Variants of beta-glucocerebrosidase for use in treating Gaucher Disease

Abstract

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

Description

of of of catalyzing catalyzing catalyzing catalyzing According to a specific embodiment, the GCase polypeptide is capable of catalyzing hydrolysis of p-NP-Glc by at least about 1.8 x 106 kCat / Km (M^min1). Methods of measuring catalytic efficiency of the GCase polypeptides described herein are known in the art and include, for example, in situ activity assay (using e.g., a substrate applied on the cells containing an active enzyme), in vitro activity assays (in which the activity of a particular enzyme is measured in a protein mixture extracted from the cells). For example, an enzyme activity assay may be performed usingp-nitrophenyl-P-D-glucopyranoside (p-NP-Glc) as the substrate [discussed in Wei R.R. et al., J. Biol. Chem. (2011) 286: 299-308, incorporated herein by reference]. As discussed in the Examples section below (see Examples 3, 6 and 9, below), GCase variants D7, D15 and D16 comprise a higher thermal stability as compared to wild-type GCase and to Cerezyme®. Specifically, at pH 6.1, D7 GCase showed an increase of about 6-7 °C when compared to wild-type GCase and of about 11 °C when compared to Cerezyme® (see Table 1, below). At the same pH level, D15 GCase showed a substantial increase of about 12-13 °C when compared to wildtype GCase and a substantial increase of about 17 °C when compared to Cerezyme® (see Tables 1 and 3, below). Similarly D16 GCase showed a substantial increase of about 19 °C when compared to Cerezyme® (see Table 5, below). According to one embodiment, the GCase polypeptide comprises a thermal stability in a temperature range 3-22 °C (e.g. 15-22 °C, 10-20 °C, 5-15 °C, 7-13 °C, 9-11 °C) higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). The terms “thermal stability” or “increased thermal stability” relative to the wild-type polypeptide means that the GCase polypeptide comprises increased heat stability, i.e. the ability to resist denaturation with increasing temperature. Standard techniques to quantify thermal stability are known in the art, including but not limited to circular dichroism, differential scanning calorimetry and surface plasmon resonance. Methods of measuring thermal stability of the GCase polypeptides described herein are known in the art and include, for example, enzyme stability assay as discussed in Wei R.R. et al., J. Biol. Chem. (2011) 286: 299-308 (incorporated herein by reference). According to one embodiment, the GCase polypeptide comprises a thermal stability under a temperature being 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 compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 5 °C higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 7 °C higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 9 °C higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 11 °C higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 13 °C higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 15 °C higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 17 °C higher compared to a wild-type polypeptide under the same conditions (e.g. at a pH of 6.1). According to one embodiment, the GCase polypeptide comprises a thermal stability under a temperature range being 3-22 °C (e.g. 15-22 °C, 10-20 °C, 5-15 °C, 7-13 °C, 9-11 °C) higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). The term “Cerezyme®” also referred to as Imiglucerase is a commercial drug for enzyme replacement therapy. According to one embodiment, the GCase polypeptide comprises a thermal stability under a temperature being 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 compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 5 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 7 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 9 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 11 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 13 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 15 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 17 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 19 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 6.1). According to one embodiment, the GCase polypeptide comprises a thermal stability under a temperature range being 15-25 °C (e.g. 17-23 °C, 19-23 °C) higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 7.4). According to one embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 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 compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 7.4). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 15 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 7.4). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 17 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 7.4). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 20 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 7.4). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 22 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 7.4). According to a specific embodiment, the GCase polypeptide comprises a thermal stability under a temperature being about 25 °C higher compared to a Cerezyme® polypeptide under the same conditions (e.g. at a pH of 7.4). As mentioned above, the GCase polypeptide is highly expressed and secreted from eukaryotic cells. Expression in eukaryotic cells enables glycosylation of GCase, which is vital for GCase activity. According to another embodiment, the GCase polypeptide is secreted from eukaryotic cells as compared to a wild-type polypeptide not being secreted under the same culture conditions. According to one embodiment, the GCase polypeptide is secreted about 1.5-10 times (e.g. about 1.5-2 times, about 2-3 times, about 3-4 times, about 4-5 times, about 5-6 times, about 6-7 times, about 8-9 times, about 9-10 times) higher from eukaryotic cells (e.g. from HEK293T cells), as compared to a wild-type GCase under the same culture conditions. According to one embodiment, the GCase polypeptide is secreted about 3 times higher from eukaryotic cells (e.g. from HEK293T cells), as compared to a wild-type GCase under the same culture conditions. According to one embodiment, the GCase polypeptide is secreted about 5 times higher from eukaryotic cells (e.g. from HEK293T cells), as compared to a wild-type GCase under the same culture conditions. According to one embodiment, the GCase polypeptide is secreted about 10 times higher from eukaryotic cells (e.g. from HEK293T cells), as compared to a wild-type GCase under the same culture conditions. According to one embodiment, the GCase polypeptide comprises about 1.3-5 times (e.g. about 1.3-2 times, about 1.3-3 times, about 2-3 times, about 2-4 times, about 3-4 times, about 4-5 times) higher intracellular expression level in eukaryotic cells as compared to a wild-type human GCase under the same culture conditions (e.g. from HEK293T cells). According to one embodiment, the GCase polypeptide comprises at least about 1.3 times higher intracellular expression level in eukaryotic cells as compared to a wild-type polypeptide under the same culture conditions. According to one embodiment, the GCase polypeptide comprises about 2 times higher intracellular expression level in eukaryotic cells as compared to a wild-type human GCase under the same culture conditions (e.g. from HEK293T cells). According to one embodiment, the GCase polypeptide comprises 3 times higher intracellular expression level in eukaryotic cells as compared to a wild-type human GCase under the same culture conditions (e.g. from HEK293T cells). According to one embodiment, the GCase polypeptide comprises about 4 times higher intracellular expression level in eukaryotic cells as compared to a wild-type human GCase under the same culture conditions (e.g. from HEK293T cells). As used herein, the phrases “level of expression” and “expression level” refers to the degree of gene expression and / or gene product activity in a biological sample (e.g. eukaryotic cell). It should be noted that the level of expression can be determined in arbitrary absolute units, or in normalized units (relative to known expression levels of a control reference). According to one embodiment, the secretion level of the GCase polypeptide from eukaryotic cells may be higher by at least about 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or 100 % as compared to that of the wild-type polypeptide under the same culture conditions. According to one embodiment, the intracellular expression level of the GCase polypeptide in eukaryotic cells may be higher by at least about 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or 100 % as compared to that of the wild-type polypeptide under the same culture conditions. According to specific embodiments the amount of expression is determined using an RNA and / or a protein detection method. Non-limiting examples of methods of detecting the level of RNA expressed in cells include Northern Blot analysis, RT-PCR analysis, RNA in situ hybridization stain, and in situ RT-PCR stain. Non-limiting examples of methods of detecting the level and / or activity of specific protein molecules in a cell include Enzyme linked immunosorbent assay (ELISA), Western blot analysis, immunoprecipitation (IP), radio-immunoassay (RIA), Fluorescence activated cell sorting (FACS), immunohistochemical analysis, in situ activity assay (using e.g., a substrate applied on the cells containing an active enzyme), in vitro activity assays (in which the activity of a particular enzyme is measured in a protein mixture extracted from the cells) and molecular weight-based approach. In case the detection of the expression level of a secreted protein is desired, ELISA assay may be performed on a cell medium of in which the cells have been cultured (i.e. which contains cell-secreted content). According to one embodiment, there is provided an isolated cell comprising at least one exogenous polynucleotide or construct (as discussed above). According to one embodiment, the isolated cell is a eukaryotic cell (as discussed above). The genetically modified human GCase of some embodiments, the isolated polynucleotide of some embodiments, the construct of some embodiments, or the cell of some embodiments of the invention, can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term "active ingredient" refers to the genetically modified human GCase accountable for the biological effect. Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which may be interchangeably used, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections. Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol 39 solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions that can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (genetically modified human GCase) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., Gaucher Disease), or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans. For example, any in vivo or in vitro assay of GCase activity may be employed such as utilizing the animal models for Gaucher disease discussed in Farfel-Becker et al. [Farfel-Becker, Vitner and Futerman, Dis Model Meeh. (2011) 4(6): 746-752]. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or in experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above. It will be appreciated that the kit may further comprise another therapeutic composition for treating Gaucher Disease, e.g. an agent for substrate reduction therapy (SRT). The genetically modified human GCase of some embodiments of the invention, the polynucleotides encoding same, the expression constructs used for their expression, or the cells of some embodiments of the invention, can be used for treating a disease associated with P-glucocerebrosidase deficiency in a subject in need thereof. The terms “treating” and “treatment” as used herein refer to abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially delaying the appearance of clinical symptoms of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition. The term “treating” as used herein further refers to extending survival or delaying death of patients inflicted with a condition. As used herein the phrases “subject” and “subject in need thereof’ which are interchangeably used herein, refer to a mammal, preferably human beings at any age or gender that suffer from the pathology. This term encompasses individuals who are at risk to develop the pathology. The subject may include e.g. neonatal, infant, juvenile, adolescent, adult and elderly adult. According to one embodiment, the subject has been diagnosed with a disease associated with the GBA gene. According to one embodiment, the subject has been diagnosed with a disease associated with reduced P-glucocerebrosidase levels and / or activity. According to one embodiment, the subject has been diagnosed with a disease associated with P-glucocerebrosidase deficiency. Exemplary diseases associated with P-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. According to a specific embodiment, the disease associated with P-glucocerebrosidase deficiency is Gaucher Disease. The terms “Gaucher's disease”, “Gaucher disease” or “GD” as interchangeably used herein, refer to a lysosomal storage disease (LSD) characterized by accumulation of glucosylceramide (GlcCer, also known as glucocerebroside) in cells, particularly in cells of the mononuclear cell lineage. Glucosylceramide can collect in the spleen, liver, kidneys, lungs, brain and bone marrow. The disease is typically caused by a deficiency of the enzyme glucocerebrosidase (also known as betaglucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, GCD or GCase; EC 3.2.1.45), a lysosomal enzyme with glucosylceramidase activity that is needed to catalyze the hydrolysis of glucosylceramide / GlcCer. GD is divided into two major types: neuropathic and non-neuropathic disease, based on the particular symptoms of the disease. In non-neuropathic disease most organs and tissues can be involved, but not the brain. In neuropathic disease (nGD) the brain is also involved. Type I (or non-neuropathic type, GDI) is the most common form of the disease, occurring in approximately 1 in 50,000 live births. It occurs most often among persons of Ashkenazi Jewish heritage. Symptoms may begin early in life or in adulthood, and include enlarged liver and grossly enlarged spleen (known together as 'hepatosplenomegaly'); the spleen can rupture and cause additional complications. Spleen enlargement and bone marrow replacement cause anemia, thrombocytopenia and leukopenia. Skeletal weakness and bone disease may be extensive. The brain is not affected pathologically, but there may be lung and, rarely, kidney impairment. Patients in this group usually bruise easily (due to low levels of platelets) and experience fatigue due to low numbers of red blood cells. Depending on disease onset and severity, GD type 1 patients may live well into adulthood. Some patients have a mild form of the disease or may not show any symptoms. Neuropathic GD (nGD) as used herein encompasses both Type 2 and Type 3 GD. GD type 2, also referred to as acute infantile neuropathic GD, typically begins within 6 months of birth and has an incidence rate around one 1 in 100,000 live births. Symptoms include an enlarged liver and spleen, extensive and progressive brain damage, eye movement disorders, spasticity, seizures, limb rigidity, and a poor ability to suck and swallow. Affected children usually die by age two. GD type 3, also referred to as chronic neuropathic GD, can begin at any time in childhood or even in adulthood, and occurs in about one in 100,000 live births. It is characterized by slowly progressive, but milder neurologic symptoms compared to the acute or type 2 version. GD Type 3 has been divided into two variants, termed Types 3b and 3a. Type 3b has earlier onset of massive livers and spleens and the patients can also experience direct involvement of the lungs and rapidly progressive bony disease. Major symptoms include an enlarged spleen and / or liver, seizures, poor coordination, skeletal irregularities, eye movement disorders, blood disorders including anemia, and respiratory problems. Patients often live into their early teen years and adulthood. According to a specific embodiment, the GD is type 1. According to one embodiment, the genetically modified human GCase of some embodiments of the invention is used for enzyme replacement therapy. As used herein "enzyme replacement therapy (ERT)" refers to the exogenous administration of P-glucocerebrosidase (GCase). According to specific embodiments, the genetically modified human GCase treatment is combined with a substrate reduction therapy agent. As used herein, the term "Substrate reduction therapy (SRT) agent" refers to an agent (e.g. small molecule) that inhibits the synthesis of the natural substrate of the GCase, i.e. glucosylceramide (or GL1). A number of health regulatory agency-approved versions of SRT are available on the market. Examples include, but are not limited to, Miglustat (Zavesca.RTM.) and Eliglustat Tartrate. It is expected that during the life of a patent maturing from this application many relevant SRT will be developed and the scope of the term SRT is intended to include all such new technologies a priori. As used herein the term “about” refers to + 10 %. The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of’ means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably, and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or an RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or an RNA sequence format. For example, SEQ ID NO: 1 is expressed in a DNA sequence format (e.g., reciting T for thymine), but it can refer to either a DNA sequence that corresponds to an GCase nucleic acid sequence, or the RNA sequence of an RNA molecule nucleic acid sequence. Similarly, though some sequences are expressed in an RNA sequence format (e.g., reciting U for uracil), depending on the actual type of molecule being described, it can refer to either the sequence of a RNA molecule comprising a dsRNA, or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the sequences disclosed with any substitutes are envisioned. EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (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., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the 47 art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. GENERAL MATERIALS AND EXPERIMENTAL PROCEDURES Materials Dulbecco's modified Eagle's 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 tag antibody [HRP] were obtained from GenScript. Nickel beads were obtained from Adar Biotech. Strep-Tactin®XT 4Flow high capacity resign, StrepMAB-Classic HRP (anti-Strep) antibody and biotin were purchased from IBA GmbH, Germany. Anti-GCase (C-terminal) antibodies produced in rabbits, Monoclonal Anti-polyHistidine-Peroxidase antibodies produced in mice, polyethyleneimine, defatted bovine serum albumin, protease inhibitor cocktail, deoxyribonuclease, Nonidet™ P 40 Substitute (NP-40) and p-nitrophenyl-P-D-glucopyranoside were all obtained from Sigma-Aldrich. Equipment for SDS-PAGE and Western blotting was 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 leftovers from treatment of patients. Generation of a more stable form of GCase See Examples section below E. coli expression and subsequent purification of GCase Wild-type (WT) and four mutant variants of human GCase, whose sequences were generated by use of PROSS, viz., D2, D4, D6, D7, were all expressed in E. coli as pET28-bd-SUMO [Zahradmk J. et al., FEBS J. (2019) 286: 3858-3873] constructs that also contained an N-terminal his-tag for purification. The expressed GCase was isolated from the E. coli lysates using standard Ni2+ chelate chromatography, and the bound GCase was released from the column using SUMO protease [Frey S. and Gorlich D., J. Chromatogr. A. (2014) 1337: 95-105]. Purity of the protein was assessed on 10 % Tris-Glycine SDS-PAGE gels stained with Coomassie blue (Instant blue, Expedeon). GCase was identified by Western blotting, using anti-GCase and anti-his-tag antibodies and by Mass Spectrometry (MS). Cell culture and transfection in eukaryotic cells Wild-type (WT) and the D7 variant DNA sequences of human GCase were cloned into a pCDNA 3.1 (Invitrogen) vector, together with an N-terminal FLAG tag for purification (Figure 2A). HEK293T cells were cultured in Dulbecco's modified Eagle medium supplemented with 10 % fetal 48 bovine serum, 100 lU / ml penicillin, 100 pg / ml streptomycin, and 110 pg / ml sodium pyruvate. The cells were transfected using polyethyleneimine reagent and 10 pg of plasmid per 10 cm culture dish. Cells and growth medium were collected 36-48 hours after transfection. GCase purification GCase was isolated either from cell pellets or from growth medium using anti-DYKDDDDK affinity resign (FLAG beads) and Strep-Tactin®XT resign (Strep beads). Growth medium was transferred to 250 ml tubes and centrifuged at 10,000 g at 4 °C for 20 minutes. 200 pl of a FLAG beads or Strep beads suspension in 150 mM NaCl / 50 mM Tris, pH 7.4, was added to a 50 ml Falcon tube filled with the growth medium, and placed on a rotator at 4 °C overnight to enable binding of the GCase to the beads. Cell pellets were lysed by sonication in the same Tris buffer, containing 1 % NP-40, 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 pl of bead suspension per 1-5 ml of supernatant). The mixture was placed on a rotator for a minimum of 2 hours at 4 °C. The beads, either FLAG or Strep beads, were then washed with an excess of the Tris buffer. GCase containing FLAG tag was released by competitive elution in 3 consecutive elution steps, using as the eluting ligand the 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 1 of double distilled water, 187 mM D-mannitol, and 0.1 % (v / v) ml Tween 80, pH was adjusted to 6.1 using citric acid). The protein was further purified and stored in the sodium citrate buffer. The eluted fractions were combined, and underwent size exclusion chromatography (SEC) on an analytical Superdex 200 column. Fractions corresponding to the monomeric peak were collected and concentrated on Amicon Ultracentrifugal filters (10 kDa cut-off, Merck Millipore). The protein concentration was determined from the absorbance at 280 nm, and extinction coefficients were calculated on the basis of amino acid sequence composition (sov-GCase = 108 290 M^crn1; 8wT-GCase = 95 800 M^cm'1). Purity was assessed on 10 % Tris-Glycine SDS-PAGE gels stained with Coomassie blue (Instant blue, Expedeon). GCase was identified by Western blotting, using anti-GCase, anti-Strep and anti-DYKDDDDK antibodies, and by mass spectrometry (MS). Differential scanning fluorimetry Differential scanning fluorimetry (DSF) was performed using a NanoDSF Prometheus NT.48 instrument (NanoTemper, Germany). Samples were heated at 1 °C / min steps in the temperature range of 20-95 °C. Fluorescence emission of tyrosine and tryptophan was recorded at 330 nm and 350 nm. Data were analyzed using a PR.ThermControl v2.1.1 instrument (NanoTemper, Germany). The melting temperature (Tm) was defined as the inflection point of the fluorescence intensity (FT) ratio curve, where R(F7) = FFsonm / FFsonm. Enzymatic activity assay using a synthetic substrate (p-NP-Glc) Specific enzyme activity was determined using p-nitrophenyl-|3-D-glucopyranoside (p-NP-Glc) as the substrate. The reaction was stopped by raising the pH to 10, at which pH the p-nitrophenol released is fully ionized, and displays a molar absorption of 20 000 M^cm1 at 405 nm. The activity assay was adopted from Wei et al [Wei R.R. et al., J. Biol. Chem. (2011) 286: 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 / 0.1 M potassium phosphate, pH 5.9, at 25 °C for 60 minutes. The reaction was stopped by 20-50-fold dilution in 1 M glycine buffer, pH 10, and the absorbance of the p-nitrophenol was measured at 405 nm, in a 1 cm cuvette, using an Agilent Cary 3500 spectrophotometer (Agilent Technologies, USA). Absorbance values were translated into p-nitrophenol concentrations, and Michaelis-Menten plots were constructed and fitted using Origin software (OriginLab). Vmax values obtained from the fits were converted to kcat values according to the equation kcat = Vmax / c, where c is the molar concentration of the GCase catalytic sites. Enzymatic activity assay using C6NBD GlcCer Variants were tested for enzymatic activity using fluorescently labelled natural substrate of GCase (NBD glucosylceramide (dl8:1 / 6:0) (CeNBD GlcCer)). Protein preparations were incubated with 20 pM C6-NBD-GlcCer, 20 pM defatted BSA in 50 mM MES buffer pH 5.5 at 37 °C for 5 minutes. Reactions were terminated by addition of 750 pl chloroform / methanol (1:2, v / v), followed by addition of 500 pl of chloroform and 730 pl of double distilled water. Samples were vortexed vigorously and centrifuged for 10 minutes at 2,000 g. The upper phase was aspirated and the lower phase, containing the extracted lipids, was dried under the N2 stream. Lipids were resuspended in chloroform / methanol (9:1, v / v), and separated by TLC using chloroform / methanol / 9.8 mM CaCh.2H2O (65 / 30 / 8, v / v / v) as the developing solvent. NBD-labeled lipids were visualized using a Typhoon 9410 variable mode imager and quantified by ImageQuantTL (GE Healthcare, Chalfont St Giles, UK). Activity values were calculated as pmol of substrate turned into product by 1 mg of enzyme in 1 minute (pmol.mg^.min1). EXAMPLE 1 PROSS-generated variants Six GCase variants (designs 2-7, i.e. D2-7, set forth in SEQ ID NOs: 4, 6, 8, 10, 12 and 14, respectively, see Figure 1A) were designed by use of the PROSS algorithm already used successfully for improving expression levels and stability of several other proteins [see, PCT / IL2016 / 050812 and Goldenzweig A. et al., Mol. Cell. (2016) 63: 337-346, incorporated herein by reference]. Four of these 50 variants, D2, D4, D6 and D7, were expressed in E. coli and tested for enzymatic activity using a synthetic substrate, p-NP-Glc (data not shown). The sequence of WT GCase is shown in Figure IB (SEQ ID NO: 2), and the line below displays the mutations that occur in the D7 variant (SEQ ID NO: 14), which bears the highest number of mutations, i.e. 30. It is also the construct displaying the highest enzymatic activity. For further work, the D7 variant was expressed in HEK293T cells, which are capable of protein glycosylation. EXAMPLE 2 Expression and purification of variant D7 GCase Both WT hGCase and the D7 variant were expressed in HEK293T cells, and isolated either from cell pellets (intracellular) or from culture medium (secreted). Both WT and D7 GCase were expressed intracellularly, but the D7 variant showed higher expression than the WT. SDS-PAGE of the three eluent fractions obtained from individual preparations, using Coomassie blue staining, is displayed in Figures 2B and 2C, GCase was identified as the principal band (marked with arrows), by Western blotting and MS. Only D7 GCase was secreted. A highly purified preparation of the secreted D7 GCase was obtained by a one-step purification using FLAG beads (Figure 2C). Subsequently, the samples were applied to a Superdex200 column. SEC revealed significant oligomerization of secreted D7 GCase (Figures 3A-B). Similar patterns were observed for intracellular WT and D7 GCase. The position of the monomer was established by calibrating the column with molecular weight markers, as the peak with the absorbance maximum at approximately 15 ml (peak 1, Figures 3A-B). The monomeric peaks were also shown to be the fractions with the highest specific activity. The fractions corresponding to the monomer were pooled, concentrated, and used for stability and activity assays. In the following, data presented were for the D7 monomer obtained by 2-step purification from the secreted fraction. Due to the extremely low yield of secreted WT GCase, the data obtained for D7 GCase were compared to similar data for recombinant WT GCase expressed in CHO cells, obtained from R&D Systems, and for Cerezyme®, produced by Sanofi Genzyme, which possesses the WT sequence with a single Arg495His substitution. EXAMPLE 3 Thermal stability of variant D7 GCase The melting temperature (Tm) of D7 GCase was determined using differential scanning fluorimetry (DSF). DSF measures the changes in fluorescence of tyrosine and tryptophan residues upon protein unfolding which results in their exposure to the aqueous environment. Average Tm values for the preparations analyzed are shown in Table 1, below. Tm values of 71.8 ± 2.4 °C and 61.4 + 1.9 °C for D7 GCase were determined in Tris buffer, pH 7.4, and citrate buffer, pH 6.1, respectively. D7 GCase displayed higher thermal stability than WT GCase. The increase in stability was of about 20 °C and about 11 °C, when compared to Cerezyme® at pH 7.4 and pH 6.1, respectively. The values that were obtained for Cerezyme® and WT GCase using DSF were in good agreement with previously reported Tm values obtained for Cerezyme® by differential scanning calorimetry, viz. 51.30 ± 0.02 °C at pH 7.1, and 57.67 + 0.04 °C at pH 5.4 [Wei R.R. et al., J. Biol. Chem. (2011) 286: 299-308, supra]. Table 1: Tm (°C) measured by differential scanning fluorimetry Tm (°C) pH 7.4 Tm (°C) pH 6.1 GCase WT - 55.1 ± 0.5 (n = 2) GCase D7 71.8 ± 2.4 (n = 3) 61.4 ± 1.9 (n > 3) Cerezyme® 49.5 ± 1.0 (n> 3) 50.6 ± 2.2 (n > 3) Values are shown for WT GCase, D7 GCase, and Cerezyme® at two different pH values. EXAMPLE 4 Specific Activity of variant D7 GCase Fitting of the Michaelis-Menten equation to the experimental data permitted to obtain Km and kcat values (Table 2, below, representative enzymatic kinetics data are shown in Figure 4). The overall catalytic efficiency of the various preparations were compared on the basis of the bimolecular rate constant, kCat / Km. The data obtained showed that the catalytic activity of D7 and the WT does not significantly differ, their kCat / Km values being 0.28 x 106 and 0.27 x 106 Mimin’1, respectively. This data thus validated the protocol employed, and the comparative analysis with the artificial substrate, p-nitrophenyl-P-D-glucopyranoside. Table 2: Kinetic parameters Km (mM) kcat (min1) kcat / Km (M 'min4) GCase WT 1.22 ± 0.38 (n=2) 347 ± 49 (n=2) 0.28 x 106 GCase D7 0.89 ± 0.26 (n=3) 244 ±102(n=3) 0.27 x 106 Of note, the kinetic parameters were obtained for the activity of GCase preparations on p-nitrophenyl-P-D-glucopyranoside by fitting the Michaelis-Menten curve to the measured data points. 52 EXAMPLE 5 Additional PROSS-generated variants Three additional variants (D13, D14 and D15) of glucosylceramidase (GCase) were designed using the PROSS algorithm previously used successfully for improving expression levels and stability of several other proteins, including GCase (design D7) [see, PCT / IL2016 / 050812 and Goldenzweig A. et al., (2016) supra, incorporated herein by reference]. GCase variants D13, D14 and D15 were expressed in HEK293T cells and isolated from culture medium. Highly purified preparations of the GCase designs were obtained by a one-step purification using FLAG tag (DYKDDDDK tag, SEQ ID NO: 16) or TwinStrep® tag (SEQ ID NO: 29). All variants were tested for enzymatic activity using fluorescently labelled analogue of GCase (NBD glucosylceramide (dl8:1 / 6:0) (CeNBD GlcCer)) (data not shown). The design with highest enzymatic activity, i.e. variant D15 GCase (as illustrated in Figures 5A-B) was used for further characterization. As discussed below, thermal stability and enzymatic activity of the new D15 GCase was compared to previously characterized D7 GCase and to Cerezyme®, produced by Sanofi Genzyme, which possesses the WT sequence with a single Arg495His substitution. All enzymes were kept in 4 °C dissolved in sodium / citrate buffer containing 187 mM D-mannitol, and 0.1% (v / v) Tween 80, pH 6.1. EXAMPLE 6 Thermal stability of variant D15 GCase The melting temperature (Tm) of D15 GCase was determined using differential scanning fluorimetry (DSF). DSF measures the changes in fluorescence of tyrosine and tryptophan residues upon protein unfolding which results in their exposure to the aqueous environment. Experiments were carried out with enzymes dissolved in citrate buffer, pH 6.1 (as discussed above). D7 GCase was shown previously to have approximately 10 °C higher temperature when compared to Cerezyme®. Further increase in melting temperature was observed for the variant D15 GCase. The average Tm values measured for D15 GCase was 17 °C higher, when compared to Cerezyme®, reflecting substantial increase in protein thermal stability (Table 3, below). The Tm value measured for Cerezyme® was in good agreement with previously reported Tm values obtained by differential scanning calorimetry, 51.30 + 0.02 °C at pH 7.1 [WeiR.R. et al., J. Biol. Chem. (2011)286:299-308, supra]. Table 3: Tm (°C) measured by differential scanning fluorimetry Tm (°C) Cerezyme® 50.6 ± 2.2 (n > 3) GCase D7 61.4+1.9 (n > 3) GCase D15 67.6+1.0 (n = 3) 53 EXAMPLE 7 Specific Activity of variant DI5 GCase Specific activity was determined by two approaches (i) using fluorescently labelled natural substrate of GCase CeNBD GlcCer (in pH 5.5) and (ii) using artificial substrate p-nitrophenyl-P-D-glucopyranoside (p-NP-Glc) (in pH 5.9). In the first approach, the substrate and product were separated by thin-layer chromatography and quantified by the NBD fluorescence. In the latter, the substrate was quantified spectroscopically, by absorption of created p-nitrophenyl at 405 nm. Specific enzymatic activity determined by both substrates was comparable for commercial Cerezyme® and D15 GCase. Activity values were calculated as pmol of substrate turned into product by 1 mg of enzyme in 1 minute (pmol.mg^.min1; Table 4A, below). Substrate concentration was 20 pM for CeNBD GlcCer assay and 0.4, 1.5 and 3 mM for p-NP-Glc assay (Figure 6). Table 4A: Specific activity (pmol.mg^.min1) C6NBD GlcCer p-NP-Glc (3mM) Cerezyme® 0.28 ± 0.12 (n = 3) 1.16 ± 0.21 (n > 3) GCase D15 0.28 ± 0.12 (n > 3) 1.29 ±0.41 (n > 3)* * Of note, GCase D15 was purified using FLAG tag A further experiment was carried out to compare the enzymatic kinetics of WT GCase, commercial Cerezyme®, D7 GCase and D15 GCase. The kinetic parameters were obtained for the activity of GCase preparations on p-nitrophenyl-P-D-glucopyranoside (p-NP-Glc) by fitting the Michaelis-Menten curve to the measured data points. The experimental data permitted to obtain Km and kcat values (Table 4B, below). The overall catalytic efficiency of the various preparations were compared on the basis of the bimolecular rate constant, kCat / Km. The data obtained showed that the catalytic activity of D15 was comparable to that of Cerezyme®, their kCat / Km values being 1.49 x 106 M^min1 and 1.53 x 106 Mimin'1, respectively, while the catalytic activity of D7 was comparable to that of WT GCase, their kCat / Km values being 0.27 x 106 M^min1 and 0.28 x 106M1min1, respectively. Table 4B: Kinetic parameters Km (mM) kcat (min1) kcat / Km (M 'min4) GCase WT (n=2) 1.22 ±0.38 347 ± 49 0.28 x 106 Cerezeme® (n=3) 0.7 ± 0.28 1071 ±26 1.53 x 106 GCase D7 (n=3) 0.89 ±0.26 244 ±102 0.27 x 106 GCase D15 (n=3) 0.90 ± 0.23 1340 ±546 1.49 x 106 54 EXAMPLE 8 Additional GCase variant - DI6 An additional variant (D16) of glucosylceramidase enzyme (GCase) was designed using the PROSS algorithm previously used successfully for improving expression levels and stability of several other proteins, including GCase (design D7 and D15) [Goldenzweig A. et al., (2016), supra, incorporated herein by reference]. D16 GCase was expressed in HEK 293T cells and isolated from culture medium. A highly purified preparation of the GCase design was obtained by a one-step purification using TwinStrep® tag (SEQ ID NO: 29). As discussed below, thermal stability and enzymatic activity of the new D16 GCase was compared to previously characterized D15 GCase, purified using TwinStrep® tag (SEQ ID NO: 29), and to Cerezyme®, produced by Sanofi Genzyme, which possesses the WT sequence with a single Arg495His substitution. All enzymes were kept in 4 °C dissolved in sodium / citrate buffer containing 187 mM D-mannitol, and 0.1% (v / v) Tween 80, pH 6.1. EXAMPLE 9 Thermal stability of variant DI6 GCase The melting temperature (Tm) of GCase was determined using differential scanning fluorimetry (DSF). DSF measures the changes in fluorescence of tyrosine and tryptophan residues upon protein unfolding which results in their exposure to the aqueous environment. Experiments were carried out with enzymes dissolved in citrate buffer, pH 6.1 (storage buffer described above). Tm values measured previously for GCase D15 were approximately 17 °C higher, when compared to Cerezyme®, reflecting substantial increase in protein thermal stability (Table 3, above, and Table 5, below). The new GCase D16 showed slight increase in Tm, namely by 2.5 °C as compared to GCase D15. The Tm value measured here for Cerezyme® was in good agreement with previously reported Tm values obtained by differential scanning calorimetry, 51.30 ± 0.02 °C at pH 7.1 [Wei R.R. et al., J. Biol. Chem. (2011) 286: 299-308, supra]. Table 5: Tm (°C) measured by differential scanning fluorimetry Tm (°C) Cerezyme® 50.6 ± 2.2 (n > 3) GCase D7 61.4+1.9 (n > 3) GCase D15 67.9 ± 0.7 (n > 3) GCase D16 70.4+1.0 (n = 3) 55 EXAMPLE 10 Specific Activity of variant DI6 GCase Specific activity was determined using artificial substrate p-nitrophenyl-P-D-glucopyranoside (p-NP-Glc) (in pH 5.9). In this assay the substrate was quantified spectroscopically, by absorption of created p-nitrophenyl at 405 nm. Substrate concentration was 3 mM. Specific enzymatic activity of the new GCase D16 variant was higher than the one determined for commercial Cerezyme® and comparable to GCase DI5. Activity values were calculated as pmol of substrate turned into product by 1 mg of enzyme in 1 minute (qmol.mg1.min1; Table 6, below). Table 6: Specific activity (pmol.mg’1.min-1) p-NP-Glc (3 mM)* Cerezyme® 1.16 ±0.21 (n > 3) GCase D15 2.67 ±1.01 (n = 3) GCase D16 2.42 ± 0.70 (n = 3) * Of note, GCases D15 and D16 were purified using TwinStrep® Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

1. A genetically modified human p-glucocerebrosidase (GCase):(i)    comprising an amino acid sequence at least 85% identical to SEQ ID NO: 2; and(ii)   comprising mutations at coordinates L34P, K224N / G, T369E and N370D, where thecoordinates correspond to said SEQ ID NO: 2; and(iii) capable of catalyzing hydrolysis of a glycolipid glucosylceramide (GlcCer).

2. The genetically modified human GCase of claim 1, further comprising:(i)    at least one of the mutations: H145K / R, I204K, E222K, T334F / Y / K and / or L372N;(ii)   at least one of the mutations: N102D / E, L165Q, Q226T, L241I, S242P, K473W and / orH495R;(iii)   at least one of the mutations: I130T, A168S and / or D263N;(iv)   at least one of the mutations: R211N and / or K303R;(v) at least one of the mutations: H60W, L103N / E / R, Q166A, H274R, N333D, N386D, R395K, I406T / A and / or L420M / I;(vi) at least one of the mutations: V78I, A95K, V191M, A322D, V343T, M361E, S364A, H374W, T410E, H451N and / or L480I;(vii) at least one of the mutations: H162K, S181A, T297S, M335F, K346H, S431A, S465D and / or A476D;(viii) at least one of the mutations: R47K, L51R, Q70H, L91I, G115E, A124G, D140N / G, S196T and / or V437S; and / or(ix) at least one of the mutations: T36Q, S38A, Q143E, T183A, L185M, T272S, H274K, N275D, L286S, K293Q, E300R, K321E, V376T, K408R, Q440E, M450Q and / or I483V.

3. The genetically modified human GCase of claim 1 or claim 2, wherein amino acids at coordinates D127, F128, W179, N234, E235, Y244, F246, Q284, Y313, E340, S345, W381, N396, where the coordinates correspond to said SEQ ID NO: 2, are not modified.

4. The genetically modified human GCase of any one of claims 1-3, wherein said amino acid2021245409  07 Mar 2025sequence is identical to a sequence selected from the group consisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 18, 20, 22 and 27.

5. The genetically modified human GCase of claim 4, wherein said amino acid sequence is as set forth in SEQ ID NO: 14.

6. The genetically modified human GCase of claim 4, wherein said amino acid sequence is as set forth in SEQ ID NO: 22.

7. The genetically modified human GCase of claim 4, wherein said amino acid sequence is as set forth in SEQ ID NO: 27.

8. The genetically modified human GCase of any one of claims 1-7, wherein:(i) the genetically modified human GCase is capable of catalyzing hydrolysis of said GlcCer by at least about 0.2 x 106 kcat / Km (M-1min-1);(ii) the genetically modified human GCase comprises a thermal stability under a temperature range being 5-20°C higher compared to a wild-type polypeptide under the same conditions;(iii) the genetically modified human GCase comprises at least 2 times higher intracellular expression level in eukaryotic cells as compared to a wild-type polypeptide under the same culture conditions; and / or(iv) the genetically modified human GCase is secreted from eukaryotic cells as compared to a wild-type polypeptide not being secreted under the same culture conditions.

9. An isolated polynucleotide comprising a nucleic acid sequence encoding the genetically modified human GCase of any one of claims 1-8.

10. The isolated polynucleotide of claim 9, comprising the nucleic acid sequence as set forth in any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 17, 19, 21, 23 or 26.

11. A nucleic acid construct comprising the isolated polynucleotide of claim 9 or claim 10, and a cis-acting regulatory element for directing expression of said nucleic acid sequence in a cell.2021245409  07 Mar 202512. An isolated cell comprising the polynucleotide of claim 9 or claim 10, or nucleic acid construct of claim 11.

13. A method of treating a disease associated with p-glucocerebrosidase deficiency in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the genetically modified human GCase of any one of claims 1-8, the isolated polynucleotide of claim 9 or claim 10, the nucleic acid construct of claim 11, or the cell of claim 12, thereby treating the disease associated with the p-glucocerebrosidase deficiency in the subject.

14. The method of claim 13, wherein the disease associated with p-glucocerebrosidase deficiency is Gaucher disease.

15. The method of claim 13 or claim 14, wherein the subject is a human being.

16. Use of the genetically modified human GCase of any one of claims 1-8, the isolatedpolynucleotide of claim 9 or claim 10, the nucleic acid construct of claim 11, or the cell of claim 12 in the manufacture of a medicament for treating a disease associated with p-glucocerebrosidase deficiency.

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