EP4626462A1

EP4626462A1 - Glucocerebrosidase (gba) polymer conjugate, preparation method and use for nanotechnological based enzyme replacement therapy

Info

Publication number: EP4626462A1
Application number: EP23813763.2A
Authority: EP
Inventors: Marta MARTÍNEZ VICENTE; Julia Lorenzo Rivera; María Jesus VICENT DOCÓN; Fernando NOVIO VÁZQUEZ; David MONTPEYO GARCIA-MORENO; Eddi PRADAS GRACIA; Inmaculada Conejos Sanchez; Pau SARLE VALLES; Daniel Ruiz Molina
Original assignee: Fundacio Hospital Universitari Vall Hebron Institut De Recerca; Universitat Autonoma de Barcelona UAB; Centro de Investigacion Principe Felipe
Current assignee: Fundacio Hospital Universitari Vall Hebron Institut De Recerca; Universitat Autonoma de Barcelona UAB; Centro de Investigacion Principe Felipe
Priority date: 2022-11-29
Filing date: 2023-11-29
Publication date: 2025-10-08
Also published as: AU2023400894A1; JP2025539926A; IL321148A; WO2024115612A1; CN120731086A; MX2025006236A

Abstract

The present invention relates to the medical field, in particular, to a nanotechnological based Enzyme Replacement Therapy, preferably for Parkinson's disease, based on the restoration of lysosomal glucocerebrosidase activity through enzyme-polymer nanoconjugation of GBA, the GBA polymer conjugate for such use, and its manufacturing method.

Description

Glucocerebrosidase (GBA) polymer conjugate, preparation method and use for nanotechnological based Enzyme Replacement Therapy

TECHNICAL FIELD

The present invention relates to the medical field, in particular, to a nanotechnological based Enzyme Replacement Therapy, preferably for Parkinson's disease and Gaucher's disease, based on the restoration of lysosomal glucocerebrosidase activity through enzyme-polymer nanoconjugation of GBA, the GBA polymer conjugate for such use, and its manufacturing method.

BACKGROUND OF THE INVENTION

The GBA1 gene encodes glucocerebrosidase (GCase) protein or GBA, a lysosomal enzyme involved in the metabolism of sphingolipids. Mutations in homozygosis in GBA causes Gaucher disease (GD), an autosomal-recessive lysosomal disorder. Heterozygous mutation carriers highly increase the risk for development of Parkinson's disease (PD). In this sense, it is known that the presence of mutations in GBA1 is the main genetic risk factor for PD. Between 10-12% of PD patients present mutations in GBA (Kinghorn, KJ. Pathological looping in the synucleinopathies: Investigating the link between Parkinson's disease and Gaucher disease. DMM Dis. Model. Meeh. 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).

PD patients carrying GBA mutations cannot clinically be distinguished from those without GBA mutations; however, they present a significantly early mean onset and higher risk for development of cognitive dysfunctional and other neuropsychiatric disorders.

PD patients with GBA mutations present a significant decrease in GCase activity in the substantia nigra (SNpc) and other brain regions. Interestingly, other sporadic PD patients without GBA mutations also present a significant reduction in GCase activity suggesting that GBA might contribute to the PD pathogenesis. The deficiency in GCase activity in PD patients is associated to an increase in alpha- synuclein (syn) levels, supporting the possible role of GBA deficiency in syn accumulation or aggregation. In cellular and animal models, the decrease in GBA/GCase activity is also linked to an increased in syn levels (Mazzulli, J.R.; Xu, Y.H.; Sun, Y.; Knight, A.L.; McLean, P.J.; Caldwell, G.A.; Sidransky, E.; Grabowski, G.A.; Krainc, D. Gaucher disease glucocerebrosidase and alpha-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell 2011, 146, 37-52). Therefore, there is a need to restore GBA activity, in particular, to restore GBA activity in lysosomes to recover the activity of the lysosomal system, avoid the sphingolipid substrates accumulation, decrease syn levels and ultimately prevent neurodegeneration. Currently, available therapies for such restoration of the GBA activity are:

Enzyme Replacement Therapy (ERT): Restoring GCase activity via recombinant GBA protein. This is the main therapy currently used to treat GD patients, this therapy is beneficial for the non-neurological manifestations since the recombinant protein itself cannot cross the BBB and reach the brain. ERT is not valid at the present time to treat GCase loss of function effect in the central nervous system of GD and PD associated with GBA.

Substrate Reduction Therapy (SRT): inhibition of the synthesis of the GCase substrate (GlcCer) to avoid its accumulation.

Molecular Chaperons (MC): pharmacological GBA-specific chaperones are able to stabilize misfolded mutant GBA retained in the ER and promote its transit to the lysosome and recovery its function. Examples: isofagomine and ambroxol.

Currently, restoration of GBA activity in humans has been only approved for Gaucher's 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.

As regards PD, currently, there is not an approved treatment for this disease targeting GBA restoration, although there are a number of ongoing clinical trials.

In the present invention, we have developed a nanotechnological based Enzyme Replacement Therapy, preferably for Parkinson's disease, based on the restoration of lysosomal glucocerebrosidase activity through enzyme-polymer nanoconjugation of GBA.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1. This figure is a scheme of how the GBA nanoconjugates of the present invention are internalized by the cells by endocytosis and sent directly to the lysosomes for their degradation, once the nanoconjugates reach the lysosome, the polypeptide coating should degrade in the acidic lysosomal environment and GBA, as a lysosomal protein, should recovered its lysosomal function, restoring its GBA activity.

FIGURE 2. ST-Q7 mouse striatal neurons were treated with 200 ng/pl of Velaglucerase loaded PLGA nanoparticles or nanoconjugates for 24 hours. An Immunocytochemical assay was performed staining Velaglucerase with anti-GBA antibody (right) and anti-LAMP-1 antibody (left) as a lysosomal marker.

FIGURE 3. GBA-PGA characterization. A) Percentage of unmodified lysine decrease after SATP modification. Unmodified Velaglucerase (GBA) and SATP modified Velaglucerase (GBA-SATP) under optimal conditions (PBS at pH 9, for 60 minutes and with 15 equivalents of SATP) were analysed for lysine levels decrease through 2,4,6-Trinitrobenzene Sulfonic Acid (TNBSA) assay. The experiment was conducted in triplicate. B) Percentage of unmodified Velaglucerase after conjugation. Velaglucerase band of silver staining was quantified for each time point, providing quantitative values of the efficiency of the reaction. Values were expressed as a percentage of Velaglucerase at GBA-SATP step, as the following samples have the same concentration of GBA-SATP (there is no Amicon washing step). C) Silver staining of conjugation time-course. Unmodified Velaglucerase (1), SATP modified Velaglucerase (2) and a time course of conjugation at 0.5h (3), lh (4), 2h (5), 4h (6) and overnight (7) without (0 IFG) and with (+ IFG) 26.9 pM Isofagomine D-Tartarate (IFG) were submitted to a nonreducing SDS-PAGE. Then, gels were stained with a silver staining protocol, showing the MW of the conjugates and the kinetics of conjugation. From top to bottom, the arrows signal the GBA-PGA band (a), the aggregation bands (P) and the Velaglucerase band (y). D) GCase activity loss during conjugation in absence or presence of IFG. The optimized protocol of conjugation was analysed for GCase activity at the different steps of conjugation: unmodified Velaglucerase (GBA, dark), SATP modified Velaglucerase (GBA-SATP, medium) and conjugated Velaglucerase (GBA-PGA, light). Two experiments were performed in quadruplicates: Conjugation in absence of IFG (IFG (-)) and in presence of 26.9 pM IFG (IFG (+)). *Statistically significant differences (p<0.0005).

Figure 4. PLGA nanocapsules images obtained by SEM. Unloaded PLGA nanocapsules (left) were synthesised as control (0 245 nm, -25 ± 5 mV) and Velaglucerase was successfully encapsulated into PLGA nanocapsules (right, 0 217 nm, -20 ± 4 mV). is herein defined as Z-potential.

Figure 5. GCase activity was tested in (light grey) GBA (Velaglucerase) after modification with SATP in different molar ratios (untreated Velaglucerase was used as a control) and in (dark grey) PGA- Velaglucerase conjugates after release via incubation with 10 mM of GSH for 1 hour. Figure 6. Interference of MCs on SATP modification. A) SATP reactivity against primary amines on proteins. B) Molecular structure of Isofagomine and Ambroxol. Circled in green the secondary amine of Isofagomine (unreactive). Circled in red the primary amine of Ambroxol (reactive).

Figure 7. Effect of IFG addition on conjugation efficiency. Velaglucerase (Vela) was modified with the SATP crosslinker in absence (Vela SATP) or presence of IFG (Vela SATP + IFG). Then, the PGA conjugation step was followed up taking samples at lh, 2h and 4h of reaction in absence (Conj) or presence of IFG (Conj +IFG).

Figure 8. IFG addition improves GCase activity upon nanoconjugation. GCase activity was measured from each step of conjugation and the obtained activity was compared to the unmodified enzyme.

Figure 9. Modification of Velaglucerase with SATP was tested at different pH and reaction times. The incorporation of thiols at each condition (A) the decrease in free lysins (B) and the variation of GCase activity (C) was measured.

Figure 10: In vitro validation. A. Stability in human blood plasma (HBP) was assessed by GCase activity assessment upon internalization. Compounds at 200 g/ml in velaglucerase citrate buffer (VCB, 50 mM of sodium citrate at pH 5.6, 5% w/v of sucrose and 0.01% v/v of Polysorbate 20) were incubated with 4 volumes of HBP up to 24 hours and diluted with opti-MEM with 1 U/ml heparin to 5 pg compound/mL. BE(2)-M17 GBA1-/- cells were incubated for 3 hours with each compound, lysed and GCase activity was assessed (data from three independent experiments in triplicates). B: Levels of Glucosyl-Sphingosine levels were analysed by LC-MS/MS after BE(2)-M17 GBA1 -/- cells were treated up to 7 days with GBA or NanoGBA. C. BE(2)-M17 GBA1 cells were treated up to 7 days with GBA or NanoGBA. Untreated cells were used as a negative control (KO) and untreated BE(2)-M17 WT cells as a positive control (WT). After treatments, cell lysates were analyzed for total GBA internalized, 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 the different treatments as a % of the WT levels (data from three independent experiments and represented as Mean ± SEM). D. Levels of alpha-synuclein protein after 7 days of treatment. Statistical differences were determined with Two-way ANOVA with multiple mean comparisons. Compound differences were determined p<0.0001. ***p<0.01 in multiple comparisons. **** p<0.0001 in multiple comparisons.

Figure 11: Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) biodistribution detected by I VIS. Figure 12: Ex vivo detection of Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) detected by IVIS (left). GCase activity at 30 min, 3h, 24h and 72h after administration of nanoGBA in different tissues (right).

Figure 13: Ex vivo detection of Cy5-5-PGA-Vela-IFG (fluorescent nanoGBA) in brain (left). GCase activity after 24h in olfactory bulb and quantificaction of the two GBA substrates (GlcCer and GlcSph or lysoGbl).

Figure 14: Circular dichroism spectra. A) GBA-SATP difference with GBA enzyme. B) Differences between NanoGBA and the other two products analysed, GBA and GBA-SATP. Data represented as As (M-l, cm-1).

Figure 15: Size exclusion chromatography in Superdex 200. GBA (Blue), GBA-SATP and NanoGBA were loaded into the column and the absorbance at 280 nm was followed along the time.

Figure 16: Analysis of the tryptic digestion. This figure shows the Coomassie staining of the tryptic digestion of GBA, GBA-SATP and NanoGBA and the relative quantification by densitometry of the GBA bands.

Figure 17: Plasma stability assay. This figure shows: A) the activity recovered inside the treated M17 GBA KO cells. The activity data is displayed as a percentage of the activity of the untreated wild type cells. Bars show the mean plus the standard deviation (n=2). B) Western Blot showing the internalised total GBA upon each treatment.

Figure 18: Uptake assay. A) Activity recovered inside the treated M17 GBA KO cells. The activity data is displayed as a percentage of the activity of the untreated wild type cells. Bars show the mean plus the standard deviation (n=3). B) Western Blot showing the internalised total GBA upon each treatment.

DESCRIPTION

DEFINITIONS

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

The term "about" when referred to a given amount or quantity is meant to include deviations of plus or minus ten, preferably five, percent.

As used herein, the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or."

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of", though less preferred.

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

In the context of the present invention, "GBA protein" refers to an enzyme (also called acid - glucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, Glucocerebrosidase, GCase or any other name listed in the BRENDA Enzyme database under entries 3.2.1.45 and 3.2.1.62) which induces glucosylceramidase activity. In the context of the present invention, "isofagomine" or "IFG" refers to a pharmacological chaperone which binds selectively to glucocerebrosidase and restores its correct conformation and enhances its activity.

In the context of the present invention, a "GBA-specific chaperone" refers to pharmacological chaperones that bind specifically to GBA and assist in the folding of the protein to prevent misfolding, enhance the enzyme activity and/or enhance accurate translocation of the protein to the lysosome. Examples of those GBA-specific chaperones include but are not limited to iminosugars with inhibitory activity and their derivatives (derivatives such as l,5-dideoxy-l,5-iminoxylitol (DIX), deoxynojirimycin (DNJ), and 1-Azafagomine), sugar analogues with inhibitory activity and their derivatives, molecules with inhibitory activity not classified in the last two categories (Ambroxol (ABX) is the main example), and allosteric enhancers for GBA.

In the context of the present invention, a "SATP-modified protein" refers to a protein modified with N-succinimidyl-S-acetylthiopropionate. SATP adds acetylated sulfhydryl groups to the amine groups present in a specific protein. It is herein noted that SATP is a heterobifunctional crosslinker containing an amine-reactive group (N-succinimidyl) and a protected sulfhydryl reactive group (S-acetyl) able to modify the lateral chain of lysine residues by incorporating a protected thiol group. Consequently, SATP-modified proteins can then be conjugated with a polymer forming a disulphide bond, a covalent bond that is responsive to reductive environment, maintaining the conjugate in an oxidizing environment but releasing the protein in a reductive one. It is herein further noted that achieving a conjugation between any protein (including GBA) and a polymer via disulfide bond from modified lysins is possible not only with SATP, but also with any other crosslinker that can modify the lateral chain of lysins and add a sulfhydryl reactive group. According to this chemistry, molecules containing similar reactive headgroups but with different central chain length could be used as an alternative to SATP (the SATA crosslinker is an example of this type of molecule). Other crosslinkers with the ability to add a pyridyl disulphide group into lysine residues, such as SPDP or SMPT, could virtually create the same conjugate with GBA by conjugating GBA with a PGA with thiols added to the lateral chain of glutamic residues (instead of pyridyl disulphide groups). Additionally, lysine lateral chains can be modified with other amine-reactive headgroups such as Sulfo-N-succinimidyl or Imidoesters, among others.

All these potential alternatives to modify GBA, which deviate from SATP-modified GBA, are covered by the present invention. Therefore, the present invention does not only cover GBA SATP-modified proteins but also GBA modified proteins (from hereinafter "GBA-modified proteins" or "GBA-modified protein") comprising lysins having a lateral chain modified with sulfhydryl reactive group. These types of GBA modified proteins can be preferably obtained by using crosslinkers such as the SATA, or crosslinkers with the ability to add a pyridyl disulphide group into lysine residues, such as SPDP or SMPT. Preferably, GBA modified proteins can be GBA SATP-modified proteins or GBA modified proteins with any other crosslinker that can modify the lateral chain of lysins and add a sulfhydryl reactive group. In this sense and according to the present invention, molecules containing similar reactive headgroups but with different spacer arm length could be used as an alternative to SATP such as those selected from the list consisting of SATA, dPEG®4-SATA, dPEG®8-SATA, dPEG®i₂-SATA, dPEG®24-SATA_and_3-Mercaptopropanyl-N-Hydroxysuccinimide. Other molecules that can also modify the lateral chain of lysins but add a pyridyl dithiol reactive group, such as those selected from the list consisting of SMPT, SPDP, LC-SPDP, PEG4- SPDP and PEG12-SPDP could virtually create similar conjugates by conjugating GBA with a PGA with thiols added to the lateral chain of glutamic residues (instead of pyridyl disulphide groups). Additionally, lysine lateral chains can be modified with other amine-reactive headgroups such as Sulfo-N-succinimidyl or Imidoesters, among others, such as those selected from the list consisting of 2-lminothiolane (Traut's reagent) and Sulfo-LC-SPDP. (see table 4)

Schematic reaction of the modification with SATP:

Lysine SATP SATP-modified lysine

Nomenclature IUPAC of SAPT and SATP alternatives thereof

SATP - (2,5-dioxopyrrolidin-l-yl) 3-acetylsulfanylpropanoate

SATA - (2,5-dioxopyrrolidin-l-yl) 2-acetylsulfanylacetate dPEG®4-SATA - (2,5-dioxopyrrolidin-l-yl) 3-[2-[2-[2-(2- acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]propanoate dPEG®8-SATA (2,5-dioxopyrrolidin-l-yl) 3-[2-[2-[2-[2-[2-[2-[2-(2- acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate dPEG®i₂-SATA

(2,5-dioxopyrrolidin-l-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth oxy]ethoxy]propanoate dPEG®24-SATA

(2,5-dioxopyrrolidin-l-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-acetylsulfanylethoxy) ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]et hoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

3-Mercaptopropanyl-N-Hydroxysuccinimide.

(2,5-dioxopyrrolidin-l-yl) 3-sulfanylpropanoate

SMPT

(2,5-dioxopyrrolidin-l-yl) 4-[l-(pyridin-2-yldisulfanyl)ethyl]benzoate

SPDP

(2,5-dioxopyrrolidin-l-yl) 3-(pyridin-2-yldisulfanyl)propanoate

LC-SPDP

(2,5-dioxopyrrolidin-l-yl) 6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate

Sulfo LC-SPDP

2,5-dioxo-l-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid

PEG4-SPDP

(2,5-dioxopyrrolidin-1 -yl) 3 - [2- [2- [2- [2- [3 -(pyrid i n-2- yldisulfanyl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate

PEG12-SPDP

(2,5-dioxopyrrolidin-l-yl) 3-[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]e thoxy]ethoxy]propanoate

2-lminothiolane (Traut's reagent) thiolan-2-imine

In the context of the present invention, the term "molar equivalents" refers to the ratio of the moles of one compound to the moles of another. Once you determine the moles of each compound you can determine the molar equivalents.

In the context of the present invention, the term "L-PGA" refers to linear Polyglutamic acid.

In the context of the present invention, the term "Isofagomine D-Tartrate" refers to a competitive inhibitor of human lysosomal P-glucosidase (Ki = 0.016-0.025 pM; IC50 = 0.06 pM)

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.

In the context of the present invention, the term "room temperature" is the average room temperature that is typically around 20°C.

In the context of the present invention the term "deacetylated" refers to a molecule to which an acetyl group was removed usually by hydrolysis. In the context of the present invention, the term "VCB" refers to Velaglucerase Citrate Buffer, a buffer containing 50 mM of sodium citrate at pH 5.6, 5% w/v of sucrose and 0.01% v/v of Polysorbate 20.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Conventional Enzyme Replacement Therapy (ERT) consists of the direct intravenous administration of a particular enzyme that is deficient or absent in the patient. This therapy is currently available and administered successfully for different 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 spontaneously internalized into the cell and reach the lysosomes where they can restore the enzymatic activity. However, ERT treatment has some relevant drawbacks such as poor stability in circulation of the enzymes (due to protease degradation or renal filtration) (Yu M, Wu J, Shi J & Farokhzad OC (2016) Nanotechnology for protein delivery: Overview and perspectives. Journal of controlled release: official journal of the Controlled Release Society 240, 24-37.), the development of immunological response against the infused protein (Turkia H Ben, Gonzalez DE, Barton NW, Zimran A, Kabra M, Lukina EA, Giraldo P, Kisinovsky I, Bavdekar A, Dridi MF Ben, Gupta N, Kishnani PS, Sureshkumar EK, Wang N, Crombez E, Bhirangi K & Mehta A (2013) Velaglucerase alfa enzyme replacement therapy compared with imiglucerase in patients with Gaucher disease. American Journal of Hematology 88, 179-184.), the impossibility to cross some biological barriers such as the blood-brain barrier (being ineffective for neurological manifestations) and its high cost of production (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.).

Given the current limitations of the ERT to delivery recombinant proteins to the CNS (central nervous system), and being ineffective to treat Parkinson's disease as well as the neurologic manifestations of Gaucher's disease, we herein propose a new therapeutic strategy for Parkinson's and Gaucher's disease based on ERT with glucocerebrosidase (GBA) improved through the nanoconjugation of the GBA enzyme with polypeptide to facilitate the BBB (blood brain barrier) crossing and improve the intracellular delivery to lysosomes. It is noted that this approach is not limited to the intravenous administration but also includes the intranasal administration as a non-invasive route of administration and an alternative to intravenous administration. In recent years, the use of nanotechnological strategies to encapsulate proteins in a drug delivery system (DDS) emerged as a possibility to overcome the mentioned limitations of ERT. DDS offer enhanced stability and in vivo protection, controlled release, and targeted delivery of drugs. Also, the use of DDS dramatically reduces the immunogenicity against drugs (Chen et al., 2016). GBA nanoconjugates are internalized by the cells via endocytosis and sent directly to the lysosome for their degradation, once the nanoconjugates reach the lysosome, the polypeptide coating is degraded in the acidic lysosomal environment and GBA, as a lysosomal protein, recovers its lysosomal function, restoring GBA activity. In addition to the main goal that is the delivery of the GBA protein into the CNS (therapeutic application in PD and GD), in the present invention we also aim to improve the ERT technology currently used to treat the non-neurological symptoms in GD.

For the above purposes, the present invention proposes the nanoconjugation of the GBA, preferably recombinant, enzyme to polypeptide polymers and the subsequent introduction of chemical modifications to improve protein stabilization, plasma protease resistance and BBB crossing. In particular, due to the high instability of the GBA protein and its great ability to lose its enzymatic activity, (GBA half-life in blood plasma ~ 10 minutes) the goal is to protect GBA using Polymer Unmasked-Masked Protein Therapy (PUMPT) conditions and avoid inactivation using molecular chaperones (MC) that improve the GBA stability due to the generation of stable GBA/chaperone nanoconjugates able to be up taken by the cells, delivered to the lysosomes and restore GBA activity with more efficacy than free GBA. The goal is also to functionalize the PGA chains with different molecules and/or biomolecules, to increase the stability and to facilitate the BBB crossing (in case of intravenous administration) or to reach the brain through intranasal administration.

The structure of the ERT delivery system proposed herein is illustrated in figure 1. In particular, as illustrated in figure 1, the proposed GBA nanoconjugates (from herein "GBA nanoconjugates of the invention") should be internalized by the cells by endocytosis and sent directly to the lysosome for their degradation, once the nanoconjugates reach the lysosome, the polypeptide coating should degrade in the acidic lysosomal environment and GBA, as a lysosomal protein, should recovered its lysosomal function, restoring its GBA activity. That is, the GBA nanoconjugates of the invention aimed to achieve the following objectives:

- Protect GBA against enzyme activity loss. The physical barrier provided by the nanoconjugate should shield the protein against harsh conditions encountered during circulation on the bloodstream. A sustained release after internalization in the cells should supply a steady source of active GBA, unharmed throughout its path through the bloodstream. - Overcome the Blood-Brain Barrier (BBB) via surface modification or intranasal administration.

As traditional ERT fails to deliver active GBA to the Central Nervous System (CNS), the neurological manifestations remain untreatable in GD type 2 and 3. Intranasal administration of encapsulated PLGA should overcome the BBB and deliver active GBA into the brain.

To achieve these objectives, and as shown in the examples of the present invention, encapsulation of GBA into PLGA nanocapsules was initially attempted via a Water in Oil in Water (W/O/W) double emulsion method as indicated in example 1. By this method velaglucerase loaded PLGA nanoconjugates were successfully synthesised and characterized. In order to test if the encapsulated protein retained enzyme activity, an in vitro assay was performed ST-Q7 cells (mouse striatal neurons). However, although the Velaglucerase successfully colocalized with LAMP-1 (a lysosome marker indicating that the protein reached its intended destination), as seen in figure 2, no enzyme activity could be detected. In order to resolve this issue, an optimization of the synthesis conditions was attempted to overcome GCase activity loss, with no success. Such optimization is described herein below.

In order to preserve GCase activity, several changes in the phases and synthesis procedure were applied to protect GBA against enzyme activity loss. We classified them according to the affected phase, and a final section with global changes.

- Modifications of W1 phase.

The modifications that affected the W1 phase were mainly changes in the buffer composition. As indicated in the examples, none of the changes resulted in nanoconjugates with active GBA.

- Modifications of W2 phase.

The surfactants that constitute the W2 are responsible for the stabilization of the nanoconjugates during the organic solvent evaporation. PVA was substituted by Polysorbate 80 and Pluronic. None of the changes resulted in nanoconjugates with active GBA.

- Modifications in the emulsification method.

The sonication times and amplitude were tested to get a protocol able to produce nanoconjugates with desired properties but reducing the amount of energy delivered to the samples, which was believed to cause protein unfolding. Shorter times at higher amplitude, longer times at lower amplitude and different conditions of continuous and intermittent sonication were tested.

Preliminary conclusions: None of the changes resulted in nanoconjugates with active GBA and PLGA was discarded as a drug delivery system. GBA was inactivated during the encapsulation process.

In view of the above results, an alternative approach to incorporate Velaglucerase into a nanoparticulate system without a complete activity loss was attempted via 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) developed by Dr Maria J. Vicent (CIPF, Valencia, Spain). PUMPT refers to a soft nanotechnological strategy in which a protein is conjugated to a biodegradable polymer conferring protection and masking its activity during transit, while enabling controlled restore of the activity at the target site due to external stimuli. In this sense, an adaptation of the PUMPT method was tested for Velaglucerase conjugation. Velaglucerase was conjugated with a Poly-Glutamic Acid (PGA) polymer via a reductionsensitive linker, which was intended to mask (and protect) protein activity in oxidizing environments (such as the bloodstream) and release it in reductive ones (such as the lysosomes).

In order to successfully conjugate Velaglucerase with PGA polymer, the conjugation protocol developed by Talelli and Vicent (Talelli and Vicent, 2014), which was optimized for lysozyme, was adapted to Velaglucerase. This conjugation procedure consisted in two main steps: (1) a modification of the surface protein with SATP (N-succinimidyl-S-acetylthiopropionate), which adds a thiol group by modifying the lateral chain of lysines; and (2) the conjugation of the SATP-modified protein with PGA modified with pyridyl dithiol groups (PGA-PD), which attack the thiol groups added onto the lysines to form a disulphide bond.

The conjugation conditions are indicated in example 1.

Although the objective of the PGA conjugation step is to mask the GCase activity, this loss of activity should be completely reversible upon release in a reductive environment. However, the SATP modification step, which irreversibly modifies surface lysine residues of the protein, causes irreversible activity loss. The three initial ratios of SATP tested for Velaglucerase conjugation showed descents in enzyme activity, proportional to SATP concentration. PGA conjugation was successful in completely masking CGase activity but upon release with 10 mM of reduced glutathione (GSH) only one of the conditions recovered GCase activity, and way below the activity levels showed by the SAPT-modified Velaglucerase, which should fully recover. The activity loss during the modification of GBA with SATP and the conjugation of the modified protein with PGA was attributed mainly to the instability of the protein in the conditions of the working buffers used for the conjugation steps. To ameliorate this activity loss, we resorted to use GBA molecular chaperones (MCs) during the synthesis procedure. MCs are small molecules with the capability to bind and stabilize a target enzyme. Usually, these MCs are competitive inhibitors that bind to the catalytic pocket of the enzyme.

In the case of GBA, there are currently two MCs that are commercially available although none of them has thus far reached the drug market as a Pharmacological Chaperone (PC) for GBA. Among them, two of the most studied are Ambroxol hydrocloride and Isofagomine D-Tartarate (IFG). IFG was chosen as the stabilizing agent as it does not contain any primary amine which would compete against lysines in the SATP modification reaction. Furthermore, IFG is a strong chaperone for GBA as it possesses a Ki of 5.8 nM at pH 7.0 implying that little amounts of the MC could be enough to stabilize GCase during conjugation. To test the lack of interference of IFG in the conjugation procedure, a comparative conjugation protocol was performed in presence or absence of the chaperone. The protocol with IFG only differed by adding an excess of IFG (25 pM, almost 1000 times the Ki) to the working buffers to ensure maximal inhibition and protection of the enzyme. Samples from each step of conjugation (SATP modification and PGA conjugation) from both protocols were taken and analysed by non-reducing SDS- PAGE coupled to silver staining. This technique permitted the determination of the molecular weight of the nanoconjugates without breaking the disulphide bonds that bind the polymer with the protein to qualitatively assess the conjugation rate.

Results from non-reducing SDS-PAGE showed no significant differences between conjugation in absence or presence of IFG. SATP modification of Velaglucerase produced the dimerization of little amounts of the enzyme. The presence of the dimerization bands on SATP-modified enzyme in the presence of IFG indirectly indicated the lack of interference at this step. The PGA conjugation with the protein was appreciated by the apparition of a smearing of protein-polymer conjugates, which presented no differences between conditions. Although no differences were observed in terms of conjugation efficiency, IFG addition during the synthesis procedure improved the GCase activity measured at each step of conjugation, demonstrating its capacity to stabilize and protect the enzyme during the conjugation. After SATP modification, no loss of GCase activity was detected in the IFG containing reaction, a fact that was attributed to complete stabilization of the enzyme at this step. The progressive decrease in GCase activity after conjugation in presence of IFG was attributed to the masking effect of the polymer on the enzymatic activity. Therefore, IFG improved the enzyme stability during the conjugation and masking process. However, despite the interesting results obtained thanks to the addition of IFG as a stabilizing agent, the nanoconjugation strategy seemed to be far from being workable. As more than 80% of Velaglucerase remained in an unmodified state after all conjugation procedures. The explanation for this poor conjugation of the enzyme was found later in the buffering conditions used for the SATP modification step. As explained before, the protocol for Velaglucerase conjugation with PGA was adapted from the work of Ta lei I i and Vicent. The critical step to adapt was the modification of the protein with SATP, as contrary to the PGA conjugation step, the SATP modification entails an irreversible loss of activity. This adaptation of the protocol turned out to be ineffective for proper Velaglucerase modification with SATP, obtaining poor conjugation yields. Exploring the chemistry of N-succinimidyl-ester reaction with primary amines (the reaction by which SATP is coupled to the protein), it was shown that this reaction is highly dependent on the pH of the reaction mixture. At neutral pH (pH 7,4), which was the one initially used for SATP modification, the reaction advanced very slowly and needed hours to reach an equilibrium. Nonetheless, the same reaction carried out at a more basic pH (pH 8,5-9) occurred rapidly, reaching equilibrium in minutes. Thiol addition was quantified with a free thiol assay kit after SATP modification and deacetylation of the protected thiols. Results were plotted in a relative ratio between the SATP-modified enzyme and the unmodified Velaglucerase. The free thiol levels doubled after 2 hours of reaction at pH 7.4. In contrast, the reactions performed at pH 8,5 and pH 9 showed a 4-fold increase in the free thiol levels, indicating a higher reactivity of SATP at these conditions.

As mentioned, thiol quantification required the deacetylation of the SATP protected thiols, which implies an extra reaction that could interfere in the measures obtained (for example by disulphide bond formation between protein molecules). To avoid this deacetylation step and taking into account that SATP only reacts with primary amines of Lysine residues, it was decided to analyse the decreasing of the free surface amines from the lysine residues instead. The free lysine residues decrease was quantified with a TNBSA assay. TNBSA reacts with primary amines producing a yellow molecule, quantifiable by absorbance. In the case of a protein, the only primary amine detectable by TNBSA is the one from exposed lysine residues (apart from the N-terminal). Therefore, TNBSA reactivity is directly proportional to the number of lysine residues in the surface of the protein. Results from lysine quantification showed no reduction of the percentage of lysine residues exposed after SATP- modification at pH 7,4, demonstrating the poor reactivity of the molecule at these conditions. However, reactions carried out at pH 8,5 and pH 9 displayed a 25-30% reduction in the lysines exposed, further confirming the huge increase in SATP reactivity at these pH conditions. In terms of GCase activity, no enzymatic activity changes were detected at pH 7.4. This lack of activity loss after SATP modification was previously observed and attributed to total stabilization of the enzyme in presence of IFG. On the other hand, GCase activity after SATP modification at pH 8,5 and pH 9 displayed a reduction of 25% and 30%, respectively. Taking into account the effect of IFG, the only explanation for the GCase activity reduction is the modification of certain essential lysine residues for enzymatic activity.

To this point, the main goal for 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 a statistical analysis while optimizing the synthesis conditions can offer more promising results (Karbasian et al., 2019). An optimization via a Central Composite Design - Response Surface Methodology (CCD-RSM) should help select the values of the independent variables that affect, to a greater extent, the output of SATP modification and GCase activity maintaining (our dependent variables). According to bibliography, the manual of SATP (Thermo Scientific, Ref. 26100) and the present pre-tests the main independent variables that mainly affected SATP modification and maintaining GCase activity were (1) pH, (2) molar ratio of SATP to protein and (3) reaction time. For CCD-RSM optimization, the range of working for independent variables needs to be defined. Again, using the information found in the bibliography, the manual of SATP and the pre-tests the pH range was set from pH 7 to pH 9, the molar ratio of SATP to protein range was set from 5 equivalents to 25 and the reaction time range was set from 30 min to 120 min.

Each numeric factor varied over five levels, calculated by the software Design Expert 11 (DX11): plus and minus alpha (axial points), plus and minus 1 (factorial points) and the centre point. The conditions that simultaneously yielded the most for SATP modification and maintaining of GCase activity were set at pH 9, a SATP to protein molar ratio of 15:1 and a reaction time of 60 minutes. The final protocol for Velaglucerase modification and conjugation with PGA was stablished as follows:

1. SATP modification step: Velaglucerase was prepared at 0.5 mg/ml in PBS pH 9 with 25 pM of IFG. 15 molar equivalents of SATP from a stock concentration of 20 mg/ml in dimethyl sulfoxide (DMSO) were added and incubated for lh at RT under mild shaking. The resulting product was washed 3 times with VCB supplemented with 25 pM IFG using a centrifugal concentration device (cutoff 10 kDa).

2. PGA conjugation step: onto the purified SATP-modified protein already in VCB 0,1 volumes of deacetylation solution (0.5 M of hydroxylamine-HCI in PBS) were added. 0,1 volumes of PGA-PD were added to a final molar ratio PGA/protein of 5:1 and incubated 4 hours at RT. The resulting product was washed 3 times with VCB supplemented with 25 pM IFG using an amicon centrifugal unit (cut-off 30 kDa).

The final SATP modification protocol, integrated in the conjugation procedure proved to successfully improve the GCase activity maintenance while having a high SATP modification rate. As seen in figure 3, there was around a 25% of lysine modification after the optimization with a higher preservation of GCase activity (figure 3 d). The addition of IFG didn't interfere with the conjugation between the SATP- modified protein and the conjugate. Compared with the batches of conjugates prepared before the SATP modification optimization, the conjugation yield was significantly higher as it reached conjugation levels over 90%.

Therefore, the modification of the GBA enzyme with the SATP crosslinker was performed to obtain protected thiol groups at the accessible surface of the enzyme. The optimization and validation of SATP modification was thoroughly detailed above. From those presented results, it derives that the GBA protein was efficiently modified with SATP, obtaining a protein with 25% of the exposed lysins modified with the SATP crosslinker. This result corresponds to 4-5 lysins modified by SATP. The product generated, GBA-SATP, was conjugated with PGA polymer with more than 90% of efficiency, obtaining the final product, NanoGBA, with high yields. Yet, it is herein noted that modifying the lysines, as discussed herein, on the surface of GBA conferred the enzyme with unexpected characteristics, which are herein detailed below.

- Physico-chemical changes at GBA-SATP step and NanoGBA

The GBA protein comprises three structural domains: domain I (residues 1-27 and 383-414), consisting of an antiparallel p-sheet with two disulfide bridges, whose function is thought to be structural; domain II (residues 30-75 and 431-497), which is an immunoglobulin-like structure, usually considered to be an interaction domain; and domain III (residues 76-381 and 416-430), which is the catalytic domain with a TIM barrel structure.

As a protein with 497 amino acids, the modification of 4-5 lysines on the enzyme's surface was initially expected not significantly affect the overall structure. Nonetheless, there is evidence of changes at the three-dimensional structure of the enzyme. In terms of secondary structure, circular dichroism (CD) spectra revel a reorganisation in the p-sheet conformation of the enzyme, indicating a switch from parallel to anti-parallel p-sheet in approximately 5% of the global structure (see example 2). In the case of NanoGBA, CD spectra display a more pronounced change in the secondary structure. In this case, there is an increase in the proportion of a-helix. This result is in accordance with PGA conformational space (PGA can be found as unfolded or as an a-helix) (see example 2).

In addition to this conformational change, there is also a substantial shift in the chromatographic elution time in a Superdex 200 gel filtration column. Specifically, the unmodified GBA enzyme is eluted after 46 minutes, whereas GBA-SATP is eluted at 35 minutes. This reduction in the retention time for GBA-SATP corroborates the unexpected conformational change in GBA after SATP modification. In this case, this change in the elution time supposes not only a change in secondary structure, but also at quaternary structure of the enzyme, strongly suggesting the dimerization of the enzyme after SATP modification. Moreover, NanoGBA in a Superdex 200 is eluted at 32 minutes. This result agrees with the increase in molecular weight due to the addition of PGA polymer to the GBA-SATP product (see example 2).

Biochemical changes derived from the structural changes

The changes demonstrated in GBA-SATP and NanoGBA structures not only confer a distinct globular structure to the protein, but also induces changes in the biochemical properties of the enzyme. Specifically, here we demonstrate a huge change in protein stability due to SATP modification, which is further exploited by the NanoGBA final product.

Firstly, when subjected to in vitro trypsin digestion, the GBA enzyme is susceptible to degradation. In contrast, both the GBA-SATP product and the NanoGBA final product demonstrate complete resistance to trypsin. This result confirms that the resistance to protease degradation is conferred by the SATP modification itself and not by the addition of the PGA polymer.

The enhanced resistance of GBA-SATP is further validated by a human blood plasma (HBP) stability assay. When exposed to HBP, GBA enzyme undergoes rapid inactivation, being totally inactivated after 1 hour in HBP. This leads to the inability of the enzyme to restore glucocerebrosidase activity after internalisation in M17 GBA KO cells after this incubation time. In contrast, notably GBA-SATP is far more resistant to HBP inactivation, maintaining about 35% of its initial capacity even after 24 hours of incubation in HBP. In this regard, NanoGBA demonstrated further improvement in stability. NanoGBA maintains almost its total capacity to restore glucocerebrosidase activity after 24 hours of HBP incubation. The increased stability of the GBA-SATP product and the evolution of this property by the Nano-GBA product signifies a substantial transformation in the behavior of the initial enzyme. This can be shown by the capacity of the different products to restore glucocerebrosidase activity in the M17 GBA KO model over time. As it can be shown in the examples, GBA enzyme restores initially 20% of the WT glucocerebrosidase activity, but this activity rapidly declines, losing this activity after 3 days. In contrast, GBA-SATP initially restores 50% of the WT activity and after 7 days of internalisation assay, it still maintains 25% of the WT activity. This substantial contrast in behavior highlights the transformative impact of the SATP modification on GBA.

Finally, the NanoGBA product exhibits a completely different behaviour from GBA and GBA-SATP products. In this case, NanoGBA restores initially 25% of the WT activity, but in contrast to the other products, NanoGBA activity increases over time, reaching WT activity levels after 24 hours of incubation and maintaining it until at least 7 days. Furthermore, western blot analysis shows that NanoGBA internalisation is higher than GBA or GBA-SATP and increases over time. This effect is exclusively attributed to the PGA polymer conjugation, which not only enhances enzyme stabilization but also augments the capacity for cellular uptake (see example 2).

Therefore, a first aspect of the invention refers to a manufacturing method for modifying the surface of a glucocerebrosidase enzyme with N-succinimidyl-S-acetylthiopropionate (SATP), by modifying the lateral chain of lysines of the protein and providing a SATP-modified protein, which comprises: a. adding an aprotic, polar solvent, miscible with water and able to dissolve SATP, preferably DMSO or DMF , comprising N-succinimidyl-S-acetylthiopropionate (SATP), and optionally a surfactant with an HLB value between 14 and 20 such as polysorbate 20, to an aqueous composition comprising the glucocerebrosidase enzyme protein and a molecular chaperone (MC), wherein the molecular chaperone is preferably a salt of isofagomine, wherein 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, a molar ratio of SATP to glucocerebrosidase enzyme from at least 5 molar equivalents up to 25 molar equivalents and a reaction time range of from at least 30 min, preferably under mild shaking (such as 100 rpm).

It is noted that, as indicated in the DEFINITIONS section, molecules containing similar reactive headgroups but with different spacer arm length could be used as an alternative to SATP such as those selected from the list consisting of SATA, dPEG®4-SATA, dPEG®8-SATA, dPEG®₁₂-SATA, dPEG®24-SATA and 3-Mercaptopropanyl-N-Hydroxysuccinimide. Other molecules that can also modify the lateral chain of lysins but add a pyridyl dithiol reactive group are those selected from the list consisting of SMPT, SPDP, LC-SPDP, PEG4-SPDP and PEG12-SPDP. Additionally, lysine lateral chains can be modified with other amine-reactive headgroups such as Sulfo-N-succinimidyl or Imidoesters, among others, such as those selected from the list consisting of 2-lminothiolane (Traut's reagent) and Sulfo-LC-SPDP. (see table 4). All of these alternatives to SATP are contemplated in the present invention to provide GBA-modified proteins.

Therefore, the first aspect of the invention does not only provide a method for modifying the surface of a glucocerebrosidase enzyme with N-succinimidyl-S-acetylthiopropionate (SATP), by modifying the lateral chain of lysines of the protein and providing a SATP-modified protein, but also for modifying the surface of a glucocerebrosidase enzyme with molecules containing similar reactive headgroups to SATP but with different spacer arm length such as any of the molecules indicated above (see also table 4), by modifying the lateral chain of lysines of the protein and providing a GBA-modified protein,

In preferred embodiment, the pH is from 8.0 to 12, more preferably from 8.5 to 12, more preferably from 9.0 to 12, more preferably from 9.5 to 12. Also preferably from 8.0 to 10, more preferably from 8.5 to 10, more preferably from about 8.5 to about 9.5.

It is noted that the aprotic, polar solvent, miscible with water and able to dissolve SATP, is much preferably DMSO.

It is noted that alternatives useful in the present invention to polysorbate 20 would be Triton X-100 (4- (l,l,3,3-Tetramethylbutyl)-phenyl-polyethylenglykol), Triton X-114 ((1, 1,3,3-

Tetramethylbutyl)phenyl-polyethylenglycol), NP-40 (nonyl phenoxypolyethoxylethanol), Brij-58 (Polyethylenglycolhexadecylether); n-Dodecyl-beta-Maltoside, or Octyl-beta-Glucoside. Preferably polysorbates (such as polysorbate 20), polyethylenes (such as NP-40), or derivatives of folic acid modified sulfobetaine.

In a preferred embodiment of the first aspect, the salt of isofagomine, or alternatively 1- deoxynorjirimycin or 1-Azafagomine, is at a concentration of at least 0.5 pM, preferably from about 0.5 to about 25 pM. In addition, further GBA-specific chaperones, either inhibitory or allosteric, can be used instead of the salt of Isofagomine. Examples of these molecular chaperones are but not limited to alkylated derivatives of isofagomine, l,5-dideoxy-l,5-iminoxylitol (DIX) and their derivatives, Deoxynojirimycin (DNJ) and their derivatives, and Ambroxol (ABX). In another preferred embodiment, the glucocerebrosidase enzyme protein is selected from the list consisting of velaglucerase, imiglucerase or uplyso or any further, preferably recombinant, glucocerebrosidase enzyme.

In another preferred embodiment of the first aspect of the invention or of any preferred embodiments therefrom, the salt of isofagomine is Isofagomine D-Tartrate and said salt of isofagomine is preferably at a concentration of about 25 pM.

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

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

In another preferred embodiment of the first aspect of the invention or of any preferred embodiments therefrom, the SATP-modified protein, GBA SATP-modified protein, or the GBA-modified protein resulting from the reaction of step (a) is isolated, and preferably purified, preferably in a Citrate Buffer. Preferably, the SATP-modified protein, GBA SATP-modified protein, or GBA-modified protein resulting from the reaction of step (a) is isolated, preferably purified, and deacetylated, preferably with a deacetylation solution such as a hydroxylamine-HCI solution (at room temperature for 4 hours).

A second aspect of the invention refers to a method to manufacture a polymer conjugate, wherein the method comprises: b) Adding the polymer to the deacetylated, and preferably purified, SATP-modified protein, GBA SATP-modified protein, or GBA-modified protein resulting from the reaction of step (a) according to the first aspect of the invention wherein the SATP-modified protein, GBA SATP-modified protein, or GBA-modified protein resulting from the reaction of step (a) is isolated, preferably purified, and deacetylated, preferably with a deacetylation solution such as a hydroxylamine-HCI solution (preferably at room temperature for about 4 hours); and wherein the polymer is selected from the group consisting of dextran, a water soluble linear polyamino acid or polypept(o)ide (including a polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosin (PSar)....), a polyethylenglycol (PEG), a polylactic acid (PLA) a polylactic-co-glycolic acid (PLGA), a poly(D,L-lactide-co-glycolide) (PLA/PLGA), a poly(hydroxyalkylmethacrylamide), a polyglycerol, a polyamidoamine (PAM AM), and a polyethylenimine (PEI), polyorthoesters, polyacetals; and c) Optionally washing the resulting product from b), preferably with an acidic buffer such as VCB optionally supplemented with a molecular chaperone such as a salt of isofagomine or any other suitable MC as cited throughout the present specification.

An alternative embodiment of the second aspect of the invention refers to a method to manufacture a polymer conjugate, wherein the method comprises: b. Adding the polymer to a non-deacetylated SATP-modified protein, non-deacetylated GBA SATP-modified protein, or non-deacetylated GBA-modified protein resulting from the reaction of step (a) in accordance with the first aspect of the invention, wherein the polymer is selected from the group consisting of dextran, a water soluble linear polyamino acid or polypept(o)ide (including a polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosin (PSar)....), a polyethylenglycol (PEG), a polylactic acid (PLA) a polylactic-co-glycolic acid (PLGA), a poly(D,L-lactide-co-glycolide) (PLA/PLGA), a poly(hydroxyalkylmethacrylamide), a polyglycerol, a polyamidoamine (PAMAM), and a polyethylenimine (PEI), polyorthoesters, polyacetals; and the GBA SATP-modified protein or GBA-modified protein is concomitantly deacetylated, preferably with a deacetylation solution such as a hydroxylamine-HCI solution; and c. Optionally washing the resulting product from b), preferably with an acidic buffer such as VCB optionally supplemented with a molecular chaperone such as a salt of isofagomine.

In a preferred embodiment of the second aspect of the invention (including its alternative embodiment), 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), wherein 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 from 25 to 150 units Glutamic acid monomers. A third aspect of the invention refers to a method of conjugation of a glucocerebrosidase enzyme to poly-L-glutamic acid (PGA), wherein the method comprises: a. the manufacturing method for modifying the surface protein of the glucocerebrosidase enzyme alfa with N-succinimidyl-S-acetylthiopropionate by modifying the lateral chain of lysines of the protein, according to the first aspect of the invention or according to any of its preferred embodiments including those providing the GBA-modified protein; b. subsequently to step a) deacetylating the product resultant from a); and c. simultaneously or subsequently conjugating a polymer to the product resultant from b) in accordance with any the second aspect of the invention or with any of its preferred embodiments.

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

A fourth aspect refers to a SATP-modified protein, GBA SATP-modified protein, or GBA-modified protein obtained according to the first aspect of the invention or to any of its referred embodiments. In some embodiments, the obtained product presents the SATP-modified protein, GBA SATP-modified protein, or GBA-modified protein in a weight percentage of at least about 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt% or 100 wt%. The term 'wt %' denotes an amount by weight, as a percentage of the total weight of the obtained product.

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

It is noted that in a sixth aspect of the invention, the present invention also provides a composition comprising or consisting of a glucocerebrosidase enzyme modified with N-succinimidyl-S- acetylthiopropionate (SATP), by modifying the lateral chain of lysines of the protein and providing a GBA SATP-modified protein, and/or a glucocerebrosidase enzyme modified with molecules containing similar reactive headgroups to SATP but with different spacer arm length such as any of the molecules indicated in table 4, by modifying the lateral chain of lysines of the protein and providing 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 further, preferably recombinant, glucocerebrosidase enzyme. It is further noted that as indicated above, molecules containing similar reactive headgroups but with different spacer arm length could be used as an alternative to SATP such as those selected from the list consisting of SATA, dPEG®4-SATA, dPEG®8-SATA, dPEG®₁₂-SATA, dPEG®24-SATA and 3-Mercaptopropanyl-N-Hydroxysuccinimide. Other molecules that can also modify the lateral chain of lysins but add a pyridyl dithiol reactive group are those selected from the list consisting of SMPT, SPDP, LC-SPDP, PEG4-SPDP and PEG12-SPDP. Additionally, lysine lateral chains can be modified with other amine-reactive headgroups such as Sulfo-N-succinimidyl or Imidoesters, among others, such as those selected from the list consisting of 2-lminothiolane (Traut's reagent) and Sulfo- LC-SPDP (see table 4). All of these alternatives to SATP are contemplated in the present invention to provide GBA-modified proteins.

A further aspect of the invention (a seventh aspect of the invention) refers to a composition comprising or consisting of a polymer conjugate of the glucocerebrosidase enzyme modified with N-succinimidyl- S-acetylthiopropionate (SATP) of the sixth aspect of the invention, or a composition comprising or consisting of a polymer conjugate of the glucocerebrosidase enzyme modified protein (GBA-modified protein) of the sixth aspect of the invention. It is noted that the polymer conjugated to the protein can be preferably selected from the group consisting of dextran, a water soluble linear polyamino acid or polypept(o)ide (including a polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosin ( PSar)....), a polyethylenglycol (PEG), a polylactic acid (PLA) a polylactic-co-glycolic acid (PLGA), a poly(D,L-lactide- co-glycolide) (PLA/PLGA), a poly(hydroxyalkylmethacrylamide), a polyglycerol, a polyamidoamine (PAM AM), and a polyethylenimine (PEI), polyorthoesters, polyacetals. 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), wherein 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 from 25 to 150 units Glutamic acid monomers. It is noted that the polymer conjugates of the seventh aspect of the invention are produced as indicated in the second and/or third aspect of the invention.

In some embodiments, in any of the compositions of the sixth or seventh aspects of the invention the modified glucocerebrosidase enzyme or the protein conjugate is present in a weight percentage of at least about 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt% or 100 wt%.

The term 'wt %' denotes an amount by weight, as a percentage of the total weight of the composition.

An eight aspect of the invention refers to a pharmaceutical composition comprising the polymer conjugate of the fifth aspect or seventh aspect, optionally further comprising excipients, vehicle, permeation enhancers and/or adjuvants.

A ninth aspect of the invention refers to the polymer conjugate of the fifth or seventh aspect or the pharmaceutical composition of the eight aspect, for use in therapy. Preferably for use in a method for treating a subject with Gaucher disease and/or Parkinson's disease. More preferably, wherein the glucocerebrosidase enzyme of the pharmaceutical composition or of the polymer conjugate is selected from velaglucerase, imiglucerase or uplyso or any recombinant glucocerebrosidase enzyme.

The following example is merely for illustrative purposed and does not limit the present invention.

EXAMPLE

Example 1. Development of a nanotechnological based Enzyme Replacement Therapy for Parkinson's and Gaucher's diseases.

Two different types of nanoformulations or nanoconjugates had been tested: encapsulation of GBA with PLGA and conjugation of GBA with PGA, both approached with several methodological lines and formulations.

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 those nanoconjugates affect the characteristics of the final capsules, such as size, polydispersity, surface potential (^) and release time among others, making them highly tuneable. Encapsulation of drugs in nanoparticulate systems allow a sustained and controlled release of the payload, which in a clinical level can translate in a reduction of the administration frequency, a reduction of the dosage and a minimisation of the drug fluctuation; thus increasing the efficacy of treatments while reducing their side effects. PLGA nanocapsules, as well as other nanoconjugates, can undergo surface modifications which allow the crossing of biological barriers and a targeted delivery of encapsulated drugs. The main objective of GBA encapsulation in PLGA nanocapsules was to overcome the main limitations of traditional ERT. Those were: a) Protect GBA (Velaglucerase) against enzyme activity loss. The physical barrier provided by the nanocapsule shields the protein against harsh conditions encountered during circulation on the bloodstream. b) Increase the cellular uptake and GBA intracellular release in a control manner. The size and morphology of the resulting nanocapsules induce an enhanced cellular uptake. A sustained release after internalization in the cells should supply a steady source of active GBA, unharmed throughout its path through the bloodstream. c) Overcome the Blood-Brain Barrier (BBB) via surface modification or intranasal administration. As traditional ERT fails to deliver active GBA to the Central Nervous System (CNS), the neurological manifestations remain untreatable in GD type 2 and 3. Intranasal administration of encapsulated PLGA should overcome the BBB and deliver active GBA into the brain.

In order to achieve the above objectives, encapsulation of Velaglucerase, a type of GBA, into PLGA nanocapsules was attempted as described in the sections below.

1.1. Initial conditions.

The procedure was a Water in Oil in Water (W/O/W) double emulsion method, with the following stages:

Wl: internal aqueous phase. Buffer: 50 mM citric/ sodium citrate at pH 5.6 with 5% sucrose and 0.01% Polysorbate 20. Velaglucerase was dissolved at 0,5 mg/ml.

O: organic phase. 25 mg of PLGA were dissolved in 750 pl of dichloromethane (DCM) and 4% v/v of Span 80 were added as a surfactant.

W2: external aqueous phase. 2% PVA and 1 M NaCI in water.

The synthesis steps were the following:

1. Preparation of the O phase: The PLGA was dissolved into the DCM under magnetic stirring. Sorbitan monooleate was added upon complete dissolution of the PLGA. 2. The W1 phase was added over the O phase and sonicated for 1 minute at 25% amplitude using a microtip in a Branson Sonifier SFX 550.

3. The emulsion was poured onto the W2 phase and sonicated in the same conditions from the step 2.

4. The final emulsion was left at room temperature under magnetic stirring until complete evaporation of the dichloromethane.

Velaglucerase loaded PLGA nanoparticles or nanoconjugates were successfully synthesised and characterized. SEM images of loaded and unloaded nanoconjugates are shown in figure 4. In order to test if the encapsulated protein retained enzyme activity, an in vitro assay was performed ST-Q7 cells (mouse striatal neurons). Although the Velaglucerase successfully colocalized with LAMP1 (a lysosome marker indicating that the protein reached its intended destination), as seen in figure 2, no enzyme activity could be detected.

An optimization of the synthesis conditions was attempted in order to overcome GCase activity loss, with no success.

1.2. Optimization attempts to preserve GCase activity.

Several changes in the phases and synthesis procedure were applied in order to protect GBA against enzyme activity loss. In the following sections, we classified them according to the affected phase, and a final section with global changes.

1.2.1. Modifications of W1 phase.

The modifications that affected the W1 phase were mainly changes in the buffer composition. In order to stabilize de protein and protect it against activity loss, attributed to denaturalization due to contact with an organic solvent and due to sonication. The changes were, individually:

1. Addition of different types of polyethylene glycol.

2. Change in the concentration of the citric/citrate buffer.

None of the changes resulted in nanoconjugates with active GBA.

1.2.2. Modifications of O phase. The modifications that affected the O phase were focussed on the reduction of the hydrophobicity of the phase, in order minimize the unfolding of GBA during the homogenization step. The PLGA polymer was partially substituted by a PLGA-PEG polymer and several ratios were assayed. Ethyl acetate was tested as an alternative of dichloromethane.

None of the changes resulted in nanoconjugates with active GBA.

1.2.3. Modifications of W2 phase.

The surfactants that constitute the W2 are responsible for the stabilization of the nanoparticles during the organic solvent evaporation. PVA was substituted by Polysorbate 80 and Pluronic.

None of the changes resulted in nanoconjugates with active GBA.

1.2.4. Modifications in the emulsification method.

The sonication times and amplitude were tested in order to get a protocol able to produce nanoparticles with desired properties but reducing the amount of energy delivered to the samples, which was believed to cause protein unfolding. Shorter times at higher amplitude, longer times at lower amplitude and different conditions of continuous and intermittent sonication were tested.

Conclusions: None of the changes resulted in nanoparticles with active GBA and PLGA was subsequently discarded as a drug delivery system.

2. Conjugation of GBA with PGA polymer.

An alternative approach to incorporate Velaglucerase into a nanoparticulate system without a complete activity loss was attempted via 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.) developed by Dr Maria J. Vicent (CIPF, Valencia, Spain). PUMPT refers to a soft nanotechnological strategy in which a protein is conjugated to a biodegradable polymer conferring protection and masking its activity during transit, while enabling controlled restore of the activity at the target site due to external stimuli. In this invention, an adaptation of the PUMPT method was tested for Velaglucerase conjugation. Velaglucerase was conjugated with a Poly-Glutamic Acid (PGA) polymer via a reduction-sensitive linker, which was intended to mask (and protect) protein activity in oxidizing environments (such as the bloodstream) and release it in reductive ones (such as the lysosomes). a. Initial attempts using the Talleli et al., 2014 protocol.

In order to successfully conjugate Velaglucerase with PGA polymer, the conjugation protocol developed by Talelli and Vicent (Talelli and Vicent, 2014), which was optimized for lysozyme, was adapted to Velaglucerase. This conjugation procedure consisted in two main steps: (1) a modification of the surface protein with SATP, which adds a thiol group by modifying the lateral chain of lysins; and (2) the conjugation of the SATP-modified protein with PGA modified with pyridyl dithiol groups (PGA- PD), which attack the thiol groups added onto the lysins to form a disulphide bond.

The conjugation conditions of the Talelli and Vicent protocol were the following:

SATP modification step: Lysozyme was dissolved in PBS at 1 mg/ml. SATP in a stock concentration of 2 mg/ml in dimethyl sulfoxide (DMSO) was added and incubated for lh at RT under mild shaking. The resulting product was washed 3 times with PBS using an amicon centrifugal unit (cut-off 3 kDa). Two molar ratios of SATP/Lysozyme were tested for this step, 10:1 and 6:1, but was the 10:1 ratio which showed a higher modification rate and thus was selected for the next step.

PGA conjugation step: the buffer of the purified SATP-modified protein (modified in a SATP/Lysozyme ratio of 10:1) was changed to ammonium acetate 300 mM pH 5 and 0.2 volumes of deacetylation solution (0.5 M of hydroxylamine-HCI in PBS) were added, to prepare the thiol groups for the conjugation. PGA-PD was added and incubated overnight at RT. The resulting product was washed 3 times with PBS using an amicon centrifugal unit. Three PGA- PD/Lysozyme ratios were assayed, 5:1, 2.5:1 and 0.5:1.

Several modifications had to be done in order to adapt this protocol for its use with Velaglucerase. Those changes and its rationale are listed below:

Velaglucerase initial concentration was changed from 1 mg/ml to 0.5 mg/ml. Our original stock of Velaglucerase was 2.5 mg/ml and higher concentrations tended to cause protein aggregation. The washing steps with amicon devices used during the SATP modification and the conjugation steps can concentrate the protein up to 5-fold the working concentration. In order to keep the protein always under 2.5 mg/ml, the working concentration was decreased to 0.5 mg/ml.

• The concentration of the SATP stock was increased from 2 mg/ml to 20 mg/ml in order to decrease the concentration of DMSO during the reaction, which can cause the Velaglucerase to unfold and aggregate. Ratios SATP/Lysozyme tested for the lysozyme modification were chosen due to the number of lysines present in the lysozyme, which are 6. A SATP/Lysozyme 6:1 molar ratio was tested because it meant it was a SATP/Lysine 1:1 ratio. The SATP/Lysozyme 10:1 ratio was to ensure an excess of SATP compared to the lysine residues. As Velaglucerase has 22 lysine residues instead of 6, 3 ratios were tested: 10:1 (to compare it with the 10:1 ratio in the lysozyme protocol) a 22:1 (to have a SATP/lysine 1:1 ratio) and 40:1 (to have an excess of SATP).

The ammonium acetate 300 mM pH 5 buffer was substituted by the original buffer of Velaglucerase (Velaglucerase Citrate Buffer or VCB, 50 mM citrate pH 5.6, 5% sucrose and 0.01% v/v of Polysorbate 20) after checking that it didn't interfere with the conjugation procedure. This change was to improve Velaglucerase stability during the conjugation.

Although the objective of the PGA conjugation step is to mask the GCase activity, this loss of activity should be completely reversible upon release in a reductive environment. However, the SATP modification step, which irreversibly modifies surface lysine residue of the protein, causes irreversible activity loss. It is thus a critical step to optimize in order to guarantee a maximum rate of modification with a minimum loss of enzyme activity. The three initial ratios of SATP tested for Velaglucerase conjugation showed descents in enzyme activity, proportional to SATP concentration (figure 5). PGA conjugation was successful in completely masking CGase activity but upon release with 10 mM of reduced glutathione (GSH) only one of the conditions recovered GCase activity, and way below the activity levels showed by the SAPT-modified Velaglucerase, which should fully recover. b. Stabilization of GBA with molecular chaperones.

The activity loss during the modification of GBA with SATP and the conjugation of the modified protein with PGA was attributed mainly to the instability of the protein in the Sorbitan monooleate conditions of the working buffers used for the conjugation steps. To ameliorate this activity loss, we resorted to use GBA molecular chaperones (MCs) during the synthesis procedure. MCs are small molecules with the capability to bind and stabilize a target enzyme. Usually, these MCs are competitive inhibitors that bind to the catalytic pocket of the enzyme.

In the case of GBA, there are currently many MCs that are commercially available although none of them has reached the drug market as a Pharmacological Chaperone (PC) for GBA. Among them, two of the most studied are Ambroxol hydrocloride and Isofagomine D-Tartarate (IFG). For our purpose, Ambroxol was discarded due to the presence of a primary amine in its structure, which interfered with the SATP modification competing against the lysine residues (figure 6). IFG was chosen as the stabilizing agent as it does not contain any primary amine. Furthermore, IFG is a strong chaperone for GBA as it possesses a Ki of 5.8 nM at pH 7.0 implying that little amounts of the MC could be enough to stabilize GCase during conjugation.

To test the lack of interference of IFG in the conjugation procedure, a comparative conjugation protocol was performed in presence or absence of the chaperone. The protocol with IFG only differed by adding an excess of IFG (25 pM, almost 1000 times the Ki) to the working buffers to ensure maximal inhibition and protection of the enzyme. Samples from each step of conjugation (SATP modification and PGA conjugation) from both protocols were taken and analysed by non-reducing SDS-PAGE coupled to silver staining (figure 7). This technique allowed us to see the molecular weight of the nanoconjugates without breaking the disulphide bonds that bind the polymer with the protein to qualitatively assess the conjugation rate.

Results from non-reducing SDS-PAGE showed no significant differences between conjugation in absence or presence of IFG (figure 7 and Figure 3C). SATP modification of Velaglucerase produced the dimerization of little amounts of the enzyme. The presence of the dimerization bands on SATP- modified enzyme in the presence of IFG indirectly indicated the lack of interference at this step. The PGA conjugation with the protein was appreciated by the apparition of a smearing of protein-polymer conjugates, which presented no differences between conditions. Although no differences were observed in terms of conjugation efficiency, IFG addition during the synthesis procedure improved the GCase activity measured at each step of conjugation, demonstrating its capacity to stabilize and protect the enzyme during the conjugation. After SATP modification, no loss of GCase activity was detected in the IFG containing reaction, a fact that was attributed to complete stabilization of the enzyme at this step. The progressive decrease in GCase activity after conjugation in presence of IFG was attributed to the masking effect of the polymer on the enzymatic activity. Conclusions: IFG improved the enzyme stability during the conjugation and masking process. In the new modified protocols, all buffers used for GBA nanoconjugation include 25 pM of IFG. c. Optimization of SATP-modification

Despite the interesting results obtained thanks to the addition of IFG as a stabilizing agent, the nanoconjugation strategy seemed to be far from being optimized. As seen in figure 7, more than 80% of Velaglucerase remained in an unmodified state after all conjugation procedures. The explanation for this poor conjugation of the enzyme was found later in the buffering conditions used for the SATP modification step

As explained in section 2a), the protocol for Velaglucerase conjugation with PGA was adapted from the work of Talelli and Vicent. The critical step to adapt was the modification of the protein with SATP, as contrary to the PGA conjugation step, the SATP modification entails an irreversible loss of activity. As seen in section 2b, this adaptation of the protocol turned out to be ineffective for proper Velaglucerase modification with SATP, obtaining poor conjugation yields. Exploring the chemistry of N-succinimidyl- ester reaction with primary amines (the reaction by which SATP is coupled to the protein), it was shown that this reaction is highly dependent on the pH of the reaction mixture. At neutral pH (pH 7,4), which was the one initially used for SATP modification, the reaction advances very slow and needs hours to reach an equilibrium. Nonetheless, the same reaction carried out at a more basic pH (pH 8,5-9) occurs rapidly, reaching equilibrium in minutes. Taking this information into account, the SATP modification step buffering conditions were modified to react at pH 8,5 or pH 9. SATP modification efficiency was evaluated in comparison to pH 7,4 in terms of relative thiol addition, relative lysine reduction and relative GCase activity maintenance.

Thiol addition was quantified with a free thiol assay kit after SATP modification and deacetylation of the protected thiols. Results were plotted in a relative ratio between the SATP-modified enzyme and the unmodified Velaglucerase. As shown in figure 9a, the free thiol levels doubled after 2 hours of reaction at pH 7,4. In contrast, the reactions performed at pH 8,5 and pH 9 showed a 4-fold increase in the free thiol levels, indicating a higher reactivity of SATP at these conditions.

As mentioned, thiol quantification required the deacetylation of the SATP protected thiols, which implies an extra reaction that could interfere in the measures obtained (for example by disulphide bond formation between protein molecules). To avoid this deacetylation step and taking in account that SATP only reacts with primary amines of Lysine residues, it was decided to analyse the decreasing of the free surface amines from the lysine residues instead. The free lysine residues decrease was quantified with a TNBSA assay. TNBSA reacts with primary amines producing a yellow molecule, quantifiable by absorbance. In the case of a protein, the only primary amine detectable by TNBSA is the one from exposed lysine residues (apart from the N-terminal). Therefore, TNBSA reactivity is directly proportional to the number of lysine residues in the surface of the protein.

Results from lysine quantification showed no reduction of the percentage of lysine residues exposed after SATP-modification at pH 7,4, demonstrating the poor reactivity of the molecule at these conditions (figure 9b). However, reactions carried out at pH 8,5 and pH 9 displayed a 25-30% reduction in the lysines exposed, further confirming the huge increase in SATP reactivity at these pH conditions.

In terms of GCase activity, no enzymatic activity changes were detected at pH 7,4 (figure 9c). This lack of activity loss after SATP modification was previously observed in section 2b and attributed to total stabilization of the enzyme in presence of IFG. On the other hand, GCase activity after SATP modification at pH 8,5 and pH 9 displayed a reduction of 25% and 30%, respectively. Taking into account the effect of IFG, the only explanation for the GCase activity reduction is the modification of certain essential lysine residues for enzymatic activity.

This consequence of SATP modification was predicted earlier but assumed as an acceptable loss of the procedure that cannot be avoided, as SATP modification is a stochastic process that can modify any exposed lysine residue in the protein's surface. d. Determination of the SATP modification step's parameters via CCD-RSM optimization.

To this point, the main goal for the SATP modification procedure was to simultaneously optimize the yield of SATP modification and the maintaining of GCase activity. Considering these two parameters together in a statistical analysis while optimizing the synthesis conditions can offer more promising results. An optimization via a Central Composite Design - Response Surface Methodology (CCD-RSM) should help select the values of the independent variables that affect, to a greater extent, the output of SATP modification and GCase activity maintaining (our dependent variables).

According to bibliography, the manual of SATP (Thermo Scientific, Ref. 26100) and our pre-tests the main independent variables that mainly affected SATP modification and maintaining GCase activity were (1) pH, (2) molar ratio of SATP to protein and (3) reaction time. For CCD-RSM optimization, the range of working for independent variables needs to be defined. Again using the information found in the bibliography, the manual of SATP and our pre-tests the pH range was set from pH 7 to pH 9, the molar ratio of SATP to protein range was set from 5 equivalents to 25 and the reaction time range was set from 30 min to 120 min.

Each numeric factor varied over five levels, calculated by the software Design Expert 11 (DX11): plus and minus alpha (axial points), plus and minus 1 (factorial points) and the centre point. The values for each factor are listed in Table 1.

Table 1. Numeric factors and their levels used for the experimental design of the CCD-RSM optimization.

The advantage of this statistical method is that instead of testing all the possible combinations of these conditions, the software randomised 16 different experimental conditions. The SATP modification step was performed 16 times, one for each of the 16 conditions of pH, molar ratio of SATP to protein and reaction time that the software calculates and measurements for the yield of modification and the maintaining of the GCase activity were made. The results were introduced in the software and analysed via analysis of variance (ANOVA). The conditions that simultaneously yielded the most for SATP modification and maintaining of GCase activity were set at pH 9, a SATP to protein molar ratio of 15:1 and a reaction time of 60 minutes. The final protocol for Velaglucerase modification and conjugation with PGA was stablished as follows:

• SATP modification step: Velaglucerase was prepared at 0.5 mg/ml in PBS pH 9 with 25 pm of IFG. 15 molar equivalents of SATP from a stock concentration of 20 mg/ml in DMSO were added and incubated for lh at RT under mild shaking. The resulting product was washed 3 times with VCB supplemented with 25 pM IFG using an amicon centrifugal unit (cutoff 10 kDa).

• PGA conjugation step: onto the purified SATP-modified protein already in VCB 0,1 volumes of deacetylation solution (0.5 M of hydroxylamine-HCI in PBS) were added. 0,1 volumes of PGA- PD were added to a final molar ratio PGA/protein of 5:1 and incubated 4 hours at RT. The resulting product was washed 3 times with VCB supplemented with 25 pM IFG using an amicon centrifugal unit (cut-off 30 kDa). Conclusions: The final SATP modification protocol, integrated in the conjugation procedure proved to successfully improve the GCase activity maintenance while having a high SATP modification rate. There was around a 25% of lysine modification after the optimization with a higher preservation of GCase activity. The addition of IFG didn't interfere with the conjugation between the SATP-modified protein and the conjugate. Compared with the batches of conjugates prepared before the SATP modification optimization, the conjugation yield was significantly higher as it reached conjugation levels over 90%.

3. In vitro validation of PG A-GBA/IFG nanoconjugate.

Velaglucerase-PGA-IFG conjugates (NanoGBA, as produced in sections 1 and 2 above) were validated in vitro in a neuronal model; BE(2)M17 cells (wild type and GBA-knock out strains) were used to study the delivery of GBA and the therapeutic effect.

We have demonstrated that NanoGBA was able to:

• Increase the stability of the GBA protein (in human blood plasma);

• Is internalized into the cell;

• Is delivered to lysosomes as an active enzyme;

• Presents a long-term recovery of GBA activity (7 days);

• Is able to reduce the accumulation of GlcSph substrate;

• Is able to reduce the accumulation of neurotoxic forms of alpha-synuclein protein (hallmark of neurodegeneration in Parkinson's disease).

Conclusions: GBA-PGA-IFG conjugates are delivered to lysosomes and are able to reduce the accumulation of GlcSph substrate based on a long-term recovery of GBA activity.

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

Mouse model: In vivo validation was tested in two mouse models: GBA-WT and GBA-D409V (presents >90% loss of GCase activity in all tissues and accumulation of substrates). • NanoGBA: in addition to the nanoGBA, a new formulation of the nanoconjugate with fluorescent PGA (Cy5.5-PGA) was generated in order to study the in vivo biodistribution after the administration by IVIS (Spectrum In Vivo Imaging System) analysis.

• Administration: o Intravenous: validate systemic and CNS activity; o Intranasal: validate CNS delivery. The intranasal administration was improved with a crosslinked hyaluronic acid-based hydrogel named HA-CP® crosspolymer (Patent EP17382498. W02019020344A1)

Table 2. Summary PGA-GBA validation in vitro. a. Intravenous administration:

Intravenous administration showed biodistribution in serum, liver, blood marrow, spleen, kidney and lungs. The nanoconjugate is active and increased of GCase activity with a peak at 30min in serum and 3h in liver, blood marrow and spleen. b. Intranasal administration:

Intranasal administration of nanoGBA showed a lack of delivery to the CNS (tested by IVIS and increase GCase activity) c. Intranasal administration with HA-CP® crosspolymer: Intranasal administration was improved using as adjuvant HA-CP® (nurturing® crosspolymer commercially available), formed by three biodegradables, biocompatible and active ingredients (hyaluronic acid, polyglutamic acid and lysine; Dr. Vicent patent co-inventor) to improve the delivery of drugs to the CNS by intranasal administration.

The intranasal administration of nanoGBA with HA-CP® is able to deliver GBA protein into brain (figure 13 left). In the olfactory bulb an increase in GCase activity is observed in GBA-D409V mutant animals and the gain of GCase activity is translated into a decrease in the accumulation of GBA substrates GlcCer and GlcSph (figure 13 right). The analysis of other brain regions is currently in progress.

Conclusions:

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

• Delivery to the CNS is effective by intranasal administration with HA-CP® crosspolymer, the nanoconjugate is detected, and GBA enzyme is active and reduces the accumulation of substrates in olfactory bulb, analysis of other brain regions is in progress.

Example 2

To assess the new properties of the SATP modified GBA and NanoGBA several assays have been performed, including circular dichroism, size exclusion chromatography, stability against tryptic digestion and human blood plasma and internalisation in a cellular model.

Experiment 1: Circular Dichroism

Objective: To compare the secondary structure of GBA, GBA-SATP and NanoGBA.

Methodology: GBA, GBA-SATP and NanoGBA were diluted in Circular Dichroism Buffer (10 mM Acetate, 0.01 % Tween-20) and their spectrum was recorded from 190 to 250 nm in a 1 mm pathlength quartz cuvette. Secondary structure analysis was performed with the free online software "BeStSel", obtaining the percentage of a-Helix, p-sheet and unfolded (unrecognised) conformations.

Results (see figure 14): NanoGBA is far different from GBA and GBA-SATP, mainly due to the increased alpha helix content in NanoGBA (19 %) with respect to GBA (14.6 %) and GBA-SATP (14.8 %). In contrast, GBA-SATP do not differs with GBA on the a-helix or p-sheet total amounts but on the p-sheet types (parallel and antiparallel), being up to 5 % more abundant the antiparallel on the GBA-SATP.

Table 3: Predicted secondary structure for GBA, GBA-SATP and NanoGBA. Data analysed by BeStSel software from 190 to 250 nn. Results presented as percentage of total structural data.

Conclusions: There are differences in the secondary structure of all 3 products. NanoGBA shows an increased presence of alpha helix, which is consistent with the possible conformations of PGA-PD. Moreover, its structure is also different from the unconjugated mixture of GBA and PGA-PD, showing that the confirmation that NanoGBA adopts once conjugated is only due to the presence of the polymer.

In addition, differences are also found between GBA and GBA-SATP, meaning that the thiol protected groups on several aminoacids can cause a conformational change on the global structure of GBA.

Experiment 2: Size exclusion chromatography

Objective: To verify that our products structurally differ from GBA.

Methodology: 75 pg of GBA, GBA-SATP and NanoGBA in Superdex 200 buffer (10 mM MES, 100 mM NaCI, pH 6) were applied to a Superdex 200 column and eluted under the same conditions in order to assess their retention.

Results (see figure 15): GBA is eluted after 46 minutes; GBA-SATP, after 35; and NanoGBA after 32.

Conclusions: The distinct behaviors observed in all three products during size exclusion chromatography indicate notable structural differences among them, resulting in reduced retention times.

Experiment 3: Limited Proteolysis

Objective: To assess the protection against tryptic digestion of the different products (GBA, GBA-SATP, NanoGBA)

Methodology: 50 pg GBA, GBA-SATP and NanoGBA were incubated at 37°C with 5pg trypsin during 0, 30, 60 and 90 minutes. After that, the reaction was stopped, adding a denaturing buffer and heating the samples at 95°C for 10 minutes. The digestion was analysed through a PAGE followed by total protein staining (coomassie).

Results (see figure 16): GBA is sensitive to trypsin digestion in a time-dependent manner leaving only 25 % of the initial amount undigested after 90 minutes. In contrast, both GBA-SATP and NanoGBA are totally resistant to the effect of trypsin.

Conclusions: GBA-SATP confers to the GBA protection against tryptic digestion, which is further maintained on the final nanoconjugated.

Experiment 4: Plasma Stability assay

Objective: To assess the stability of the different products in human blood plasma (HBP). In order to evaluate the capacity of the different products to recover glucocerebrosidase activity inside the cells after being incubated with human blood plasma, GBA, GBA-SATP and NanoGBA were incubated in HBP for 0, 1, 3, 6 and 24 hours before treating M17 GBA KO cells. These cells were treated during 3 hours with 5ug/ml of the different products (GBA, GBA-SATP, NanoGBA) priorly incubated in HBP. After that, cell lysates were collected, and the internalised protein was assessed by Activity assay and Western Blot.

Results (see f ii : GBA was near to total inactivation upon incubation of 1 hour or more in HBP, leading only up to 5 % of WT glucocerebrosidase activity recovered of the treated cells. Instead, GBA- SATP and NanoGBA showed a high stability, leading to treatments 10 times more effective in recovering the activity. Regarding GBA-SATP, after being incubated 24 hours in HBP it maintains a third of its initial activity and in the case of NanoGBA up to an 83 % of the initial activity is preserved.

Conclusions Although the HBP incubation does not seem to differently affect the internalisation capacity of our products (total GBA internalisation, determined by Western Blot), differences are found in their ability to restore GCase activity once internalised, thus showing an increase in the stability in plasma conferred by our modifications. Both GBA-SATP and NanoGBA are far more stable in HBP than GBA, being the latter even more stable specially after longer incubations. in M17 GBA KO cells he capacity of the different products to be internalised along time in a cellular BA KO cells were treated with 5ug/ml of the different products (GBA, GBA-SATP,

NanoGBA) during 1, 3, 6, 24, 72 and 168 hours. After that, cell lysates were collected, and the internalised protein was assessed by Activity assay and Western Blot.

Results (see fi: : Despite its internalisation, after 1 hour of "Naked" GBA treatment only recovers up to 20% of the WT glucocerebrosidase activity. Moreover, after this time, glucocerebrosidase activity inside the cells decreases over time reaching negligible values after 3-7 days. In contrast, after 1 hour of GBA-SATP treatment, cells reach 50% of the WT activity, which is maintained over time until 7 days, when the activity drops to 25%. In the case of NanoGBA, a clear increase over time is observed, reaching levels of 150% after 3 days and even showing more activity than the WT after 7 days of treatment.

Conclusions: The nternalisation of the three products is clearly different. SATP modification of GBA confers the protein a major stabilisation over time in the cell culture media, which results in a major activity recovery over time, being in the order of 2-fold more effective. Furthermore, the nanoconjugation enhances even more this stabilisation and internalisation capacity, showing the best results. Table 4 of commercially available crosslinkers that can emulate SATP function:

Nomenclature IUPAC

SATP - (2,5-dioxopyrrolidin-l-yl) 3-acetylsulfanylpropanoate

3-Mercaptopropanyl-N-Hydroxysuccinimide. (2,5-dioxopyrrolidin-l-yl) 3-sulfanylpropanoate

SMPT

(2,5-dioxopyrrolidin-l-yl) 4-[l-(pyridin-2-yldisulfanyl)ethyl]benzoate

SPDP

(2,5-dioxopyrrolidin-l-yl) 3-(pyridin-2-yldisulfanyl)propanoate

LC-SPDP

Sulfo LC-SPDP

PEG4-SPDP

PEG12-SPDP

2-lminothiolane (Traut's reagent) thiolan-2-imine

Claims

1. A manufacturing method for modifying the surface of a glucocerebrosidase enzyme with N- succinimidyl-S-acetylthiopropionate (SATP), by modifying the lateral chain of lysines of the protein and providing a SATP-modified protein, which comprises: a. adding a polar, aprotic solvent, miscible with water and able to dissolve SATP comprising N-succinimidyl-S-acetylthiopropionate (SATP), and optionally a surfactant with an HLB value between 14 and 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 greater than 8, at a molar ratio of SATP to glucocerebrosidase enzyme from at least 5 molar equivalents up to 25 molar equivalents and for reaction time in the range of from at least 30 min.

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

3. The manufacturing method according to claim 1, wherein the MC is the salt of Isofagomine D- Tartrate.

4. The manufacturing method according to any of claims 1 to 3, wherein the reaction of step (a) is carried out at a molar ratio of SATP to glucocerebrosidase enzyme from 5 molar equivalents to 25 molar equivalents and a reaction time range set from 30 min to 120 min.

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

6. The manufacturing method according to any of claims 1 to 5, wherein the SATP-modified protein resulting from the reaction of step (a) is isolated, and preferably purified, preferably in a Citrate Buffer.

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

8. A method to manufacture a polymer conjugate, wherein the method comprises: b) Adding a polymer to a deacetylated, and preferably purified, SATP-modified protein, , wherein the modified protein is a glucocerebrosidase enzyme, resulting from the reaction of step (a) according to claim 7, wherein the polymer is selected from the group consisting of dextran, a water soluble linear polyamino acid or polypept(o)ide (including a polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosin (PSar)....), a polyethylenglycol (PEG), a polylactic acid (PLA) a polylactic-co-glycolic acid (PLGA), a poly(D,L-lactide-co-glycolide) (PLA/PLGA), a poly(hydroxyalkylmethacrylamide), a polyglycerol, a polyamidoamine (PAM AM), and a polyethylenimine (PEI), polyorthoesters, polyacetals; and c) Optionally washing the resulting product from b), preferably with an acidic buffer (Citrate Buffer) optionally supplemented with a molecular chaperone such as a salt of isofagomine.

9. A method to manufacture a polymer conjugate, wherein the method comprises: b. Adding a polymer to a SATP-modified protein resulting from the reaction step (a) according to any of claims 1 to 6, wherein the modified protein is a glucocerebrosidase enzyme, and wherein the polymer is selected from the group consisting of dextran, a water soluble linear polyamino acid or polypept(o)ide (including a polyglutamic acid (PGA), polyaspartic acid (pAsp), polysarcosin (PSar)....), a polyethylenglycol (PEG), a polylactic acid (PLA) a polylactic-co-glycolic acid (PLGA), a poly(D,L-lactide-co- glycolide) (PLA/PLGA), a poly(hydroxyalkylmethacrylamide), a polyglycerol, a polyamidoamine (PAMAM), and a polyethylenimine (PEI), polyorthoesters, polyacetals; and the SATP-modified protein is concomitantly deacetylated, preferably with a deacetylation solution such as a hydroxylamine-HCI solution; and c. Optionally washing the resulting product from b), preferably with an acidic buffer such as VCB optionally supplemented with a molecular chaperone such as a salt of isofagomine.

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

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), wherein 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. The method of claim 10, wherein the polymer is L-PGA having from 25 to 250 units of glutamic acid monomers.

13. A method of conjugation of a glucocerebrosidase enzyme to poly-L-glutamic acid (PGA), wherein the method comprises: a. the manufacturing method for modifying the surface protein of the glucocerebrosidase enzyme alfa with (N-succinimidyl-S-acetylthiopropionate) by modifying the lateral chain of lysines of the protein, according to any of claims 1 to 7; b. subsequently to step a) deacetylating the product resultant from a); and c. simultaneously or subsequently conjugating a polymer to the product resultant from b) in accordance with any of claims 8 to 12.

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

15. A SATP-modified protein obtained according to any of claims 1 to 6, wherein the modified protein is a glucocerebrosidase enzyme.

16. A SATP-modified protein obtained according to claim 7, wherein the modified protein is a glucocerebrosidase enzyme.

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

18. A pharmaceutical composition comprising the polymer conjugate of claim 17, optionally further comprising excipients, vehicle, permeation enhancers and/or adjuvants.

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

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

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

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