WO2019165369A2

WO2019165369A2 - Improving enzyme augmentation therapies by modifying glycosylation

Info

Publication number: WO2019165369A2
Application number: PCT/US2019/019420
Authority: WO
Inventors: Kelsey Moody; Aaron WOLFE; Adam BLANDEN; Kris GROHN; Kathleen Kelly; Jennifer DEROSA
Original assignee: Ichor Therapeutics, Inc.
Priority date: 2018-02-26
Filing date: 2019-02-25
Publication date: 2019-08-29
Also published as: WO2019165369A3

Abstract

Disclosed herein are enzymes with tunable pH dependence for use in treating various diseases, disorders, and conditions. In some embodiments, the enzyme is a peroxidase having a glycosylation pattern that is altered from that of the native enzyme. The enzyme can be administered to a patient with a lysosomal storage disorder, such as macular degeneration, wherein the enzyme is less active in compartments and organelles having physiologic pH, and is relatively more active in compartments and organelles having a pH that is lower than physiologic pH.

Description

IMPROVING ENZYME AUGMENTATION THERAPIES BY MODIFYING

GLYCOSYLATION

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of priority pursuant to 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/635,333, filed February 26, 2018 and entitled“ENZYME AUGMENTATION THERAPIES BY MODIFYING GLYCOSYLATION,” the entirety of which is hereby incorporated by reference for all purposes.

FIELD

[0002] The disclosed processes, methods, and systems are directed to modified enzymes, compositions thereof, and methods of making and using same. The disclosed modified enzymes are useful in, and as, therapeutic treatments of various disorders and indications. In some embodiments, the disorders and indications may involve

accumulations and/or aggregations of compounds in various cellular and tissue

compartments. In one embodiment, the disclosed modified enzymes are administered to a patient suffering from a disease of indication related to lysosomal storage. The disclosed modifications may include“editing” of one or more glycans or saccharides on a protein. Editing may involve adding and/or subtracting glycosylation sites (e.g. amino acid changes) in the protein’s amino acid sequence, and/or by post-translational editing (e.g. adding, subtracting saccharides) of the protein, for example through use of transferases, endoglycosidases, and/or exoglycosidases.

SUMMARY

[0003] Disclosed herein are modified proteins having a glycosylation pattern that differs from that of a native protein (as used herein a native protein has an amino acid sequence that is unmodified and/or a glycosylation pattern that is produced by a eukaryote expressing that protein), wherein the modified protein may be an enzyme with a catalytic activity, at a pH between 4 and 8, that is different than a catalytic activity of the native enzyme. Also disclosed are methods of altering the catalytic activity of an enzyme at a pH between 4 and 8, the method comprising: altering the glycosylation pattern of the enzyme to create a modified enzyme. Also disclosed are methods of treating patient suffering from a disease or condition associated with altered lysosomal storage, the method comprising; modifying a glycosylation pattern of an enzyme to create a modified enzyme; administering the modified enzyme to a patient suffering from the disease or disorder; allowing the modified enzyme to accumulate in the cytoplasm or a cytoplasmic organelle (in one embodiment the lysosome); allowing the modified enzyme to degrade one or more compounds, thereby treating the patient. The glycosylation pattern of the modified enzyme may be modified by: changing one or more amino acid residues within the amino acid sequence of the native enzyme;

contacting the native protein with one or more glycosidases; contacting the native protein with one or more glycotransferases; or a combination thereof. In some embodiments, the modified enzyme is a peroxidase, for example a manganese peroxidase, such as a manganese peroxidase from a fungus that may be Pleurotus ostreatus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 is a graph showing relative specific activity versus pH for three types of one embodiment of a disclosed enzyme that has been: freshly thawed, PNGasF treated, and PNGaseF mock-treated.

[0005] FIG. 2 is a bar graph showing relative specific activity of embodiment of the disclosed enzyme treated with and without PNGaseF wherein the substrate is A2E.

[0006] FIG. 3 is a bar graph showing relative specific activity of enzyme treated with and without PNGaseF, and BSA, wherein the substrate is 2,6-dimethoxyphenol (DMP).

[0007] FIG. 4 is a photograph of a coomassie-stained protein gel showing relative molecular masses of untreated native enzyme, native enzyme treated with PNGaseF, denatured enzyme treated with PNGase F, and PNGase F alone. Left lane shows molecular mass standards.

[0008] FIG. 5 shows ENDO H digestion pattern schematic (upper left), coomassie blue stained SDS PAGE study of rMnP digestion (upper right), and rMnP +/- digestion activity on DMP substrate.

[0009] FIG. 6 shows B14 digestion pattern schematic (upper left), coomassie blue stained SDS PAGE study of rMnP digestion (upper right), and rMnP +/- digestion activity on DMP substrate.

[0010] FIG. 7 shows A(1-2,3,6) digestion pattern schematic (upper left), coomassie blue stained SDS PAGE study of rMnP digestion (upper right), and rMnP +/- digestion activity on DMP substrate.

[0011] FIG. 8 shows A(1-6) digestion pattern schematic (upper left), coomassie blue stained SDS PAGE study of rMnP digestion (upper right), and rMnP +/- digestion activity on DMP substrate. [0012] FIG. 9 graph of activity of rMnP +/- A(1-2,3,6) digestion over 4.5 to 7.8 pH.

[0013] FIG. 10 point graphs of Absorbance vs. Time for non-modified rMnP (top) and

A(1-2,3,6) treated rMnP (bottom graph) over 4.5 to 7.8 pH.

[0014] FIG. 11 graph of triplicate assays for activity of rMnP +/- A(1 -2,3,6) digestion over 4.5 to 7.8 pH; top graph is activity and bottom graph is normalized reaction velocity.

[0015] FIG. 12 graph of activity of rMnP +/- A(1-6) digestion over 4.5 to 7.8 pH; top graph is activity and bottom graph is normalized reaction velocity.

[0016] FIG. 13 bar graphs of normalized reaction velocity for rMnP, rMnP treated with A(1-2,3,6), and A(1-6) at pH 5.1 (top), pH 7.2 (middle), and pH 7.5 (bottom).

[0017] FIG. 14 graph comparing normalized reaction velocity pH profiles for rMnP (blue line), rMnP treated with A(1-2,3,6) (red line), and A(1-6) (grey line).

[0018] FIG. 15 graph of activity of rMnP +/- ENDO H digestion over 4.5 to 7.8 pH; top graph is activity and bottom graph is normalized reaction velocity.

[0019] FIG. 16 graph of second activity assay for rMnP +/- ENDO H digestion over 4.5 to 7.8 pH; top graph is activity and bottom graph is normalized reaction velocity.

[0020] FIG. 17 graph of activity of rMnP +/- A(1-2) digestion over 4.5 to 7.8 pH; top graph is activity and bottom graph is normalized reaction velocity.

DETAILED DESCRIPTION

[0021] Disclosed herein are compounds, compositions, methods, and systems for creating and using modified enzyme compositions for therapeutic use in treating a disease, condition, or indication. In some embodiments, the disease or condition is related to aggregation or accumulation of materials within a tissue compartment or cellular organelle.

In many embodiments, the diseases, conditions, and indications may be associated with accumulation of one or more products (for example an aggregate) in an organelle or tissue compartment. In most embodiments, the organelle or compartment is characterized by a pH that is not physiologic - that is, the pH is less than about 7 and greater than about 7.5. In one embodiment, the disclosed compounds and compositions may be useful in the treatment of aggregate accumulation in lysosomes. In one embodiment the aggregate is lipofuscin, for example lipofuscin build-up in the lysosome of cells in the eye. In most embodiments, lipofuscin comprises the bis-retinoid A2E (N-retinylidene-N- retinylethanolamine). In one embodiment, the disclosed compound is a modified enzyme having a catalytic activity optimized for lysosomal pH, which is typically less than about pH 6. [0022] Disclosed herein are enzymes with altered glycosylation patters, and methods and systems for using them. In many embodiments, the glycosylation pattern of an enzyme may aid in determining the catalytic activity of the enzyme at a given pH. In some embodiments, altering the glycosylation pattern of an enzyme may raise or lower the enzyme’s activity at a specific pH. In many embodiments, the disclosed enzymes are designed to optimize their activity at a pH found in the lysosome.

[0023] In many embodiments, the glycosylation pattern of the enzyme may be altered by changing one or more amino acids in the sequence of the enzyme. Typically, glycans are attached to a protein at specific amino acids (e.g. asparagine, arginine, serine, etc.). Thus, in these embodiments, amino acid changes (e.g. a conservative change such as asparagine to glutamine) may be used to remove or add glycosylation sites within the amino acid sequence of the enzyme. In other embodiments, the glycosylation pattern may be altered with glycosidases, and/or glycotransferases. In some embodiments, altering the

glycosylation pattern may include adding or removing carbohydrates from an amino acid, and/or trimming, reshaping, or altering the oligosaccharide at one or more amino acids of the enzyme.

[0024] The glycosylation pattern of the disclosed enzymes may be modified by removing glycans (one or more saccharides attached to an amino acid side chain), by adding glycans, and/or changing the identity of one or more glycans attached to the enzyme. Thus in various embodiments, the total number of glycosylation sites can be modified and/or the identity of one or more of the glycans attached at those sites may be altered. For example, in mammals N-linked glycans may be of three forms: mannose, hybrid, and complex oligosaccharides. In some embodiments, modifying the glycosylation pattern may include altering the form of the glycan at a specific residue, for example changing a mannose to a complex oligosaccharide.

[0025] In some embodiments, for example in the case of N-linked glycans, use of one or more N-glycosidase may be used to remove one or more N-linked glycans from the enzyme. In other embodiments, one or more glycosyltransferases may be used to add glycans or saccharides to the enzyme. In many embodiments, the amino acid sequence of the enzyme may be altered to alter the pattern of glycosylation.

[0026] In one embodiment, the glycosylation state of the enzyme manganese

peroxidase (MnP) is modified. In these embodiments, modifying the glycosylation of manganese peroxidase may modify the pH profile of manganese peroxidase, for example modification of the glycosylation pattern of manganese peroxidase may alter the enzyme’s activity at a given pH. In these embodiments, modification may result in an enzyme activity that is higher at pH below 6.0 than above 6.0, while the unmodified enzyme may have maximum activity around 7.0. The disclosed modifications may help aid in degradation of the retinoid A2E in organelles or tissue compartments with a pH that is lower than physiologic pH, for example the lysosomes of retinal pigmented epithelial (RPE) cells, where build-up of A2E is associated with age-related macular degeneration (AMD) and Stargardt’s macular degeneration (SMD). The present methods, compounds, and systems may be useful in the treatment of other diseases or disorders associated with lysosomal storage diseases, as well as other conditions.

Glycosylation

[0027] Glycosylation refers to the process of adding sugar molecules to a protein and/or the pattern of sugar molecules attached to a protein. Sugars, or saccharides, may be added to various amino acid side chains, for example: asparagine (‘Asn’ or‘N’), arginine (‘Arg,’ or “R), serine (‘Ser’ or‘S’), threonine (Thr’ or T), tyrosine (Tyr’ or Ύ’), hydroxy-lysine (‘Hyl’ is a modified derivative of lysine‘Lys’ or‘K’), hydroxy-proline (ΉrG is a modified derivative of proline‘Pro’ or‘P’), tryptophan (‘Trp’ or ) and combinations thereof. In most

embodiments, the glycosylation is /V-linked (attached via a side-chain nitrogen) or O-linked (attached to a side-chain oxygen), but other types of glycosylation are known to those of skill in the art, and may be used in modifying the disclosed proteins and enzymes (e.g. C-linked, carbon, and phopho-linked).

[0028] Eukaryotes possess various pathways for glycosylating proteins. Other organisms, including Archea, are also able to glycosylate proteins. In most cases, bacteria do not possess the necessary enzymes or pathways for glycosylating proteins, but can, in some cases, be modified to glycosylate proteins. In some cases the glycosylation patterns of a protein may be the same among various organisms.

[0029] Where a saccharide is attached to an Asn residue, the Asn may be accessible on the surface of the protein, and may be found in the target sequence, for example N-X-S-N- X-T/C (SEQ ID NO:2), or Asn-X (any amino acid)-Ser (serine), Asn-X-Thr (threonine), or Asn-X-Cys (cysteine). In many embodiments, /V-linked glycosylation results from attaching the oligosaccharide to a side chain nitrogen atom of Asn or Arg side-chains. In many embodiments, O-linked glycosylation involves attaching an oligosaccharide to the hydroxyl oxygen of Ser, Thr, Tyr, hydroxy-Lys, hydroxy-Pro. In some embodiments, C-linked glycosylation involves attaching an oligosaccharide to a carbon on a tryptophan side-chain. The present disclosure is directed to methods that modify the glycosylation pattern of a protein, for example an enzyme. In many embodiments, the modification may include changes to the amino acid sequence of the protein and/or changes in the identity or amount of saccharides attached to the protein. In some embodiments, the amino acid that accepts the glycan may remain, but amino acids surrounding that residue may be changed to remove a glycosylation site. In some embodiments, glycosylation sites may be added by introducing an amino acid change that creates a new glycosylation site.

Glycosyltransferases

[0030] Glycosyltransferases are enzymes that aid in attaching saccharides (sugars or carbohydrates) to nitrogen, oxygen, or carbon atoms. In many embodiments,

glycosyltransferases may be useful in building polymeric saccharides (for example linear and branched oligosaccharides and polysaccharides), that is linking two or more saccharide molecules, or they may be used to transfer one or more saccharides to a protein (e.g. an enzyme).

[0031] Disclosed herein are methods of using glycosyltransferases to alter the glycosylation pattern of a protein. In some embodiments, glycosyltransferases may be used to add saccharides to amino acid side chains and/or add saccharides to saccharides that have been previously added to an amino acid side chain. Various glycosyltransferases are known in the art.

[0032] The use of glycosyltransferases may modify a proteins activity at a given pH. In these embodiments, the protein’s activity may be higher at one pH relative to a second pH. In other embodiments, the protein’s activity at one pH may modified to be higher or lower than that of a protein that has not been treated with a glycosyltransferase.

Glycosidases

[0033] Glycosidases may be used to modify the glycosylation pattern of a protein by removing one or more oligosaccharides. In many embodiments, glycosidase treatment may result in altering the proteins activity at one or more pH levels. In some embodiments, this may be used to modify an enzyme’s pH profile. For example, an enzyme’s activity at a given pH may be increased or decreased, relative to that enzyme not treated with a glycosidase. In one embodiment, glycosidase treatment may help to modify a disclosed enzyme’s activity to be high in a low pH environment, for example the lysosome, and lower in an environment with physiologic pH. [0034] Various glycosidases are known in the art. Exoglycosidase hydrolyzes the glycosidic bond at the terminal sugar residue, while nndoglycosidases cleave

polysaccharide chains between residues that are not the terminal residue. For example: a(1 ,2)-Mannosidase specifically cleaves a(1-2)-linked mannose from the non-reducing terminus of glycans; a(1-2,3,6)-Mannosidase releases non-reducing terminal a(1-2,3,6)- linked mannose from oligosaccharides; b-Mannosidase hydrolyzes terminal, non-reducing b- D-mannose residues in b-D-mannosides; Endoglycosidase H hydrolyzes N-linked oligosaccharides; and a1-6 Mannosidase removes unbranched a1-6 linked mannose residues from oligosaccharides. Another glycosidase that may be used to remove or trim N- linked glycosylation is Peptide: N-Glycosidase F, commonly referred to as PNGase F.

PNGase F is an enzyme that catalyzes hydrolysis of amids into a carboxylic acid and NH₃. PNGase F belongs to a class of enzymes referred to as peptide-N4-(N-acetyl-beta- glucosaminyl) asparagine amidases. PNGase F cleaves the glycan between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins and glycopeptides. One of skill in the art may use the disclosed or other glycosidases, alone or in combination, to practice the disclosed method of modifying a protein.

Chemoselective ligation of glycans

[0035] Various chemical transformations have been reported that can chemo-, regio-, and/or diastereoselectively ligate properly protected, functionalized, and activated glycan chains to specific amino acid moieties in proteins. Such chemoenzymatic strategies are often biorthogonal and typically involve the formation of a carbon-heteroatom bond (e.g. transglycosylation reaction). Site-specific bioconjugation reactions target side chains of specific amino acids such as asparagine, lysine, arginine, histidine, tryptophan, cysteine, or tyrosine. In some cases, the /V-terminal amine of peptides or proteins can be selectively targeted for chemical modification as well. Many such reactions are known in the art, and one of skill in the art may use the disclosed or other reactions, alone or in combinations, to practice the disclosed method of modifying a protein.

Physiologic pH

[0036] Most enzymes have optimal catalytic activity at or about physiological pH, which in most cases is about 7.2-7.5. For example the pH of blood is normally about 7.3-7.5, and is normally maintained between about pH 7.35 and 7.45. The intracellular, cytosolic pH of most cells is about 7.2. However, other compartments and fluids may have a pH that is other than physiological. For example, in most cases, the lysosomal pH is lower than intracellular or extracellular pH, for example about 4.5, while the aqueous humor of the eye is about 7.5-7.6.

[0037] pH is a measure of the acidity or basicity of a solution and is written as the negative log of the molar concentration of hydrogen ions in solution. Acidic solutions have a pH less than 7, while basic solutions have a pH above 7. A pH of 7.0 is said to be neutral. Pure water has a pH of about 7.0. pH may be measured electrically or by the use of color changing solutions or compounds. In some cases a colormetric solution may be added dropwise to a test solution, or a test solution may be applied to various papers containing a colormetric compound/solution (test strips). pH may also be assayed electronically by pH meter and comparison to solutions of known pH. pH values may vary +/- 0.1. Thus, a pH of 7.6 may include a range from about 7.5 to about 7.7.

[0038] The disclosed compositions may include one or more proteins that are modified to be more active in one pH compared to another. In many embodiments, the modified protein may be an enzyme with lower activity at physiologic pH compared to non-physiologic pH. In most embodiments, the non-physiologic pH may be 2.5-7.0 or 7.5-10.0, for example less than about 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, or 3.0 and greater than about 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5, or greater than about 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 and less than about 10.5, 10.0, 9.5, 9.0, 8.5, 8.0 and 7.5.

[0039] The difference in activity at physiologic pH versus non-physiologic pH may be 10%-200%. Protein activity may be measured using various methods. For example, where the protein is an enzyme, the maximal velocity (Vmax molecules/time) may be measured. In these embodiments, the Vmax of the modified enzyme at a non-physiological pH greater than 2%, 5%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,

150%, and 200%, and less than about 250%, 200%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. In some embodiments, the difference may be relative to the non-modified protein.

[0040] The disclosed proteins may have pH profile that varies with pH. In most embodiments, the disclosed modified proteins may have a maximal activity at a pH less than about 7 and a minimal activity greater than pH 7.

Tissue Compartments and Cellular Organelles

[0041] The disclosed modified proteins may have enhanced activity in one or more subcellular or tissue compartments. In some embodiments, the modified protein may have enhanced activity, relative to non-modified protein, in the cytoplasm, lysosome, endosome, golgi, secretory vesicle, or mitochondria, and reduced activity, relative to the non-modified protein (or the modified protein at non-physiological pH), at physiological pH. Thus, in many embodiments, the modified proteins may have low activity in compartments, tissues, and organelles, with physiologic pH and higher activity in compartments, tissues, and organelles, with non-physiologic pH. In one embodiment, the modified protein is an enzyme with enhanced catalytic activity at a non-physiologic pH, and/or a greater activity at non- physiologic pH, relative to physiologic pH.

[0042] The disclosed modified proteins may have enhanced activity at one or more tissues. For example the disclosed modified proteins may be useful for treating a disease of the

Lysosomes

[0043] The lysosome is a membrane-bound cellular organelle found in nearly all animal cells. Lysosomes generally contain about 60 different enzymes to metabolize biomolecules within the lysosome’s lumen. The lumen, or interior, of the lysosome has an acidic pH in the range of about 4.5 - 6.5, for example 4.5 - 5.0. Most lysosomal enzymes are optimized to catalyze reactions at this acidic pH. The lysosome is typically thought of as a waste disposal system for the cell, and its enzymes help digest unwanted materials found with the in and around the cell. These materials may come from outside of the cell, or may be the cell’s own materials that have become damaged or are no longer needed. Extracellular material found in the lysosome may be taken-up through endocytosis, and intracellular material through autophagy.

[0044] Large amounts of one compound (e.g. molecule, protein, etc.) may accumulate within the lysosome or other organelle. If the enzymes within that organelle cannot metabolize the compound (or metabolize it inefficiently or slowly), the concentration of the compound or metabolite within that lysosome may accumulate and hinder function of the lysosome.

Lysosomal Storage Diseases

[0045] Lysosomal storage diseases are generally inherited metabolic diseases. In most cases, these diseases and disorders result from the build-up of various materials in a cell’s lysosome. In some cases, these disorders are due to a deficiency in one or more enzymes, or in proteins involved in membrane transport. Due to these deficiencies, large amounts of material may build up in the lysosome, causing it to swell and eventually kill the cell. [0046] Lysosomal storage disorders can affect many different systems and tissues, including the skeleton, skin, heart, and central nervous system. New lysosomal storage disorders continue to be identified. In some cases, a disease may not be, traditionally, considered as a lysosomal storage diseases, but may function similar to that of a lysosomal storage disease. For example, age-related macular degeneration (AMD) is associated with the buildup of A2E, and atherosclerosis is associated with the buildup of cholesterol and 7- keto cholesterol (7KC).

[0047] Because lysosomal storage diseases are caused, generally, by insufficient or ineffective enzymes (due to insufficient quantities, missing, mutant, or damaged enzymes), they can, in some cases, be treated by replacing or augmenting the enzyme. In some embodiments, replacement may be accomplished by expressing a recombinant version of the missing or damaged enzyme. This may be referred to as enzyme replacement therapy, or ERT.

AMD

[0048] Macular degeneration (MD) is the loss of vision in the center of the visual field. It is typically seen in older people and is the major cause of vision loss and blindness in this population. This type of MD is usually referred to as age-related macular degeneration, or AMD. There are three types of MD: early, intermediate, and late. Late type MD has two forms‘dry’ and‘wet’. The dry form accounts for the majority of macular degeneration cases, and is typically the less serious form, caused by the loss of light-sensing cells

(photoreceptors) in the macula. This form results from the buildup of cellular debris in the macula. Specifically, the debris accumulates in an area between the retina and an underlying vascular layer, the choroid. This buildup can result in atrophy of cells in the region, as well as scarring of the retina.

[0049] Retinal pigmented epithelial (RPE) cells are essential support cells found in the macula. The RPE cells are important in that they support the light sensitive photoreceptor cells. In MD, RPE cells experience an accumulation of debris, termed lipofuscin, within the cell’s lysosomes. Enlargement of the lysosomes, due to build-up of lipofuscin, affects the ability of RPE cells to properly support the photoreceptor cells. Eventually the RPE cells may die. Failure and death of RPE cells, in turn leads to death of the photoreceptors and thus a progressive loss of vision. Extracellular accumulations of debris, termed drusen, also increases as MD progresses. Lipofuscin mediated RPE cell death is thought to be a contributing factor to the formation of drusen. As drusen accumulates, it too can destabilize the macular region by contributing to inflammation, complement activation, and other processes. Thus, over time, dry MD progresses to the wet form of macular degeneration, also referred to as neovascular macular degeneration.

[0050] Lipofuscin comprises the bisretinoid N-retinylidene-N-retinylethanolamine (A2E; Figure 1 top) and its photoisomers, which have adverse effects due to their amphiphillicity and photoreactivity. In addition to MD, lysosomal A2E accumulation is also seen in patients with Stargardt disease (SD), and Best vitelliform macular dystrophy.

Atherosclerosis

[0051] One aspect of aging is associated with deterioration of metabolic capacity. This may lead to a reduction in the ability of cells to break down some waste biomaterial, which will accumulate and aggregate over time. Atherosclerosis is associated with the buildup of cholesterol and its oxidized derivatives (particularly 7-ketocholesterol or 7KC) in the lysosome. One way to treat this disease may be to restore or augment the body’s ability to metabolize cholesterol and/or 7-ketocholesterol.

Enzymes

[0052] Enzymes are catalytic proteins. In most cases, a specific enzyme is optimized to catalyze reactions in a specific environment. For example, some enzymes are optimized to survive and be active in the stomach (which is very acidic - 1.5-3.5 pH), while other enzymes are optimized for activity at very high or low temperatures (e.g. taq polymerase is active at 75-80 °C). In most cases, an enzyme that is optimized for one environment will not work, or will work poorly (slowly and or indiscriminantly) in a different environment. Thus, an enzyme intended to catalyze a reaction at physiologic pH may function poorly in the stomach or in a lysosome.

[0053] The disclosed modified enzymes may be optimized to catalyze reactions in an environment that is different than the native, non-modified enzyme’s environment. In many embodiments, the modified enzymes are optimized for activity in the lysosome (i.e. less than about 7.0) while being inactive, or less active, at physiologic pH (i.e. 7.0 - 7.5).

Manganese Peroxidase

[0054] Generally, peroxidases catalyze the oxidation of a molecule using hydrogen peroxide. Various peroxidases are well known in the art. Some peroxidases, for example horse radish peroxidase, and manganese peroxidase are useful in oxidizing aromatic compounds. Aromatic compounds include biomolecules with one or more aromatic carbon ring structure. Manganese peroxidase oxidizes Mnll (Mn²⁺) to form Mnlll (Mn³⁺) in the presence of hydrogen peroxide (H2O2). The Mnlll is stabilized by organic acid to form a diffusible complex that attacks a variety of compounds including aromatic compounds.

[0055] In some embodiments, the disclosed manganese peroxidase is obtained from a fungus, for example Pleurotus ostreatus or Phanerochaete crysosporium. In other embodiments, the peroxidase genes may be from a mammalian or plant source.

Modifying glycosylation by mutagenesis

[0056] The amino acid sequence of the disclosed enzymes may be altered, relative to the native enzyme’s amino acid sequence. In some embodiments, the amino acid sequence is altered to help alter the modified enzyme’s glycosylation pattern. In some embodiments, for example where /V-linked glycosylation is modified, one or more asparagine residues in the native sequence may be changed to a different amino acid (for example to a similarly charged amino acid - e.g. glutamine - or a smaller residue, e.g. alanine or glycine; these may be referred to as conservative mutations) to remove one or more glycosylation sites. In other embodiments, residues other than asparagine may be changed to asparagine to add a glycosylation site. Other target residues may also be changed to add or remove a glycosylation site.

Mutations to or from a glycosylated residue (e.g. Asn) may include non-conservative substitutions or conservative substitutions at those specific positions (conservative/non conservative substitutions may be based on charge, hydrophobicity, size, etc). One such weighting algorithm may be found at homology database server www.clustal.org/clustal2/, among others. Conservative amino acid changes involve substitution of one type of amino acid for the same type of amino acid. For example, where charge is being conserved, changing lysine to arginine is a conservative change, whereas changing lysine to glutamic acid is non-conservative. Where size is being conserved, a change from glutamic acid to glutamine may be conservative, while a change from glutamic acid to glycine may be non conservative. It will be appreciated by one skilled in the art that the above classifications are not absolute and that an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behavior and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids.

[0057] Methods for synthesizing mutated protein sequences are well known in the art. For example, the gene sequence for the disclosed modified enzymes may be synthesized, in vitro mutagenesis and selection, site-directed mutagenesis, error prone PCR,“gene shuffling” or other means can be employed to obtain random and/or directed changes in the protein sequences of naturally occurring genes coding for disclosed enzymes. These methods permit production of a polypeptide having a modified glycosylation pattern relative to native polypeptides. If desired, specific regions of the disclosed enzymes important for glycan attachment can be identified, and targeted for amino acid changes that would result in modifications to the glycosylation pattern. Mutants may also include deletions, insertions and point mutations, or combinations thereof.

Enzyme Augmentation Therapy

[0058] Disclosed herein are methods, compounds, compositions, and systems for treating lysosomal storage diseases. In some embodiments, enzymes may be identified that destroy the material building up in the lysosome. In many embodiments, an exogenous enzyme may be engineered to function in the lysosome and then targeted to the lysosomes of affected cells. This method may be referred to as enzyme augmentation therapy (EAT).

[0059] Targeting one or more enzymes to a cell suffering from lysosomal storage disorder may help to alleviate the disorder. In some cases, a native lysosomal enzyme may be targeted to the lysosome to compensate for a deficient and/or mutant enzyme. In other cases, such as those disclosed here, non-lysosomal enzymes may aid in alleviating lysosomal storage disorders. In these embodiments, the enzyme may not be optimized to work in the acidic environment of the lysosome. In these embodiments, enzymes may be targeted to the lysosome by expressing recombinant modified enzymes with lysosomal targeting tags, or by importing modified enzymes from outside the cell.

[0060] Some enzymes may be modified for high activity at lysosomal pH, and inactive at physiologic pH. This may help to avoid the enzyme from targeting unintended substrate materials. In some embodiments, enzyme optimization may increase the safety, efficacy, and/or stability. In many embodiments, modification to the glycosylation state of the enzyme may be useful in tune the stability and activity of the enzyme at one or more pH.

[0061] The nucleic acids of the present invention can be produced by any synthetic or recombinant process such as is well known in the art. Nucleic acids according to the invention can further be altered to provide for the disclosed modified enzymes. In some embodiments, the nucleic acid sequences coding for the disclosed enzymes may be modified by adding one or more sequences that code for a lysosomal targeting sequence. [0062] The disclosed nucleic acid sequences can include one or more portions of nucleotide sequence that are non-coding for the disclosed enzyme. In some embodiments, the nucleic acid sequences may include one or more regions that aid in expression of the gene in a particular host organism. Host cells, for expressing the disclosed genes, include various organisms that do or do not support glycosylation including bacterial cells such as E. coli, yeast and fungi such as Pichia pastoris, and mammalian cells such as Chinese hamster ovary (CHO) cells and myeloma cells.

EXAMPLES

Example 1 - Comparison of pH dependence of MnP activity +/- glycosylase treatment rMnP production

[0063] Recombinant Manganese peroxide from Phanerochaete crysosporium was expressed in, and purified from, pichia pastoris. Expression was from pGAPZalpha

(Invitrogen) under GAP promoter control in the presence of heme (0.1-1 g/L). The rMnP was engineered to remove the endogenous fungal secretion signal and stop codon, and to include a His tag and a Pichia pastoris secretion tag. Secretion tag exports it into the media, purified by NiNTA.

[0064] The sequence of the MnP gene expressed in P. crysosporium is:

MAFKSLIAFVALAAAVRAAPTAVCPDGTRVSHAACCAFIPLAQDLQETIFQNECGEDAHEVI RLTFHDAIAISRSQGPKAGGGADGSMLLFPTVEPNFSANNGIDDSVNNLIPFMQKHNTISAA DLVQFAGAVALSNCPGAPRLEFLAGRPNKTIAAVDGLIPEPQDSVTKI LQRFEDAGGFTPFE VVSLLASHSVARADKVDQTIDAAPFDSTPFTFDTQVFLEVLLKGVGFPGSANNTGEVASPLP LGSGSDTGEMRLQSDFALAHDPRTACIWQGFVNEQAFMAASFRAAMSKLAVLGHNRNSLI DCSDVVPVPKPATGQPAMFPASTGPQDLELSCPSERFPTLTTQPGASQSLIAHCPDGSMS CPGVQFNGPA, (SEQ ID NO:1).

PNGase treatment of rMnP

[0065] Native rMnP was trimmed using a PNGase F kit purchased from New England Biolabs, Inc (Ipswich, MA) according to the manufacturer’s instructions. Briefly, 30 pL of 17.85 pg/pL rMNP was combined with 53.5 pL of the supplied 10x GlycoBuffer 2, 30 pL of the supplied PNGaseF, and 422 pL of house deionized water. In parallel, 5.6 pL of 17.85 pg/pL rMNP was combined with 10 pL supplied GlycoBuffer2 and 84.4 pL house deionized water as a temperature only control. [0066] As a positive control for deglycosylation by SDS-PAGE, the manufacturer’s PNGase F denaturing protocol was followed. Briefly, 1.1 pl_ of 17.85 pg/pL rMNP was combined with 1 mI_ of supplied 10x denaturing buffer and 8 mI_ of house deionized water, and heated to 100 °C for 10 min. The sample was then mixed with 2 mI_ supplied

GlycoBuffer2, 2 mI_ supplied 10% NP-40, 5 mI_ house deionized water, and 1 mI_ supplied PNGaseF.

[0067] As an rMNP control for the gel and activity assays, 1.1 pL supplied PNGase F, 2 mI_ supplied 10x GlycoBuffer2, and 17 pL house deionized water were mixed.

[0068] All samples were incubated at 37 °C for 24 hours in a thermostated water bath. After 24 hours, samples were run on SDS-PAGE (10% Tris-Glycine), as well as being assayed for activity, as described below.

A2E activity Assay

Buffer Preparation

[0069] A combined 50 mM sodium phosphate/50 mM sodium acetate buffering system containing 150 mM NaCI and 0.2% triton X-100 was prepared as follows: 1.1 g monosodium phosphate, 1 1.2g disodium phosphate, 4.102g sodium acetate, and 8.79 g of NaCI were added to 800 ml_ of deionized water, after mixing the dried material into solution, the volume was brought up to 1 L. 2 M HCI was added continuously in small volumes to reach the following pH conditions: 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5. At each of the specified pH conditions, 50 ml_ were drawn and placed into 50 ml_ conical tubes. From each tube, 100 mI_ of buffer were removed and 100 mI_ of Triton X-100 were added and mixed by repeated inversion until in solution. Buffer conditions were then retested to confirm pH stability prior to use.

Activity assay

[0070] In separate 1.5 ml_ microcentrifuge tubes, 3.47 mI_ of 288 mM rMNP were added to 497 mI_ of each buffer pH condition to get 500 mI_ of 2 mM rMNP solution for each of the 7 pH conditions (4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5). These initial 2 mM mixtures were then serially diluted 1 : 1 with the appropriate buffer to get 10 final enzyme concentrations (2 mM, 1 mM,

0.5 mM 0.25 mM, 0.125 mM, 0.0625 mM, 0.0313 mM, 0.0156 mM, 0.0078 mM, and 0.0039 mM rMNP) for each of the 7 buffers. This protocol was repeated with PNGaseF treated rMNP as above. In addition, 3 enzyme activity curves were assessed using BSA, PNGase-only as described above, and non-PNGaseF rMNP that was incubated in parallel with the PNGaseF treated rMNP. There was no measurable activity in the BSA or PNGase only controls. Samples were relocated immediately to a darkroom under dim red light conditions and 2.2 pl_ of 1.5 mM A2E in DMSO was added to each sample and vortexed to mix. Samples were then incubated at 37 °C on a shaking platform inside a cell culture incubator for 4 hours. Extraction

[0071] Each sample was treated with 250 mI of 2:1 dichloromethane: methanol (166 mI_ dichloromethane, 83 mI_ methanol) then vortex mixed and inverted by hand to stop the reaction by denaturing the rMnP. Samples were centrifuged on a tabletop centrifuge for 10 min. Using a pipette equilibrated with dichloromethane, the lower organic phase of each sample containing dichloromethane and A2E was drawn up and placed into new 1.5 ml_ microcentrifuge tubes. The tubes containing the organic phase were then centrifuged in a speed-vac vacuum concentration system at 37 °C under low vacuum for 45 min to remove the solvent from each sample. 400 mI_ of methanol were then added to each sample, which were thoroughly vortexed for 1 min to dissolve the dried residue. Each sample was centrifuged to pellet any insoluble matter that may damage the HPLC system prior to injection.

Analysis

[0072] A PerkinElmer LC Flexar HPLC system, equipped with a UV/VIS detector and run with Chromera software was used for HPLC analysis (PerkinElmer, Inc. Waltham, Massachusetts, U.S). A reverse phase C18 column (4.6*150mm, 5mM, Cosmosil 5C18-AR- II, Nacalai, Japan) was used for the entire analysis. A mixed solvent system of methanol and water with trifluoroacetic acid (TFA) was used for the mobile phase, run isocratically with a composition of 90% Methanol/10% water with 0.1 % TFA with a flow rate of 1.0 ml/min for a total run time of 10 min. Injection volumes were 10 pL and the column was held at a constant temperature of 26 °C. Detection was performed at 430 nm. Enzyme activity was determined by fitting the linear portion of the A2E concentration vs protein concentration curve and taking the slope. Activity was normalized to the maximum activity observed.

Example 2 - Endoglycosidase H

Digestion of recombinant MnP (rMnP) with Endoglycosidase H (Endo H)

[0073] Synthesis of rMnP and enzyme digestion was carried out as described above. Briefly, rMnP was digested in the presence of enzyme for 1 h, 2h, 3h, 4h, and 5h, with a second 5h digestion in the presence of EDTA at 50 mM. [0074] FIG. 5 includes a diagram of the digestion pattern for Endo H (upper) on an oligosaccharide; green balls are mannose, purple squares are N-acetylglucosamine (GlcNAc), Asn is asparagine on the protein backbone, and P is phosphate. The site of cleavage is shown as a single red line between the two GlcNAc residues. Digestion products were separated by SDS-PAGE (lower) as above (lane 1 , Ladder, 4 uL; Iane2, Predigested rMnP, 10 uL; lane 3, rMnP with ENDO H (1 hour), 10 uL; Iane4, rMnP with ENDO H (2 hours), 10 uL; lane 5, rMnP with ENDO H (3 hours), 10 uL; lane 6, rMnP with ENDO H (4 hours), 10 uL; lane 7, rMnP with ENDO H (5 hours), 10 uL; lane 8, rMnP with ENDO H (5 hours + 50mM EDTA added at T=0), 10 uL; lane 9, Pre SEC, 10 uL; lane 10, Post SEC, 20 uL).

Example 3 - b-Mannosidase

Digestion of recombinant MnP with 3(1 ,4)-Mannosidase (B14)

[0075] Synthesis of rMnP and enzyme digestion was carried out as described above. Briefly, rMnP was digested in the presence of enzyme for 1 h, 2h, 3h, 4h, and 5h, with a second 5h digestion in the presence of EDTA at 50 mM.

[0076] FIG. 6 includes a diagram of the digestion pattern for B14 (upper) on an oligosaccharide; green balls are mannose, purple squares are N-acetylglucosamine (GlcNAc),‘Asn’ is asparagine on the protein backbone, and‘P’ is phosphate. The site of cleavage is shown as a single red line between the second GlcNAc residue and first mannose. Digestion products were separated by SDS-PAGE (bottom) as above (lane 1 , Predigested rMnP, 10 uL; lane 2, rMnP with B(1 ,4) Mannosidase (1 hour), 10 uL; lane 3, Ladder, 4 uL; lane 4, rMnP with B(1 ,4) Mannosidase (2 hours), 10 uL; lane 5, rMnP with B(1 ,4) Mannosidase (3 hours), 10 uL; lane 6, rMnP with B(1 ,4) Mannosidase (4 hours), 10 uL; lane 7, rMnP with B(1 ,4) Mannosidase (5 hours), 10 uL; lane 8, rMnP with B(1 ,4) Mannosidase (5 hours+ 50mM EDTA added at T=0), 10 uL; lane 9, Pre SEC, 10 uL; lane 10, Post SEC, 20 uL).

Example 4 - a(1-2,3,6)-Mannosidase

Digestion of recombinant MnP with a(1 ,-2,3,6)-Mannosidase (A1236)

[0077] Synthesis of rMnP and enzyme digestion was carried out as described above. Briefly, rMnP was digested in the presence of enzyme for 1 h, 2h, 3h, 4h, and 5h, with a second 5h digestion in the presence of EDTA at 50 mM. [0078] FIG. 7 includes a diagram of the digestion pattern for Endo H (upper) on an oligosaccharide; green balls are mannose, purple squares are N-acetylglucosamine

(GlcNAc), Asn is asparagine on the protein backbone, and P is phosphate. The sites of cleavage are shown as red lines between a1-2, a1-3, and a1-6 linked mannose residues. Digestion products were separated by SDS-PAGE (lower) as above (lane 1 , Predigested rMnP, 10 uL; lane 2, rMnP with A(1-2,3,6) Mannosidase (1 hour), 10 uL; lane 3, rMnP with A(1-2,3,6) Mannosidase (2 hours), 10 uL; lane 4, Ladder, 4 uL; lane 5, rMnP with A(1-2,3,6) Mannosidase (3 hours), 10 uL; lane 6, rMnP with A(1-2,3,6) ) Mannosidase (4 hours), 10 uL; lane 7, rMnP with A(1-2,3,6) Mannosidase (5 hours), 10 uL; lane 8, rMnP with A(1-2,3,6) Mannosidase (5 hours + 50mM EDTA added at T=0), 10 uL; lane 9, Pre SEC, 10 uL; lane 10, Post SEC, 20 uL).

Example 5 - a(1-2)-Mannosidase

Digestion of recombinant MnP with a(1-6)-Mannosidase (A16)

[0079] Synthesis of rMnP and enzyme digestion was carried out as described above. Briefly, rMnP was digested in the presence of enzyme for 1 h, 2h, 3h, 4h, and 5h, with a second 5h digestion in the presence of EDTA at 50 mM.

[0080] FIG. 8 includes a diagram of the digestion pattern for Endo H (upper) on an oligosaccharide; green balls are mannose, purple squares are N-acetylglucosamine

(GlcNAc), Asn is asparagine on the protein backbone, and P is phosphate. The site of cleavage is shown as red lines between a1-6 linked mannose residues. Digestion products were separated by SDS-PAGE (lower) as above (lane 1 , Predigested rMnP, 10 uL; lane 2, Ladder, 4 uL; lane 3, rMnP with A(16) Mannosidase (1 hour), 10 uL; lane 4, rMnP with A(16) Mannosidase (2 hours), 10 uL; lane 5, rMnP with A(1-2) Mannosidase (3 hours), 10 uL; lane 6, rMnP with A(16) Mannosidase (4 hours), 10 uL; lane 7, rMnP with A(16) Mannosidase (5 hours), 10 uL; lane 8, rMnP with A(16) Mannosidase (5 hours + 50mM EDTA added at T=0), 10 uL; lane 9, Pre SEC, 10 uL; lane 10, Post SEC, 20 uL).

Example 6 - pH Profile of A2E Digestion by rMnP +/- a(1-2,3,6)-Mannosidase

[0081] Activity of enzymes before and after glycosidase treatment was tested on the substrate A2E as described above in Example 1. Briefly, 30 uL of 182 uM MnP was mixed with 20 pL of 5X A2E activity buffer (250mM Sodium acetate, 250mM Bis-Tris, 750mM NaCI 0.2% Tween 20 pH 7.8-4.5) and 50 pL 90 pM A2E in 0.2% Tween 20. Digestion was for 2 hours at 37 C as follows: rMNP control contained 300 ul 242 pM rMnP + 80 ul 5x activity buffer + 20 ul water; and glycosidase digestion contained 300 ul 242 mM rMnP P +80 ul 5X activity buffer + 20 ul glycosidase (1 Unit of enzyme).

[0082] Results are graphed for pH 4.5 to 7.8 at FIG. 9. This showed that, compared to non-modified enzyme, there was reduced activity of A(1-2,3,6) mannosidase cleaved rMnP at pH conditions greater than about 6.8, and similar activity at pH of 4.5 to 6.0.

[0083] FIG. 10 shows Absorbance vs Time graphs of individual activity studies for the graph shown in FIG. 9. Top graph is undigested rMnP and bottom graph is enzyme treated rMnP.

[0084] FIG. 11 is shows activity and normalized activity, as above, based on three studies where rate was determined over 40-50 minutes.

[0085] These studies confirmed decreased activity at pH above about 6.8, relative to non-modified enzyme and relative to modified enzyme at lower pH.

Example 7 - pH Profile of A2E Digestion by rMnP +/- a(1-6)-Mannosidase

[0086] Activity of enzymes before and after glycosidase treatment was tested on the substrate A2E as described above in Examples 1 and 6. Briefly, 30 uL of 182 uM MnP was mixed with 20 mI_ of 5X A2E activity buffer (250mM Sodium acetate, 250mM Bis-Tris, 750mM NaCI 0.2% Tween 20 pH 7.8-4.5) and 50 pL 90 mM A2E in 0.2% Tween 20.

Digestion was for 2 hours at 37 C as follows: rMNP control contained 300 ul 242 mM rMnP + 80 ul 5x activity buffer + 20 ul water; and glycosidase digestion contained 300 ul 242 mM rMnP P +80 ul 5X activity buffer + 20 ul glycosidase (40 Units of enzyme).

[0087] Results are graphed for pH 4.5 to 7.8 at FIG. 12. In contrast to A(1-2,3,6) treatment rMnP treated with A(1-6) showed similar activity relative to non-modified enzyme and modified enzyme across tested pH.

Example 8 - Comparison of A(1-2,3,6) to A(1-6) at Lysosomal and Physiological pH

[0088] FIG. 13 shows three bar graphs with non-modified rMnP (light grey), A(1 -2,3,6) treated rMnP (grey), and A(1-6) treated rMnP (black). The top graph shows normalized reaction velocity at pH 5.1 , middle graph shows normalized reaction velocity at pH 7.2, and bottom graph shows normalized reaction velocity at pH 7.5.

Example 9 - Comparison of Non-Modified rMnP, A(1 -2,3,6) treated rMnP, and A(1-6) treated rMnP

[0089] FIG. 14 shows all normalized reaction velocities on one graph. Blue line is non- modified rMnP, red line is A(1-2,3,6) treated rMnP, and grey line is A(1-6) treated rMnP. Example 10 - pH Profile of A2E Digestion by rMnP +/- ENDO H

[0090] Activity of enzymes before and after glycosidase treatment was tested on the substrate A2E as described above in Examples 1 and 6. Briefly, 30 uL of 182 uM MnP was mixed with 20 mI_ of 5X A2E activity buffer (250mM Sodium acetate, 250mM Bis-Tris,

750mM NaCI 0.2% Tween 20 pH 7.8-4.5) and 50 mI_ 90 mM A2E in 0.2% Tween 20.

Digestion was for 2 hours at 37 C as follows: rMNP control contained 300 ul 242 mM rMnP + 80 ul 5x activity buffer + 20 ul water; and glycosidase digestion contained 300 ul 242 mM rMnP P +80 ul 5X activity buffer + 20 ul glycosidase (0.1 unit of enzyme).

[0091] Results are graphed for pH 4.5 to 7.8 at FIGs. 15 and 16 (upper graph is activity vs. pH, and lower graph is normalized reaction velocity). As with A(1-6), Example 7, while the two studies showed variability, they demonstrate similar activity relative to non-modified enzyme and modified enzyme across tested pH.

Example 11 - pH Profile of A2E Digestion by rMnP +/- A(1-2)

[0092] Activity of enzymes before and after glycosidase treatment was tested on the substrate A2E as described above in Examples 1 and 6. Briefly, 30 uL of 182 uM MnP was mixed with 20 mI_ of 5X A2E activity buffer (250mM Sodium acetate, 250mM Bis-Tris,

750mM NaCI 0.2% Tween 20 pH 7.8-4.5) and 50 mI_ 90 mM A2E in 0.2% Tween 20.

[0093] Results are graphed for pH 4.5 to 7.8 at FIG. 17 (upper graph is activity vs. pH, and lower graph is normalized reaction velocity). As with A(1-6) and ENDO H, these studies showed similar activity relative to non-modified enzyme and modified enzyme across tested pH.

[0094] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention.

Accordingly, the detailed description is to be regarded as illustrative in nature and not restrictive.

[0095] All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each was included at its citation, in its entirety. In case of conflict between reference and specification, the present specification, including definitions, will control.

[0096] Although the present disclosure has been described with a certain degree of particularity, it is understood the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims.

Claims

CLAIMS We claim:

1. A composition comprising a modified enzyme:

the modified enzyme having a catalytic activity at a pH between 4 and 8 that is different than a catalytic activity of a native, unmodified enzyme with a sequence identity that is greater than 80% identical to the modified enzyme.

2. The composition of claim 1 , wherein the modified enzyme comprises a modified glycosylation pattern that is different than the native glycosylation pattern of the native enzyme.

3. The composition of claim 2, wherein the modified glycosylation pattern of the modified enzyme is the result of removing one or more glycans from the glycosylation pattern of the native glycosylation pattern.

4. The composition of claim 2, wherein the modified glycosylation pattern of the modified enzyme is the result of adding one or more glycans to a native enzyme lacking glycans.

5. The composition of any of claims 2-4, wherein the modified glycosylation pattern is the result of contacting the native protein with one or more glycotransferases and/or glycosidases.

6. The composition of claim 2, wherein the native enzyme comprises an amino acid sequence that is less than 100% identical to an amino acid sequence of the modified enzyme.

7. The composition of any of claims 1-6, wherein the enzyme is a peroxidase.

8. The composition of any of claims 1-7, wherein the enzyme is a manganese peroxidase.

9. The composition of any of claims 1-8, wherein the enzyme is manganese peroxidase from a fungus.

10. The composition of any of claims 1-9, wherein the enzyme is manganese peroxidase from Phanerochaete crysosporium.

11. The composition of any of claims 1-10, wherein the enzyme has an amino acid sequence greater than 80% identical to SEQ ID NO: 1.

12. The composition of any of claims 1-10, wherein the enzyme has a catalytic activity at first pH that is at least 10% lower than a catalytic activity at a second pH.

13. The composition of claim 12, wherein first pH is greater than 7.0, and second pH is less than 6.0.

14. The composition of any of claims 3 or 5, wherein the glycosidase is a(1-2,3,6) mannosidase.

15. A method of creating a therapeutic enzyme for use in a pH environment that is less than about 7.0, comprising:

obtaining an enzyme from a cell;

altering the glycosylation pattern of the enzyme; thereby

creating a therapeutic enzyme for use in a pH environment that is less than about 7.0.

16. The method of claim 15, wherein altering the glycosylation pattern of the enzyme comprises contacting the enzyme with a glycosidase.

17. The method of claim 15, wherein altering the glycosylation pattern of the enzyme comprises contacting the enzyme with a glycotransferase.

18. The method of any of claims 16-17, wherein altering the glycosylation pattern includes removal of one or more glycans.

19. The method of any of claims 16-17, wherein altering the glycosylation pattern includes addition of one or more glycans.

20. The method of claim 16, wherein the enzyme comprises an amino acid sequence that is less than 100% identical to an amino acid sequence of a native enzyme.

21. The method of any of claims 16-20, wherein the enzyme is a peroxidase.

22. The method of any of claims 16-21 , wherein the enzyme is a manganese

peroxidase.

23. The method of any of claims 16-22, wherein the enzyme is manganese peroxidase from a fungus.

24. The method of any of claims 16-23, wherein the enzyme is manganese peroxidase from Phanerochaete crysosporium.

25. The method of any of claims 16-24, wherein the enzyme has an amino acid sequence greater than 80% identical to SEQ ID NO: 1.

26. The method of any of claims 16-25, wherein the enzyme has an amino acid sequence greater than 80% identical to SEQ ID NO: 1.

27. The method of any of claims 16 or 18, wherein the glycosidase is a(1-2,3,6) mannosidase.

28. A method of treating patient suffering from a disease or condition associated with altered lysosomal storage, comprising:

administering a modified enzyme to a patient suffering from the disease or disorder, wherein the modified enzyme has lower catalytic activity at about pH 7.2 than at about pH 6; allowing the modified enzyme to accumulate in the lysosome;

allowing the modified enzyme to metabolize one or more compounds in the lysosome;

lowering the concentration of the metabolized compound in the lysosome, thereby treating the patient suffering from a disease or condition associated with altered lysosomal storage

29. The method of claim 28, wherein the modified enzyme comprises a glycosylation pattern that is different from the glycosylation pattern of a native enzyme.

30. The method of claim 29, wherein the glycosylation pattern of the modified enzyme results from contacting the native enzyme with a glycotransferase.

31. The method of claim 29, wherein the glycosylation pattern of the modified enzyme results from contacting the native enzyme with a glycosidase

32. The method of any of claims 29-31 , wherein the glycosylation pattern of the modified enzyme results from adding one or more glycans to the native enzyme.

33. The method of any of claims 29-31 , wherein the glycosylation pattern of the modified enzyme results from removing one or more glycans from the native enzyme.

34. The method of claim 29, wherein the modified enzyme comprises an amino acid sequence that is less than 100% identical to the amino acid sequence of the native enzyme.

35. The method of any of claims 28-34, wherein the enzyme is a peroxidase.

36. The method of any of claims 28-35, wherein the enzyme is a manganese

peroxidase.

37. The method of any of claims 28-36, wherein the enzyme is manganese peroxidase from a fungus.

38. The method of any of claims 28-37, wherein the enzyme is manganese peroxidase from Phanerochaete crysosporium.

39. The method of any of claims 28-38, wherein the enzyme has an amino acid sequence greater than 80% identical to SEQ ID NO: 1.

40. The method of any of claims 29 or 31 , wherein the glycosidase is a(1-2,3,6) mannosidase.

41. The method of any of claims 28 or 40, wherein the lysosome is a lysosome of a retinal pigmented epithelial (RPE) cell.

42. The method of any of claims 28 or 41 , wherein the compound in the lysosome is A2E.

43. The method of any of claims 28 or 42, wherein the disease or condition is macular degeneration.

44. The method of any of claims 28 or 43, wherein the disease or condition is age- related macular degeneration (AMD).

45. The method of any of claims 28 or 44, wherein the disease or condition is Stargardt’s macular degeneration (SMD).