Glucocerebrosidase (gba) polymer conjugates, methods of preparation and uses for nanotechnology-based enzyme replacement therapy

Abstract

The present invention relates to the field of medicine, in particular to: a nanotechnology-based enzyme replacement therapy based on recovery of lysosomal glucocerebrosidase activity by enzyme-polymer nanoconjugation of GBA, preferably for Parkinson's disease; a GBA polymer conjugate for use in such a use; and a method for manufacturing the same.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to nanotechnology-based enzyme replacement therapy, preferably for Parkinson's disease and Gaucher's disease, based on restoring lysosomal glucocerebrosidase activity through enzyme-polymer nanoconjugation of GBA, GBA polymer conjugates for such use, and methods for preparing the same. Background Art

[0002] The GBA1 gene encodes the glucocerebrosidase (GCase) protein, or GBA, a lysosomal enzyme involved in sphingolipid metabolism. Homozygous mutations in GBA cause Gaucher disease (GD), an autosomal recessive lysosomal disorder. Heterozygous mutation carriers are at a highly increased risk of developing Parkinson's disease (PD). Therefore, the presence of GBA1 mutations is known to be a major genetic risk factor for PD. GBA mutations are present in 10% to 12% of PD patients (Kinghorn, KJ Pathological looping in the synucleinopathies: Investigating the link between Parkinson's disease and Gaucher disease. DMM Dis. Model. Mech. 2011, 4, 713–715. Stojkovska, I.; Krainc, D.; Mazzulli, J RM Polecular mechanisms of -synuclein and GBA1 in Parkinson's disease. Cell Tissue Res. 2018, 373, 51–60).

[0003] PD patients with GBA mutations are clinically indistinguishable from those without GBA mutations; however, they have a significantly earlier mean onset of disease and are at higher risk for cognitive impairment and other neuropsychiatric disorders.

[0004] Patients with Parkinson's disease (PD) who harbor GBA mutations have significantly reduced GCase activity in the substantia nigra (SNpc) and other brain regions. Interestingly, other sporadic PD patients who do not harbor GBA mutations have significantly reduced GCase activity, suggesting that GBA may contribute to PD pathogenesis. Deficit in PD patients is associated with increased levels of α-synuclein (syn), supporting a possible role for GBA deficiency in syn accumulation or aggregation. Reduced GBA / GCase activity has also been associated with increased syn levels in cell and animal models (Mazzulli, JR; Xu, YH; Sun, Y.; Knight, AL; McLean, PJ; Caldwell, GA; Sidransky, E.; Grabowski, GA; Krainc, D. Gaucher disease glucocerebrosidase and alpha-synuclein form a bidirectional pathogenic loopin synucleinopathies. Cell 2011, 146, 37–52).

[0005] Therefore, it is necessary to restore GBA activity, especially in lysosomes, to restore the activity of the lysosomal system, avoid the accumulation of sphingolipid substrates, reduce syn levels and ultimately prevent neurodegeneration. Currently, available therapies to restore GBA activity include:

[0006] Enzyme replacement therapy (ERT) restores GCase activity with recombinant GBA protein. This is currently the mainstay of treatment for patients with GD. It benefits non-neurological manifestations because the recombinant protein itself cannot cross the BBB and reach the brain. ERT is currently ineffective for treating the central nervous system effects of GCase loss in GBA-related GD and Parkinson's disease.

[0007] -Substrate reduction therapy (SRT): Inhibits the synthesis of GCase substrate (GlcCer) to prevent its accumulation.

[0008] - Molecular chaperones (MC): Pharmacological GBA-specific molecular chaperones can stabilize misfolded mutant GBA retained in the ER and promote its transport to lysosomes, restoring its function. Examples include isofagomine and ambroxol.

[0009] Currently, restoration of GBA activity in humans is only approved for Gaucher disease:

[0010] - ERT: Cerezyme (imiglucerase) from Sanofi-Genzyme, VPRIV (velaglucerase alfa) from Takeda, and Elelyso (taliglucerase alfa) from Pfizer; and

[0011] - SRT: Cerdelga (eliglustat) from Sanofi-Genzyme and Zepilca (miglustat) from Actelion.

[0012] As for PD, currently, there are no approved treatments for GBA recovery, although there are some ongoing clinical trials.

[0013] In the present invention, we have developed a nanotechnology-based enzyme replacement therapy, preferably for Parkinson's disease, based on the restoration of lysosomal glucocerebrosidase activity by enzyme-polymer nanoconjugation of GBA. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 .This figure is a schematic diagram of how the GBA nanoconjugates of the present invention are internalized by cells through endocytosis and sent directly to lysosomes for degradation. Once the nanoconjugates reach the lysosomes, the polypeptide coating should be degraded in the acidic lysosomal environment, and GBA, as a lysosomal protein, should restore its lysosomal function, thereby restoring GBA activity.

[0015] Figure 2 Striatal neurons from ST-Q7 mice were treated with PLGA nanoparticles or nanoconjugates loaded with 200 ng / μl velaglucerase for 24 hours. Immunocytochemistry was performed to stain velaglucerase using anti-GBA antibody (right) and anti-LAMP-1 antibody (left) as lysosomal markers.

[0016] Figure 3.GBA-PGA characterization. A) Percentage reduction of unmodified lysine after SATP modification. The reduction in lysine levels of unmodified glucerase (GBA) and SATP-modified glucerase (GBA-SATP) was analyzed by 2,4,6-trinitrobenzenesulfonic acid (TNBSA) assay under optimal conditions (PBS at pH 9 for 60 minutes with 15 equivalents of SATP). The experiment was performed in triplicate. B) Percentage of unmodified glucerase after conjugation. Silver-stained glucerase bands at each time point were quantified to provide quantitative values ​​for the reaction efficiency. Because the following samples had the same concentration of GBA-SATP (no Amicon wash step), the values ​​are expressed as a percentage of glucerase at the GBA-SATP step. C) Silver staining of the conjugation time course. Unmodified glucerase (1) and SATP-modified glucerase (2) were incubated in the absence of 26.9 μM isofagomine D-tartrate (IFG) Non-reducing SDS-PAGE was performed with a conjugation time course of 0.5 h (3), 1 h (4), 2 h (5), 4 h (6) and overnight (7) in the presence of IFG (+IFG). The gel was then stained with a silver staining protocol to show the MW of the conjugates and the conjugation kinetics. From top to bottom, arrows indicate the GBA-PGA band (α), the aggregation band (β) and the velaglucerase band (γ). D) Loss of GCase activity during conjugation in the absence or presence of IFG. The GCase activity of the optimized conjugation protocol was analyzed at different conjugation steps: unmodified velaglucerase (GBA, dark), SATP-modified velaglucerase (GBA-SATP, moderate) and conjugated velaglucerase (GBA-PGA, bright). Two experiments were performed in quadruplicate: conjugation in the absence of IFG (IFG(-)) and in the presence of 26.9 μM IFG (IFG(+)). *Statistically significant difference (p<0.0005).

[0017] Figure 4 .Image of PLGA nanocapsules obtained by SEM. Nanocapsules without PLGA loading (left) were synthesized as a control ( ζ-25±5mV), and successfully encapsulated velaglucerase into PLGA nanocapsules (right, In this article, ζ is defined as the zeta potential.

[0018] Figure 5 GCase activity was tested in GBA (velaglucerase) after modification with different molar ratios of SATP (light grey) (untreated velaglucerase was used as control) and in PGA-velaglucerase conjugates (dark grey) after release by incubation with 10 mM GSH for 1 h.

[0019] Figure 6 MC interference with SATP modification. A) SATP reactivity with primary amines on proteins. B) Molecular structures of isofagomine and ambroxol. The green circle represents the secondary amine (non-reactive) of isofagomine. The red circle represents the primary amine (reactive) of ambroxol.

[0020] Figure 7 Effect of IFG addition on conjugation efficiency. Velaglucerase (Vela) was modified with the SATP crosslinker in the absence of IFG (Vela SATP) or in the presence of IFG (Vela SATP+IFG). Samples were then collected at 1, 2, and 4 hours of reaction time for the PGA conjugation step in the absence of IFG (Conj) or in the presence of IFG (Conj+IFG).

[0021] Figure 8 IFG addition enhanced GCase activity after nanoconjugation. GCase activity was measured at each step of the conjugation and compared with that of the unmodified enzyme.

[0022] Figure 9 Velaglucerase modified with SATP was tested at different pH and reaction times. Sulfhydryl incorporation (A), reduction in free lysine (B), and changes in GCase activity (C) were measured under each condition.

[0023] Figure 10: In vitro validation. A. Stability in human plasma (HBP) was assessed by GCase activity after internalization. 200 μg / ml of compound in velaglucerase citrate buffer (VCB, 50 mM sodium citrate, pH 5.6, 5% w / v sucrose and 0.01% v / v polysorbate 20) was incubated with 4 volumes of HBP for up to 24 hours and diluted to 5 μg compound / mL in opti-MEM containing 1 U / ml heparin. BE(2)-M17 GBA1- / - cells were incubated with each compound for 3 hours, lysed and GCase activity assessed (data from three independent experiments performed in triplicate). B: Glucosyl-sphingosine levels were analyzed by LC-MS / MS after treatment of BE(2)-M17GBA1- / - cells with GBA or NanoGBA for up to 7 days. C. BE(2)-M17 GBA1- / - cells were treated with GBA or NanoGBA for up to 7 days. Untreated cells were used as negative control (KO), and untreated BE(2)-M17WT cells were used as positive control (WT). After treatment, cell lysates were analyzed for total GBA internalization, GCase specific activity (GCase activity / unit protein), and GCase activity recovery (GCase activity / unit GCase). The GCase specific activity (left axis, columns) and GCase activity recovery (right axis, dots and lines) of different treatments were expressed as % relative to WT levels (data from three independent experiments, expressed as mean ± SEM). D. α-synuclein levels after 7 days of treatment. Statistical differences were determined using two-way ANOVA and multiple mean comparisons. Composite differences were determined at p < 0.0001. In multiple comparisons *** p<0.01. Multiple comparisons **** p<0.0001.

[0024] Figure 11 : The biodistribution of Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) was detected by IVIS.

[0025] Figure 12 : In vitro detection of Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) by IVIS (left). GCase activity in different tissues 30 min, 3 h, 24 h, and 72 h after nanoGBA administration (right).

[0026] Figure 13 : In vitro detection of Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) in the brain (left). Quantification of GCase activity and two GBA substrates (GlcCer and GlcSph or lysoGb1) in the olfactory bulb after 24 h.

[0027] Figure 14 Circular dichroism spectra. A) Difference between GBA-SATP and GBA enzyme. B) Difference between NanoGBA and the other two products analyzed, GBA and GBA-SATP. Data are expressed as Δε (M-1, cm-1).

[0028] Figure 15 : Size exclusion chromatography using Superdex 200. GBA (Blue), GBA-SATP, and NanoGBA were loaded onto the column, and the absorbance at 280 nm was followed over time.

[0029] Figure 16 : Trypsin digestion analysis. The figure shows Coomassie staining of trypsin digests of GBA, GBA-SATP, and NanoGBA and relative quantification by densitometry of GBA bands.

[0030] Figure 17 : Plasma stability assay. The figure shows: A) Activity recovered in treated M17 GBA KO cells. Activity data are presented as a percentage of activity relative to untreated wild-type cells. The bar graph shows the mean plus standard deviation (n=2). B) Western blot showing total GBA internalized after each treatment.

[0031] Figure 18 : Uptake assay. A) Activity was restored in treated M17 GBA KO cells. Activity data are presented as a percentage of activity relative to untreated wild-type cells. Bar graphs show mean values ​​plus standard deviations (n=3). B) Western blots show total GBA internalized after each treatment. Summary of the Invention

[0032] definition

[0033] It must be noted that, unless the context clearly indicates otherwise, as used herein, the singular forms "a," "an," and "the" include plural reference. Furthermore, unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0034] When referring to a given amount or quantity, the term "about" is intended to include a deviation of plus or minus 10%, preferably 5%.

[0035] As used herein, the term "and / or" connecting multiple referenced elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first element and the second element being applicable together. Any of these options is understood to fall within this meaning and, therefore, satisfies the requirements of the term "and / or" as used herein. The simultaneous application of more than one option is also understood to fall within this meaning and, therefore, satisfies the requirements of the term "and / or."

[0036] Throughout the specification and claims, unless the context requires otherwise, the word "comprising" and variations thereof will be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprising" may be replaced by the terms "containing" or "including," or sometimes when used herein, by the term "having." Any of the above terms (comprising, containing, including, having), whenever used in the context of an aspect or embodiment of the invention, may be replaced by the term "consisting of," although this is less preferred.

[0037] When used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0038] In the context of the present invention, "GBA protein" refers to an enzyme that induces glucosylceramidase activity (also known as acid β-glucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, glucocerebrosidase, GCase or any other name listed in the BRENDA enzyme database under entries 3.2.1.45 and 3.2.1.62).

[0039] In the context of the present invention, "isofagomin" or "IFG" refers to a pharmacological chaperone that selectively binds to glucocerebrosidase and restores its correct conformation and enhances its activity.

[0040] In the context of the present invention, "GBA-specific chaperone" refers to a pharmacological chaperone that specifically binds to GBA and helps protein folding to prevent misfolding, enhance enzyme activity, and / or enhance accurate protein translocation to lysosomes. Examples of these GBA-specific molecular chaperones include, but are not limited to, iminosugars and their derivatives with inhibitory activity (such as 1,5-dideoxy-1,5-iminoxylitol (DIX), deoxynojirimycin (DNJ), and 1-azafagomine derivatives), sugar analogs and their derivatives with inhibitory activity, molecules with inhibitory activity that are not classified into the latter two categories (the main example is ambroxol (ABX)), and allosteric enhancers of GBA.

[0041] In the context of the present invention, "SATP-modified proteins" refer to proteins modified with N-succinimidyl-S-acetylthiopropionate. SATP adds acetylated sulfhydryl groups to amino groups present in specific proteins. This article describes SATP as a heterobifunctional crosslinker containing an amine-reactive group (N-succinimidyl) and a protective sulfhydryl-reactive group (S-acetyl), capable of modifying the side chains of lysine residues by incorporating protective sulfhydryl groups. Consequently, SATP-modified proteins can be conjugated to polymers to form disulfide bonds, covalent bonds that respond to reducing environments, allowing the conjugate to persist in oxidizing environments but release the protein in reducing ones. This article further describes that conjugation between any protein (including GBA) and a polymer by modifying the disulfide bonds of lysine can be achieved using not only SATP, but any other crosslinker that can modify the side chains of lysine and add sulfhydryl-reactive groups. Based on this chemistry, molecules containing similar reactive head groups but different central chain lengths can be used as alternatives to SATP (SATA crosslinkers are examples of this type of molecule). Other cross-linkers capable of adding pyridyldisulfide groups to lysine residues (e.g., SPDP or SMPT) can also be used to prepare the same conjugate with GBA by conjugating GBA to PGA, which adds a sulfhydryl group (instead of a pyridyldisulfide group) to the side chain of the glutamic acid residue. In addition, the lysine side chain can be modified with other amine-reactive head groups (e.g., sulfo-N-succinimidyl or imidoester).

[0042] The present invention covers all potential alternatives to these modified GBAs that are different from SATP-modified GBAs. Therefore, the present invention covers not only SATP-modified GBA proteins, but also GBA-modified proteins comprising lysine having a side chain modified with a thiol-reactive group (hereinafter referred to as "GBA-modified proteins"). These types of GBA-modified proteins can preferably be obtained by using a cross-linking agent (such as SATA) or a cross-linking agent (such as SPDP or SMPT) that is capable of adding a pyridyldisulfide group to a lysine residue. Preferably, the GBA-modified protein can be a SATP-modified GBA protein or a GBA-modified protein modified with any other cross-linking agent that modifies the side chain of lysine and adds a thiol-reactive group. In this sense and according to the present invention, molecules containing similar reactive head groups but different spacer arm lengths can be used as alternatives to SATP, for example selected from SATA 、 4-SATA, 8-SATA, 12 -SATA, 24 -SATA and Other molecules that can also modify the side chain of lysine but add a pyridyldithiol-reactive group are those selected from SMPT, SPDP, LC-SPDP, PEG4-SPDP, and PEG12-SPDP. In fact, similar conjugates can be produced by conjugating GBA to PGA, which adds a thiol group (rather than a pyridyldisulfide group) to the side chain of the glutamic acid residue. In addition, the lysine side chain can be modified with other amine-reactive head groups, such as sulfo-N-succinimidyl or imidoesters, especially those selected from 2-iminothiolane (Traut's reagent) and sulfo-LC-SPDP (see Table 4).

[0043] use SATP Schematic representation of modification reaction :

[0044]

[0045] IUPAC nomenclature of SAPT and its SATP alternatives

[0046] SATP-(2,5-dioxopyrrolidin-1-yl) 3-acetylthiopropionate

[0047] SATA-(2,5-dioxopyrrolidin-1-yl) 2-acetylthioacetate

[0048] 4-SATA-(2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]propanoate

[0049] 8-SATA(2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0050] 12 -SATA

[0051] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0052] 24 -SATA

[0053] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0054] 3-Mercaptopropyl-N-hydroxysuccinimide

[0055] (2,5-Dioxopyrrolidin-1-yl) 3-thiopropanoate

[0056] SMPT

[0057] (2,5-Dioxopyrrolidin-1-yl) 4-[1-(pyridin-2-yldisulfanyl)ethyl]benzoate

[0058] SPDP

[0059] (2,5-Dioxopyrrolidin-1-yl) 3-(pyridin-2-yldithio) propionate

[0060] LC-SPDP

[0061] (2,5-Dioxopyrrolidin-1-yl)6-[3-(pyridin-2-yldisulfanyl)propionylamino]hexanoate

[0062] Sulfo-LC-SPDP

[0063] 2,5-Dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propionylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid

[0064] PEG4-SPDP

[0065] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0066] PEG12-SPDP

[0067] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0068] 2-Iminothiolane (Traut's reagent)

[0069] Sulfane-2-imine

[0070] In the context of the present invention, the term "molar equivalent" refers to the ratio of the number of moles of a compound to the number of moles of another compound. Once the number of moles of each compound is determined, the molar equivalent can be determined.

[0071] In the context of the present invention, the term "L-PGA" refers to linear polyglutamic acid.

[0072] In the context of the present invention, the term "isofagomine D-tartrate" refers to a competitive inhibitor of human lysosomal β-glucosidase (Ki = 0.016 μM-0.025 μM; IC50 = 0.06 μM).

[0073] In the context of the present invention, the term "molar ratio" refers to the ratio of any two substances in a chemical reaction, or the ratio of two coefficients in a balanced chemical equation.

[0074] In the context of the present invention, the term "room temperature" is the average room temperature, which is typically around 20°C.

[0075] In the context of the present invention, the term "deacetylated" refers to molecules from which the acetyl group has been removed, typically by hydrolysis.

[0076] In the context of the present invention, the term "VCB" refers to velaglucerase citrate buffer, which is a buffer containing 50 mM sodium citrate (pH 5.6), 5% w / v sucrose, and 0.01% v / v polysorbate 20. DETAILED DESCRIPTION

[0077] Traditional enzyme replacement therapy (ERT) involves administering the specific enzyme that is deficient or missing directly into the patient's vein. This therapy is currently available and has been successfully used for various lysosomal storage diseases, including Gaucher disease, by intravenously administering recombinant GBA (Platt FM. Emptying the stores: lysosomal diseases and therapeutic strategies. Nat Rev Drug Discov. 2018 Feb; 17(2): 133-150. doi: 10.1038 / nrd.2017.214. Epub 2017 Nov 17. PMID: 29147032.). The recombinant lysosomal proteins are spontaneously internalized into the cell and reach the lysosomes, where they can restore enzyme activity.However, ERT therapy has some associated disadvantages: for example, poor enzyme circulation stability (due to protease degradation or renal filtration) (Yu M, Wu J, Shi J Farokhzad OC (2016) Nanotechnology for protein delivery: Overview and perspectives. Journal of controlled release: official journal of the Controlled Release Society 240, 24–37.); the development of immune responses against the infused protein (Turkia H Ben, Gonzalez DE, Barton NW, Zimran A, Kabra M, Lukina EA, Giraldo P, Kisinovsky I, Bavdekar A, Dridi MF Ben, Gupta N, Kishnani PS, Sureshkumar EK, Wang N, Crombez E, Bhirangi K & Mehta A (2013) Velaglucerase alfa enzyme replacement therapy compared with imiglucerase in patients with Gaucher disease. American Journal of Hematology 88, 179–184.); cannot cross some biological barriers, such as the blood-brain barrier (being ineffective for neurological manifestations); and high production costs (Marcucci G, Zimran A, Bembi B, Kanis J, Reginster JY, Rizzoli R, Cooper C & Brandi ML (2014) Gaucher Disease and Bone Manifestations. Calcified Tissue International 95, 477–494.).

[0078] Given the current limitations of ERT for delivering recombinant proteins to the CNS (central nervous system) and its ineffectiveness in treating the neurological manifestations of Parkinson's disease and Gaucher disease, this paper proposes a new ERT-based therapeutic strategy for Parkinson's disease and Gaucher disease, in which glucocerebrosidase (GBA) is modified by nanoconjugating it with a peptide to promote BBB (blood-brain barrier) crossing and improve its intracellular delivery to lysosomes. It should be noted that this approach is not limited to intravenous administration, but also includes intranasal administration, which is a non-invasive route of administration and an alternative to intravenous administration.

[0079] In recent years, the use of nanotechnology strategies to encapsulate proteins in drug delivery systems (DDS) has made it possible to overcome the above-mentioned limitations of ERT. DDS provides enhanced stability and in vivo protection, controlled release, and targeted delivery of drugs. In addition, the use of DDS significantly reduces immunogenicity against drugs (Chen et al., 2016). GBA nanoconjugates are internalized by cells through endocytosis and sent directly to lysosomes for degradation. Once the nanoconjugates reach the lysosomes, the polypeptide coating is degraded in the acidic lysosomal environment. GBA, as a lysosomal protein, restores its lysosomal function, thereby restoring GBA activity. In addition to the main goal of delivering GBA proteins into the CNS (therapeutic applications in PD and GD), the present invention also aims to improve the ERT technology currently used to treat the non-neurological symptoms of GD.

[0080] For the above purposes, the present invention proposes nanoconjugation of GBA enzyme (preferably recombinant) with polypeptide polymers, and the subsequent introduction of chemical modifications to improve protein stability, plasma protease resistance, and BBB crossing. In particular, due to the high instability of GBA protein and its easy loss of enzymatic activity (GBA half-life in plasma is about 10 minutes), the goal is to use polymer unmasked-masked protein therapy (MMPT) conditions to protect GBA and use molecular chaperones (MC) to avoid inactivation. The molecular chaperone (MC) improves GBA stability by generating stable GBA / chaperone nanoconjugates that can be taken up by cells and delivered to lysosomes, restoring GBA activity more efficiently than free GBA. The goal is also to functionalize PGA chains with different molecules and / or biomolecules to increase stability and promote BBB crossing (in the case of intravenous administration) or to reach the brain via intranasal administration.

[0081] The structure of the ERT delivery system proposed in this paper is Figure 1 In particular, Figure 1As shown, the proposed GBA nanoconjugate (hereinafter referred to as "GBA nanoconjugate of the present invention") should be internalized by cells through endocytosis and sent directly to lysosomes for degradation. Once the nanoconjugate reaches the lysosome, the polypeptide coating should be degraded in the acidic lysosomal environment, and GBA, as a lysosomal protein, should restore its lysosomal function and GBA activity. In other words, the GBA nanoconjugate of the present invention is intended to achieve the following objectives:

[0082] Protect GBA from loss of enzymatic activity. The physical barrier provided by the nanoconjugate should protect the protein from the harsh conditions encountered during bloodstream circulation. Sustained release following internalization in cells should provide a steady source of active GBA, unharmed throughout its path through the bloodstream.

[0083] - Overcoming the blood-brain barrier (BBB) ​​through surface modification or intranasal administration. Since traditional ERT cannot deliver active GBA to the central nervous system (CNS), the neurological manifestations of type 2 and type 3 GD remain untreatable. Intranasal administration of encapsulated PLGA should overcome

[0084] BBB and deliver active GBA into the brain.

[0085] To achieve these objectives, and as shown in the examples of the present invention, an initial attempt was made to encapsulate GBA into PLGA nanocapsules by a water-in-oil-in-water (W / O / W) double emulsion method as shown in Example 1. By this method, PLGA nanoconjugates loaded with velaglucerase were successfully synthesized and characterized. To test whether the encapsulated protein retained enzymatic activity, in vitro assays were performed in ST-Q7 cells (mouse striatal neurons). However, although velaglucerase successfully co-localized with LAMP-1 (a lysosomal marker indicating that the protein reached its intended destination), as shown in Example 1, the encapsulated protein was not encapsulated. Figure 2 As shown, no enzyme activity was detected. To address this issue, attempts to optimize the synthesis conditions to overcome the loss of GCase activity were unsuccessful. This optimization is described below.

[0086] To preserve GCase activity, several changes were made during the phase and synthesis process to protect GBA from loss of enzyme activity. These changes are categorized according to the phase affected, with the final section covering overall changes.

[0087] -Modification of W1 phase.

[0088] The modifications affecting the W1 phase were mainly changes in the buffer composition. As shown in the examples, none of these changes resulted in active GBA nanoconjugates.

[0089] -Modifications to the W2 stage.

[0090] The surfactant that constitutes W2 is responsible for the stabilization of the nanoconjugate during evaporation of the organic solvent. Polysorbate 80 and Pluronic were used instead of PVA. None of these changes resulted in nanoconjugates with active GBA.

[0091] - Modification of emulsification method.

[0092] Sonication times and amplitudes were tested to obtain a protocol that produced nanoconjugates with the desired properties but reduced the amount of energy delivered to the sample that would cause protein unfolding. Different conditions of shorter times at higher amplitudes, longer times at lower amplitudes, and continuous and intermittent sonication were tested.

[0093] Preliminary conclusion: None of these changes resulted in active GBA nanoconjugates, and the use of PLGA as a drug delivery system was abandoned. GBA was inactivated during the encapsulation process.

[0094] In light of these results, an alternative approach was attempted to incorporate velaglucerase into nanoparticle systems without completely losing activity, using polymer unmasking–masked protein therapeutics (PUMPT), developed by Dr. María J. Vicent (CIPF, Valencia, Spain) (Talelli M & Vicent MJ (2014) Reduction Sensitive Poly(l-glutamic acid) (PGA)-Protein Conjugates Designed for Polymer Masked–Unmasked Protein Therapy. Biomacromolecules 15, 4168–4177. Available at: https: / / pubs.acs.org / doi / 10.1021 / bm5011883). PUMPT refers to a soft nanotechnology strategy in which proteins are conjugated to biodegradable polymers that provide protection and mask protein activity during transport, while enabling controlled restoration of activity at the target site due to external stimuli. In this context, an adaptation of the PUMPT approach for velaglucerase conjugation was tested. Velaglucerase is conjugated to a polyglutamic acid (PGA) polymer via a reduction-sensitive linker, which is intended to mask (and protect) the protein activity in oxidative environments (such as the bloodstream) and release it into reducing environments (such as lysosomes).

[0095] To successfully conjugate velaglucerase to PGA polymers, the conjugation protocol developed by Talelli and Vicent (Talelli and Vicent, 2014), which was optimized for lysozyme, was adapted for velaglucerase. The conjugation procedure consists of two main steps: (1) modification of the surface protein with SATP (N-succinimidyl-S-acetylthiopropionate), which adds sulfhydryl groups by modifying lysine side chains; and (2) conjugation of PGA modified with pyridyldithiol groups (PGA-PD) to the SATP-modified protein, which attacks the sulfhydryl groups added to lysine to form disulfide bonds.

[0096] Conjugation conditions are indicated in Example 1.

[0097] Although the purpose of the PGA conjugation step is to mask GCase activity, this loss of activity should be fully reversible after release in a reducing environment. However, the SATP modification step, which irreversibly modifies the surface lysine residues of the protein, results in irreversible activity loss. The three initial ratios of SATP used for the velaglucerase conjugation test showed a decrease in enzyme activity, proportional to the SATP concentration. PGA conjugation successfully completely masked GCase activity, but after release with 10mM reduced glutathione (GSH), only one condition restored GCase activity, and the restored activity was much lower than the activity level that should be fully restored by the SAPT-modified velaglucerase. The activity loss during the modification of GBA with SATP and the conjugation of the modified protein with PGA is mainly attributed to the instability of the protein under the conditions of the working buffer used for the conjugation step. In order to reduce this activity loss, a GBA molecular chaperone (MC) is used in the synthesis process. MC is a small molecule with the ability to bind and stabilize the target enzyme. Typically, these MCs are competitive inhibitors that bind to the catalytic pocket of the enzyme.

[0098] In the case of GBA, there are currently two commercially available MCs, although to date, neither has reached the pharmaceutical market as a pharmacological chaperone (PC) for GBA. Of these, the two most studied are ambroxol hydrochloride and isofagomine D-tartrate (IFG). IFG was chosen as a stabilizer because it does not contain any primary amines that compete with lysine in the SATP modification reaction. Furthermore, IFG is a strong chaperone for GBA, with a Ki of 5.8 nM at pH 7.0, suggesting that a small amount of MC may be sufficient to stabilize GCase during conjugation. To test that IFG did not interfere with the conjugation process, conjugation protocols were compared in the presence or absence of the chaperone. The protocol using IFG differed only in that an excess of IFG (25 μM, almost 1000 times the Ki) was added to the working buffer to ensure maximal enzyme inhibition and protection. Samples were taken from each conjugation step (SATP modification and PGA conjugation) in both protocols and analyzed by non-reducing SDS-PAGE coupled with silver staining. This technique allows the determination of the molecular weight of the nanoconjugates without disrupting the disulfide bonds that bind the polymer to the protein, thereby qualitatively assessing the conjugation yield.

[0099] The results of non-reducing SDS-PAGE showed that there was no significant difference in conjugation in the absence or presence of IFG. SATP modification of velaglucerase resulted in dimerization of a small amount of the enzyme. In the presence of IFG, a dimerization band was present on the SATP-modified enzyme, indirectly indicating that there was no interference in this step. Conjugation of PGA to the protein was observed by the tailing effect of the protein-polymer conjugate, which did not differ between the different conditions. Although no differences were observed in the conjugation efficiency, the addition of IFG during the synthesis process increased the GCase activity measured in each step of the conjugation, demonstrating the ability of IFG to stabilize and protect the enzyme during conjugation. After SATP modification, no loss of GCase activity was detected in the reaction containing IFG, a fact attributed to the complete stabilization of the enzyme at this step. In the presence of IFG, the gradual decrease in GCase activity after conjugation was attributed to the masking effect of the polymer on the enzyme activity.

[0100] Thus, IFG improves the stability of the enzyme during the conjugation and masking processes. However, despite the interesting results obtained due to the addition of IFG as a stabilizer, the nanoconjugation strategy seems to be still far from being practical. Because after all conjugation procedures, more than 80% of the velaglucerase remained unmodified. The explanation for this poor conjugation of the enzyme was subsequently found in the buffer conditions used for the SATP modification step. As mentioned above, the protocol for conjugating velaglucerase with PGA was adapted from the study of Talelli and Vicent. The key step of the adaptation is the modification of the protein with SATP, which, in contrast to the PGA conjugation step, leads to irreversible loss of activity. This protocol adaptation is ineffective for proper modification of velaglucerase with SATP, and the conjugation yield obtained is poor. By studying the chemical reaction of N-succinimidyl esters with primary amines (the reaction of SATP coupling to proteins), it was shown that the reaction is highly dependent on the pH of the reaction mixture. At neutral pH (pH 7.4), where SATP modification was initially carried out, the reaction proceeded very slowly and took several hours to reach equilibrium. However, the same reaction carried out at a more alkaline pH (pH 8.5-pH 9) occurs rapidly, reaching equilibrium within a few minutes. Following SATP modification and deacetylation of the protective sulfhydryl groups, sulfhydryl addition was quantified using a free sulfhydryl assay kit. The results were plotted as relative ratios between the SATP-modified enzyme and the unmodified velaglucerase. After a 2-hour reaction at pH 7.4, free sulfhydryl levels doubled. In contrast, reactions carried out at pH 8.5 and pH 9 showed a 4-fold increase in free sulfhydryl levels, indicating that SATP is more reactive under these conditions.

[0101] As mentioned above, sulfhydryl quantification requires deacetylation of the sulfhydryl groups protected by SATP, which means that additional reactions (such as disulfide bond formation between protein molecules) may interfere with the measurement results obtained. In order to avoid this deacetylation step and considering that SATP only reacts with the primary amine of lysine residues, it was decided to analyze the reduction of free surface amines in lysine residues instead. The reduction of free lysine residues was determined quantitatively with TNBSA. TNBSA reacts with primary amines to produce yellow molecules, which can be quantified by absorbance. In the case of proteins, the only primary amines that can be detected by TNBSA are those from exposed lysine residues (except the N-terminus). Therefore, TNBSA reactivity is directly proportional to the number of lysine residues on the protein surface. The results of lysine quantification show that the percentage of lysine residues exposed after SATP modification does not decrease under pH 7.4, proving that the reactivity of molecules under these conditions is poor. However, the reaction performed under pH 8.5 to pH 9 shows that the exposed lysine reduces by 25% to 30%, further confirming the greatly increased reactivity of SATP under these pH conditions.

[0102] Regarding GCase activity, no changes were detected at pH 7.4. Previously, no activity loss was observed after SATP modification, which was attributed to the complete stabilization of the enzyme in the presence of IFG. On the other hand, GCase activity decreased by 25% and 30% after SATP modification at pH 8.5 and pH 9, respectively. Considering the effects of IFG, the only explanation for the decreased GCase activity is the modification of certain lysine residues that are critical for enzyme activity.

[0103] At this point, the main goal of the SATP modification process is to simultaneously optimize the yield of SATP modification and maintain GCase activity. Considering both parameters simultaneously in the statistical analysis and optimizing the synthesis conditions can provide more promising results (Karbasian et al., 2019). Optimization by central composite design–response surface methodology (CCD-RSM) should help to select the values ​​of the independent variables that have a greater impact on the output of SATP modification and GCase activity maintenance (dependent variables). According to the reference, the manual of SATP (ThermoScientific, Ref. 26100) and this preliminary experiment, the main independent variables that mainly affect SATP modification and maintenance of GCase activity are (1) pH, (2) the molar ratio of SATP to protein and (3) reaction time. For CCD-RSM optimization, it is necessary to limit the working range of the independent variables. Again using information from references, SATP handbooks, and preliminary experiments, the pH range was set to pH 7 to pH 9, the molar ratio of SATP to protein was set to range from 5 equivalents to 25 equivalents, and the reaction time was set to range from 30 min to 120 min.

[0104] Each numerical factor was varied across five levels, calculated using the Design Expert 11 (DX11) software: plus or minus α (axial points), plus or minus 1 (factor points), and a central point. The conditions that simultaneously maximized SATP modification and GCase activity were pH 9, a SATP:protein molar ratio of 15:1, and a reaction time of 60 minutes. The final protocol for velaglucerase modification and PGA conjugation is as follows:

[0105] 1. SATP Modification Step: Prepare a 0.5 mg / ml solution of velaglucerase in PBS (pH 9) with 25 μM IFG. Add 15 molar equivalents of a 20 mg / ml stock solution of SATP in dimethyl sulfoxide (DMSO) and incubate at room temperature with gentle shaking for 1 hour. Wash the resulting product three times with VCB supplemented with 25 μM IFG using a centrifugal concentrator (10 kDa cutoff).

[0106] 2. PGA conjugation step: To the purified SATP-modified protein solution in VCB, add 0.1 volume of deacetylation solution (0.5 M hydroxylamine-HCl in PBS). Add 0.1 volume of PGA-PD to a final PGA / protein molar ratio of 5:1 and incubate at room temperature for 4 hours. The resulting product is washed three times with VCB supplemented with 25 μM IFG using an amicon centrifugal unit (cutoff 30 kDa).

[0107] The final SATP modification scheme integrated into the conjugation procedure was shown to successfully improve GCase activity maintenance with a high SATP modification rate. Figure 3 As shown in Figure 2, approximately 25% of lysine modifications were present after optimization, and GCase activity was highly preserved ( Figure 3 d) The addition of IFG did not affect the conjugation of the SATP-modified protein to the conjugate. Compared to the conjugate batch prepared before the SATP modification optimization, the conjugation yield was significantly higher, as conjugation levels exceeding 90% were achieved.

[0108] Therefore, the GBA enzyme was modified with a SATP crosslinker to obtain protective sulfhydryl groups on the accessible surface of the enzyme. The optimization and validation of the SATP modification are described in detail above. From these presented results, it can be inferred that the GBA protein was effectively modified with SATP, resulting in a protein with 25% of exposed lysines modified by the SATP crosslinker. This result corresponds to 4 to 5 lysines modified by SATP. The resulting product (GBA-SATP) was conjugated to the PGA polymer with an efficiency exceeding 90%, obtaining a high yield of the final product, NanoGBA. However, it is noted herein that, as discussed herein, modification of the lysines on the GBA surface imparts unexpected characteristics to the enzyme, which are detailed below.

[0109] -GBA-SATP step and physicochemical changes of NanoGBA

[0110] The GBA protein contains three domains: domain I (residues 1 to residue 27 and residue 383 to residue 414), which is composed of an antiparallel β-sheet with two disulfide bonds and whose function is believed to be structural; domain II (residues 30 to residue 75 and residue 431 to residue 497), which is an immunoglobulin-like structure and is generally considered to be an interaction domain; and domain III (residues 76 to residue 381 and residue 416 to residue 430), which is a catalytic domain with a TIM barrel structure.

[0111] As a protein with 497 amino acids, it was initially expected that modification of 4 to 5 lysine residues on the surface of the enzyme would not significantly affect the overall structure. Nevertheless, there is evidence that the three-dimensional structure of the enzyme has been altered. In terms of secondary structure, circular dichroism (CD) spectroscopy revealed a reorganization of the enzyme's β-sheet conformation, indicating a transition from parallel β-sheets to antiparallel β-sheets in approximately 5% of the overall structure (see Example 2).

[0112] In the case of NanoGBA, the CD spectrum shows a more pronounced change in secondary structure. In this case, the proportion of α-helices increases. This result is consistent with the conformational space of PGA (PGA can be found to be unfolded or α-helical) (see Example 2).

[0113] In addition to this conformational change, the chromatographic elution time in the Superdex 200 gel filtration column also undergoes a significant shift. Specifically, the unmodified GBA enzyme eluted after 46 minutes, while GBA-SATP eluted at 35 minutes. The reduction in the retention time of GBA-SATP confirms the unexpected conformational change of GBA after SATP modification. In this case, this change in elution time is not only speculated to have changed the secondary structure of the enzyme, but also to have changed the quaternary structure of the enzyme, which strongly suggests that the enzyme dimerizes after SATP modification. In addition, NanoGBA eluted at 32 minutes in the Superdex 200. This result is consistent with the increase in molecular weight caused by adding PGA polymer to the GBA-SATP product (see Example 2).

[0114] Biochemical changes caused by structural changes

[0115] The changes revealed in the GBA-SATP and NanoGBA structures not only confer a unique globular structure to the protein but also induce alterations in the enzyme's biochemical properties. Specifically, this demonstrates a dramatic shift in protein stability due to SATP modification, which is further exploited by the NanoGBA end product.

[0116] First, GBA was readily degraded by trypsin in vitro. In contrast, both the GBA-SATP product and the NanoGBA final product showed complete resistance to trypsin. This result confirms that resistance to protease degradation is conferred by the SATP modification, rather than the addition of the PGA polymer.

[0117] The enhanced resistance of GBA-SATP was further validated by a human plasma (HBP) stability assay. Upon exposure to HBP, the GBA enzyme undergoes rapid inactivation, being completely inactivated after 1 hour in HBP. This incubation time results in the enzyme being unable to restore glucocerebrosidase activity after internalization in M17 GBAKO cells. Notably, GBA-SATP was much more resistant to inactivation by HBP, retaining approximately 35% of its initial capacity even after 24 hours of incubation in HBP. In this regard, NanoGBA's stability was further enhanced. After 24 hours of incubation in HBP, NanoGBA maintained nearly its full capacity to restore glucocerebrosidase activity.

[0118] The increased stability of the GBA-SATP product and the evolution of this property in the Nano-GBA product suggest a substantial shift in the behavior of the initial enzyme. This is demonstrated by the ability of the different products to restore glucocerebrosidase activity over time in the M17 GBA KO model. As shown in the Examples, the GBA enzyme initially restored 20% of WT glucocerebrosidase activity, but this activity rapidly declined, becoming inactive after three days. In contrast, GBA-SATP initially restored 50% of WT activity and, after seven days in the internalization assay, still retained 25% of WT activity. This striking contrast in behavior highlights the transformative impact of SATP modification on GBA.

[0119] Finally, the NanoGBA product exhibited distinct properties from those of the GBA and GBA-SATP products. In this case, NanoGBA initially recovered 25% of WT activity, but in contrast to the other products, NanoGBA activity increased over time, reaching WT activity levels after 24 hours of incubation and maintaining this level for at least 7 days. Furthermore, Western blot analysis revealed that NanoGBA internalization was higher than that of GBA or GBA-SATP and increased over time. This effect was entirely attributable to the PGA polymer conjugation, which not only enhanced enzyme stability but also increased cellular uptake (see Example 2).

[0120] Therefore, the first aspect of the present invention relates to a method for modifying the surface of glucocerebrosidase with N-succinimidyl-S-acetylthiopropionate (SATP) by modifying the lysine side chain of the protein and providing a SATP-modified protein, the method comprising:

[0121] a. adding an aprotic polar solvent (preferably DMSO or DMF) containing N-succinimidyl-S-acetylthiopropionate (SATP) that is miscible with water and capable of dissolving SATP, and optionally a surfactant (e.g., polysorbate 20) having an HLB value between 14 and 20, to an aqueous composition containing a glucocerebrosidase protein and a molecular chaperone (MC), wherein the molecular chaperone is preferably a salt of isofagomine, wherein the reaction is carried out under the following conditions: an alkaline pH greater than 8, preferably greater than 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5; a molar ratio of SATP to glucocerebrosidase of at least 5 molar equivalents to at most 25 molar equivalents; a reaction time range of at least 30 min; and preferably gentle shaking (e.g., 100 rpm).

[0122] It should be noted that, as indicated in the definitions section, molecules containing similar reactive head groups but with different spacer arm lengths can be used as alternatives to SATP, for example, selected from SATA, 4-SATA, 8-SATA, 12 -SATA, Other molecules that can also modify the lysine side chain but add a pyridyldithiol-reactive group are selected from SMPT, SPDP, LC-SPDP, PEG4-SPDP, and PEG12-SPDP. In addition, the lysine side chain can be modified with other amine-reactive head groups, such as sulfo-N-succinimidyl or imidoesters, especially those selected from 2-iminothiolane (Traut's reagent) and sulfo-LC-SPDP. (See Table 4). All of these alternatives to SATP are contemplated by the present invention to provide GBA-modified proteins.

[0123] Thus, the first aspect of the present invention provides not only a method for modifying the surface of glucocerebrosidase with N-succinimidyl-S-acetylthiopropionate (SATP) by modifying the lysine side chains of the protein and providing SATP-modified proteins, but also a method for modifying the surface of glucocerebrosidase with a molecule containing a reactive head group similar to SATP but having a different spacer arm length (e.g., any of the above-mentioned molecules (see also Table 4)) by modifying the lysine side chains of the protein and providing GBA-modified proteins,

[0124] In a preferred embodiment, the pH is 8.0 to 12, more preferably 8.5 to 12, more preferably 9.0 to 12, more preferably 9.5 to 12. Also preferred is 8.0 to 10, more preferably 8.5 to 10, more preferably about 8.5 to about 9.5.

[0125] Note that the aprotic polar solvent that is miscible with water and capable of dissolving SATP is more preferably DMSO.

[0126] It should be noted that alternatives to polysorbate 20 that can be used in the present invention include Triton X-100 (4-(1,1,3,3-tetramethylbutyl)-phenyl-polyethylene glycol), Triton X-114 ((1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol), NP-40 (nonylphenoxypolyhydroxyethylethanol), Brij-58 (polyethylene glycol hexadecyl ether); n-dodecyl-β-maltoside or octyl-β-glucoside. Derivatives of polysorbates (e.g., polysorbate 20), polyethylene (e.g., NP-40), or folic acid-modified sulfobetaine are preferred.

[0127] In a preferred embodiment of the first aspect, the concentration of a salt of isofagomine (or alternatively 1-deoxynojirimycin or 1-azafagomine) is at least 0.5 μM, preferably about 0.5 μM to about 25 μM. In addition, other inhibitory or allosteric GBA-specific chaperones can be used instead of salts of isofagomine. Examples of such chaperones are (but are not limited to) alkylated derivatives of isofagomine, 1,5-dideoxy-1,5-inositol (DIX) and its derivatives, deoxynojirimycin (DNJ) and its derivatives, and ambroxol (ABX).

[0128] In another preferred embodiment, the glucocerebrosidase protein is selected from velaglucerase, imiglucerase or uplyso or any other (preferably recombinant) glucocerebrosidase.

[0129] In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the salt of isofagovine is isofagovine D-tartrate, and the salt of isofagovine is preferably at a concentration of about 25 μM.

[0130] In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the reaction of step (a) is carried out at a molar ratio of SATP to glucocerebrosidase of about 5 molar equivalents to about 25 molar equivalents and a reaction time range set to about 30 min to about 120 min.

[0131] In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the reaction of step (a) is carried out at a molar ratio of SATP to glucocerebrosidase of about 10 molar equivalents to about 20 molar equivalents, preferably about 15 molar equivalents, and a reaction time ranging from about 50 minutes to about 70 minutes, preferably 60 minutes.

[0132] In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the SATP-modified protein, SATP-modified GBA protein, or GBA-modified protein obtained by the reaction in step (a) is isolated, preferably purified, and the purification is preferably performed in a citric acid buffer. Preferably, the SATP-modified protein, SATP-modified GBA protein, or GBA-modified protein obtained by the reaction in step (a) is isolated, preferably purified, and deacetylated, preferably with a deacetylation solution (e.g., hydroxylamine-HCl solution) (at room temperature for 4 hours).

[0133] A second aspect of the present invention relates to a method of making a polymer conjugate, wherein the method comprises:

[0134] b) adding a polymer to the deacetylated and preferably purified SATP-modified protein, SATP-modified GBA protein or GBA-modified protein obtained by the reaction of step (a) according to the first aspect of the present invention, wherein the SATP-modified protein, SATP-modified GBA protein or GBA-modified protein obtained by the reaction of step (a) is isolated, preferably purified, and deacetylated, preferably deacetylated with a deacetylation solution (e.g., hydroxylamine-HCl solution) (preferably at room temperature for about 4 hours); and wherein the polymer is selected from dextran, water-soluble linear polyamino acids or polypeptides / polypept(o)ides (including polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosine (PSar)...), polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolide) (PLA / PLGA), poly(hydroxyalkylmethacrylamide), polyglycerol, polyamidoamine (PAMAM) and polyethyleneimine (PEI), polyorthoesters, polyacetals; and

[0135] c) Optionally, washing the product obtained from b), preferably with an acidic buffer (such as VCB), optionally supplemented with a molecular chaperone (such as a salt of isofagomine or any other suitable MC referred to throughout the specification).

[0136] An alternative embodiment of the second aspect of the present invention relates to a method of making a polymer conjugate, wherein the method comprises:

[0137] b. adding a polymer to the non-deacetylated SATP-modified protein, the non-deacetylated SATP-modified GBA protein or the non-deacetylated GBA-modified protein obtained by the reaction of step (a) according to the first aspect of the present invention, wherein the polymer is selected from dextran, a water-soluble linear polyamino acid or a polypeptide / polypeptide-like polypeptide (including polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosine (PSar)...), polyethylene glycol (PEG), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolide) (PLA / PLGA), poly(hydroxyalkyl methacrylamide), polyglycerol, polyamidoamine (PAMAM) and polyethyleneimine (PEI), polyorthoester, polyacetal; and simultaneously deacetylating the SATP-modified GBA protein or GBA-modified protein, preferably using a deacetylation solution (e.g., hydroxylamine-HCl solution); and

[0138] c. Optionally, the product obtained from b) is washed, preferably with an acidic buffer (eg VCB), which is optionally supplemented with a molecular chaperone (eg a salt of isofagomine).

[0139] In preferred embodiments of the second aspect of the invention (including alternative embodiments thereof), the polymer is polyglutamic acid (PGA). Preferably, the polymer is a polyglutamic acid selected from poly(L-glutamic acid), poly(D-glutamic acid), poly(D,L-glutamic acid), poly(L-γ-glutamic acid), poly(D-γ glutamic acid) and poly(D,L-γ glutamic acid), wherein optionally, the polyglutamic acid comprises at least 50% glutamic acid backbone units, and optionally comprises 60%, 70%, 80%, 90% or 100% glutamic acid backbone units. More preferably, the polymer is L-PGA having 25 to 150 glutamic acid monomer units.

[0140] A third aspect of the present invention relates to a method for conjugating glucocerebrosidase to poly-L-glutamic acid (PGA), wherein the method comprises:

[0141] a. A method for producing a surface protein of glucocerebrosidase α modified with N-succinimidyl-S-acetylthiopropionate by modifying the lysine side chain of the protein according to the first aspect of the present invention or according to any one of the preferred embodiments comprising providing a GBA modified protein;

[0142] b. after step a), deacetylation of the product obtained by a); and

[0143] c. According to any second aspect of the present invention or any of its preferred embodiments, simultaneously or subsequently conjugating a polymer to the product obtained from b).

[0144] In a preferred embodiment of the second or third aspect, the method further comprises isolating and / or purifying the obtained or obtained polymer conjugate.

[0145] A fourth aspect relates to a SATP-modified protein, a SATP-modified GBA protein, or a GBA-modified protein obtained according to the first aspect of the present invention or any of its embodiments. In some embodiments, the product obtained contains at least about 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt%, or 100 wt% of the SATP-modified protein, the SATP-modified GBA protein, or the GBA-modified protein. The term "wt%" refers to an amount by weight, expressed as a percentage of the total weight of the obtained product.

[0146] A fifth aspect of the present invention relates to a polymer conjugate obtained according to the second or third aspects of the present invention or any preferred embodiment thereof. In some embodiments, the product obtained contains at least about 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt%, or 100 wt% of the polymer conjugate by weight. The term "wt%" indicates an amount by weight, expressed as a percentage of the total weight of the obtained product.

[0147] It should be noted that in the sixth aspect of the present invention, the present invention also provides a composition comprising or consisting of: glucocerebrosidase modified with N-succinimidyl-S-acetylthiopropionate (SATP) by modifying the lysine side chain of the protein and providing a SATP-modified GBA protein; and / or glucocerebrosidase modified with a molecule containing a reactive head group similar to SATP but with a different spacer arm length (e.g., any one of the molecules shown in Table 4) by modifying the lysine side chain of the protein and providing a GBA-modified protein. Preferably, the GBA protein can be any glucocerebrosidase protein selected from velaglucerase, imiglucerase or uplyso, or any other (preferably recombinant) glucocerebrosidase. It should also be noted that, as described above, molecules containing similar reactive head groups but with different spacer arm lengths can be used as substitutes for SATP, for example, selected from SATA, 4-SATA, 8-SATA, 12 -SATA, 24-SATA and 3-mercaptopropyl-N-hydroxysuccinimide. Other molecules that can also modify the lysine side chain but add a pyridyldithiol reactive group are selected from SMPT, SPDP, LC-SPDP, PEG4-SPDP and PEG12-SPDP. In addition, the lysine side chain can be modified with other amine-reactive head groups, such as sulfo-N-succinimidyl or imidoesters, especially those selected from 2-iminothiolane (Traut's reagent) and sulfo-LC-SPDP (see Table 4). All of these alternatives to SATP are contemplated by the present invention to provide GBA-modified proteins.

[0148] Another aspect of the present invention (the seventh aspect of the present invention) relates to: a composition comprising or consisting of: a polymer conjugate of glucocerebrosidase modified with N-succinimidyl-S-acetylthiopropionate (SATP) according to the sixth aspect of the present invention; or a composition comprising or consisting of: a polymer conjugate of the glucocerebrosidase-modified protein (GBA-modified protein) according to the sixth aspect of the present invention. It should be noted that the polymer conjugated to the protein can preferably be selected from dextran, water-soluble linear polyamino acids or polypeptides / polypeptide-like peptides (including polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosine (PSar), etc.), polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolide) (PLA / PLGA), poly(hydroxyalkylmethacrylamide), polyglycerol, polyamidoamine (PAMAM), polyethyleneimine (PEI), polyorthoesters, and polyacetals. Preferably, the polymer is polyglutamic acid (PGA). Preferably, the polymer is a polyglutamic acid selected from poly(L-glutamic acid), poly(D-glutamic acid), poly(D,L-glutamic acid), poly(L-γ-glutamic acid), poly(D-γ glutamic acid) and poly(D,L-γ glutamic acid), wherein optionally, the polyglutamic acid comprises at least 50% glutamic acid backbone units, and optionally comprises 60%, 70%, 80%, 90% or 100% glutamic acid backbone units. More preferably, the polymer is L-PGA having 25 to 150 glutamic acid monomer units. It should be noted that the polymer conjugate of the seventh aspect of the invention is produced in the manner shown in the second aspect and / or the third aspect of the invention.

[0149] In some embodiments, in any of the compositions of the sixth or seventh aspects of the invention, the modified glucocerebrosidase or protein conjugate is present in a weight percentage of at least about 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt% or 100 wt%. The term "wt%" indicates an amount by weight, expressed as a percentage of the total weight of the composition.

[0150] The eighth aspect of the present invention relates to a pharmaceutical composition comprising the polymer conjugate of the fifth aspect or the seventh aspect, and optionally further comprising an excipient, a carrier, a penetration enhancer and / or an adjuvant.

[0151] The ninth aspect of the present invention relates to the polymer conjugate of the fifth or seventh aspect or the pharmaceutical composition of the eighth aspect for use in treatment. Preferably, the method is for treating a subject suffering from Gaucher disease and / or Parkinson's disease. More preferably, the glucocerebrosidase of the pharmaceutical composition or the glucocerebrosidase of the polymer conjugate is selected from velaglucerase, imiglucerase, uplyso, or any recombinant glucocerebrosidase.

[0152] The following examples are for illustrative purposes only and are not intended to limit the present invention.

[0153] Example

[0154] Example 1. Development of Nanotechnology-Based Enzyme Replacement Therapy for Parkinson's Disease and Gaucher Disease

[0155] Two different types of nanoformulations or nanoconjugates have been tested: encapsulation of GBA with PLGA and conjugation of GBA with PGA, both using several process routes and formulations.

[0156] 1. Encapsulation of GBA into PLGA nanoconjugates.

[0157] Poly(lactic-co-glycolic acid) or PLGA is an organic polymer used to produce biocompatible nanocapsules. The synthesis parameters of these nanoconjugates affect the properties of the final capsules, such as size, polydispersity, surface potential (ζ), and release time, making them highly tunable. The encapsulation of drugs in nanoparticle systems allows for sustained and controlled release of the payload, which at the clinical level can translate into reduced administration frequency, reduced dosage, and minimized drug fluctuations; thereby increasing the efficacy of the treatment while reducing its side effects. PLGA nanocapsules, as well as other nanoconjugates, can be surface modified to allow for crossing biological barriers and targeted delivery of the encapsulated drug.

[0158] The main objectives of GBA encapsulation in PLGA nanocapsules are to overcome the main limitations of traditional ERT. These objectives are:

[0159] a) Protection of GBA (velaglucerase) from loss of enzymatic activity. The physical barrier provided by the nanocapsules protects the protein from the harsh conditions encountered during blood circulation.

[0160] b) Increased cellular uptake and intracellular release of GBA in a controlled manner. The size and morphology of the resulting nanocapsules induce enhanced cellular uptake. Sustained release following cellular internalization should provide a stable source of active GBA that remains unharmed throughout its path through the bloodstream.

[0161] c) Overcoming the blood-brain barrier (BBB) ​​through surface modification or intranasal administration. Neurological manifestations of GD types 2 and 3 remain untreatable because traditional ERT cannot deliver active GBA to the central nervous system (CNS). Intranasal administration of encapsulated PLGA should overcome the BBB and deliver active GBA to the brain.

[0162] To achieve the above objectives, we will attempt to encapsulate velaglucerase (GBA type) into PLGA nanocapsules as described in the next section.

[0163] 1.1. Initial conditions.

[0164] The process is a water-in-oil-in-water (W / O / W) double emulsion method with the following stages:

[0165] W1: inner aqueous phase. Buffer: 50 mM citric acid / sodium citrate (pH 5.6) with 5% sucrose and 0.01% polysorbate 20. 0.5 mg / ml velaglucerase was dissolved.

[0166] O: organic phase. 25 mg of PLGA was dissolved in 750 μl of dichloromethane (DCM), and 4% v / v Span 80 was added as a surfactant.

[0167] W2: External aqueous phase: 2% PVA and 1M NaCl in water.

[0168] The synthesis steps are as follows:

[0169] 1. Preparation of Phase O: Dissolve PLGA in DCM under magnetic stirring. Add sorbitan monooleate after PLGA is completely dissolved.

[0170] 2. Add phase W1 onto phase O and sonicate in a Branson Sonifier SFX 550 using a microprobe at 25% amplitude for 1 minute.

[0171] 3. Pour the emulsion into phase W2 and sonicate under the same conditions as step 2.

[0172] 4. The final emulsion was stirred magnetically at room temperature until the dichloromethane was completely evaporated.

[0173] PLGA nanoparticles or nanoconjugates loaded with velaglucerase were successfully synthesized and characterized. SEM images of loaded and unloaded nanoconjugates are shown in Figure 4 To test whether the encapsulated protein retained enzymatic activity, an in vitro assay was performed in ST-Q7 cells (mouse striatal neurons). Although velaglucerase successfully colocalized with LAMP1 (a lysosomal marker indicating that the protein reached its intended destination), as Figure 2 As shown, no enzyme activity was detected.

[0174] Attempts to optimize the synthesis conditions to overcome the loss of GCase activity were unsuccessful.

[0175] 1.2. Optimization attempts to preserve GCase activity.

[0176] To protect GBA from loss of enzyme activity, several changes were made in the phases and synthesis process. In the following sections, these changes are categorized according to the phase affected, with the final section covering the overall changes.

[0177] 1.2.1.Modification of phase W1.

[0178] The modifications affecting the W1 phase were mainly changes in the buffer composition. This was done to stabilize the protein and protect it from activity loss due to denaturation caused by contact with organic solvents and by sonication. These changes were:

[0179] 1. Add different types of polyethylene glycol.

[0180] 2. Change the concentration of citric acid / citrate buffer.

[0181] None of these changes resulted in nanoconjugates with active GBA.

[0182] 1.2.2. Modification of phase O.

[0183] Modifications affecting the O phase focused on reducing the hydrophobicity of the phase to minimize GBA unfolding during the homogenization step. The PLGA polymer was partially replaced with a PLGA-PEG polymer, and several ratios were tested. Ethyl acetate was tested as an alternative to dichloromethane.

[0184] None of these changes resulted in nanoconjugates with active GBA.

[0185] 1.2.3. Modifications to the W2 stage.

[0186] The surfactants constituting W2 are responsible for the stabilization of the nanoparticles during the evaporation of the organic solvent. Polysorbate 80 and Pluronic were used instead of PVA.

[0187] None of these changes resulted in nanoconjugates with active GBA.

[0188] 1.2.4. Modification of emulsification method.

[0189] Sonication times and amplitudes were tested to obtain a protocol that produced nanoparticles with the desired properties but reduced the amount of energy delivered to the sample that causes protein unfolding. Various conditions were tested, including shorter times at higher amplitudes, longer times at lower amplitudes, and continuous and intermittent sonication.

[0190] Conclusion: None of these changes resulted in active GBA nanoparticles, and PLGA was subsequently abandoned as a drug delivery system.

[0191] 2. Conjugation of GBA and PGA polymers.

[0192] An alternative approach to incorporating velaglucerase into a nanoparticle system without complete loss of activity was attempted through polymer unmasking–masked protein therapy (PUMPT), developed by Dr. María J. Vicent (CIPF, Valencia, Spain) (Talelli M & Vicent MJ (2014) Reduction Sensitive Poly(L-glutamic acid) (PGA)-Protein Conjugates Designed for Polymer Masked–Unmasked Protein Therapy. Biomacromolecules 15, 4168–4177. Available at: https: / / pubs.acs.org / doi / 10.1021 / bm5011883.). PUMPT refers to a soft nanotechnology strategy in which proteins are conjugated to biodegradable polymers that provide protection and mask protein activity during transport while enabling controlled recovery of activity at the target site due to external stimuli. In the present invention, an adaptation of the PUMPT approach for velaglucerase conjugation was tested. Velaglucerase is conjugated to a polyglutamic acid (PGA) polymer via a reduction-sensitive linker, which is intended to mask (and protect) the protein activity in oxidative environments (such as the bloodstream) and release it into reducing environments (such as lysosomes).

[0193] a. Initial attempt using the Talleli et al., 2014 protocol.

[0194] To successfully conjugate velaglucerase to PGA polymers, the conjugation protocol developed by Talelli and Vicent (Talelli and Vicent, 2014), which was optimized for lysozyme, was adapted for velaglucerase. The conjugation procedure consists of two main steps: (1) modification of the surface protein with SATP, which adds sulfhydryl groups by modifying lysine side chains; and (2) conjugation of the SATP-modified protein with PGA modified with pyridyldithiol groups (PGA-PD), which attack the sulfhydryl groups added to lysine to form disulfide bonds.

[0195] The conjugation conditions of the Talelli and Vicent protocol are as follows:

[0196] - SATP modification step: 1 mg / ml lysozyme was dissolved in PBS. A 2 mg / ml stock solution of SATP in dimethyl sulfoxide (DMSO) was added and incubated at room temperature with gentle shaking for 1 h. The resulting product was washed three times with PBS using an amicon centrifugal unit (cutoff 3 kDa). Two molar ratios of SATP / lysozyme (10:1 and 6:1) were tested for this step, but the 10:1 ratio showed a higher modification rate and was therefore selected for the next step.

[0197] -PGA conjugation step: The buffer of the purified SATP-modified protein (modified with a SATP / lysozyme ratio of 10:1) was changed to 300mM ammonium acetate (pH 5) and 0.2 volumes of deacetylation solution (0.5M hydroxylamine-HCl in PBS) were added to prepare sulfhydryl groups for conjugation. PGA-PD was added and incubated overnight at room temperature. The resulting product was washed three times with PBS using an amicon centrifugal unit. Three PGA-PD / lysozyme ratios (5:1, 2.5:1, and 0.5:1) were measured.

[0198] Several modifications were necessary to adapt this protocol for use with velaglucerase. These changes and their rationale are as follows:

[0199] The initial velaglucerase concentration was changed from 1 mg / ml to 0.5 mg / ml. The initial stock concentration of velaglucerase was 2.5 mg / ml; higher concentrations tend to cause protein aggregation. The wash steps of the amicon apparatus used during the SATP modification and conjugation steps can concentrate the protein to up to 5 times the working concentration. To maintain the protein below 2.5 mg / ml, the working concentration was reduced to 0.5 mg / ml.

[0200] The concentration of the SATP stock solution was increased from 2 mg / ml to 20 mg / ml to reduce the concentration of DMSO during the reaction, which could cause the velaglucerase to unfold and aggregate. The SATP / lysozyme ratio tested for lysozyme modification was chosen based on the number of lysines present in lysozyme, i.e. 6. A 6:1 molar ratio of SATP / lysozyme was tested as this would imply a SATP / lysine ratio of 1:1. The 10:1 SATP / lysozyme ratio was to ensure an excess of SATP compared to the lysine residues. Since velaglucerase has 22 lysine residues instead of 6, the following three ratios were tested: 10:1 (compared to the 10:1 ratio of the lysozyme protocol), 22:1 (SATP / lysine 1:1 ratio) and 40:1 (with excess SATP).

[0201] After checking that it did not interfere with the conjugation procedure, the 300 mM ammonium acetate buffer at pH 5 was replaced with the original buffer of velaglucerase (velaglucerase citrate buffer or VCB, 50 mM citrate pH 5.6, 5% sucrose and 0.01% v / v polysorbate 20). This change was made to improve the stability of velaglucerase during conjugation.

[0202] Although the purpose of the PGA conjugation step is to mask GCase activity, this loss of activity should be fully reversible after release in a reducing environment. However, the SATP modification step, which irreversibly modifies the surface lysine residues of the protein, leads to irreversible activity loss. Therefore, optimization is a key step to ensure maximum modification rate and minimum enzyme activity loss. The three initial ratios of SATP used for the velaglucerase conjugation test show a decrease in enzyme activity that is proportional to the SATP concentration ( Figure 5 PGA conjugation successfully completely masked GCase activity, but after release with 10 mM reduced glutathione (GSH), only one condition restored GCase activity, and the restored activity was far lower than the activity level that should be fully restored by SAT-modified velaglucerase.

[0203] b. Molecular chaperones stabilize GBA.

[0204] Activity loss during modification of GBA with SATP and conjugation of the modified protein to PGA is primarily attributed to protein instability in the sorbitan monooleate working buffer used in the conjugation step. To mitigate this activity loss, GBA chaperones (MCs) are used during the synthesis process. MCs are small molecules with the ability to bind and stabilize the target enzyme. Typically, these MCs are competitive inhibitors that bind to the enzyme's catalytic pocket.

[0205] In the case of GBA, there are currently many commercially available MCs, although none of them have entered the pharmaceutical market as a pharmacological partner (PC) for GBA. Of these, the two most studied are ambroxol hydrochloride and isofagomine D-tartrate (IFG). For our purposes, ambroxol was abandoned due to the presence of primary amines in its structure, which compete with lysine residues and interfere with SATP modification. Figure 6 IFG was chosen as a stabilizer because it does not contain any primary amines. In addition, IFG is a strong partner of GBA as it has a Ki of 5.8 nM at pH 7.0, which means that a small amount of MC may be sufficient to stabilize GCase during conjugation.

[0206] To test that IFG did not interfere with the conjugation process, the conjugation protocols were compared in the presence or absence of the partner. The protocol using IFG differed only in that an excess of IFG (25 μM, almost 1000 times the Ki) was added to the working buffer to ensure maximum inhibition and protection of the enzyme. Samples were taken from each conjugation step (SATP modification and PGA conjugation) of both protocols and analyzed by non-reducing SDS-PAGE coupled with silver staining ( Figure 7 ). This technique allows the determination of the molecular weight of the nanoconjugates without disrupting the disulfide bonds that bind the polymer to the protein, thereby qualitatively assessing the conjugation yield.

[0207] The results of non-reducing SDS-PAGE showed that there was no significant difference in conjugation in the absence or presence of IFG ( Figure 7 and Figure 3 C). SATP modification of velaglucerase results in dimerization of a small amount of the enzyme. In the presence of IFG, a dimerization band was present on the SATP-modified enzyme, indirectly indicating that there was no interference in this step. Conjugation of PGA to the protein was observed by the tailing effect of the protein-polymer conjugate, which did not differ between the different conditions. Although no differences were observed in the conjugation efficiency, the addition of IFG during the synthesis process increased the GCase activity measured in each step of the conjugation, demonstrating the ability of IFG to stabilize and protect the enzyme during conjugation. After SATP modification, no loss of GCase activity was detected in the reaction containing IFG, a fact attributed to the complete stabilization of the enzyme at this step. In the presence of IFG, the gradual decrease in GCase activity after conjugation was attributed to the masking effect of the polymer on the enzyme activity.

[0208] Conclusion: IFG improves enzyme stability during conjugation and masking. In the new modified protocol, all buffers used for GBA nanoconjugation included 25 μM IFG.

[0209] c. Optimization of SATP modification

[0210] Despite the interesting results obtained due to the addition of IFG as a stabilizer, the nanoconjugation strategy seems to be still far from being optimized. Figure 7 As shown, more than 80% of velaglucerase remained unmodified after all conjugation procedures. The explanation for this poor conjugation of the enzyme was subsequently found in the buffer conditions used for the SATP modification step.

[0211] As described in Section 2a), the protocol for conjugating velaglucerase to PGA was adapted from the work of Talelli and Vicent. The key step in the adaptation is the modification of the protein with SATP, which, in contrast to the PGA conjugation step, results in irreversible loss of activity. As seen in Section 2b, this protocol adaptation is ineffective for proper modification of velaglucerase with SATP, and the conjugation yields obtained are poor. By studying the chemical reaction of N-succinimidyl esters with primary amines (the reaction of SATP conjugation to proteins), it was shown that the reaction is highly dependent on the pH of the reaction mixture. At the neutral pH (pH 7.4) where SATP modification was initially performed, the reaction proceeded very slowly, requiring several hours to reach equilibrium. However, the same reaction performed at a more alkaline pH (pH 8.5-pH 9) occurred rapidly, reaching equilibrium within a few minutes. Taking this information into account, the buffer conditions for the SATP modification step were modified to react at pH 8.5 or pH 9. The SATP modification efficiency was assessed in terms of relative thiol addition, relative lysine reduction, and relative maintenance of GCase activity compared to pH 7.4.

[0212] After SATP modification and deacetylation of the protected thiol groups, thiol addition was quantified using a free thiol assay kit. The results were plotted as the relative ratio between the SATP-modified enzyme and the unmodified velaglucerase. Figure 9 As shown in (a), free thiol levels doubled after 2 hours of reaction at pH 7.4. In contrast, reactions performed at pH 8.5 and pH 9 showed a 4-fold increase in free thiol levels, indicating that SATP is more reactive under these conditions.

[0213] As mentioned above, sulfhydryl group quantitatively needs to be deacetylated to the sulfhydryl group of SATP protection, which means that additional reaction (such as by disulfide bond formation between protein molecules) may interfere with the measurement result obtained. In order to avoid this deacetylation step and considering that SATP only reacts with the primary amine of lysine residues, decision is changed to analyze the minimizing of free surface amine in lysine residues. Free lysine residues reduce and measure quantitatively with TNBSA. TNBSA produces yellow molecule with primary amine reaction, can be quantitatively measured by absorbance. In the case of protein, the only primary amine that can be detected by TNBSA is the primary amine (except N-terminus) from the lysine residue exposed. Therefore, TNBSA reactivity is directly proportional to the quantity of the lysine residues on protein surface.

[0214] The results of lysine quantification showed that the percentage of exposed lysine residues did not decrease after SATP modification at pH 7.4, demonstrating the poor reactivity of the molecule under these conditions ( Figure 9 b) However, reactions performed at pH 8.5 to pH 9 showed a 25% to 30% decrease in exposed lysines, further confirming the strong increase in SATP reactivity under these pH conditions.

[0215] As for GCase activity, no change in enzyme activity was detected at pH 7.4 ( Figure 9 c) The lack of activity loss after SATP modification, previously observed in Section 2b, was attributed to complete enzyme stabilization in the presence of IFG. On the other hand, GCase activity decreased by 25% and 30% after SATP modification at pH 8.5 and pH 9, respectively. Considering the effects of IFG, the only explanation for the decreased GCase activity is modification of certain lysine residues that are critical for enzyme activity.

[0216] The consequences of this SATP modification were predicted early on but were considered an unavoidable and acceptable cost in the process because SATP modification is a stochastic process that can modify any exposed lysine residue on the protein surface.

[0217] d. Parameters of the SATP modification step were optimized by CCD-RSM.

[0218] At this point, the primary goal of the SATP modification process is to simultaneously optimize the yield of SATP modification and maintain GCase activity. Considering both parameters simultaneously in statistical analysis while optimizing the synthesis conditions can provide more promising results. Optimization via central composite design–response surface methodology (CCD-RSM) should help select values ​​for the independent variables that have a greater impact on the outputs of SATP modification and GCase activity maintenance (dependent variables).

[0219] According to the reference, the SATP manual (Thermo Scientific, Ref. 26100), and this pilot study, the main independent variables that affect SATP modification and maintenance of GCase activity are (1) pH, (2) the molar ratio of SATP to protein, and (3) reaction time. For CCD-RSM optimization, it is necessary to limit the working range of the independent variables. Again using the information from the reference, the SATP manual, and the pilot study, the pH range was set to pH 7 to pH 9, the molar ratio of SATP to protein was set to 5 equivalents to 25 equivalents, and the reaction time range was set to 30 min to 120 min.

[0220] Each numerical factor varies at five levels, which are calculated by the software Design Expert 11 (DX11): plus or minus α (axial points), plus or minus 1 (factor points), and the center point. The values ​​of each factor are listed in Table 1.

[0221]

[0222] Table 1 Numerical factors and their levels used for CCD-RSM optimization experimental design.

[0223] The advantage of this statistical method is that the software randomly selects 16 different experimental conditions instead of testing all possible combinations of these conditions. The SATP modification step was performed 16 times, and the software calculation was performed once for each of the 16 conditions of pH, molar ratio of SATP to protein and reaction time, and the yield of modification and maintenance of GCase activity were measured. The results were introduced into the software and analyzed by analysis of variance (ANOVA). The conditions that had the greatest impact on SATP modification and maintenance of GCase activity were set to pH 9, a molar ratio of SATP to protein of 15:1, and a reaction time of 60 minutes. The final scheme for modification of velaglucerase and conjugation with PGA is as follows:

[0224] SATP modification step: Prepare a 0.5 mg / mL solution of velaglucerase in PBS (pH 9) with 25 μM IFG. Add 15 molar equivalents of a 20 mg / mL stock solution of SATP in DMSO and incubate at room temperature with gentle shaking for 1 hour. Wash the resulting product three times with VCB supplemented with 25 μM IFG using an amicon centrifugal unit (10 kDa cutoff).

[0225] PGA conjugation step: Add 0.1 volume of deacetylation solution (0.5 M hydroxylamine-HCl in PBS) to a solution of the purified SATP-modified protein in VCB. Add 0.1 volume of PGA-PD to a final PGA / protein molar ratio of 5:1 and incubate at room temperature for 4 hours. Wash the resulting product three times with VCB supplemented with 25 μM IFG using an amicon centrifugal unit (30 kDa cutoff).

[0226] Conclusion: The final SATP modification scheme, integrated into the conjugation procedure, demonstrated successful improvement in GCase activity maintenance while maintaining a high SATP modification yield. Approximately 25% lysine modification was present after optimization, demonstrating high preservation of GCase activity. The addition of IFG did not affect conjugation of the SATP-modified protein to the conjugate. Compared to conjugate batches prepared before SATP modification optimization, conjugation yields were significantly higher, achieving conjugation levels exceeding 90%.

[0227] 3. In vitro validation of PGA-GBA / IFG nanoconjugate.

[0228] Velaglucerase-PGA-IFG conjugate (NanoGBA, as generated in Sections 1 and 2 above) was validated in vitro in a neuronal model; BE(2)M17 cells (wild type and GBA knockout strains) were used to study the delivery and therapeutic effects of GBA.

[0229] NanoGBA has been shown to:

[0230] Increased stability of GBA protein (in human plasma);

[0231] Internalized into cells;

[0232] Delivered to lysosomes as active enzymes;

[0233] Demonstrated long-term recovery of GBA activity (7 days);

[0234] Can reduce the accumulation of GlcSph substrates;

[0235] Can reduce the accumulation of neurotoxic forms of alpha-synuclein, a hallmark of neurodegeneration in Parkinson's disease.

[0236] Conclusion: GBA-PGA-IFG conjugate is delivered to lysosomes and is able to reduce the accumulation of GlcSph substrates based on the long-term restoration of GBA activity.

[0237] 4. In vivo validation of PGA-GBA / IFG nanoconjugate (nanoGBA).

[0238] Mouse Models: In vivo validation testing was performed in two mouse models: GBA-WT and GBA-D409V (which exhibit >90% loss of GCase activity and substrate accumulation in all tissues).

[0239] • NanoGBA: In addition to nanoGBA, a new formulation of nanoconjugate with fluorescent PGA was generated (Cy5.5-PGA) to study the in vivo biodistribution after administration by IVIS (spectral in vivo imaging system) analysis.

[0240] Application:

[0241] o Intravenous: Verify systemic and CNS activity;

[0242] o Intranasal: Validation of CNS delivery. Cross-linked polymers) improve intranasal administration (patent EP17382498.WO2019020344A1)

[0243]

[0244] Table 2. Summary of in vitro PGA-GBA validation.

[0245] a. Intravenous administration:

[0246] After intravenous administration, the nanoconjugate was biodistributed in serum, liver, bone marrow, spleen, kidney, and lung. The nanoconjugate was active and increased GCase activity, reaching a peak at 30 min in serum and at 3 h in liver, blood, bone marrow, and spleen.

[0247] b. Intranasal administration:

[0248] Intranasal administration of nanoGBA showed lack of delivery to the CNS (tested by IVIS and increased GCase activity)

[0249] c. Intranasal administration of HA- Cross-linked polymers:

[0250] Using HA- As an adjuvant, it improves the effect of intranasal administration. It is formed from three biodegradable, biocompatible active ingredients (hyaluronic acid, polyglutamic acid, and lysine; Dr. Vicent patent co-inventor) to improve drug delivery to the CNS via intranasal administration.

[0251] Intranasal administration of HA- nanoGBA can deliver GBA protein to the brain ( Figure 13In the olfactory bulb, an increase in GCase activity was observed in GBA-D409V mutant animals, and this increase in GCase activity was translated into a decrease in the accumulation of the GBA substrates GlcCer and GlcSph ( Figure 13 Right). Analyses of other brain regions are currently underway.

[0252] in conclusion:

[0253] • Systemic delivery of nanoGBA was effective by intravenous administration, the nanoconjugate was detected in serum, liver and spleen and the GBA enzyme was active.

[0254] · By intranasal administration of HA- The cross-linked polymers were detected, delivery to the CNS was efficient, the nanoconjugates were tested, and the GBA enzyme was active and reduced substrate accumulation in the olfactory bulb; analysis of other brain regions is ongoing.

[0255] Example 2

[0256] To evaluate the novel properties of SATP-modified GBA and NanoGBA, several assays have been performed, including circular dichroism, size exclusion chromatography, stability to trypsin digestion and human plasma, and internalization in cell models.

[0257] Experiment 1: Circular Dichroism

[0258] Purpose : Comparison of the secondary structures of GBA, GBA-SATP and NanoGBA.

[0259] Research Methodology: GBA, GBA-SATP, and NanoGBA were diluted in circular dichroism buffer (10 mM acetate, 0.01% Tween-20) and their spectra were recorded from 190 nm to 250 nm in a 1 mm pathlength quartz cuvette. Secondary structure analysis was performed using the free online software "BeStSel" to obtain the percentages of α-helical, β-sheet, and unfolded (unidentified) conformations.

[0260] Results (see Figure 14 ) NanoGBA differs significantly from GBA and GBA-SATP, primarily due to an increased α-helix content in NanoGBA (19%) relative to GBA (14.6%) and GBA-SATP (14.8%). In contrast, GBA-SATP does not differ from GBA in total α-helix or β-sheet content, but does differ in β-sheet type (parallel and antiparallel), with the antiparallel type being up to 5% more abundant in GBA-SATP.

[0261] Table 3: Predicted secondary structures of GBA, GBA-SATP, and NanoGBA. Data from 190 nm to 250 nm were analyzed using BeStSel software. Results are expressed as a percentage of the total structural data.

[0262]

[0263] in conclusion All three products exhibited differences in their secondary structures. NanoGBA exhibited an increased presence of α-helices, consistent with the possible conformation of PGA-PD. Furthermore, the structure of NanoGBA differed from that of the unconjugated mixture of GBA and PGA-PD, suggesting that the conformation of NanoGBA after conjugation is solely due to the presence of the polymer.

[0264] In addition, differences between GBA and GBA-SATP were also found, implying that the thiol protecting groups on several amino acids can cause conformational changes in the overall structure of GBA.

[0265] Experiment 2: Size Exclusion Chromatography

[0266] Purpose: Verify that this product is structurally different from GBA.

[0267] Research Methodology: A solution of 75 μg of GBA, GBA-SATP and NanoGBA in Superdex 200 buffer (10 mM MES, 100 mM NaCl, pH 6) was applied to a Superdex 200 column and eluted under the same conditions to evaluate their retention.

[0268] Results (see Figure 15 ): GBA eluted after 46 minutes; GBA-SATP eluted after 35 minutes; NanoGBA eluted after 32 minutes.

[0269] in conclusion : The different behaviors observed in all three products during size exclusion chromatography indicate that there are significant structural differences between them, resulting in shortened retention times.

[0270] Experiment 3: restricted proteolysis

[0271] Purpose : Evaluate the protective effect of different products (GBA, GBA-SATP, NanoGBA) against trypsin digestion.

[0272] Research Methods: 50 μg of GBA, GBA-SATP, and NanoGBA were incubated with 5 μg of trypsin at 37°C for 0, 30, 60, and 90 minutes. Afterwards, the reaction was stopped, denaturation buffer was added, and the samples were heated at 95°C for 10 minutes. Digestion was analyzed by PAGE followed by total protein (Coomassie) staining.

[0273] result (See Figure 16 ) : GBA was sensitive to trypsin digestion in a time-dependent manner, with only 25% of the initial amount remaining undigested after 90 minutes. In contrast, both GBA-SATP and NanoGBA were completely resistant to trypsin.

[0274] in conclusion : GBA-SATP confers protection to GBA against trypsin digestion, and this protection is further maintained on the final nanoconjugate.

[0275] Experiment 4: Plasma stability assay

[0276] Purpose : Evaluation of the stability of different products in human plasma (HBP).

[0277] Research Methods To evaluate the ability of different products to restore intracellular glucocerebrosidase activity after incubation with human plasma, GBA, GBA-SATP, and NanoGBA were incubated in HBP for 0, 1, 3, 6, and 24 hours prior to treatment of M17 GBA KO cells. These cells were treated with 5 μg / ml of the different products (GBA, GBA-SATP, NanoGBA) pre-incubated in HBP for 3 hours. Afterwards, cell lysates were collected and internalized proteins were assessed by activity assays and Western blotting.

[0278] Results (see Figure 17 ) After incubation in HBP for 1 hour or longer, GBA is nearly completely inactivated, resulting in treated cells recovering only up to 5% of WT glucocerebrosidase activity. In contrast, GBA-SATP and NanoGBA exhibit high stability, resulting in a 10-fold improvement in activity recovery. GBA-SATP retained one-third of its initial activity after 24 hours of incubation in HBP, while NanoGBA preserved up to 83% of the initial activity.

[0279] in conclusionAlthough HBP incubation did not appear to have a differential effect on the internalization capacity of the products (total GBA internalization, as determined by Western blot), differences were observed in their ability to recruit GCase once internalized, demonstrating that the modifications confer increased stability in plasma. GBA-SATP and NanoGBA were significantly more stable in HBP than GBA, with NanoGBA being even more stable, especially after longer incubations.

[0280] Experiment 5: Uptake in M17 GBA KO cells

[0281] Purpose : Evaluate the ability of different products to be internalized over time in cell models.

[0282] Research Methods M17 GBA KO cells were treated with 5 μg / ml of different products (GBA, GBA-SATP, NanoGBA) at 1, 3, 6, 24, 72, and 168 hours. Cell lysates were then collected and internalized proteins were assessed by activity assays and Western blotting.

[0283] Results (see Figure 18 ) Despite internalization, only a maximum of 20% of WT glucocerebrosidase activity was restored after a 1-hour "naked" GBA treatment. Furthermore, intracellular glucocerebrosidase activity decreased over time, reaching negligible values ​​between 3 and 7 days. In contrast, after 1 hour of GBA-SATP treatment, cells reached 50% of WT activity, a level maintained over time until 7 days, at which point activity dropped to 25%. In the case of NanoGBA, a significant increase was observed over time, reaching 150% of WT activity after 3 days, and even exhibiting higher activity than WT after 7 days of treatment.

[0284] in conclusion Internalization of the three products differed significantly. SATP modification of GBA resulted in significant stability of the protein over time in cell culture, leading to a significant recovery of activity over time (a 2-fold improvement). Furthermore, nanoconjugation enhanced this stabilization and internalization capacity even more, showing the best results.

[0285] Table 4 Commercially available cross-linkers that can mimic SATP functionality:

[0286]

[0287] IUPAC nomenclature

[0288] SATP-(2,5-dioxopyrrolidin-1-yl) 3-acetylthiopropionate

[0289] SATA-(2,5-dioxopyrrolidin-1-yl) 2-acetylthioacetate

[0290] 4-SATA-(2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]propanoate

[0291] 8-SATA(2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0292] 12 -SATA

[0293] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0294] 24 -SATA

[0295] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-acetylthioethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0296] 3-Mercaptopropyl-N-hydroxysuccinimide

[0297] (2,5-Dioxopyrrolidin-1-yl) 3-thiopropanoate

[0298] SMPT

[0299] (2,5-Dioxopyrrolidin-1-yl) 4-[1-(pyridin-2-yldisulfanyl)ethyl]benzoate

[0300] SPDP

[0301] (2,5-Dioxopyrrolidin-1-yl) 3-(pyridin-2-yldithio) propionate

[0302] LC-SPDP

[0303] (2,5-Dioxopyrrolidin-1-yl)6-[3-(pyridin-2-yldisulfanyl)propionylamino]hexanoate

[0304] Sulfo-LC-SPDP

[0305] 2,5-Dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propionylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid

[0306] PEG4-SPDP

[0307] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0308] PEG12-SPDP

[0309] (2,5-Dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

[0310] 2-Iminothiolane (Traut's reagent)

[0311] Sulfane-2-imine

Claims

1. A method for modifying the surface of glucocerebrosidase by modifying the lysine side chain of a protein with N-succinimidyl-S-acetylthiopropionate (SATP) and providing a SATP-modified protein, the method comprising: a. adding a polar aprotic solvent miscible with water and capable of dissolving N-succinimidyl-S-acetylthiopropionate (SATP), and optionally a surfactant having an HLB value between 14 and 20, to an aqueous composition comprising a protein, glucocerebrosidase, and a molecular chaperone (MC), wherein the reaction is carried out at an alkaline pH greater than 8, a molar ratio of SATP to glucocerebrosidase of at least 5 molar equivalents to at most 25 molar equivalents, and a reaction time in the range of at least 30 minutes.

2. The preparation method according to claim 1, wherein The MC is a salt of isofagomine at a concentration of at least 0.5 μM.

3. The preparation method according to claim 1, wherein The MC is the salt of isofagormine D-tartaric acid.

4. The preparation method according to any one of claims 1 to 3, wherein The reaction of step (a) is carried out at a molar ratio of SATP to glucocerebrosidase of 5 to 25 molar equivalents and a reaction time range of 30 to 120 minutes.

5. The preparation method according to any one of claims 1 to 3, wherein The reaction of step (a) is carried out at a molar ratio of SATP to glucocerebrosidase of 10 to 20 molar equivalents, preferably about 15 molar equivalents, and a reaction time ranging from 50 to 70 minutes, preferably 60 minutes.

6. The preparation method according to any one of claims 1 to 5, wherein The SATP modified protein obtained from the reaction of step (a) is isolated, preferably purified, and the purification is preferably carried out in a citrate buffer.

7. The preparation method according to any one of claims 1 to 6, wherein The SATP modified protein obtained from the reaction of step (a) is separated, preferably purified, and deacetylated. The deacetylation is preferably performed using a deacetylation solution such as a hydroxylamine-HCl solution.

8. A method for producing a polymer conjugate, wherein: The method comprises: b) adding a polymer to the deacetylated and preferably purified SATP modified protein obtained by the reaction of step (a) according to claim 7, wherein the modified protein is glucocerebrosidase, wherein the polymer is selected from the group consisting of dextran, water-soluble linear polyamino acids or polypeptides / polypeptides (including polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosine (PSar)...), polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolide) (PLA / PLGA), poly(hydroxyalkylmethacrylamide), polyglycerol, polyamidoamine (PAMAM) and polyethyleneimine (PEI), polyorthoesters, polyacetals; and c) Optionally, washing the product obtained from b), preferably with an acidic buffer (citrate buffer), optionally supplemented with a salt of a molecular chaperone, such as isofagomine.

9. A method for producing a polymer conjugate, wherein: The method comprises: b. adding a polymer to the SATP modified protein obtained by the reaction step (a) according to any one of claims 1 to 6, wherein the modified protein is glucocerebrosidase, and wherein the polymer is selected from dextran, a water-soluble linear polyamino acid or a polypeptide / polypeptide (including polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosine (PSar) ...), polyethylene glycol (PEG), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolide) (PLA / PLGA), poly(hydroxyalkylmethacrylamide), polyglycerol, polyamidoamine (PAMAM) and polyethyleneimine (PEI), polyorthoester, polyacetal; and simultaneously deacetyling the SATP modified protein, wherein the deacetylation is preferably carried out using a deacetylation solution (e.g., hydroxylamine-HCl solution); and c. Optionally, washing the product obtained from b), preferably with an acidic buffer such as VCB, optionally supplemented with a salt of a molecular chaperone such as isofagomine.

10. The method according to any one of claims 8 or 9, wherein The polymer is polyglutamic acid (PGA).

11. The method according to claim 10, wherein: The polymer is a polyglutamate selected from poly(L-glutamic acid), poly(D-glutamic acid), poly(D,L-glutamic acid), poly(L-γ-glutamic acid), poly(D-γ glutamic acid) and poly(D,L-γ glutamate), wherein optionally, the polyglutamate comprises at least 50% glutamic acid backbone units, and optionally comprises 60%, 70%, 80%, 90% or 100% glutamic acid backbone units.

12. The method according to claim 10, wherein: The polymer is L-PGA having 25 to 250 glutamic acid monomer units.

13. A method for conjugating glucocerebrosidase to poly-L-glutamic acid (PGA), wherein the method comprises: a. A method for producing a surface protein of glucocerebrosidase α modified by modifying the lysine side chain of the protein with (N-succinimidyl-S-acetylthiopropionate) according to any one of claims 1 to 7; b. after step a), deacetylation of the product obtained by a); and c. Conjugating a polymer to the product obtained from b) simultaneously or subsequently according to any one of claims 8 to 12.

14. The method according to any one of claims 8 to 13, wherein The method further comprises isolating and / or purifying the obtained or obtained polymer conjugate.

15. A SATP modified protein obtained according to any one of claims 1 to 6, wherein The modified protein is glucocerebrosidase.

16. A SATP modified protein obtained according to claim 7, wherein The modified protein is glucocerebrosidase.

17. A polymer conjugate obtained according to any one of claims 8 to 14, wherein The SATP-modified protein of the polymer conjugate is glucocerebrosidase.

18. A pharmaceutical composition comprising the polymer conjugate according to claim 17, optionally further comprising an excipient, a carrier, a penetration enhancer and / or an adjuvant.

19. The polymer conjugate according to claim 17 or the pharmaceutical composition according to claim 18 for use in therapy.

20. The polymer conjugate of claim 17 or the pharmaceutical composition of claim 18 for use in a method of treating a subject suffering from Gaucher disease and / or Parkinson's disease.

21. The method according to any one of claims 1 to 7, wherein The glucocerebrosidase is selected from velaglucerase, imiglucerase or uplyso or any recombinant glucocerebrosidase.

22. The polymer conjugate according to claim 17 or the pharmaceutical composition according to claim 18, wherein The glucocerebrosidase is selected from velaglucerase, imiglucerase or uplyso or any recombinant glucocerebrosidase.

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