polypeptide can retain at least 40% of its activity after being incubated at pH 5.6 and 37°C for 7 days.Modified GCase polypeptides can retain at least 60% activity after incubation at pH 5.6 and 37°C for 7 days. Modified GCase polypeptides can retain at least 80% activity after incubation at pH 5.6 and 37°C for 7 days. Activity may be measured after incubation at pH 7.4. Activity may be measured after incubation at 37°C. Activity may be measured after incubation at pH 7.4 and 37°C. Incubation may be performed in PBS. Incubation may be performed in serum or plasma. Modified GCase polypeptides can retain at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 65%, at least 60%, at least 65%, at least 60%, at least 75%, at least 70%, at least 75%, at least 80%, or at least 85% activity after incubation at pH 7.4 and 37°C for at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 120 minutes, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In some embodiments, modified GCase polypeptides retain at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, or at least 3 times higher activity than the reference GCase polypeptide (wild-type GCase polypeptide, etc.) when measured under the same conditions (e.g., the activity of the modified GCase polypeptide and the reference (e.g., wild-type) GCase were measured after incubation at pH 7.4 and 37°C for 120 minutes, respectively). The reference to “same conditions” indicates that any variability acceptable in terms of experimental tolerance is included.Modified GCase polypeptides can retain at least 50 times, at least 60 times, at least 70 times, at least 80 times, or at least 90 times higher activity than the reference GCase polypeptide when measured under the same conditions. Modified GCase polypeptides can retain at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 2.5 times, at least 2.8 times, or at least 3 times higher activity than the reference GCase polypeptide when measured after incubation at pH 7.4 and 37°C for 120 minutes. Modified GCase polypeptides can retain at least 1.1 to 10 times, 2 to 8 times, or 2 to 5 times higher activity than the reference GCase polypeptide when measured after incubation at pH 7.4 and 37°C for 120 minutes. Modified GCase polypeptides can retain at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes. Modified GCase polypeptides can retain at least 70%, at least 75%, at least 80%, or at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes. Modified GCase polypeptides can retain at least 50 times, at least 60 times, at least 70 times, at least 80 times, or at least 90 times higher activity than the reference GCase polypeptide after incubation at pH 7.4 and 37°C for 72 hours. Modified GCase polypeptides can retain 1.1 to 150 times, 2 to 120 times, or 2 to 100 times higher activity than the reference GCase polypeptide after incubation at pH 7.4 and 37°C for 72 hours.Modified GCase polypeptides can retain at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of activity after incubation at pH 7.4 and 37°C for 72 hours. Modified GCase polypeptides can retain at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% of activity after incubation at pH 7.4 and 37°C for 72 hours. Modified GCase polypeptides can retain at least 1.1 times, at least 1.2 times, at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, or at least 90 times higher activity than the reference GCase polypeptide after incubation at pH 7.4 and 37°C for 7 days. Modified GCase polypeptides, when measured after incubation at pH 7.4 and 37°C for 7 days, can retain 1.1 to 150 times, 2 to 120 times, or 2 to 100 times higher activity than the reference GCase polypeptide. Modified GCase polypeptides can retain at least 15% activity after incubation at pH 7.4 and 37°C for 7 days. Modified GCase polypeptides can retain at least 20% activity after incubation at pH 7.4 and 37°C for 7 days. Modified GCase polypeptides can retain at least 40% activity after incubation at pH 7.4 and 37°C for 7 days. Incubation may be performed in serum or plasma. Incubation may be performed in PBS. Modified GCase polypeptides can retain at least 60% activity after incubation at pH 7.4 and 37°C for 7 days. Modified GCase polypeptides can retain at least 80% activity after incubation at pH 7.4 and 37°C for 7 days. Activity may be measured after incubation in human plasma.Modified GCase polypeptides, when measured after incubation in human plasma at 37°C for 120 minutes, can retain at least 1.1 times, at least 1.2 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, or at least 5 times higher activity than the reference GCase polypeptide. Modified GCase polypeptides, when measured after incubation in human plasma at 37°C for 120 minutes, can retain at least 1.1 to 10 times, 2 to 8 times, or 4 to 8 times higher activity than the reference GCase polypeptide. Modified GCase polypeptides, when measured after incubation in human plasma at 37°C for 120 minutes, can retain at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% of activity. Modified GCase polypeptides can retain at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times higher activity than reference GCase polypeptides when measured after incubation in human plasma at 37°C for 72 hours. Modified GCase polypeptides can retain at least 10 to 100 times, 20 to 100 times, or 20 to 80 times higher activity than reference GCase polypeptides when measured after incubation in human plasma at 37°C for 72 hours. Modified GCase polypeptides can retain at least 20%, at least 30%, at least 40%, or at least 50% of activity when measured after incubation in human plasma at 37°C for 72 hours. Modified GCase polypeptides can retain at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times higher activity than reference GCase polypeptides when incubated in human plasma at 37°C for 7 days. Modified GCase polypeptides, when incubated in human plasma at 37°C for 7 days and then measured, can retain activity 10 to 100 times, 20 to 100 times, or 20 to 80 times higher than that of the reference GCase polypeptide.

[0080] Modified GCase polypeptides can retain at least 20% activity after incubation in human plasma at 37°C for 7 days. Modified GCase polypeptides can retain at least 30% activity after incubation in human plasma at 37°C for 7 days. Modified GCase polypeptides can retain at least 40% activity after incubation in human plasma at 37°C for 7 days. Activity may also be measured after incubation in human serum. Modified GCase polypeptides can retain at least 1.1 times, at least 1.2 times, at least 1.5 times, at least 1.8 times, or at least 2 times higher activity than the reference GCase polypeptide after incubation in human serum at 37°C for 120 minutes. Modified GCase polypeptides can retain 1.1 to 10 times, 1.1 to 8 times, or 1.1 to 5 times higher activity than the reference GCase polypeptide after incubation in human serum at 37°C for 120 minutes. Modified GCase polypeptides can retain at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% activity when measured after incubation in human serum at 37°C for 120 minutes. Modified GCase polypeptides can retain at least 1.1 times, at least 1.2 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, or at least 40 times higher activity than the reference GCase polypeptide when measured after incubation in human serum at 37°C for 72 hours. Modified GCase polypeptides can retain at least 1.1 to 100 times, 2 to 80 times, or 5 to 80 times higher activity than the reference GCase polypeptide when incubated in human serum at 37°C for 72 hours.Modified GCase polypeptides can retain at least 2%, at least 5%, at least 20%, at least 30%, or at least 40% activity after incubation in human serum at 37°C for 72 hours. Modified GCase polypeptides can retain at least 10 times, at least 20 times, at least 30 times, or at least 40 times higher activity than the reference GCase polypeptide after incubation in human serum at 37°C for 7 days. Modified GCase polypeptides can retain at least 10 to 100 times, 20 to 100 times, or 20 to 80 times higher activity than the reference GCase polypeptide after incubation in human serum at 37°C for 7 days. Modified GCase polypeptides can retain at least 20% activity after incubation in human serum at 37°C for 7 days. Modified GCase polypeptides can retain at least 30% activity after incubation in human serum at 37°C for 7 days. Modified GCase polypeptides can retain at least 40% of their activity after being incubated in human serum at 37°C for 7 days and then measured.

[0081] The modified GCase polypeptide, when measured after incubation at pH 7.4 (optionally in PBS) and 37°C for 120 minutes, can retain at least 1.5 times, at least 1.7 times, or at least 1.8 times higher activity than the reference GCase polypeptide, the reference GCase polypeptide containing a histidine-to-leucine substitution at the position corresponding to position 184 of SEQ ID NO: 1. Optionally, the reference GCase polypeptide is SEQ ID NO: 4. The modified GCase polypeptide, when measured after incubation at pH 7.4 (optionally in PBS) and 37°C for 120 minutes, can retain at least 1.1 times or at least 1.2 times higher activity than the reference GCase polypeptide, the reference GCase polypeptide containing a histidine-to-leucine substitution at the position corresponding to position 184 of SEQ ID NO: 1, and a lysine-to-asparagine substitution at the position corresponding to position 360 of SEQ ID NO: 1. Optionally, the reference GCase polypeptide is SEQ ID NO: 5. The modified GCase polypeptide can retain at least 1.5 times or at least 1.7 times higher activity than the reference GCase polypeptide of SEQ ID NO: 3 when measured after incubation at pH 7.4 (optionally in PBS) and 37°C for 120 minutes. The modified GCase polypeptide can retain at least 50 times, at least 60 times, at least 70 times, at least 80 times, or at least 90 times higher activity than the reference GCase polypeptide when incubated at pH 7.4 (optionally in PBS) and 37°C for 72 hours, wherein the reference GCase polypeptide contains a histidine-to-leucine substitution at the position corresponding to position 184 of SEQ ID NO: 1. Optionally, the reference GCase polypeptide is SEQ ID NO: 4.The modified GCase polypeptide, when measured after incubation at pH 7.4 (optionally in PBS) at 37°C for 72 hours, can retain at least 50, 60, 70, 80, or 90 times higher activity than the reference GCase polypeptide, the reference GCase polypeptide comprising a histidine-to-leucine substitution at the position corresponding to position 184 of SEQ ID NO: 1, and a lysine-to-asparagine substitution at the position corresponding to position 360 of SEQ ID NO: 1. Optionally, the reference GCase polypeptide is SEQ ID NO: 5. The modified GCase polypeptide, when measured after incubation at pH 7.4 (optionally in PBS) at 37°C for 72 hours, can retain at least 50, 60, 70, 80, or 90 times higher activity than the reference GCase polypeptide of SEQ ID NO: 3. The modified GCase polypeptide, when measured after incubation at pH 7.4 (optionally in PBS) and 37°C for 7 days, can retain at least 1.1 times, at least 1.5 times, at least 2 times, at least 5 times, or at least 10 times higher activity than the reference GCase polypeptide, the reference GCase polypeptide includes a histidine-to-leucine substitution at the position corresponding to position 184 of SEQ ID NO: 1. Optionally, the reference GCase polypeptide is SEQ ID NO: 4. The modified GCase polypeptide, when measured after incubation at pH 7.4 (optionally in PBS) and 37°C for 7 days, can retain at least 1.1 times, at least 1.5 times, at least 2 times, at least 5 times, or at least 10 times higher activity than the reference GCase polypeptide, the reference GCase polypeptide includes a histidine-to-leucine substitution at the position corresponding to position 184 of SEQ ID NO: 1, and a lysine-to-asparagine substitution at the position corresponding to position 360 of SEQ ID NO: 1. Optionally, the reference GCase polypeptide is SEQ ID NO: 5.Modified GCase polypeptides, when incubated at pH 7.4 (optionally in PBS) at 37°C for 7 days, can retain at least 1.1 times, at least 1.5 times, at least 2 times, at least 5 times, or at least 10 times higher activity than the reference GCase polypeptide of SEQ ID NO: 3. Modified GCase polypeptides, when incubated in human plasma at 37°C for 120 minutes, can retain at least 3 times, at least 4 times, or at least 5 times higher activity than the reference GCase polypeptide of SEQ ID NO: 3. Modified GCase polypeptides, when incubated in human serum at 37°C for 120 minutes, can retain at least 1.2 times, at least 1.5 times, at least 1.8 times, or at least 2 times higher activity than the reference GCase polypeptide of SEQ ID NO: 3.

[0082] Modified GCase polypeptides may retain at least 30%, at least 35%, at least 40%, at least 45%, at least 5, at least 6, or at least 7 days of incubation at pH 5.6 and 37°C before measurement. Optionally, modified GCase polypeptides may retain at least 40%, at least 45%, at least 50%, at least 55%, at least 70%, at least 75%, at least 80%, or at least 85% of activity after incubation in PBS at pH 7.4 at 37°C for 72 hours before measurement. Optionally, modified GCase polypeptides may retain at least 50% of activity after incubation in PBS at pH 7.4 at 37°C for 72 hours before measurement. Modified GCase polypeptides can retain at least 10 times, at least 15 times, at least 20 times, at least 30 times, or at least 50 times higher activity than reference GCase polypeptides when measured after incubation at pH 5.6 and 37°C for 72 hours. For example, modified GCase polypeptides can retain at least 30 times higher activity than reference GCase polypeptides when measured after incubation at pH 5.6 and 37°C for 72 hours. Modified GCase polypeptides can retain at least 10 times, at least 15 times, at least 20 times, at least 30 times, or at least 50 times higher activity than reference GCase polypeptides when incubated in PBS at pH 7.4 at 37°C for 72 hours. For example, modified GCase polypeptides can retain at least 30 times higher activity than reference GCase polypeptides when incubated in PBS at pH 7.4 at 37°C for 72 hours. Optionally, the modified GCase polypeptide retains at least 80% activity after incubation at pH 5.6 and 37°C for 72 hours.

[0083] In some embodiments, at least one mutation provides structural stabilization at physiological pH (e.g., pH 7.4). Exemplary mutations that provide structural stabilization are disclosed herein. "At least one mutation provides structural stabilization at physiological pH" means that a modified GCase polypeptide containing at least one mutation is structurally more stable at physiological pH than a reference GCase polypeptide that does not contain the at least one mutation but is otherwise identical to the modified GCase polypeptide containing the at least one mutation. For example, if at least one mutation provides structural stabilization at physiological pH, and the modified GCase polypeptide containing at least one mutation is otherwise identical to the wild-type GCase polypeptide except that it contains at least one mutation (e.g., mutations W351C and A380C), then the modified GCase polypeptide containing at least one mutation is structurally more stable at physiological pH than the wild-type GCase polypeptide. In some embodiments, a “test” polypeptide identical to SEQ ID NO: 1 except for at least one mutation is structurally more stable at physiological pH compared to the polypeptide of SEQ ID NO: 1, and optionally, if the “test” polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, at least one mutation “provides structural stabilization at physiological pH.” A modified GCase polypeptide containing at least one mutation does not necessarily have to be structurally more stable at physiological pH than the wild-type GCase polypeptide. For example, a modified GCase polypeptide containing at least one mutation may contain one or more further modifications (such as deletions) that reduce the structural stability of the polypeptide at physiological pH.In such cases, the modified GCase polypeptide may not have the same structural stability at physiological pH as the wild-type GCase polypeptide. Nevertheless, at least one mutation disclosed herein may help to make the modified GCase polypeptide more structurally stable at physiological pH than a reference GCase polypeptide that is otherwise identical to the modified GCase polypeptide except for lacking at least one mutation. Alternatively, at least one mutation may provide structural stability at physiological pH that restores the structural stability of the modified GCase polypeptide to the level of structural stability of the wild-type GCase polypeptide.

[0084] In some embodiments, modified GCase polypeptides containing at least one mutation are more structurally stable at physiological pH (e.g., pH 7.4).

[0085] In some embodiments, at least one mutation provides structural stabilization at pH 7.4. In some embodiments, a modified GCase polypeptide containing at least one mutation is more structurally stable at pH 7.4. In some embodiments, a modified GCase polypeptide containing at least one mutation is more structurally stable at physiological pH (e.g., pH 7.4) than the reference GCase polypeptide described herein. For example, the reference GCase polypeptide may be a wild-type GCase polypeptide. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 4 or 5. Optionally, a modified GCase polypeptide containing at least one mutation is more structurally stable at physiological pH than the reference GCase polypeptide. Optionally, a modified GCase polypeptide containing at least one mutation is more structurally stable at pH 7.4 than the reference GCase polypeptide. In some embodiments, the structural stability of the modified GCase at physiological pH is compared to the structural stability of the reference GCase polypeptide at physiological pH. This specification discloses exemplary mutations that provide structural stabilization at physiological pH of a modified GCase polypeptide containing at least one mutation, such as a mutation that enables the formation of disulfide crosslinks.

[0086] The structural stability of a GCase polypeptide at pH 7.4 can be determined using any of the methods herein for determining the stability or half-life of a GCase polypeptide. In such a method, any incubation of a sample containing a GCase polypeptide is performed at a physiological pH (e.g., pH 7.4).

[0087] To determine whether a modified GCase polypeptide is structurally more stable at physiological pH than a reference GCase polypeptide (such as a wild-type GCase polypeptide, as described herein), the structural stability of the modified GCase polypeptide and the reference GCase polypeptide at physiological pH is determined by the same method, and the results are compared. The method may be any of the methods described herein for determining stability or half-life.

[0088] Polypeptide stability or structural stability is typically mediated by interactions between amino acid side chains and / or interactions between amino acid side chains and the protein backbone. These interactions may be non-covalent, or they may form covalent bonds (e.g., disulfide bonds). Therefore, GCase polypeptides can be stabilized (e.g., compared to a reference GCase polypeptide) by amino acid substitutions (substitutional mutations) that stabilize the interactions between amino acid side chains and / or between amino acid side chains and the protein backbone. For example, disulfide bonds formed between cysteines at positions 351 and 380 of SEQ ID NO: 1 in a modified GCase polypeptide may provide increased stability or structural stability. A modified GCase polypeptide with disulfide bonds formed between cysteines at positions 351 and 380 of SEQ ID NO: 1 may have increased stability or structural stability compared to a reference GCase polypeptide.

[0089] In some embodiments, at least one mutation provides a longer half-life. Exemplary mutations that provide a longer half-life are disclosed herein. "At least one mutation provides a longer half-life" means that a modified GCase polypeptide containing at least one mutation has a longer half-life than a reference GCase polypeptide that does not contain the at least one mutation but is otherwise identical to the modified GCase polypeptide containing the at least one mutation. For example, if at least one mutation provides a longer half-life, and the modified GCase polypeptide containing at least one mutation is otherwise identical to the wild-type GCase polypeptide except that it contains at least one mutation (e.g., mutations W351C and A380C), then the modified GCase polypeptide containing at least one mutation has a longer half-life than the wild-type GCase polypeptide. In some embodiments, if a “test” polypeptide that is identical to SEQ ID NO: 1 except that it contains at least one mutation has a longer half-life compared to the polypeptide of SEQ ID NO: 1, and optionally, if the “test” polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, then at least one mutation “provides a longer half-life.” A modified GCase polypeptide containing at least one mutation does not necessarily have to have a longer half-life than a wild-type GCase polypeptide. For example, a modified GCase polypeptide containing at least one mutation may contain one or more further modifications (such as deletions) that shorten the polypeptide's half-life. In such cases, the modified GCase polypeptide may not have the same half-life as a wild-type GCase polypeptide, but nevertheless, the at least one mutation disclosed herein may help to give the modified GCase polypeptide a longer half-life, such that it has a longer half-life than a reference GCase polypeptide that is otherwise identical to the modified GCase polypeptide except for the lack of at least one mutation. Alternatively, the at least one mutation may provide a longer half-life that restores the half-life of the modified GCase polypeptide to the typical half-life of a wild-type GCase polypeptide.

[0090] In some embodiments, a modified GCase polypeptide containing at least one mutation has a longer half-life. In some embodiments, a modified GCase polypeptide has a longer half-life than a reference GCase polypeptide described herein. For example, the reference GCase polypeptide may be a wild-type GCase polypeptide. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 4 or 5. In some embodiments, the half-life of a modified GCase polypeptide containing at least one mutation is compared to the half-life of a reference GCase polypeptide. Exemplary mutations that extend the half-life of a modified GCase polypeptide containing at least one mutation are disclosed herein. In some embodiments, the stability or structural stability of a modified GCase polypeptide containing at least one mutation can be determined by determining its half-life.

[0091] Optionally, a modified GCase polypeptide may have a half-life at least 1.2 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times longer than the half-life of the reference GCase polypeptide. In other words, a modified GCase polypeptide may have a longer half-life at least 1.2 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times longer than the half-life of the reference GCase polypeptide. Optionally, a modified GCase polypeptide may have a half-life at least 1.2 to 30 times or at least 1.2 to 25 times longer than the half-life of the reference GCase polypeptide. In other words, a modified GCase polypeptide may have a longer half-life at least 1.2 to 30 times or at least 1.2 to 25 times longer than the half-life of the reference GCase polypeptide. Optionally, a modified GCase polypeptide may have a half-life 1.2 to 20 times, 1.2 to 15 times, or 2 to 15 times longer than that of a reference GCase polypeptide. In other words, a modified GCase polypeptide may have a longer half-life 1.2 to 20 times, 1.2 to 15 times, or 2 to 15 times longer than that of a reference GCase polypeptide. A longer half-life may be a longer half-life at physiological pH, for example, pH 7.4. Optionally, the half-life may be measured at pH 5.6 or pH 7.4. A longer half-life may be a longer half-life at pH 7.4. For example, a modified GCase polypeptide may have a half-life at pH 7.4 that is at least 1.8 times longer than that of a reference GCase polypeptide. A longer half-life may be a longer half-life at lysosomal pH. A longer half-life may be a longer half-life at pH 5.6. For example, a modified GCase polypeptide may have a half-life at pH 5.6 that is at least 20 times longer than that of a reference GCase polypeptide. Optionally, the half-life may be measured in a suitable matrix, such as serum (e.g., human or mouse) or plasma (e.g., human or mouse). A longer half-life may be a longer half-life in serum or plasma.Preferably, when the half-life is measured in serum (e.g., human), the half-life is at least 5 times longer than the half-life of the reference GCase polypeptide. The half-life may be measured using a fluorescence assay. The half-life can be determined by any method known in the art. The half-life can be determined by measuring the residual enzyme activity in the sample after a series of specified periods have elapsed. Optionally, the sample is obtained by transfecting host cells (e.g., Expi293F cells) with an expression vector containing a nucleotide sequence encoding the GCase polypeptide and collecting the GCase polypeptide from the culture medium. Optionally, the GCase polypeptide is collected from the culture medium 4 days after transfection. Optionally, the sample is obtained according to the method described in the chapters of Example 1 titled “Amplification of Expi293F Cells,” “Day Before Transfection of Expi293F Cells,” “Transfection of Expi293F Cells,” and “Collection of Transfected Expi293F Cells.” A sample containing GCase polypeptides may be transferred to a matrix (such as AB buffer at pH 5.6, PBS at pH 7.4, serum, or plasma) and incubated. The pH of the matrix may be selected to determine the half-life at a particular pH. The incubation temperature may be selected to determine the half-life at a particular temperature. Incubation may be performed at 37°C. Aliquots of the incubated sample containing GCase polypeptides may be taken at suitable time points (e.g., 0 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 24 hours, 48 ​​hours, 120 hours, and 144 hours). The initial time point (i.e., 0 minutes or 0 hours) is the time when the sample is transferred to the matrix for incubation. By comparing the GCase activity at a given time point with the GCase activity at the initial time point, the residual GCase activity at that given time point can be determined.Optionally, residual enzyme activity is measured in the sample at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 24, 48, 120, and 144 hours, according to the chapter titled “Stability Assessment” in Example 1. The activity of GCase polypeptide in aliquots can be measured according to the fluorescence assay protocol described herein. For example, GCase polypeptide may be incubated with 4-MUG at 37°C for 30 minutes. Optionally, GCase polypeptide is incubated with 4-MUG at 37°C for 1 hour in the presence and absence of an irreversible GCase inhibitor, such as conzlitol B epoxide. For example, a portion of the sample is incubated in the presence of an irreversible GCase inhibitor, such as conzlitol B epoxide, and a portion of the sample is incubated in its absence, and the results are compared to obtain the GCase activity level. Optionally, GCase polypeptide is incubated at pH 5.6 or pH 5.75. The levels of the generated fluorescence products can be measured using emission wavelengths of 365 nm and excitation wavelengths of 445 nm, respectively. Optionally, the activity of the GCase polypeptide in aliquots can be measured according to the fluorescence assay protocol described in the chapter titled "Determination of GCase Activity" in Example 1 or the paragraph beginning with "GCase Activity Assay" in the chapter titled "Method" in Example 13. This value can be applied to one-phase disintegration models, such as those outlined in Example 5. Optionally, the GCase polypeptide may be purified before determining its half-life. The GCase polypeptide may be purified as described herein, for example, according to the chapter titled "Purification of GCase Protein" in Example 1. Optionally, the half-life of the GCase polypeptide can be determined using the methodology of Example 5.

[0092] To determine whether a modified GCase polypeptide has a longer half-life than a reference GCase polypeptide (such as a wild-type GCase polypeptide, as described herein), the half-lives of the modified GCase polypeptide and the reference GCase polypeptide are determined by the same method, and the results are compared. The method may be any of the methods described herein for determining half-lives.

[0093] The effective activity, stability, residual activity, or half-life of the modified GCase polypeptide may be determined at pH 5, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, or pH 8, or from pH 5 to pH 6, from pH 5.3 to pH 5.7, from pH 5.6 to pH 5.8, from pH 6.8 to pH 7.8, from pH 7 to pH 7.6, from pH 7.2 to pH 7.5, or from pH 7.3 to pH 7.8. Optionally, the pH is lysosomal pH (e.g., pH 5.6) or physiological pH (e.g., pH 7.4). Optionally, the pH is pH 7.35 to pH 7.45. Preferably, the pH is physiological pH, e.g., pH 7.4. Optionally, higher effective activity, increased stability, higher residual activity, or longer half-life of the modified GCase polypeptide is observed at pH 5, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, p This refers to higher effective activity, increased stability, higher residual activity, or a longer half-life at pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, or pH 8, or at pH 5-6, pH 5.3-5.7, pH 5.6-5.8, pH 6.8-7.8, pH 7-7.6, pH 7.2-7.5, or pH 7.3-7.8. Optionally, pH is lysosomal pH (e.g., pH 5.6) or physiological pH (e.g., pH 7.4). Optionally, pH is pH 7.35-7.45. Preferably, pH is physiological pH, for example, pH 7.4.

[0094] The active activity, stability, residual activity, or half-life of the modified GCase polypeptide may be determined at 18°C, 20°C, 25°C, 30°C, 35°C, or 37°C. Preferably, the active activity, stability, residual activity, or half-life of the modified GCase polypeptide is determined at 37°C. Optionally, higher active activity, increased stability, increased residual activity, or longer half-life of the modified GCase polypeptide refers to higher active activity, increased stability, increased residual activity, or longer half-life at 18°C, 20°C, 25°C, 30°C, 35°C, or 37°C, respectively. Preferably, higher active activity, increased stability, increased residual activity, or longer half-life refers to higher active activity, increased stability, increased residual activity, or longer half-life at 37°C, respectively.

[0095] Modified GCase polypeptides may exhibit increased stability in liquid (i.e., in solution). Optionally, GCase polypeptides may have a longer half-life in liquid. Optionally, the liquid is a conditional medium, such as a conditional medium for culturing host cells expressing the GCase polypeptide. Optionally, the liquid is a biological sample. The biological sample may be blood, serum, or plasma. Optionally, modified GCase polypeptides may exhibit higher stability in plasma. Optionally, modified GCase polypeptides may have a longer half-life in plasma.

[0096] In some embodiments, at least one mutation provides increased thermal stability. Exemplary mutations that provide increased thermal stability are disclosed herein. "At least one mutation provides increased thermal stability" means that a modified GCase polypeptide containing at least one mutation has increased thermal stability compared to a reference GCase polypeptide that does not contain the at least one mutation but is otherwise identical to the modified GCase polypeptide containing the at least one mutation. For example, if at least one mutation provides increased thermal stability and the modified GCase polypeptide containing at least one mutation is otherwise identical to the wild-type GCase polypeptide except that it contains at least one mutation (e.g., mutations W351C and A380C), then the modified GCase polypeptide containing at least one mutation has increased thermal stability compared to the wild-type GCase polypeptide. In some embodiments, if a "test" polypeptide that is identical to SEQ ID NO: 1 except that it contains at least one mutation has increased thermal stability compared to the polypeptide of SEQ ID NO: 1, and optionally, if the "test" polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, then at least one mutation "provides increased thermal stability". A modified GCase polypeptide containing at least one mutation does not necessarily have increased thermal stability compared to a wild-type GCase polypeptide. For example, a modified GCase polypeptide containing at least one mutation may contain one or more further modifications (such as deletions) that reduce the polypeptide's thermal stability. In such cases, the modified GCase polypeptide may not have the same thermal stability as a wild-type GCase polypeptide, but nevertheless, the at least one mutation disclosed herein may help provide increased thermal stability to the modified GCase polypeptide so that it has higher thermal stability than a reference GCase polypeptide that is otherwise identical to the modified GCase polypeptide except for the lack of at least one mutation.Alternatively, at least one mutation may provide increased thermal stability, restoring the thermal stability of the modified GCase polypeptide to the typical thermal stability of the wild-type GCase polypeptide.

[0097] Thermal stability can be measured by determining the melting temperature (also known as the central denaturation temperature or the temperature at which half of the polypeptide denatures). Polypeptides with higher melting temperatures are more thermally stable than polypeptides with lower melting temperatures. Therefore, in some embodiments, a “test” polypeptide that is identical to SEQ ID NO: 1 except for at least one mutation has a higher melting temperature compared to the polypeptide of SEQ ID NO: 1, and optionally, if the “test” polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, at least one mutation “provides increased thermal stability.” In some embodiments, the modified GCase polypeptide has a melting temperature of at least 55°C, at least 56°C, or at least 57°C at pH 5.75. Optionally, the modified GCase polypeptide has a melting temperature of at least 53°C or at least 54°C at pH 5.75. Optionally, the modified GCase polypeptide has a melting temperature of at least 52°C, at least 53°C, or at least 53.5°C at pH 7.

[0098] In some embodiments, the modified GCase polypeptide has increased thermal stability compared to the reference GCase polypeptide described herein. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 4 or 5. In some embodiments, the thermal stability of the modified GCase polypeptide containing at least one mutation is compared to the thermal stability of the reference GCase polypeptide. Exemplary mutations that increase the thermal stability of the modified GCase polypeptide containing at least one mutation are disclosed herein.

[0099] Optionally, the melting temperature of the modified GCase polypeptide is at least 1°C, at least 2°C, at least 3°C, at least 4°C, at least 5°C, or at least 5.4°C higher than that of the reference GCase polypeptide at pH 5.75. Optionally, the melting temperature of the modified GCase polypeptide is at least 1°C, at least 2°C, or at least 2.5°C higher than that of the reference GCase polypeptide at pH 7.

[0100] Optionally, the melting temperature is calculated at modified GCase polypeptide concentrations of 1.5 μM, 3 μM, or 6 μM. Optionally, the melting temperature is calculated at modified GCase polypeptide concentrations of 1.5 μM or 3 μM. Optionally, the melting temperature is calculated at modified GCase polypeptide concentration of 3 μM.

[0101] Thermal stability can be measured using a thermal shift assay. Measuring the thermal stability of a polypeptide (such as the modified GCase polypeptide or reference GCase polypeptide described herein) using a thermal shift assay essentially involves incubating the polypeptide at an increasing temperature and monitoring the unfolding of the polypeptide as the temperature rises. For example, the polypeptide (such as the modified GCase polypeptide or reference GCase polypeptide) may be held at 25°C for a certain period (e.g., 2 minutes), and then the temperature may be gradually increased over time. Optionally, the temperature may be increased by 1°C per minute. Optionally, the temperature may be increased by 1°C per minute until the temperature at which all of the modified GCase polypeptide has denatured, for example, 95°C. Optionally, the thermal shift assay may involve incubating the modified GCase polypeptide at a temperature that increases by 1°C per minute. Optionally, a thermal shift assay includes measuring the unfolding of a modified GCase polypeptide or a reference GCase polypeptide when incubated at a temperature increasing by 1°C per minute; i.e., the melting temperature is calculated by measuring the unfolding of a modified GCase polypeptide or a reference GCase polypeptide when incubated at a temperature increasing by 1°C per minute.

[0102] Polypeptide unfolding can be monitored using fluorescent dyes that are sensitive to changes in protein structure. For example, dyes such as SYPRO® Orange Protein Gel Stain bind to hydrophobic protein regions and emit fluorescence when the protein unfolds. When using fluorescent dyes to monitor polypeptide unfolding, the fluorescent dye is added to the culture medium in which the polypeptide is incubated during a heat shift assay, and the fluorescence is monitored.

[0103] A melting curve may be created by plotting the fluorescence level (in relative fluorescence units or RFU) on the Y-axis and temperature on the X-axis. The melting temperature corresponds to the minimum derivative of the negative first derivative of the melting curve. Optionally, the melting temperature can be measured using the assay described under the heading "Thermal Stability Test" in Example 1.

[0104] Optionally, before performing thermal stability assays such as a heat shift assay, the modified GCase polypeptide and / or reference GCase polypeptide are stored in pH 5.75 buffer (such as sodium citrate buffer). Optionally, the reference GCase polypeptide may be a wild-type GCase polypeptide such as VPRIV. Optionally, VPRIV is reconstituted with nuclease-free water before performing thermal stability assays such as a heat shift assay.

[0105] In some embodiments, at least one mutation reduces the number of human leukocyte antigen (HLA)-I and / or HLA-II binders. Exemplary mutations that reduce the number of HLA-I and / or HLA-II binders are disclosed herein. References to a reduction in HLA-I and / or HLA-II binders, such as at least one mutation "reduces the number of HLA-I and / or HLA-II binders" or a modified GCase polypeptide "has a reduced number of HLA-I and / or HLA-II binders," should be interpreted as meaning that a reduction in the number of HLA-I and / or HLA-II binders is expected. The number of HLA-I and / or HLA-II binders can be predicted as described herein. "At least one mutation reduces the number of HLA-I and / or HLA-II binders" means that a modified GCase polypeptide containing at least one mutation has fewer HLA-I and / or HLA-II binders than a reference GCase polypeptide that does not contain the at least one mutation but is otherwise identical to the modified GCase polypeptide containing the at least one mutation. For example, if at least one mutation reduces the number of HLA-I and / or HLA-II binders, and a modified GCase polypeptide containing at least one mutation is otherwise identical to a wild-type GCase polypeptide except that it contains at least one mutation (e.g., mutations W351C and A380C), then the modified GCase polypeptide containing at least one mutation has fewer HLA-I and / or HLA-II binders than the wild-type GCase polypeptide. In some embodiments, a “test” polypeptide identical to SEQ ID NO: 1 except for at least one mutation has fewer HLA-I and / or HLA-II binders compared to the polypeptide of SEQ ID NO: 1, and optionally, if the “test” polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, at least one mutation “reduces the number of HLA-I and / or HLA-II binders”.

[0106] In some embodiments, at least one mutation reduces the number of HLA-I and / or HLA-II binders. In some embodiments, a modified GCase polypeptide containing at least one mutation has a reduced number of HLA-I and / or HLA-II binders. Optionally, the HLA-I and / or HLA-II binders are potent binders. Optionally, the HLA-I binder is a potent binder. Optionally, the HLA-II binder is a potent binder. An "HLA-I binder" is a polypeptide-derived fragment that can bind to HLA-I. An "HLA-II binder" is a polypeptide-derived fragment that can bind to HLA-II. A lower number of polypeptide-derived HLA-I and / or HLA-II binders (especially "potent" binders) may reduce the risk of the polypeptide inducing an immune response. The number of HLA-I and HLA-II binders (especially "strong" binders) can be predicted as will be discussed further below. As will be discussed further below, a "strong" binder can be defined as having an IC50 cutoff range of 0 to ≤ 50 nM. In some embodiments, the number of HLA-I and / or HLA-II binders is predicted using IC50 binding affinity. Preferably, a reduced number of HLA-I and / or HLA-II binders are strong binders. A reduced number of HLA-I binders may be strong binders. A reduced number of HLA-II binders may be strong binders. In some embodiments, a reduced number of HLA-I and / or HLA-II binders (e.g., strong binders) is compared to the number of HLA-I and / or HLA-II binders (e.g., strong binders) for each reference GCase polypeptide described herein. For example, the reference GCase polypeptide may be a wild-type GCase polypeptide. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 3. The reference GCase polypeptide may be the polypeptide of SEQ ID NO: 4 or 5. At least one mutation can reduce the number of HLA-I binders (e.g., a potent binder).A modified GCase polypeptide containing at least one mutation may have a reduced number of HLA-I binders (e.g., potent binders) compared to the number of HLA-I binders (e.g., potent binders) in a reference GCase polypeptide. At least one mutation may reduce the number of HLA-II binders (e.g., potent binders). A modified GCase polypeptide containing at least one mutation may have a reduced number of HLA-II binders (e.g., potent binders) compared to the number of HLA-II binders (e.g., potent binders) in a reference GCase polypeptide. Exemplary mutations that reduce the number of HLA-I and / or HLA-II binders in a modified GCase polypeptide are disclosed herein.

[0107] The immunological risk profile of GCase polypeptides can be evaluated using known in silico analysis or prediction. For example, the data may be input for calculations using the Immune Epitope database and the Analysis Resource database. The prediction methods used may be NetMHCpan EL 4.0 (for HLA-I) and NetMHCIIpan 3.2 (for HLA-II). For example, the immunogenicity risk profile of a GCase polypeptide can be evaluated according to the methodology of Example 7. For example, peptide fragments of 9 amino acid length and 15 amino acid length (to calculate the binding affinity to the HLA-I and HLA-II receptor molecules, respectively) from the region extending 50 amino acids upstream and 50 amino acids downstream of the relevant amino acid substitution of the modified GCase polypeptide (e.g., from 50 amino acids upstream of W351C to 50 amino acids downstream of A380C) may be used as input for calculations using the Immune Epitope database and the Analysis Resource database. Possible prediction methods include NetMHCpan EL 4.0 (for HLA-I) and NetMHCIIpan 3.2 (for HLA-II). The IC50 value can be used as a measure of peptide binding affinity. The IC50 value can be used to classify binders as "strong" or "moderate" binders. A lower IC50 value predicts better binding. For "strong" binders, an IC50 cutoff range of 0 to ≤50 nM may be used, while for "moderate" binders, an IC50 cutoff range of >50 to ≤500 nM may be used.

[0108] To determine whether a modified GCase polypeptide has a reduced number of HLA-I and / or HLA-II binders compared to a reference GCase polypeptide (such as a wild-type GCase polypeptide as described herein), the number of HLA-I and / or HLA-II binders in the modified GCase polypeptide and the reference GCase polypeptide is predicted using the same method, and the results are compared. The method may be any of the methods described herein for predicting the number of HLA-I and / or HLA-II binders.

[0109] In some embodiments, at least one mutation may provide reduced immunogenicity. In some embodiments, a modified GCase polypeptide containing at least one mutation has reduced immunogenicity. In some embodiments, the level of immunogenicity of a modified GCase polypeptide containing at least one mutation is compared to the level of a reference GCase polypeptide described herein. In some embodiments, a modified GCase polypeptide containing at least one mutation has reduced immunogenicity compared to the immunogenicity of a reference GCase polypeptide described herein. Exemplary mutations that can reduce the immunogenicity of a modified GCase polypeptide are disclosed herein.

[0110] "At least one mutation provides reduced immunogenicity" means that a modified GCase polypeptide containing at least one mutation has reduced immunogenicity compared to a reference GCase polypeptide that does not contain the at least one mutation but is otherwise identical to the modified GCase polypeptide containing the at least one mutation. For example, if at least one mutation reduces immunogenicity, and the modified GCase polypeptide containing at least one mutation is otherwise identical to the wild-type GCase polypeptide except that it contains at least one mutation (e.g., mutations W351C and A380C), then the modified GCase polypeptide containing at least one mutation has reduced immunogenicity compared to the wild-type GCase polypeptide. In some embodiments, if a "test" polypeptide that is identical to SEQ ID NO: 1 except for at least one mutation has reduced immunogenicity compared to the polypeptide of SEQ ID NO: 1, and optionally, if the "test" polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line, then at least one mutation "provides reduced immunogenicity".

[0111] In some embodiments, the immunogenicity of a GCase polypeptide is assessed by predicting the number of HLA-I and / or HLA-II binders, as described herein.

[0112] In some embodiments, the immunogenicity of a GCase polypeptide is evaluated by measuring the immune response to the GCase polypeptide. For example, the GCase polypeptide may be administered to test animals such as mice, and the immune response in the test animals may be measured and compared to the immune response in equivalent test animals administered with a reference GCase polypeptide. The immune response can be measured by examining inflammatory markers in the blood, such as cytokines or acute-phase proteins. Examples of cytokines that can be measured are TNF-α, IL-1β, and / or INF-γ. Examples of acute-phase proteins that can be measured are C-reactive proteins, mannan-binding lectins, and / or complement factors. A decrease in inflammatory markers can be used to indicate lower immunogenicity.

[0113] In some embodiments, the immunogenicity of GCase polypeptides is evaluated using a T cell proliferation assay that tests the level of T cell activation in human peripheral blood mononuclear cells (PMBCs) when incubated with the whole protein (e.g., wild-type GCase polypeptide or the modified GCase polypeptide of the present invention). For example, the ProMap® naive CFSE T cell proliferation assay may be used (https: / / www.proimmune.com / promap-t-cell-proliferation-assays / ).

[0114] To determine whether a modified GCase polypeptide has reduced immunogenicity compared to a reference GCase polypeptide (such as a wild-type GCase polypeptide as described herein), the immunogenicity of the modified GCase polypeptide and the reference GCase polypeptide is evaluated using the same method, and the results are compared. The method may be any of the methods described herein for evaluating immunogenicity (e.g., predicting the number of HLA-I and / or HLA-II binders).

[0115] The no-observed-adverse-effect level (NOAEL) may be calculated for the modified GCase polypeptide of the present invention. A high NOAEL suggests that the modified GCase polypeptide of the present invention has low immunogenicity. For example, the GCase polypeptide may be administered to test animals such as mice in escalating doses and monitored to determine whether the mice experience adverse events. The lowest dose that causes adverse events is the NOAEL. As will be discussed in more detail below, the modified GCase polypeptide of the present invention may also be administered as part of viral particles, for example, in escalating doses, e.g., 1 × 10⁻⁶ doses. 8 , 1 x 10 9 , 5×10 9 , 1 x 10 10 , 5×10 10Mice may be administered viral particles encoding GCase polypeptides in doses such as 1 × 10⁻¹⁶. Optionally, the modified GCase polypeptide is encoded by a modified GBA nucleotide sequence, and the GBA nucleotide sequence is administered in viral particles containing a recombinant genome with polynucleotides including the GBA nucleotide sequence, and the NOAEL is at least 1 × 10⁻¹⁶. 12 vg / kg, at least 5 × 10 12 vg / kg, at least 1 × 10⁻⁶ 13 vg / kg, or at least 2 × 10⁻⁶ 13 It is vg / kg.

[0116] The enzymatic efficiency of the modified GCase polypeptide of the present invention may be the same as that of the wild-type GCase polypeptide. The statement that the enzymatic efficiency (i.e., the ability to process a substrate) of the first GCase polypeptide (e.g., the modified GCase polypeptide) is "the same" as that of the second GCase polypeptide (e.g., the wild-type GCase polypeptide) means that the enzymatic efficiency of the first GCase polypeptide may be identical to that of the second GCase polypeptide by plus or minus 10%, more specifically by plus or minus 5%, or more specifically by plus or minus 1%. For the purposes of the present invention, "enzymatic efficiency" refers to the ability of a GCase polypeptide to process a substrate, such as methylumbelliferyl-β-D-glucopyranosideuronic acid (4-MUG) or its natural substrate, glucocerebroside. While the enzymatic efficiency of the modified GCase polypeptide of the present invention may be the same as that of the wild-type GCase polypeptide, the effective activity or residual activity of the modified GCase polypeptide of the present invention may be higher than that of the wild-type GCase polypeptide. This finding may be a result of the modified GCase polypeptide of the present invention being more stable and therefore more active over a longer period. Thus, the modified GCase polypeptide of the present invention has the same substrate handling capacity as the wild-type GCase polypeptide, but can maintain its activity over a longer period, resulting in higher effective activity and / or higher residual activity.

[0117] The enzymatic efficiency of GCase polypeptides can be measured by determining the values ​​of Km and / or Kcat. The values ​​of Km and / or Kcat can be determined by standard methods in the art. Enzymatic efficiency can be measured by treating 4-methylumbelliferyl-β-D-glucopyranosideuronic acid (4-MUG) by the method described in Example 6, for example. GCase polypeptides may be purified as described herein, for example, in Example 1. The purified protein (e.g., 1 nM or 3.5 nM) is then increased in AB buffer (e.g., 2.5 × 10⁻⁶). 6 nM, 5×10 6 nM, 7.5 × 10 6 nM, or 1 × 10⁻⁶ 7 The protein may be incubated with nM 4-MUG. Optionally, the purified protein is incubated with 4-MUG in a pH 5.75 composition. The formation of 4-methylumbelliferone (4-MU) may be monitored, for example, over 15 minutes, by measuring the resulting fluorescence (excitation wavelength: 365 nm, emission wavelength: 445 nm). The 4-MU formation rate at each 4-MUG concentration may be plotted against the 4-MUG concentration and fitted using the Michaelis-Menten model. The Kcat value is 32s -1 ~40s -1 , 32.8s -1 ~39.2s -1 , 33s -1 ~39s -1 , or 35s -1 ~37s -1 That's fine. The Kcat value is 36.0s -1 The Km value of the modified GCase polypeptide of the present invention may be around 1.4 mM to 2.5 mM, 1.6 mM to 2.2 mM, or 1.8 mM to 2.2 mM. Optionally, the Km value of the modified GCase polypeptide may be around 2.0 mM.

[0118] The modified GCase polypeptide of the present invention may have higher effective activity and / or increased stability and / or structural stabilization at physiological pH and / or a longer half-life. The modified GCase polypeptide of the present invention may have a reduced number of HLA-I and / or HLA-II binders. The modified GCase polypeptide of the present invention may have reduced immunogenicity. The modified GCase polypeptide of the present invention may have increased thermal stability. The modified GCase polypeptide of the present invention may be expressed in host cells at higher levels than the reference GCase polypeptide described herein.

[0119] The modified GCase polypeptides of the present invention contain at least one mutation. In some embodiments, at least one mutation (i.e., one or more substitutional mutations) is located at a position selected from the group consisting of 351, 380, 272, 262, 313, 404, 407, 482, 484, 490, 494, 503, and 534 of SEQ ID NO: 1. At least one mutation (such as a mutation at a position corresponding to one of the positions listed above) may provide one or more of the properties discussed herein (e.g., higher active activity, increased stability, structural stabilization at physiological pH, longer half-life, higher residual activity, reduced number of HLA-I and / or HLA-II binders, reduced immunogenicity, or increased thermal stability). Modified GCase polypeptides containing at least one mutation (such as a mutation at a position corresponding to one of the positions listed above) may have one or more of the properties discussed herein (e.g., higher active activity, increased stability, structural stabilization at physiological pH, longer half-life, higher residual activity, reduced number of HLA-I and / or HLA-II binders, reduced immunogenicity, increased thermal stability, etc.).

[0120] In some embodiments, at least one mutation may involve a substitution with an amino acid that acts as a proton donor at pH 7.4.

[0121] Optionally, at least one mutation is located at the position corresponding to position 272 of sequence number 1. Preferably, the mutation is a glutamine substitution. For example, the mutation may be E272Q.

[0122] Optionally, at least one mutation is located at the position corresponding to position 262 of SEQ ID NO: 1. The mutation may be a substitution with asparagine or tyrosine. Preferably, the mutation is a substitution with asparagine. For example, the mutation may be H262N.

[0123] Optionally, at least one mutation includes a tyrosine mutation at the position corresponding to position 262 of sequence number 1, e.g., H262Y.

[0124] Optionally, at least one mutation is located at the position corresponding to position 313 of SEQ ID NO: 1. Preferably, the mutation is an asparagine substitution. For example, the mutation may be H313N.

[0125] Optionally, at least one mutation is located at the position corresponding to position 404 of SEQ ID NO: 1. Preferably, the mutation is a lysine substitution. For example, the mutation may be H404K.

[0126] Optionally, at least one mutation is located at the position corresponding to position 490 of SEQ ID NO: 1. Preferably, the mutation is a lysine substitution. For example, the mutation may be H490K.

[0127] Optionally, at least one mutation is located at the position corresponding to position 534 of SEQ ID NO: 1. Preferably, the mutation is an asparagine substitution. For example, the mutation may be R534N.

[0128] Optionally, at least one mutation in the modified GCase polypeptide includes two mutations, each of which is a cysteine ​​substitution enabling the formation of a disulfide crosslink. The formation of the disulfide crosslink can stabilize the structure of the modified GCase, for example, at physiological pH. The formation of disulfide bonds can be determined by mass spectrometry. For example, the formation of disulfide bonds can optionally be determined by analyzing the fragmentation pattern of polypeptides suspected of containing disulfide bonds after limited protein degradation, as outlined, for example, Gorman et al. 2002, Mass Spectrometry Reviews 21, 183-216. Alternatively, the formation of disulfide bonds can also be determined by limited protein degradation of the polypeptide and, optionally combined with N-terminal sequencing, by analyzing the resulting protein fragments by SDS-PAGE under both reducing and non-reducing conditions.

[0129] Optionally, at least one mutation (e.g., two mutations) includes (i) a mutation at the position corresponding to position 482 of SEQ ID NO: 1 and (ii) a mutation at the position corresponding to position 503 of SEQ ID NO: 1. Optionally, (i) the mutation at position 482 of SEQ ID NO: 1 is a cysteine ​​substitution, and (ii) the mutation at position 503 of SEQ ID NO: 1 is a cysteine ​​substitution. Optionally, (i) the mutation at position 482 of SEQ ID NO: 1 is an aspartic acid to cysteine ​​substitution, and (ii) the mutation at position 503 of SEQ ID NO: 1 is a serine to cysteine ​​substitution. For example, the mutation may be D482C / S503C.

[0130] Optionally, at least one mutation (e.g., including two mutations) includes (i) a mutation at the position corresponding to position 494 of SEQ ID NO: 1 and (ii) a mutation at the position corresponding to position 534 of SEQ ID NO: 1. Optionally, (i) the mutation at the position corresponding to position 494 of SEQ ID NO: 1 is a cysteine ​​substitution, and (ii) the mutation at the position corresponding to position 534 of SEQ ID NO: 1 is a cysteine ​​substitution. Optionally, (i) the mutation at the position corresponding to position 494 of SEQ ID NO: 1 is a serine-to-cysteine ​​mutation, and (ii) the mutation at the position corresponding to position 534 of SEQ ID NO: 1 is an arginine-to-cysteine ​​mutation. For example, the mutation may be S494C / R534C.

[0131] Optionally, at least one mutation (e.g., two mutations) includes (i) a mutation at the position corresponding to position 351 of SEQ ID NO: 1 and (ii) a mutation at the position corresponding to position 380 of SEQ ID NO: 1. Optionally, (i) the mutation at position 351 of SEQ ID NO: 1 is a cysteine ​​substitution, and (ii) the mutation at position 380 of SEQ ID NO: 1 is a cysteine ​​substitution. Optionally, (i) the mutation at position 351 of SEQ ID NO: 1 is a tryptophan to cysteine ​​substitution, and (ii) the mutation at position 380 of SEQ ID NO: 1 is an alanine to cysteine ​​substitution. For example, the mutation may be W351C / A380C.

[0132] Optionally, at least one mutation (e.g., two mutations) includes (i) a mutation at the position corresponding to position 407 of SEQ ID NO: 1 and (ii) a mutation at the position corresponding to position 484 of SEQ ID NO: 1. Optionally, (i) the mutation at the position corresponding to position 407 of SEQ ID NO: 1 is a cysteine ​​substitution, and (ii) the mutation at the position corresponding to position 484 of SEQ ID NO: 1 is a cysteine ​​substitution. Optionally, (i) the mutation at the position corresponding to position 407 of SEQ ID NO: 1 is an isoleucine to cysteine ​​substitution, and (ii) the mutation at the position corresponding to position 484 of SEQ ID NO: 1 is an aspartic acid to cysteine ​​substitution. For example, the mutation may be I407C / D484C.

[0133] Optionally, at least one mutation (e.g., including three mutations) includes (i) a mutation at the position corresponding to position 272 of SEQ ID NO: 1, (ii) a mutation at the position corresponding to position 351 of SEQ ID NO: 1, and (iii) a mutation at the position corresponding to position 380 of SEQ ID NO: 1. Optionally, (i) the mutation at the position corresponding to position 272 of SEQ ID NO: 1 is a glutamine substitution, (ii) the mutation at the position corresponding to position 351 of SEQ ID NO: 1 is a cysteine ​​substitution, and (iii) the mutation at the position corresponding to position 380 of SEQ ID NO: 1 is a cysteine ​​substitution. Optionally, (i) the mutation at the position corresponding to position 272 of SEQ ID NO: 1 is a glutamate-to-glutamine substitution, (ii) the mutation at the position corresponding to position 351 of SEQ ID NO: 1 is a tryptophan-to-cysteine ​​substitution, and (iii) the mutation at the position corresponding to position 380 of SEQ ID NO: 1 is an alanine-to-cysteine ​​substitution. For example, the mutation could be E272Q / W351C / A380C.

[0134] Optionally, at least one mutation includes a glutamine substitution at the position corresponding to position 272 of SEQ ID NO: 1, and the modified GCase polypeptide exhibits increased stability compared to the reference GCase polypeptide described herein (such as the wild-type GCase polypeptide), retaining at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes. Optionally, at least one mutation includes (i) a cysteine ​​substitution at the position corresponding to position 351 of SEQ ID NO: 1 (e.g., a tryptophan to cysteine ​​substitution), and (ii) a cysteine ​​substitution at the position corresponding to position 380 of SEQ ID NO: 1 (e.g., an alanine to cysteine ​​substitution), and the modified GCase polypeptide exhibits increased stability compared to the reference GCase polypeptide described herein (such as the wild-type GCase polypeptide), retaining at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes. In one embodiment, at least one mutation includes a glutamine substitution at the position corresponding to position 272 of SEQ ID NO: 1, and the modified GCase polypeptide has increased stability compared to the reference GCase polypeptide, retaining at least 60% activity when measured after incubation at 37°C for 72 hours in PBS at pH 7.4, and optionally the reference GCase polypeptide is the wild-type GCase polypeptide. In another embodiment, at least one mutation includes a glutamine substitution at the position corresponding to position 272 of SEQ ID NO: 1, and the modified GCase polypeptide has increased stability compared to the reference GCase polypeptide, retaining at least 50% or at least 55% activity when measured after incubation at 37°C for 72 hours at pH 5.6, and optionally the reference GCase polypeptide is the wild-type GCase polypeptide.

[0135] Optionally, at least one mutation includes (i) a glutamine substitution at the position corresponding to position 272 of SEQ ID NO: 1 (e.g., E272Q); (ii) a cysteine ​​substitution at the position corresponding to position 351 of SEQ ID NO: 1 (e.g., a tryptophan to cysteine ​​mutation); and (iii) a cysteine ​​substitution at the position corresponding to position 380 of SEQ ID NO: 1 (e.g., an alanine to cysteine ​​mutation). Optionally, the modified GCase polypeptide has increased stability compared to the reference GCase polypeptide described herein (e.g., wild-type GCase polypeptide), retaining at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes. In one embodiment, at least one mutation is: (i) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of sequence number 1; and (ii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of SEQ ID NO: 1 The modified GCase polypeptide, comprising [specific components], exhibits increased stability compared to the reference GCase polypeptide, retaining at least 60% activity after incubation in PBS at pH 7.4 at 37°C for 72 hours, and optionally, the reference GCase polypeptide is a wild-type GCase polypeptide. In one embodiment, at least one mutation is [specific component]. (i) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of sequence number 1; and (ii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of SEQ ID NO: 1 The modified GCase polypeptide, comprising [specific components], exhibits increased stability compared to the reference GCase polypeptide, retaining at least 50% or at least 55% activity after incubation at pH 5.6 and 37°C for 72 hours, and optionally, the reference GCase polypeptide is a wild-type GCase polypeptide. In one embodiment, at least one mutation is [specific component]. (i) Glutamine substitution at the position corresponding to position 272 of sequence number 1, optionally E272Q; and (ii) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of SEQ ID NO: 1; and (iii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of Sequence ID No. 1 The modified GCase polypeptide, comprising [specific components], exhibits increased stability compared to the reference GCase polypeptide, retaining at least 60% activity after incubation in PBS at pH 7.4 at 37°C for 72 hours, and optionally, the reference GCase polypeptide is a wild-type GCase polypeptide. In one embodiment, at least one mutation is [specific component]. (i) Glutamine substitution at the position corresponding to position 272 of sequence number 1, optionally E272Q; and (ii) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of SEQ ID NO: 1; and (iii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of Sequence ID No. 1 The modified GCase polypeptide, containing the modified GCase polypeptide, exhibits increased stability compared to the reference GCase polypeptide, retaining at least 50% or at least 55% activity after incubation at pH 5.6 and 37°C for 72 hours, and optionally the reference GCase polypeptide is the wild-type GCase polypeptide.

[0136] Optionally, at least one mutation comprises (i) a cysteine ​​substitution at the position corresponding to position 351 of SEQ ID NO: 1 (e.g., a tryptophan to cysteine ​​substitution), and (ii) a cysteine ​​substitution at the position corresponding to position 380 of SEQ ID NO: 1 (e.g., an alanine to cysteine ​​substitution), and the modified GCase polypeptide has increased stability compared to the reference GCase polypeptide described herein (e.g., wild-type GCase polypeptide), and retains at least 15% or at least 20% activity after incubation at pH 7.4 and 37°C for 7 days. In one embodiment, at least one mutation comprises a glutamine substitution at the position corresponding to position 272 of SEQ ID NO: 1, and the modified GCase polypeptide has increased stability compared to the reference GCase polypeptide, and retains at least 15% or at least 20% activity after incubation at 37°C in PBS at pH 7.4 for 7 days, and optionally, the reference GCase polypeptide is the wild-type GCase polypeptide. In one embodiment, at least one mutation is, (i) Glutamine substitution at the position corresponding to position 272 of sequence number 1, optionally E272Q; and (ii) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of SEQ ID NO: 1; and (iii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of Sequence ID No. 1 The modified GCase polypeptide, containing the modified GCase polypeptide, exhibits increased stability compared to the reference GCase polypeptide, retaining at least 60% or at least 80% of its activity when measured after incubation at 37°C for 7 days in pH 7.4 PBS, and optionally, the reference GCase polypeptide is the wild-type GCase polypeptide.

[0137] Optionally, the modified GCase polypeptide has higher effective activity and / or increased stability compared to the reference GCase polypeptide described herein. Optionally, the reference GCase polypeptide is otherwise identical to the modified GCase polypeptide, but does not contain at least one mutation. Optionally, the reference GCase polypeptide is selected from any one of SEQ ID NOs: 1-5.

[0138] Optionally, at least one mutation does not contain one or more mutations selected from H184L, E272L, W351C, K360N, and A380T located at positions 184, 272, 351, 360, and 380 of SEQ ID NO: 1, 272, 351, 360, and 380, respectively. Optionally, at least one mutation does not contain the H184L mutation (located at position 184 of SEQ ID NO: 1). Optionally, at least one mutation does not contain the H184L mutation and the K360N mutation (located at positions 184 and 360 of SEQ ID NO: 1, 272, 351, 360, and 380, respectively). Optionally, the modified GCase polypeptide does not contain, and does not have, the amino acid sequence of SEQ ID NO: 4 or 5.

[0139] Optionally, at least one mutation does not contain the A380T mutation (which is located at position 380 of SEQ ID NO: 1). Optionally, at least one mutation does not contain the R534H mutation (which is located at position 534 of SEQ ID NO: 1).

[0140] Optionally, at least one mutation does not contain one or more mutations selected from H184L, H184F, F355A, L356F, K360N, and K360A located at positions 184, 355, 356, and 360 of SEQ ID NO: 1. Optionally, at least one mutation does not contain one or more mutations selected from H184F, F355A, L356F, and K360A located at positions 184, 355, 356, and 360, respectively of SEQ ID NO: 1. Optionally, at least one mutation does not contain the F355A mutation and the L356F mutation (these are located at positions 355 and 356, respectively, of SEQ ID NO: 1). Optionally, at least one mutation does not include the F355A, L356F, and K360N mutations (which are located at positions 355, 356, and 360 of SEQ ID NO: 1, respectively).

[0141] Optionally, the modified GCase polypeptide contains an amino acid sequence described in any one of SEQ ID NOs. 41-46, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs. Optionally, the modified GCase polypeptide contains an amino acid sequence described in SEQ ID NOs. 42 or 45.

[0142] Typically, GCase polypeptides are first expressed as a precursor "immature" form (e.g., the GCase polypeptide of SEQ ID NO: 1) containing a signal peptide (e.g., amino acid residues 1-39 of SEQ ID NO: 1) and a mature GCase polypeptide region. After processing, the "mature" form of the GCase polypeptide lacks the signal peptide. The terms "mature GCase" or "mature GCase polypeptide" refer to GCase polypeptides that do not contain a signal peptide, such as the GCase polypeptide of SEQ ID NO: 2.

[0143] The sequences of wild-type human GCase polypeptides are described in SEQ ID NO: 1 (immature) and SEQ ID NO: 2 (mature). Modified GCase polypeptides may contain amino acid sequences that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% identical to the SEQ ID NO: 1 fragment. Modified GCase polypeptides may contain amino acid sequences that are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, or at least 99.7% identical to the SEQ ID NO: 2 fragment. Optionally, the fragment is at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, or at least 450 amino acids, or 300-497 amino acids, 400-497 amino acids, or 450-497 amino acids. Optionally, the modified GCase polypeptide contains at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical amino acid sequence to the SEQ ID NO: 1 or SEQ ID NO: 2 fragment, which contains at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, 300-497 amino acids, 400-497 amino acids, or 450-497 amino acids. Optionally, the modified GCase polypeptide contains at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical amino acid sequence to SEQ ID NO: 1 or SEQ ID NO: 2.

[0144] Optionally, the modified GCase polypeptide contains an amino acid sequence that is at least 98% identical to the 400-536 amino acid fragment of SEQ ID NO: 1.

[0145] Optionally, the modified GCase polypeptide contains an amino acid sequence at least 98% identical to the 400-497 amino acid fragment of SEQ ID NO: 2.

[0146] Optionally, the modified GCase polypeptide contains an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8% identical to SEQ ID NO: 1. Optionally, the modified GCase polypeptide contains an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, or at least 99.7% identical to SEQ ID NO: 2. Preferably, the modified GCase polypeptide contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. Preferably, the modified GCase polypeptide contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 2.

[0147] Preferably, the modified GCase polypeptide may contain an amino acid sequence identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified GCase polypeptide contains at least one mutation (i.e., one or more substitutional mutations) as defined herein. In other words, the modified GCase polypeptide may contain a GCase amino acid sequence that differs from the amino acid sequence described in SEQ ID NO: 1 or 2 by only at least one mutation. Preferably, the modified GCase polypeptide contains an amino acid sequence identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified GCase polypeptide contains a mutation at the position corresponding to position 272 of SEQ ID NO: 1. Preferably, the modified GCase polypeptide contains a cysteine ​​substitution at the position corresponding to position 351 and position 380 of SEQ ID NO: 1, and optionally contains an amino acid sequence identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that it contains a mutation at the position corresponding to position 272 of SEQ ID NO: 1.

[0148] Optionally, a modified GCase polypeptide contains a GCase amino acid sequence that differs from the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 by only 20 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 substitution mutation. Optionally, a modified GCase polypeptide contains a GCase amino acid sequence that differs from the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 by only 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 substitution mutation. Optionally, a modified GCase polypeptide contains a GCase amino acid sequence that differs from the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 by only 3 or fewer substitution mutations. Optionally, a modified GCase polypeptide contains a GCase amino acid sequence that differs from the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 by only 2 or fewer substitution mutations. Optionally, a modified GCase polypeptide contains a GCase amino acid sequence that differs from the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 by at least one, at least two, at least three, at least four, at least five, at least eight, at least ten, at least fifteen, or at least twenty substitution mutations. Optionally, a GCase polypeptide may contain a GCase amino acid sequence that differs from the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 by at least 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 to 6, 3 to 8, 5 to 10, 8 to 13, 10 to 15, 13 to 18, or 15 to 20 substitution mutations. Optionally, the substitution mutations are non-conservative substitutions.

[0149] When the amino acid sequence of a modified GCase polypeptide is compared to the wild-type GCase amino acid sequence, any or all of the modified GCase polypeptides may contain one or more further modifications, such as substitutions, insertions, and / or deletions, in addition to at least one mutation as defined herein (i.e., one or more substitutional mutations). One or more further modifications may be one or more substitutions. One or more further modifications may be one or more conservative substitutions. One or more further modifications may be one or more non-conservative substitutions. At least one mutation may be located within one or more further modifications. For example, a contiguous portion of a GCase polypeptide (e.g., 50 or fewer, 20 or fewer, or 10 or fewer amino acids) may be replaced by another portion containing at least one mutation.

[0150] Polynucleotides containing the glucocerebrosidase (GBA) nucleotide sequence The present invention provides a polynucleotide comprising a modified glucocerebrosidase (GBA) nucleotide sequence encoding the modified GCase polypeptide of the present invention. The term "modified" means that the nucleotide sequence has at least one difference compared to a wild-type GBA nucleotide sequence, for example, that a mutation has been introduced.

[0151] The terms “nucleic acid molecule,” “polynucleotide,” and “nucleotide sequence” are intended to refer to polymer chains of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, or analogs thereof. For example, a nucleic acid molecule, polynucleotide, or nucleotide sequence may contain DNA (deoxyribonucleotide) or RNA (ribonucleotide). A nucleic acid molecule, polynucleotide, or nucleotide sequence may consist of DNA. A nucleic acid molecule, polynucleotide, or nucleotide sequence may contain mRNA. Since a nucleic acid molecule, polynucleotide, or nucleotide sequence may contain RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).

[0152] In some embodiments, the term "nucleotide sequence" can be replaced with the term "nucleic acid molecule."

[0153] The GBA nucleotide sequence codes for a GCase polypeptide. The polynucleotide of the present invention comprises a modified GBA nucleotide sequence, which codes for the modified GCase polypeptide of the present invention. The terms “sequence coding for” or “sequence coding for” refer to a nucleotide sequence that includes an open reading frame containing codons coding for the encoded polypeptide. For example, a nucleotide sequence coding for a GCase polypeptide includes codons coding for the amino acid sequence of the GCase polypeptide. An example of a GBA nucleotide sequence coding for wild-type GCase is provided in Sequence ID No. 40.

[0154] Codons that code for polypeptides are also called "coding nucleotides." While GBA nucleotide sequences may be interrupted by non-coding nucleotides (e.g., introns), only the nucleotides that code for polypeptides (i.e., coding nucleotides) should be considered part of the GBA nucleotide sequence. For example, a GBA nucleotide sequence coding for a GCase polypeptide contains any codon (i.e., coding nucleotide) that codes for an amino acid that forms part of the GCase polypeptide, regardless of whether the codons are sequential or separated by one or more non-coding nucleotides. In other words, a GBA polynucleotide containing a sequence of coding nucleotides interrupted by a sequence of non-coding nucleotides is considered to contain a "GBA nucleotide sequence" consisting of a discontinuous coding extension immediately adjacent to it (i.e., excluding the sequence of non-coding nucleotides). However, in this specification, the nucleotide of a stop codon is considered a coding nucleotide.

[0155] The GBA nucleotide sequence encoding a GCase polypeptide may also contain a sequence encoding a signal peptide. Several proteins, particularly those transported to different tissues, are well known to be expressed in association with a signal peptide. The signal peptide may be located at the N-terminus of the protein sequence (in this case, the 5' end of the coding sequence), and many signal peptides are cleaved after intracellular processing. Therefore, as used herein, a mature protein or polypeptide (such as a mature GCase protein or polypeptide) is considered to be the protein or polypeptide obtained after the signal peptide has been processed and removed / cleaved (and thus no longer forms part of the polypeptide sequence).

[0156] The table below shows the codons that code for each amino acid.

[0157] [Table 2]

[0158] The corresponding RNA codon contains U instead of T in the table above.

[0159] The present invention provides a polynucleotide comprising a modified GBA nucleotide sequence encoding the modified GCase polypeptide of the present invention. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 1000, at least 1200, at least 1300, at least 1400, at least 1494, 1494 or less, 1611 or less, 1000-1494, 1000-1611, 1300-1494, 1300-1611, or approximately 1494 nucleotides identical by at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to any one of SEQ ID NOs. 6-29, specifically a fragment of at least 750, at least 850, at least 950, at least 1000, at least 1000, at least 1200, at least 1300, at least 1400, at least 1494, at least 1494, at least 1494, at least 1494, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, 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 750 nucleotides, at least 850 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1200 nucleotides, at least 1400 nucleotides, or at least 1494 nucleotides from any one of SEQ ID NOs. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to a nucleotide sequence of at least 96%,6%, at least 99.8%, at least 99.9%, or 100% identical toOptionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, 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 the nucleotide sequences of SEQ ID NOs. 6-29. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the nucleotide sequence of SEQ ID NOs. 6 or 10. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 10. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 18. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 18.Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 26. Optionally, the modified GBA nucleotide sequence includes a sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8

[0160] The modified GBA nucleotide sequence of the present invention may include a codon-optimized portion. The codon-optimized portion may correspond to a sequence encoding part or all of the modified GCase polypeptide. Optionally, the codon-optimized portion of the GBA nucleotide sequence is a contiguous portion. The modified GBA nucleotide sequence of the present invention may be codon-optimized. The modified GBA nucleotide sequence may be codon-optimized for expression in human hepatocytes. Optionally, the human hepatocytes are Huh-7 cells. As previously noted, the genetic code is degenerate, and many amino acids may be encoded by more than one alternative codon. However, the genetic code of various organisms, tissues, or cells may be biased towards the use of certain codons to encode specific amino acids. A “codon-optimized” nucleotide sequence can be optimized for expression in a specific host cell or organism, for example, in human hepatocytes. Preferably, the codon-optimized modified GBA nucleotide sequence is modified compared to the non-codon-optimized modified GBA nucleotide sequence, but the amino acid sequences encoded by the codon-optimized modified GBA nucleotide and the non-codon-optimized modified GBA nucleotide sequence are the same.

[0161] The modified GBA nucleotide sequences of the present invention may include one or more alternative codons instead of wild-type codons, where the "alternative" codons have a different sequence from the wild-type codons but encode the same amino acid as the wild-type codons (i.e., degenerate codons). Table 2 shows the codons encoding each amino acid.

[0162] A codon-optimized nucleotide sequence may contain at least one more "preferred" codon than the corresponding uncodon-optimized nucleotide sequence. A codon-optimized nucleotide sequence may contain a higher proportion of "preferred" codons than the corresponding uncodon-optimized nucleotide sequence. A codon-optimized nucleotide sequence may contain at least one fewer "unpreferred" codon than the corresponding uncodon-optimized nucleotide sequence. A codon-optimized nucleotide sequence may contain a lower proportion of "unpreferred" codons than the corresponding uncodon-optimized nucleotide sequence. For example, when codon-optimizing a nucleotide sequence for expression in human liver, the nucleotide sequence is modified to increase the number of codons that may be preferred in human liver (meaning that such codons correspond to tRNA species that are more abundant than other tRNA species specific to the same amino acid). As a further example, when codon-optimizing a nucleotide sequence for expression in human hepatocytes, the nucleotide sequence may be modified to increase the number of codons that may be preferred in human hepatocytes (meaning that such codons correspond to tRNA species that are more abundant than other tRNA species specific to the same amino acid). Those skilled in the art will understand that codon optimization does not require changing all codons, particularly because "preferred codons" may already exist in several positions.

[0163] Such codon optimization may also be influenced by other factors. For example, since the presence of CpG (i.e., CG dinucleotide) is known to negatively affect expression, a user may decide not to use a preferred codon at a location where doing so would introduce a CpG into the sequence, and this is also considered codon optimization. In embodiments, preferred codons ending in C nucleotides are not included in portions of codon-optimized coding sequences where the next codon in the sequence begins with G. For example, the codon CTC codes for leucine. In a scheme where CTC is a preferred codon, it should not be used to code for leucine at locations where the next codon in the sequence begins with G, such as the codon GTT (or, if possible, the next codon may be selected to avoid the first position being G).

[0164] Even if a nucleotide sequence is described as "codon-optimized," not all codons need to be optimized. Therefore, a codon-optimized portion of a modified GBA nucleotide sequence may contain one or more substituted codons in place of wild-type codons compared to the corresponding portion of a wild-type GBA nucleotide sequence. If a modified GBA nucleotide sequence contains a codon-optimized portion, the polypeptide encoded by the GBA nucleotide sequence can be expressed at a higher level than the polypeptide encoded by the wild-type GBA nucleotide sequence. A codon-optimized modified GBA nucleotide sequence or portion of a modified GBA nucleotide sequence can be codon-optimized for expression in human hepatocytes. Therefore, a modified GCase polypeptide encoded by a modified GBA nucleotide sequence can be expressed at a higher level than the polypeptide encoded by an equivalent, non-codon-optimized modified GBA nucleotide sequence when the sequence is expressed in human hepatocytes. Optionally, human hepatocytes are Huh-7 cells. An "equivalent" non-codon-optimized modified GBA nucleotide sequence is identical (i.e., codes for the same GCase polypeptide and contains the same transcriptional regulatory element, etc.) except that the codon used to encode the GCase polypeptide corresponds to the corresponding codon in the wild-type GCase sequence, such as the one in SEQ ID NO: 40. Optionally, the sequence (e.g., codon) encoding at least one mutation in the modified GCase polypeptide of the present invention is not codon-optimized. Optionally, the modified GBA nucleotide sequence is codon-optimized except for the sequence encoding at least one mutation. Typically, the codon-optimized portion of a modified GBA nucleotide sequence does not contain a stop codon.

[0165] Polypeptides encoded by codon-optimized GBA nucleotide sequences can be expressed at higher levels in human hepatocytes compared to non-codon-optimized GBA nucleotide sequences. The modified GCase polypeptide of the present invention, encoded by the codon-optimized modified GBA nucleotide sequence of the present invention, can be expressed at higher levels in human hepatocytes compared to GCase polypeptides encoded by non-codon-optimized GBA nucleotide sequences. When the sequence of the modified GCase polypeptide encoded by the codon-optimized modified GBA nucleotide sequence of the present invention is expressed in human hepatocytes, it can be expressed at higher levels than GCase polypeptides encoded by non-codon-optimized GBA nucleotide sequences. For example, the modified GCase polypeptide encoded by the codon-optimized modified GBA nucleotide sequence can be expressed at least 1.1 times, at least 1.2 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times more in human hepatocytes compared to a non-codon-optimized reference GBA nucleotide sequence. The “uncodon-optimized reference GBA nucleotide sequence” may be any GBA nucleotide sequence that uses wild-type codons to encode a GCase polypeptide, such as the GBA nucleotide sequence of SEQ ID NO: 40. Optionally, the uncodon-optimized reference GBA nucleotide sequence is an “equivalent” GBA nucleotide sequence, i.e., the uncodon-optimized reference GBA nucleotide sequence encodes the same GCase polypeptide as the GBA nucleotide sequence being compared. The modified GCase polypeptide of the present invention encoded by the codon-optimized modified GBA nucleotide sequence of the present invention can be expressed at higher levels in human hepatocytes compared to the modified GCase polypeptide encoded by the uncodon-optimized modified GBA nucleotide sequence.The modified GCase polypeptide encoded by the codon-optimized modified GBA nucleotide sequence of the present invention can be expressed at a higher level in human hepatocytes than the modified GCase polypeptide encoded by a non-codon-optimized modified GBA nucleotide sequence. Optionally, the human hepatocytes are Huh-7 cells.

[0166] In some embodiments, a portion of the modified GBA nucleotide sequence is not codon-optimized; for example, a portion of the coding sequence is not codon-optimized for expression in the liver. In some embodiments, the uncodon-optimized portion is at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, 200 nucleotides or less, 170 nucleotides or less, 140 nucleotides or less, or around 117 nucleotides. In some embodiments, the uncodon-optimized portion of the modified GBA nucleotide sequence codes for a signal peptide. Optionally, a codon-optimized portion of the GBA nucleotide sequence codes for a mature modified GCase polypeptide. In some embodiments, a codon-optimized portion of the modified GBA nucleotide sequence does not code for all or part of the signal peptide. For example, the modified GBA nucleotide sequence may code for an immature GCase polypeptide (i.e., including the signal peptide), while a codon-optimized portion of the modified GBA nucleotide sequence codes for a mature GCase protein and does not code for all or part of the signal peptide. In some embodiments, a portion of the codon-optimized modified GBA nucleotide sequence encodes all or part of the signal peptide. For example, the modified GBA nucleotide sequence may encode an immature modified GCase polypeptide (i.e., including the signal peptide), while a portion of the codon-optimized modified GBA nucleotide sequence encodes all or part of the mature modified GCase polypeptide and the signal peptide. In some embodiments, the modified GBA nucleotide sequence encoding the entire mature modified GCase polypeptide sequence is codon-optimized.

[0167] Optionally, a modified GBA nucleotide sequence comprises the sequence of SEQ ID NO: 59 or 60, except that the modified GCase polypeptide encoded by the modified GBA nucleotide sequence contains at least one mutation described herein. For example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may contain a cysteine ​​substitution, optionally W351C, at the position corresponding to position 351 of SEQ ID NO: 1, and a cysteine ​​substitution, optionally A380C, at the position corresponding to position 380 of SEQ ID NO: 1. As a further example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may contain a mutation, optionally E272Q, at the position corresponding to position 272 of SEQ ID NO: 1. As a further example, a modified GCase polypeptide encoded by a modified GBA nucleotide sequence may contain a cysteine ​​substitution, optionally W351C, at the position corresponding to position 351 of SEQ ID NO: 1, and a cysteine ​​substitution, optionally A380C, at the position corresponding to position 380 of SEQ ID NO: 1. A modified GCase polypeptide encoded by a modified GBA nucleotide sequence may also contain a mutation, optionally E272Q, at the position corresponding to position 272 of SEQ ID NO: 1.

[0168] Optionally, a modified GBA nucleotide sequence comprises the sequence of SEQ ID NO: 40 or SEQ ID NO: 64, except that the modified GCase polypeptide encoded by the modified GBA nucleotide sequence contains at least one mutation described herein. For example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may contain a cysteine ​​substitution, optionally W351C, at the position corresponding to position 351 of SEQ ID NO: 1, and a cysteine ​​substitution, optionally A380C, at the position corresponding to position 380 of SEQ ID NO: 1. As a further example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may contain a mutation, optionally E272Q, at the position corresponding to position 272 of SEQ ID NO: 1. As a further example, a modified GCase polypeptide encoded by a modified GBA nucleotide sequence may contain a cysteine ​​substitution, optionally W351C, at the position corresponding to position 351 of SEQ ID NO: 1, and a cysteine ​​substitution, optionally A380C, at the position corresponding to position 380 of SEQ ID NO: 1. A modified GCase polypeptide encoded by a modified GBA nucleotide sequence may also contain a mutation, optionally E272Q, at the position corresponding to position 272 of SEQ ID NO: 1.

[0169] Optionally, a modified GBA nucleotide sequence includes the sequence of SEQ ID NO: 15 of International Publication No. 2019 / 070893 or International Publication No. 2019 / 070894, except that the modified GCase polypeptide encoded by the modified GBA nucleotide sequence includes at least one mutation described herein. For example, the modified GCase polypeptide encoded by the modified GBA nucleotide may include a mutation at the position corresponding to position 272 of SEQ ID NO: 1. As a further example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may include cysteine ​​substitutions at the positions corresponding to position 351 and position 380 of SEQ ID NO: 1. As yet another example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may include cysteine ​​substitutions at the positions corresponding to position 351 and position 380 of SEQ ID NO: 1, and the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may include a mutation at the position corresponding to position 272 of SEQ ID NO: 1.

[0170] Optionally, a modified GBA nucleotide sequence may include the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16 of International Publication No. 2020 / 012149 or International Publication No. 2020 / 012164, except that the modified GCase polypeptide encoded by the modified GBA nucleotide sequence contains at least one mutation described herein. For example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may contain a mutation at the position corresponding to position 272 of SEQ ID NO: 1. As a further example, the modified GCase polypeptide encoded by the modified GBA nucleotide sequence may contain cysteine ​​substitutions at the positions corresponding to position 351 and position 380 of SEQ ID NO: 1. As a further example, a modified GCase polypeptide encoded by a modified GBA nucleotide sequence may contain cysteine ​​substitutions at the position corresponding to position 351 of SEQ ID NO: 1 and position corresponding to position 380 of SEQ ID NO: 1, and a modified GCase polypeptide encoded by a modified GBA nucleotide sequence may contain a mutation at the position corresponding to position 272 of SEQ ID NO: 1.

[0171] The polynucleotide of the present invention may contain a transcriptional regulatory element.

[0172] Any suitable transcriptional regulatory element can be used, such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, or LSP, all of which are 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. The transcriptional regulatory elements may include liver-specific promoters.

[0173] Optionally, a transcriptional regulatory element is "liver-specific" if it drives a higher level of expression in hepatocytes compared to other cells. For example, a person skilled in the art can determine whether a transcriptional regulatory element is liver-specific by comparing the expression of a polynucleotide in hepatocytes (e.g., Huh 7 cells) with the expression of a polynucleotide in cells from other tissues (e.g., kidney cells, e.g., HEK293T cells). If the expression level is higher in hepatocytes compared to cells from other tissues, then the transcriptional regulatory element is liver-specific. Optionally, a liver-specific transcriptional regulatory element does not drive a level of expression that is recognizable in cells other than liver cells.

[0174] The transcriptional regulatory element may include promoters and / or enhancers such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, or LSP-derived promoter and / or enhancer elements. Each of these transcriptional regulatory elements includes a promoter, an enhancer, and optionally other nucleotides.

[0175] In some embodiments, the transcriptional regulatory element includes a promoter or fragment thereof that is a human α-1 antitrypsin promoter (A1AT; Miao et al (2000), Molecular Therapy 1(6):522). In embodiments, the A1AT promoter fragment is at least 100 nucleotides long, at least 120 nucleotides long, at least 150 nucleotides long, at least 180 nucleotides long, 255 nucleotides or less long, 100 to 255 nucleotides long, 150 to 225 nucleotides long, 150 to 300 nucleotides long, or 180 to 255 nucleotides long. Optionally, the A1AT promoter fragment is 150 to 300 nucleotides long. Optionally, the A1AT promoter fragment is 180 to 255 nucleotides long. In the embodiment, the A1AT promoter fragment is at least 200 nucleotides long, at least 250 nucleotides long, at least 300 nucleotides long, 500 nucleotides or less long, 200 to 500 nucleotides long, 250 to 500 nucleotides long, or 350 to 450 nucleotides long. Optionally, the A1AT promoter fragment is 350 to 450 nucleotides long.

[0176] Suitable A1AT promoter fragments are described in SEQ ID NOs. 30 and 31. Optionally, the transcriptional regulatory element comprises a promoter of at least 100 nucleotides, at least 120 nucleotides, at least 150 nucleotides, at least 180 nucleotides, 255 nucleotides or less, 100 to 255 nucleotides, 150 to 300 nucleotides, or 180 to 255 nucleotides, wherein 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. 30. Optionally, the transcriptional regulatory element comprises a promoter of 180 to 255 nucleotides, wherein the promoter 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. 30. Optionally, the polynucleotide includes 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: 30. Optionally, the polynucleotide includes 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: 30. Optionally, the polynucleotide includes 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: 30. Optionally, the polynucleotide includes the promoter of SEQ ID NO: 30. Optionally, the transcriptional regulatory element is 418 nucleotides or less in length, 255 nucleotides or less in length, or 185 nucleotides or less in length, and includes a fragment of the A1AT promoter containing SEQ ID NO: 30.

[0177] The transcriptional regulatory element may include a promoter of at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, 500 nucleotides or less, 200 to 500 nucleotides, 250 to 500 nucleotides, 350 to 450 nucleotides, or approximately 418 nucleotides in length, wherein the promoter contains 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: 31. Optionally, the transcriptional regulatory element may include a promoter of 350 to 450 nucleotides in length, wherein the promoter contains 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: 31. Optionally, the polynucleotide includes 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 at least 350 nucleotide fragments of SEQ ID NO: 31. Optionally, the polynucleotide includes 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: 31. Optionally, the polynucleotide includes 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: 31. Optionally, the polynucleotide includes the promoter of SEQ ID NO: 31.

[0178] Optionally, the transcription control element may include a minimal nephrine promoter NPHS1 or a podosin promoter NPHS2.

[0179] The transcriptional regulatory element may include an enhancer. In some embodiments, the transcriptional regulatory element includes an enhancer or fragment thereof that is a human apolipoprotein E (ApoE) liver locus regulatory region (HCR; Miao et al (2000), Molecular Therapy 1(6):522). In embodiments, the transcriptional regulatory element includes a fragment of an HCR enhancer that is at least 80 nucleotides long, at least 90 nucleotides long, at least 100 nucleotides long, 192 nucleotides or less, 80 to 192 nucleotides long, 90 to 192 nucleotides long, 100 to 250 nucleotides long, or 117 to 192 nucleotides long. Optionally, the fragment of the HCR enhancer is 100 to 250 nucleotides long. Optionally, the fragment of the HCR enhancer is 117 to 192 nucleotides long. In the embodiment, the HCR enhancer fragment is a fragment of at least 150 nucleotides, at least 190 nucleotides, at least 230 nucleotides, 400 nucleotides or less, 150 to 400 nucleotides, 190 to 370 nucleotides, 230 to 340 nucleotides, 250 to 340 nucleotides, or approximately 321 nucleotides. Optionally, the HCR enhancer fragment is 250 to 340 nucleotides long.

[0180] Suitable HCR enhancer element fragments are defined in SEQ ID NOs. 32 and 33. Optionally, the transcriptional regulatory element comprises an enhancer having a length of at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, 192 nucleotides or less, 80 to 192 nucleotides, 90 to 192 nucleotides, 100 to 250 nucleotides, or 117 to 192 nucleotides, wherein 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. 32. Optionally, the transcriptional regulatory element comprises an enhancer having a length of 117 to 192 nucleotides, wherein 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. 32. Optionally, the transcriptional regulatory element includes 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: 32. Optionally, the polynucleotide includes 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: 32. Optionally, the polynucleotide includes 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: 32. Optionally, the polynucleotide includes an enhancer of SEQ ID NO: 32. Optionally, the transcriptional regulatory element is 321 nucleotides or less in length, 192 nucleotides or less in length, or 117 nucleotides or less, and includes a fragment of the HCR enhancer containing SEQ ID NO: 32.

[0181] In some embodiments, the transcriptional regulatory element includes an enhancer that is at least 150 nucleotides long, at least 190 nucleotides long, at least 230 nucleotides long, less than 400 nucleotides long, 150 to 400 nucleotides long, 190 to 370 nucleotides long, 230 to 340 nucleotides long, 250 to 340 nucleotides long, or approximately 321 nucleotides long, and the enhancer includes 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: 33. Optionally, the transcriptional regulatory element includes an enhancer of 250 to 340 nucleotides long, and the enhancer includes 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: 33. Optionally, the transcriptional regulatory element includes 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 the at least 250-nucleotide fragment of SEQ ID NO: 33. Optionally, the polynucleotide includes 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: 33. Optionally, the polynucleotide includes 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: 33. Optionally, the polynucleotide includes an enhancer of SEQ ID NO: 33.

[0182] In the 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: 34. In the embodiment, the polynucleotide includes 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: 34. Optionally, the polynucleotide includes a transcriptional regulatory element that is at least 98% identical to SEQ ID NO: 34. Optionally, the transcriptional regulatory element includes a sequence that is 100% identical to SEQ ID NO: 34. Optionally, the polynucleotide includes a transcriptional regulatory element that is 100% identical to SEQ ID NO: 34. Optionally, the polynucleotide includes the transcriptional regulatory element of SEQ ID NO: 34. Optionally, the polynucleotide includes a transcriptional regulatory element consisting of SEQ ID NO: 34.

[0183] In embodiments, 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: 35. In embodiments, the polynucleotide includes 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: 35. Optionally, the polynucleotide includes a transcriptional regulatory element that is at least 98% identical to SEQ ID NO: 35. Optionally, the transcriptional regulatory element includes a sequence that is 100% identical to SEQ ID NO: 35. Optionally, the polynucleotide includes a transcriptional regulatory element that is 100% identical to SEQ ID NO: 35. Optionally, the polynucleotide includes the transcriptional regulatory element of SEQ ID NO: 35. Optionally, the polynucleotide includes a transcriptional regulatory element consisting of SEQ ID NO: 35.

[0184] In embodiments, the polynucleotide of the present invention comprises a woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof. For example, the polynucleotide of the present invention comprises a variant WPRE sequence described in Zanta-Boussif et al (2009), Gene Therapy, 16:605-619. Optionally, the woodchuck hepatitis post-transcriptional regulatory element is at least 75%, at least 80%, at least 85%, at least 90%, 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 any one of SEQ ID NOs. 61-63. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to any one of SEQ ID NOs. 61-63. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element that is at least 98% identical to any one of SEQ ID NOs. 61-63. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element having the sequence of any one of SEQ ID NOs. 61-63. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element of any one of SEQ ID NOs. 61-63. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element consisting of any one of SEQ ID NOs. 61-63. In another embodiment, the polynucleotide of the present invention does not contain a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

[0185] The polynucleotide of the present invention may further comprise one or two ITRs. In one embodiment, the nucleotide sequence of the ITR or each ITR is less than 157 nucleotides long, less than 154 nucleotides long, or approximately 145 nucleotides long. Optionally, the ITR or each ITR is a wild-type ITR. Optionally, the ITR or each ITR is an AAV2 ITR.

[0186] The polynucleotide of the present invention may further contain a poly(A) sequence. The poly(A) sequence may be located downstream of the modified GBA nucleotide sequence encoding the modified GCase polypeptide of the present invention. The poly(A) sequence may be the bovine growth hormone poly(A) sequence (bGHpA-SEQ ID NO: 58). The poly(A) sequence may be 250 to 270 nucleotides long.

[0187] The polynucleotide of the present invention may further contain an intron sequence, for example, a viral intron sequence, and optionally an SV40 intron sequence (SEQ ID NO: 57).

[0188] In some embodiments, a modified GCase polypeptide containing at least one mutation can be expressed in host cells at a higher level than the reference GCase polypeptide described herein. For example, a modified GCase polypeptide containing at least one mutation can be expressed at least 1.1 times, at least 1.2 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, or at least 5 times more than the reference GCase polypeptide. Optionally, a modified GCase polypeptide containing at least one mutation can be expressed at 1.1 to 5 times, 1.2 to 5 times, 1.5 to 5 times, 1.8 to 5 times, 2 to 5 times, 2.5 to 5 times, 3 to 5 times, 3.5 to 5 times, 4 to 5 times, or 4.5 to 5 times more than the reference GCase polypeptide.

[0189] The expression level of GCase polypeptides (and therefore whether the modified GCase polypeptide is expressed at a higher level in host cells than the reference GCase polypeptide) can usually be determined by measuring the level of GCase polypeptide in a sample. The expression level of the modified GCase polypeptide of the present invention in host cells may be compared with the expression level of the reference GCase polypeptide in host cells. This can be determined quantitatively. For example, the expression level of GCase polypeptides can be determined by an ELISA assay as described above. Alternatively, the expression level of GCase polypeptides can be determined semi-quantitatively, for example, by SDS-PAGE electrophoresis or Western blotting.

[0190] The level of GCase polypeptide secreted by the host cell can be used to determine the expression level of the GCase polypeptide (i.e., the level of GCase polypeptide retained intracellularly by the host cell). Optionally, the GCase polypeptide of the present invention is secreted by the host cell at a higher level than the reference GCase polypeptide. For example, a modified GCase polypeptide containing at least one mutation may be secreted at a level at least 1.1 times, at least 1.2 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, or at least 5 times higher than the reference GCase polypeptide. Optionally, a modified GCase polypeptide containing at least one mutation may be secreted at a level 1.1 to 5 times, 1.2 to 5 times, 1.5 to 5 times, 1.8 to 5 times, 2 to 5 times, 2.5 to 5 times, 3 to 5 times, 3.5 to 5 times, 4 to 5 times, or 4.5 to 5 times higher than the reference GCase polypeptide.

[0191] The level of GCase polypeptide secreted by host cells can be determined, for example, by separating cells (e.g., host cells) from a liquid containing GCase polypeptide (e.g., a biological sample such as blood, serum, or plasma, or a culture medium such as a conditional medium in which host cells expressing GCase polypeptide are cultured) and determining the expression level of GCase polypeptide in the liquid. For example, cells may be separated by centrifugation or filtration, and / or the liquid may be decanted from the cells (e.g., by pipetting). The expression level of GCase polypeptide may be determined in a biological sample such as blood, serum, or plasma, or in a culture medium such as a conditional medium in which host cells expressing GCase polypeptide are cultured.

[0192] The host cell may be any eukaryotic cell, such as a mammalian cell or an insect cell. The host cell may be any eukaryotic host cell expressing the modified GCase polypeptide (or reference GCase polypeptide) of the present invention. Optionally, the host cell may be an insect cell expressing the modified GCase polypeptide (or reference GCase polypeptide) of the present invention. Typically, the host cell may be a mammalian host cell expressing the modified GCase polypeptide (or reference GCase polypeptide) of the present invention. Examples of mammalian host cells include human cells (e.g., Expi293F human embryonic kidney cells), dog cells, pig cells, mouse cells, hamster cells, or guinea pig cells. More specifically, the host cell may be a mammalian hepatocyte, and more specifically, a human hepatocyte. Optionally, the host cell may be a Huh7 cell. Optionally, the host cell may be a host cell within an organism. Optionally, expression may be in vivo. Optionally, expression may be in vivo, and expression in plasma may be sought.

[0193] Virus particles containing polynucleotides The present invention further provides viral particles comprising a recombinant genome containing the polynucleotides of the present invention. For the purposes of the present invention, the term “viral particle” refers to all or part of a virion. For example, a viral particle comprises a recombinant genome and may further comprise a capsid. A viral particle may also be a gene therapy vector. In this specification, the terms “viral particle” and “vector” are used interchangeably. For the purposes of the present application, a “gene therapy” vector is a viral particle that can be used in gene therapy, i.e., a viral particle that contains all the functional elements necessary to express a transgene, such as a GBA nucleotide sequence, in a host cell after administration.

[0194] Suitable viral particles include parvoviruses, retroviruses, lentiviruses, or herpes simplex viruses. Parvoviruses may be adeno-associated viruses (AAVs). Optionally, the viral particles are AAV, adenovirus, or lentivirus viral particles. The viral particles are preferably recombinant AAV vectors or lentiviral vectors. More preferably, the viral particles are AAV viral particles. The terms AAV and rAAV are used interchangeably herein unless the context clearly indicates otherwise.

[0195] The genomic makeup of all known AAV serotypes is remarkably similar. The AAV genome is a linear single-stranded DNA molecule less than approximately 5,000 nucleotides long. Reverse terminal repeats (ITRs) are adjacent to coding nucleotide sequences specific to non-structural replication proteins (Rep) and structural proteins (VP). VP proteins (VP1, VP2, and VP3) form a capsid. The approximately 145 nt (ITR) at the end is self-complementary and constructed to allow for the formation of energetically stable intramolecular double helices that form a T-shaped hairpin. These hairpin structures function as origins for viral DNA replication and as primers for intracellular DNA polymerase complexes. After infection of mammalian cells with wild-type (wt) AAV, the Rep genes (i.e., encoding the Rep78 and Rep52 proteins) are expressed from the P5 promoter and P19 promoter, respectively, and both Rep proteins have a function in viral genome replication. As a result of splicing events in Rep ORFs, four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40) are expressed. However, in mammalian cells, it has been shown that unspliced ​​mRNA encoding Rep78 and Rep52 proteins is sufficient for AAV vector generation. Similarly, in insect cells, Rep78 and Rep52 proteins are sufficient for AAV vector generation.

[0196] The recombinant viral genome of the present invention may contain ITRs. The recombinant genome or viral particle may contain one or more ITRs, for example, the ITRs described above. The AAV vector of the present invention can function with only one ITR. Therefore, the viral genome typically contains at least one ITR, but more typically contains two ITRs (generally one at each end of the viral genome, i.e., one at the 5' end and one at the 3' end). There may be interposing sequences between the polynucleotide of the present invention and one or more ITRs. The polynucleotide may be incorporated into a viral particle located between two normal ITRs, or it may be located on both sides of an ITR genetically engineered with two D regions.

[0197] The AAV sequences that can be used in the present invention to construct AAV vectors may be derived from the genome of any AAV serotype. Generally, AAV serotypes have genomic sequences with significant homology at the amino acid and nucleic acid levels, provide the same set of gene functions, produce essentially physically and functionally equivalent virions, and are replicated and assembled by substantially the same mechanisms. For an overview of the genomic sequences and genomic similarities of various AAV serotypes, see, for example, GenBank accession number U89790; GenBank accession number J01901; GenBank accession number AF043303; GenBank accession number AF085716; Chiorini et al, 1997; Srivastava et al, 1983; Chiorini et al, 1999; Rutledge et al, 1998; and Wu et al, 2000. In this invention, AAV serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, or 12 can be used. Sequences from the AAV serotypes may be mutated or genetically modified when used to construct gene therapy vectors.

[0198] Optionally, the AAV vector includes an ITR sequence derived from AAV1, AAV2, AAV4, and / or AAV6. Preferably, the ITR sequence is the AAV2 ITR sequence. In this specification, the term AAVx / y refers to a viral particle having at least a genomic component such as an ITR derived from AAVx (where x is the AAV serotype number) and a capsid derived from AAVy (where y is the same or a different serotype number). For example, an AAV2 / 8 vector may include a portion of the viral genome containing an ITR derived from the AAV2 strain and a capsid derived from the AAV8 strain.

[0199] In embodiments, the viral particle is an AAV viral particle containing a capsid. The AAV capsid is generally formed from three proteins: VP1, VP2, and VP3. The amino acid sequence of VP1 contains the amino acid sequence of VP2. The portion of VP1 that does not form part of VP2 is referred to as VP1unique or VP1U. The amino acid sequence of VP2 contains the sequence of VP3. The portion of VP2 that does not form part of VP3 is referred to as VP2unique or VP2U. Optionally, the viral particle contains a liver-targeting or CNS-targeting capsid. Whether a viral particle (capsid) is tissue-targeting can be evaluated, for example, by administering such particles expressing a marker gene such as luciferase and imaging them in vivo 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 the liver or CNS tissues are considered liver or CNS-specific, especially if their expression is significantly lower in other tissues.

[0200] In some embodiments, the liver-targeting capsid may be a capsid derived from AAV3, AAV3B, AAV5, or AAV8. Optionally, the liver-targeting capsid may be a capsid derived from AAV3, AAV3B, or AAV8. Optionally, the liver-targeting capsid may be a capsid derived from AAV3 or AAV3B. Optionally, the liver-targeting capsid contains a sequence at least 98%, at least 99%, or at least 99.5% identical to the fragment of at least 600 amino acids, at least 650 amino acids, at least 700 amino acids, 600-736 amino acids, 650-736 amino acids, or 700-736 amino acids of SEQ ID NO: 36, 37, or 38. Optionally, the liver-targeting capsid contains a sequence at least 99% identical to SEQ ID NO: 36. Optionally, the liver-targeting capsid contains a sequence at least 99% identical to SEQ ID NO: 37. Optionally, the liver-targeting capsid contains a sequence at least 99% identical to SEQ ID NO: 38. Optionally, the liver-targeting capsid contains a sequence 100% identical to at least 600 amino acids, at least 650 amino acids, at least 700 amino acids, 600-736 amino acids, 650-736 amino acids, or 700-736 amino acid fragments of SEQ ID NO: 36, 37, or 38. Optionally, the liver-targeting capsid contains a sequence 100% identical to SEQ ID NO: 36. Optionally, the liver-targeting capsid contains a sequence 100% identical to SEQ ID NO: 37. Optionally, the liver-targeting capsid contains a sequence 100% identical to SEQ ID NO: 38. Optionally, the CNS-directed capsid contains a sequence that is at least 98%, at least 99%, or at least 99.5% identical to at least 600 amino acids, at least 650 amino acids, at least 700 amino acids, 600-736 amino acids, 650-736 amino acids, or a fragment of 700-736 amino acids of SEQ ID NO: 39. Optionally, the CNS-directed capsid contains a sequence that is 100% identical to at least 600 amino acids, at least 650 amino acids, at least 700 amino acids, 600-736 amino acids, 650-736 amino acids, or a fragment of 700-736 amino acids of SEQ ID NO: 39. Optionally, the CNS-directed capsid contains a sequence that is at least 99% identical to SEQ ID NO: 39. Optionally, the CNS-directed capsid contains a sequence that is 100% identical to SEQ ID NO: 39.In some embodiments, the CNS-targeted capsid may be a capsid derived from AAV9 or AAVrh.10. The viral particles of the present invention may be “hybrid” particles in which the viral ITR and viral capsid are derived from different parvoviruses, e.g., different AAV serotypes. Preferably, the viral ITR and capsid are derived from different AAV serotypes, in which case such viral particles are known as capsid-converted or pseudotyped. Similarly, the parvovirus may have a “chimeric” capsid (e.g., containing sequences derived from different parvoviruses, preferably different AAV serotypes) or a “targeted” capsid (e.g., directed tropism).

[0201] In some embodiments, the recombinant AAV genome contains intact ITRs containing functional end separation sites (TRS). Such an AAV genome may contain one or two degradable ITRs, i.e., ITRs containing functional TRS that can undergo site-specific nicking to create free 3' hydroxyl groups that can be used as substrates for DNA polymerase to unwind and copy the ITR.

[0202] Preferably, the recombinant genome is single-stranded (i.e., packaged in a single-stranded form within the viral particle). Optionally, the recombinant genome is not packaged in a self-complementary configuration; i.e., the genome does not contain a single covalently bonded polynucleotide chain having a substantially self-complementary portion that anneals within the viral particle. Alternatively, the recombinant genome may be packaged in a "monomer double-stranded" form. A "monomer double-stranded" form is described in International Publication No. 2011 / 122950. The genome may be packaged as two substantially complementary but non-covalently bonded polynucleotides that anneal within the viral particle.

[0203] As described above, the polynucleotide of the present invention may contain a polyA nucleotide sequence. Therefore, recombinant genomes or viral particles may contain polyA sequences such as the polyA sequence described above.

[0204] As described above, the polynucleotide of the present invention may contain intron sequences. Therefore, the recombinant genome or viral particle of the present invention may contain intron sequences such as the above-mentioned intron sequences.

[0205] In some embodiments, the viral particle comprises a polynucleotide sequence including a transcriptional regulatory element (e.g., including a promoter and / or enhancer), a modified GBA nucleotide sequence, and a poly-A sequence such as a bGHpA sequence. In such embodiments, the poly-A sequence, such as a bGHpA sequence, may be located downstream of the modified GBA nucleotide sequence. In some embodiments, the viral particle comprises an AAV2 ITR and a poly-A sequence (such as a bGHpA sequence).

[0206] In some embodiments, the viral particle includes a polynucleotide sequence comprising a transcriptional regulatory element (e.g., including a promoter and / or enhancer), an intron sequence such as an SV40 intron sequence, a modified GBA nucleotide sequence, and a poly-A sequence such as a bGHpA sequence. In such embodiments, the intron sequence such as an SV40 intron sequence may be located between the transcriptional regulatory element and the modified GBA nucleotide sequence. In such embodiments, the poly-A sequence such as a bGHpA sequence may be located downstream of the modified GBA nucleotide sequence. In some embodiments, the viral particle includes an AAV2 ITR and a poly-A sequence (such as a bGHpA sequence), and / or an intron (such as an SV40 intron).

[0207] In some embodiments, after transduction of the viral particles of the present invention into host cells (e.g., Huh-7 cells), the effective activity of the modified GCase polypeptide is the same as or higher than that of the reference GCase polypeptide after transduction of otherwise identical viral particles, including the GBA nucleotide sequence encoding the reference GCase polypeptide, into host cells (e.g., Huh-7 cells). In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 41 or 44. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 41 or 44. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 6 or 10. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 43 or 46. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 43 or 46. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 22 or 26. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 42 or 45. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 42 or 45. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide contains the nucleotide sequence of SEQ ID NO: 14 or 18. In some embodiments, the reference GCase polypeptide is the wild-type GCase polypeptide. Optionally, the reference GCase polypeptide contains the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 1 or 2. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide contains the nucleotide sequence of SEQ ID NO: 59 or 60. In some embodiments, the effective activity of the modified GCase polypeptide is higher than that of the reference GCase polypeptide.In some embodiments, the effective activity of the modified GCase polypeptide is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, or at least 70 times higher than the effective activity of the reference GCase polypeptide. Optionally, the effective activity of the modified GCase polypeptide is at least 50 times, at least 60 times, or at least 70 times higher than the effective activity of the reference GCase polypeptide. Optionally, the effective activity of the modified GCase polypeptide is up to 50 times, up to 60 times, up to 70 times, up to 80 times, or up to 100 times higher than the effective activity of the reference GCase polypeptide. Optionally, the effective activity of the modified GCase polypeptide is 2 to 100 times, 3 to 100 times, 5 to 100 times, 15 to 100 times, 20 to 100 times, 30 to 100 times, 40 to 100 times, 50 to 100 times, or 70 to 100 times higher than the effective activity of the reference GCase polypeptide. Optionally, the effective activity of the modified GCase polypeptide is 2 to 80 times, 5 to 80 times, 20 to 80 times, or 50 to 80 times higher than the effective activity of the reference GCase polypeptide. Optionally, the effective activity of the modified GCase polypeptide is 2 to 70 times, 2 to 10 times, 3 to 20 times, 5 to 10 times, 15 to 30 times, 20 to 40 times, 30 to 50 times, 40 to 60 times, or 50 to 70 times higher than the effective activity of the reference GCase polypeptide. In some embodiments, the reference GCase polypeptide is a wild-type GCase polypeptide. Optionally, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 1 or 2. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 59 or 60. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 41 or 44. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 41 or 44. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 6 or 10.In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 43 or 46. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 43 or 46. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 22 or 26. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 42 or 45. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 42 or 45. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 14 or 18. Optionally, the effective activity of the modified GCase polypeptide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the effective activity of the GCase polypeptide encoded by the sequence of SEQ ID NO: 14 or 18. Optionally, the host cell is a Huh-7 cell. Optionally, the viral particles of the present invention and other viral particles identical in other respects comprise the capsid of SEQ ID NO: 37.

[0208] The statement that the effective activity of the modified GCase polypeptide is "the same" as the effective activity of the reference GCase polypeptide indicates that the effective activity of the modified GCase polypeptide can be identical to the effective activity of the reference GCase polypeptide plus or minus 10%, more specifically plus or minus 5%, or more specifically plus or minus 1%.

[0209] "Other respects identical" viral particles comprising the GBA nucleotide sequence encoding the reference GCase polypeptide are viral particles identical to the viral particles of the present invention (i.e., the viral particles contain the same capsid, the recombinant genome contains the same transcriptional control elements, etc.) except that the GBA nucleotide sequences are different. For example, different GBA nucleotide sequences encoding different GCase polypeptides being compared are operably linked to the same promoter sequence.

[0210] In some embodiments, after administration of the viral particles of the present invention, the effective activity of the modified GCase polypeptide is the same as or higher than that of the reference GCase polypeptide after administration of viral particles otherwise identical, including a GBA nucleotide sequence encoding the reference GCase polypeptide, in the liver, plasma, leukocytes, spleen, bone marrow, lung tissue, and / or any other Gaucher disease-affected organ. In some embodiments, after administration of the viral particles of the present invention, the effective activity of the modified GCase polypeptide is the same as or higher than that of the reference GCase polypeptide after administration of viral particles otherwise identical, including a GBA nucleotide sequence encoding the reference GCase polypeptide, in the liver. In some embodiments, after administration of the viral particles of the present invention, the effective activity of the modified GCase polypeptide is the same as or higher than that of the reference GCase polypeptide after administration of viral particles otherwise identical, including a GBA nucleotide sequence encoding the reference GCase polypeptide, in plasma, leukocytes, spleen, bone marrow, lung tissue, and / or any other Gaucher disease-affected organ. In some embodiments, the effective activity of the modified GCase polypeptide is the same as or higher than that of the reference GCase polypeptide in bone marrow, spleen, and / or plasma. In some embodiments, the effective activity of the modified GCase polypeptide is the same as or higher than that of the reference GCase polypeptide in spleen, lung, and / or bone marrow. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 41 or 44. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 41 or 44. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 6 or 10. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 43 or 46. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 43 or 46. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 22 or 26.In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 42 or 45. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 42 or 45. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 14 or 18. In some embodiments, the reference GCase polypeptide is the wild-type GCase polypeptide. Optionally, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 1 or 2. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 59 or 60. In some embodiments, the effective activity of the modified GCase polypeptide is higher than that of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, or at least 70 times higher than the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, or at least 10 times higher than the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, or at least 10 times higher in plasma than the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, or at least 40 times higher than the effective activity of the reference GCase polypeptide.In some embodiments, the effective activity of the modified GCase polypeptide is at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, or at least 40 times higher in plasma compared to the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is at least 2 times, at least 3 times, or at least 4 times higher in plasma compared to the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is at least 2 times, at least 3 times, or at least 4 times higher in the spleen compared to the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is at least 2 times, at least 3 times, or at least 4 times higher in the bone marrow compared to the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is 2 to 5 times, 2 to 10 times, 3 to 7 times, 3 to 20 times, 5 to 10 times, 15 to 30 times, 20 to 40 times, 30 to 50 times, 40 to 60 times, or 50 to 70 times higher than the effective activity of the reference GCase polypeptide. In some embodiments, the effective activity of the modified GCase polypeptide is 2 to 20 times, 2 to 15 times, 2 to 10 times, 2 to 7 times, 2 to 5 times, or 3 to 5 times higher than the effective activity of the reference GCase polypeptide. In some embodiments, the reference GCase polypeptide is a wild-type GCase polypeptide. Optionally, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 1 or 2. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 59 or 60. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 41 or 44. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 41 or 44. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 6 or 10. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 43 or 46.Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 43 or 46. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 22 or 26. In some embodiments, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 42 or 45. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 42 or 45. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 14 or 18. In some embodiments, the effective activity of the modified GCase polypeptide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the effective activity of the GCase polypeptide encoded by the sequence of SEQ ID NO: 14 or 18. Optionally, the viral particles of the present invention and other viral particles identical in other respects comprise an AAV8 capsid.

[0211] In some embodiments, effective activity is measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration of the viral particles of the present invention. Preferably, effective activity is measured at 12 weeks or 12 weeks. For example, the effective activity at at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of the viral particles of the present invention may be compared to the levels at at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of viral particles that are otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide, respectively. As a specific example, the effective activity level may be measured at least 12 weeks after administration of the viral particles of the present invention (e.g., at week 12) and compared to the level measured at least 12 weeks after administration of viral particles that are otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide (e.g., at week 12). Preferably, the effective activity level is measured at the same time point after administration using the same assay.

[0212] In some embodiments, GCase active activity is measured using a fluorescence assay, such as those described herein, using a GCase-specific fluorescent substrate. The fluorescent substrate may be as described herein.

[0213] In some embodiments, the effective activity of the modified GCase polypeptide is the same as or higher than that of GCase enzyme replacement therapy in plasma, liver, leukocytes, spleen, bone marrow, lung tissue, and / or any other Gaucher disease-affected organs. In some embodiments, the effective activity of the modified GCase polypeptide is the same as or higher than that of GCase enzyme replacement therapy in liver, leukocytes, spleen, bone marrow, lung tissue, and / or any other Gaucher disease-affected organs. In one embodiment, the effective activity of the modified GCase polypeptide is the same as or higher than that of GCase enzyme replacement therapy in bone marrow and / or lung tissue.

[0214] In one embodiment, the effective activity of the modified GCase polypeptide is higher than that of GCase enzyme replacement therapy. In one embodiment, the effective activity of the modified GCase polypeptide is at least 1.1 times, at least 1.2 times, or at least 1.3 times higher than that of GCase enzyme replacement therapy. In one embodiment, the effective activity of the modified GCase polypeptide is at least 1.5 times or at least 2 times higher in lung tissue compared to that of GCase enzyme replacement therapy.

[0215] In one embodiment, when the effective activity is preferably measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after the start of treatment in the subject, the effective activity of the modified GCase polypeptide (in the subject) is higher than that of GCase enzyme replacement therapy. Preferably, the effective activity is measured at 12 weeks or after 12 weeks.

[0216] "GCase enzyme replacement therapy" may refer to any therapy involving the administration of a GCase polypeptide to a subject. The GCase polypeptide may be wild-type, for example, a GCase polypeptide having the amino acid sequence of SEQ ID NO: 3 and / or veraglucerase alpha. The GCase polypeptide may be administered in any preferred dose, optionally at doses of 40-100 U / kg BW, 50-80 U / kg BW, 60-70 U / kg BW, or around 60 U / kg BW. The GCase polypeptide may be administered via any suitable route, optionally by intravenous or subcutaneous injection. The effective activity of the GCase enzyme replacement therapy is preferably measured 2 hours after administration of the dose of the GCase enzyme replacement therapy (e.g., the previous dose). As an example, if the subject is given GCase enzyme replacement therapy every 2 weeks, the measurement of the effective activity of the GCase enzyme replacement therapy at 12 weeks involves obtaining a tissue sample from the subject 2 hours after administration of the dose at 12 weeks (i.e., the 7th dose). Optionally, the measurement of the effective activity of GCase enzyme replacement therapy includes administering a dose of GCase enzyme replacement therapy every two weeks. Optionally, the measurement of the effective activity of GCase enzyme replacement therapy includes administering a dose of GCase enzyme replacement therapy for at least 12 weeks or over a period of 12 weeks. Optionally, the measurement of the effective activity of GCase enzyme replacement therapy includes administering a dose of GCase enzyme replacement therapy every two weeks, and the effective activity of GCase enzyme replacement therapy is measured 2 hours after the administration of the last dose of GCase enzyme replacement therapy.

[0217] In one embodiment, after administration of viral particles, the levels of hexosylsphingosine and / or hexosylceramide are lower in plasma, liver, spleen, bone marrow, lung tissue, and / or any other Gaucher disease-affected organs compared to the levels of hexosylsphingosine and / or hexosylceramide after the GCase enzyme replacement therapy described above. In a related embodiment, after administration of viral particles, the levels of hexosylsphingosine or hexosylceramide are lower in plasma, liver, spleen, bone marrow, lung tissue, and / or any other Gaucher disease-affected organs compared to the levels of hexosylsphingosine and / or hexosylceramide in subjects with Gaucher disease (e.g., the same or equivalent organs). Optionally, after administration of viral particles, the levels of hexosylsphingosine and / or hexosylceramide are less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, or 0% to 25% of the levels of hexosylsphingosine and / or hexosylceramide after GCase enzyme replacement therapy. Optionally, after administration of viral particles, the levels of hexosylsphingosine and / or hexosylceramide are less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, or 0% to 25% of the levels of hexosylsphingosine and / or hexosylceramide in subjects with Gaucher disease (e.g., identical or equivalent organs). In one embodiment, the levels of hexosylsphingosine and / or hexosylceramide are measured by mass spectrometry, for example, the method described in Example 13. The subject with Gaucher disease may be a model organism for Gaucher disease, such as a model mouse for Gaucher disease (e.g., a 9V / null mouse).

[0218] In some embodiments, after administration of viral particles, levels of hexosisphingosine and / or hexosylceramide are lower in plasma, liver, spleen, bone marrow, lung tissue, and / or any other Gaucher disease-affected organs compared to levels of hexosisphingosine and / or hexosylceramide after administration of viral particles otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide. Optionally, after administration of viral particles, levels of hexosylsphingosine and / or hexosylceramide are less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, or 0% to 25% of levels of hexosylsphingosine and / or hexosylceramide after administration of viral particles otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide. Optionally, the reference GCase polypeptide is a wild-type GCase polypeptide. Optionally, the reference GCase polypeptide contains the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 1 or 2. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide includes the nucleotide sequence of SEQ ID NO: 59 or 60. Optionally, after administration of viral particles, the levels of hexosylsphingosine and / or hexosylceramide are less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, or 0% to 25% of the levels of hexosylsphingosine and / or hexosylceramide in a subject with Gaucher disease (e.g., the same or equivalent organ). In one embodiment, the levels of hexosylsphingosine and / or hexosylceramide are measured by mass spectrometry, e.g., the method described in Example 13. The subject with Gaucher disease may be a model organism of Gaucher disease, e.g., a model mouse of Gaucher disease (e.g., a 9V / null mouse).

[0219] In one embodiment, after administration of viral particles, the number of storage cells or the level of activated macrophages in the liver or lung tissue (and / or any other Gaucher disease-affected organ) is lower than the number of storage cells or the level of activated macrophages after the GCase enzyme replacement therapy described above. Optionally, the number of storage cells is less than 50%, less than 40%, less than 30%, less than 25%, less than 15%, or 0% to 25% of the number of storage cells after GCase enzyme replacement therapy. Optionally, the level of activated macrophages is less than 50%, less than 40%, less than 30%, less than 25%, less than 15%, or 0% to 25% of the level of activated macrophages after GCase enzyme replacement therapy.

[0220] In one embodiment, levels of hexosylsphingosine, hexosylceramide, storage cells, and / or activated macrophages are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration of viral particles. Preferably, levels of hexosylsphingosine, hexosylceramide, storage cells, and / or activated macrophages are measured at 12 weeks or after 12 weeks.

[0221] In some embodiments, levels of hexosylsphingosine and / or hexosylceramide are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration of viral particles. Preferably, levels of hexosylsphingosine and / or hexosylceramide are measured at 12 weeks or after 12 weeks. For example, the levels of hexosylsphingosine and / or hexosylceramide at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of the viral particles of the present invention may be compared to the levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of viral particles that are otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide. Specifically, the levels of hexosylsphingosine and / or hexosylceramide at least 12 weeks (e.g., week 12) after administration of the viral particles of the present invention may be measured and compared to the levels measured at least 12 weeks (e.g., week 12) after administration of viral particles that are otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide. Preferably, the levels of hexosylsphingosine and / or hexosylceramide are measured at the same time point after administration using the same assay.

[0222] When measuring hexosylsphingosine levels, hexosylceramide levels, storage cell counts, and / or activated macrophage levels after GCase enzyme replacement therapy, these levels are preferably measured 2 hours after administration of the GCase enzyme replacement therapy dose (e.g., the previous dose). For example, if GCase enzyme replacement therapy is administered to a subject every two weeks, measuring hexosylsphingosine levels, hexosylceramide levels, storage cell counts, and / or activated macrophage levels at week 12 involves obtaining a tissue sample from the subject 2 hours after administration of the week 12 (i.e., the 7th) dose. Optionally, measuring hexosylsphingosine levels, hexosylceramide levels, storage cell counts, and / or activated macrophage levels after GCase enzyme replacement therapy involves administering the GCase enzyme replacement therapy dose every two weeks. Optionally, the measurement of hexosylsphingosine levels, hexosylceramide levels, storage cell count, and / or activated macrophage levels after GCase enzyme replacement therapy includes administering doses of GCase enzyme replacement therapy for at least 12 weeks or over a period of 12 weeks. Optionally, the measurement of hexosylsphingosine levels, hexosylceramide levels, storage cell count, and / or activated macrophage levels after GCase enzyme replacement therapy includes administering doses of GCase enzyme replacement therapy every two weeks, with the levels of hexosylsphingosine levels, hexosylceramide levels, storage cell count, and / or activated macrophage levels being measured two hours after the administration of the last dose of GCase enzyme replacement therapy.

[0223] In one embodiment, the number of storage cells is measured by microscopic examination. In one embodiment, the level of activated macrophages is detected by measuring CD68 density using immunohistochemical testing. Storage cells and / or CD68 陽性Cell identification can be carried out by methods known in the art, such as immunohistochemical testing or microscopic examination, for example, the method described in Example 13.

[0224] For example, a decrease in hexosylceramide and / or hexosylsphingosine levels may represent a decrease in glucosylceramide and / or glucosylsphingosine levels, respectively. For instance, a decrease in hexosylceramide may represent a decrease in glucosylceramide. As a further example, a decrease in hexosylsphingosine levels may represent a decrease in glucosylsphingosine levels.

[0225] Composition, method, and use In a further aspect of the present invention, a composition is provided comprising a modified GCase polypeptide, polynucleotide, or vector / viral particle of the present invention and a pharmaceutically acceptable excipient.

[0226] Pharmaceutically acceptable excipients may include carriers, diluents, and / or other drugs, pharmaceuticals, or adjuvants. Optionally, pharmaceutically acceptable excipients may include physiological saline. Optionally, pharmaceutically acceptable excipients may include human serum albumin.

[0227] The present invention further provides modified GCase polypeptides, polynucleotides, vectors / viral particles, or compositions for use in therapeutic methods. Optionally, a therapeutic method comprises administering an effective amount of the modified GCase polypeptides, polynucleotides, vectors / viral particles, or compositions to a patient.

[0228] The present invention further provides a therapeutic method comprising administering an effective amount of the modified GCase polypeptide, polynucleotide, vector / viral particle, or composition of the present invention to a patient.

[0229] The present invention further provides the use of a modified GCase polypeptide, polynucleotide, vector / viral particle, or composition of the present invention in the manufacture of a medicament for use in a treatment method. Optionally, the treatment method includes administering to a patient an effective amount of a modified GCase polypeptide, polynucleotide, vector / viral particle, or composition of the present invention.

[0230] Optionally, the treatment method is gene therapy. "Gene therapy" includes administering a vector / viral particle of the present invention capable of expressing a transgene (such as a GBA nucleotide sequence) to a host (such as a patient) to which it is administered.

[0231] Optionally, the treatment method is GCase enzyme replacement therapy. "GCase enzyme replacement therapy" can refer to any therapy that includes administering a GCase polypeptide to a subject. A modified GCase polypeptide of the present invention may be administered. The modified GCase polypeptide may be administered at any suitable dose, optionally at a dose of about 40-100 U / kg BW, 50-80 U / kg BW, 60-70 U / kg BW, or about 60 U / kg BW. The GCase polypeptide may be administered through any appropriate route, optionally through intravenous injection or subcutaneous injection.

[0232] Optionally, the treatment method is a method for treating a disease associated with GCase deficiency. As discussed above, when GCase is deficient, accumulation of glucocerebroside in macrophages infiltrating many important organs may occur, which can cause various diseases including synucleinopathy or Parkinson's disease (as discussed in WO 08 / 144591). Optionally, the treatment method is a method for treating Parkinson's disease or synucleinopathy. Optionally, the treatment method is a method for treating Parkinson's disease. Optionally, the treatment method is a method for treating Lewy body dementia, peripheral neuropathy, multiple system atrophy, or pure autonomic failure.

[0233] Optionally, the treatment method is a method for treating lysosomal storage disorders such as Gaucher disease (GD), e.g., type I, type II, or type III GD. Preferably, the lysosomal storage disorder is characterized by bruising, fatigue, anemia, decreased platelet count, and hepatomegaly and splenomegaly. Optionally, the treatment method is a method for treating GD. Optionally, the treatment method is a method for treating type I GD. Optionally, the treatment method is a method for treating type II GD. Optionally, the treatment method is a method for treating type III GD. In some embodiments, the patient is a patient suffering from GD. In some embodiments, the patient is a patient suffering from type I GD. In some embodiments, the patient is a patient suffering from type II GD. In some embodiments, the patient is a patient suffering from type III GD. Optionally, the patient has antibodies or inhibitors against recombinant GCases (e.g., imiglucerase, veraglucerase alfa, or taliglucerase alfa) previously treated as part of enzyme replacement therapy.

[0234] Optionally, the treatment method is a method for treating Niemann-Pick disease, such as type C Niemann-Pick (NPC).

[0235] Optionally, modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions are administered intravenously. Optionally, modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions are intended for single-dose administration to a patient.

[0236] Optionally, modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions are administered directly to the target CNS, for example, by direct injection into the target brain and / or spinal cord. Optionally, direct injection is intracerebral, intraventricular, intracisional, intraparenchymal, intrathecal, or any combination thereof. Optionally, convection-enhanced delivery (CED) is used for direct injection into the target CNS. CED is a therapeutic strategy that involves surgically exposing the brain, placing a small-diameter catheter directly into a target region of the brain, and then directly injecting the therapeutic agent (e.g., modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions described herein) into the target brain. CED is described, for example, in Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27. Optionally, modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions are administered peripherally to the target, for example, by peripheral injection. Optionally, peripheral injection may be subcutaneous, intravenous, intra-arterial (e.g., injection into the carotid artery of the subject), intraperitoneal, or any combination thereof. Optionally, modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions may be administered directly to the periphery and CNS of the subject. For example, in some embodiments, the composition is administered to the subject by intra-arterial injection (e.g., injection into the carotid artery) and intraciscular injection (e.g., intraciscular injection via CED). Optionally, direct and peripheral injections to the CNS may be performed simultaneously. Optionally, direct injection may be performed before peripheral injection (e.g., 1 minute to 1 week or more before). Optionally, direct injection may be performed after peripheral injection (e.g., 1 minute to 1 week or more after).

[0237] Optionally, modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions are administered by injection into the renal artery. Optionally, modified GCase polypeptides, polynucleotides, vectors / viral particles, and / or compositions are administered retrogradely, for example, via the ureter using a urinary catheter.

[0238] When a disease or disorder (e.g., GD such as type I GD) is “treated” as discussed herein (e.g., in the methods or uses of the present invention), this means that one or more symptoms of the disease or disorder (e.g., GD such as type I GD) go into remission. This does not mean that the symptoms of the disease or disorder (e.g., GD such as type I GD) are completely cured and as a result no longer exist in the patient, although in some methods this may be the case. Therefore, in all examples, the terms “treated” or “to treat” can be replaced with the terms “remission” or “to go into remission,” respectively. The methods or uses of the present invention (treatment methods or treatments, etc.) can reduce the severity of one or more symptoms of the disease or disorder (e.g., GD such as type I GD) to a level lower than before treatment. Optionally, compared to the pre-administration situation, the methods or uses of the present invention (treatment methods or treatments, etc.) increase the amount / concentration of circulating GCase in the patient’s blood and / or increase the overall level of detectable GCase activity in a given volume of the patient’s blood and / or macrophages. In embodiments, the method or use of the present invention (treatment method or treatment, etc.) results in one or more of the following, relative to the pre-administration condition: an increase in hemoglobin concentration; an increase in platelet count; a decrease in spleen size; or a decrease in liver size.

[0239] Furthermore, the methods or uses of the present invention can also "prevent" diseases such as Gaucher disease. Gaucher disease is generally associated with the accumulation of glucocerebrosidase in various tissues, and when the methods or uses of the present invention are applied to young subjects (teenagers, young adults, children, or infants, etc.), it should be possible to prevent the establishment of Gaucher disease. Accordingly, in all examples, the terms "treat" or "treat" can be replaced with the terms "prevent" or "prevent" respectively.

[0240] "Effective dose" refers to the amount and duration of administration that is effective in achieving the desired therapeutic outcome, such as increasing the level of functional GCase in the subject (to the extent that a sufficient level of functional GCase is produced to alleviate the symptoms of a disease or disorder such as GD, e.g., type I GD).

[0241] Optionally, 1 x 10 per kg of patient's body weight. 11 Less than 1 vector genome (vg / kg), 2 × 10⁻¹⁶ 11 Less than vg / kg, 1 × 10 12 Less than vg / kg, 5 x 10 12 Less than vg / kg, 2 × 10 12 Less than vg / kg, 1.5 × 10 12 Less than vg / kg, 3 x 10 12 Less than vg / kg, 1 × 10 13 Less than vg / kg, 2 × 10 13 Less than vg / kg, or 3 × 10 13 A dose of vector / viral particles less than vg / kg is administered. Optionally, the dose of vector / viral particles administered is selected to elicit GCase expression at levels of 10%–90%, 20%–80%, 30%–70%, 25%–50%, 20–150%, 30–140%, 40%–130%, 50–120%, 60%–110%, or 70–100% of those levels in healthy, non-GD-affected subjects.

[0242] The present invention further provides a modified GCase polypeptide, polynucleotide, viral particle, or composition for use in the treatment of a disease, wherein the modified GCase polypeptide or the encoded modified GCase polypeptide may have increased stability at pH 7.4 compared to a wild-type GCase polypeptide, for example, the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40. Optionally, the modified GCase polypeptide or the encoded modified GCase polypeptide may retain at least 1.2 times, at least 1.5 times, at least 1.8 times, or at least 2 times higher activity than the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40, when measured after incubation at pH 7.4 and 37°C for 120 minutes. Optionally, the modified GCase polypeptide or the encoded modified GCase polypeptide retains at least 3 times, at least 5 times, at least 7 times, at least 10 times, or at least 15 times higher activity than the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40, when measured after incubation at pH 7.4 and 37°C for 120 minutes.

[0243] The present invention further provides a method for treating a disease by administering a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention, wherein the modified GCase polypeptide or encoded modified GCase polypeptide has increased stability at pH 7.4 compared to a reference wild-type GCase polypeptide, for example, the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40. Optionally, the modified GCase polypeptide or encoded modified GCase polypeptide retains at least 1.2 times, at least 1.5 times, at least 1.8 times, or at least 2 times higher activity than the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40, when measured after incubation at pH 7.4 and 37°C for 120 minutes. Optionally, the modified GCase polypeptide or encoded modified GCase polypeptide retains at least 3 times, at least 5 times, at least 7 times, at least 10 times, or at least 15 times higher activity than the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40, when measured after incubation at pH 7.4 and 37°C for 120 minutes.

[0244] The present invention further provides the use of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention in the treatment of a disease, wherein the modified GCase polypeptide or encoded modified GCase polypeptide has increased stability at pH 7.4 compared to a wild-type GCase polypeptide, for example, the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40. Optionally, the modified GCase polypeptide or encoded modified GCase polypeptide retains at least 1.2 times, at least 1.5 times, at least 1.8 times, or at least 2 times higher activity than the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40, when measured after incubation at pH 7.4 and 37°C for 120 minutes. Optionally, the modified GCase polypeptide or encoded polypeptide retains at least 3 times, at least 5 times, at least 7 times, at least 10 times, or at least 15 times higher activity than the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40, when measured after incubation at pH 7.4 and 37°C for 120 minutes.

[0245] The present invention further provides a modified GCase polypeptide, polynucleotide, viral particle, or composition for use in the treatment of a disease, comprising administering a lower dose of the modified GCase polypeptide, polynucleotide, viral particle, or composition compared to the dose required for administering an equivalent polypeptide, polynucleotide, viral particle, or composition comprising or encoding the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0246] The present invention further provides a method for treating a disease by administering a GCase polypeptide, polynucleotide, viral particle, or composition of the present invention, wherein the treatment of the disease comprises administering a lower dose of the modified GCase polypeptide, polynucleotide, viral particle, or composition compared to the dose required for administering an equivalent polypeptide, polynucleotide, viral particle, or composition comprising or encoding the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0247] The present invention further provides a use of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention in the treatment of a disease, wherein the treatment of the disease involves administering a lower dose of the modified GCase polypeptide, polynucleotide, viral particle, or composition compared to the dose required for administering an equivalent polypeptide, polynucleotide, viral particle, or composition comprising or encoding the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40.

[0248] In relation to the expression "treating a disease," "disease" may be any one of the diseases or disorders described above. Therefore, treatment of a disease may be treatment of lysosomal storage disorder in a subject or patient with Gaucher disease (GD), such as type I, type II, or type III GD. Preferably, lysosomal storage disorder is characterized by bruising, fatigue, anemia, decreased platelet count, and hepatomegaly and splenomegaly. Optionally, "treating a disease" may refer to treatment of GD, such as type I GD, in a subject or patient. In some embodiments, the subject or patient is a subject or patient suffering from GD, such as type I GD.

[0249] In relation to "an equivalent polypeptide, polynucleotide, viral particle, or composition comprising or encoding a GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40," the "equivalent" polypeptide comprises (or is) the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40. The "equivalent" polynucleotide is the same as the polynucleotide of the present invention compared to the equivalent, except that the "equivalent" polynucleotide encodes the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40 rather than the modified GCase polypeptide of the present invention. The "equivalent" viral particle is the same as the viral particle of the present invention compared to the equivalent, except that the "equivalent" viral particle encodes the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40 rather than the modified GCase polypeptide of the present invention. The "equivalent" composition is the same as the composition of the present invention compared to the equivalent, except that the "equivalent" composition comprises or encodes the GCase polypeptide encoded by the GBA nucleotide sequence of SEQ ID NO: 40 rather than the modified GCase polypeptide of the present invention.

[0250] In one embodiment, in a subject suffering from a disease or condition related to GCase deficiency, (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. The use of a modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention in the manufacture of pharmaceuticals is provided.

[0251] In one embodiment, by administering the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention to subjects suffering from a disease or condition related to GCase deficiency, (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. A method is provided. Optionally, (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. This allows for the treatment of diseases or conditions associated with GCase deficiency.

[0252] In one embodiment, in a subject suffering from a disease or condition related to GCase deficiency, (iv) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (v) Reduce the number of storage cells; and / or (vi) Reduce the level of activated macrophages. A modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention for use in the method, Optional (iv) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (v) Reduce the number of storage cells; and / or (vi) Reduce the level of activated macrophages. A modified GCase polypeptide, polynucleotide, viral particle, or composition that leads to the treatment of a disease or condition associated with the GCase deficiency.

[0253] Optionally, levels of hexosylceramide and / or hexosylsphingosine are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or 12 weeks after administration. Levels of hexosylceramide and / or hexosylsphingosine can be reduced by more than 2, 3, 4, 5, 6, 2-3, 2-4, 2-5, 2-6, or 3-5 times compared to the (starting) levels of hexosylceramide and / or hexosylsphingosine at the time of administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention. For example, after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention (for example, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or 12 weeks after administration), the levels of hexosylceramide and / or hexosylsphingosine in the patient may be 50% or less, 40% or less, 30% or less, 25% or less, or 20% or less compared to the levels of (starting) hexosylceramide and / or hexosylsphingosine at the time of administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention. Optionally, patients may have elevated levels of hexosylceramide and / or hexosylsphingosine compared to healthy subjects or subjects without GCase deficiency-related diseases or conditions; that is, prior to administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention, patients have higher levels of hexosylceramide and / or hexosylsphingosine compared to healthy subjects or subjects without GCase deficiency-related diseases or conditions. For example, levels of hexosylceramide and / or hexosylsphingosine are measured in the lungs, spleen, liver, and / or bone marrow of the patient / subject. Optionally, the measurement of levels of hexosylceramide and / or hexosylsphingosine in the lungs, spleen, liver, and / or bone marrow may involve estimating levels of hexosylceramide and / or hexosylsphingosine in the lungs, spleen, liver, and / or bone marrow based on data obtained in mouse studies.For example, a GCase polypeptide can be tested to see if it can reduce the level of hexosylsphingosine in a patient's spleen by testing whether it can reduce the level of hexosylsphingosine in the spleen of a mouse. The levels of hexosylceramide and / or hexosylsphingosine may be measured in the serum and / or leukocytes (e.g., macrophages) of the patient / subject. The levels of hexosylceramide and / or hexosylsphingosine may be measured in the plasma of the patient / subject. Methods for measuring the levels of hexosylceramide and / or hexosylsphingosine are known in the art, and the levels of hexosylceramide and / or hexosylsphingosine are preferably measured using mass spectrometry (LC / MS analysis), for example, by the method described in Example 13.

[0254] If, optionally, preferably, levels of hexosylceramide and / or hexosylsphingosine are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after the start of treatment, the decrease in levels of hexosylceramide and / or hexosylsphingosine (e.g., in the patient / subject's lungs, serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow; further examples include in the patient / subject's plasma) is greater than the decrease achieved by GCase enzyme replacement therapy. For example, levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention may be compared to levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after the first administration of GCase enzyme replacement therapy, respectively. Specifically, levels of hexosylceramide and / or hexosylsphingosine may be measured at least 12 weeks (e.g., week 12) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention and compared to levels measured at least 12 weeks (e.g., week 12) after the first administration of GCase enzyme replacement therapy. Preferably, the levels of hexosylceramide and / or hexosylsphingosine are measured at the same time after administration using the same assay. Optionally, GCase enzyme replacement therapy may be administered every two weeks. Optionally, if the levels of hexosylsphingosine and / or hexosylceramide are measured at least 6 weeks, 8 weeks, 10 weeks, or 12 weeks after the start of treatment, the levels of hexosylsphingosine and / or hexosylceramide will decrease to less than 50%, 40%, 30%, 25%, 10%, or 5%-25% of the levels of hexosylsphingosine and / or hexosylceramide achieved in subjects receiving GCase enzyme replacement therapy.If hexosylsphingosine and / or hexosylceramide levels are optionally measured at least 6 weeks, 8 weeks, 10 weeks, or 12 weeks after the start of treatment, the levels of hexosylsphingosine and / or hexosylceramide will decrease to less than 50%, 40%, 30%, 25%, 10%, or 5%-25% of the levels of hexosylsphingosine and / or hexosylceramide achieved in subjects administered an effective dose of GCase enzyme replacement therapy. If hexosylsphingosine and / or hexosylceramide levels are measured in subjects after administration of an effective dose of GCase enzyme replacement therapy, the levels of hexosylsphingosine and / or hexosylceramide are preferably measured 2 hours after administration of the dose of GCase enzyme replacement therapy (e.g., the previous dose). For example, when GCase enzyme replacement therapy is administered to a subject every two weeks, measuring the levels of hexosylsphingosine and / or hexosylceramide in the serum at, for example, week 12, involves obtaining a serum sample from the subject two hours after the administration of the week 12 (i.e., the 7th) dose.

[0255] Optionally, the decrease in hexosylsphingosine and / or hexosylceramide levels (e.g., in the lungs, plasma, serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) following administration of the viral particles of the present invention is greater than the decrease achieved in subjects otherwise identical to the viral particles, including the GBA nucleotide sequence encoding the reference GCase polypeptide.

[0256] Optionally, if the modified GCase polypeptide or polynucleotide of the present invention is provided as part of a viral particle encoding the modified GCase polypeptide or a viral particle containing the polynucleotide of the present invention, it will cause a greater reduction in the levels of hexosphingosine and / or hexosylceramide (e.g., in the patient / subject's lungs, plasma, serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) compared to a reference GCase polypeptide; that is, the reduction in the levels of hexosylsphingosine and / or hexosylceramide (e.g., in the patient / subject's lungs, plasma, serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) after administration of a viral particle encoding the modified GCase polypeptide or a viral particle containing the polynucleotide of the present invention will be greater than the reduction achieved in a subject administered with a viral particle otherwise identical, including a GBA nucleotide sequence encoding the reference GCase polypeptide.

[0257] In such a configuration, viral particles that are "otherwise identical" containing a GBA nucleotide sequence encoding a reference GCase polypeptide are, except for having different GBA nucleotide sequences, (i) The virus particles of the present invention; (ii) Viral particles encoding the modified GCase polypeptide of the present invention; or (iii) Virus particles containing the polynucleotide of the present invention; These are viral particles that are identical (i.e., the viral particles contain the same capsid, the recombinant genome contains the same transcriptional regulatory elements, etc.). For example, different GBA nucleotide sequences encoding different GCase polypeptides are operably linked to the same promoter sequence.

[0258] Preferably, levels of hexosylceramide and / or hexosylsphingosine are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration. For example, levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of (i) viral particles of the present invention; (ii) viral particles encoding a modified GCase polypeptide of the present invention; or (iii) viral particles containing a polynucleotide of the present invention may be compared to levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of viral particles otherwise identical, including a GBA nucleotide sequence encoding a reference GCase polypeptide, respectively. As a specific example, levels of hexosylceramide and / or hexosylsphingosine may be measured at least 12 weeks (e.g., at week 12) after administration of (i) viral particles of the present invention; (ii) viral particles encoding a modified GCase polypeptide of the present invention; or (iii) viral particles containing a polynucleotide of the present invention, and compared to levels measured at least 12 weeks (e.g., at week 12) after administration of viral particles otherwise identical, including a GBA nucleotide sequence encoding a reference GCase polypeptide. Preferably, levels of hexosylceramide and / or hexosylsphingosine are measured at the same time point after administration using the same assay. If hexosylceramide and / or hexosylsphingosine levels are optionally measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration, the levels of hexosylsphingosine and / or hexosylceramide will decrease to less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, or 5% to 25% of the levels of hexosylsphingosine and / or hexosylceramide achieved in subjects administered with viral particles otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide.In some embodiments, the reference GCase polypeptide is a wild-type GCase polypeptide. Optionally, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 1 or 2. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 59 or 60.

[0259] Optionally, levels of hexosylceramide and / or hexosylsphingosine are measured in the plasma of the subject. Optionally, levels of hexosylceramide and / or hexosylsphingosine are measured in the bone marrow of the subject. Optionally, levels of hexosylceramide and / or hexosylsphingosine are measured in the spleen of the subject. Optionally, levels of hexosylceramide and / or hexosylsphingosine are measured in the liver of the subject. Optionally, levels of hexosylceramide and / or hexosylsphingosine are measured in the lungs of the subject. Methods for measuring levels of hexosylceramide and / or hexosylsphingosine are known in the art, and the levels of hexosylceramide and / or hexosylsphingosine are preferably measured using mass spectrometry (LC / MS analysis), for example, by the method described in Example 13.

[0260] Optionally, a decrease in hexosylsphingosine and / or hexosylceramide levels in a subject (or patient) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention is defined as the level of hexosylsphingosine and / or hexosylceramide (e.g., in bone marrow, lung, serum, leukocytes (e.g., macrophages), liver, and / or spleen; further examples include in the plasma of the patient / subject) being 200%, 150%, or 125% or less of the level of hexosylsphingosine and / or hexosylceramide measured in a healthy subject or a subject not suffering from any disease or condition associated with GCase deficiency. In one example, a decrease in hexosylceramide and / or hexosylsphingosine levels may represent a decrease in glucosylceramide and / or glucosylsphingosine levels, respectively. For example, a decrease in hexosylceramide may represent a decrease in glucosylceramide. As a further example, a decrease in hexosylsphingosine levels may indicate a decrease in glucosylsphingosine levels.

[0261] In one example, a decrease in hexosylceramide and / or hexosylsphingosine levels corresponds to a decrease in glucosylceramide and / or glucosylsphingosine, respectively. In other words, if glucosylceramide and / or glucosylsphingosine levels are measured preferably at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or 12 weeks after administration, patients administered with the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention (e.g., patients suffering from a disease or condition associated with GCase deficiency) may have decreased levels of glucosylceramide and / or glucosylsphingosine after administration. The levels of glucosylceramide and / or glucosylsphingosine can be reduced by 2 to 3 times, 4 to 4 times, 5 to 6 times, 2 to 3 times, 2 to 4 times, 2 to 5 times, 2 to 6 times, or 3 to 5 times compared to the levels of (starting) glucosylceramide and / or glucosylsphingosine at the time of administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention. For example, after administration of the polynucleotide, viral particle, or composition of the present invention (e.g., at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or 12 weeks after administration), the levels of glucosylceramide and / or glucosylsphingosine in the patient may be 50% or less, 40% or less, 30% or less, 25% or less, or 20% or less compared to the levels of (starting) glucosylceramide and / or glucosylsphingosine at the time of administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention. Optionally, (prior to administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention) the patient may have elevated levels of glucosylceramide and / or glucosylsphingosine compared to a healthy subject or a subject without GCase deficiency-related disease or condition. For example, levels of glucosylceramide and / or glucosylsphingosine are measured in the lungs, spleen, liver, and / or bone marrow of the patient / subject.The levels of glucosylceramide and / or glucosylsphingosine may be measured in the patient's / subject's serum and / or leukocytes (e.g., macrophages). The levels of glucosylceramide and / or glucosylsphingosine may be measured in the patient's / subject's plasma. Methods for measuring the levels of glucosylceramide and / or glucosylsphingosine are known in the art, and the levels of glucosylceramide and / or glucosylsphingosine are preferably measured using mass spectrometry (LC / MS analysis), for example, by the method described in Example 13. If, optionally, preferably, levels of glucosylceramide and / or glucosylsphingosine are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after the start of treatment, the decrease in levels of glucosylceramide and / or glucosylsphingosine (e.g., in patient / subject serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow; further examples include in patient / subject plasma) is greater than the decrease achieved by GCase enzyme replacement therapy. For example, levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention may be compared to levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after the first administration of GCase enzyme replacement therapy, respectively. Specifically, levels of glucosylceramide and / or glucosylsphingosine may be measured at least 12 weeks (e.g., week 12) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention and compared to levels measured at least 12 weeks (e.g., week 12) after the first administration of GCase enzyme replacement therapy. Preferably, glucosylceramide and / or glucosylsphingosine levels are measured at the same time after administration using the same assay. Optionally, GCase enzyme replacement therapy may be administered every two weeks.When measuring the levels of glucosylceramide and / or glucosylsphingosine achieved in a subject after administration of an effective dose of GCase enzyme replacement therapy, the levels of glucosylsphingosine and / or glucosylceramide are preferably measured 2 hours after administration of the dose of GCase enzyme replacement therapy (e.g., the previous dose). For example, if GCase enzyme replacement therapy is administered to a subject every two weeks, measuring the levels of glucosylsphingosine and / or glucosylceramide in the serum at, for example, week 12, involves obtaining a serum sample from the subject 2 hours after administration of the week 12 (i.e., the 7th) dose.

[0262] Optionally, the decrease in glucosylceramide and / or glucosylsphingosine levels (e.g., in the lungs, plasma, serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) following administration of the viral particles of the present invention is greater than the decrease achieved in subjects otherwise identical to the viral particles, including the GBA nucleotide sequence encoding the reference GCase polypeptide.

[0263] Optionally, if the modified GCase polypeptide or polynucleotide of the present invention is provided as part of a viral particle encoding the modified GCase polypeptide or a viral particle containing the polynucleotide of the present invention, it will cause a greater reduction in glucosylceramide and / or glucosylsphingosine levels (e.g., in the patient / subject's lungs, plasma, serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) compared to a reference GCase polypeptide; that is, the reduction in glucosylceramide and / or glucosylsphingosine levels (e.g., in the patient / subject's lungs, plasma, serum, leukocytes (e.g., macrophages), spleen, liver, and / or bone marrow) after administration of a viral particle encoding the modified GCase polypeptide or a viral particle containing the polynucleotide of the present invention will be greater than the reduction achieved in a subject administered with a viral particle otherwise identical, including a GBA nucleotide sequence encoding the reference GCase polypeptide.

[0264] In such a configuration, viral particles that are "otherwise identical" containing a GBA nucleotide sequence encoding a reference GCase polypeptide are, except for having different GBA nucleotide sequences, (i) The virus particles of the present invention; (ii) Viral particles encoding the modified GCase polypeptide of the present invention, or (iii) Virus particles containing the polynucleotide of the present invention; These are identical viral particles (i.e., the viral particles contain the same capsid, the recombinant genome contains the same transcriptional regulatory elements, etc.). For example, different GBA nucleotide sequences encoding different GCase polypeptides are operably linked to the same promoter sequence.

[0265] Preferably, levels of glucosylceramide and / or glucosylsphingosine are measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration. For example, levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of (i) viral particles of the present invention; (ii) viral particles encoding a modified GCase polypeptide of the present invention; or (iii) viral particles containing a polynucleotide of the present invention may be compared to levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of viral particles otherwise identical, including a GBA nucleotide sequence encoding a reference GCase polypeptide, respectively. As a specific example, levels of glucosylceramide and / or glucosylsphingosine may be measured at least 12 weeks (e.g., at week 12) after administration of (i) viral particles of the present invention; (ii) viral particles encoding a modified GCase polypeptide of the present invention; or (iii) viral particles containing a polynucleotide of the present invention, and compared to levels measured at least 12 weeks (e.g., at week 12) after administration of viral particles otherwise identical, including a GBA nucleotide sequence encoding a reference GCase polypeptide. Preferably, levels of glucosylceramide and / or glucosylsphingosine are measured at the same time point after administration using the same assay. If glucosylceramide and / or glucosylsphingosine levels are optionally measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after administration, the glucosylceramide and / or glucosylsphingosine levels will be reduced to less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, or 5% to 25% of the levels achieved in subjects administered with viral particles otherwise identical, including the GBA nucleotide sequence encoding the reference GCase polypeptide.In some embodiments, the reference GCase polypeptide is a wild-type GCase polypeptide. Optionally, the reference GCase polypeptide comprises the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 1 or 2. Optionally, the GBA nucleotide sequence encoding the reference GCase polypeptide comprises the nucleotide sequence of SEQ ID NO: 59 or 60.

[0266] Optionally, the decrease in glucosylceramide levels in a subject (or patient) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention is such that the glucosylceramide levels (e.g., in serum, leukocytes (e.g., macrophages), liver, and / or spleen) are 200%, 150%, or 125% or less of the glucosylceramide levels measured in a healthy subject or a subject not suffering from any disease or condition related to GCase deficiency.

[0267] If cells are counted optionally, preferably at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration, patients administered with the modified GCase polypeptide, polynucleotide, viral particles, or composition of the present invention (e.g., patients suffering from a disease or condition associated with GCase deficiency) may show a reduced number of storage cells and / or activated macrophages in the target liver or lung (e.g., lung tissue) after administration. A reduction in the number of storage cells and / or activated macrophages in the liver or lung (e.g., lung tissue) may be an indicator of reduced inflammation and, consequently, therapeutic effect. The number of activated macrophages is measured by CD68 陽性 This can be indicated or estimated by measuring the number of cells. Storage cells and / or CD68 陽性 Cell identification can be carried out by methods known in the art, such as immunohistochemical testing or microscopic examination, for example, the method described in Example 13.

[0268] Optionally, measuring the number of activated macrophages and / or storage cells in the liver and / or lungs includes estimating the number of activated macrophages and / or storage cells in the liver and / or lungs based on data obtained in mouse studies. For example, a GCase polypeptide can be tested to see if it can reduce the number of storage cells in a patient's liver by testing whether it can reduce the number of storage cells in the liver of a mouse.

[0269] If the number of storage cells and / or activated macrophages in the liver or lung (e.g., lung tissue) is optionally measured at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks after the start of treatment, the decrease in storage cells and / or activated macrophages in the liver or lung (e.g., lung tissue) of the subject is greater than the decrease achieved by GCase enzyme replacement therapy. For example, levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention may be compared to levels at least 6 weeks (e.g., week 6), at least 8 weeks (e.g., week 8), at least 10 weeks (e.g., week 10), or at least 12 weeks (e.g., week 12) after the first administration of GCase enzyme replacement therapy, respectively. As a specific example, the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) may be measured at least 12 weeks (e.g., 12 weeks) after administration of the modified GCase polypeptide, polynucleotide, viral particle, or composition of the present invention, and compared to the levels measured at least 12 weeks (e.g., 12 weeks) after the first administration of GCase enzyme replacement therapy. Preferably, the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) are measured at the same time point after administration using the same assay. Optionally, GCase enzyme replacement therapy may be administered every two weeks.If, optionally, the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) are measured at least 6 weeks, 8 weeks, 10 weeks, or 12 weeks after the start of treatment, the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) will decrease to less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, 0-25%, or 5-25% of the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) achieved in subjects receiving GCase enzyme replacement therapy. If, optionally, the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) are measured at least 6 weeks, 8 weeks, 10 weeks, 12 weeks, or 12 weeks after the start of treatment, the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) will decrease to less than 50%, less than 40%, less than 30%, less than 25%, less than 10%, 0-25%, or 5-25% of the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) achieved in subjects treated with an effective dose of GCase enzyme replacement therapy. When measuring the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) achieved in a subject after administration of an effective dose of GCase enzyme replacement therapy, it is preferable to measure the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) two hours after administration of the dose of GCase enzyme replacement therapy (e.g., the previous dose). For example, if GCase enzyme replacement therapy is administered to a subject every two weeks, measuring the levels (e.g., number) of storage cells and / or activated macrophages in the liver or lungs (e.g., lung tissue) at week 12 involves obtaining a tissue sample from the subject two hours after administration of the week 12 (i.e., the 7th) dose.

[0270] "GCase enzyme replacement therapy" may refer to any therapy that involves administering a GCase polypeptide to a subject. The GCase polypeptide may be wild-type, for example, a GCase polypeptide having the amino acid sequence of SEQ ID NO: 3, for example, veraglucerase alfa. The GCase polypeptide may be administered in any preferred dose, optionally at doses of 40-100 U / kg BW, 50-80 U / kg BW, 60-70 U / kg BW, or around 60 U / kg BW. The GCase polypeptide may be administered via any suitable route, optionally by intravenous or subcutaneous injection.

[0271] Embodiments of the Invention The present invention will be further described in the following embodiments.

[0272] 1. A modified β-glucocerebrosidase (GCase) polypeptide comprising at least one mutation, wherein the at least one mutation is (i) Provides higher effective activity; and / or (ii) to provide increased stability; and / or (iii) Provides structural stabilization at physiological pH; and / or (iv) Provides a longer half-life; and / or (v) Provides increased thermal stability Polypeptide.

[0273] 2. A modified β-glucocerebrosidase (GCase) polypeptide having at least one mutation at a position selected from the group consisting of 351, 380, 272, 262, 313, 404, 407, 482, 484, 490, 494, 503, and 534 of SEQ ID NO: 1.

[0274] 3. A modified GCase polypeptide according to embodiment 1 or 2, wherein at least one mutation provides higher effective activity.

[0275] 4. A modified GCase polypeptide according to any one of embodiments 1 to 3, wherein at least one mutation provides increased stability.

[0276] 5. A modified GCase polypeptide according to any one of embodiments 1 to 4, wherein at least one mutation provides structural stabilization at physiological pH.

[0277] 6. A modified GCase polypeptide according to any one of embodiments 1 to 5, wherein at least one mutation provides a longer half-life.

[0278] 7. A modified GCase polypeptide according to any one of embodiments 1 to 6, having higher effective activity compared to the reference GCase polypeptide.

[0279] 8. The modified GCase polypeptide according to embodiment 7, wherein the reference GCase polypeptide is a wild-type GCase polypeptide, and optionally, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3.

[0280] 9. The modified GCase polypeptide according to Embodiment 7, wherein the reference GCase polypeptide is the polypeptide of SEQ ID NO: 4 or 5.

[0281] 10. A modified GCase polypeptide according to any one of embodiments 7 to 9, wherein the effective activity of the modified GCase polypeptide is at least 1.2 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 10 times, at least 15 times, at least 20 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times higher than the effective activity of the reference GCase polypeptide.

[0282] 11. A modified GCase polypeptide according to any one of embodiments 1 to 10, wherein at least one mutation provides increased stability, and the increased stability is at pH 7.4.

[0283] 12. A modified GCase polypeptide according to any one of embodiments 1 to 11, wherein at least one mutation provides increased stability, which is measured after incubation at pH 7.4 and 37°C for 120 minutes.

[0284] 13. A modified GCase polypeptide according to any one of embodiments 1 to 12, wherein at least one mutation provides structural stabilization at pH 7.4.

[0285] 14. A modified GCase polypeptide according to any one of embodiments 1 to 13, wherein the modified GCase polypeptide is more structurally stable at physiological pH than the reference GCase polypeptide, and optionally the pH is 7.4.

[0286] 15. A modified GCase polypeptide according to any one of embodiments 1 to 14, wherein it has increased stability compared to a reference GCase polypeptide, and optionally, the increased stability is increased stability at pH 7.4.

[0287] 16. A modified GCase polypeptide according to an...

Claims

1. A modified β-glucocerebrosidase (GCase) polypeptide comprising at least one mutation, wherein the at least one mutation is (i) A mutation at the position corresponding to position 351 of sequence number 1, which is a cysteine ​​substitution; and (ii) A mutation at the position corresponding to position 380 of SEQ ID NO: 1, which is a cysteine ​​substitution. Includes, The modified GCase polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and The modified GCase polypeptide has increased stability compared to the reference GCase polypeptide, and the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3, 4, or 5. Modified GCase polypeptide.

2. (i) The modified GCase polypeptide has higher active activity than the reference GCase polypeptide, and the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3, 4, or 5; and / or (ii) The modified GCase polypeptide has higher active activity than the reference GCase polypeptide, the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3, 4, or 5, and the active activity of the modified GCase polypeptide is at least 1.2 times higher than the active activity of the reference GCase polypeptide. A modified GCase polypeptide according to claim 1.

3. (i) The increased stability is the increased stability at pH 7.4; and / or (ii) The modified GCase polypeptide retains at least 30% of its activity compared to its initial activity when measured after incubation at pH 7.4 and 37°C for at least 10 minutes; and / or (iii) When the modified GCase polypeptide is incubated at pH 7.4 and 37°C for 120 minutes, it exhibits activity at least 1.1 times higher than that of the reference GCase polypeptide, wherein the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3, 4, or 5. A modified GCase polypeptide according to claim 1 or 2.

4. The modified GCase polypeptide is (i) When measured after incubation at pH 7.4 and 37°C for 120 minutes, it retains at least 70% of its activity compared to its initial activity; and / or (ii) When measured after incubation at pH 7.4 and 37°C for 72 hours, it retains at least 40% of its activity compared to its initial activity; and / or (iii) When measured after incubation at pH 7.4 and 37°C for 72 hours, it retains at least 40% of its initial activity and is incubated in PBS; or (iv) When measured after incubation at pH 7.4 and 37°C for 7 days, it retains at least 15% or at least 20% of its activity compared to its initial activity; and / or (v) When incubated at pH 7.4 and 37°C for 7 days, it retains at least 15% or at least 20% of its activity compared to the initial activity, and is incubated in serum or plasma; or (vi) When measured after incubation at pH 7.4 and 37°C for 7 days, it retains at least 40% of its activity compared to its initial activity; and / or (vii) When measured after incubation at pH 7.4 and 37°C for 7 days, it retains at least 40% of its initial activity and is incubated in serum or plasma; or (viiii) When measured after incubation at pH 7.4 and 37°C for 7 days, it retains at least 60% of its activity compared to its initial activity; and / or (ix) When measured after incubation at pH 7.4 and 37°C for 7 days, it retains at least 80% of its activity compared to its initial activity; and / or (x) Activity, effective activity, and / or stability are determined using a fluorescence assay. A modified GCase polypeptide according to any one of claims 1 to 3.

5. A modified GCase polypeptide according to any one of claims 1 to 4, having a longer half-life than the reference GCase polypeptide, (i) The reference GCase polypeptide is the polypeptide of SEQ ID NO: 3, 4, or 5; or (ii) The reference GCase polypeptide is the polypeptide of SEQ ID NO: 3, 4, or 5, and the modified GCase polypeptide has a half-life at least 1.2 times longer than the half-life of the reference GCase polypeptide. Modified GCase polypeptide.

6. a. A longer half-life than the above is a longer half-life at pH 7.4 or pH 5.6; and / or b. The modified GCase polypeptide has a half-life at pH 5.6 that is at least 20 times longer than the half-life of the reference GCase polypeptide; and / or c. A longer half-life than the aforementioned is a longer half-life in serum or plasma; and / or d. The half-life is determined using a fluorescence assay. The modified GCase polypeptide according to claim 5.

7. (i) The at least one mutation includes a mutation at the position corresponding to position 272 of Sequence ID No. 1; or (ii) The at least one mutation includes a mutation at the position corresponding to position 272 of SEQ ID NO: 1, wherein the mutation at the position corresponding to position 272 of SEQ ID NO: 1 is a glutamine substitution; or (iii) The at least one mutation includes a mutation at the position corresponding to position 272 of SEQ ID NO: 1, wherein the mutation at the position corresponding to position 272 of SEQ ID NO: 1 is E272Q. A modified GCase polypeptide according to any one of claims 1 to 6.

8. (a) The at least one mutation is (i) A mutation at the position corresponding to position 482 of sequence number 1; and (ii) Mutation at position 503 of sequence number 1 Including; or (b) The at least one mutation is (i) A mutation at the position corresponding to position 482 of SEQ ID NO: 1 is (I) a substitution by cysteine, or (II) a mutation from aspartic acid to cysteine; and (ii) Mutation at position 503 of Sequence ID No. 1, wherein the mutation at position 503 of Sequence ID No. 1 is (I) a substitution by cysteine, or (II) a mutation from serine to cysteine. Including; and / or (c) The at least one mutation is (i) A mutation at the position corresponding to position 494 of sequence number 1; and (ii) Mutation at position 534 of sequence number 1 Including; or (d) The at least one mutation is (i) A mutation at the position corresponding to position 494 of Sequence ID No. 1 is (I) a substitution by cysteine, or (II) a mutation from serine to cysteine; and (ii) The mutation at the position corresponding to position 534 of SEQ ID NO: 1 includes a mutation at the position corresponding to position 534 of SEQ ID NO: 1, wherein the mutation is (I) a substitution with cysteine, or (II) a mutation from arginine to cysteine; and / or (e) (i) The mutation at the position corresponding to position 351 of Sequence ID No. 1 is a mutation from tryptophan to cysteine; and (ii) The mutation at the position corresponding to position 380 of Sequence ID No. 1 is a mutation from alanine to cysteine; and / or (f) The at least one mutation is (i) A mutation at the position corresponding to position 407 of sequence number 1; and (ii) Mutation at position 484 of sequence number 1 Including; or (g) The at least one mutation is (i) A mutation at the position corresponding to position 407 of SEQ ID NO: 1 is (I) a substitution by cysteine, or (II) a mutation from isoleucine to cysteine; and (ii) A mutation at the position corresponding to position 484 of SEQ ID NO: 1, wherein the mutation at the position corresponding to position 484 of SEQ ID NO: 1 is (I) a substitution by cysteine, or (II) a mutation from aspartic acid to cysteine; and / or (h) The at least one mutation is (i) Glutamine substitution at the position corresponding to position 272 of (I) Sequence ID No. 1, or (II) E272Q; and (ii) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of sequence number 1; and (iii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of sequence number 1. including A modified GCase polypeptide according to any one of claims 1 to 7.

9. (a) The at least one mutation is a glutamine substitution at the position corresponding to position 272 of SEQ ID NO: 1, the modified GCase polypeptide has increased stability compared to the reference GCase polypeptide, retains at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes, and the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3; and / or (b) The at least one mutation is (i) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of Sequence ID No. 1; and (ii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of sequence number 1; The modified GCase polypeptide comprises the modified GCase polypeptide having increased stability compared to the reference GCase polypeptide, retaining at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes, and the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3; and / or (c) The at least one mutation is (i) Glutamine substitution at the position corresponding to position 272 of (I) Sequence ID No. 1, or (II) E272Q; and (ii) A mutation from tryptophan to cysteine ​​at the position corresponding to position 351 of sequence number 1; and (iii) A mutation from alanine to cysteine ​​at the position corresponding to position 380 of sequence number 1. The modified GCase polypeptide comprises the modified GCase polypeptide having increased stability compared to the reference GCase polypeptide, retaining at least 85% activity after incubation at pH 7.4 and 37°C for 120 minutes, and the reference GCase polypeptide is the polypeptide of SEQ ID NO: 3; and / or (d) The modified GCase polypeptide has higher active activity and / or increased stability compared to the reference GCase polypeptide, and the reference GCase polypeptide is selected from any one of SEQ ID NOs: 1 to 5. A modified GCase polypeptide according to any one of claims 1 to 8.

10. The modified GCase polypeptide is (i) Containing an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 2; or (ii) Having an amino acid sequence that is at least 98% identical to SEQ ID NO: 1; or (iii) Containing an amino acid sequence that is at least 98% identical to SEQ ID NO: 2; or (iv) comprising an amino acid sequence identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that it comprises at least one mutation described in any one of claims 1 to 9; or (v) an amino acid sequence identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that it includes cysteine ​​substitutions at the positions corresponding to position 351 and position 380 of SEQ ID NO: 1; or (vi) an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that it includes cysteine ​​substitutions at positions corresponding to position 351 and position 380 of SEQ ID NO: 1, and a mutation at position 272 of SEQ ID NO:

1. A modified GCase polypeptide according to any one of claims 1 to 9.

11. A polynucleotide comprising a modified glucocerebrosidase (GBA) nucleotide sequence encoding a modified GCase polypeptide according to any one of claims 1 to 10.

12. The polynucleotide according to claim 11, wherein the modified GBA nucleotide sequence includes a sequence that is at least 90% identical to the nucleotide sequence described in any one of SEQ ID NOs: 14 to 29.

13. (i) The modified GBA nucleotide sequence includes a sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 18; or (ii) The modified GBA nucleotide sequence includes a sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO:

26. The polynucleotide according to claim 11 or 12.

14. The polynucleotide according to any one of claims 11 to 13, wherein the modified GBA nucleotide sequence includes a sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO:

18.

15. A viral particle comprising a recombinant genome containing a polynucleotide as described in any one of claims 11 to 14.

16. The aforementioned virus particles (i) (I) a viral particle of AAV, adenovirus, or lentivirus, or (II) an AAV viral particle; and / or (ii) comprising a liver-targeting or CNS-targeting capsid; or (iii) comprising a liver-targeting or CNS-targeting capsid, wherein the liver-targeting capsid contains a sequence that is at least 98% identical to sequence number 36, 37, or 38; and / or (iv) a) AAV2 ITR; b) Poly-A sequences; and / or c) Intron This further includes; and / or (v) The recombinant genome is single-stranded; and / or (vi) The effective activity of the modified GCase polypeptide is at least 50% of the effective activity of the GCase polypeptide encoded by the sequence of SEQ ID NO: 14 or 18. The virus particle according to claim 15.

17. (i) The virus particle is an AAV virus particle and comprises a CNS-targeted capsid; (ii) The polynucleotide further comprises a control element; (iii) The modified GBA nucleotide sequence comprises a sequence that is codon-optimized and / or is at least 90% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 18; and (iv) The modified GBA nucleotide sequence includes an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that it includes cysteine ​​substitutions at the positions corresponding to position 351 and position 380 of SEQ ID NO:

1. The virus particle according to claim 15 or 16.

18. (i) The virus particle is an AAV virus particle, (a) Liver-targeting capsid; or (b) A liver-targeting capsid containing a sequence that is 100% identical to sequence number 37. Including; (ii) the polynucleotide further comprises a transcriptional regulatory element, (a) the transcriptional regulatory element comprises a liver-specific promoter, and / or (b) the transcriptional regulatory element comprises a sequence that is 100% identical to SEQ ID NO: 35; and (iii) The modified GBA nucleotide sequence is codon-optimized and / or includes a sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO:

18. The virus particle according to claim 15 or 16.

19. A composition comprising a modified GCase polypeptide, polynucleotide, or virus particle according to any one of claims 1 to 18, and a pharmaceutically acceptable excipient.

20. A modified GCase polypeptide, polynucleotide, virus particle, or composition according to any one of claims 1 to 19 for use in a therapeutic method, (i) The treatment method is a method for treating synucleinopathy; or (ii) The treatment method is a method for treating Parkinson's disease; or (iii) The treatment method is a method for treating Gaucher disease; or (iv) The treatment method is a method for treating Gaucher disease, and the Gaucher disease is type I, type II, or type III Gaucher disease; or (v) The treatment method is a method for treating Gaucher disease, wherein the patient has antibodies or inhibitors against recombinant GCase to which the patient was previously treated as part of enzyme replacement therapy; or (vi) The treatment method is a method for treating Gaucher disease, wherein the patient has antibodies or inhibitors against recombinant GCase to which the patient was previously treated as part of enzyme replacement therapy, and the Gaucher disease is type I, type II, or type III Gaucher disease. Modified GCase polypeptides, polynucleotides, virus particles, or compositions.

21. In subjects suffering from diseases or conditions related to GCase deficiency, (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. The use of a modified GCase polypeptide, polynucleotide, virus particle, or composition according to any one of claims 1 to 19 in the manufacture of a pharmaceutical product for the purpose of, (i) The disease or condition associated with the GCase deficiency is Gaucher disease; or (ii) The disease or condition associated with the GCase deficiency is Gaucher disease, and the Gaucher disease is type I, type II, or type III Gaucher disease; or (iii) The disease or condition associated with the GCase deficiency is Parkinson's disease; or (iv) The disease or condition associated with the GCase deficiency is synucleinopathy.

22. (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages; The use according to claim 21, wherein the use is used to treat a disease or condition related to GCase deficiency, (i) The disease or condition associated with the GCase deficiency is Gaucher disease; or (ii) The disease or condition associated with the GCase deficiency is Gaucher disease, and the Gaucher disease is type I, type II, or type III Gaucher disease; or (iii) The disease or condition associated with the GCase deficiency is Parkinson's disease; or (iv) The disease or condition associated with the GCase deficiency is synucleinopathy.

23. In subjects suffering from diseases or conditions related to GCase deficiency, administration to the subject will result in (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. A modified GCase polypeptide, polynucleotide, virus particle, or composition according to any one of claims 1 to 19, for use in the method, (i) The disease or condition associated with the GCase deficiency is Gaucher disease; or (ii) The disease or condition associated with the GCase deficiency is Gaucher disease, and the Gaucher disease is type I, type II, or type III Gaucher disease; or (iii) The disease or condition associated with the GCase deficiency is Parkinson's disease; or (iv) The disease or condition associated with the GCase deficiency is synucleinopathy. Modified GCase polypeptides, polynucleotides, virus particles, or compositions.

24. (i) Reduce the levels of hexosylsphingosine and / or hexosylceramide; (ii) Reduce the number of storage cells; and / or (iii) Reduce the level of activated macrophages. A modified GCase polypeptide, polynucleotide, viral particle, or composition for use according to claim 23, which treats a disease or condition associated with GCase deficiency, (i) The disease or condition associated with the GCase deficiency is Gaucher disease; or (ii) The disease or condition associated with the GCase deficiency is Gaucher disease, and the Gaucher disease is type I, type II, or type III Gaucher disease; or (iii) The disease or condition associated with the GCase deficiency is Parkinson's disease; or (iv) The disease or condition associated with the GCase deficiency is synucleinopathy. Modified GCase polypeptides, polynucleotides, virus particles, or compositions.

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