WO2019040507A1 - Modified lysosomal enzymes - Google Patents

Abstract

Provided herein are lysosomal enzymes comprising modifications that increase stability, pharmaceutical compositions thereof, and methods of using such compounds for the treatment of disorders associated with lysosomal enzyme deficiency.

Description

MODIFIED LYSOSOMAL ENZYMES

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application Serial No. 62/547,915, filed on August 21, 2017, entitled "MODIFIED LYSOSOMAL ENZYMES", the entire contents of which are incorporated herein by reference.

BACKGROUND

Defects in lyosomal enzymes can lead to disease. For example, Gaucher disease is a rare inborn error of glycosphingolipid metabolism due to deficiency of lysosomal acid β- glucocerebrosidase; the condition has totemic significance for the development of orphan drugs. A designer therapy, which harnesses the mannose receptor to complement the functional defect in macrophages, ameliorates the principal clinical manifestations in hematopoietic bone marrow and viscera. While several aspects of Gaucher disease (particularly those affecting the skeleton and brain) are refractory to treatment, enzyme replacement therapy has become a pharmaceutical blockbuster. Human β-glucocerebrosidase was originally obtained from placenta and was subsequently developed as a recombinant product. After purification, the enzyme is modified to reveal terminal mannose residues which facilitate selective uptake of the protein, imiglucerase (Cerezyme®), in macrophage-rich tissues. Two related agents, velaglucerase-alfa, and taliglucerase-alfa, are now approved or in late-phase clinical development.

SUMMARY

In some aspects, the disclosure relates to modified lysosomal enzymes. In some embodiments, a lysosomal enzyme (e.g., a modified lysosomal enzyme) is glucocerebrosidase (GBA). In some embodiments, a lysosomal enzyme (e.g., a modified lysosomal enzyme is recombinant GBA, for example Cerezyme®).

In some embodiments, the disclosure provides a modified lysosomal enzyme comprising a mannose residue, wherein the mannose residue is modified to comprise a first protecting group that is cleavable under physiological conditions. In an embodiment, the first protecting group comprises a water-soluble polymer that is conjugated to the mannose residue by a first linker, wherein the first linker is cleavable under physiological conditions.

In some embodiments, a modified lysosomal enzyme comprises a thiol residue, wherein the thiol residue is modified to comprise a protecting group (e.g. , a first protecting group or second protecting group) that is cleavable under physiological conditions. In some embodiments, a second protecting group comprises a water-soluble polymer that is conjugated to the thiol residue by a second linker, wherein the second linker is cleavable under

physiological conditions.

In some embodiments, a modified lysosomal enzyme comprises a thioether residue, wherein the thioether residue is modified to comprise a protecting group (e.g. , a first, second or third protecting group) that is cleavable under physiological conditions. In some embodiments, a third protecting group comprises a water-soluble polymer that is conjugated to the thioether residue by a third linker, wherein the third linker is cleavable under physiological conditions.

In some embodiments, a protecting group (e.g., a first protecting group, a second protecting group, and/or a third protecting group) comprises a water-soluble polymer. In some embodiments, the polymer is non-peptidic. In some embodiments, the polymer is linear. In some embodiments, the polymer is branched.

In some embodiments, the polymer comprises a poly(alkylene)oxide, poly(oxyalkylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and/or combinations thereof.

In some embodiments, the polymer is comprises a poly(alkylene)oxide. In some embodiments, the poly(alkylene)oxide is polyethyleneglycol (PEG).

In some embodiments, a protecting group is cleavable under pH-dependent conditions (e.g., acidic conditions, neutral conditions, basic conditions, etc.). In some embodiments, a protecting group is cleavable under reductive conditions (e.g., non-oxidative conditions).

In some embodiments, a linker (e.g., a first linker, second linker, and/or third linker) comprises a moiety selected from: ketone, amide, imide, sulfone, 2-pyridine, ester, orthoester, carbonate, carbamate, acetal, or hemiaminal. In some embodiments, a linker (e.g., a first linker, second linker, and/or third linker) comprises a maleimide linker.

In some embodiments, a linker is cleavable under pH-dependent conditions (e.g., acidic conditions, neutral conditions, basic conditions, etc.). In some embodiments, a linker is cleavable under pH-dependent conditions reductive conditions (e.g., non-oxidative conditions).

In some embodiments, a modified lysosomal enzyme comprises a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of mannose residues. In some embodiments, one or more of the mannose residues is a terminal mannose residue. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the mannose residues are modified to comprise a protecting group (e.g., a first protecting group). In some embodiments, each mannose residue comprises 1, 2, 3 or 4 conjugated water-soluble polymers.

In some embodiments, a modified lysosomal enzyme comprises a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of thiol residues. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the thiol residues are modified to comprise a protecting group (e.g., a second protecting group). In some embodiments, each thiol residue comprises 1, 2, 3 or 4 conjugated water-soluble polymers.

In some embodiments, a modified lysosomal enzyme comprises a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of thioether residues. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the thioether residues are modified to comprise a protecting group (e.g., a second protecting group). In some embodiments, each thiol residue comprises 1, 2, 3 or 4 conjugated water-soluble polymers.

In some aspects, the disclosure provides a pharmaceutical composition comprising a modified lysosomal enzyme as described herein, such as a modified lysosomal enzyme comprising a mannose residue is modified to comprise a first protecting group, and/or a thiol residue is modified to comprise a second protecting group, and/or a thioether residue modified to comprise a third protecting group. In some embodiments, each of the first, second, and/or third protecting groups is conjugated to the residue by a linker, such as a linker that is cleavable under physiological conditions. In some embodiments, each of the first, second, and/or third protecting groups is conjugated to the residue by a permanent linker (e.g. , a linker that is stable under physiological conditions).

In some aspects, the disclosure provides a pharmaceutical composition comprising a modified lysosomal enzyme as described herein and a chaperone molecule (e.g. , a

pharmaceutical chaperone, for example a chaperone inhibitor of GBA), wherein the chaperone molecule stabilizes the conformation of the enzyme.

In some embodiments, the lysosomal enzyme is GBA and the chaperone molecule is isofagomine or ambroxol, 1-Deoxynojirimycin (DNJ), DIX, or 2-deoxy-2-fluoro-D-glucose (2FGlc). In some embodiments, a chaperone comprises or consists of a structure selected from:

In some aspects, the disclosure provides a method of treating a disorder associated with lysosomal enzyme deficiency, comprising administering to a subject in need of such treatment a modified lysosomal enzyme or a pharmaceutical composition as described herein. In some embodiments, the disorder is selected from Lewy body dementia, Parkinson's disease (PD), corticobasal ganglionic degeneration (CBGD), prion disease, Alzheimer' s disease, Amyotrophic lateral sclerosis (ALS), or Gaucher disease.

In some embodiments, the disorder is Gaucher disease, for example type 1, type 2 or type 3 Gaucher disease.

In some embodiments, a modified lysosomal enzyme or a pharmaceutical composition as described herein is administered to the subject peripherally (e.g., not directly to the central nervous system). In some embodiments, peripheral administration is intravenous (IV) injection or subcutaneous (SQ) injection.

In some embodiments, a modified lysosomal enzyme or pharmaceutical composition as described herein is administered to the central nervous system (CNS) of a subject. In some embodiments, CNS administration is intrathecal (IT) injection, intracerebroventricular (ICV) injection, or intracisternal injection. In some embodiments, a modified lysosomal enzyme or pharmaceutical composition as described herein is administered intraparenchymally by direct injection.

In some embodiments, administration to a subject comprises convection enhanced delivery (CED).

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

FIG. 1A is a deconvoluted spectrum of a whole-protein mass spectrometry analysis of a single batch of Cerezyme^®.

FIG. IB is a size exclusion chromatography analysis of a single batch of Cerezyme^®. The batch included 96.8% Cerezyme^® monomer and 2.3% Cerezyme^® dimer or trimer.

FIG. 2A shows the enzymatic activity of different batches of Cerezyme^® stored under varying conditions. Samples of Cerezyme^® in buffer at pH 6.3 were stored at different temperatures or underwent freeze-thaw cycles. Further, a sample of Cerezyme^® was stored in buffer at pH 7.4 at 25°C.

FIG. 2B is an overlay of size exclusion chromatography analyses of Cerezyme^® samples stored in buffer at pH 7.4 at 25°C. Cerezyme^® samples that are incubated for T=90 minutes (shown in red) and T=320 minutes (shown in green) show increased dimer formation relative to Cerezyme^® samples at T=0 minutes (shown in blue).

FIG. 3 shows SDS-PAGE analysis of an exemplary PEG-GBA conjugate. SDS- PAGE samples from left-to-right are (1) Cerezyme^® starting material (1 μg); (2) Conjugation mixture (1 μg); (3) Pre-stained High Molecular Weight Protein Standard; (4) Cerezyme^® starting material (5 μg); and (5) Conjugation mixture (5 μg).

FIG. 4 shows the enzymatic activity and percent conjugation of exemplary PEG- GBA conjugation reactions.

DETAILED DESCRIPTION

Aspects of this document relate to a recognition that certain lysosomal enzymes, such as

Cerezyme^® and biosimilars, exhibit poor pharmacokinetics, pharmacodynamics and

biodistribution due to (i) too rapid mannose uptake through the mannose receptor, (ii) poor intrinsic stability due to oxidation of reactive thiol residues, and/or (iii) limited stability once delivered to lysosomes. In some embodiments, improving intrinsic stability may not be enough due to rapid uptake kinetics due to terminal mannose residues. However, in some embodiments, without mannose residues there is no uptake, and therapeutic lysosomal enzymes, such

Cerezyme^®, may be too unstable in plasma at neutral pH to be effective. Accordingly, aspects of this document relate to lysosomal enzymes, such as modified acid β-glucosidase, having improved kinetics and/or biodistribution. The disclosure is based, in part, on modified lysosomal enzymes (e.g., modified GBA) comprising a modification selected from a mannose residue (e.g., a terminal mannose residue), a thiol residue, and/or a thioether residue, wherein one or more of the residues are attached to a protecting group, for example a water soluble protecting group such as one or more PEG molecules. In some aspects, modified lysosomal enzymes described by the disclosure are present in a pharmaceutical composition that comprises a pharmaceutical chaperone (e.g., a chaperone inhibitor of GBA, such as ambroxol, isofagomine, 1-Deoxynojirimycin (DNJ), DIX, 2-deoxy-2-fluoro-D-glucose (2FGlc), etc.). Structure and function of glucocerebrosida.se: GBA

Acid β-glucosidase (GCase, glucocerebrosidase, EC 3.2.1.45) is a membrane-associated lysosomal hydrolase whose defective function leads to Gaucher disease (Liou et ah, 2006). The mature glycoprotein has 497 amino acids that are derived from 517- or 536-amino acid precursors containing leader sequences that are removed during transit through the endoplasmic reticulum membrane.

Cotranslational glycosylation occurs at four of five N-glycosylation sites. This glycosylation is essential for the development of a catalytically active conformer, specifically with N-glycosylation of the first sequon. The primary sequence does not provide clues to the tight membrane association of GCase, since typical hydrophobic transmembrane domains are not present in the mature sequence. The newly synthesized enzyme is trafficked to the lysosome via mannose 6-phosphate and oligosaccharide-independent pathways; the peptide sequence responsible for this trafficking remains to be defined. Once resident in the lysosome, the enzyme has a half-life of ~60 h in fibroblasts, and glycosylation is important to maintaining this survival. GCase cleaves a variety of glucosyl ceramides and synthetic β-glucosides using detergent or phospholipid-based systems for in vitro assays. The major natural substrate is the glycosphingolipid, glucosyl ceramide, with the deacylated analogue, glucosylsphingosine, being a minor, but pathologically important, substrate. GCase defects lead to accumulation of these substrates in a tissue- specific manner.

GCase is a typical retaining β-glucosidase, whose catalytic cycle proceeds through a two- step reaction mechanism requiring glucosylation of the active site by substrate followed by deglucosylation with a release of β-glucose. The nucleophile in this active site function is

Glu 340 , and the presumptive acid/base is Glu 235. The Oglucosidic bond of substrates is protonated by the acid/base (Glu 325 ) and is then attacked by the nucleophile (Glu 340 ). Glucose becomes covalently attached, and the leaving group is removed. This is followed by deprotonation of water by the acid/base with attack of the enzyme-glucose complex by the liberated nucleophile and release of β-glucose. The enzyme requires membrane interfaces, with preference for negatively charged phospholipids, to enhance the in vitro enzyme activity from near zero in a completely delipidated form. The presence of a naturally occurring 80-amino acid activator protein, saposin C, has additional effects on the activity of the negatively charged phospholipid-activated GCase. The exact activation mechanisms of the negatively charged phospholipids and/or saposin C remain to be determined, but the lipids appear to alter the enzyme into a catalytically active conformer that is receptive to the function of saposin C in vivo. The genetic absence of saposin C leads to a deficiency of glucosyl ceramide cleavage and a Gaucher-like disease.

About 200 different point mutations encoding amino acid substitutions have been identified in patients with Gaucher disease. Some such mutations have been subjected to functional assessment. The vast majority of point mutations occur as compound heterozygotes with two different mutant alleles expressed in the same cells. Many of these mutations involve amino acid substitutions that are predicted to be highly disruptive (e.g., large charge and/or side chain changes), whereas others do not (e.g., substitutions of two different branched-chain amino acids). The relationships of genotype to phenotype in Gaucher disease may be based on known disruptive functional changes derived from mutations. For example, highly disruptive mutations could produce more significant effects on catalytic function and/or enzyme instability, leading to more severe or progressive disease manifestations. Also, knowledge of the distribution of the mutations and their functional effects on catalytic or stability properties of GCase could have significant implications for the design and/or development of new therapeutic agents for enzyme, chaperone, or genic therapeutic approaches. In some embodiments, patients range from having compound heterozygous severe mutations to heterozygous mild mutations such as 370S.

Cerezyme and terminal mannose residues.

In general mannose residues promote rapid uptake of conjugated proteins, particularly into myeloid/innate immune cells: macrophage, dendritic cells, microglia, kupfer cells, langerhans cells, but also other cell types, including neurons. Terminal mannose residues on Cerezyme^® allow the uptake of the modified GBA through Mannose receptors such as the cation-independent mannose 6 phosphate receptor (Van Patten et ah, 2007) on macrophage and other cells.

For commercial production, GBA is typically overexpressed in CHO or HEK293 cells in vitro. The generation of terminal mannoses can be achieved by enzymatic treatment as is done for Cerezyme^®, or using carrot cells that naturally leave such residues, or by treating mammalian cells with an inhibitor of glycosylation during production. Each of these lead to somewhat different sugar structures and thus the products are not identical, but seem to have similar properties (Van Patten et ah, 2007). Such residues can be immunogenic and a subset of patients develop antibodies that may neutralize activity.

Terminal mannoses are not typically present on endogenous GBA and usually trafficked to lysosomes from Golgi and endosomes through LIMP-2 (gene is SCARB2) binding (Reczek et ah, 2007), a receptor present on lysosome membranes. There is some limited uptake from plasma of endogenous GBA but this is very limited.

Poor PK/PD biodistribution of cerezyme and related drugs

Because of uptake through mannose receptors on macrophage and dendritic cells and other myeloid cells, most of Cerezyme^® delivered intravenously has an extremely short half-life, and most is cleared from blood biexponentially, with a half-life of 4-6 min for 72% of the tracer and half-life of 88-210 min for the remainder. In patients with enlarged spleens, very little

Cerezyme^® gets to the liver, so the pharmacokinetics are essentially first-pass.

In some embodiments, GBA is taken through endosomes to lysosomes, where the half- life in tissue is l-2hrs (measured as bound radioactivity). In some embodiments, some fractions appears more stable with a half-life of 30-40 hours.

Another potential aspect of PK/PD is binding of mannose residues to MBP in serum.

This appears to be differentially the case with the different commercial forms of the therapeutic enzyme (Van Patten et ah, 2007).

In the absence of terminal mannoses, the half-life of GBA delivered in blood is longer, likely hours. Although circulating half-life is longer for GBA without terminal mannose residues, activity is quickly lost at neutral pH in plasma and the protein fails to enter cells in time

Provided herein, in some embodiments, is a solution to the issue of circulating half-lives. Specifically, provided herein is a modified lysosomal enzyme comprising a mannose residue, wherein the mannose residue is modified to comprise a first protecting group, e.g., a conjugated water-soluble polymer, that is cleavable under physiological conditions. Such a modified enzyme exhibits greatly increased half-life, PK, PD and distribution of Cerezyme^®/GBA. The modified enzyme is stable ex vivo, but is slowly decomposed/degraded in vivo, leading to exposure of terminal mannose residues in a regulated slow fashion. High chronic levels are thereby achieved, leading to improved efficacy and patient tolerance. GlycoPEGylation of a glycoprotein generally involves 3 steps: (Turecek, Bossard, Schoetens, & Ivens, 2016) (1) removal of the carbohydrate terminal sialic acids using a sialidase (neuraminidase) exposing galactose residues, (2) enzymatic PEGylation at the galactose using an enzyme that attaches sialic acid-gly-PEG, (3) capping any residual galactose residues with sialic acid using the enzyme ST3Gal III and cytidine monophosphate sialic acid. This approach was previously used on the coagulation enzyme rFVIIa. (Stennicke et ah, 2008) (Turecek et ah, 2016).

Intrinsic instability at neutral pH

At neutral pH, the mechanism for loss of activity and of aggregation and degradation appears to be oxidation of thiols (Bobst, Thomas, Salinas, Savickas, & Kaltashov, 2010). In some embodiments, such oxidation can happen theoretically at up to 113 amino acids on GBA, but occurs most prominently on thiols on 3 methionines, and to a lesser extent on 2 cysteines. The specific sites most affected by oxidation are M53, M335, and M450 (Bobst et ah, 2010). Noticeable oxidation was also observed at two other sites: oxidation of C126 and C342 to sulfinic acid (Bobst et ah, 2010). Of these Cysteines, C342 is important for activity and quite near the active site nucleophile Glu340.

Provided herein, in some embodiments, is a solution to this issue. Specifically, provided herein is a modified lysosomal enzyme comprising a thiol residue, wherein the thiol residue is modified to comprise a second protecting group, e.g., a conjugated water-soluble polymer, that is cleavable under physiological conditions. In some embodiments, the water-soluble polymer is polyethylene glycol (PEG). In some embodiments, the thiol residue is located in a methionine or a cysteine of an amino acid sequence encoding GBA. In some embodiments, the thiol is located on M53, M335, or M450 of GBA. Protection of thiols with polymers such as PEG stabilize the reduced form of proteins containing cysteines. Thiol pegylation has been described (Turecek et ah, 2016). PEG reagents may be thiol selective or N-terminal selective. (Roberts et al, 2002)

Uptake to lysosomes and stability in lysosomes

GBA is transported typically from ER to lysosomes through interactions with

LIMP2/SCARB2. Exogenous cerezyme is in contrast transported through the mannose receptor pathway to lysosomes. In the low-pH environment of lysosomes the protein is likely to be more stable. In this relatively reduced environment, it is possible that thiol containing residues, and particularly cysteines, play some structural roles. Binding to substrate key aspect in stabilization and conformational dynamics is the presence of substrate (Kornhaber et al. Shen et ah, BBRC).

In some embodiments, an important feature of GBA is the conformational flexibility of the loopl domain, that is next to the E340 catalytic residue, which becomes stabilized by the presence of substrate or inhibitor "chaperones" that interact with the active site. These chaperone inhibitors stabilize the GBA protein in both neutral pH environments and in the cell. Examples of such inhibitory chaperones are isofagomine and Ambroxol. Additional examples of inhibitors include the following, which may be used as non-covalent inhibitors or covalent inhibitors.

In some embodiments, an inhibitor of GBA selectively binds GBA at neutral pH of plasma. In some embodiments, a chaperone inhibitor of GBA stabilizes the exogenous form of GBA but does not inhibit GBA function or activity in cells {e.g., in the lysosome of cells). In some embodiments, delivery of enzyme replacement therapy (ERT) or polymer- stabilized ERT is improved when delivered together with one or more chaperone inhibitors.

Experience with PEG-GBA

Certain previously utilized PEG-GBA products lacked terminal mannose residues.

These residues generally allow for uptake into macrophage and other myeloid cells, which especially in spleen and liver get congested with substrate (glucosylsphingosine,

Glucosylceramide) in Gaucher patients leading to the formation of swollen "Gaucher" cells. GBA without these terminal mannose residues has a longer half-life but does not get into the right cells. Without terminal mannoses, thiols are also oxidized and then the protein activity is lost, and ultimately aggregation and degradation occur, thereby reducing protein half-life. In some embodiments, PEGylation of GBA leads to a longer serum half-life, relative to a GBA that has not been PEGylated. There is a critical unmet need in children with neuronopathic Gaucher. Most cases of GBA are type 1, but20% are types 2 and 3, which presents very early in life (<2yo in type 2) or later in type 3 disease. The most severe cases have the lowest enzyme activity and can present prenatally. Weaker mutations can present late in life.

As little as 1% or less of enzyme activity may be sufficient to rescue CNS activity.

Thus, elevating the endogenous levels of GBA in plasma sufficiently may be effective even in type 2 and 3.

Carriers of only one mutant allele are at high risk of Parkinson's disease or Lewy Body Dementia (LBD). The more severe the enzymatic activity loss of the mutation, the more rapid the progression of the disease. Furthermore, PD patients with mutations in both alleles of GBA progress more quickly and have a worse presentation than one allele. In essence it is hypothesized that there is a continuum between severe Gaucher and increased PD risk, all essentially the same pathological mechanism at different degrees. In fact some patients with 2 mutant alleles are undiagnosed, or have mild neutropenia or mild hepatospenomegaly. In some embodiments, differences in presentation may be due to other genetic or environmental factors. Some aspects of the disease can be stopped or reversed by ERT in any of these neuropathic or non-neuropathic forms.

Definitions

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or

configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. 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. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

As used herein, the term "oxygen protecting group" or "oxygen protecting groups" includes, but is not limited to, -R^aa, -N(R^bb)₂, -C(=0)SR^aa, -C(=0)R^aa, -CO^, - C(=0)N(R^bb)₂, -C(=NR^bb)R^aa, -C(=NR^bb)OR^aa, -C(=NR^bb)N(R^bb)₂, -S(=0)R^aa, -SO^, - Si(R^aa)₃ -P(R^CC)₂, -P(R^CC) , -Ρ(=0)^, -P(=0)(R^aa)₂, -P(=0)(OR^cc)₂, -P(=0)₂N(R^bb)₂, and - P(=0)(NR^bb)₂, wherein R^aa, R^bb, and R^cc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 edition, John Wiley & Sons, 1999, incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to, methyl,

methoxylmethyl (MOM), methylthiomethyl (MTM), i-butylthiomethyl,

(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), /?- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), i-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-

(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4- methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2- yl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-l- benzyloxyethyl, l-methyl-l-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2- trimethylsilylethyl, 2-(phenylselenyl)ethyl, i-butyl, allyl, /?-chlorophenyl, /?-methoxyphenyl,

2,4-dinitrophenyl, benzyl (Bn), /?-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p- nitrobenzyl, /?-halobenzyl, 2,6-dichlorobenzyl, /?-cyanobenzyl, /?-phenylbenzyl, 2-picolyl, 4- picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p '-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, /?-methoxyphenyldiphenylmethyl, di( ?-methoxyphenyl)phenylmethyl, tri( ?-methoxyphenyl)methyl, 4-(4 ' - bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl,

4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-l- yl)bis(4',4"-dimethoxyphenyl)methyl, l,l-bis(4-methoxyphenyl)-l '-pyrenylmethyl, 9-anthryl,

9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1 ,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldimethylsilyl (TBDMS), i-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), i-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, /?-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate

(levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, i-butyl carbonate (BOC or Boc), /?-nitrophenyl carbonate, benzyl carbonate, /?-methoxybenzyl carbonate, 3,4- dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, /?-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4- azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro^4- (1,1 ,3 ,3-tetramethylbutyl)phenoxyacetate, 2,4-bis( 1 , l-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o- (methoxyacyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

As used herein, the terms "sulfur protecting group" and "sulfur protecting groups" include, but are not limited to, -R^aa, -N(R^bb)₂, -C(=0)SR^aa, -C(=0)R^aa, -CO^, - C(=0)N(R^bb)₂, -C(=NR^bb)R^aa, -C(=NR^bb)OR^aa, -C(=NR^bb)N(R^bb)₂, -S(=0)R^aa, -SO^, - Si(R^aa)₃ -P(R^CC)₂, -P(R^CC)₃, -Ρ(=0)^, -P(=0)(R^aa)₂, -P(=0)(OR^cc)₂, -P(=0)₂N(R^bb)₂, and - P(=0)(NR^bb)₂, wherein R^aa, R^bb, and R^cc are as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic

Synthesis, T. W. Greene and P. G. M. Wuts, 3 edition, John Wiley & Sons, 1999, incorporated herein by reference.

As used herein, each instance of is, independently, selected from Ci_io alkyl, Ci_io perhaloalkyl, C₂-io alkenyl, C₂-io alkynyl, heteroCi_io alkyl, heteroC₂-ioalkenyl, heteroC₂- _loalkynyl, C₃_io carbocyclyl, 3-14 membered heterocyclyl, Ce-i₄ aryl, and 5-14 membered heteroaryl, or two groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^dd groups.

As used herein, each instance of R^bb is, independently, selected from hydrogen, -OH, - OR"", -N(R^CC)₂, -CN, -C(=0)R^aa, -C(=0)N(R^cc)₂, -CO^, -SO^, -C(=NR^cc)OR^aa, - C(=NR^CC)N(R^CC)₂, -S0₂N(R^cc)₂, -S0₂R^cc, -S0₂OR^cc, -SOR^aa, -C(=S)N(R^CC)₂, -C(=0)SR^cc, - C(=S)SR^CC, -P(=0)₂R^aa, -P(=0)(R^aa)₂, -P(=0)₂N(R^cc)₂, -P(=0)(NR^cc)₂, Ci_io alkyl, Cno perhaloalkyl, C₂_io alkenyl, C₂_io alkynyl, heteroCi_ioalkyl, heteroC₂_ioalkenyl, heteroC₂_ _loalkynyl, C₃_io carbocyclyl, 3-14 membered heterocyclyl, Ce-i₄ aryl, and 5-14 membered heteroaryl, or two R^bb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^dd groups.

As used herein, each instance of R^cc is, independently, selected from hydrogen, Ci_io alkyl, Ci_io perhaloalkyl, C₂_io alkenyl, C₂_io alkynyl, heteroCi_io alkyl, heteroC₂_io alkenyl, heteroC₂_io alkynyl, C₃_io carbocyclyl, 3-14 membered heterocyclyl, Ce-i₄ aryl, and 5-14 membered heteroaryl, or two R^cc groups are joined to form a 3-14 membered heterocyclyl or 5- 14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^dd groups.

As used herein, each instance of R^dd is, independently, selected from halogen, -CN, - N0₂, -N₃, -S0₂H, -S0₃H, -OH, -OR^ee, -ON(R^ff)₂, -N(R^ff)₂, -N(R^ff)₃ ⁺X , -N(OR^ee)R^ff, -SH, - SR^ee, -SSR^ee, -C(=0)R^ee, -C0₂H, -C0₂R^ee, -OC(=0)R^ee, -OC0₂R^ee, -C(=0)N(R^ff)₂, - OC(=0)N(R^ff)₂, -NR^ffC(=0)R^ee, -NR^ffC0₂R^ee, -NR^ffC(=0)N(R^ff)₂, -C(=NR^ff)OR^ee, - OC(=NR^ff)R^ee, -OC(=NR^ff)OR^ee, -C(=NR^ff)N(R^ff)₂, -OC(=NR^ff)N(R^ff)₂, - NR^ffC(=NR^ff)N(R^ff)₂,-NR^ffS0₂R^ee, -S0₂N(R^ff)₂, -S0₂R^ee, -S0₂OR^ee, -OS0₂R^ee, -S(=0)R^ee, - Si(R^ee)₃, -OSi(R^ee)₃, -C(=S)N(R^ff)₂, -C(=0)SR^ee, -C(=S)SR^ee, -SC(=S)SR^ee, -P(=0)₂R^ee, - P(=0)(R^ee)₂, -OP(=0)(R^ee)₂, -OP(=0)(OR^ee)₂, Ci_₆ alkyl, Ci_₆ perhaloalkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, heteroCi_₆alkyl, heteroC₂_₆alkenyl, heteroC₂_₆alkynyl, C₃_io carbocyclyl, 3-10 membered heterocyclyl, Ce-io aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^gg groups, or two geminal R^dd substituents can be joined to form =0 or =S. As used herein, each instance of R^ee is, independently, selected from Ci_₆ alkyl, Ci_₆ perhaloalkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, heteroCi_₆ alkyl, heteroC₂_₆alkenyl, heteroC₂_₆ alkynyl, C₃_io carbocyclyl, Ce-io aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^gg groups.

As used herein, each instance of R ff is, independently, selected from hydrogen, Ci_₆ alkyl, Ci_₆ perhaloalkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, heteroCi_6alkyl, heteroC₂_6alkenyl, heteroC₂_ ₆alkynyl, C₃_io carbocyclyl, 3-10 membered heterocyclyl, Ce-io aryl and 5-10 membered heteroaryl, or two R ff groups are joined to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^gg groups.

As used herein, each instance of R^gg is, independently, halogen, -CN, -N0₂, -N₃, - S0₂H, -S0₃H, -OH, -OCi_₆ alkyl, -ON(Ci_₆ alkyl)₂, -N(Ci_₆ alkyl)₂, -N(Ci_₆ alkyl)₃ ⁺X- - NH(Ci_6 alkyl)₂ ⁺X-, -NH₂(Ci_₆ alkyl) ⁺X^~, -NH₃ ⁺X , -N(OCi_₆ alkyl)(Ci_₆ alkyl), -N(OH)(Ci_₆ alkyl), -NH(OH), -SH, -SCi_₆ alkyl, -SS(Ci_₆ alkyl), -C(=0)(Ci_₆ alkyl), -C0₂H, -C0₂(Ci ₆ alkyl), -OC(=0)(Ci_₆ alkyl), -OC0₂(Ci ₆ alkyl), -C(=0)NH₂, -C(=0)N(Ci_₆ alkyl)₂, - OC(=0)NH(Ci_6 alkyl), -NHC(=0)( Ci_e alkyl), -N(Ci_₆ alkyl)C(=0)( Ci_₆ alkyl), - NHC0₂(Ci_6 alkyl), -NHC(=0)N(Ci_₆ alkyl)₂, -NHC(=0)NH(Ci_₆ alkyl), -NHC(=0)NH₂, - C(=NH)0(Ci^, alkyl),-OC(=NH)(Ci_6 alkyl), -OC(=NH)OCi_₆ alkyl, -C(=NH)N(Ci_₆ alkyl)₂, - C(=NH)NH(Ci_6 alkyl), -C(=NH)NH₂, -OC(=NH)N(Ci_₆ alkyl)₂, -OC(NH)NH(Ci_₆ alkyl), - OC(NH)NH₂, -NHC(NH)N(Ci_₆ alkyl)₂, -NHC(=NH)NH₂, -NHS0₂(Ci^, alkyl), -S0₂N(Ci_₆ alkyl)₂, -S0₂NH(Ci^, alkyl), -S0₂NH₂,-S0₂Ci^ alkyl, -S0₂OCi_₆ alkyl, -OS0₂Ci^, alkyl, - SOCi_6 alkyl, -Si(Ci_₆ alkyl)₃, -OSi(Ci^, alkyl)₃ -C(=S)N(Ci_₆ alkyl)₂, C(=S)NH(Ci_6 alkyl), C(=S)NH₂, -C(=0)S(Ci^, alkyl), -C(=S)SCi_6 alkyl, -SC(=S)SCi^, alkyl, -P(=0)₂(Ci_₆ alkyl), - P(=0)(Ci_₆ alkyl)₂, -OP(=0)(Ci_₆ alkyl)₂, -OP(=0)(OCi_₆ alkyl)₂, Ci_₆ alkyl, Ci_₆ perhaloalkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, heteroCi_₆alkyl, heteroC₂_₆alkenyl, heteroC₂_₆alkynyl, C₃_io carbocyclyl, C6 io aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal R^gg substituents can be joined to form =0 or =S; wherein X^~ is a counterion.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are

physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

Modified Lysosomal Enzymes

In one aspect, provided herein is a modified lysosomal enzyme comprising a mannose residue, wherein the mannose residue is modified to comprise a first protecting group that is cleavable under physiological conditions.

In an embodiment, the first protecting group is an oxygen protecting group.

In an embodiment, the first protecting group comprises a water-soluble polymer that is conjugated to the mannose residue by a first linker, wherein the first linker is cleavable under physiological conditions.

In an embodiment, the polymer is non-peptidic. In an embodiment, the polymer is linear. In an embodiment, the polymer is branched. In an embodiment, the polymer comprises a poly(alkylene)oxide, poly(oxyalkylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a- hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N- acryloylmorpholine), and combinations thereof. In a particular embodiment, the polymer is comprises a poly(alkylene)oxide. In a preferred embodiment, the poly(alkylene)oxide is polyethyleneglycol. In a preferred embodiment, the polyethyleneglycol has an average molecular weight in the range of 100-40000 g/mol.

Compositions (e.g., modified lysosomal enzymes, such as GBA) described herein may be PEGylated in certain embodiments. The PEGylation may be permanent or transient

PEGylation. For example, in some embodiments, a modified lysosomal enzyme is linked to one or more PEG molecules, which may independently be the same or different sizes (e.g., as measured by molecular weight or number of polymer subunits). In embodiments cases, the PEGylation may be relatively permanent. For example, the PEG moieties may be covalently attached or linked (e.g. , via a linker that is stable under physiological conditions) to the modified lysosomal enzyme.

In some cases, the PEGylation may be transient or temporary. For example, the PEG moieties may be transiently or noncovalently attached or linked (e.g. , via a linker that is cleavable under physiological conditions) to the modified lysosomal enzyme.

Any suitable method may be used to attach a PEG moiety to the enzyme. Non-limiting examples include glutaraldehyde, NHS-esters (N-hydroxysuccinimide) (e.g. ,

dithiobis(succinimidylpropionate), dithiobis(sulfosuccinimidylpropionate), etc.), imidoesters (e.g. , dimethyl adipimidate, dimethyl suberimidate, dimethyl pimelimidate, etc.), maleimides, pyridyls, carbodiimide, isocyanate, or the like. Those of ordinary skill in the art will be familiar with methods of cross-linking or conjugating PEG moieties to enzymes and other moieties.

The PEG moiety may have any suitable molecular weight, and if more than one PEG moiety is present, the PEG moieties may independently have the same or different molecular weights. For example, the molecular weight (Mn) of a PEG moiety may be at least 1,000, at least 2,000, at least 3,000, at least 5,000 at least 10,000, at least 20,000, at least 30,000, at least 50,000, or at least 100,000, and/or no more than 100,000, no more than 50,000, no more than 30,000, no more than 20,000, no more than 10,000, no more than 5,000, no more than 3,000, no more than 2,000, no more than 1,000, etc.

PEG or PEGylation residues are generally water-soluble polymers characterized by repeating units. Suitable polymers useful for PEGylation may be selected from the group consisting of polyalkyloxy polymers, hyaluronic acid and derivatives thereof, polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(ortho esters), polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides, polyacry-lates, polymethacrylates, polyorganophosphazenes, polysiloxanes, polyvinylpyrrolidone, polycyanoacrylates, and polyesters.

In some embodiments, PEG chains may consist of an interconnecting moiety, a polymer moiety, and an end group.

The term "PEG load" herein is understood as a descriptor of the molecular mass of a polymer chain of a number of repeating units. Total PEG load is understood as the total molecular mass of all polymeric carrier chains attached on a molecular basis.

In some embodiments the total PEG load amounts to at least 25 kDa. Generally the total PEG load will be less than 1000 kDa. In some cases, the PEG load is at least 25 kDa and at most 500 kDa, or at least 30 kDa and at most 250 kDa, or at least 30 kDa and at most 120 kDa, or at least 40 kDa and at most 100 kDa, or at least 40 kDa and at most 90 kDa. PEG may be attached through one or more anchoring points. In case of one anchoring point, the corresponding PEG may be branched in some cases and contain at least 3 chains. In case of more than one anchoring point, such as in a bisconjugate, the corresponding PEG may be branched or linear in some cases. Bisconjugates may contain one or two transient linkages, and PEG may be linear or branched or contain a mixture of one linear and one branched chain. In case the bisconjugate contains one transient linkage and one linear and one branched chain the transient linkage may be on either chain.

In case a branched PEG chain is used, there may be one or more branching units.

A branched PEG is a PEG molecule with a branching point connecting two or more PEG chains, to form a molecule with one anchoring point for attachment. This could be, for example, two 20 kDa PEG chains joined to form one branched 40 kDa PEG molecule. In the case where the molecule contains two or three branching points, the molecule can be referred to 3 and 4 armed PEG, respectively.

The PEG polymer is not limited to a particular structure and can be linear, branched, or multi-armed (e.g. , forked PEG or PEG attached to a polyol core), dendritic, or with degradable linkers.

Without being limited to theory the PEG load is intended to provide a suitable molecular mass to get the required relatively low activity and not having a too high molecular mass of the PEG that could create other problems.

The term "linker" is frequently used in publications in the field of bioconjugation and broadly describes chemical structures used to connect two molecular entities. Such connectivity may be of permanent or transient nature.

The term "transient linkage" or "transient linker" herein is understood as describing the lability of the linkage with PEG. In such transient linkages, the PEG may be released or detached, e.g. , with an in vivo linker half-life of up to 1200 hours.

Thus, a transient linker may be a linker in which the conjugation to PEG is or can be reversible. This implies that cleavage of the linker releases the target (e.g. , a modified lysosomal enzyme) in its native and active form. To structurally differentiate a transient linker unit from the polymer carrier (e.g. , PEG) may be difficult in the case of carrier prodrugs in some cases, e.g. , if the polymer is permanently attached to the linker and the linker-related degradation product is therefore not released as a consequence of prodrug cleavage. Structural

characterization of a linker is may also be challenging if the linker is functioning both as an auto-cleavage inducing group and a branching unit. The term "permanent linker" refers to a PEG conjugate via the formation of an aliphatic amide or aliphatic carbamate. If conventional PEGylation reagents are used, the resulting conjugates are usually very stable against hydrolysis and the rate of cleavage of the amide or carbamate bond would generally be very slow. If such stable linkages are to be employed, cleavage of the functional group may in some cases be facilitated using enzymatic catalysis.

In an embodiment, the first linker comprises a moiety selected from: ketone, amide, imide, sulfone, 2-pyridine, ester, orthoester, carbonate, carbamate, acetal, or hemiaminal. The first linker is stable ex vivo, but is capable of enzymatic or hydrolytic degradation in vivo, thereby exposing the unmodified mannose residue. In an embodiment, the first linker is cleavable under pH-dependent conditions.

In an embodiment, the modified lysosomal enzyme comprises a plurality of mannose residues. In an embodiment, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the mannose residues are modified to comprise the first protecting group (e.g. , a water-soluble polymer conjugated to the mannose residue by a first linker).

In an embodiment, each conjugated mannose residue comprises 1, 2, 3 or 4 conjugated water-soluble polymers. In a preferred embodiment, each conjugated mannose residue comprises 1 conjugated water-soluble polymer.

In an embodiment, the modified lysosomal enzyme further comprises a thiol residue, wherein the thiol residue is modified to comprise a second protecting group that is cleavable under physiological conditions.

In an embodiment, the second protecting group is a sulfur protecting group.

In an embodiment, the second protecting group comprises a water-soluble polymer that is conjugated to the thiol residue by a second linker, wherein the second linker is cleavable under physiological conditions. In an embodiment, the polymer is non-peptidic. In an embodiment, the polymer is linear. In an embodiment, the polymer is branched. In an embodiment, the polymer comprises a poly(alkylene)oxide, poly(oxyalkylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and combinations thereof. In a preferred embodiment, the polymer is comprises a poly(alkylene)oxide. In a preferred embodiment, the poly(alkylene)oxide is polyethyleneglycol. In a preferred embodiment, the polyethyleneglycol has an average molecular weight in the range of 100-40000 g/mol. In an embodiment, the second linker comprises a moiety selected from: ketone, amide, imide, ester, sulfone, 2-pyridine, thioamide, thioester, orthothioester, thiocarbonate,

thiocarbamate, thioacetal, thioaminal or hemithioaminal. The second linker is stable ex vivo, but is capable of enzymatic or hydrolytic degradation in vivo, thereby exposing the unmodified thiol residue. In an embodiment, the second linker is cleavable under pH-dependent conditions. In an embodiment, the second protecting group is cleavable under pH-dependent conditions. In an embodiment, the second linker is capable of lysosomal cleavage. In an embodiment, the half- life of the second linker under lysosomal cleavage conditions is less than 1 day, less than 5 days, less than 10 days, or less than 1 month.

In an embodiment, the thiol residue is a cysteine residue. In an embodiment, the modified lysosomal enzyme comprises a plurality of thiol residues. In an embodiment, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the thiol residues are modified to comprise the second protecting group (e.g. , a water-soluble polymer conjugated to the thiol residue by a second linker).

In an embodiment, the modified lysosomal enzyme further comprises a thioether residue, wherein the thioether residue is modified to comprise a third protecting group that is cleavable under physiological conditions.

In an embodiment, the third protecting group is a sulfur protecting group, e.g. , a sulfimide as described by Lin et al. (Science 355 (2017), 597-602; incorporated herein by reference).

In an embodiment, the third protecting group comprises a water-soluble polymer that is conjugated to the thioether residue by a third linker, wherein the third linker is cleavable under physiological conditions. In an embodiment, the polymer is non-peptidic. In an embodiment, the polymer is linear. In an embodiment, the polymer is branched. In an embodiment, the polymer comprises a poly(alkylene)oxide, poly(oxyalkylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly (hydroxy alky lmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and combinations thereof. In a preferred embodiment, the polymer is comprises a poly(alkylene)oxide. In a preferred embodiment, the poly(alkylene)oxide is polyethyleneglycol. In a preferred embodiment, the polyethyleneglycol has an average molecular weight in the range of 100-40000 g/mol.

In an embodiment, the third linker comprises a sulfimide (i.e. , (R)(R')S=N-R") moiety. The third linker is stable ex vivo, but is capable of enzymatic, hydrolytic or reductive

degradation in vivo, thereby exposing the unmodified thioether residue. In an embodiment, the second linker is cleavable under pH-dependent conditions. In an embodiment, the second protecting group is cleavable under pH-dependent conditions. In an embodiment, the second linker is capable of lysosomal cleavage. In an embodiment, the half-life of the second linker under lysosomal cleavage conditions is less than 1 day, less than 5 days, less than 10 days, or less than 1 month.

In an embodiment, the thioether residue is a methionine residue, for example M53, M335, or M450 of GBA (or a modified GBA).

In an embodiment, the modified lysosomal enzyme comprises a plurality of thioether residues. In an embodiment, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the thioether residues are modified to comprise the third protecting group.

In a preferred embodiment, the lysosomal enzyme is glucocerebrosidase (GBA). In some embodiments, the lysosomal enzyme is Cerezyme^®. Pharmaceutical Compositions

In another aspect, provided herein is a pharmaceutical composition comprising a modified lysosomal enzyme as described herein, and a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition further comprises a chaperone molecule, wherein the chaperone molecule stabilizes the conformation of the enzyme. In a preferred embodiment, the enzyme is GBA and the chaperone molecule is isofagomine. In a preferred embodiment, the enzyme is GBA and the chaperone molecule is ambroxol.

Chaperone co-therapy is particularly effective in conjunction with the modified lysosomal enzymes described herein. Without the stabilization that results from the

modification of mannose, thiol and/or thioether residues, the half-life of the unmodified enzyme is too short. If the chaperone compound is selectively bound at neutral pH it will not affect stability in cells. Chaperone/inhibitor treatment is typically intermittent {e.g., one week on one week off), so as to allow stabilization and then activity. Additional molecules have been generated and described that appear to stabilize without inhibition in an allosteric fashion {Sidransky, LTI).

In certain embodiments, the modified lysosomal enzyme described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is an amount effective for treating a disorder associated with a lysosomal enzyme deficiency. In certain embodiments, the effective amount is an amount effective for reducing the risk of developing a disease in a subject in need thereof.

In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g. , mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g. , transgenic mice and transgenic pigs).

Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the compound described herein (e.g. , the "active ingredient") into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one- half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of provided

pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g. , cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral

administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer' s solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

Modified lysosomal enzymes provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

The compounds (e.g. , modified lysosomal enzymes) and compositions (e.g. ,

pharmaceutical compositions) provided herein can be administered by any route, including enteral (e.g. , oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g. , systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g. , its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g. , whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of

administration, and the like. An effective amount may be included in a single dose (e.g. , single oral dose) or multiple doses (e.g. , multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g. , a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.

Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. In certain embodiments, a dose described herein is a dose to an adult human whose body weight is 70 kg. A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g. , therapeutically and/or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g. , activity (e.g. , potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, and/or in inhibiting the activity of a protein kinase in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional

pharmaceutical agent, but not both.

The compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which are different from the compound or composition and may be useful as, e.g. , combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include

prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g. , compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins,

mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g. , proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

The additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, cytotoxic agents, anti-angiogenesis agents, anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol- lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, and pain- relieving agents. In certain embodiments, the additional pharmaceutical agent is an antiproliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti- cancer agent. In certain embodiments, the additional pharmaceutical agent is an anti- viral agent. In certain embodiments, the additional pharmaceutical agent is a binder or inhibitor of a protein kinase. In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g. , DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g. , taxanes and vinca alkaloids), hormone receptor modulators (e.g. , estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g. , tyrosine protein kinase inhibitors), modulators of protein stability (e.g. , proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g. , stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.

Methods of Treatment

In another aspect, provided herein is a method of treating a disorder associated with lysosomal enzyme deficiency, comprising administering to a subject in need of such treatment a modified lysosomal enzyme a pharmaceutical composition as described herein.

In another aspect, provided herein is a method of treating the symptoms of a disorder associated with lysosomal enzyme deficiency, comprising administering to a subject in need of such treatment a modified lysosomal enzyme a pharmaceutical composition as described herein.

In certain embodiments of the above aspects, the disorder is selected from Lewy body dementia, Parkinson's disease (PD), corticobasal ganglionic degeneration (CBGD), prion disease, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), or Gaucher disease. In a preferred embodiment, the disorder is Gaucher disease. In a preferred embodiment, the Gaucher disease is type 1, type 2 or type 3.

However, in some embodiments, modified lysosomal enzymes (e.g., modified GBA) provided herein are useful for treating synucleinopathies, neurodegeneration or a Gaucher-like disease. For example, a synucleinopathy treated according to methods or compositions provided herein can be any one or more of: Parkinson' s disease (PD); sporadic or heritable dementia with Lewy bodies (DLB); pure autonomic failure (PAF) with synuclein deposition; multiple system atrophy (MSA); hereditary neurodegeneration with brain iron accumulation; and incidental Lewy body disease of advanced age. In other embodiments, the synucleinopathy can be any one or more of: Alzheimer's disease of the Lewy body variant; Down's syndrome; progressive supranuclear palsy; essential tremor with Lewy bodies; familial parkinsonism with or without dementia; tau gene and progranulin gene-linked dementia with or without parkinsonism;

Creutzfeldt Jakob disease; bovine spongiform encephalopathy; secondary Parkinson disease; parkinsonism resulting from neurotoxin exposure; drug-induced parkinsonism with (X-synuclein deposition; sporadic or heritable spinocerebellar ataxia; amyotrophic lateral sclerosis (ALS); and idiopathic rapid eye movement sleep behavior disorder.

Other conditions that may be treated, in some embodiments, include Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Tay Sachs, Farber, Niemann-Pick Types A, B & C, GMlGangliosidosis, GM2 Gangliosidosis, and MPS-I. Still, in other embodiments, the condition may be selected from the group consisiting of:

adrenoleukodystrophy, AIDS and AIDS -related dementia, Agryophilic grain disease,

Alzheimer's disease, amyotrophic lateral sclerosis (Parkinsonism-dementia complex of Guam or Lytico-Bodigdisease), aortic medial amyloid, apathy, atherosclerosis, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism, autoimmune vasculitis, B 12 deficiency, bipolar disorder, bovine spongiform encephalopathy, brain neoplasms, brain lesions, cardiac arrythmias, cerebrovascular disease, cerebral amyloid angiopathy (and Icelandic type), cognitive impairment due to electroconvulsive shock therapy, cognitive impairment due to chemotherapy, cognitive impairment due to a history of drug abuse, cognitive impairment during waking hours due to sleep apnea, complications post anoxia, complications from intracerebral hemorrhage, corticobasal degeneration, dementia with Lewy bodies, dementia pugilistica, dentatorubropallidouysian atrophy, depression, diabetes mellitus type 2, dialysis related amyloidosis, diffuse Lewy body disease, Down's syndrome, dyslexia, epilepsy, familial amyloid polyneuropathy, Finnish amyloidosis, folic acid deficiency, Fragile X syndrome, Fragile X associated tremor/ataxia syndrome, Fragile XE mental retardation, frontal lobe syndrome, frontotemporal dementia with Parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Friedrich's ataxia, ganglioglioma, hallervorden-spatz disease, hepatic conditions, hereditary non-neuropathic systemic amyloidosis, hypoglycemia,

hypercalcemia, hypothyroidism, hydrocephalus, inclusion body myositis, infectious vasculitis, Kufs' disease, Kufor Rakeb disease, isolated atrial amyloidosis, lattice corneal dystrophy, lead enphalapathy, Lewy body disease, Lewy body mutant of Alzheimer' s disease, Lipofuscinosis, Lyme disease, malnutrition, maple syrup urine disease, medullary carcinoma of the thyroid, meningioangiomatosis, metabolic diseases, mild cognitive impairment, multi-infarct dementia, multiple sclerosis, multiple system atrophy, myasthenia gravis, Myotonic dystrophy,

neurofibromatosis, neurosyphiUis, neurodegeneration with brain iron accumulation type I, niacin deficiency, Parkinson's disease and Parkinson's disease dementia, Pick's disease,

phenylketonuria, polymyalgia rheumatica, post-traumatic stress disorder, prion disease

(Creutzfeldt-Jakob disease), prolactinomas, post coronary artery by-pass graft surgery, progressive supranuclear palsy, protein and lipid accumulation due to normal aging, Rett's syndrome, Rheumatoid arthritis, schizophrenia, systemic lupus erythematosus, spinocerebellar ataxis (types 1-8, 10-14, 16-29), spinobulbar muscular atrophy (Kennedy's disease), sporadic inclusion body myositis, storage diseases, stroke, subacute sclerosing panencephalitis, syphillis, systemic AL amyloidosis, thiamine deficiency, traumatic brain injury, Tourette's syndrome, transmissible spongiform encephalopathy, Tuberous sclerosis, and vascular dementia.

Symptoms of Lewy body demential include, for example, fluctuations in alertness, visual hallucinations, slowness of movement, trouble walking, rigidity, excessive movement during sleep, and mood changes such as depression are also common.

Symptoms of Parkinson' s disease include, for example, motor dysfunction (e.g. , shaking, rigidity, slowness of movement, difficulty with walking), cognitive dysfunction (e.g. , dementia, depression, anxiety), emotional and behavioral dysfunction.

In an embodiment, the modified lysosomal enzyme or pharmaceutical composition is administered to the subject peripherally. In an embodiment, the peripheral administration is rV or SQ. In an embodiment, the modified lysosomal enzyme or pharmaceutical composition is administered to the CNS (central nervous system) of the subject. In an embodiment, the CNS administration is ΓΤ, ICV, or intracysternal. In an embodiment, the modified lysosomal enzyme or pharmaceutical composition is administered intraparenchymally by direct injection.

In an embodiment, administration comprises convection enhanced delivery. In an embodiment, administration is chronic. In an embodiment, the modified lysosomal enzyme or pharmaceutical composition is administered using a delivery pump. Preferably, the pharmaceutical composition is stabilized in the tubing and in the vials, in contrast to existing therapies. Stabilized forms could thus be more convenient to patients who must endure IV treatment once or twice monthly.

In some embodiments, administration of a modified lysosomal enzyme or

pharmaceutical composition results a therapeutically effective increase in circulating enzyme levels or half-life. For example, in some embodiments, (e.g., a dose in a range of 0.5 to 5 mg/kg or 1 to 1.5 mg/kg) mean serum half-lives are in the range of 3 to 20 minutes, 5 to 15 minutes, or 3 to 12 minutes as measure in enzyme levels or activity in serum. However, in some embodiments, improvements in half-life are in the range of greater than 1 hour, greater than 2 hours, greater than 3 hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 7 hours, greater than 24 hours, greater than 7 days. In some embodiments, improvements of half-life are in the range of 6 hours to 48 hours, 1 day to 7 or more days, 1 day to 5 days, 2 days to 10 days, or 1 day to 3 days.

In some embodiments, modified lyosomal enzymes provided herein exhibit improved biodistribution. For example, in some embodiments, IV administration of a lysosomal enzyme provided herein results in greater than 0.5%, greater than 1%, greater than 2% or more distribution to brain or CNS tissue. In some embodiments, e.g., treatment of a pediatric patient by IV administrations, results in greater than 0.5% (e.g., about 1%), greater than 1 %, greater than 2% or more of the enzyme in CSF relative to plasma.

Methods of Manufacture

In one aspect, modified lysosomal enzymes may be prepared without undue

experimentation using known methods for chemo selective modification of proteins (see, e.g., Turecek et ah, J. Pharm. Sci., 105 (2016) 460-475; Roberts et ah, Advanced Drug Delivery Reviews 54 (2002) 459-476; Lin et al, Science 355 (2017), 597-602, incorporated herein by reference) by orthogonal modification of mannose, thiol and thioether residues of the enzyme.

In another aspect, modified lysosomal enzymes may be prepared without undue experimentation from non-glycosylated lysosomal enzymes by orthogonal modification of thiol and thioether residues, followed by N-glycosylation of the enzyme using a protected mannose residue (e.g., a mannose residue comprising a water-soluble polymer conjugated to the mannose residue by a first linker).

Unmodified lysosomal enzymes may be obtained from natural sources or by known methods of recombinant synthesis. EXAMPLES

Example 1: In vitro assays for stability, activity

This Example describes in vitro investigation of the stability and activity of modified proteins, such as modified lysosomal enzymes {e.g., modified GBA) as described by the disclosure.

Stability of modified lysosomal enzymes {e.g., modified GBA) is investigated using in vitro assays described by Liou et al. (2006) J. Biol. Chem. 281(7): 4242-4253, the entire contents of which are incorporated herein by reference.

Example 2: Cell based assays of uptake, activity in reducing substrate

This Example describes cell-based assays for uptake and activity of modified proteins, such as modified lysosomal enzymes {e.g., modified GBA) as described by the disclosure, in reducing substrate.

Uptake and activity of modified lysosomal enzymes {e.g., modified GBA) in reducing substrate is investigated using cell-based assays described by Liou et al. (2006) J. Biol. Chem. 281(7): 4242-4253, the entire contents of which are incorporated herein by reference.

Example 3: In vivo assays using mutant mice

This Example describes in vivo assays of modified proteins, such as modified lysosomal enzymes {e.g., modified GBA) as described by the disclosure, using mutant mice.

In vivo studies of modified lysosomal enzymes {e.g., modified GBA) in mutant mice are performed using assays described, for example, by Liou et al. (2006) J. Biol. Chem. 281(7): 4242-4253, Sun et al. (2005) J. Lipid Res. 46:2102-2113, and Farfel-Becker et al. (2011) Dis. Model Mech. 4(6):746-752, the entire contents of each of which are incorporated herein by reference.

Example 4: Chemical models of disease

This Example describes in vivo assays of modified proteins, such as modified lysosomal enzymes {e.g., modified GBA) as described by the disclosure, using a chemically-induced mouse model of Gaucher disease {e.g., the CBE mouse model).

In vivo studies of modified lysosomal enzymes {e.g., modified GBA) in a chemically- induced mouse model of Gaucher disease are performed using assays described, for example, by Vardi et al. (2016) J Pathol. 239(4):496-509, the entire contents of which are incorporated herein by reference. Example 5: Clinical trials in PD, LBD, Gaucher disease patients

In some embodiments, patients having certain forms of Gaucher disease (e.g., GDI) have an increased risk of developing Parkinson's disease (PD) or Lewy body dementia (LBD). This Example describes clinical trials to assess the safety and efficacy of modified proteins, such as modified lysosomal enzymes (e.g., modified GBA) as described by the disclosure, in patients having Gaucher disease, PD and/or LBD.

Clinical trials of modified lysosomal enzymes (e.g., modified GBA) for treatment of Gaucher disease, PD and/or LBD are performed using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(l):33-39, the entire contents of which are incorporated herein by reference.

Example 6: Treatment of peripheral disease

In some embodiments, patients having certain forms of Gaucher disease exhibit symptoms of peripheral neuropathy, for example as described in Biegstraaten et al. (2010) Brain 133(10):2909-2919, the entire contents of which are incorporated herein by reference.

This Example describes administration of modified proteins, such as modified lysosomal enzymes (e.g., modified GBA) as described by the disclosure, for treatment of peripheral neuropathy associated with Gaucher disease (e.g., Type 1 Gaucher disease). Briefly, Type 1 Gaucher disease patients identified as having signs or symptoms of peripheral neuropathy are administered a modified lysosomal enzyme (e.g., modified GBA) as described by the disclosure. In some embodiments, the peripheral neuropathic signs and symptoms of the subject are monitored, for example using methods described in Biegstraaten et al., after administration of the modified lysosomal enzyme.

Levels of modified lysosomal enzymes (e.g., modified GBA) as described by the disclosure present in patients (e.g., in serum of a patient, in peripheral tissue (e.g., liver tissue, spleen tissue, etc.) of a patient) are assayed, for example by Western blot analysis, enzymatic functional assays, or imaging studies. Example 7: Treatment of CNS forms

This Example describes administration of modified proteins, such as modified lysosomal enzymes (e.g., modified GBA) as described by the disclosure, for treatment of CNS forms of

Gaucher disease. Briefly, Gaucher disease patients identified as having a CNS form of Gaucher disease (e.g., Type 2 or Type 3 Gaucher disease) are administered a modified lysosomal enzyme (e.g., modified GBA) as described by the disclosure. Levels of modified lysosomal enzymes (e.g., modified GBA) as described by the disclosure present in the CNS of patients (e.g., in serum of the CNS of a patient, in cerebrospinal fluid (CSF) of a patient, or in CNS tissue of a patient) are assayed, for example by Western blot analysis, enzymatic functional assays, or imaging studies.

Example 8: Biomarkers

This example describes detection and/or quantification of biomarkers associated with Gaucher disease. Examples of biomarkers associated with Gaucher disease include but are not limited to chitotriosidase, CCL18, and glucosylceramide (GL1), aSyn and Tau (e.g., for

Gaucher-associated Parkinson's disease), for example as described in Aerts et al. (2005) Acta Paediatr. Suppl. 94(447):43-6, Murugesan et al. (2016) Am. J. Hematol. 91(11): 1082-1089, and Magdalinou et al. (2014) J. Neurol. Neurosurg Psychiatry 85(10): 1065-1075, the entire contents of each of which are incorporated herein by reference. Further biomakers include GPNMB (Transmembrane glycoprotein^ SCTSS (Isoform 2 of Cathepsin), IGKC (Ig kappa chain V-III region), LYZ (Lysozyme C), CFD (Complement factor D), PLD3 (Phospholipase D3), and glycolipid substrates, such as glucosylsphingosine and glucosylceramide.

Biomarkers are identified or quantified using any suitable method. For example, chitotriosidase level of a subject may be measured by obtaining a serum sample from the subject and performing a functional assay, as described for example by Schoonhoven et al. (2007) Clin. Chim. Acta. 381(2): 136-9, the entire contents of which are incorporated by reference herein. Imaging studies for Gaucher disease are carried out according to methods known in the art, for example those described by Simpson et al. (2014) World J. Radiol. 6(9): 657-668.

Biomakers for Parkinson's include, in CSF or blood, aSynuclein, phospho-Synuclein, oligomeric aSynuclein, Tau, p-Tau, and neurofilament light (NFL).

Example 9: Methods

Analytical Size Exclusion Chromatography. Analytical size exclusion chromatography analysis was performed on a AKTA Explorer (GE Healthcare) system. Samples were analyzed using a Superdex 200 or a Sepharose column (10x300 mm). 20 mM sodium phosphate, 135 mM sodium chloride, pH 7.4 was used as mobile phase. The flow rate for the column was 0.75 ml/min and the eluted proteins were detected at 280 nm.

Activity Determination of GBA. Samples of GBA (e.g., Cerezyme^®) were assayed for enzymatic activity to hydrolyze the native substrate glucosylceramide. GBA was incubated with glucosylceramide for a defined period of time (e.g., 30 minutes at 37 °C) before the reaction was quenched, e.g., by boiling for 3 min at 100 °C. The samples were then centrifuged before being analyzed for their glucose content, e.g., using a Glucose Assay Kit from Biovision according to the manufacturer's instructions.

SDS-PAGE Analysis. Proteins and protein conjugates were analyzed by SDS-PAGE using NuPAGE® Novex Tris-Acetate gels (1.5 mm thick, 15 lanes), NuPAGE Tris-Acetate SDS-Running Buffer, HiMark™ Pre-stained High Molecular Weight Protein Standard and Simply Blue™ SafeStain (Invitrogen). Electrophoresis and subsequent staining steps were performed according to the manufacturer's instructions.

Example 10: Characterization of GBA

Cerezyme^®, a commercially available form of GBA, was subjected to whole-protein mass spectrometry. A protein sample in buffer (1 μΐ) was mixed with acetonitrile/0.1% trifluoroacetic acid (9 μΐ) before being analyzed using electrospray ionization with mass spectrometric detection (ESI-MS). As shown in FIG. 1A, a deconvoluted mass of 60206.74 Da was observed. This detected mass of Cerezyme^® does not match the reported MW of GBA (60430 Da).

Cerezyme^® was subjected to analysis by analytical size exclusion chromatography, as described above. As shown in FIG. IB, a sample of Cerezyme^® comprised 96.8% Cerezyme^® monomer, 2.3% Cerezyme^® dimer or trimer, and 0.9% small molecule impurity.

The stability of Cerezyme^® under different conditions was further analyzed. Cerezyme^® was incubated in buffer at pH 6.3 at varying temperatures (5 °C, 25 °C, and 37 °C) for four days. An additional Cerezyme^® sample was frozen before being thawed on day four. Further, a Cerezyme^® sample was incubated in buffer at pH 7.4 for 320 minutes. Time points of

Cerezyme^® samples were collected at the beginning of the incubation period, at 90 minutes, at 320 minutes, at 1 day, and at 4 days. All collected time points were then analyzed for enzymatic activity. Notably, all Cerezyme^® samples maintained high levels of enzymatic activity up through the last time point (FIG. 2A). This indicates that Cerezyme^® is stable at temperatures ranging from 5 °C to 37 °C, stable in buffers at pH 6.3 and 7.4, and can undergo freeze-thaw cycles. However, while these activity assays did not suggest a significant drop in enzymatic activity for storage of Cerezyme^® at pH 7.4, size exclusion chromatography analysis indicated that longer time periods of storage at elevated pH did lead to an increase in the formation of protein dimers (FIG. 2B). Example 11. Synthesis of PEG-GBA conjugates

Cerezyme^®, a commercially available form of GBA, was exchanged into a pH 6.3 buffer for conjugation reactions. The concentration of Cerezyme^® in all conjugation reactions was approximately 2.5 mg/ml. As shown in Table 1, a PEG-containing reagent was mixed with Cerezyme^® for up to 24.5 hours at either 15 °C or 25 °C. Following the conjugation reaction, each conjugation mixture was analyzed by SDS-PAGE (FIG. 3), size exclusion

chromatography, and assayed for enzymatic activity. Percent conjugation, as defined by the amount of formed PEG-GBA conjugate divided to the total amount of GBA, was determined according to analysis of the size exclusion chromatograms. Enzymatic activity of each conjugation was compared to the enzymatic activity of a Cerezyme^® control experiment. The enzymatic activity relative to the Cerezyme^® control experiment and the percent conjugation of select conjugation reactions are shown in FIG. 4.

Table 1

Claims

CLAIMS What is claimed is:

1. A modified lysosomal enzyme comprising a mannose residue, wherein the mannose residue is modified to comprise a first protecting group that is cleavable under physiological conditions.

2. The enzyme of claim 1, wherein the first protecting group comprises a water-soluble polymer that is conjugated to the mannose residue by a first linker, wherein the first linker is cleavable under physiological conditions.

3. The enzyme of claim 2, wherein the polymer is non-peptidic.

4. The enzyme of claim 2, wherein the polymer is linear.

5. The enzyme of claim 2, wherein the polymer is branched.

6. The enzyme of claim 2, wherein the polymer comprises a poly(alkylene)oxide,

poly(oxyalkylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),

poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and combinations thereof.

7. The enzyme of claim 6, wherein the polymer is comprises a poly(alkylene)oxide.

8. The enzyme of claim 7, wherein the poly(alkylene)oxide is polyethyleneglycol.

9. The enzyme of claim 2, wherein the first linker comprises a moiety selected from: ketone, amide, imide, sulfone, 2-pyridine, ester, orthoester, carbonate, carbamate, acetal, or hemiaminal.

10. The enzyme of claim 1 or claim 2, comprising a plurality of mannose residues.

11. The enzyme of claim 10, wherein about 10%, about 20%, about 30%, about 40%, about

50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the mannose residues are modified to comprise the first protecting group.

12. The enzyme of claim 11, wherein each conjugated mannose residue comprises 1, 2, 3 or

4 conjugated water-soluble polymers.

13. The enzyme of claim 1, wherein the first protecting group is cleavable under pH- dependent conditions.

14. The enzyme of claim 2, wherein the first linker is cleavable under pH-dependent conditions.

15. The enzyme of claim 1 or claim 2, further comprising a thiol residue, wherein the thiol residue is modified to comprise a second protecting group that is cleavable under physiological conditions.

16. The enzyme of claim 15, wherein the second protecting group comprises a water-soluble polymer that is conjugated to the thiol residue by a second linker, wherein the second linker is cleavable under physiological conditions.

17. The enzyme of claim 15, wherein the polymer is non-peptidic.

18. The enzyme of claim 15, wherein the polymer is linear.

19. The enzyme of claim 15, wherein the polymer is branched.

20. The enzyme of claim 15, wherein the polymer comprises a poly(alkylene)oxide,

poly(oxyalkylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),

poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and combinations thereof.

21. The enzyme of claim 20, wherein the polymer is comprises a poly(alkylene)oxide.

22. The enzyme of claim 21, wherein the poly(alkylene)oxide is polyethyleneglycol.

23. The enzyme of claim 16, wherein the second linker comprises a moiety selected from: ketone, amide, imide, ester, sulfone, 2-pyridine, thioamide, thioester, orthothioester, thiocarbonate, thiocarbamate, thioacetal, thioaminal or hemithioaminal.

24. The enzyme of claim 15, wherein the thiol residue is a cysteine residue.

25. The enzyme of claim 15, comprising a plurality of thiol residues.

26. The enzyme of claim 25, wherein about 10%, about 20%, about 30%, about 40%, about

50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the thiol residues are modified to comprise the second protecting group.

27. The enzyme of claim 15, wherein the second protecting group is cleavable under pH- dependent conditions.

28. The enzyme of claim 16, wherein the second linker is cleavable under pH-dependent conditions.

29. The enzyme of claim 16, wherein the second linker is capable of lysosomal cleavage.

30. The enzyme of claim 29, wherein the half-life of the second linker under lysosomal

cleavage conditions is less than 1 day, less than 5 days, less than 10 days, or less than 1 month.

31. The enzyme of any one of claims 1, 2 and 15, further comprising a thioether residue, wherein the thioether residue is modified to comprise a third protecting group that is cleavable under physiological conditions

32. The enzyme of claim 31, wherein the third protecting group comprises a water-soluble polymer that is conjugated to the thioether residue by a third linker, wherein the third linker is cleavable under physiological conditions.

33. The enzyme of claim 32, wherein the polymer is non-peptidic.

34. The enzyme of claim 32, wherein the polymer is linear.

35. The enzyme of claim 32, wherein the polymer is branched.

36. The enzyme of claim 32, wherein the polymer comprises a poly(alkylene)oxide,

poly(oxyalkylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),

poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and combinations thereof.

37. The enzyme of claim 36, wherein the polymer is comprises a poly(alkylene)oxide.

38. The enzyme of claim 37, wherein the poly(alkylene)oxide is polyethyleneglycol.

39. The enzyme of claim 32, wherein the third linker comprises a sulfimide moiety.

40. The enzyme of claim 31, wherein the thioether residue is a methionine residue.

41. The enzyme of claim 31, comprising a plurality of thioether residues.

42. The enzyme of claim 41, wherein about 10%, about 20%, about 30%, about 40%, about

50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the thioether residues are modified to comprise the third protecting group.

43. The enzyme of claim 31, wherein the third protecting group is cleavable under reductive conditions.

44. The enzyme of claim 32, wherein the third linker is cleavable under reductive conditions.

45. The enzyme of claim 32, wherein the third linker is capable of lysosomal cleavage.

46. The enzyme of claim 45, wherein the half-life of the third linker under lysosomal

cleavage conditions is less than 1 day, less than 5 days, less than 10 days, or less than 1 month.

47. The enzyme of any one of claims 1-46, wherein the enzyme is glucocerebrosidase

(GBA).

48. A composition comprising a modified lysosomal enzyme of any one of claims 1-47.

49. The composition of claim 48, further comprising a chaperone molecule, wherein the chaperone molecule stabilizes the conformation of the enzyme.

50. The composition of claim 49, wherein the enzyme is GBA and wherein the chaperone molecule is isofagomine or ambroxol.

51. A method of treating a disorder associated with lysosomal enzyme deficiency,

comprising administering to a subject in need of such treatment a modified lysosomal enzyme of any one of claims 1-47 or a pharmaceutical composition of any one of claims 48-50.

52. The method of claim 51, wherein the disorder is selected from Lewy body dementia,

Parkinson's disease (PD), corticobasal ganglionic degeneration (CBGD), prion disease, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), or Gaucher disease.

53. The method of claim 52, wherein the disorder is Gaucher disease.

54. The method of claim 53, wherein the Gaucher disease is type 1, type 2 or type 3.

55. The method of claim 52, wherein the modified lysosomal enzyme or composition is administered to the subject peripherally.

56. The method of claim 55, wherein the peripheral administration is IV or SQ.

57. The method of claim 52, wherein the modified lysosomal enzyme or composition is administered to the CNS (central nervous system) of the subject.

58. The method of claim 57, wherein the CNS administration is via intrathecal (IT) injection, intracerebroventricular (ICV) injection, or intracysternal injection.

59. The method of claim 52, wherein the modified lysosomal enzyme or composition is administered intraparenchymally by direct injection.

60. The method of claim 52, wherein administration comprises convection enhanced delivery.

61. The method of claim 52, wherein administration is chronic.

62. The method of claim 52, wherein the modified lysosomal enzyme or composition administered using a delivery pump.