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

Abstract

The present invention relates to the medical field, in particular to nanotechnology-based enzyme replacement therapy, preferably for Parkinson's disease, based on the restoration of lysosomal glucocerebrosidase activity by enzyme-polymer nanoconjugation of GBA, GBA-polymer conjugates for such use, and methods for their preparation.

Description

[Technical Field]

[0001] The present invention relates to the medical field, in particular to nanotechnology-based enzyme replacement therapy, preferably for Parkinson's disease and Gaucher disease, based on the restoration of lysosomal glucocerebrosidase activity by enzyme-polymer nanoconjugation of GBA, GBA-polymer conjugates for such use, and methods for their preparation. [Background technology]

[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 greatly increased risk for developing Parkinson's disease (PD). In this sense, the presence of mutations in GBA1 is known to be the main genetic risk factor for PD. 10-12% of PD patients show mutations in GBA (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.R. Molecular 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 PD patients without GBA mutations; however, they have a significantly earlier mean onset and higher risk of developing cognitive impairment and other neuropsychiatric disorders.

[0004] PD patients with GBA mutations exhibit significantly reduced GCase activity in the substantia nigra (SNpc) and other brain regions. Interestingly, other sporadic PD patients without GBA mutations also exhibit significantly reduced GCase activity, suggesting that GBA may contribute to PD pathogenesis. Defective GCase activity in PD patients is associated with increased alpha-synuclein (syn) levels, supporting the possibility that GBA deficiency may be involved in syn accumulation or aggregation. In cell and animal models, reduced GBA / GCase activity is also associated with elevated syn levels (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 loop in synucleinopathies. Cell 2011, 146, 37-52).

[0005] Therefore, there is a need to restore GBA activity, particularly in the lysosomal system, to prevent the accumulation of sphingolipid substrates, reduce syn levels, and ultimately prevent neurodegeneration. Currently, therapies available for such restoration of GBA activity are as follows: - Enzyme replacement therapy (ERT): Restoration of GCase activity via recombinant GBA protein. This is the main treatment currently used to treat GD patients, and this treatment is beneficial for non-neurological symptoms because the recombinant protein itself cannot cross the BBB and reach the brain. ERT is not currently effective in treating the loss-of-function effects of GCase in the central nervous system in GBA-associated GD and PD. - Substrate reduction therapy (SRT): inhibition of the synthesis of the GCase substrate (GlcCer) to avoid its accumulation. -Molecular chaperones (MC): Pharmacological GBA-specific chaperones can stabilize misfolded mutant GBA retained in the ER, promote its translocation to lysosomes, and restore its function. Examples: isofagomine and ambroxol.

[0006] Currently, restoration of GBA activity in humans is approved only for Gaucher disease by: ERT: Cerezyme (imiglucerase) from Sanofi-Genzyme, VPRIV (velaglucerase alfa) from Takeda, and Elelyso (taliglucerase alfa) from Pfizer; and SRT: Cerdelga (eliglustat) from Sanofi-Genzyme and Zavesca (miglustat) from Actelion.

[0007] Regarding PD, there are currently no approved treatments for this disease that target restoration of GBA, although there are several ongoing clinical trials. Summary of the Invention

[0008] In the present invention, the inventors 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 explanation of the drawings]

[0009] [Figure 1] This figure is a scheme of how the GBA nanoconjugates of the present invention are internalized by cells by endocytosis and sent directly to the lysosomes for their degradation; once the nanoconjugates reach the lysosomes, the polypeptide coating should degrade in the acidic lysosomal environment, and GBA, as a lysosomal protein, should regain its lysosomal function and its GBA activity should be restored. [Figure 2]ST-Q7 mouse striatal neurons were treated with 200 ng / μl of velaglucerase-loaded PLGA nanoparticles or nanoconjugates for 24 hours. Immunocytochemistry assays were performed to detect velaglucerase using anti-GBA (right) and anti-LAMP-1 (left) antibodies as lysosomal markers. [Figure 3]GBA-PGA characterization. A) Reduction in the percentage of unmodified lysine after SATP modification. Unmodified velaglucerase (GBA) and SATP-modified velaglucerase (GBA-SATP) under optimal conditions (pH 9, 60 min, and 15 equivalents of SATP in PBS) were analyzed for lysine level reduction by 2,4,6-trinitrobenzenesulfonic acid (TNBSA) assay. Experiments were performed in triplicate. B) Percentage of unmodified velaglucerase after conjugation. Silver-stained velaglucerase bands were quantified for each time point to obtain a quantitative value for the efficiency of the reaction. Since the following samples had the same concentration of GBA-SATP (no Amicon wash step), values ​​were expressed as the percentage of velaglucerase at the GBA-SATP step. C) Silver staining over the conjugation time course. Unmodified velaglucerase (1), SATP-modified velaglucerase (2), and conjugation time courses of 0.5 hours (3), 1 hour (4), 2 hours (5), 4 hours (6), and overnight (7) without (ΦIFG) and with 26.9 μM isofagomine D-tartrate (IFG) (+IFG) were subjected to non-reducing SDS-PAGE. The gels were then stained with a silver staining protocol to show the MW of the conjugates and the kinetics of conjugation. 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 over the course of conjugation in the absence or presence of IFG. The optimized conjugation protocol was analyzed for GCase activity at different steps of the conjugation: unmodified velaglucerase (GBA, dark), SATP-modified velaglucerase (GBA-SATP, medium), and conjugated velaglucerase (GBA-PGA, light). Two experiments were repeated four times: conjugation in the absence of IFG (IFG(-)) and in the presence of 26.9 μM IFG (IFG(+)). *Statistically significantly different (p<0.0005). [Figure 4]PLGA nanocapsule images obtained by SEM. Non-encapsulated PLGA nanocapsules (left) were synthesized as a control (Φ 245 nm, ζ -25 ± 5 mV), and velaglucerase was successfully encapsulated in PLGA nanocapsules (right, Φ 217 nm, ζ -20 ± 4 mV). ζ is defined herein as the Z potential. [Figure 5] GCase activity was tested on GBA (velaglucerase) after modification with various molar ratios of SATP (light grey) (untreated velaglucerase was used as a control) and on PGA-velaglucerase conjugate after release by incubation with 10 mM GSH for 1 h (dark grey). [Figure 6] Interference of MC with SATP modification. A) SATP reactivity towards primary amines on proteins. B) Molecular structures of isofagomine and ambroxol. The secondary amine of isofagomine (non-reactive) is circled in green. The primary amine of ambroxol (reactive) is circled in red. [Figure 7] Effect of IFG addition on conjugation efficiency. Velaglucerase (Vela) was modified with the SATP crosslinker in the absence (Vela SATP) or presence (Vela SATP + IFG) of IFG. The PGA conjugation step was then monitored by taking samples at 1, 2, and 4 hours in the absence (Conj) or presence (Conj + IFG) of IFG. [Figure 8] The addition of IFG improves GCase activity during nanoconjugation. GCase activity was measured from each conjugation step and the resulting activity was compared to that of the unmodified enzyme. [Figure 9] The modification of velaglucerase by SATP was examined at various pH values ​​and reaction times. The changes in thiol incorporation (A), the decrease in free lysine (B), and the change in GCase activity (C) were measured under each condition. [Figure 10]In vitro validation. A. Stability in human plasma (HBP) was assessed by GCase activity assessment upon internalization. Compounds at 200 μg / ml in Velacrylase citrate buffer (VCB, 50 mM sodium citrate, 5% w / v sucrose, and 0.01% v / v polysorbate 20, pH 5.6) were incubated with 4 volumes of HBP for up to 24 hours and diluted to 5 μg of 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). B: Glucosyl-sphingosine levels were analyzed by LC-MS / MS after treating BE(2)-M17 GBA1- / - cells with GBA or NanoGBA for 7 days. C. BE(2)-M17 GBA1- / - cells were treated with GBA or NanoGBA for up to 7 days. Untreated cells served as a negative control (KO), and untreated BE(2)-M17 WT cells served as a positive control (WT). After treatment, cell lysates were analyzed for total internalized GBA, GCase specific activity (GCase activity / unit protein), and GCase activity recovery (GCase activity / unit GCase). Double representation of GCase specific activity (left axis, bars) and GCase activity recovery (right axis, dots and lines) of various treatments as % of WT levels (data from three independent experiments and presented as mean ± SEM). D. Alpha-synuclein protein levels after 7 days of treatment. Statistical differences were determined using two-way ANOVA with multiple mean comparisons. Differences between compounds were determined with p<0.0001. ***p<0.01 for multiple comparisons. ****p<0.0001 for multiple comparisons. [Figure 11] Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) biodistribution detected by IVIS. [Figure 12] Ex vivo detection of Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) detected by IVIS (left). GCase activity in various tissues 30 min, 3 h, 24 h, and 72 h after administration of nanoGBA (right). [Figure 13]Ex vivo 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 hours. [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). [Figure 15] Size exclusion chromatography on Superdex 200. GBA (blue), GBA-SATP, and NanoGBA were loaded onto the column and the absorbance at 280 nm was followed over time. [Figure 16] Analysis of trypsin digestion. This figure shows Coomassie staining of trypsin digests of GBA, GBA-SATP, and NanoGBA and relative quantification by densitometry of the GBA bands. [Figure 17] Plasma stability assay. This figure shows A) the activity recovered inside treated M17 GBA KO cells. Activity data are shown as a percentage of the activity of untreated wild-type cells. Bars represent the mean + standard deviation (n=2). B) Western blot showing total GBA internalized at each treatment. [Figure 18] Uptake assay. A) Activity recovered in treated M17 GBA KO cells. Activity data are shown as a percentage of activity in untreated wild-type cells. Bars represent mean + standard deviation (n=3). B) Western blot showing total GBA internalized at each treatment.

[0010] explanation definition It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element of 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.

[0011] The term "about" with respect to a given amount or quantity is meant to include a deviation of ±10 percent, preferably ±5 percent.

[0012] As used herein, the conjunctive term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first option without the second option. The second option refers to the applicability of the second option without the first option. The third option refers to the applicability of the first and second options together. Any one of these options is understood to fall within the meaning and thus meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more options is also understood to fall within the meaning and thus meet the requirements of the term "and / or."

[0013] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to mean 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. As used herein, the term "comprising" may be replaced with the terms "containing" or "including," or, sometimes, as used herein, with the term "having." Any of the foregoing terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the invention, may also, but is less preferred, be replaced with the term "consisting of."

[0014] As used herein, "consisting of" excludes elements, steps, or ingredients not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0015] In the context of the present invention, "GBA protein" refers to an enzyme that induces glucosylceramidase activity (also called acid β-glucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, glucocerebrosidase, GCase, or any other name listed in entries 3.2.1.45 and 3.2.1.62 of the BRENDA enzyme database).

[0016] In the context of the present invention, "isofagomine" or "IFG" refers to a pharmacological chaperone that selectively binds to glucocerebrosidase, restoring its correct conformation and enhancing its activity.

[0017] In the context of the present invention, the term "GBA-specific chaperone" refers to a pharmacological chaperone that specifically binds to GBA, assists in protein folding to prevent misfolding, enhances enzymatic activity, and / or enhances the correct transport of proteins to lysosomes. Examples of these GBA-specific chaperones include, but are not limited to, inhibitory imino analogs and their derivatives (e.g., 1,5-dideoxy-1,5-iminoxylitol (DIX), deoxynojirimycin (DNJ), 1-azafagomine, etc.), inhibitory sugar analogs and their derivatives, molecules with inhibitory activity that do not fall into the last two categories (ambroxol (ABX) is a prime example), and allosteric enhancers of GBA.

[0018] In the context of the present invention, "SATP-modified protein" refers to a protein modified with N-succinimidyl-S-acetylthiopropionate. SATP adds an acetylated sulfhydryl group to the amine groups present in certain proteins. It should be noted herein that SATP is a heterobifunctional crosslinker containing an amine-reactive group (N-succinimidyl) and a protected sulfhydryl-reactive group (S-acetyl), which can modify the side chain of lysine residues by incorporating a protected thiol group. As a result, SATP-modified proteins can be conjugated with polymers that form a disulfide bond, a covalent bond that responds to a reducing environment, maintaining the conjugate in an oxidizing environment but releasing the protein in a reducing environment. It should be further noted herein that conjugation between any protein (including GBA) and a polymer via a disulfide bond from the modified lysine can be achieved not only with SATP, but also with any other crosslinker that can modify the side chain of lysine and add a sulfhydryl-reactive group. According to this chemistry, molecules containing similar reactive head groups but with different central chain lengths can be used as substitutes for SATP (SATA crosslinker is an example of this type of molecule). Other crosslinkers capable of adding pyridyl disulfide groups to lysine residues, such as SPDP or SMPT, could produce essentially the same conjugate as GBA by conjugating GBA and PGA with a thiol (instead of a pyridyl disulfide group) added to the side chain of the glutamic acid residue. Furthermore, the lysine side chain could be modified with other amine-reactive head groups, such as sulfo-N-succinimidyl or imidoester, among others.

[0019] All of these potential alternatives for modifying GBA deviate from the SATP-modified GBA and are encompassed by the present invention. Therefore, the present invention encompasses not only GBA SATP-modified proteins, but also GBA-modified proteins containing lysines whose side chains have been modified with sulfhydryl-reactive groups (hereinafter, "GBA-modified proteins" or "GBA-modified proteins"). These types of GBA-modified proteins can be obtained by using a crosslinker such as SATA, or a crosslinker capable of adding a pyridyl disulfide group to lysine residues, such as SPDP or SMPT. Preferably, the GBA-modified protein can be a GBA SATP-modified protein or a GBA-modified protein with any other crosslinker capable of modifying the side chain of lysine and adding a sulfhydryl-reactive group. In this sense, and according to the present invention, SATA, dPEG®4-SATA, dPEG®8-SATA, dPEG® 12 -SATA, dPEG (registered trademark) 24 Molecules containing similar reactive head groups but with different spacer arm lengths, such as those selected from the list consisting of -SATA and 3-mercaptopropanyl-N-hydroxysuccinimide, can be used as substitutes for SATP. Other molecules that modify the lysine side chain but add a pyridyldithiol-reactive group, such as those selected from the list consisting of SMPT, SPDP, LC-SPDP, PEG4-SPDP, and PEG12-SPDP, can conjugate GBA with PGA to create substantially similar conjugates with a thiol (instead of a pyridyl disulfide group) added to the side chain of a glutamic acid residue. Furthermore, the lysine side chain can be modified with other amine-reactive head groups, such as sulfo-N-succinimidyl or imidoesters, particularly those selected from the list consisting of 2-iminothiolane (Traut's reagent) and Sulfo-LC-SPDP. (See Table 4.)

[0020] General reaction diagram of modification and SATP: [ka]

[0021] Nomenclature of SAPT by IUPAC and its SATP alternatives SATP-(2,5-dioxopyrrolidin-1-yl) 3-acetylsulfanylpropanoate SATA-(2,5-dioxopyrrolidin-1-yl)2-acetylsulfanyl acetate dPEG® 4-SATA-(2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]propanoate dPEG® 8-SATA (2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate dPEG (registered trademark) 12 -SATA (2,5-Dioxopyrrolidin-1-yl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate dPEG (registered trademark) 24 -SATA (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-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-acetylsulfanylethoxy] ... 3-Mercaptopropanyl-N-hydroxysuccinimide. (2,5-dioxopyrrolidin-1-yl) 3-sulfanylpropanoate SMPT (2,5-dioxopyrrolidin-1-yl)4-[1-(pyridin-2-yldisulfanyl)ethyl]benzoate SPDP (2,5-Dioxopyrrolidin-1-yl)3-(pyridin-2-yldisulfanyl)propanoate LC-SPDP (2,5-dioxopyrrolidin-1-yl)6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate Sulfo-LC-SPDP 2,5-Dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid PEG4-SPDP (2,5-Dioxopyrrolidin-1-yl)3-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate PEG12-SPDP (2,5-Dioxopyrrolidin-1-yl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)]propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate 2-Iminothiolane (Traut's reagent) Thiolane-2-imine

[0022] In the context of the present invention, the term "molar equivalent" refers to the ratio of moles of one compound to moles of another compound. Once the moles of each compound are determined, the molar equivalent can be determined.

[0023] In the context of the present invention, the term "L-PGA" refers to linear polyglutamic acid.

[0024] In the context of the present invention, the term "isofagomine D-tartrate" refers to a competitive inhibitor of human lysosomal β-glucosidase (Ki=0.016-0.025 μM; IC50=0.06 μM).

[0025] In the context of the present invention, the term "molar ratio" refers to the ratio between any two substances in a chemical reaction or the ratio between two coefficients in a balanced chemical equation.

[0026] In the context of the present invention, the term "room temperature" refers to average room temperature, which is typically about 20°C.

[0027] In the context of the present invention, the term "deacetylated" refers to a molecule that has had the acetyl group removed, usually by hydrolysis.

[0028] In the context of the present invention, the term "VCB" refers to Velaglucerase Citrate Buffer, a buffer containing 50 mM sodium citrate, 5% w / v sucrose and 0.01% v / v polysorbate 20 at pH 5.6. DETAILED DESCRIPTION OF THE INVENTION

[0029] Description of the embodiments of the invention Traditional enzyme replacement therapy (ERT) consists of the direct intravenous administration of specific enzymes that are deficient or absent in patients. This treatment is currently available and has been successfully administered for various lysosomal storage diseases, including Gaucher disease, via intravenous administration of 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). Recombinant lysosomal proteins are naturally internalized within cells and can reach lysosomes, where they can restore enzyme activity.However, ERT treatment has several drawbacks, including poor stability of the enzyme in circulation (due to proteolytic 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.), and the development of an immune response to the administered 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.), but have several associated drawbacks, such as their inability to penetrate biological barriers, such as the blood-brain barrier (making them ineffective against neurological symptoms) and their 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).

[0030] Considering the current limitations of ERT for delivering recombinant proteins to the CNS (central nervous system) and its ineffectiveness in treating the neurological symptoms of Parkinson's disease and Gaucher disease, we propose a new therapeutic strategy for Parkinson's disease and Gaucher disease based on ERT with glucocerebrosidase (GBA), improved by nanoconjugation of the GBA enzyme with a polypeptide to facilitate BBB (blood-brain barrier) crossing and improve intracellular delivery to lysosomes. Note that this approach is not limited to intravenous administration but also includes non-invasive administration routes and intranasal administration as an alternative to intravenous administration.

[0031] In recent years, the use of nanotechnology strategies to encapsulate proteins into drug delivery systems (DDS) has emerged as a potential solution to overcome the aforementioned limitations of ERT. DDSs offer improved drug stability and in vivo protection, controlled release, and targeted delivery. Furthermore, the use of DDSs dramatically reduces drug immunogenicity (Chen et al., 2016). GBA nanoconjugates are internalized by cells via endocytosis and delivered directly to lysosomes for degradation. Once the nanoconjugates reach the lysosome, the polypeptide coating is degraded in the acidic lysosomal environment, allowing GBA, as a lysosomal protein, to regain its lysosomal function and restore GBA activity. In addition to the primary goal of delivering GBA protein to the CNS (therapeutic applications in PD and GD), this invention also aims to improve ERT technologies currently used to treat non-neurological symptoms of GD.

[0032] To achieve the above objectives, the present invention proposes nanoconjugation of GBA, preferably recombinant, from an enzyme to a polypeptide polymer, followed by the introduction of chemical modifications to improve protein stabilization, plasma protease resistance, and BBB penetration. In particular, due to the high instability of the GBA protein and its significant ability to lose its enzymatic activity (GBA half-life in plasma is approximately 10 minutes), the goal is to protect GBA using polymer unmasked-masked protein therapy (PUMPT) conditions and avoid inactivation using molecular chaperones (MCs), thereby improving GBA stability by generating stable GBA / chaperone nanoconjugates that can be taken up by cells, delivered to lysosomes, and restore GBA activity with greater efficacy than free GBA. The goal is also to functionalize the PGA chains with various molecules and / or biomolecules to enhance stability and facilitate BBB penetration (in the case of intravenous administration) or reach the brain via intranasal administration.

[0033] The structure of the ERT delivery system proposed herein is shown in Figure 1. In particular, as shown in Figure 1, the proposed GBA nanoconjugate (hereinafter referred to herein as the "GBA nanoconjugate of the present invention") should be internalized by cells by endocytosis and delivered directly to lysosomes for their degradation; once the nanoconjugate reaches the lysosome, the polypeptide coating should degrade in the acidic lysosomal environment, and GBA, as a lysosomal protein, should restore its lysosomal function and restore its GBA activity. That is, the GBA nanoconjugate of the present invention aims to achieve the following objectives: -Prevents degradation of GBA enzymatic activity. The physical barrier provided by the nanoconjugate should shield the protein from the harsh conditions encountered during circulation in the bloodstream. Sustained release after internalization within cells should provide a steady source of active GBA that remains intact throughout its passage through the bloodstream. - Overcoming the blood-brain barrier (BBB) ​​by surface modification or intranasal administration. Because traditional ERT cannot deliver active GBA to the central nervous system (CNS), neurological symptoms remain untreatable in GD types 2 and 3. Intranasal administration of encapsulated PLGA should overcome the BBB and deliver active GBA into the brain.

[0034] To achieve these goals, and as shown in the examples of the present invention, we first attempted to encapsulate GBA in PLGA nanocapsules using a water-in-oil-in-water (W / O / W) double emulsion method, as shown in Example 1. Using this method, velaglucerase-loaded PLGA nanoconjugates were successfully synthesized and characterized. To test whether the encapsulated protein retained its enzymatic activity, an in vitro assay was performed in ST-Q7 cells (mouse striatal neurons). However, as seen in Figure 2, although velaglucerase successfully colocalized with LAMP-1 (a lysosomal marker indicating that the protein had reached its intended destination), no enzymatic activity could be detected. To resolve this issue, we attempted to optimize the synthesis conditions to overcome the loss of GCase activity, but without success. Such optimization is described herein below.

[0035] To preserve GCase activity, several modifications of phases and synthesis procedures were applied to protect GBA from the loss of enzymatic activity. They were classified according to the affected phases and the final section with the overall changes.

[0036] -W1 phase modification. The modifications that affected the W1 phase were primarily changes in buffer composition, and as shown in the Examples, none of these changes resulted in nanoconjugates with active GBA.

[0037] -W2 phase modification. The surfactants that make up W2 stabilize the nanoconjugates during evaporation of the organic solvent. PVA was replaced with polysorbate 80 and Pluronic. Neither change resulted in nanoconjugates with active GBA.

[0038] -Modification of emulsification method Sonication time and amplitude were tested to obtain a protocol that could produce nanoconjugates with the desired properties but reduce the amount of energy delivered to the sample that was thought to cause protein unfolding. Various conditions were tested: shorter times at higher amplitudes, longer times at lower amplitudes, and continuous and intermittent sonication.

[0039] Preliminary conclusion: None of the modifications resulted in nanoconjugates with active GBA and PLGA, which were discarded as drug delivery systems. GBA was inactivated during the encapsulation process.

[0040] Considering the above results, an alternative approach to incorporate velaglucerase into nanoparticle systems without complete loss of activity was attempted by Dr. Maria J. Vicent (CIPF, Valencia, Spain) using Polymer Unmasked-Masked Protein Therapy (PUMPT) (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. In this strategy, the protein is protected and its activity is masked during transport, while its activity is designed to be restored in a controlled manner upon external stimulation at the target site. In this sense, the adaptation of the PUMPT method was tested for velaglucerase conjugation: velaglucerase was conjugated to a polyglutamic acid (PGA) polymer via a reduction-sensitive linker, which was intended to mask (and protect) the protein activity in oxidizing environments (e.g., bloodstream) and release it in reducing environments (e.g., lysosomes).

[0041] To successfully conjugate velaglucerase with PGA polymers, we adapted the conjugation protocol developed by Talelli and Vicent (Talelli and Vicent, 2014) optimized for lysozyme. This conjugation procedure consisted of two major steps: (1) modification of the surface protein with SATP (N-succinimidyl-S-acetylthiopropionate), which adds a thiol group by modifying the side chain of lysine; (2) conjugation of the SATP-modified protein with PGA (PGA-PD), which attacks the thiol group added to the lysine to form a disulfide bond.

[0042] The conjugation conditions are shown in Example 1.

[0043] The purpose of the PGA conjugation step is to mask GCase activity, but this loss of activity should be fully reversible upon release in a reducing environment. However, the SATP modification step, which irreversibly modifies the surface lysine residues of the protein, causes irreversible activity loss. The three initial ratios of SATP tested for velaglucerase conjugation resulted in a decrease in enzyme activity proportional to the SATP concentration. Although PGA conjugation successfully masked GCase activity, only one of the conditions restored GCase activity upon release with 10 mM reduced glutathione (GSH), significantly below the activity level exhibited by the SAPT-modified velaglucerase, which should have fully restored GCase activity. The loss of activity during modification of GBA with SATP and conjugation of the modified protein with PGA was primarily due to the instability of the protein in the working buffer conditions used for the conjugation step. To ameliorate this activity loss, we resorted to the use of a GBA molecular chaperone (MC) during the synthesis procedure. MC is a small molecule that has the ability to bind to and stabilize the target enzyme. Typically, these MCs are competitive inhibitors that bind to the catalytic pocket of the enzyme.

[0044] In the case of GBA, there are currently two commercially available MCs, but none have reached the drug market as pharmacological chaperones (PCs) for GBA to date. Among these, two of the 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 would compete with lysines in the SATP modification reaction. Furthermore, IFG is a potent chaperone for GBA, with a K 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 the lack of interference of IFG in the conjugation procedure, comparative conjugation protocols were performed in the presence or absence of the chaperone. The protocol using IFG differed only by adding an excess of IFG (25 μM, nearly 1000-fold the K) to the working buffer to ensure maximal inhibition and protection of the enzyme. Samples from each step of the conjugation from both protocols (SATP modification and PGA conjugation) were collected and analyzed by non-reducing SDS-PAGE combined with silver staining. This technique allowed us to determine the molecular weight of the nanoconjugates without cleaving the disulfide bond connecting the polymer to the protein, thereby qualitatively assessing the conjugation rate.

[0045] Results from non-reducing SDS-PAGE showed no significant differences between conjugations in the absence or presence of IFG. SATP modification of velaglucerase resulted in a small amount of enzyme dimerization. The presence of a dimerization band on the SATP-modified enzyme in the presence of IFG indirectly indicated the lack of interference in this step. PGA conjugation with the protein was recognized by the appearance of a smeared protein-polymer conjugate, which showed no difference between conditions. Although no difference was observed in conjugation efficiency, the addition of IFG during the synthesis procedure improved the GCase activity measured at each step of the conjugation, demonstrating its ability to stabilize and protect the enzyme during conjugation. After SATP modification, no loss of GCase activity was detected in IFG-containing reactions, a fact attributed to the complete stabilization of the enzyme at this step. The gradual decrease in GCase activity after conjugation in the presence of IFG was attributed to the masking effect of the polymer on enzyme activity.

[0046] Thus, IFG improved enzyme stability during the conjugation and masking processes. However, despite the intriguing results obtained thanks to the addition of IFG as a stabilizer, the nanoconjugation strategy seemed far from feasible. After all conjugation procedures, over 80% of velaglucerase remained unmodified. The explanation for this insufficient conjugation of the enzyme was later found to be the buffer conditions used in the SATP modification step. As previously described, the protocol for conjugating velaglucerase with PGA was adapted from the work of Talelli and Vicent. The key step for adaptation was the modification of the protein with SATP, which, in contrast to the PGA conjugation step, is accompanied by an irreversible loss of activity. Adapting this protocol proved ineffective for proper modification of velaglucerase with SATP, resulting in low conjugation yields. Examination of the chemistry of the N-succinimidyl ester reaction with primary amines (the reaction by which SATP binds to proteins) showed that this reaction is highly dependent on the pH of the reaction mixture. At neutral pH (pH 7.4), the pH initially used for SATP modification, the reaction proceeded very slowly, requiring several hours to reach equilibrium. Nevertheless, the same reaction performed at more basic pHs (pH 8.5-9) occurred rapidly, reaching equilibrium within minutes. After SATP modification and deacetylation of the protected thiol, thiol addition was quantified using a free thiol assay kit. Results were plotted relative to the SATP-modified enzyme and unmodified velaglucerase. Free thiol levels doubled after 2 hours of reaction at pH 7.4. In contrast, reactions performed at pH 8, 5, and 9 showed a four-fold increase in free thiol levels, indicating higher reactivity of SATP under these conditions.

[0047] As mentioned, thiol quantification requires deacetylation of SATP-protected thiols, suggesting additional reactions that may interfere with the resulting measurements (e.g., by disulfide bond formation between protein molecules). To avoid this deacetylation step and consider that SATP reacts only with primary amines of lysine residues, we instead analyzed the reduction of free surface amines from lysine residues. The reduction of free lysine residues was quantified with the TNBSA assay. TNBSA reacts with primary amines to produce a yellow molecule, which can be quantified by absorbance. In proteins, the only primary amines detectable by TNBSA are from exposed lysine residues (away from the N-terminus). Therefore, TNBSA reactivity is directly proportional to the number of lysine residues on the surface of the protein. Results from lysine quantification demonstrated that the proportion of exposed lysine residues did not decrease after SATP modification at pH 7.4, demonstrating insufficient reactivity of the molecule at these conditions. However, reactions performed at pH 8.5 and pH 9 resulted in a 25–30% reduction in exposed lysines, further confirming the significant increase in SATP reactivity at these pH conditions.

[0048] Regarding GCase activity, no change in enzyme activity was detected at pH 7.4. This lack of activity loss after SATP modification was previously observed and attributed to complete stabilization of the enzyme in the presence of IFG. On the other hand, GCase activity after SATP modification at pH 8.5 and pH 9 showed a 25% and 30% decrease, respectively. Considering the effect of IFG, the only explanation for the decrease in GCase activity is modification of specific lysine residues essential for enzymatic activity.

[0049] In this regard, the primary goal of the SATP modification procedure was to simultaneously optimize the yield of SATP modification and the maintenance of GCase activity. Considering these two parameters together in statistical analysis while optimizing synthesis conditions may provide more promising results (Karbasian et al., 2019). Optimization by central composite design-response surface methodology (CCD-RSM) should help select values ​​for the independent variables (our dependent variables) that significantly affect the outputs of SATP modification and GCase activity maintenance. According to the literature, the SATP manual (Thermo Scientific, Ref. 26100), and our preliminary studies, the main independent variables that primarily affected SATP modification and maintained GCase activity were (1) pH, (2) SATP-to-protein molar ratio, and (3) reaction time. For CCD-RSM optimization, it is necessary to define the operating ranges for the independent variables. Again, using information found in the literature, the SATP manual, and prior testing, the pH range was set from pH 7 to pH 9, the molar ratio of SATP to protein ranged from 5 equivalents to 25, and the reaction time range was set from 30 min to 120 min.

[0050] Each numerical factor was varied across five levels calculated by the software Design Expert 11 (DX11): plus and minus alpha (axis points), plus and minus one (factor points), and the center point. The conditions that best achieved simultaneous SATP modification and maintenance of GCase activity were set at pH 9, a 15:1 molar ratio of SATP to protein, and a 60-minute reaction time. The final protocol for velaglucerase modification and PGA conjugation was established as follows: 1. SATP modification step: Velaglucerase was prepared at 0.5 mg / ml in PBS pH 9 containing 25 μM IFG. 15 molar equivalents of SATP from a stock concentration of 20 mg / ml in dimethyl sulfoxide (DMSO) was added and incubated at room temperature for 1 hour with gentle shaking. The resulting product was washed three times with VCB supplemented with 25 μM IFG using a centrifugal concentrator (cutoff 10 kDa). 2. PGA conjugation step: One volume of deacetylation solution (0.5 M hydroxylamine-HCl in PBS) was added to the purified SATP-modified protein already in VCB. To the resulting solution, one volume of PGA-PD was added to a final molar ratio of 5:1 PGA / protein, and the mixture was incubated at room temperature for 4 hours. The resulting product was washed three times with VCB supplemented with 25 μM IFG using an Amicon centrifuge unit (cutoff 30 kDa).

[0051] The final SATP modification protocol incorporated into the conjugation procedure proved to successfully improve GCase activity maintenance while maintaining a high SATP modification rate. As shown in Figure 3, approximately 25% of lysines were modified after optimization, resulting in higher GCase activity preservation (Figure 3d). The addition of IFG did not interfere with conjugation between the SATP-modified protein and the conjugate. Compared to the batch of conjugate prepared before SATP modification optimization, the conjugation yield was significantly higher, reaching a conjugation level of over 90%.

[0052] Therefore, the GBA enzyme was modified with the SATP crosslinker to provide protected thiol groups on the enzyme's accessible surface. The optimization and validation of the SATP modification were detailed above. These results demonstrate that the GBA protein was efficiently modified with SATP, resulting in a protein in which 25% of the exposed lysines were modified with the SATP crosslinker. This result corresponds to 4–5 lysines modified by SATP. The resulting product, GBA-SATP, was conjugated with PGA polymer with greater than 90% efficiency to obtain the final product, NanoGBA, in high yield. However, as discussed herein, it should be noted that modifying the lysines on the surface of GBA confers the enzyme with unexpected characteristics, which are detailed herein below.

[0053] Physicochemical changes in the GBA-SATP process and NanoGBA GBA proteins contain three structural domains: domain I (residues 1–27 and 383–414), consisting of an antiparallel β-sheet with two disulfide bridges, whose function is thought to be structural; domain II (residues 30–75 and 431–497), an immunoglobulin-like structure usually considered an interaction domain; and domain III (residues 76–381 and 416–430), a catalytic domain with a TIM barrel structure.

[0054] As a 497-amino acid protein, modification of four to five lysines on the surface of the enzyme was initially expected not to significantly affect the overall structure. Nevertheless, there is evidence of a change in the enzyme's three-dimensional structure. Regarding secondary structure, circular dichroism (CD) spectroscopy reveals a rearrangement in the enzyme's β-sheet conformation, showing a switch from parallel to antiparallel β-sheet in approximately 5% of the overall structure (see Example 2).

[0055] In the case of NanoGBA, the CD spectrum shows a more pronounced change in secondary structure, with an increased proportion of α-helices. This result is in accordance with the conformational space of PGA, which can be found in an unfolded state or as an α-helix (see Example 2).

[0056] In addition to this conformational change, there is also a substantial shift in chromatographic elution time on a Superdex 200 gel filtration column. Specifically, the unmodified GBA enzyme elutes after 46 minutes, whereas GBA-SATP elutes at 35 minutes. This decrease in retention time for GBA-SATP confirms an unexpected conformational change in GBA after SATP modification. In this case, the change in elution time suggests not only a change in secondary structure but also a change in the quaternary structure of the enzyme, strongly suggesting enzyme dimerization after SATP modification. Furthermore, NanoGBA elutes at 32 minutes in Superdex 200. This result is consistent with the increase in molecular weight due to the addition of PGA polymer to the GBA-SATP product (see Example 2).

[0057] Biochemical changes resulting from structural changes The changes demonstrated in the GBA-SATP and NanoGBA structures not only confer different globular structures to the proteins but also induce changes in the biochemical properties of the enzymes. Specifically, we demonstrate here that SATP modifications result in significant changes in protein stability, which are further utilized by the NanoGBA end product.

[0058] First, the GBA enzyme is susceptible to degradation when subjected to in vitro trypsin digestion. In contrast, both the GBA-SATP product and the NanoGBA final product are completely resistant to trypsin. This result confirms that resistance to protease degradation is conferred by the SATP modification itself, not by the addition of PGA polymer.

[0059] The enhanced resistance of GBA-SATP was further verified by a human plasma (HBP) stability assay. Upon exposure to HBP, the GBA enzyme was rapidly inactivated, becoming completely inactivated after 1 h in HBP. This resulted in the enzyme internalized in M17 GBA KO cells being unable to restore glucocerebrosidase activity after this incubation period. In contrast, GBA-SATP was significantly more resistant to HBP inactivation, maintaining approximately 35% of its initial capacity even after 24 h of incubation in HBP. In this regard, NanoGBA demonstrated further improvements in stability. NanoGBA maintained nearly its full capacity to restore glucocerebrosidase activity after 24 h of HBP incubation.

[0060] The increased stability of the GBA-SATP product and the evolution of this property by the Nano-GBA product represent a substantial transformation in the behavior of the initial enzyme. This can be demonstrated by the ability of various products to restore glucocerebrosidase activity in the M17 GBA KO model over time. As shown in the Examples, the GBA enzyme initially restores 20% of WT glucocerebrosidase activity, but this activity rapidly declines and is lost after 3 days. In contrast, GBA-SATP initially restores 50% of WT activity and still maintains 25% of WT activity after 7 days of internalization assay. This substantial contrast in behavior highlights the transformative impact of the SATP modification on GBA.

[0061] Finally, the NanoGBA product exhibits behavior quite different from that of the GBA and GBA-SATP products. In this case, NanoGBA initially recovers 25% of WT activity, but in contrast to the other products, NanoGBA activity increases over time, reaching WT activity levels after 24 h of incubation and remaining at that level for at least 7 days. Furthermore, Western blot analysis shows that the internalization of NanoGBA is higher than that of GBA or GBA-SATP and increases over time. This effect is solely due to PGA polymer conjugation, which not only enhances enzyme stabilization but also enhances cellular uptake (see Example 2).

[0062] Accordingly, a first aspect of the present invention provides a method for modifying the surface of a glucocerebrosidase enzyme with N-succinimidyl-S-acetylthiopropionate (SATP) by modifying the lysine side chains of the protein to provide an SATP-modified protein, comprising: The present invention relates to a production method comprising adding an aprotic polar solvent, preferably DMSO or DMF, which is miscible with water and capable of dissolving N-succinimidyl-S-acetylthiopropionate (SATP) and, optionally, a surfactant having an HLB value of 14 to 20, such as polysorbate 20, to an aqueous composition containing a glucocerebrosidase enzyme protein and a molecular chaperone (MC), wherein the molecular chaperone is preferably a salt of isofagomine, and the reaction is carried out at a basic 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, at a molar ratio of SATP to glucocerebrosidase enzyme of at least 5 to 25 molar equivalents, and for a reaction time of at least 30 minutes, preferably with gentle shaking (e.g., 100 rpm).

[0063] As shown in the definitions section, SATA, dPEG®4-SATA, dPEG®8-SATA, dPEG® 12 It should be noted that molecules containing similar reactive head groups but with different spacer arm lengths, such as those selected from the list consisting of -SATA, dPEG®24-SATA, and 3-mercaptopropanyl-N-hydroxysuccinimide, can be used as substitutes for SATP. Other molecules to which pyridyldithiol-reactive groups can be added, while also modifying the lysine side chain, are those selected from the list consisting of SMPT, SPDP, LC-SPDP, PEG4-SPDP, and PEG12-SPDP. Furthermore, the lysine side chain can be modified with other amine-reactive head groups, such as sulfo-N-succinimidyl or imidoesters, particularly those selected from the list consisting of 2-iminothiolane (Traut's reagent) and Sulfo-LC-SPDP (see Table 4). All of these embodiments for SATP are contemplated in the present invention to provide GBA-modified proteins.

[0064] Thus, the first aspect of the present invention not only provides a method for modifying the surface of a glucocerebrosidase enzyme with N-succinimidyl-S-acetylthiopropionate (SATP) by modifying the lysine side chains of the protein, and a method for providing an SATP-modified protein, but also a method for modifying the surface of a glucocerebrosidase enzyme with a molecule containing a reactive head group similar to SATP but with a different spacer arm length, such as any of the molecules described above (see also Table 4), by modifying the lysine side chains of the protein, and a method for providing a GBA-modified protein.

[0065] 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, the pH is preferably 8.0 to 10, more preferably 8.5 to 10, more preferably about 8.5 to about 9.5.

[0066] It should be noted that the aprotic polar solvent that is miscible with water and capable of dissolving SATP is highly preferably DMSO.

[0067] It should be noted that alternatives useful in the present invention to polysorbate 20 are 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 (nonylphenoxypolyethoxyethanol), Brij-58 (polyethylene glycol-hexadecyl ether), n-dodecyl-β-maltoside or octyl-β-glucoside. Preferably, the derivatives are polysorbates (such as polysorbate 20), polyethylenes (such as NP-40), or folate-modified sulfobetaines.

[0068] In a preferred embodiment of the first aspect, the salt of isofagomine, or the salt of 1-deoxynorjirimycin or 1-azafagomine, is at a concentration of at least 0.5 μM, preferably about 0.5 to about 25 μM. Additionally, additional GBA-specific chaperones, either inhibitory or allosteric, may be used in place of the salt of isofagomine. Examples of these molecular chaperones include, but are not limited to, alkylated derivatives of isofagomine, 1,5-dideoxy-1,5-iminoxylitol (DIX) and its derivatives, deoxynojirimycin (DNJ) and its derivatives, and ambroxol (ABX).

[0069] In another preferred embodiment, the glucocerebrosidase enzyme protein is selected from the list consisting of velaglucerase, imiglucerase or applyso or any further, preferably recombinant glucocerebrosidase enzyme.

[0070] In another preferred embodiment of the first aspect of the invention or any preferred embodiment derived therefrom, the salt of isofagomine is isofagomine D-tartrate, and said salt of isofagomine is preferably at a concentration of about 25 μM.

[0071] In another preferred embodiment of the first aspect of the present invention or any preferred embodiment therefrom, the reaction of step (a) is carried out at a molar ratio of about 5 molar equivalents to about 25 molar equivalents of SATP to glucocerebrosidase enzyme and a reaction time range set at about 30 minutes to about 120 minutes.

[0072] In another preferred embodiment of the first aspect of the present invention or any preferred embodiment therefrom, the reaction of step (a) is carried out at a molar ratio of SATP to glucocerebrosidase enzyme of about 10 to about 20 molar equivalents, preferably about 15 molar equivalents, and a reaction time range of about 50 to about 70 minutes, preferably 60 minutes.

[0073] In another preferred embodiment of the first aspect of the invention or any preferred embodiment derived therefrom, the SATP-modified protein, GBA SATP-modified protein or GBA-modified protein resulting from the reaction of step (a) is isolated, preferably purified, preferably in a citrate buffer. Preferably, the SATP-modified protein, GBA SATP-modified protein or GBA-modified protein obtained in the reaction of step (a) is isolated, preferably purified and deacetylated, preferably using a deacetylation solution such as hydroxylamine-HCl solution (at room temperature for 4 hours).

[0074] A second aspect of the present invention is a method for producing a polymer conjugate, comprising: b) adding a polymer to the deacetylated and preferably purified SATP-modified protein, GBA SATP-modified protein or GBA-modified protein obtained from the reaction of step (a) according to the first aspect of the present invention (wherein preferably the SATP-modified protein, GBA SATP-modified protein or GBA-modified protein obtained from the reaction of step (a) The SATP-modified protein or GBA-modified protein is isolated, preferably purified, and deacetylated, preferably using a deacetylation solution such as hydroxylamine-HCl solution (preferably for about 4 hours at room temperature), and the polymer is selected from the group consisting of dextran, water-soluble linear polyamino acids or polypeptides (polypeptoids) (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), and polyethyleneimine (PEI), polyorthoesters, polyacetals, and the like. c) optionally washing the product obtained from b), preferably with an acidic buffer such as VCB, optionally with the addition of a molecular chaperone such as a salt of isofagomine or other suitable MC as described throughout this specification.

[0075] Another embodiment of the second aspect of the invention is a method for producing a polymer conjugate, comprising: b. adding a polymer to the non-deacetylated SATP modified protein, non-deacetylated GBA SATP modified protein, or non-deacetylated GBA modified protein obtained from the reaction of step (a) according to the first aspect of the present invention, wherein the polymer is selected from the group consisting of dextran, water-soluble linear polyamino acids or polypeptides (polypeptoids) (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), and polyethyleneimine (PEI), polyorthoester, and polyacetal, and the GBA SATP modified protein or GBA modified protein is simultaneously deacetylated, preferably using a deacetylation solution such as hydroxyamine-HCl solution; and c. Optionally, washing the product obtained from b) preferably with an acidic buffer such as VCB, optionally with the addition of a molecular chaperone such as a salt of isofagomine.

[0076] In preferred embodiments of the second aspect of the present invention (including alternative embodiments thereof), the polymer is polyglutamic acid (PGA). Preferably, the polymer is a polyglutamic acid selected from the group consisting of poly(L-glutamic acid), poly(D-glutamic acid), poly(D,L-glutamic acid), poly(L-gamma glutamic acid), poly(D-gamma glutamic acid), and poly(D,L-gamma glutamic acid), optionally the polyglutamic acid comprises at least 50% of its backbone units as glutamic acid, and optionally comprises 60, 70, 80, 90, or 100% of its backbone units as glutamic acid. More preferably, the polymer is L-PGA having 25 to 150 glutamic acid monomer units.

[0077] A third aspect of the present invention is a method for conjugating a glucocerebrosidase enzyme to poly-L-glutamic acid (PGA), comprising: a. A method for modifying the surface protein of glucocerebrosidase enzyme alpha with N-succinimidyl-S-acetylthiopropionate by modifying the side chains of lysines of the protein according to the first aspect of the present invention or any of its preferred embodiments, including those providing GBA modified proteins; b. following step a), deacetylating the product obtained from a); and c. According to any second aspect of the invention or any of its preferred embodiments, the method comprises simultaneously or subsequently conjugating a polymer to the product obtained from b).

[0078] In a preferred embodiment of the second or third aspect, the method further comprises isolating and / or purifying the obtained or resulting polymer conjugate.

[0079] The fourth aspect refers to a SATP-modified protein, a GBA SATP-modified protein, or a GBA-modified protein obtained according to the first aspect of the invention or any of its mentioned embodiments. In some embodiments, the obtained product presents a weight percent of SATP-modified protein, GBA SATP-modified protein, or GBA-modified protein of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% by weight. The term "wt%" refers to weight as a percentage of the total weight of the obtained product.

[0080] A fifth aspect of the present invention relates to a polymer conjugate obtained according to the second or third aspect of the present invention or any of its preferred embodiments. In some embodiments, the resulting product presents at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% by weight of the polymer conjugate. The term "wt%" refers to weight as a percentage of the total weight of the resulting product.

[0081] In a sixth aspect of the present invention, the present invention also provides a composition comprising or consisting of a glucocerebrosidase enzyme modified with N-succinimidyl-S-acetylthiopropionate (SATP), whereby the side chains of the lysines of the protein are modified to provide a GBA SATP-modified protein, and / or a glucocerebrosidase enzyme modified with a molecule containing a reactive head group similar to SATP but with a different spacer arm length, such as any of the molecules shown in Table 4, whereby the side chains of the lysines of the protein are modified to provide a GBA-modified protein. Preferably, the GBA protein can be any glucocerebrosidase enzyme protein selected from the list consisting of velaglucerase, imiglucerase, or uplyso, or any additional, preferably recombinant, glucocerebrosidase enzyme. As mentioned above, SATA, dPEG®4-SATA, dPEG®8-SATA, dPEG® 12It should be further noted that molecules containing similar reactive head groups but with different spacer arm lengths can be used as substitutes for SATP, such as those selected from the list consisting of -SATA, dPEG®24-SATA, and 3-mercaptopropanyl-N-hydroxysuccinimide. Other molecules to which pyridyldithiol-reactive groups can be added, although the lysine side chains can also be modified, are those selected from the list consisting of SMPT, SPDP, LC-SPDP, PEG4-SPDP, and PEG12-SPDP. Furthermore, the lysine side chains can be modified with other amine-reactive head groups, such as sulfo-N-succinimidyl or imidoesters, particularly those selected from the list consisting of 2-iminothiolane (Traut's reagent) and Sulfo-LC-SPDP (see Table 4). All of these embodiments for SATP are contemplated in the present invention to provide GBA-modified proteins.

[0082] A further aspect of the present invention (seventh aspect of the present invention) refers to a composition comprising or consisting solely of a polymer conjugate of a glucocerebrosidase enzyme modified with N-succinimidyl-S-acetylthiopropionate (SATP) according to the sixth aspect of the present invention, or a composition comprising or consisting solely of a polymer conjugate of a glucocerebrosidase enzyme 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 may preferably be selected from the group consisting of dextran, water-soluble linear polyamino acids or polypeptides (polypeptoids) (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), and polyethyleneimine (PEI), polyorthoester, and polyacetal. Preferably, the polymer is polyglutamic acid (PGA). Preferably, the polymer is a polyglutamic acid selected from the group consisting of poly(L-glutamic acid), poly(D-glutamic acid), poly(D,L-glutamic acid), poly(L-gamma glutamic acid), poly(D-gamma glutamic acid), and poly(D,L-gamma glutamic acid), optionally comprising at least 50% of its backbone units as glutamic acid, and optionally comprising 60, 70, 80, 90, or 100% of its backbone units as glutamic acid. More preferably, the polymer is L-PGA having 25 to 150 glutamic acid monomer units. Note that the polymer conjugate of the seventh aspect of the present invention is prepared as set forth in the second and / or third aspects of the present invention.

[0083] In some embodiments, in any of the compositions of the sixth or seventh aspects of the invention, the modified glucocerebrosidase enzyme or protein conjugate is present in a weight percent of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% by weight, where the term "% by weight" refers to weight as a percentage of the total weight of the composition.

[0084] An eighth aspect of the present invention relates to a pharmaceutical composition comprising the polymer conjugate of the fifth or seventh aspect, and optionally an excipient, vehicle, permeation enhancer and / or adjuvant.

[0085] A 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 therapy, preferably for use in a method of treating a subject with Gaucher disease and / or Parkinson's disease. More preferably, the glucocerebrosidase enzyme contained in the pharmaceutical composition or polymer conjugate is selected from velaglucerase, imiglucerase, or applyso, or any recombinant glucocerebrosidase enzyme.

[0086] The following examples are for illustrative purposes only and are not intended to limit the invention. [Example]

[0087] Example 1. Development of nanotechnology-based enzyme replacement therapy for Parkinson's disease and Gaucher disease. Two different types of nanoformulations or nanoconjugates were tested: GBA encapsulated in PLGA and GBA conjugated with PGA, both of which were investigated using multiple methodological approaches and formulations.

[0088] 1. Encapsulation of GBA into PLGA nanoconjugates. 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, particularly their size, polydispersity, surface potential (ζ), and release time, making them highly tunable. Encapsulation of drugs in nanoparticle systems allows for sustained and controlled release of the payload, which at the clinical level may lead to reduced administration frequency, reduced dosage, and minimized drug variability, thereby increasing therapeutic efficacy while reducing side effects. PLGA nanocapsules, as well as other nanoconjugates, can undergo surface modifications that enable penetration of biological barriers and targeted delivery of the encapsulated drug.

[0089] The main objectives of encapsulating GBA in PLGA nanocapsules were to overcome the major limitations of conventional ERT. These were: a) Protecting GBA (velaglucerase) against loss of enzymatic activity: The physical barrier provided by the nanocapsules protects the protein from the harsh conditions encountered during circulation in the bloodstream. 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. The sustained release following internalization within the cells should provide a steady source of active GBA that remains intact throughout its passage through the bloodstream. c) Overcoming the blood-brain barrier (BBB) ​​by surface modification or intranasal administration. Because traditional ERT cannot deliver active GBA to the central nervous system (CNS), neurological symptoms remain untreatable in GD types 2 and 3. Intranasal administration of encapsulated PLGA should overcome the BBB and deliver active GBA into the brain.

[0090] To achieve the above objectives, we attempted to encapsulate velaglucerase, a type of GBA, into PLGA nanocapsules as described in the following section.

[0091] 1.1. Initial conditions. The procedure was a water-in-oil-in-water (W / O / W) double emulsion method with the following steps: W1: inner aqueous phase. Buffer: 50 mM citric acid / sodium citrate, pH 5.6, containing 5% sucrose and 0.01% polysorbate 20. Velaglucerase was dissolved at 0.5 mg / ml. O: Organic phase. 25 mg of PLGA was dissolved in 750 μl of dichloromethane (DCM) and 4% v / v of Span80 was added as a surfactant. W2: outer aqueous phase. 2% PVA and 1M NaCl in water.

[0092] The synthesis steps were as follows: 1. Preparation of O phase: PLGA was dissolved in DCM under magnetic stirring. Sorbitan monooleate was added when PLGA was completely dissolved. 2. W1 phase was added onto O phase and sonicated for 1 minute at 25% amplitude using a microtip in a Branson Sonifier SFX550. 3. The emulsion was poured into W2 phase and ultrasonicated under the same conditions as in step 2. 4. The final emulsion was left under magnetic stirring at room temperature until the dichloromethane had completely evaporated.

[0093] We successfully synthesized and characterized velaglucerase-encapsulated PLGA nanoparticles or nanoconjugates. SEM images of the encapsulated and non-encapsulated nanoconjugates are shown in Figure 4. To test whether the encapsulated protein retained enzymatic activity, we performed an in vitro assay in ST-Q7 cells (mouse striatal neurons). As shown in Figure 2, velaglucerase successfully colocalized with LAMP1 (a lysosomal marker, indicating that the protein had reached its intended destination), but no enzymatic activity was detectable.

[0094] Attempts to optimize the synthesis conditions to overcome the loss of GCase activity were unsuccessful.

[0095] 1.2. Optimization efforts to maintain GCase activity. To protect GBA from the loss of enzymatic activity, several modifications of the phases and synthetic procedures were applied. In the following sections, they are classified according to the affected stages, and the final section with the overall changes.

[0096] 1.2.1.W1 phase modification. The modifications that affected the W1 phase were primarily changes in buffer composition. To stabilize the protein and protect it from activity loss due to denaturation upon contact with organic solvents and sonication, the following specific changes were made: 1. Addition of different types of polyethylene glycol; 2. Varying the concentration of citric acid / citrate buffer.

[0097] Neither change resulted in nanoconjugates with active GBA.

[0098] 1.2.2.O Phase Modification. To minimize GBA unfolding during the homogenization process, modifications affecting the O phase focused on reducing the hydrophobicity of the phase. PLGA polymer was partially replaced with PLGA-PEG polymer, and several ratios were assayed. Ethyl acetate was tested as an alternative to dichloromethane.

[0099] Neither change resulted in nanoconjugates with active GBA.

[0100] 1.2.3.W2 phase modification. The surfactants that make up W2 are responsible for stabilizing the nanoparticles during organic solvent evaporation: PVA was replaced with polysorbate 80 and Pluronic.

[0101] Neither change resulted in nanoconjugates with active GBA.

[0102] 1.2.4. Modifications in the emulsification method Sonication time and amplitude were tested to obtain a protocol that would reduce the amount of energy delivered to the sample, which would cause protein unfolding, but would still produce nanoparticles with the desired properties. Various conditions were tested: shorter times at higher amplitudes, longer times at lower amplitudes, and continuous and intermittent sonication.

[0103] Conclusion: None of the modifications resulted in nanoparticles with active GBA and PLGA, which were subsequently discarded as drug delivery systems.

[0104] 2. Conjugation of GBA with PGA polymer. An alternative approach to incorporate velaglucerase into nanoparticle systems without complete loss of activity was developed through Polymer Unmasked-Masked Protein Therapy (PUMPT) (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 from: https: / / pubs.acs.org / doi / 10.1021 / bm5011883.) developed by Dr. Maria J. Vicent (CIPF, Valencia, Spain). PUMPT refers to a soft nanotechnology strategy in which proteins are conjugated to biodegradable polymers. In this strategy, the protein is protected and its activity is masked during transport, while its activity is designed to be restored in a controlled manner upon external stimulation at the target site. In this invention, the application of the PUMPT method was tested for velaglucerase conjugation. Velaglucerase was conjugated to a polyglutamic acid (PGA) polymer via a reduction-sensitive linker, which was intended to mask (and protect) the protein activity in oxidizing environments (such as the bloodstream) and release it in reducing environments (such as the lysosomes).

[0105] a. First attempt using the Talleli et al., 2014 protocol. To successfully conjugate velaglucerase with PGA polymers, we adapted the conjugation protocol developed by Talelli and Vicent (Talelli and Vicent, 2014) optimized for lysozyme. This conjugation procedure consisted of two major steps: (1) modification of the surface protein with SATP, which adds a thiol group by modifying the side chain of lysine; (2) conjugation of the SATP-modified protein with pyridyldithiol-modified PGA (PGA-PD), which attacks the thiol group added to lysine to form a disulfide bond.

[0106] The conjugation conditions for the Talelli and Vicent protocol were as follows: -SATP modification step: Lysozyme was dissolved in PBS at 1 mg / ml. SATP at a stock concentration of 2 mg / ml in dimethyl sulfoxide (DMSO) was added and incubated at room temperature for 1 hour with gentle shaking. The resulting product was washed three times with PBS using an Amicon centrifuge unit (cutoff 3 kDa). Two molar ratios of SATP / lysozyme, 10:1 and 6:1, were tested for this step, but 10:1 showed the highest modification rate and was therefore selected for the next step. -PGA conjugation step: The buffer of purified SATP-modified protein (modified at a 10:1 SATP / lysozyme ratio) was changed to 300 mM ammonium acetate, pH 5, and 0.2 volumes of deacetylation solution (0.5 M hydroxylamine-HCl in PBS) was added to prepare thiol 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 centrifuge unit. Three PGA-PD / lysozyme ratios were assayed: 5:1, 2.5:1, and 0.5:1.

[0107] In order to adapt this protocol for use with velaglucerase, several modifications had to be made, and those changes and their rationale are listed below.

[0108] The initial concentration of velaglucerase was changed from 1 mg / ml to 0.5 mg / ml. Our original stock of velaglucerase was 2.5 mg / ml; higher concentrations tended to cause protein aggregation. The washing step with the Amicon device used during the SATP modification and conjugation steps can concentrate the protein up to five times the working concentration. To keep the protein always below 2.5 mg / ml, the working concentration was reduced to 0.5 mg / ml.

[0109] To reduce the concentration of DMSO in the reaction, which can cause velaglucerase to unfold and aggregate, the concentration of the SATP stock was increased from 2 mg / ml to 20 mg / ml. The SATP / lysozyme ratio tested for lysozyme modification was chosen based on the number of lysines present in lysozyme (6). A 6:1 molar ratio of SATP / lysozyme was tested because it corresponds to a 1:1 SATP / lysine ratio. The 10:1 SATP / lysozyme ratio was used to ensure an excess of SATP relative to the lysine residues. Because velaglucerase has 22 rather than 6 lysine residues, three ratios were tested: 10:1 (to compare with the 10:1 ratio in the lysozyme protocol), 22:1 (to achieve a 1:1 SATP / lysine ratio), and 40:1 (to achieve an excess of SATP).

[0110] After confirming that it did not interfere with the conjugation procedure, the ammonium acetate 300 mM buffer 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 to improve the stability of velaglucerase during conjugation.

[0111] The purpose of the PGA conjugation step is to mask GCase activity, but this loss of activity should be fully reversible upon release in a reducing environment. However, the SATP modification step, which irreversibly modifies the surface lysine residues of the protein, causes irreversible activity loss. Therefore, optimizing this step to ensure maximum modification while minimizing loss of enzyme activity is a critical step. Three initial ratios of SATP tested for velaglucerase conjugation showed a decrease in enzyme activity proportional to the SATP concentration (Figure 5). While PGA conjugation was successful in completely masking GCase activity, upon release with 10 mM reduced glutathione (GSH), only one of the conditions restored GCase activity, significantly below the activity level exhibited by the SAPT-modified velaglucerase, which should have fully restored GCase activity.

[0112] b. Stabilization of GBA by molecular chaperones. The loss of activity during modification of GBA with SATP and conjugation of the modified protein with PGA was primarily due to the instability of the protein in the sorbitan monooleate conditions of the working buffer used in the conjugation step. To ameliorate this activity loss, we resorted to the use of GBA molecular chaperones (MCs) during the synthesis procedure. MCs are small molecules capable of binding to and stabilizing target enzymes. Typically, these MCs are competitive inhibitors that bind to the catalytic pocket of the enzyme.

[0113] In the case of GBA, there are currently many commercially available MCs, although none have yet reached the market as pharmacological chaperones (PCs) for GBA. Among these, two of the most studied are ambroxol hydrochloride and isofagomine D-tartrate (IFG). For our purposes, ambroxol was discarded due to the presence of a primary amine in its structure, which prevents SATP modification by competing with lysine residues (Figure 6). IFG was selected as a stabilizer because it does not contain a primary amine. Furthermore, IFG is a potent chaperone for GBA, with a K of 5.8 nM at pH 7.0, suggesting that a small amount of MC may be sufficient to stabilize GCase during conjugation.

[0114] To test the lack of interference of IFG in the conjugation procedure, comparative conjugation protocols were performed in the presence or absence of chaperones. The protocol using IFG differed only by adding excess IFG (25 μM, nearly 1000-fold the Ki) to the working buffer to ensure maximal inhibition and protection of the enzyme. Samples from each step of the conjugation from both protocols (SATP modification and PGA conjugation) were collected and analyzed by non-reducing SDS-PAGE combined with silver staining (Figure 7). This technique allowed us to confirm the molecular weight of the nanoconjugate without cleaving the disulfide bond connecting the polymer and protein, thereby qualitatively assessing the conjugation rate.

[0115] Results from non-reducing SDS-PAGE showed no significant differences between conjugations in the absence or presence of IFG (Figures 7 and 3C). SATP modification of velaglucerase resulted in a small amount of enzyme dimerization. The presence of a dimerization band on the SATP-modified enzyme in the presence of IFG indirectly indicated the lack of interference in this step. PGA conjugation with the protein, recognized by the appearance of a smeared protein-polymer conjugate, showed no difference between conditions. Although no differences were observed in conjugation efficiency, the addition of IFG during the synthesis procedure improved the GCase activity measured at each step of the conjugation, demonstrating its ability to stabilize and protect the enzyme during conjugation. After SATP modification, no loss of GCase activity was detected in IFG-containing reactions, a fact attributed to the complete stabilization of the enzyme during this step. The gradual decrease in GCase activity after conjugation in the presence of IFG was attributed to the masking effect of the polymer on the enzyme activity.

[0116] Conclusion: IFG improved enzyme stability during the conjugation and masking processes. In the new modified protocol, all buffers used for GBA nanoconjugation contain 25 μM IFG.

[0117] c. Optimization of SATP modification Despite the interesting results obtained with the addition of IFG as a stabilizer, the nanoconjugation strategy appeared to be far from being optimized. As shown in Figure 7, after all conjugation steps, over 80% of the velaglucerase remained unmodified. The explanation for this insufficient conjugation of the enzyme was later found to be the buffer conditions used for the SATP modification step.

[0118] As described in section 2a), the protocol for velaglucerase conjugation with PGA was adapted from the work of Talelli and Vicent. The key step for adaptation was the modification of the protein with SATP, which, in contrast to the PGA conjugation step, results in irreversible loss of activity. As shown in section 2b, adapting this protocol proved ineffective for proper velaglucerase modification with SATP, and the conjugation yield was low. Examining the chemistry of the N-succinimidyl ester reaction with primary amines (the reaction by which SATP binds to proteins) showed that this reaction is highly dependent on the pH of the reaction mixture. At the neutral pH (pH 7.4) initially used for SATP modification, the reaction proceeded very slowly, requiring several hours to reach equilibrium. Nevertheless, the same reaction performed at more basic pHs (pH 8.5-9) occurred rapidly, reaching equilibrium within minutes. Taking this information into account, the buffer conditions for the SATP modification step were modified to allow reactions at pH 8, 5, or 9. SATP modification efficiency was evaluated relative to pH 7.4 in terms of relative thiol addition, relative lysine reduction, and relative GCase activity retention.

[0119] After SATP modification and deacetylation of the protected thiol, thiol addition was quantified using a free thiol assay kit. Results were plotted relative to the SATP-modified enzyme and unmodified velaglucerase. As shown in Figure 9a, free thiol levels doubled after 2 hours of reaction at pH 7.4. In contrast, reactions performed at pH 8, 5, and 9 showed a four-fold increase in free thiol levels, indicating the higher reactivity of SATP under these conditions.

[0120] As mentioned, thiol quantification requires deacetylation of SATP-protected thiols, which suggests additional reactions that may interfere with the resulting measurements (e.g., by disulfide bond formation between protein molecules). To avoid this deacetylation step and consider that SATP reacts only with primary amines of lysine residues, we instead analyzed the reduction of free surface amines from lysine residues. The reduction of free lysine residues was quantified with the TNBSA assay. TNBSA reacts with primary amines to produce a yellow molecule, which can be quantified by absorbance. In proteins, the only primary amines detectable by TNBSA are from exposed lysine residues (away from the N-terminus). Therefore, TNBSA reactivity is directly proportional to the number of lysine residues on the surface of the protein.

[0121] Results from lysine quantification demonstrated that the percentage of exposed lysine residues did not decrease after SATP modification at pH 7.4, demonstrating the insufficient reactivity of the molecule at these conditions (Figure 9b). However, reactions performed at pH 8.5 and pH 9 resulted in a 25–30% reduction in exposed lysines, further confirming the significant increase in SATP reactivity at these pH conditions.

[0122] Regarding GCase activity, no change in enzyme activity was detected at pH 7 or 4 (Figure 9c). This lack of activity loss after SATP modification was previously observed in section 2b and attributed to the complete stabilization of the enzyme in the presence of IFG. On the other hand, GCase activity after SATP modification at pH 8.5 and pH 9 showed a 25% and 30% decrease, respectively. Considering the effect of IFG, the only explanation for the decrease in GCase activity is modification of specific lysine residues essential for enzymatic activity.

[0123] This outcome of SATP modification was predicted previously but was hypothesized to be an unavoidable stepwise loss of activity because SATP modification is a stochastic process that can modify surface-exposed lysine residues in proteins.

[0124] d. Determination of parameters of SATP modification process by CCD-RSM optimization. In this regard, the main objective of the SATP modification procedure was to simultaneously optimize the yield of SATP modification and the maintenance of GCase activity. Considering these two parameters together in statistical analysis while optimizing the synthesis conditions may provide more promising results. Optimization by central composite design-response surface methodology (CCD-RSM) should help select the values ​​of the independent variables (our dependent variables) that significantly affect the output of SATP modification and GCase activity maintenance.

[0125] According to the literature, the SATP manual (Thermo Scientific, Ref. 26100), and this preliminary study, the main independent variables that primarily affected SATP modification and maintained GCase activity were (1) pH, (2) the molar ratio of SATP to protein, and (3) reaction time. For CCD-RSM optimization, it was necessary to define the range of operation for the independent variables. Again, using information found in the literature, the SATP manual, and the preliminary study, the pH range was set to pH 7 to pH 9, the molar ratio of SATP to protein ranged from 5 equivalents to 25, and the reaction time range was set to 30 min to 120 min.

[0126] Each numerical factor varied across five levels calculated by Software Design Expert 11 (DX11): plus and minus alpha (axis points), plus and minus 1 (factor points), and center points. The values ​​for each factor are listed in Table 1.

[0127] [Table 1]

[0128] The advantage of this statistical method is that instead of testing all possible combinations of these conditions, the software randomized 16 different experimental conditions. The SATP modification process was performed 16 times for each of the 16 conditions of pH, SATP to protein molar ratio, and reaction time calculated by the software, and the modification yield and maintenance of GCase activity were measured. The results were imported into the software and analyzed by analysis of variance (ANOVA). The conditions that best achieved both SATP modification and maintenance of GCase activity simultaneously were set to pH 9, SATP to protein molar ratio 15:1, and reaction time 60 minutes. The final protocol for velaglucerase modification and PGA conjugation was established as follows:

[0129] SATP modification step: Velaglucerase was prepared at 0.5 mg / ml in PBS pH 9 containing 25 μM IFG. 15 molar equivalents of SATP from a stock concentration of 20 mg / ml in DMSO was added and incubated for 1 hour at room temperature with gentle shaking. The resulting product was washed three times with VCB supplemented with 25 μM IFG using an Amicon centrifuge unit (cutoff 10 kDa).

[0130] PGA conjugation step: One volume of deacetylation solution (0.5 M hydroxylamine-HCl in PBS) was added to the purified SATP-modified protein already in VCB. One volume of PGA-PD was added to a final PGA / protein molar ratio of 5:1 and incubated at room temperature for 4 hours. The resulting product was washed three times with VCB supplemented with 25 μM IFG using an Amicon centrifuge unit (cutoff 30 kDa).

[0131] Conclusion: The final SATP modification protocol incorporated into the conjugation procedure proved to successfully improve GCase activity maintenance while maintaining a high SATP modification rate. Approximately 25% of lysines were modified after optimization, resulting in higher GCase activity preservation. The addition of IFG did not interfere with the conjugation between the SATP-modified protein and the conjugate. Compared to the batch of conjugate prepared before SATP modification optimization, the conjugation yield was significantly higher, reaching a conjugation level of over 90%.

[0132] 3. In vitro validation of PGA-GBA / IFG nanoconjugates. The velaglucerase-PGA-IFG conjugate (NanoGBA, produced 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 test the delivery and therapeutic efficacy of GBA.

[0133] The inventors have found that NanoGBA: Increased stability of GBA protein (in human plasma); ·Internalized into cells; ·Delivered to lysosomes as an active enzyme; Prolonged recovery of GBA activity (7 days); Reduces accumulation of GlcSph substrates; May reduce the accumulation of neurotoxic forms of the alpha-synuclein protein (a hallmark of neurodegeneration in Parkinson's disease).

[0134] Conclusion: The GBA-PGA-IFG conjugate can be delivered to lysosomes and reduce the accumulation of GlcSph substrates based on the long-term restoration of GBA activity.

[0135] 4. In vivo validation of PGA-GBA / IFG nanoconjugate (nanoGBA). Mouse models: In vivo validation was tested in two mouse models: GBA-WT and GBA-D409V, which showed a >90% loss of GCase activity and substrate accumulation in all tissues. · NanoGBA: In addition to nanoGBA, we generated a new formulation of nanoconjugates containing fluorescent PGA (Cy5.5-PGA) to test its in vivo biodistribution after administration by IVIS (Spectrum In Vivo Imaging System) analysis. Administration o Intravenous: verify systemic and CNS activity; Intranasal: To validate CNS delivery, intranasal administration was improved with a cross-linked hyaluronic acid-based hydrogel called HA-CP® Crosspolymer (EP17382498, WO2019020344).

[0136] [Table 2]

[0137] a. Intravenous administration: Intravenous administration showed biodistribution in serum, liver, bone marrow, spleen, kidney, and lung. The nanoconjugate was active and showed increased GCase activity, peaking at 30 minutes in serum and 3 hours in liver, bone marrow, and spleen.

[0138] b. Intranasal administration: Intranasal administration of nanoGBA showed a lack of delivery to the CNS (tested by IVIS and increased GCase activity).

[0139] c. Intranasal Administration with HA-CP® Crosspolymer: To improve drug delivery to the CNS via intranasal administration, the adjuvant HA-CP® (a commercially available crosspolymer from nurturing®), formed by three biodegradable, biocompatible, and active components (hyaluronic acid, polyglutamic acid, and lysine; patent co-inventors of Dr. Vicent), was used to improve intranasal administration.

[0140] Intranasal administration of nanoGBA with HA-CP® can deliver GBA protein into the brain (Figure 13, left). In the olfactory bulb, increased GCase activity was observed in GBA-D409V mutant animals, and this increased GCase activity translated into decreased accumulation of the GBA substrates GlcCer and GlcSph (Figure 13, right). Analysis of other brain regions is currently underway.

[0141] Conclusion: Systemic delivery of nanoGBA is effective via intravenous administration, and the nanoconjugate is detectable and the GBA enzyme is active in serum, liver, and spleen. Delivery to the CNS was effective via intranasal administration using HA-CP® crosspolymer, and the nanoconjugate was detected, GBA enzyme was active, and reduced substrate accumulation in the olfactory bulb; analysis of other brain regions is ongoing.

[0142] Example 2 To evaluate the novel properties of SATP-modified GBA and NanoGBA, several assays were performed, including circular dichroism, size-exclusion chromatography, stability to trypsin digestion and human plasma, and internalization in cell models.

[0143] Experiment 1: Circular dichroism Objective: To compare the secondary structures of GBA, GBA-SATP and NanoGBA.

[0144] Methods: 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 to 250 nm in a quartz cuvette with a 1 mm path length. Secondary structure analysis was performed using the free online software "BeStSel," which yielded the percentages of α-helix, β-sheet, and unfolded (unrecognized) conformations.

[0145] Results (see Figure 14): NanoGBA differs significantly from GBA and GBA-SATP, primarily due to increased alpha-helical content in NanoGBA (19%) compared to GBA (14.6%) and GBA-SATP (14.8%). In contrast, GBA-SATP does not differ from GBA in the total amount of alpha-helices or beta-sheets, but differs in the beta-sheet forms (parallel and antiparallel), with antiparallel being up to 5% more abundant in GBA-SATP.

[0146] [Table 3]

[0147] Conclusion: There are differences in the secondary structure of all three products. NanoGBA shows an increased presence of alpha helices, which is consistent with the possible conformation of PGA-PD. Furthermore, the structure also indicates that, unlike the unconjugated mixture of GBA and PGA-PD, the confirmation that NanoGBA adopts a conjugated structure is only due to the presence of the polymer.

[0148] Furthermore, differences were also observed between GBA and GBA-SATP, implying that the thiol protecting groups on some amino acids may cause conformational changes in the overall structure of GBA.

[0149] Experiment 2: Size exclusion chromatography Objective: To verify that our product is structurally distinct from GBA.

[0150] Methods: 75 μg of GBA, GBA-SATP and NanoGBA in Superdex 200 buffer (10 mM MES, 100 mM NaCl, pH 6) were applied to a Superdex 200 column and eluted under the same conditions to assess their retention.

[0151] Results (see Figure 15): GBA elutes after 46 minutes; GBA-SATP, from 35 onwards; and NanoGBA from 32 onwards.

[0152] Conclusion: The distinct behavior observed for all three products during size exclusion chromatography indicates significant structural differences between them, resulting in shorter retention times.

[0153] Experiment 3: Limited proteolysis Objective: To evaluate the protection of different products (GBA, GBA-SATP, NanoGBA) against trypsin digestion.

[0154] Methods: 50 μg of GBA, GBA-SATP, and NanoGBA were incubated with 5 μg of trypsin for 0, 30, 60, and 90 minutes at 37°C. The reaction was then stopped, denaturing buffer was added, and the samples were heated to 95°C for 10 minutes. Digestions were analyzed by PAGE followed by total protein staining (Coomassie).

[0155] Results (see Figure 16): GBA is susceptible 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 are completely resistant to the effects of trypsin.

[0156] Conclusion: GBA-SATP confers GBA protection against trypsin digestion, which is further maintained on the final nanoconjugate.

[0157] Experiment 4: Plasma stability assay Objective: To evaluate the stability of different products in human plasma (HBP).

[0158] Methods: To evaluate the ability of various products to recover intracellular glucocerebrosidase activity after incubation with human plasma, M17 GBA KO cells were treated with GBA, GBA-SATP, and NanoGBA in HBP for 0, 1, 3, 6, and 24 hours. These cells were treated with 5 μg / ml of various products (GBA, GBA-SATP, NanoGBA) preincubated in HBP for 3 hours. Cell lysates were then collected, and internalized protein was assessed by activity assay and Western blot.

[0159] Results (see Figure 17): GBA was almost completely inactivated after incubation in HBP for 1 hour or more, with only a maximum of 5% of WT glucocerebrosidase activity recovered from treated cells. Instead, GBA-SATP and NanoGBA exhibited high stability, resulting in a 10-fold increase in activity recovery. GBA-SATP maintained one-third of its initial activity after 24 hours of incubation in HBP, while NanoGBA preserved up to 83% of its initial activity.

[0160] Conclusion: Although HBP incubation does not appear to differentially affect the internalization capacity of our products (total GBA internalization as determined by Western blot), differences are observed in their ability to restore GCase activity once internalized, thus demonstrating the increased stability in plasma afforded by our modifications. Both GBA-SATP and NanoGBA are much more stable in HBP than GBA, with the latter being particularly stable after longer incubations.

[0161] Experiment 5: Uptake in M17 GBA KO cells Objective: To evaluate the ability of different products to be internalized over time in a cell model.

[0162] Methods: M17 GBA KO cells were treated with 5 μg / ml of various products (GBA, GBA-SATP, NanoGBA) for 1, 3, 6, 24, 72, and 168 hours. Cell lysates were then collected and internalized proteins were assessed by activity assay and Western blot.

[0163] Results (see Figure 18): Despite its internalization, "naked" GBA treatment after 1 hour only recovers a maximum of 20% of WT glucocerebrosidase activity. Furthermore, after this time, intracellular glucocerebrosidase activity decreases over time, reaching negligible values ​​after 3–7 days. In contrast, after 1 hour of GBA-SATP treatment, cells reach 50% of WT activity, which is maintained over time until 7 days, when activity drops to 25%. In the case of NanoGBA, a clear increase over time was observed, reaching levels of 150% after 3 days and even demonstrating higher activity than WT after 7 days of treatment.

[0164] Conclusion: The internalization of the three products is clearly different. The SATP modification of GBA confers major stabilization to the protein over time in cell culture medium, which results in major recovery of activity over time, approximately two-fold more effective. Furthermore, nanoconjugation further enhances this stabilization and internalization ability, demonstrating the best results.

[0165] [Table 4]

[0166] Nomenclature IUPAC SATP-(2,5-dioxopyrrolidin-1-yl) 3-acetylsulfanylpropanoate SATA-(2,5-dioxopyrrolidin-1-yl)2-acetylsulfanyl acetate dPEG® 4-SATA-(2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]propanoate dPEG® 8-SATA (2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate dPEG (registered trademark) 12 -SATA (2,5-Dioxopyrrolidin-1-yl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate dPEG (registered trademark) 24 -SATA (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-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-acetylsulfanylethoxy] ... 3-Mercaptopropanyl-N-hydroxysuccinimide. (2,5-dioxopyrrolidin-1-yl) 3-sulfanylpropanoate SMPT (2,5-dioxopyrrolidin-1-yl)4-[1-(pyridin-2-yldisulfanyl)ethyl]benzoate SPDP (2,5-Dioxopyrrolidin-1-yl)3-(pyridin-2-yldisulfanyl)propanoate LC-SPDP (2,5-dioxopyrrolidin-1-yl)6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate Sulfo-LC-SPDP 2,5-Dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid PEG4-SPDP (2,5-Dioxopyrrolidin-1-yl)3-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate PEG12-SPDP (2,5-Dioxopyrrolidin-1-yl)3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(pyridin-2-yldisulfanyl)]propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate 2-Iminothiolane (Traut's reagent) Thiolane-2-imine

Claims

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

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

3. The method according to claim 1, wherein the MC is isofagomine D-tartrate.

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

5. 4. The process according to any one of claims 1 to 3, wherein the reaction in step (a) is carried out at a molar ratio of SATP to glucocerebrosidase enzyme of 10 molar equivalents to 20 molar equivalents, preferably about 15 molar equivalents, and a reaction time in the range of 50 to 70 minutes, preferably 60 minutes.

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

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

8. 1. A method for producing a polymer conjugate, comprising: b) adding a polymer to the deacetylated, preferably purified SATP modified protein, wherein the modified protein is the glucocerebrosidase enzyme obtained from the reaction of step (a) of claim 7, and the polymer is selected from the group consisting of dextran, water-soluble linear polyamino acids or polypeptides (polypeptoids) (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), and polyethyleneimine (PEI), polyorthoester, polyacetal; and c) optionally washing the product obtained from b), preferably with an acidic buffer (citrate buffer), and optionally adding a molecular chaperone such as a salt of isofagomine. A method comprising:

9. 1. A method for producing a polymer conjugate, comprising: b. adding a polymer to the SATP modified protein obtained from reaction step (a) according to any one of claims 1 to 6, wherein the modified protein is a glucocerebrosidase enzyme, the polymer is selected from the group consisting of dextran, water-soluble linear polyamino acids or polypeptides (polypeptoids) (including polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosine (PSar) and the like), 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), polyorthoester, and polyacetal, and the SATP modified protein is simultaneously deacetylated, preferably using a deacetylation solution such as a hydroxyamine-HCl solution; and c. Optionally, washing the product obtained from b) preferably with an acidic buffer such as VCB, optionally with the addition of a molecular chaperone such as a salt of isofagomine.

10. 10. The method of claim 8 or 9, wherein the polymer is polyglutamic acid (PGA).

11. 11. The method of claim 10, wherein the polymer is a polyglutamic acid selected from the group consisting of poly(L-glutamic acid), poly(D-glutamic acid), poly(D,L-glutamic acid), poly(L-gamma glutamic acid), poly(D-gamma glutamic acid) and poly(D,L-gamma glutamic acid), optionally the polyglutamic acid comprises at least 50% of its backbone units as glutamic acid, and optionally comprises 60, 70, 80, 90 or 100% of its backbone units as glutamic acid.

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

13. 1. A method for conjugating a glucocerebrosidase enzyme to poly-L-glutamic acid (PGA), comprising: a. A method for the preparation of the surface protein of glucocerebrosidase enzyme alpha by modifying the lysine side chains of said protein with (N-succinimidyl-S-acetylthiopropionate) according to any one of claims 1 to 7; b. following step a), deacetylating the product obtained from a); and c) A method according to any one of claims 8 to 12, comprising simultaneously or subsequently conjugating a polymer to the product obtained from b).

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

15. 7. A modified SATP protein obtainable by the method of any one of claims 1 to 6, wherein the modified protein is a glucocerebrosidase enzyme.

16. 8. A SATP modified protein obtained by the method of claim 7, wherein the modified protein is a glucocerebrosidase enzyme.

17. A polymer conjugate obtainable by the method of any one of claims 8 to 14, wherein the SATP modified protein of the polymer conjugate is a glucocerebrosidase enzyme.

18. 20. A pharmaceutical composition comprising the polymer conjugate of claim 17, optionally further comprising an excipient, vehicle, permeation enhancer and / or adjuvant.

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

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

21. 8. The method of any of claims 1 to 7, wherein the glucocerebrosidase enzyme is selected from velaglucerase, imiglucerase, or applyso, or any recombinant glucocerebrosidase enzyme.

22. 19. The polymer conjugate of claim 17 or the pharmaceutical composition of claim 18, wherein the glucocerebrosidase enzyme is selected from velaglucerase, imiglucerase, or applyso or any recombinant glucocerebrosidase enzyme.

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