Beta-glucocerebrosidase variant for use in the treatment of Gaucher disease

Abstract

A genetically modified human beta-glucocerebrosidase (GCase) is disclosed. The genetically modified GCase comprises an amino acid sequence at least 85% identical to SEQ ID NO:2; contains mutations at positions L34P, K224N / G, T369E, and N370D corresponding to SEQ ID NO:2; and is capable of catalyzing the hydrolysis of the glycolipid glucosylceramide (GlcCer). Pharmaceutical compositions comprising the genetically modified GCase and therapeutic methods using the same are also disclosed.

Description

[Technical Field]

[0001] Related applications This application claims priority to Israeli Patent Application No. 273684, filed on 29 March 2020, and U.S. Provisional Patent Application No. 63 / 049,685, filed on 9 July 2020, the contents of which are incorporated herein by reference as a whole.

[0002] Sequence List An ASCII file consisting of 81,406 bytes, created on March 29, 2021, and titled 86504SequenceListing.txt, which was filed concurrently with the filing of this application, is incorporated herein by reference.

[0003] In some embodiments, the present invention relates to variants of β-glucocerebrosidase (GCase), more specifically, but not exclusively, their use for the treatment of β-glucocerebrosidase deficiencies, including Gaucher disease. [Background technology]

[0004] Lysosomal storage disorders (LSDs) encompass approximately 50 different genetic disorders. They are caused by deficiencies in lysosomal enzymes or transporters, resulting in the accumulation of undegraded metabolites within lysosomes. Among LSDs, Gaucher disease (GD) is the most common, and it is caused by mutations in the GBA1 gene. The GBA1 gene encodes beta-glucocerebrosidase (also called acid beta-glucosidase, D-glucosyl-N-acyl sphingosingle cohydrolase, or GCase), a lysosomal enzyme with glucosylceramidase activity that needs to be cleaved by hydrolysis of the beta-glucosidic bond of glucosylceramide (GlcCer, also called glucocerebroside), an intermediate in glycolipid metabolism. As a result, cells accumulate large amounts of GlcCer and eventually die.

[0005] From a clinical perspective, GD can be divided into three subtypes based on the age of onset and signs of neurological complications. The most common form of the disease, type 1 GD, is characterized by major symptoms of splenic and hepatic dilation, anemia, thrombocytopenia, and skeletal damage. Types 2 and 3 GD, which are neuropathic forms (nGD), are classified according to the time of onset and the rate of progression of neurological symptoms. Type 2, the acute neuropathic form, typically presents with neurological abnormalities before 6 months of age and death occurs between 2 and 4 years of age, referring to children. In type 3, the subacute, chronic neuropathic form, patients present with symptoms similar to those observed in type 2, but with a later onset and greater severity.

[0006] Patients with type 1 GD are typically treated with enzyme replacement therapy (ERT). Ceredase® (alglucerase) (placental-derived product), the first GD-targeted ERT drug, was approved by the FDA in 1991 and has been withdrawn from the market due to the approval of similar drugs produced using recombinant DNA technology, including imiglucerase (Cerezyme®), approved in 1995; veraglucerase alfa (VPRIV®), approved in 2010; and taliglucerase alfa (Elelyso®), approved in 2012. These therapies do not cure GD; that is, they do not correct the underlying gene deficiency. Therefore, symptomatic patients need to continue ERT for life in order to benefit from the treatment.

[0007] In addition to the aforementioned ERT treatment, miglustat (OGT 918, N-butyl-deoxynojirimycin) (Zavesca®), an orally available small molecule drug approved in 2002, provides substrate reduction therapy (SRT) for the treatment of GD. Zavesca® reduces the harmful accumulation of sphingoglycolipids (GSLs) in the body by reducing the amount of GSLs produced by the body. In addition, eliglustat (Cerdelga®), approved in 2014, is also a small molecule used for the treatment of GD. Cerdelga® is thought to work by inhibiting glucosylceramide synthase.

[0008] A retrospective analysis of miglustat in type 1 glandular dysplasia (GD) found that combination therapy may provide better disease regulation in GD patients (by utilizing more than one mechanism of action on glucosylceramide accumulation in cells) and may be cost-effective and lead to an acceptable quality of life by enabling the use of reduced doses of both ERT and miglustat [Machaczka M. et al., Upsala Journal of Medical Sciences (2012) 117, pp. 28-34]. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] PCT / IL2016 / 050812 [Patent Document 2] U.S. Patent No. 4,666,828 [Patent Document 3] U.S. Patent No. 4,683,202 [Patent Document 4] U.S. Patent No. 4,801,531 [Patent Document 5] U.S. Patent No. 5,192,659 [Patent Document 6] U.S. Patent No. 5,272,057 [Patent Document 7] U.S. Patent No. 3,791,932 [Patent Document 8] U.S. Patent No. 3,839,153 [Patent Document 9] U.S. Patent No. 3,850,752 [Patent Document 10] U.S. Patent No. 3,850,578 [Patent Document 11] U.S. Patent No. 3,853,987 [Patent Document 12] U.S. Patent No. 3,867,517 [Patent Document 13] U.S. Patent No. 3,879,262 [Patent Document 14] U.S. Patent No. 3,901,654 [Patent Document 15] U.S. Patent No. 3,935,074 [Patent Document 16] U.S. Patent No. 3,984,533 [Patent Document 17] U.S. Patent No. 3,996,345 [Patent Document 18] U.S. Patent No. 4,034,074 [Patent Document 19] U.S. Patent No. 4,098,876 [Patent Document 20] U.S. Patent No. 4,879,219 [Patent Document 21] U.S. Patent No. 5,011,771 [Patent Document 22] U.S. Patent No. 5,281,521 [Non-patent literature]

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[0011] According to some embodiments of the present invention, a genetically modified human β-glucocerebrosidase (GCase) is provided which (i) has an amino acid sequence that is at least 85% identical to SEQ ID NO: 2; (ii) has mutations at positions L34P, K224N / G, T369E and N370D corresponding to SEQ ID NO: 2; and (iii) has the ability to catalyze the hydrolysis of the glycolipid glucosylceramide (GlcCer).

[0012] According to some embodiments of the present invention, isolated polynucleotides are provided that include nucleic acid sequences encoding genetically modified human GCases of some embodiments of the present invention.

[0013] According to some embodiments of the present invention, a nucleic acid construct is provided comprising an isolated polynucleotide of some embodiment of the present invention and a cis-acting regulatory element for directing the expression of a nucleic acid sequence in a cell.

[0014] According to some embodiments of the present invention, isolated cells comprising a polynucleotide of some embodiment of the present invention or a construct of some embodiment of the present invention are provided.

[0015] According to some embodiments of the present invention, a pharmaceutical composition is provided comprising, as an active ingredient, a genetically modified human GCase of some embodiments of the present invention, an isolated polynucleotide of some embodiments of the present invention, a construct of some embodiments of the present invention, or cells of some embodiments of the present invention, and a pharmaceutically acceptable carrier or diluent.

[0016] According to aspects of certain embodiments of the present invention, a method is provided for treating a disease associated with β-glucocerebrosidase deficiency in a subject requiring such treatment, comprising the steps of administering to the subject a therapeutically effective amount of a genetically modified human GCase of certain embodiments of the present invention, an isolated polynucleotide of certain embodiments of the present invention, a construct of certain embodiments of the present invention, or cells of certain embodiments of the present invention, thereby treating a disease associated with β-glucocerebrosidase deficiency in the subject.

[0017] According to aspects of certain embodiments of the present invention, therapeutically effective amounts of genetically modified human GCase of certain embodiments of the present invention, isolated polynucleotides of certain embodiments of the present invention, constructs of certain embodiments of the present invention, or cells of certain embodiments of the present invention are provided for use in the treatment of diseases associated with β-glucocerebrosidase deficiency in subjects requiring such treatment.

[0018] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the mutations: H145K / R, I204K, E222K, T334F / Y / K and / or L372N, the position corresponding to SEQ ID NO: 2.

[0019] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W and / or H495R, the position corresponding to SEQ ID NO: 2.

[0020] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the mutations:I130T,A168S and / or D263N, the position corresponding to SEQ ID NO: 2.

[0021] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one mutation:R211N and / or K303R, the position of which corresponds to SEQ ID NO: 2.

[0022] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A and / or L420M / I, the position corresponding to SEQ ID NO: 2.

[0023] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the following mutations: V78I, A95K, V191M, A322D, V343T, M361E, S364A, H374W, T410E, H451N and / or L480I, the position corresponding to SEQ ID NO: 2.

[0024] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the mutations: H162K, S181A, T297S, M335F, K346H, S431A, S465D and / or A476D, the position corresponding to SEQ ID NO: 2.

[0025] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the following mutations: R47K, L51R, Q70H, L91I, G115E, A124G, D140N / G, S196T, and / or V437S, the position of which corresponds to SEQ ID NO: 2.

[0026] According to some embodiments of the present invention, the genetically modified human GCase further comprises at least one of the following mutations: T36Q, S38A, Q143E, T183A, L185M, T272S, H274K, N275D, L286S, K293Q, E300R, K321E, V376T, K408R, Q440E, M450Q, and / or I483V, the position corresponding to SEQ ID NO: 2.

[0027] According to some embodiments of the present invention, the amino acids at positions D127, F128, W179, N234, E235, Y244, F246, Q284, Y313, E340, S345, W381, and N396, corresponding to Sequence ID No. 2, remain unchanged.

[0028] According to some embodiments of the present invention, the amino acid sequence is identical to a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 18, 20, 22, and 27.

[0029] According to some embodiments of the present invention, the amino acid sequence is described in Sequence ID No. 14.

[0030] According to some embodiments of the present invention, the amino acid sequence is described in Sequence ID No. 22.

[0031] According to some embodiments of the present invention, the amino acid sequence is described in Sequence ID No. 27.

[0032] According to some embodiments of the present invention, genetically modified human GCase has the ability to catalyze the hydrolysis of the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc).

[0033] According to some embodiments of the present invention, the genetically modified human GCase is at least about 0.2 × 10⁻⁶ 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of GlcCer.

[0034] According to some embodiments of the present invention, the genetically modified human GCase has the ability to catalyze the hydrolysis of GlcCer at at least about 0.5×10 6 k cat / K m (M -1 min -1 ).

[0035] According to some embodiments of the present invention, the genetically modified human GCase has the ability to catalyze the hydrolysis of GlcCer at at least about 1.5×10 6 k cat / K m (M -1 min -1 ).

[0036] According to some embodiments of the present invention, the genetically modified human GCase has thermal stability in a temperature range that is 5 to 20 °C higher compared to the GCase polypeptide under the same conditions.

[0037] According to some embodiments of the present invention, the genetically modified human GCase has thermal stability in a temperature range that is 5 to 20 °C higher compared to the wild-type polypeptide under the same conditions.

[0038] According to some embodiments of the present invention, the genetically modified human GCase has thermal stability in a temperature range that is at least 5 °C higher compared to the wild-type GCase polypeptide under the same conditions.

[0039] According to some embodiments of the present invention, the genetically modified human GCase has thermal stability in a temperature range that is at least 10 °C higher (e.g., at least 1 °C higher) compared to the wild-type GCase polypeptide under the same conditions high).

[0040] According to some embodiments of the present invention, the genetically modified human GCase has thermal stability in a temperature range that is 5 to 20 °C higher compared to the Cerezyme® polypeptide under the same conditions.

[0041] According to some embodiments of the present invention, genetically modified human GCases exhibit thermal stability over a temperature range at least 10°C higher (e.g., at least 11°C higher) compared to Cerezyme® polypeptides under the same conditions.

[0042] According to some embodiments of the present invention, genetically modified human GCases exhibit thermal stability over a temperature range at least 15°C higher (e.g., at least 17°C higher) compared to Cerezyme® polypeptides under the same conditions.

[0043] According to some embodiments of the present invention, genetically modified human GCases exhibit at least twice the intracellular expression level in eukaryotic cells compared to wild-type polypeptides under the same conditions.

[0044] According to some embodiments of the present invention, genetically modified human GCases are secreted from eukaryotic cells compared to wild-type polypeptides that are not secreted under the same culture conditions.

[0045] According to some embodiments of the present invention, the isolated polynucleotide comprises a nucleic acid sequence described in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 17, 19, 21, 23, or 26.

[0046] According to some embodiments of the present invention, the cis-acting control element includes a promoter.

[0047] According to some embodiments of the present invention, a disease associated with β-glucocerebrosidase deficiency is Gaucher disease.

[0048] According to some embodiments of the present invention, the subject is a human being.

[0049] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Similar or equivalent methods and materials may be used in carrying out or testing embodiments of the present invention, but exemplary methods and / or materials are described below. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not necessarily intended to limit the scope of this invention.

[0050] Some embodiments of the present invention are described herein only as examples, with reference to the accompanying drawings. It is emphasized that the specifics shown herein, with particular reference to the detailed drawings, are for illustrative purposes only and for illustrative purposes of the embodiments of the present invention. In this regard, the description provided, together with the drawings, will make it clear to those skilled in the art how embodiments of the present invention can be carried out. [Brief explanation of the drawing]

[0051] [Figure 1A] This table shows the amino acid sequences of the wild type (WT, SEQ ID NO: 2, Cerezyme®, including the single R495H mutation shown by Sanofi Gennzyme) and variants D2-D7 GCase (described in SEQ ID NOs: 4, 6, 8, 10, 12, and 14, respectively). The active site residues of the enzymes are underlined in each sequence. All mutations introduced into the D2-D7 GCase variant sequences by PROSS are highlighted in bold. [Figure 1B] This table shows the amino acid sequences of wild-type (WT) and variant D7 GCase. The complete amino acid sequence of GCase WT is shown in the top column (SEQ ID NO: 2), and the mutation introduced into the D7 sequence by PROSS is shown in the bottom column (SEQ ID NO: 14). [Figure 2]Figure 2A. A schematic diagram of the GCase sequence location within the pCDNA3.1 vector used for GCase expression in HEK293T cells. Figure 2B. A photograph illustrating the sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE) of three elution fractions obtained by purification of GCase isolated from WT and GCase pellets. Note that only the D7 variant produced the secreted enzyme. The arrows indicate the location of the GCase band identified by mass spectrometry (MS). Figure 2C. A photograph illustrating the SDS-PAGE of secreted GCase purified on FLAG beads. Note that only the D7 variant produced the secreted enzyme. The arrows indicate the location of the GCase band identified by mass spectrometry (MS). [Figure 3] Figure 3A. This graph illustrates the size exclusion chromatography (SEC) of D7 GCase after one-step purification using FLAG beads. The protein fraction eluted from FLAG beads was pooled using FLAG peptide and applied to a Superdex200 column. The protein was monitored by absorbance at 280 nm. The fractions corresponding to each peak were collected, concentrated, and analyzed by SDS-PAGE. Note that peak 1 corresponds to monomeric GCase. Figure 3B. This photograph illustrates the size exclusion chromatography (SEC) of D7 GCase after one-step purification using FLAG beads. The protein fraction eluted from FLAG beads was pooled using FLAG peptide and applied to a Superdex200 column. The protein was monitored by absorbance at 280 nm. The fractions corresponding to each peak were collected, concentrated, and analyzed by SDS-PAGE. Note that peak 1 corresponds to monomeric GCase. [Figure 4] This graph illustrates representative Michaelis-Menten plots for WT GCase (triangle, solid line) and D7 GCase (circle, dotted line). The rate of substrate-product conversion (y-axis) is normalized to 1 μg / ml protein. [Figure 5A]This table shows the amino acid sequences of the wild type (WT, SEQ ID NO: 2) and variants D7, D13, D14, and D15 GCases (described in SEQ ID NOs: 14, 18, 20, and 22, respectively). The active site residues of the enzymes are underlined in each sequence. All mutations introduced into the sequences of the D7, D13, D14, and D15 GCase variants by PROSS are highlighted in bold. [Figure 5B] This table illustrates a comparison of the amino acid sequences of the wild-type GCase (top sequence, shown in SEQ ID NO: 2) and the PROSS-designed variant D15 GCase (bottom sequence, shown in SEQ ID NO: 22). Mutated amino acids are highlighted in bold, and amino acids corresponding to the enzyme catalytic site are underlined. [Figure 6] This graph illustrates the specific activity of variant D15 GCase (white circles) and Cerezyme® (black circles) as predicted using p-NP-Glc as a substrate. The activity was determined at substrate concentrations of 0.4, 1.5, and 3 mM p-Np-Glc. [Figure 7] This table shows the amino acid sequences of the wild type (WT, SEQ ID NO: 2, Cerezyme®, including the single R495H mutation presented by Sanofi Genzyme), the wild type (WT, SEQ ID NO: 25), and variants D7, D13, D14, D15, and D16 GCase (described in SEQ ID NOs: 14, 18, 20, 22, and 27, respectively). The active site residues of the enzymes are underlined in each sequence. All mutations introduced into the sequences of D7, D13, D14, D15, and D16 GCase variants by PROSS are highlighted in bold. [Modes for carrying out the invention]

[0052] In some embodiments, the present invention relates to variants of β-glucocerebrosidase (GCase), more specifically, but not exclusively, to the use thereof for the treatment of β-glucocerebrosidase deficiencies, including Gaucher disease (GD).

[0053] The principles and operation of the present invention can be better understood by referring to the drawings and accompanying description.

[0054] Before describing in detail at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited to its application to the details described or illustrated by the examples below. The present invention may be in other embodiments or may be implemented or carried out in various ways. It should also be understood that the technical terms and terminology used herein are for illustrative purposes only and should not be considered limiting.

[0055] GD is a hereditary lysosomal storage disorder caused by a functional deficiency of β-glucocerebrosidase (GCase), resulting in dysfunction of multiple organs. GCase catalyzes the hydrolysis of glucocerebroside to ceramide and glucose. In GD, the enzyme deficiency leads to the accumulation of excessive glucocerebroside in the lysosomal compartment of Gaucher cells (tissue macrophages), as well as the accumulation of these cells in visceral tissues (liver, spleen, and bone marrow).

[0056] Patients with GD are typically treated with enzyme replacement therapy (ERT). These enzymes are commercially available for the treatment of GD with ERT. These include imiglucerase (Cerezyme®), veraglucerase alpha (VPRIV®), and taliglucerase alpha (Elelyso®). One drawback associated with current ERT treatment is the undesirably low in vivo physiological activity of the enzymes. This is due, for example, to low thermal stability, low uptake, reduced targeting to lysosomes in certain cells where the substrate accumulates, and / or a short functional in vivo half-life in lysosomes.

[0057] While reducing the requirements for carrying out the present invention, the inventors have generated novel GCases for Gaucher disease ERT that maintain enzyme activity (compared to wild-type human GCase) while exhibiting improved properties, namely, higher expression levels (compared to wild-type human GCase) and higher thermal stability (compared to wild-type human GCase and / or Cerezyme®). Specifically, the inventors generated six novel GCase polypeptide variants by using the PROSS algorithm described, for example, PCT / IL2016 / 050812 and Goldenzweig A. et al., Mol. Cell. (2016) 63: pp. 337-346, which are incorporated herein by reference (designs 2-7, i.e., D2-7, described in SEQ ID NOs: 4, 6, 8, 10, 12, and 14, respectively; see Figure 1A). Four of these GCase variants, D2, D4, D6, and D7, were expressed in Escherichia coli (E. coli) and demonstrated enzymatic activity toward the synthetic substrate p-NP-Glc (data not shown). Recombinant human glucosylceramidase (WT human GCase described in SEQ ID NO: 2) and the D7 variant with 30 mutations (described in SEQ ID NO: 14) were further expressed in human embryonic kidney cells (HEK293T cells) capable of glycosylation of proteins. As explained in Figures 2B-2C, D7 GCase showed higher intracellular expression levels compared to WT hGCase, while only D7 GCase was secreted from HEK293T cells. Regarding thermal stability, D7 GCase showed higher thermal stability up to approximately 11°C and 20°C compared to Cerezyme® at pH 6.1 and pH 7.4, respectively, and up to approximately 7°C compared to wild-type human GCase at pH 6.1 (see Table 1 below). Regarding enzyme activity, D7 GCase and WT hGCase exhibited similar k cat / K mThe values ​​were obtained (see Table 2 below). In summary, the novel GCase variants (D2-7 GCases) were conferred with higher expression levels and greater thermal stability compared to wild-type human GCases while maintaining enzyme activity, and with greater thermal stability compared to Cerezyme®. Therefore, they offer promising new potential for use in enzyme replacement therapy (ERT) for the treatment of GD.

[0058] The inventors further generated four novel GCase polypeptide variants using the PROSS algorithm (designs 13-16, i.e., D13-16, described in SEQ ID NOs. 18, 20, 22, and 27, respectively). GCase variants D13, D14, D15, and D16 were expressed in HEK293T cells, isolated from culture medium, and tested for enzyme activity using a fluorescently labeled GCase analog (NBD glucosylceramide (d18:1 / 6:0) (C6NBD GlcCer)). The designs with the highest enzyme activity, i.e., variants D15 and D16 GCase, were used for further characterization. As evidenced from Examples 6 and 9 below, variants D15 and D16 GCase exhibited higher thermal stability up to 17-20°C compared to Cerezyme® at pH 6.1. Furthermore, the enzyme activity of GCase, determined by both its natural substrate (C6NBD GlcCer) and synthetic substrate (p-NP-Glc), was equivalent to that of Cerezyme® and variants D15 and D16 (see Examples 7 and 10 below).

[0059] Accordingly, according to one aspect of the present invention, a genetically modified human β-glucocerebrosidase (GCase) is provided which (i) has an amino acid sequence that is at least 85% identical to SEQ ID NO: 2; (ii) has mutations at positions L34P, K224N / G, T369E and N370D corresponding to SEQ ID NO: 2; and (iii) has the ability to catalyze the hydrolysis of the glycolipid glucosylceramide (GlcCer).

[0060] As used herein, the terms “beta-glucocerebrosidase” or “glucocerebrosidase” (EC 3.2.1.45), also referred to as glucosylceramidase, acid beta-glucosidase, D-glucosyl-N-acylsphingosinglecohydrolase, GCase or GBA, refer to an enzyme having glucosylceramidase activity. The β-glucocerebrosidase (GCase) of this embodiment of the present invention is a human GCase that catalyzes the hydrolysis of glucosylceramide / GlcCer (an intermediate in glycolipid metabolism) to ceramide and glucose.

[0061] According to one embodiment, the protein to be modified includes the sequence described in Sequence ID No. 2.

[0062] According to one embodiment, the protein to be modified includes the sequence described in Sequence ID No. 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0063] It will be understood that the sequence described in Sequence ID No. 2 contains one modification at amino acid position 495 compared to human wild-type GCase; namely, the arginine (R) at position 495 of human wild-type GCase is replaced with histidine (H), resulting in Sequence ID No. 2. The enzymatic activity of GCase is not affected by this modification.

[0064] According to a particular embodiment, the sequence of the human GCase protein is described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)). The mutation sites between SEQ ID NO: 2 and SEQ ID NO: 25 (Human) are identical.

[0065] As used herein, the term “catalytic domain” of GCase refers to the amino acid residues involved in catalyzing the hydrolysis of glucosylceramide / GlcCer. The 3D structure of the catalytic domain forms the active site, and therefore GCase must be correctly folded to be active. For example, the catalytic domain of GCase includes amino acid positions D127, F128, W179, N234, E235, Y244, F246, Q284, Y313, E340, S345, W381, N396 of SEQ ID NO: 2 or SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0066] As used herein, the term “glycosylation site” refers to the asparagine residue of a GCase to which a sugar side chain is post-translationally bound, and whose presence enhances the activity of the enzyme. In human GCase, there are five candidate sites: N19, N59, N146, N270, and N462. N462 is typically unoccupied. Berg-Fussman and collaborators (Berg-Fussman, Grace, Ionnou & Grabowski

[1993] J Biol Chem 268:14861-14866) have already shown that when these asparagines are mutated to glutamine, thereby preventing glycosylation, GCase activity is significantly reduced, but not completely. While the sugar side chains differ (in the various recombinant forms expressed), the proximal sugar is an N-acetylglucosamine moiety to which several mannose residues are bound.

[0067] According to one embodiment, a genetically modified human GCase (also referred to as a variant or polypeptide) contains an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0068] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 84% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0069] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0070] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 86% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0071] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 88% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0072] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0073] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0074] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0075] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 97% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0076] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0077] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 99% identical to SEQ ID NO: 2 or SEQ ID NO: 25.

[0078] Homology (e.g., percent homology, sequence identity + sequence similarity) can be determined using any homology comparison software or computer-operated sequence alignment.

[0079] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes a reference to residues in the two sequences that are identical when aligned. When the percentage of sequence identity is used in reference to a protein, non-identical residue positions are often recognized as differing by conserved amino acid substitutions, where the amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and therefore does not change the functional properties of the molecule. If sequences differ by conserved substitutions, the percentage of sequence identity may be adjusted upward to correct the conservative nature of the substitutions. Sequences that differ by such conservative substitutions are considered to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as parts rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if identical amino acids are assigned a score of 1 and non-conservative substitutions are assigned a score of 0, then conservative substitutions are assigned scores between 0 and 1. The scoring of conservative substitutions is calculated according to the algorithm described, for example, Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 1992, 89(22): 10915-9].

[0080] Identity (e.g., percent homology) can be determined using any homology comparison software, including, for example, the BlastN software from the National Center of Biotechnology Information (NCBI), by using default parameters.

[0081] According to certain embodiments of the present invention, identity is general identity, i.e., identity over the entire amino acid or nucleic acid sequence of the present invention, and not identity over a portion thereof.

[0082] According to some embodiments of the present invention, the term “homology” or “homologous” means the identity of two or more nucleic acid sequences; or the identity of two or more amino acid sequences; or the identity of one or more nucleic acid sequences with respect to an amino acid sequence.

[0083] According to some embodiments of the present invention, homology is general homology, that is, homology over the entire amino acid or nucleic acid sequence of the present invention, and not homology over a portion thereof.

[0084] The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools. The following is a non-limiting description of such tools that can be used in conjunction with some embodiments of the present invention.

[0085] Pair general alignment is defined by S.B. Needleman and CD Wunsch, "A general method applicable to the search of similarities in the amino acid sequence of two proteins," Journal of Molecular Biology, 1970, pp. 443-453, Vol. 48.

[0086] When starting with polypeptide sequences and comparing them to polynucleotide sequences, the OneModel FramePlus algorithm [Halperin, E., Faigler, S., and Gill-More, R. (1999) - FramePlus: aligning DNA to protein sequences. Bioinformatics, 15, pp. 867-873] (available from biocceleration.com / Products.html) can be used.

[0087] When starting with a polynucleotide sequence and comparing it to other polynucleotide sequences, the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available from emboss.sourceforge.net / apps / cvs / emboss / apps / needle.html) can be used.

[0088] According to some embodiments, determining the degree of homology further requires the use of the Smith-Waterman algorithm (for comparing proteins or nucleotides).

[0089] According to certain embodiments of the present invention, general homology is performed on sequences pre-selected by local homology (e.g., 60% identity over 60% of the sequence length) to the target polypeptide or polynucleotide before performing general homology (e.g., 80% general homology across the entire sequence) to the target polypeptide or polynucleotide. For example, homologous sequences are selected using BLAST software, with the Blastp and tBlastn algorithms as first-stage filters and Needle (EMBOSS package) or Frame+ algorithm alignment for the second stage. Local identity (Blast alignment) is used only as a filter in the general alignment stage and is therefore defined using a very generous cutoff of -60% identity over a 60% span of the sequence length. In this particular embodiment (when local identity is used), the default filtering of the Blast package is not utilized (by setting the parameter "-FF"). In the second stage, homologs are defined based on at least 80% general identity to the core gene polypeptide sequence.

[0090] According to some embodiments of the present invention, the GCase polypeptide has a length of 470 to 520 amino acids.

[0091] According to some embodiments of the present invention, the GCase polypeptide has a length of 480 to 510 amino acids.

[0092] According to some embodiments of the present invention, the GCase polypeptide has a length of 490 to 510 amino acids.

[0093] According to some embodiments of the present invention, the GCase polypeptide has a length of 495 to 500 amino acids.

[0094] According to a particular embodiment, the GCase polypeptide comprises an amino acid sequence including amino acid residues 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, or 505.

[0095] According to a particular embodiment, the GCase polypeptide comprises an amino acid sequence containing 497 amino acid residues.

[0096] As used herein, the term “polypeptide” encompasses modifications that enable higher expression, greater stability in both in vitro and in vivo, in animal or human bodies, and greater cell penetration, compared to the natural GCase sequence, i.e., SEQ ID NO: 2 or SEQ ID NO: 25.

[0097] Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, polypeptide linkage modifications, skeletal modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin, Pergamon Press (1992), which are incorporated herein by reference as if they were fully described. Further details relating thereto are provided herein below.

[0098] The term "isolated" refers to being separated, at least partially, from a natural environment, such as the human body. According to one embodiment, an isolated polypeptide is essentially free from contaminating cellular components, such as carbohydrates, lipids, or other proteinaceous impurities whose properties are related to the polypeptide. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or, instead, glycosylated or derivatized forms.

[0099] The term “amino acid (one or more)” is understood to include 20 naturally occurring amino acids; for example, those amino acids that are often modified post-translation in vivo, including hydroxyproline, phosphoserine, and osphosreonine; as well as other non-ordinary amino acids, including but not limited to 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

[0100] According to one embodiment, an amino acid is a "homogeneous amino acid residue." A homogeneous amino acid residue refers to an amino acid residue that has the ability to substitute another amino acid residue in a polypeptide without substantially changing the structure and / or functionality of the polypeptide (e.g., the ability to catalyze the hydrolysis of glucosylceramide / GlcCer). Therefore, homogeneous amino acids have similar properties such as side chain bulk, side chain polarity (polar or nonpolar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic), and carbon molecule side chain organization (aromatic / aliphatic). Thus, a "homogeneous amino acid residue" can be considered a "conservative amino acid substitution."

[0101] In the sense of the term "equal amino acid substitution," an amino acid may be substituted with another amino acid within the group of amino acids shown below in this specification: i) Amino acids with polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, Cys); ii) Amino acids with nonpolar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, Met); iii) Amino acids with nonpolar aliphatic side chains (Gly, Ala, Val, Leu, Ile); iv) Amino acids with cyclic side chains (Phe, Tyr, Trp, His, Pro); v) Amino acids with aromatic side chains (Phe, Tyr, Trp); vi) Amino acids with acidic side chains (Asp, Glu); vii) Amino acids with basic side chains (Lys, Arg, His); viii) Amino acids with amide side chains (Asn, Gln); ix) Amino acids with hydroxyl side chains (Ser, Thr); x) Amino acids with sulfur-containing side chains (Cys, Met); xi) Mesozoic, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr); xii) Hydrophilic amino acids (Arg, Asn, Asp, Glu, Gln, His, Lys, Ser, Thr, Tyr); and xiii) Hydrophobic amino acids (Ala, Cys, Gly, Ile, Leu, Met, Phe, Pro, Trp, Val) xiv) Charged amino acids (Arg, Lys, Asp, Glu).

[0102] Since the polypeptide is used in therapeutic agents that require the peptide to be in a soluble form, the polypeptides of some embodiments of the present invention preferably contain one or more non-natural or natural polar amino acids, including but not limited to serine, which have the ability to increase the solubility of the polypeptide due to their hydroxyl-containing side chains.

[0103] According to a particular embodiment, the amino acid sequence of the GCase variant includes mutations, such as substitutions, compared to SEQ ID NO: 2.

[0104] According to a particular embodiment, the amino acid sequence of the GCase variant includes mutations, such as substitutions, compared to SEQ ID NO: 25.

[0105] According to one embodiment, the mutation is located on sequence number 25.

[0106] Polypeptides of some embodiments of the present invention may include mutations described herein, provided that the modified region is not part of the catalytic domain and does not, for example, form an active site (as discussed above), does not alter the 3D structure of the catalytic domain, or is a glycosylation site (as discussed above), i.e., part of SEQ ID NO: 2 or SEQ ID NO: 25.

[0107] According to one embodiment, the GCase polypeptide has an amino acid sequence in the sequence described in SEQ ID NO: 2 or SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)) of 4-80, 4-75, 4-70, 4-60, 4-50, 4-45, 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 4-10, 4-5, 6-80, 6-75, 6-70, 6-60, 6-50, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-10, 9-80, 9-75, 9-70, 9-60, 9-50, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-10, 12-80, 12-75, 12-70, 12-60, 12-50, 12-40, 12-35, 12-30, 12-25, 12-20, 12-15, 16-80, 16-75, 16-70, 16-60, 16-50, 16-40, 16-35, 16-30, 16-25, 16-20, 19-80, 19-75, 19-70, 19-60, 19-50, 19-40, 19-35, 19-30, 19-25, 19-20, 21-80, 21-75, 21-70, 21-60, 21-50, 21-45, 21-40, 21-35, 21-30, 21-2 Contains 5, 25-80, 25-75, 25-70, 25-60, 25-50, 25-45, 25-40, 25-35, 25-30, 30-80, 30-75, 30-70, 30-60, 30-50, 30-45, 30-40, 30-35, 40-80, 40-70, 40-60, 40-50, 50-80, 50-70, 50-55, 55-60, 60-70, or 70-80 mutations.

[0108] According to one embodiment, the GCase polypeptide has an amino acid sequence in the sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, Contains 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 mutations.

[0109] According to a particular embodiment, the GCase polypeptide contains four mutations in the amino acid sequence described in Sequence ID No. 2.

[0110] According to a particular embodiment, the GCase polypeptide contains nine mutations in the amino acid sequence described in SEQ ID NO: 2.

[0111] According to a particular embodiment, the GCase polypeptide contains 16 mutations in the amino acid sequence described in SEQ ID NO: 2.

[0112] According to a particular embodiment, the GCase polypeptide contains 15 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0113] According to a particular embodiment, the GCase polypeptide contains 19 mutations in the amino acid sequence described in Sequence ID No. 2.

[0114] According to a particular embodiment, the GCase polypeptide contains 18 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0115] According to a particular embodiment, the GCase polypeptide contains 21 mutations in the amino acid sequence described in Sequence ID No. 2.

[0116] According to a particular embodiment, the GCase polypeptide contains 20 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0117] According to a particular embodiment, the GCase polypeptide contains 30 mutations in the amino acid sequence described in Sequence ID No. 2.

[0118] According to a particular embodiment, the GCase polypeptide contains 29 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0119] According to a particular embodiment, the GCase polypeptide contains 36 mutations in the amino acid sequence described in SEQ ID NO: 2.

[0120] According to a particular embodiment, the GCase polypeptide contains 35 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0121] According to a particular embodiment, the GCase polypeptide contains 46 mutations in the amino acid sequence described in SEQ ID NO: 2.

[0122] According to a particular embodiment, the GCase polypeptide contains 45 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0123] According to a particular embodiment, the GCase polypeptide contains 56 mutations in the amino acid sequence described in SEQ ID NO: 2.

[0124] According to a particular embodiment, the GCase polypeptide contains 55 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0125] According to a particular embodiment, the GCase polypeptide contains 73 mutations in the amino acid sequence described in SEQ ID NO: 2.

[0126] According to a particular embodiment, the GCase polypeptide contains 72 mutations in the amino acid sequence described in SEQ ID NO: 25 (UniProtKB-P04062(GLCM_HUMAN)).

[0127] As discussed above, the GCase polypeptides of some embodiments of the present invention include mutants L34P, K224N / G, T369E, and N370D, whose positions correspond to SEQ ID NO: 2. The amino acid positions can be fitted by a skilled worker through amino acid sequence alignment, which may be performed manually or using specific bioinformatics tools such as FASTA, L-ALIGN, and Protein Blast.

[0128] According to one embodiment, the GCase polypeptide further comprises at least one of the mutations: H145K / R, I204K, E222K, T334F / Y / K, or L372N, the position corresponding to SEQ ID NO: 2.

[0129] According to one embodiment, the GCase polypeptide further comprises two of the mutations: H145K / R, I204K, E222K, T334F / Y / K, or L372N, with the positions corresponding to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide may further comprise the mutations: H145K / R and I204K; H145K / R and E222K; H145K / R and T334F / Y / K; H145K / R and L372N; I204K and E222K; I204K and T334F / Y / K; I204K and L372N; E222K and T334F / Y / K; E222K and L372N; or T334F / Y / K and L372N, with the positions corresponding to SEQ ID NO: 2.

[0130] According to one embodiment, the GCase polypeptide further comprises three of the following mutations: H145K / R, I204K, E222K, T334F / Y / K, or L372N, the positions corresponding to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide may further contain the mutations: H145K / R, I204K and E222K; H145K / R, I204K and T334F / Y / K; H145K / R, I204K and L372N; H145K / R, E222K and T334F / Y / K; H145K / R, E222K and L372N; H145K / R, T334F / Y / K and L372N; I204K, E222K and T334F / Y / K; I204K, E222K and L372N; I204K, T334F / Y / K and L372N; or E222K, T334F / Y / K and L372N, where the position corresponds to SEQ ID NO: 2.

[0131] According to one embodiment, the GCase polypeptide further comprises four of the mutations: H145K / R, I204K, E222K, T334F / Y / K, or L372N, with the positions corresponding to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide may further comprise the mutations: I204K, E222K, T334F / Y / K, and L372N; H145K / R, E222K, T334F / Y / K, and L372N; H145K / R, I204K, T334F / Y / K, and L372N; H145K / R, I204K, E222K, and L372N; or H145K / R, I204K, E222K, and T334F / Y / K, with the positions corresponding to SEQ ID NO: 2.

[0132] According to one embodiment, the GCase polypeptide further comprises all of the mutations: H145K / R, I204K, E222K, T334F / Y / K and L372N, whose positions correspond to SEQ ID NO: 2.

[0133] According to one embodiment, the GCase polypeptide includes all of the mutations: L34P, H145K / R, I204K, E222K, K224N / G, T334F / Y / K, T369E, N370D, and L372N, and their positions correspond to SEQ ID NO: 2.

[0134] According to one embodiment, the GCase polypeptide further comprises at least one of the mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W, or H495R, the position corresponding to SEQ ID NO: 2.

[0135] According to a particular embodiment, the H495R modification in SEQ ID NO: 2 reverses the single arginine (R) in SEQ ID NO: 2 to the histidine (H) modification in SEQ ID NO: 2 (i.e., it returns to the WT sequence described in SEQ ID NO: 25).

[0136] According to one embodiment, the GCase polypeptide further comprises two of the mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W, or H495R, the positions of which correspond to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide may contain the mutations: N102D / E and L165Q; N102D / E and Q226T; N102D / E and L241I; N102D / E and S242P; N102D / E and K473W; N102D / E and H495R; L165Q and Q226T; L165Q and L241I; L165Q and S242P; L165Q and K473W; It may further include L165Q and H495R; Q226T and L241I; Q226T and S242P; Q226T and K473W; Q226T and H495R; L241I and S242P; L241I and K473W; L241I and H495R; S242P and K473W; S242P and H495R; or K473W and H495R, the position corresponding to sequence number 2.

[0137] According to one embodiment, the GCase polypeptide further comprises three of the following mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W, or H495R, the positions of which correspond to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide is: N102D / E, L165Q and Q226T; N102D / E, L165Q and L241I; N102D / E, L165Q and S242P; N102D / E, L165Q and K473W; N102D / E, L165Q and H495R; N102D / E, Q226T and L241I; N102D / E, Q226T and S242P; N102 D / E, Q226T and K473W; N102D / E, Q226T and H495R; N102D / E, L241I and S242P; N102D / E, L241I and K473W; N102D / E, L241I and H495R; N102D / E, S242P and K473W; N102D / E, S242P and H495R; N102D / E, K473W and H495R; L165Q, Q226T and L241I; L165Q, Q226T and S242P; L165Q, Q226T and K473W; L165Q, Q226T and H495R; L165Q, L241I and S242P; L165Q, L241I and K473W; L165Q, L241I and H495R; L165Q, S242P and K473W; L165Q, S242P and H495R; L165Q, K473W and H495R; Q226T, L241I and S242P; Q226T, L241I and K473W; Q226T, L241I and H495R; Q226T, S242P and K473W; Q226T, S242P and H495R; L241I, S242P and K473W; L241I, S242P and H495R; or S242P, K473W and H495R may further be included, with their positions corresponding to sequence number 2.

[0138] According to one embodiment, the GCase polypeptide further comprises four of the following mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W, or H495R, the positions of which correspond to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide is: mutations: N102D / E, L165Q, Q226T and L241I; N102D / E, L165Q, Q226T and S242P; N102D / E, L165Q, Q226T and K473W; N102D / E, L165Q, Q226T and H495R; N1 02D / E, Q226T, L241I and S242P; N102D / E, Q226T, L241I and K473W; N102D / E, Q226T, L241I and H495R; N102D / E, L241I, S242P and K473W; N102D / E, L241I, S242P and H495R; N102 D / E, S242P, K473W and H495R; L165Q, Q226T, L241I and S242P; L165Q, Q226T, L241I and K473W; L165Q, Q226T, L241I and H495R; L165Q, L241I, S242P and K473W; L165Q, L241I, S2 42P and H495R; L165Q, S242P, K473W and H495R; Q226T, L241I, S242P and K473W; Q226T, L241I, S242P and H495R; or L241I, S242P, K473W and H495R may further be included, with their positions corresponding to sequence number 2.

[0139] According to one embodiment, the GCase polypeptide further comprises five of the following mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W, or H495R, the positions of which correspond to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide may contain the following mutations: N102D / E, Q226T, L241I, S242P, and K473W; N102D / E, Q226T, L241I, S242P, and H495R; N102D / E, Q226T, L241I, K473W, and H495R; N102D / E, L241I, S2 42P, K473W and H495R; N102D / E, L165Q, S242P, K473W and H495R; N102D / E, L165Q, L241I, K473W and H495R; N102D / E, L165Q, L241I, S242P and H495R; N102D / E, L165Q, L241I, S242P and K473W; N102D / E, L165Q, Q226T, K473W and H495R;N102D / E, L165Q, Q226T, S242P and H495R;N102D, L165Q, Q226T, S242P and K473W;N102D, L165Q, Q226T, L241I and H495R;N102D / E, L165Q, Q226T, L241I and K473W;Q226T, L241I, S242P, K473W and H495R;L165Q, L 241I, S242P, K473W and H495R; L165Q, Q226T, S242P, K473W and H495R; L165Q, Q226T, L241I, K473W and H495R; L165Q, Q226T, L241I, S242P and H495R; or L165Q, Q226T, L241I, S242P and K473W may further be included, the position of which corresponds to sequence number 2.

[0140] According to one embodiment, the GCase polypeptide further comprises six of the mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W, or H495R, the positions of which correspond to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide may contain the mutations: N102D / E, Q226T, L241I, S242P, K473W, and H495R; N102D / E, L165Q, Q226T, S242P, K473W, and H495R; N102D / E, L165Q, Q226 T, L241I, K473W and H495R; N102D / E, L165Q, Q226T, L241I, S242P and H495R; N102D / E, L165Q, Q226T, L241I, S242P and K473W; or L165Q, Q226T, L241I, S242P, K473W and H495R may further be included, with their positions corresponding to sequence number 2.

[0141] According to one embodiment, the GCase polypeptide further comprises all of the mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W, and H495R, whose positions correspond to SEQ ID NO: 2.

[0142] According to one embodiment, the GCase polypeptide includes all of the mutations: L34P, N102D / E, H145K / R, L165Q, I204K, E222K, K224N / G, Q226T, L241I, S242P, T334F / Y / K, T369E, N370D, L372N, K473W, and H495R, and their positions correspond to SEQ ID NO: 2.

[0143] According to one embodiment, the GCase polypeptide further comprises at least one of the mutations:I130T,A168S, orD263N, the position corresponding to SEQ ID NO: 2.

[0144] According to one embodiment, the GCase polypeptide further comprises two of the mutations: I130T, A168S, or D263N, with the positions corresponding to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide further comprises the mutations: I130T and A168S; I130T and D263N; or A168S and D263N, with the positions corresponding to SEQ ID NO: 2.

[0145] According to one embodiment, the GCase polypeptide further comprises all of the mutations: I130T, A168S and D263N, whose positions correspond to SEQ ID NO: 2.

[0146] According to one embodiment, the GCase polypeptide includes all of the mutations: L34P, N102D / E, I130T, H145K / R, L165Q, A168S, I204K, E222K, K224N / G, Q226T, L241I, S242P, D263N, T334F / Y / K, T369E, N370D, L372N, K473W, and H495R, and their positions correspond to SEQ ID NO: 2.

[0147] According to one embodiment, the GCase polypeptide further comprises at least one mutation: R211N or K303R, the position corresponding to SEQ ID NO: 2.

[0148] According to one embodiment, the GCase polypeptide further comprises both mutations:R211N and K303R, the positions corresponding to SEQ ID NO: 2.

[0149] According to one embodiment, the GCase polypeptide includes all of the mutations: L34P, N102D / E, I130T, H145K / R, L165Q, A168S, I204K, R211N, E222K, K224N / G, Q226T, L241I, S242P, D263N, K303R, T334F / Y / K, T369E, N370D, L372N, K473W, and H495R, and their positions correspond to SEQ ID NO: 2.

[0150] According to one embodiment, the GCase polypeptide further comprises at least one of the mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, the position corresponding to SEQ ID NO: 2.

[0151] According to one embodiment, the GCase polypeptide further comprises two of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, where the position corresponds to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide comprises the following mutations: H60W and L103N / E / R; H60W and Q166A; H60W and H274R; H60W and N333D; H60W and N386D; H60W and R395K; H60W and I406T / A; H60W and L420M / I; L103N / E / R and Q16 6A;L103N / E / R and H274R;L103N / E / R and N333D;L103N / E / R and N386D;L103N / E / R and R395K;L103N / E / R and I406T / A;L103N / E / R and L420M / I;Q166A and H274R;Q166A and N333D;Q166A and N3 86D;Q166A and R395K;Q166A and I406T / A;Q166A and L420M / I;H274R and N333D;H274R and N386D;H274R and R395K;H274R and I406T / A;H274R and L420M / I;N333D and N386D;N333D and R395K;N3 It may further include 33D and I406T / A; N333D and L420M / I; N386D and R395K; N386D and I406T / A; N386D and L420M / I; R395K and I406T / A; R395K and L420M / I; or I406T / A and L420M / I, where the position corresponds to sequence number 2.

[0152] According to one embodiment, the GCase polypeptide further comprises three of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, the position corresponding to SEQ ID NO: 2. Therefore, for example, GCase polypeptide has the following mutations: H60W, L103N / E / R and Q166A; H60W, L103N / E / R and H274R; H60W, L103N / E / R and N333D; H60W, L103N / E / R and N386D; H60W, L103N / E / R and R395K; H60W, L103N / E / R and I406T / A; H60W, L103N / E / R and L420M / I; H60W, Q166A and H274R; H60W, Q166A and N33 3D;H60W, Q166A and N386D;H60W, Q166A and R395K;H60W, Q166A and I406T / A;H60W, Q166A and L420M / I;H60W, H274R and N333D;H60W, H274R and N386D;H60W, H274R and R395K;H60W, H274R and I406T / A;H60W, H274R and L420M / I;H60W, N333D and N386D;H60W, N333D and R395K;H60W, N333 D and I406T / A; H60W, N333D and L420M / I; H60W, N386D and R395K; H60W, N386D and I406T / A; H60W, N386D and L420M / I; H60W, R395K and I406T / A; H60W, R395K and L420M / I; H60W, I406T / A and L420M / I; L103N / E / R, Q166A and H274R; L103N / E / R, Q166A and N333D; L103N / E / R, Q166A and N38 6D;L103N / E / R, Q166A and R395K;L103N / E / R, Q166A and I406T / A;L103N / E / R, Q166A and L420M / I;L103N / E / R, H274R and N333D;L103N / E / R, H274R and N386D;L103N / E / R, H274R and R395K;L103N / E / R, H274R and I406T / A;L103N / E / R, H274R and L420M / I;L103N / E / R, N333D and N386D;L103N / E / R, N333D and R395K; L103N / E / R, N333D and I406T / A; L103N / E / R, N333D and L420M / I; L103N / E / R, N386D and R395K; L103N / E / R, N386D and I406T / A; L103N / E / R, N386D and L420M / I; L103N / E / R, R395K and I406T / A; L103N / E / R, R395K and L420M / I; L103N / E / R, I406T / A and L420M / I; Q166A, H274R and N 333D;Q166A, H274R and N386D;Q166A, H274R and R395K;Q166A, H274R and I406T / A;Q166A, H274R and L420M / I;Q166A, N333D and N386D;Q166A, N333D and R395K;Q166A, N333D and I406T / A;Q166A, N333D and L420M / I;Q166A, N386D and R395K;Q166A, N386D and I406T / A;Q166A, N386D and L420M / I;Q166A, R395K and I406T / A;Q166A, R395K and L420M / I;H274R, N333D and N386D;H274R, N333D and R395K;H274R, N333D and I406T / A;H274R, N333D and L420M / I;H274R, N386D and R395K;H274R, N386D and I406T / A;H274R, N386D and L420M / I;H274R, R395K and I406T / A;H274R, R395K and L420M / I;H274R, I406T / A and L420M / I;N3 33D, N386D and R395K; N333D, N386D and I406T / A; N333D, N386D and L420M / I; N333D, R395K and I406T / A; N333D, R395K and L420M / I; N333D, I406T / A and L420M / I; N386D, R395K and I406T / A; N386D, R395K and L420M / I; N386D, I406T / A and L420M / I; or R395K, I406T / A and L420M / I may further be included, with the position corresponding to sequence number 2.

[0153] According to one embodiment, the GCase polypeptide further comprises four of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, the positions corresponding to SEQ ID NO: 2. Therefore, for example, GCase polypeptides include mutations: H60W, L103N / E / R, Q166A and H274R; H60W, L103N / E / R, Q166A and N333D; H60W, L103N / E / R, Q166A and N386D; H60W, L103N / E / R, Q166A and R395K; H60W, L103N / E / R, Q166A and I406T / A; H60W, L103N / E / R, Q166A and L420M / I; H60W, Q166A, H274R and N333D; H60W, Q166A, H274R and N386D; H60W, Q166A, H274R and R395K; H60W, Q166A, H274R and I406T / A; H60W, Q166A, H274R and L420M / I; H60W, Q166A, N333D and N386D; H60W, Q166A, N333D and R395K; H60W, Q166A, N333D and I406T / A; H60W, Q166A, N333D and L420M / I; H60W, Q166 A, N386D and R395K; H60W, Q166A, N386D and I406T / A; H60W, Q166A, N386D and L420M / I; H60W, Q166A, R395K and I406T / A; H60W, Q166A, R395K and L420M / I; H60W, Q166A, I406T / A and L420M / I; H60W, H274R, N333D and N386D; H60W, H274R, N333D and R395K; H60W, H274R, N333D and I40 6T / A; H60W, H274R, N333D and L420M / I; H60W, N333D, N386D and R395K; H60W, N333D, N386D and I406T / A; H60W, N333D, N386D and L420M / I; H60W, N386D, R395K and I406T / A; H60W, N386D, R395K and L420M / I; H60W, R395K, I406T / A and L420M / I; L103N / E / R, Q166A, H274R and N333D;L103N / E / R, Q166A, H274R and N386D; L103N / E / R, Q166A, H274R and R395K; L103N / E / R, Q166A, H274R and I406T / A; L103N / E / R, Q166A, H274R and L420M / I; L103N / E / R, H274R, N333D and N386D; L103N / E / R, H274R, N333D and R395K; L103N / E / R, H274R, N333D and I406T / A; L103N / E / R, H274R, N333D and L420M / I; L103N / E / R, N333D, N386D and R395K; L103N / E / R, N333D, N386D and I406T / A; L103N / E / R, N333D, N386D and L420M / I; L103N / E / R, N386D, R395K and I406T / A; L103N / E / R, N386D, R395K and L420M / I; L103N / E / R, R395K, I406T / A and L420M / I; or may further include N386D, R395K, I406T / A and L420M / I, with their positions corresponding to sequence number 2.

[0154] According to one embodiment, the GCase polypeptide further comprises five of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, the positions corresponding to SEQ ID NO: 2. Therefore, for example, GCase polypeptides include mutations: H60W, L103N / E / R, Q166A, H274R and N333D; H60W, L103N / E / R, Q166A, H274R and N386D; H60W, L103N / E / R, Q166A, H274R and R395K; H60W, L103N / E / R, Q166A, H274R and I406T / A; H60W, L103N / E / R, Q166A, H274R and L420M / I; H60W, Q166A, H274R, N33 3D and N386D; H60W, Q166A, H274R, N333D and R395K; H60W, Q166A, H274R, N333D and I406T / A; H60W, Q166A, H274R, N333D and L420M / I; H60W, H274R, N333D, N386D and R395K; H60W, H274R, N333D, N386D and I406T / A; H60W, H274R, N333D, N386D and L420M / I; H60W, N333D, N386D, R395K and I406T / A;H60W, N333D, N386D, R395K and L420M / I;H60W, N386D, R395K, I406T / A and L420M / I;L103N / E / R, Q166A, H274R, N333D and N386D;L103N / E / R, Q166A, H274R, N333D and R395K;L103N / E / R, Q166A, H274R, N333D and I406T / A;L103N / E / R, Q166A, H274R, N333D and L420M / I;L 103N / E / R, H274R, N333D, N386D and R395K; L103N / E / R, H274R, N333D, N386D and I406T / A; L103N / E / R, H274R, N333D, N386D and L420M / I; L103N / E / R, N333D, N386D, R395K and I406T / A; L103N / E / R, N333D, N386D, R395K and L420M / I; L103N / E / R, N386D, R395K, I406T / A and L420M / I;Q166A, H274R, N333D, N386D and R395K; Q166A, H274R, N333D, N386D and I406T / A; Q166A, H274R, N333D, N386D and L420M / I; Q166A, N333D, N386D, R395K and I406T / A; Q166A, N333D, N386D, R395K and L420M / I ;Q166A, N386D, R395K, I406T / A and L420M / I;H274R, N333D, N386D, R395K and I406T / A;H274R, N333D, N386D, R395K and L420M / I;or may further include N333D, N386D, R395K, I406T / A and L420M / I, in position corresponding to sequence number 2.

[0155] According to one embodiment, the GCase polypeptide further comprises six of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, the positions corresponding to SEQ ID NO: 2. Therefore, for example, GCase polypeptides include mutations: H60W, L103N / E / R, Q166A, H274R, N333D and N386D; H60W, L103N / E / R, Q166A, H274R, N333D and R395K; H60W, L103N / E / R, Q166A, H274R, N333D and I406T / A; H60W, L103N / E / R, Q166A, H274R, N333D and L420M / I; H60W, Q166A, H274 R, N333D, N386D and R395K; H60W, Q166A, H274R, N333D, N386D and I406T / A; H60W, Q166A, H274R, N333D, N386D and L420M / I; H60W, H274R, N333D, N386D, R395K and I406T / A; H60W, H274R, N333D, N386D, R395K and L420M / I; H60W, N333D, N386D, R395K, I406T / A and L420M / I; L103N / E / R, Q166A, H274R, N333D, N386D and R395K; L103N / E / R, Q166A, H274R, N333D, N386D and I406T / A; L103N / E / R, Q166A, H274R, N333D, N386D and L420M / I; L103N / E / R, H274R, N333D, N386D, R395K and I406T / A; L103N / E / R, H274R, N333D, N386D , R395K and L420M / I; L103N / E / R, N333D, N386D, R395K, I406T / A and L420M / I; Q166A, H274R, N333D, N386D, R395K and I406T / A; Q166A, H274R, N333D, N386D, R395K and L420M / I; or H274R, N333D, N386D, R395K, I406T / A and L420M / I may further be included, the position of which corresponds to sequence number 2.

[0156] According to one embodiment, the GCase polypeptide further comprises seven of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, the positions corresponding to SEQ ID NO: 2. Therefore, for example, GCase polypeptides include mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D and R395K; H60W, L103N / E / R, Q166A, H274R, N333D, N386D and I406T / A; H60W, L103N / E / R, Q166A, H274R, N333D, N386D and L420M / I; H60W, Q166A, H274R, N333D, N38 6D, R395K and I406T / A; H60W, Q166A, H274R, N333D, N386D, R395K and L420M / I; H60W, H274R, N333D, N386D, R395K, I406T / A and L420M / I; L103N / E / R, Q166A, H274R, N333D, N386D, R395K and I406T / A; L103N / E / R, Q166A, H274R, N333D, N386D, R39 5K and L420M / I;L103N / E / R, H274R, N333D, N386D, R395K, I406T / A and L420M / I;L103N / E / R, Q166A, N333D, N386D, R395K, I406T / A and L420M / I;L103N / E / R, Q166A, H274R, N386D, R395K, I406T / A and L420M / I;L103N / E / R, Q166A, H274R, N333D, R 395K, I406T / A and L420M / I; L103N / E / R, Q166A, H274R, N333D, N386D, I406T / A and L420M / I; L103N / E / R, Q166A, H274R, N333D, N386D, R395K and L420M / I; or Q166A, H274R, N333D, N386D, R395K, I406T / A and L420M / I may further be included, with their positions corresponding to sequence number 2.

[0157] According to one embodiment, the GCase polypeptide further comprises eight of the following mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, or L420M / I, where the position corresponds to SEQ ID NO: 2. Therefore, for example, the GCase polypeptide is: mutations: L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A, and L420M / I; H60W, Q166A, H274R, N333D, N386D, R395K, I406T / A, and L420M / I; H 60W, L103N / E / R, H274R, N333D, N386D, R395K, I406T / A and L420M / I; H60W, L103N / E / R, Q166A, N333D, N386D, R395K, I406T / A and L420M / I; H60W, L103N / E / R, Q166A, H274R , N386D, R395K, I406T / A and L420M / I; H60W, L103N / E / R, Q166A, H274R, N333D, R395K, I406T / A and L420M / I; H60W, L103N / E / R, Q166A, H274R, N333D, N386D, I406T / A and L42 0M / I; may further include H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K and L420M / I; H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K and I406T / A, the position corresponding to sequence number 2.

[0158] According to one embodiment, the GCase polypeptide further comprises all of the mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A and L420M / I, whose positions correspond to SEQ ID NO: 2.

[0159] According to one embodiment, the GCase polypeptide is a variant of: L34P, H60W, N102D, L103N, I130T, H145K / R, L165Q, Q166A, A168S, I204K, R211N, E222K, K224N, Q226T, L241I, S242P, D263N, H274R, K303R, N333D, T334F / The array includes at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of Y, T369E, N370D, L372N, N386D, R395K, I406T, L420M, K473W, H495R, and the position corresponds to array number 2.

[0160] According to one embodiment, the GCase polypeptide includes all of the mutations: L34P, H60W, N102D, L103N, I130T, H145K / R, L165Q, Q166A, A168S, I204K, R211N, E222K, K224N, Q226T, L241I, S242P, D263N, H274R, K303R, N333D, T334F / Y, T369E, N370D, L372N, N386D, R395K, I406T, L420M, K473W, and H495R, and their positions correspond to SEQ ID NO: 2.

[0161] According to one embodiment, the GCase polypeptide further comprises at least one of the mutations: V78I, A95K, V191M, A322D, V343T, M361E, S364A, H374W, T410E, H451N, or L480I, the position corresponding to SEQ ID NO: 2.

[0162] According to one embodiment, the GCase polypeptide further comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations: V78I, A95K, V191M, A322D, V343T, M361E, S364A, H374W, T410E, H451N, or L480I, the position corresponding to SEQ ID NO: 2.

[0163] According to one embodiment, the GCase polypeptide further comprises all of the mutations: V78I, A95K, V191M, A322D, V343T, M361E, S364A, H374W, T410E, H451N and L480I, the positions corresponding to SEQ ID NO: 2.

[0164] According to one embodiment, the GCase polypeptide is a variant of: L34P, H60W, V78I, A95K, N102E, L103E, I130T, H145K / R, L165Q, Q166A, A168S, V191M, I204K, R211N, E222K, K224G, Q226T, L241I, S242P, D263N, A322D, T334K, V343T, M361E, S364A, T369E , including at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 of N370D, L372N, H374W, I406A, T410E, L420I, H451N, K473W, L480I and / or H495R, the position of which corresponds to sequence number 2.

[0165] According to one embodiment, the GCase polypeptide includes all of the mutations: L34P, H60W, V78I, A95K, N102E, L103E, I130T, H145K / R, L165Q, Q166A, A168S, V191M, I204K, R211N, E222K, K224G, Q226T, L241I, S242P, D263N, A322D, T334K, V343T, M361E, S364A, T369E, N370D, L372N, H374W, I406A, T410E, L420I, H451N, K473W, L480I, and H495R, and their positions correspond to SEQ ID NO: 2.

[0166] According to one embodiment, the GCase polypeptide further comprises at least one of the mutations: H162K, S181A, T297S, M335F, K346H, S431A, S465D, or A476D, the position corresponding to SEQ ID NO: 2.

[0167] According to one embodiment, the GCase polypeptide further comprises at least 2, 3, 4, 5, 6, or 7 mutations: H162K, S181A, T297S, M335F, K346H, S431A, S465D, or A476D, the positions of which correspond to SEQ ID NO: 2.

[0168] According to one embodiment, the GCase polypeptide further comprises all of the mutations: H162K, S181A, T297S, M335F, K346H, S431A, S465D and A476D, the positions corresponding to SEQ ID NO: 2.

[0169] According to one embodiment, the GCase polypeptide is a variant of: L34P, H60W, V78I, A95K, N102E, L103E, I130T, H145K / R, H162K, L165Q, Q166A, A168S, S181A, V191M, I204K, R211N, E222K, K224G, Q226T, L241I, S242P, D263N, T297S, A322D, T334K, M335F, V343T, K346H, M361E, S364A, T369E, N370D, L372N, H3 Includes at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of 74W, N386D, R395K, I406A, T410E, L420I, S431A, H451N, S465D, K473W, A476D, L480I and / or H495R, the position of which corresponds to sequence number 2.

[0170] According to one embodiment, the GCase polypeptide is a variant of: L34P, H60W, V78I, A95K, N102E, L103E, I130T, H145K / R, H162K, L165Q, Q166A, A168S, S181A, V191M, I204K, R211N, E222K, K224G, Q226T, L241I, S242P, D263N, T297S This includes all of the following: A322D, T334K, M335F, V343T, K346H, M361E, S364A, T369E, N370D, L372N, H374W, N386D, R395K, I406A, T410E, L420I, S431A, H451N, S465D, K473W, A476D, L480I, and H495R, and their positions correspond to sequence number 2.

[0171] According to one embodiment, the GCase polypeptide further comprises at least one of the mutations: R47K, L51R, Q70H, L91I, G115E, A124G, D140N / G, S196T, or V437S, the position corresponding to SEQ ID NO: 2.

[0172] According to one embodiment, the GCase polypeptide further comprises at least 2, 3, 4, 5, 6, 7, or 8 mutations: R47K, L51R, Q70H, L91I, G115E, A124G, D140N / G, S196T, or V437S, the positions of which correspond to SEQ ID NO: 2.

[0173] According to one embodiment, the GCase polypeptide further comprises all of the mutations: R47K, L51R, Q70H, L91I, G115E, A124G, D140N / G, S196T, and V437S, whose positions correspond to SEQ ID NO: 2.

[0174] According to one embodiment, the GCase polypeptide is a variant of: L34P, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102E, L103E, G115E, A124G, I130T, D140N / G, H145K / R, H162K, L165Q, Q166A, A168 S, S181A, V191M, S196T, I204K, R211N, E222K, K224G, Q226T, L241I, S242P, D263N , T297S, A322D, N333D, T334K, M335F, V343T, K346H, M361E, S364A, T369E, N370D, At least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 of L372N, H374W, N386D, R395K, I406A, T410E, L420M, S431A, V437S, H451N, S465D, K473W, A476D, L480I and / or H495R , including 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55, whose position corresponds to sequence number 2.

[0175] According to one embodiment, the GCase polypeptide is a mutant of: L34P, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102E, L103E, G115E, A124G, I130T, D140N / G, H145K / R, H162K, L165Q, Q166A, A168S, S181A, V191M, S196T, I204K, R211N, E222K, K224G, Q226T, L241I, This includes all of S242P, D263N, T297S, A322D, N333D, T334K, M335F, V343T, K346H, M361E, S364A, T369E, N370D, L372N, H374W, N386D, R395K, I406A, T410E, L420M, S431A, V437S, H451N, S465D, K473W, A476D, L480I, and H495R, and their positions correspond to sequence number 2.

[0176] According to one embodiment, the GCase polypeptide further comprises at least one of the following mutations: T36Q, S38A, Q143E, T183A, L185M, T272S, H274K, N275D, L286S, K293Q, E300R, K321E, V376T, K408R, Q440E, M450Q and / or I483V, the position corresponding to SEQ ID NO: 2.

[0177] According to one embodiment, the GCase polypeptide further comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of the mutations: T36Q, S38A, Q143E, T183A, L185M, T272S, H274K, N275D, L286S, K293Q, E300R, K321E, V376T, K408R, Q440E, M450Q and / or I483V, the position corresponding to SEQ ID NO: 2.

[0178] According to one embodiment, the GCase polypeptide further comprises all of the mutations: T36Q, S38A, Q143E, T183A, L185M, T272S, H274K, N275D, L286S, K293Q, E300R, K321E, V376T, K408R, Q440E, M450Q and / or I483V, the positions corresponding to SEQ ID NO: 2.

[0179] According to one embodiment, the GCase polypeptide is a variant of: L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102E, L103R, G115E, A124G, I130T, D140G, Q143E, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, L185M, V19 1M, S196T, I204K, R211N, E222K, K224G, Q226T, L241I, S242P, D263N, T272S, H274K, N275D, L286S, K293 Q, T297S, E300R, K321E, A322D, N333D, T334K, M335F, V343T, K346H, M361E, S364A, T369E, N370D, L372N , H374W, V376T, N386D, R395K, I406A, K408R, T410E, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L480I, I483V and H495R at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2 Includes 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72, and the position corresponds to sequence number 2. The position corresponds to sequence number 2.

[0180] According to one embodiment, the GCase polypeptide is a mutant of: L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102E, L103R, G115E, A124G, I130T, D140G, Q143E, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, L185M, V191M, S196T, I204K, R211N, E222K, K224G, Q226T, L241I, S242P, D263N, T272S, H274K, This includes all of N275D, L286S, K293Q, T297S, E300R, K321E, A322D, N333D, T334K, M335F, V343T, K346H, M361E, S364A, T369E, N370D, L372N, H374W, V376T, N386D, R395K, I406A, K408R, T410E, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L480I, I483V, and H495R, and their positions correspond to sequence number 2.

[0181] As mentioned above, the amino acids in the enzyme's catalytic domain have not been modified.

[0182] According to one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the amino acids at positions D127, F128, W179, N234, E235, Y244, F246, Q284, Y313, E340, S345, W381, and N396 of the GCase polypeptide are unchanged, and the positions correspond to SEQ ID NO: 2.

[0183] According to one embodiment, all amino acids at the following positions D127, F128, W179, N234, E235, Y244, F246, Q284, Y313, E340, S345, W381, and N396 of the GCase polypeptide are unmodified, and their positions correspond to SEQ ID NO: 2.

[0184] According to one embodiment, the GCase polypeptide contains an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 18, 20, 22, and 27. Such a determination can be made, for example, using NCBI's standard protein-protein BLAST [blastp] software.

[0185] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 85% identical to SEQ ID NOs. 4, 6, 8, 10, 12, 14, 18, 20, 22, or 27.

[0186] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 90% identical to sequence numbers 4, 6, 8, 10, 12, 14, 18, 20, 22, or 27.

[0187] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 95% identical to SEQ ID NOs. 4, 6, 8, 10, 12, 14, 18, 20, 22, or 27.

[0188] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 96% identical to SEQ ID NOs. 4, 6, 8, 10, 12, 14, 18, 20, 22, or 27.

[0189] According to a particular embodiment, the genetically modified human GCase contains an amino acid sequence that is at least 97% identical to sequence numbers 4, 6, 8, 10, 12, 14, 18, 20, 22, or 27.

[0190] According to certain embodiments, the genetically modified human GCase comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, 6, 8, 10, 12, 14, 18, 20, 22 or 27.

[0191] According to certain embodiments, the genetically modified human GCase comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 4, 6, 8, 10, 12, 14, 18, 20, 22 or 27.

[0192] According to one embodiment, the GCase polypeptide comprises an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 18, 20, 22 and 27.

[0193] According to one embodiment, the GCase polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 14.

[0194] According to one embodiment, the GCase polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0195] According to one embodiment, the GCase polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22.

[0196] According to one embodiment, the GCase polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0197] According to one embodiment, the GCase polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 27.

[0198] According to one embodiment, the GCase polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0199] The polypeptides of some embodiments of the present invention may be synthesized by any technique of polypeptide synthesis known to those skilled in the art. According to one embodiment, the polypeptides of the present invention can be synthesized using recombinant DNA technology.

[0200] The polypeptides of the present invention are preferably produced using recombinant technology. Such recombinant technology is described in Bitter et al. (1987) Methods in Enzymol. 153: pp. 516-544, Studier et al. (1990) Methods in Enzymol. 185: pp. 60-89, Brisson et al. (1984) Nature 310: pp. 511-514, Takamatsu et al. (1987) EMBO J. 6: pp. 307-311, Coruzzi et al. (1984) EMBO J. 3: pp. 1671-1680, and Brogli et al. (1984) Science 224: pp. 838-843, Gurley et al. (1986) Mol. Cell. Biol. 6: pp. 559-565, and Weissbach and Weissbach, 1988, Methods for Plant Molecular Biology, Academic. This is described in Press, NY, Section VIII, pp. 421-463.

[0201] To generate the polypeptide of the present invention using recombinant technology, the polynucleotide encoding the polypeptide of the present invention is ligated to a nucleic acid expression construct and comprises the polynucleotide sequence under transcriptional regulation of a cis-regulatory (e.g., promoter) sequence suitable for supporting constitutive or inducible transcription in a host cell, as further described herein.

[0202] This instruction also provides nucleic acid sequences encoding genetically modified human GCase polypeptides.

[0203] As used herein, the term “isolated polynucleotide” refers to a single-stranded or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a complex polynucleotide sequence (e.g., a combination of the above).

[0204] Exemplary polynucleotide sequences for expressing the polypeptide of the present invention are described in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 17, 19, 21, 23, or 26.

[0205] In addition to containing elements essential for the transcription and translation of the inserted coding sequence, the expression construct of the present invention may also include sequences (i.e., tags) manipulated to enhance the stability, generation, purification, yield, or reduced toxicity of the expressed polypeptide. Such fusion proteins can be designed so that the fusion protein can be readily isolated by affinity chromatography, e.g., by immobilization on a heterologous protein-specific column. If the cleavage site is manipulated between the peptide moiety and the heterologous protein, the peptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].

[0206] Polypeptide-coding sequences can be expressed using various prokaryotic or eukaryotic cells as host expression systems.

[0207] Prokaryotic cells include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing polypeptide coding sequences.

[0208] Eukaryotic cells include, but are not limited to, unicellular and multicellular organisms, any cell of a eukaryote. Unicellular eukaryotes include, but are not limited to, yeasts, protozoa, slime molds, and algae. Multicellular eukaryotes include, but are not limited to, animals (e.g., mammals, insects, invertebrates, nematodes, birds, fish, reptiles, and crustaceans), plants, fungi, and algae (e.g., brown algae, red algae, and green algae).

[0209] According to one embodiment, the cell is a plant cell.

[0210] The plant cell may be derived from any plant tissue, such as fruit, flower, root, leaf, embryo, embryonic cell suspension, callus or seeding tissue.

[0211] According to one embodiment, the eukaryotic cell is not a plant cell.

[0212] According to one embodiment, the eukaryotic cell is an animal cell.

[0213] According to one embodiment, the eukaryotic cell is a vertebrate cell.

[0214] According to one embodiment, the eukaryotic cell is an invertebrate cell.

[0215] According to certain embodiments, the invertebrate cell is a cell of an insect, snail, bivalve, octopus, starfish, sea urchin, jellyfish, and worm.

[0216] According to certain embodiments, the invertebrate cell is a crustacean cell. Exemplary crustaceans include, but are not limited to, shrimp, prawn, crab, lobster and crayfish.

[0217] According to certain embodiments, the invertebrate cell is a fish cell. Exemplary fish include, but are not limited to, salmon, tuna, pollock, catfish, cod, haddock, sea bass, tilapia, Arctic char and koi.

[0218] A mammalian expression system can also be used to express the polypeptide of the present invention. A cell system capable of glycosylating the GCase polypeptide is advantageous.

[0219] According to one embodiment, the eukaryotic cell is a mammalian cell.

[0220] According to certain embodiments, mammalian cells are, without limitation, cells of non-human organisms such as rodents, rabbits, pigs, goats, ruminants (e.g., cattle, sheep, antelopes, deer, and giraffes), dogs, cats, horses, and non-human primates.

[0221] According to certain embodiments, eukaryotic cells are human cells.

[0222] According to one embodiment, eukaryotic cells include primary cells, cell lines, somatic cells, germ cells, stem cells, embryonic stem cells, adult stem cells, hematopoietic stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPS cells), gamete cells, zygote cells, placental cells, embryos, fetuses, and / or donor cells.

[0223] According to a particular embodiment, the cells are human embryonic cells.

[0224] According to a particular embodiment, the cells are human embryonic kidney cells.

[0225] According to a particular embodiment, the cells are HEK293T cells.

[0226] According to certain embodiments, cells have the ability to glycosylate proteins (i.e., modify human GCases).

[0227] Eukaryotic cells may be transformed using recombinant expression vectors containing polypeptide coding sequences. For example, yeast transformed using a recombinant yeast expression vector containing a polypeptide coding sequence; plant cell systems transformed using recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or recombinant plasmid expression vectors containing polypeptide coding sequences, such as Ti plasmids.

[0228] In any case, transformed cells are cultured under effective conditions that allow for the expression of large amounts of recombinant polypeptide. Effective culture conditions include, but are not limited to, effective media, bioreactors, temperature, pH, and oxygen conditions that allow for protein production. Effective media refers to any medium used to culture cells to produce the recombinant polypeptide of the present invention. Such media typically contain aqueous solutions with assimilated carbon, nitrogen, and phosphate sources, as well as other nutrients such as appropriate salts, minerals, metals, and vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shaker flasks, test tubes, microtiter dishes, and petri plates. Culture can be carried out at temperature, pH, and oxygen content appropriate for recombinant cells. Such culture conditions are within the know-how of those skilled in the art.

[0229] Depending on the vector and host system used for production, the resulting protein may remain within recombinant cells; be secreted into fermentation media; be secreted into spaces between two cell membranes, such as the perimembrane space in E. coli; or be retained on the outer surface of a cell or viral membrane.

[0230] GCase polypeptides of some embodiments of the present invention are conferred from cells (e.g., the host expression system discussed above) at higher expression levels and / or higher secretion levels compared to wild-type human GCase.

[0231] The term "wild type" refers to human glucosylceramidase (human GCase), for example, as described in Sequence ID No. 2 or Sequence ID No. 25.

[0232] According to one embodiment, higher expression levels and / or higher secretion levels are approximately 5-25%, 10-50%, 10-100%, 20-90%, 25-75%, 30-80%, 40-50%, 50-60%, 60-70%, 70-80%, 90-99%, or 95-100% compared to wild-type human GCase (e.g., under the same culture conditions).

[0233] According to one embodiment, higher expression levels and / or higher secretion levels are approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or higher compared to wild-type human GCase (e.g., under the same culture conditions).

[0234] According to one embodiment, the GCase polypeptide exhibits an intracellular expression level approximately 1.3 to 5 times (e.g., 3 times) higher in eukaryotic cells compared to wild-type human GCase (e.g., from HEK293T cells) under the same culture conditions, as discussed below.

[0235] According to one embodiment, GCase polypeptides are secreted from eukaryotic cells (e.g., HEK293T cells) compared to wild-type GCase, which is not secreted under the same culture conditions, as discussed below.

[0236] Methods for evaluating expression levels are discussed below.

[0237] After a certain period of time in the culture, the recombinant protein is recovered. The phrase "recovery of recombinant protein" refers to collecting the entire fermentation medium containing the protein and does not necessarily imply any further steps of separation or purification. The proteins of the present invention can be purified using a variety of standard protein purification techniques, including but not limited to affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed-phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubilization.

[0238] Regardless of how the GCase variant polypeptide is generated, genetically modified human GCase maintains the catalytic activity of human GCase. Furthermore, genetically modified human GCase exhibits increased thermal stability. The sustained catalytic activity of the GCase polypeptide, along with higher expression levels and thermal stability, is advantageous for enzyme replacement therapy (ERT), particularly for the treatment of Gaucher disease (discussed below).

[0239] As discussed in the following Examples section (see Examples 4, 7, and 10 below), GCase variants D7, D15, and D16 possess equivalent enzymatic activity compared to wild-type GCase and Cerezyme®, respectively (see Tables 2, 4, and 6 below).

[0240] According to one embodiment, the GCase polypeptide has the ability to catalyze the hydrolysis of GlcCer, similar to wild-type GCase under the same conditions (i.e., the same experimental conditions, e.g., the same buffer, temperature, pH, etc.).

[0241] According to one embodiment, the GCase polypeptide is approximately 0.1 to 2.5 × 10 6 k cat / K m (M -1 minutes -1 ), for example, 0.15~2.0 × 10 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of GlcCer, for example, the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc).

[0242] According to one embodiment, the GCase polypeptide is at least about 0.1 × 10 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of GlcCer, for example, the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc).

[0243] According to one embodiment, the GCase polypeptide is at least about 0.2 × 10 6 k cat / K m (M -1 minutes -1) has the ability to catalyze the hydrolysis of GlcCer, for example, the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc).

[0244] According to one embodiment, the GCase polypeptide is at least about 0.3 × 10 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of GlcCer, for example, the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc).

[0245] According to one embodiment, the GCase polypeptide is at least about 0.5 × 10 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of GlcCer, for example, the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc).

[0246] According to a particular embodiment, the GCase polypeptide is at least about 1.0 × 10⁻⁶ 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of p-NP-Glc.

[0247] According to a particular embodiment, the GCase polypeptide is at least about 1.5 × 10⁻⁶ 6 k cat / K m (M -1 minutes -1 ) It has the ability to catalyze the hydrolysis of p-NP-Glc.

[0248] According to a particular embodiment, the GCase polypeptide is at least about 1.6 × 10⁻⁶ 6 k cat / K m (M -1 minutes -1) has the ability to catalyze the hydrolysis of p-NP-Glc.

[0249] According to a particular embodiment, the GCase polypeptide is at least about 1.7 × 10⁻⁶ 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of p-NP-Glc.

[0250] According to a particular embodiment, the GCase polypeptide is at least about 1.8 × 10⁻⁶ 6 k cat / K m (M -1 minutes -1 ) has the ability to catalyze the hydrolysis of p-NP-Glc.

[0251] Methods for measuring the catalytic efficiency of GCase polypeptides described herein are known in the art and include, for example, insight activity assays (e.g., using a substrate applied to cells containing the active enzyme) and in vitro activity assays (in which the activity of a particular enzyme is measured in a protein mixture extracted from cells). For example, an enzyme activity assay may be performed using p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc) as a substrate [discussed in Wei RR et al., J. Biol. Chem. (2011) 286:299-308, which is incorporated herein by reference].

[0252] As discussed in the following Examples section (see Examples 3, 6, and 9 below), GCase variants D7, D15, and D16 exhibit higher thermal stability compared to wild-type GCase and Cerezyme®. Specifically, at pH 6.1, D7 GCase showed an increase of approximately 6-7°C compared to wild-type GCase and an increase of approximately 11°C compared to Cerezyme® (see Table 1 below). At the same pH level, D15 GCase showed a substantial increase of approximately 12-13°C compared to wild-type GCase and a substantial increase of approximately 17°C compared to Cerezyme® (see Tables 1 and 3 below). Similarly, D16 GCase showed a substantial increase of approximately 19°C compared to Cerezyme® (see Table 6 below).

[0253] According to one embodiment, the GCase polypeptide exhibits thermal stability over a higher temperature range of 3 to 22°C (e.g., 15 to 22°C, 10 to 20°C, 5 to 15°C, 7 to 13°C, 9 to 11°C) compared to the wild-type polypeptide under the same conditions (e.g., pH 6.1).

[0254] The term "thermal stability" or "increased thermal stability" compared to wild-type polypeptides means that the GCase polypeptide possesses increased thermal stability, i.e., the ability to resist denaturation as the temperature increases. Standard techniques for quantifying thermal stability, including but not limited to circular dichroism, differential scanning calorimeters, and surface plasmon resonance, are known in the art.

[0255] The methods for measuring the thermal stability of GCase polypeptides described herein are known in the art and include, for example, the enzyme stability assay discussed in Wei RR et al., J. Biol. Chem. (2011) 286: pp. 299-308 (incorporated herein by reference).

[0256] According to one embodiment, the GCase polypeptide exhibits thermal stability at temperatures at least about 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C higher than the wild-type polypeptide under the same conditions (e.g., pH 6.1).

[0257] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 5°C higher than that of the wild-type polypeptide under the same conditions (e.g., pH 6.1).

[0258] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 7°C higher than that of the wild-type polypeptide under the same conditions (e.g., pH 6.1).

[0259] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 9°C higher than that of the wild-type polypeptide under the same conditions (e.g., pH 6.1).

[0260] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 11°C higher than that of the wild-type polypeptide under the same conditions (e.g., pH 6.1).

[0261] According to certain embodiments, GCase polypeptides exhibit thermal stability at temperatures approximately 13°C higher than wild-type polypeptides under the same conditions (e.g., pH 6.1).

[0262] According to certain embodiments, GCase polypeptides exhibit thermal stability at temperatures approximately 15°C higher than wild-type polypeptides under the same conditions (e.g., pH 6.1).

[0263] According to certain embodiments, GCase polypeptides exhibit thermal stability at temperatures approximately 17°C higher than wild-type polypeptides under the same conditions (e.g., pH 6.1).

[0264] According to one embodiment, the GCase polypeptide exhibits thermal stability over a higher temperature range of 3 to 22°C (e.g., 15 to 22°C, 10 to 20°C, 5 to 15°C, 7 to 13°C, 9 to 11°C) compared to the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0265] The term "Cerezyme®," also known as imiglucerase, is a commercially available drug used for enzyme replacement therapy.

[0266] According to one embodiment, the GCase polypeptide exhibits thermal stability at temperatures at least about 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C higher than Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0267] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 5°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0268] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 7°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0269] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 9°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0270] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 11°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0271] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 13°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0272] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 15°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0273] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 17°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0274] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 19°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 6.1).

[0275] According to one embodiment, the GCase polypeptide exhibits thermal stability over a higher temperature range of 15-25°C (e.g., 17-23°C, 19-23°C) compared to the Cerezyme® polypeptide under the same conditions (e.g., pH 7.4).

[0276] According to one embodiment, the GCase polypeptide exhibits thermal stability at temperatures approximately 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C higher than Cerezyme® polypeptide under the same conditions (e.g., pH 7.4).

[0277] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 15°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 7.4).

[0278] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 17°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 7.4).

[0279] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 20°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 7.4).

[0280] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 22°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 7.4).

[0281] According to certain embodiments, the GCase polypeptide exhibits thermal stability at a temperature approximately 25°C higher than that of the Cerezyme® polypeptide under the same conditions (e.g., pH 7.4).

[0282] As mentioned above, GCase polypeptides are highly expressed and secreted from eukaryotic cells. Expression in eukaryotic cells enables GCase glycosylation, which is important for GCase activity.

[0283] According to another embodiment, GCase polypeptides are secreted from eukaryotic cells compared to wild-type polypeptides that are not secreted under the same culture conditions.

[0284] According to one embodiment, GCase polypeptides are secreted from eukaryotic cells (e.g., from HEK293T cells) at approximately 1.5 to 10 times higher levels (e.g., approximately 1.5 to 2 times, approximately 2 to 3 times, approximately 3 to 4 times, approximately 4 to 5 times, approximately 5 to 6 times, approximately 6 to 7 times, approximately 8 to 9 times, approximately 9 to 10 times) compared to wild-type GCase under the same culture conditions.

[0285] According to one embodiment, GCase polypeptides are secreted approximately three times more frequently from eukaryotic cells (e.g., from HEK293T cells) than wild-type GCase under the same culture conditions.

[0286] According to one embodiment, GCase polypeptides are secreted approximately five times more frequently from eukaryotic cells (e.g., from HEK293T cells) compared to wild-type GCase under the same culture conditions.

[0287] According to one embodiment, GCase polypeptides are secreted approximately 10 times more frequently from eukaryotic cells (e.g., from HEK293T cells) compared to wild-type GCase under the same culture conditions.

[0288] According to one embodiment, the GCase polypeptide exhibits approximately 1.3 to 5 times (e.g., approximately 1.3 to 2 times, approximately 1.3 to 3 times, approximately 2 to 3 times, approximately 2 to 4 times, approximately 3 to 4 times, approximately 4 to 5 times) higher intracellular expression levels in eukaryotic cells compared to wild-type human GCase under the same culture conditions (e.g., from HEK293T cells).

[0289] According to one embodiment, the GCase polypeptide exhibits at least approximately 1.3 times higher intracellular expression levels in eukaryotic cells compared to wild-type GCase under the same culture conditions.

[0290] According to one embodiment, the GCase polypeptide exhibits approximately twice the intracellular expression level in eukaryotic cells compared to wild-type human GCase under the same culture conditions (e.g., from HEK293T cells).

[0291] According to one embodiment, the GCase polypeptide exhibits a three-fold higher intracellular expression level in eukaryotic cells compared to wild-type human GCase under the same culture conditions.

[0292] According to one embodiment, the GCase polypeptide exhibits approximately four times higher intracellular expression levels in eukaryotic cells compared to wild-type human GCase under the same culture conditions (e.g., from HEK293T cells).

[0293] As used herein, the terms “expression level” and “expression level” refer to the degree of gene expression and / or gene product activity in a biological sample (e.g., eukaryotic cells).

[0294] It should be noted that the level of expression can be determined in arbitrary absolute units or in normalized units (compared to known expression levels of a control reference).

[0295] According to one embodiment, the secretion level of GCase polypeptide from eukaryotic cells can be at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of wild-type polypeptide under the same culture conditions.

[0296] According to one embodiment, the intracellular expression level of GCase polypeptides in eukaryotic cells may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of wild-type polypeptides under the same culture conditions.

[0297] According to a particular embodiment, the expression level is determined using an RNA and / or protein detection method.

[0298] Non-exclusive examples of methods for detecting the level of RNA expressed in cells include Northern blotting, RT-PCR analysis, RNA insights hybridization staining, and insights RT-PCR staining.

[0299] Non-limiting examples of methods for detecting the level and / or activity of specific protein molecules in cells include enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitation (IP), radioimmunoassays (RIA), fluorescence-activated cell sorting (FACS), immunohistochemistry, insight activity assays (e.g., using substrates applied to cells containing active enzymes), in vitro activity assays (where the activity of a specific enzyme is measured in a protein mixture extracted from cells), and molecular weight-based approaches. If detection of the expression level of secreted proteins is desired, the ELISA assay may be performed in the cell medium in which the cells were cultured (i.e., containing the contents secreted by the cells).

[0300] According to one embodiment, an isolated cell containing at least one exogenous polynucleotide or construct is provided (as discussed above).

[0301] According to one embodiment, the isolated cells are eukaryotic cells (as discussed above).

[0302] Genetically modified human GCases of certain embodiments of the present invention, isolated polynucleotides of certain embodiments of the present invention, constructs of certain embodiments of the present invention, or cells of certain embodiments of the present invention can be administered to an organism by themselves or in a pharmaceutical composition in which they are mixed with a suitable carrier or excipient.

[0303] As used herein, “pharmaceutical composition” refers to a preparation of one or more active ingredients described herein, in combination with other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism.

[0304] In this specification, the term "active ingredient" refers to genetically modified human GCases involved in biological action.

[0305] In this specification, the terms “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” are interchangeable and refer to carriers or diluents that do not cause significant irritation to the organism and do not inhibit the biological activity and properties of the administered compound. Adjuvants are included in these terms.

[0306] In this specification, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further enhance the administration of the active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and certain starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycol.

[0307] Drug formulation and administration techniques can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0308] Preferred routes of administration may include, for example, oral, rectal, transmucosal, parenteral delivery including nasal, intestinal or intramuscular, subcutaneous and intrathecal injection, as well as intraarachnoid, direct intraventricular, intracardiac, for example, right or left ventricular cavity contour, general coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0309] Conventional approaches to drug delivery to the central nervous system (CNS) include neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular manipulation of drugs in attempts to leverage one of the endogenous transport pathways of the blood-brain barrier (e.g., creation of chimeric fusion proteins containing transport peptides with affinity for endothelial cell surface molecules, combined with drugs that themselves cannot cross the BBB); pharmacological strategies designed to increase the lipid solubility of drugs (e.g., conjugation of water-soluble drugs to lipid or cholesterol carriers); and transient disruption of BBB integrity by hyperosmotic disruption (resulting from carotid injection of mannitol solution or the use of bioactive drugs such as angiotensin peptides). However, each of these strategies has limitations, such as the inherent risks associated with invasive surgical procedures, size limitations imposed on the inherent limitations of the endogenous transport system, potentially undesirable biological side effects associated with systemic administration of chimeric molecules consisting of carrier motifs that may be active outside the CNS, and the potential risk of brain damage in the region of the brain where the BBB is disrupted, which gives a suboptimal delivery method.

[0310] Alternatively, the pharmaceutical composition may be administered in a topical rather than systemic manner, for example, by direct injection of the pharmaceutical composition into a tissue area of ​​the patient.

[0311] Pharmaceutical compositions of some embodiments of the present invention may be produced by processes well known in the art, for example, by conventional mixing, dissolving, granulation, sugar coating, wet grinding, emulsification, encapsulation, or freeze-drying processes.

[0312] Accordingly, pharmaceutical compositions for use according to certain embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the active ingredient into a pharmaceutically usable preparation. The correct formulation depends on the selected route of administration.

[0313] For injection, the active ingredient of the pharmaceutical composition may be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or a physiological salt buffer. For transmucosal administration, a penetrating agent suitable for the barrier to be permeated is used in the formulation. Such penetrating agents are generally known in the art.

[0314] For oral administration, pharmaceutical compositions can be readily formulated by combining an active compound with a pharmaceutically acceptable carrier known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by patients. Pharmaceutical preparations for oral use can be prepared by using a solid excipient, optionally grinding the resulting mixture, and, if necessary to obtain a tablet or sugar-coated tablet core, adding a suitable adjuvant and then processing the granular mixture. Suitable excipients include fillers such as sugars containing lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or sodium alginate salts thereof may be added.

[0315] The sugar-coated tablet core is provided with a suitable coating. For this purpose, a concentrated sugar solution may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, Carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablets or sugar-coated tablets for the identification or characterization of different combinations of active compound dosing.

[0316] Pharmaceutical compositions for oral administration include push-type capsules made of gelatin and soft, sealable capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-type capsules may contain the active ingredient in a mixture with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer may be added. All formulations for oral administration should be in a dosage suitable for the selected route of administration.

[0317] For buccal administration, the composition may take the form of tablets or lozenges formulated in a conventional manner.

[0318] For administration by nasal inhalation, the active ingredient for use according to some embodiments of the present invention is conventionally delivered in the form of an aerosol spray from a compressed pack or nebulizer using a suitable high-pressure gas, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for delivering a measured amount. For example, gelatin capsules and cartridges for use in dispensers may be formulated containing the compound and a suitable powder mixture based on lactose or starch.

[0319] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous intravenous infusion. Formulations for injection may, optionally, be presented in unit dosage forms in ampoules or multi-dose containers, together with added preservatives. The compositions may be suspensions, solutions or emulsions in oily or water-soluble vehicles and may contain compounding agents for suspending, stabilizing, and / or dispersing the drugs.

[0320] For parenteral administration, the pharmaceutical composition comprises an aqueous solution of the active preparation in a water-soluble form. In addition, the suspension of the active ingredient may be prepared as a suitable oil or water-based injectable suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. The aqueous injectable suspension may also contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain a suitable stabilizer or agent that increases the solubility of the active ingredient, enabling the preparation of a highly concentrated solution.

[0321] Alternatively, the active ingredient may be in powder form for composition with a suitable vehicle, such as a sterile pyrogen-free water-based solution, before use.

[0322] The pharmaceutical compositions of some embodiments of the present invention may also be formulated as suppositories or rectal compositions such as retained enemas, using conventional suppository bases such as cocoa butter or other glycerides.

[0323] Pharmaceutical compositions suitable for use in the context of certain embodiments of the present invention include compositions in which an active ingredient is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient (genetically modified human GCase) that is effective in preventing, alleviating, or improving the symptoms of a disorder (e.g., Gaucher disease), or in prolonging the survival of the subject being treated.

[0324] Determining the therapeutically effective dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosures provided herein.

[0325] For any preparation used in the method of the present invention, the therapeutically effective dose or amount can first be estimated from in vitro and cell culture assays. For example, the dose can be formulated in an animal model to achieve the desired concentration or titer. Using such information, a useful dose in humans can be determined more accurately.

[0326] For example, any in vivo or in vitro assay of GCase activity may be used, such as utilizing an animal model of Gaucher disease, as discussed by Farfel-Becker et al. [Farfel-Becker, Vitner, and Futerman, Dis Model Mech. (2011) 4(6): pp. 746-752].

[0327] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmacokinetic procedures in vitro and in cell culture or in experimental animals. Data obtained from these in vitro and cell culture assays, as well as from animal studies, can be used to formulate dosage ranges for human use. Dosages may vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage may be selected by the individual physician, taking into account the patient's condition. (See, for example, Fingl et al., 1975, "The Pharmacological Basis of Therapeutics," Chapter 1, page 1).

[0328] The dosage and interval of the active ingredient may be individually adjusted to provide an effective amount (minimum effective concentration, MEC) that induces or inhibits a biological effect. While the MEC will vary for each preparation, it can be estimated from in vitro data. The dosage required to achieve the MEC will depend on individual characteristics and the route of administration. Plasma concentrations can be determined using detection assays.

[0329] Depending on the severity and responsiveness of the condition being treated, medication may be administered as a single dose or multiple doses over a course of treatment ranging from several days to several weeks, or until a cure is achieved or a reduction in the disease state is attained.

[0330] The amount of the composition to be administered will, of course, depend on the patient being treated, the severity of the pain, the method of administration, the prescribing physician's judgment, and so on.

[0331] Compositions of certain embodiments of the present invention may be presented in packs or dispenser devices, such as FDA-approved kits, which may contain, if desired, one or more unit dosage forms containing the active ingredient. Packs may include, for example, metal or plastic foil, such as blister packs. Packs or dispenser devices may enclose instructions for administration. Packs or dispensers may also contain warnings associated with the container in the form prescribed by government agencies that regulate the manufacture, use or sale of pharmaceuticals, and the warnings reflect agency approval of the composition or the form of human or veterinary administration. Such warnings may, for example, be labels or product inserts approved by the U.S. Food and Drug Administration for prescription drugs. Compositions containing preparations of the present invention formulated in a suitable pharmaceutical carrier may also be prepared, placed in appropriate containers, and labeled for treatment in the indicated conditions as described in further detail above.

[0332] It will be understood that the kit may further include other therapeutic compositions for treating Gaucher disease, such as agents for substrate reduction therapy (SRT).

[0333] Genetically modified human GCases of certain embodiments of the present invention, polynucleotides encoding them, expression constructs used for their expression, or cells of certain embodiments of the present invention can be used to treat β-glucocerebrosidase deficiency and related diseases in subjects requiring it.

[0334] As used herein, the terms “to treat” and “treatment” mean stopping, substantially inhibiting, delaying or reversing the progression of a condition, substantially delaying the onset of the clinical symptoms of a condition, substantially improving the clinical symptoms of a condition, or substantially preventing the onset of the clinical symptoms of a condition. As used herein, the term “to treat” also means extending the survival of a patient suffering from a condition or delaying death.

[0335] As used herein, the terms “subject” and “subject requiring it,” as used interchangeably herein, refer to a mammal, preferably a human, of any age or sex, suffering from a pathological condition. This term encompasses individuals at risk of developing the pathological condition. Subjects may include, for example, newborns, infants, boys, adolescents, adults, and the elderly.

[0336] According to one embodiment, the subject is diagnosed with a disease associated with the GBA gene.

[0337] According to one embodiment, the subject is diagnosed with a disease associated with reduced β-glucocerebrosidase levels and / or activity.

[0338] According to one embodiment, the subject is diagnosed with a disease associated with β-glucocerebrosidase deficiency.

[0339] Exemplary diseases associated with β-glucocerebrosidase deficiency include, but are not limited to, Gaucher disease, GBA-associated Parkinson's disease, GBA-associated dementia with Lewy bodies, and GBA-associated multiple system atrophy.

[0340] According to certain embodiments, the disease associated with β-glucocerebrosidase deficiency is Gaucher disease.

[0341] In this specification, the terms “Gaucher disease,” “Gaucher disease,” or “GD” refer to lysosomal storage disorders (LSD) characterized by the accumulation of glucosylceramide (GlcCer, also known as glucocerebroside) in cells, particularly in cells of mononuclear cell lineages. Glucosylceramide can be accumulated in the spleen, liver, kidneys, lungs, brain, and bone marrow. The disease is typically caused by a deficiency of the enzyme glucocerebrosidase (beta-glucosidase, also known as D-glucosyl-N-acyl sphingosingle cohydrolase, GCD, or GCase; EC 3.2.1.45), a lysosomal enzyme having the glucosylceramidase activity required to catalyze the hydrolysis of glucosylceramide / GlcCer.

[0342] GDs are divided into two main types based on the specific symptoms of the disease: neuropathic and non-neuropathic diseases. In non-neuropathic diseases, most organs and tissues may include, but are not limited to, the brain. In neuropathic diseases (nGDs), the brain is also included.

[0343] Type I (or non-neuropathic type, GD1) is the most common form of the disease, occurring in approximately 1 in 50,000 births. It is most frequently observed in individuals of Ashkenazi Jewish genetic traits. Symptoms can begin in early life or adulthood and include an enlarged liver and a significantly enlarged spleen (both known as "hepatosplenomegaly"), the spleen which can rupture and lead to further complications. Splenomegaly and bone marrow replacement cause anemia, thrombocytopenia, and leukopenia. Skeletal weakness and bone disease can be widespread. The brain is pathologically unaffected, but lung and, rarely, kidney dysfunction may be present. Patients in this group usually experience fatigue due to being easily injured (resulting in low platelet levels) and having a low red blood cell count. Depending on the onset and severity of the disease, patients with type I GD can live well into adulthood. Some patients have a mild form of the disease or show no symptoms at all.

[0344] As used herein, neuropathic gingival dysplasia (nGD) encompasses both type 2 and type 3 GD.

[0345] Type 2 GD, also known as acute infantile neuropathic GD, typically begins within the first six months of life and has an incidence of approximately 1 in 100,000 births. Symptoms include enlarged liver and spleen, extensive and progressive brain injury, oculomotor dysfunction, convulsions, seizures, limb rigidity, and poor sucking and swallowing ability. Affected children usually die by the age of two.

[0346] Also known as chronic neuropathic GD, type 3 GD can begin at any time, in childhood or even adulthood, occurring in approximately 1 in 100,000 births. It is characterized by a slow progression but milder neurological symptoms compared to the acute or type 2 version. Type 3 GD is divided into two variants, called type 3b and type 3a. Type 3b has an early onset of widespread hepatic and splenic lesions, and patients may also experience direct complications of pulmonary and rapidly progressing bone disease. Major symptoms include enlarged spleen and / or liver, seizures, poor coordination, skeletal irregularities, oculomotor disorders, blood disorders including anemia, and respiratory problems. Patients often live into their early teens and into adulthood.

[0347] According to a particular embodiment, GD is of type 1.

[0348] According to one embodiment, a genetically modified human GCase of a certain embodiment of the present invention is used for enzyme replacement therapy.

[0349] As used herein, “enzyme replacement therapy (ERT)” refers to the exogenous administration of β-glucocerebrosidase (GCase).

[0350] According to certain embodiments, genetically modified human GCase treatment is combined with a substrate reduction therapy agent.

[0351] As used herein, the term “substrate reduction therapy (SRT) agent” refers to a drug (e.g., a small molecule) that inhibits the synthesis of GCase, i.e., the natural substrate of glucosylceramide (or GL1). Several versions of SRTs approved by insurance regulatory authorities are commercially available. Examples include, but are not limited to, miglustat (Zavesca. RTM.) and eliglustat tartrate.

[0352] It is anticipated that during the term of this patent, which expires from this application, many appropriate SRTs will be developed, and that the scope of the term SRT will deductively be intended to include all such novel technologies.

[0353] As used herein, the term "approximately" refers to ±10%.

[0354] The terms "comprises," "comprising," "includes," "including," "having," and their cognates all mean "to include, but not limited to."

[0355] The term "consisting of" means "including and limited to these."

[0356] The term "essentially consisting of" means that the composition, method, or structure may include further components, steps, and / or parts, but the further components, steps, and / or parts do not materially alter the fundamental and novel features of the claimed composition, method, or structure.

[0357] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, the term “compound” or “at least one compound” may include multiple compounds, including mixtures thereof.

[0358] Throughout this application, various embodiments of the present invention may be presented in the form of a range. It should be understood that the range form is merely for convenience and brevity and should not be interpreted as an immutable limitation of the scope of the present invention. Therefore, a range description should be understood to include all possible subranges specifically disclosed and the individual numerical values ​​within those ranges. For example, a range description such as 1 to 6 should be understood to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0359] Whenever a numerical range is shown herein, it means that it includes any cited number (fraction or integer) within the range shown. The phrases “in range” between the first specified number and the second specified number, and “in range” from the first specified number to the second specified number, are used interchangeably herein and mean that they include the first and second specified numbers as well as all fractions and integers between them.

[0360] As used herein, the term “method” means a method for performing a given task, including, but not limited to, methods, means, techniques and techniques that are publicly known to technicians in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or that are readily developed by them from known methods, means, techniques and techniques.

[0361] For clarity, it is understood that certain characteristics of the Invention described in the context of a separate embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various characteristics of the Invention described in the context of a single embodiment may also be provided separately, in any preferred sub-combination, or as preferred in any other described embodiment of the Invention. Certain characteristics described in the context of various embodiments should not be considered essential characteristics of that embodiment unless the embodiment is carried out without its components.

[0362] Various embodiments and aspects of the present invention described above and claimed in the following claims section will find experimental support in the following examples.

[0363] It is understood that any sequence identifier (SEQ ID NO) disclosed in this application may refer to either a DNA sequence or an RNA sequence, depending on the context in which the SEQ ID NO is stated, even if it is expressed only in DNA sequence format or RNA sequence format. For example, SEQ ID NO: 1 is expressed in DNA sequence format (e.g., T represents thymine), but it may refer to either a DNA sequence corresponding to a GCase nucleic acid sequence or an RNA sequence of an RNA molecule nucleic acid sequence. Similarly, some sequences may be expressed in RNA sequence format (e.g., U represents uracil), depending on the actual type of molecule being described, but they may refer to either a sequence of an RNA molecule containing dsRNA or a sequence of a DNA molecule corresponding to the RNA sequence being shown. In any case, both DNA and RNA molecules having the disclosed sequences, including any substitutions, are depicted. [Examples]

[0364] The present invention will be described in a non-limiting manner with reference to the following examples, along with the above description.

[0365] In general, the scientific nomenclature used herein and the research methods utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA technologies. Such technologies are thoroughly described in the literature. For example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Volumes I-III, edited by Ausubel, RM, (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinat DNA," Scientific American Books, New York; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual Series," Volumes 1-4, Cold Spring Harbor Laboratory Press, New York. (1998); Methodologies described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook," Vols. I-III, edited by Cellis, JE. (1994); "Current Protocols in Immunology," Vols. I-III, edited by Coligan, JE. (1994); Stites et al. (eds.), "Basic and Clinical Immunology" (Vol. 8), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Selected Methods in Cellular Immunology," WH Freeman and Co.New York (1980); available immunoassays widely described in patents and scientific literature, e.g., U.S. Patents No. 3,791,932; No. 3,839,153; No. 3,850,752; No. 3,850,578; No. 3,853,987; No. 3,867,517; No. 3,879,262; No. 3,901,654; No. 3,935,074; No. 3,984,533; No. 3,996,345; No. 4,034,074; No. 4,098,876; No. 4,879,219; No. 5,011,771 and No. 5,281,521; “Oligonucleotide Synthesis” Gait, MJ ed., (1984); “Nucleic Acid See "Hybridization," Hames, BD, and Higgins, SJ (eds.), (1985); "Transcription and Translation," Hames, BD, and Higgins, SJ (eds.), (1984); "Animal Cell Culture," Freshney, RI (ed.), (1986); "Immobilized Cells and Enzymes," IRL Press, (1986); "A Practical Guide to Molecular Cloning," Perbal, B., (1984) and "Methods in Enzymology," Vols. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications," Academic Press, San Diego, CA (1990); and Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996); all of these are incorporated by reference as if they were fully described herein. Other general references are provided throughout this document. The methods described herein are considered to be well-known in the relevant technical field and are provided for the convenience of the reader. All information contained herein is incorporated herein by reference.

[0366] General materials and experimental techniques material Dulbecco's modified Eagle medium and fetal bovine serum were obtained from Gibco. Penicillin, streptomycin, and sodium pyruvate for cell culture were obtained from Biological Industries. Anti-DYKDDDDK G1 affinity resin, DYKDDDDK peptide (SEQ ID NO: 16), and anti-DYKDDDDK tagged antibody [HRP] were obtained from GenScript. Nickel beads were obtained from Adar Biotech. Strep-Tactin® XT 4Flow high-performance resin, StrepMAB-Classic HRP (anti-Strep) antibody, and biotin were purchased from IBA GmbH, Germany. Rabbit-produced anti-GCase (C-terminal) antibody, mouse-produced monoclonal anti-polyhistidine-peroxidase antibody, polyethyleneimine, defatted bovine serum albumin, protease inhibitor cocktail, deoxyribonuclease, Nonidet® P 40 substituent (NP-40), and p-nitrophenyl-β-D-glucopyranoside were all purchased from Sigma-Aldrich. SDS-PAGE and Western blotting equipment were supplied by BioRad. Recombinant human glucosylceramidase (WT GCase), expressed in CHO cells, was obtained from R&D Systems, Minneapolis, USA. Imiglucerase (Cerezyme®, Sanofi Genzyme) was obtained as a remainder from patient treatment.

[0367] Generation of more stable forms of GCase See the Examples section below.

[0368] GCase expression in E. coli and subsequent purification The sequences were generated using PROSS, namely D2, D4, D6, and D7, and the wild-type (WT) and four mutant variants of human GCase were all expressed in E. coli as the pET28-bD-SUMO [Zahradnik J. et al., FEBS J. (2019) 286: pp. 3858-3873] construct, which also included the N-terminal hist-tag for purification. The expressed GCase was then subjected to standard Ni 2+ GCase was isolated from E. coli lysates using chelate chromatography, and the bound GCase was released from the column using SUMOprotease [Frey S. and Gorlich D., J. Chromatogr. A. (2014) 1337: pp. 95-105]. Protein purity was evaluated on a 10% tris-glycine SDS-PAGE gel stained with Coomassie blue (Instant blue, Expedeon). GCase was identified by Western blotting and mass spectrometry (MS) using anti-GCase and anti-his-tag antibodies.

[0369] Gene transfer in cell culture and eukaryotic cells Human GCase wild-type (WT) and D7 variant DNA sequences were cloned into pCDNA3.1 (Invitrogen) vectors along with an N-terminal FLAG tag for purification (Figure 2A). HEK293T cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 100 IU / ml penicillin, 100 μg / ml streptomycin, and 110 μg / ml sodium pyruvate. Cells were transfected using polyethyleneimine reagent and 10 μg of plasmid per 10 cm culture dish. Cells and growth medium were collected 36–48 hours after transfection.

[0370] GCase purification GCase was isolated from either the cell pellet or growth medium using anti-DYKDDDDK affinity resin (FLAG beads) and Strep-Tactin® XT resin (Strep beads). The growth medium was transferred to a 250 ml tube and centrifuged at 10,000 g at 4°C for 20 minutes. A suspension of FLAG beads or Strep beads in 200 μl of 150 mM NaCl / 50 mM Tris, pH 7.4 was added to a 50 ml Falcon tube filled with growth medium and left on a rotator overnight at 4°C to allow GCase to bind to the beads. The cell pellet was dissolved by sonication in the same Tris buffer containing 1% Np-40, a protease inhibitor cocktail (1:500), and deoxyribonuclease (1:200). The lysate was centrifuged at 16,000 g at 4°C for 20 minutes. The pellet was discarded, and FLAG beads were added to the supernatant (50-150 μl of bead suspension per 1-5 ml of supernatant). The mixture was rotated at 4°C for a minimum of 2 hours. Then, either the FLAG or Strep beads were washed with an excess of Tris buffer. GCase containing the FLAG tag was released by competitive elution in three consecutive elution steps using DYKDDDDK peptide (FLAG peptide, SEQ ID NO: 16) dissolved in sodium citrate buffer (10.4 g trisodium citrate, 3.6 g disodium hydrogen citrate dissolved in 1 l of redistilled water, 187 mM D-mannitol, and 0.1% (v / v) ml Tween 80, pH adjusted to 6.1 using citrate) as the elution ligand. The protein was further purified and stored in sodium citrate buffer. The eluted fractions were combined and subjected to size exclusion chromatography (SEC) on a Superdex 200 column. Fractions corresponding to monomeric peaks were collected and concentrated using an Amicon Ultracentrifugal filter (10 kDa cutoff, Merck Millipore). Protein concentration was determined from absorbance at 280 nm, and the extinction coefficient was calculated based on the amino acid sequence composition (ε). D7-GCase =108 290M -1 cm -1 ;ε WT-GCase=95 800M -1 cm -1 Purity was evaluated using a 10% Tris-glycine SDS-PAGE gel stained with Coomassie blue (Instant blue, Expedeon). GCase was identified by Western blotting and mass spectrometry (MS) using anti-GCase, anti-Strep, and anti-DYKDDDDK antibodies.

[0371] Differential scanning fluorescence quantification Differential scanning fluorescence (DSF) was performed using a NanoDSF Prometheus NT.48 instrument (NanoTemper, Germany). The sample was heated at a rate of 1°C / min within the temperature range of 20–95°C. Tyrosine and tryptophan fluorescence emission was recorded at 330 nm and 350 nm. The data was analyzed using a PR.ThermControl v2.1.1 instrument (NanoTemper, Germany). Melting temperature (T m ) is the fluorescence intensity (FI) ratio curve (R(FI)=FI 350nm / FI 330nm It was defined as the inflection point of ).

[0372] Enzyme activity assay using synthetic substrate (p-NP-Glc) The specific enzyme activity was determined using p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc) as a substrate. The reaction was stopped by raising the pH to 10, and at that pH, the released p-nitrophenol was completely ionized and refracted at 20,000 M at 405 nm. -1 cm -1 This shows the molar absorption.

[0373] The activity assay was adapted from Wei et al. [Wei RR et al., J. Biol. Chem. (2011) 286: pp. 299-308]. Briefly, an aliquot of the enzyme was incubated with 0.2-4 mM p-NP-Glc in 0.1% BSA / 0.125% sodium taurocholate / 0.162% Triton X-100 / 0.02% sodium azide / 0.1M potassium phosphate, pH 5.9 at 25°C for 60 minutes. The reaction was stopped with 1 M glycine buffer diluted 20-50 times, pH 10, and the absorbance of p-nitrophenol was measured at 405 nm in a 1 cm cuvette using an Agilent Cary 3500 spectrophotometer (Agilent Technologies, USA). The absorbance values ​​were converted to p-nitrophenol concentrations, a Michaeli-Smenten plot was constructed, and the results were approximated using Origin software (OriginLab). The V obtained from the approximation was... max The value of expression k cat =V max k cat It was converted to a value.

[0374] Enzyme activity assay using C6NBD GlcCer The variants were tested for enzyme activity using a fluorescently labeled natural substrate for GCase (NBD glucosylceramide (d18:1 / 6:0) (C6NBD GlcCer)).

[0375] The protein preparation was incubated with 20 μM C6-NBD-GlcCer, 20 μM defatted BSA in 50 mM MES buffer, pH 5.5 at 37 °C for 5 min. The reaction was terminated by the addition of 750 μl chloroform / methanol (1:2, v / v), followed by the addition of 500 μl chloroform and 730 μl redistilled water. The sample was vortexed vigorously and centrifuged at 2,000 g for 10 min. The upper phase was aspirated and the lower phase containing the extracted lipids was dried under a stream of N2. The lipids were resuspended in chloroform / methanol (9:1, v / v) and separated by TLC using chloroform / methanol / 9.8 mM CaCl2·2H2O (65 / 30 / 8, v / v / v) as the developing solvent. The NBD-labeled lipids were visualized using a Typhoon 9410 variable mode imager and quantified using ImageQuantTL (GE Healthcare, Chalfont St Giles, UK). The activity value was calculated as μmol of substrate converted to product by 1 mg of enzyme per minute (μmol·mg -1 .min -1 ).

[0376] (Example 1) Variants generated by PROSS Six GCase variants (designs 2-7, i.e., D2-7, described in SEQ ID NOs. 4, 6, 8, 10, 12, and 14 respectively; see Figure 1A) were designed using the PROSS algorithm, which has already been successfully used to improve the expression levels and stability of several other proteins [incorporated herein by reference; see PCT / IL2016 / 050812 and Goldenzweig A. et al., Mol. Cell. (2016) 63: pp. 337-346]. Four of these variants, D2, D4, D6, and D7, were expressed in E. coli and tested for enzyme activity using the synthetic substrate p-NP-Glc (data not shown). The sequence of the WT GCase is shown in Figure 1B (SEQ ID NO: 2), with underlining indicating the mutation occurring in the D7 variant (SEQ ID NO: 14), which produces the largest number of mutations, i.e., 30. It is also the construct that exhibits the highest enzyme activity. For further research, the D7 variant was expressed in HEK293T cells, which have the ability to glycosylate proteins.

[0377] (Example 2) Expression and purification of variant D7 GCase Both WT hGCase and the D7 variant were expressed in HEK293T cells and isolated from either the cell pellet (intracellular) or the culture medium (secreted). Both WT and D7 GCase were expressed intracellularly, but the D7 variant showed higher expression than WT. Using Coomassie blue staining, SDS-PAGE of three elution fractions obtained from individual preparations is shown in Figures 2B and 2C, and GCase was identified as the dominant band (indicated by arrows) by Western blotting and MS. Only D7 GCase was secreted. A highly purified preparation of the secreted D7 GCase was obtained by one-step purification using FLAG beads (Figure 2C).

[0378] Next, the sample was applied to a Superdex200 column. SEC revealed significant oligomerization of the secreted D7 GCase (Figures 3A-3B). Similar patterns were observed for intracellular WT and D7 GCase. The monomer location was established by calibrating the column with a molecular weight marker to the peak with maximum absorbance at approximately 15 ml (Peak 1, Figures 3A-3B). The monomer peak also indicated that it was the fraction with the highest specific activity. The fractions corresponding to the monomers were pooled, concentrated, and used for stability and activity assays. The data presented below are for D7 monomers obtained from the secreted fraction by two-step purification. Due to the extremely low yield of secreted WT GCase, the data obtained for D7 GCase were compared with similar data for recombinant WT GCase expressed in CHO cells obtained from R&D Systems, and for Cerezyme®, a WT sequence produced by Sanofi Genzyme, which has a single Arg495His substitution.

[0379] (Example 3) Thermal stability of variant D7 GCase Melting temperature of D7 GCase (T m The following was determined using differential scanning fluorescence (DSF). DSF measures the changes in fluorescence of tyrosine and tryptophan residues in the protein unfolded state, which leads to their exposure to an aqueous solution environment.

[0380] Average T for the analyzed preparations m The values ​​are shown in Table 1 below. T values ​​for D7 GCase at 71.8±2.4℃ and 61.4±1.9℃. mValues were determined in Tris buffer, pH 7.4, and citrate buffer, pH 6.1, respectively. D7 GCase showed higher thermal stability than WT GCase. The increase in stability was about 20 °C and about 11 °C at pH 7.4 and pH 6.1, respectively, compared to Cerezyme®. Using DSF, the values obtained for Cerezyme® and WT GCase were in good agreement with the previously reported T m values obtained for Cerezyme® by differential scanning calorimetry, i.e., 51.30 ± 0.02 °C at pH 7.1 and 57.67 ± 0.04 °C at pH 5.4 [supra, Wei R.R. et al., J. Biol. Chem. (2011) 286: 299 - 308].

[0381]

Table 1

[0382] (Example 4) Specific activity of variant D7 GCase By fitting the Michaelis - Menten equation to the experimental data, K m and k cat values could be obtained (Table 2 below, representative enzyme kinetic data are shown in Figure 4). The overall catalytic efficiency of various preparations was compared based on the bimolecular rate constant k cat / K m . The data obtained showed that the catalytic activities of D7 and WT were not significantly different, being 0.28 × 10 6 and 0.27 × 10 6 M -1 min -1 respectively. Thus, this data verified the protocol used and the comparative analysis using the artificial substrate p - nitrophenyl - β - D - glucopyranoside.

[0383]

Table 2

[0384] (Example 5) Further variants generated by PROSS Three further variants of glucosylceramidase (GCase) (D13, D14, and D15) were designed using the PROSS algorithm, which has been previously used successfully to improve the expression levels and stability of several other proteins, including GCase (design D7) [see PCT / IL2016 / 050812 and Goldenzweig A. et al., Mol. Cell. (2016), cited above, incorporated herein by reference]. GCase variants D13, D14, and D15 were expressed in HEK293T cells and isolated from the culture medium. Highly purified preparations of the GCase designs were obtained by one-step purification using a FLAG tag (DYKDDDDK tag, SEQ ID NO: 16) or a TwinStrep® tag (SEQ ID NO: 29). All variants were tested for enzyme activity using a fluorescently labeled analog of GCase (NBD glucosylceramide (d18:1 / 6:0) (C6NBD GlcCer)) (data not shown). The design with the highest enzyme activity, i.e., variant D15 GCase (as described in Figures 5A-5B), was used for further characterization. As discussed below, the thermal stability and enzyme activity of the novel D15 GCase were compared with the already characterized D7 GCase and Cerezyme®, a Sanofi Genzyme product with a WT sequence containing a single Arg495His substitution. All enzymes dissolved in sodium / citrate buffer containing 187 mM D-mannitol and 0.1% (v / v) Tween 80, pH 6.1 were maintained at 4°C.

[0385] (Example 6) Thermal stability of variant D15 GCase Melting temperature of D15 GCase (T mThe melting temperature was determined using differential scanning fluorescence (DSF). DSF measures the change in fluorescence of tyrosine and tryptophan residues in the protein unfolded state, which leads to their exposure to an aqueous solution environment. Experiments were performed using enzymes dissolved in citrate buffer, pH 6.1 (as discussed above). D7 GCase has already been shown to have a melting temperature approximately 10°C higher than Cerezyme®. A further increase in melting temperature was observed for the variant D15 GCase. The average T measured for D15 GCase was m The value was 17°C higher compared to Cerezyme®, which reflects a substantial increase in protein thermal stability (see Table 3 below). m The values ​​were obtained by differential scanning calorimeter, as previously reported T m The value of 51.30±0.02℃ at pH 7.1 was in good agreement [see above, Wei RR et al., J. Biol. Chem. (2011) 286: pp. 299-308].

[0386] [Table 3]

[0387] (Example 7) Specific activity of variant D15 GCase Specific activity was determined by two approaches: (i) using a fluorescently labeled native substrate of GCase C6NBD GlcCer (at pH 5.5), and (ii) using the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc) (at pH 5.9). In the first approach, the substrate and product were separated by thin-layer chromatography and quantified by NBD fluorescence. In the latter, the substrate was spectroscopically quantified by the absorption of the generated p-nitrophenyl at 405 nm. The specific enzyme activity determined by both substrates was equivalent to that of commercially available Cerezyme® and D15 GCase. Activity values ​​were expressed as μmol of substrate converted to product by 1 mg of enzyme in 1 minute (μmol / mg). -1 .minutes -1 The following calculations were performed as shown in Table 4A (Table 4). The substrate concentrations were 20 μM for the C6NBD GlcCer assay and 0.4, 1.5, and 3 mM for the p-Np-Glc assay (Figure 6).

[0388] [Table 4]

[0389] Further experiments were conducted to compare the enzyme kinetics of WT GCase, commercially available Cerezyme®, D7 GCase, and D15 GCase. By fitting the Michaelis-Menten curve to the measured data points, kinetic parameters for the activity of GCase preparations of p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc) were obtained. Based on the experimental data, K m and k cat It became possible to obtain the value (see Table 4B (Table 5) below). The overall catalytic efficiency k of various preparations. cat / K m These were compared based on the steady state of bimolecular rates. The obtained data showed that the catalytic activity of D15 was equivalent to that of Cerezyme®, and their k cat / K m The values ​​are, respectively, 1.49 × 10 6 M-1 minutes -1 and 1.53 × 10 6 M -1 minutes -1 On the other hand, the catalytic activity of D7 is equivalent to that of WT GCase, and their k cat / K m The values ​​are, respectively, 0.27 × 10 6 M -1 minutes -1 and 0.28 × 10 6 M -1 minutes -1 This demonstrated that...

[0390] [Table 5]

[0391] (Example 8) Further GCase variant-D16 A further variant (D16) of the glucosylceramidase enzyme (GCase) was designed using the PROSS algorithm, which has been previously used successfully to improve the expression levels and stability of several other proteins, including GCases (designs D7 and D15) [see Goldenzweig A. et al., (2016), cited above, incorporated herein by reference]. D16 GCase was expressed in HEK293T cells and isolated from the culture medium. Highly purified preparations of the GCase designs were obtained by one-step purification using the TwinStrep® tag (SEQ ID NO: 29).

[0392] As discussed below, the thermal stability and enzyme activity of the novel D16 GCase were compared with the previously characterized D15 GCase purified using the TwinStrep® tag (SEQ ID NO: 29), and with Cerezyme®, a WT sequence produced by Sanofi Genzyme, which has a single Arg495His substitution. All enzymes dissolved in sodium / citrate buffer containing 187 mM D-mannitol and 0.1% (v / v) Tween 80, pH 6.1 were maintained at 4°C.

[0393] (Example 9) Thermal stability of variant D16 GCase Melting temperature of GCase (T m The following was determined using differential scanning fluorescence (DSF). DSF measures the changes in fluorescence of tyrosine and tryptophan residues in the protein unfolded state, which leads to their exposure to an aqueous solution environment.

[0394] The experiment was conducted using enzymes dissolved in citrate buffer and pH 6.1 (the storage buffer described above). The T values ​​for GCase D15 had already been measured. m The value was approximately 17°C higher compared to Cerezyme®, which reflects a substantial increase in protein thermal stability (see Table 3 above and Table 6 below). The novel GCase D16 is T m A slight increase was observed, namely 2.5°C compared to GCase D15. The T measured here for Cerezyme® was also observed. m The values ​​were obtained by differential scanning calorimeter, as previously reported T m The values ​​were in good agreement, with a temperature of 51.30±0.02℃ at pH 7.1 [see above, Wei RR et al., J. Biol. Chem. (2011) 286: pp. 299-308].

[0395] [Table 6]

[0396] (Example 10) Specific activity of variant D16 GCase Specific activity was determined using the artificial substrate p-nitrophenyl-β-D-glucopyranoside (p-NP-Glc) (at pH 5.9). In this assay, the substrate was quantified spectroscopically by the absorption of the generated p-nitrophenyl at 405 nm. The substrate concentration was 3 mM. The specific enzymatic activity of the novel GCase D16 variant was higher than that determined for the commercially available Cerezyme® and equivalent to that of GCase D15. The activity value was expressed as the amount of substrate converted to the product by 1 mg of enzyme in 1 minute (μmol / mg). -1 .minutes -1 The calculations were performed as shown in Table 6 (Table 7) below.

[0397] [Table 7]

[0398] Although the present invention is described in conjunction with its specific embodiments, it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications, and variations, which are in the spirit and broad scope of the appended claims.

[0399] All publications, patents, and patent applications described herein are incorporated herein in their entirety to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated herein by reference. In addition, any citation or identification of references in this application shall not be construed as an acknowledgment that such references are available as prior art of the present invention. To the extent that section titles are used, they shall not be construed as essential limitations. In addition, any priority documents of this application are incorporated herein in their entirety by reference.

Claims

1. A genetically modified human β-glucocerebrosidase (GCase) as described in Sequence ID No. 14, 22, or 27.

2. An isolated polynucleotide comprising a nucleic acid sequence encoding a genetically modified human GCase as described in claim 1.

3. An isolated polynucleotide according to claim 2, comprising the nucleic acid sequence described in any one of sequence numbers 13, 21, or 26.

4. A nucleic acid construct comprising an isolated polynucleotide according to claim 2 or 3, and a cis-acting regulatory element for directing the expression of the nucleic acid sequence in a cell.

5. The nucleic acid construct according to claim 4, wherein the cis-acting control element includes a promoter.

6. An isolated cell comprising the polynucleotide described in claim 2 or 3, or the construct described in claim 4 or 5.

7. A pharmaceutical composition comprising, as an active ingredient, a genetically modified human GCase as described in claim 1, an isolated polynucleotide as described in claim 2 or 3, a construct as described in claim 4 or 5, or a cell as described in claim 6, and a pharmaceutically acceptable carrier or diluent.

8. A therapeutically effective amount of the genetically modified human GCase according to claim 1, the isolated polynucleotide according to claim 2 or 3, the construct according to claim 4 or 5, or the cell according to claim 6, for use in the treatment of β-glucocerebrosidase deficiency and related diseases in subjects requiring it.

9. The disease associated with β-glucocerebrosidase deficiency is Gaucher disease, according to claim 8, a therapeutically effective amount of genetically modified human GCase, isolated polynucleotide, construct or cell for use.

10. The subject is human, and the therapeutically effective amount of genetically modified human GCase, isolated polynucleotide, construct, or cell for use according to claim 8 or 9.

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