WO2016198687A1 - Methods and compositions for treating metabolic disorders resulting in sulfite accumulation - Google Patents

Abstract

The present disclosure relates to, inter alia, a pharmaceutical composition comprising sulfite oxidase, including PEGylated sulfite oxidase, mammalian sulfite oxidase variant lacking the heme domain, and mammalian oxygen-reactive sulfite oxidase variant, and methods of their use in treating a sulfite oxidase deficiency or reduced sulfite oxidase activity resulting from molybdenum cofactor deficiency in a subject, or in reducing sulfite level in a subject.

Description

Methods and compositions for treating metabolic disorders resylting in sulfite accumulation

DESCRIPTION

SEQUENCE LISTING

{0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] This application relates to the field of metabolic disorders relating to sulfite accumulation.

BACKGROUND

[0003 ] Sulfite oxidase is responsible for the oxidation of sulfite to sulfate, a reaction that is the final step in the degradation of sulfur-containing metabolites including the amino acids cysteine and methionine.

[0004] Without active sulfite oxidase, sulfite accumulates and reacts

enzymatically with cystine to form S-sutfocysteine and with beta-mercaptopyruvate and/or hydrogen sulfide to form thiosu!fate. Consequently, S-sutfocysteine and thiosulfate accumulate in patients lacking active sulfite oxidase, while in healthy individuals levels of these markers are very Sow or not detectable. Elevated sulfite levels are also found in patients with pneumonia and in patients with chronic renal failure [Mitsuhashi, H. et al., (2004), Shock 21(2) 99-102; Kajiyama, H., et al., (2000) J Am Soc Nephrol. 1 , 923-927]. Affected individuals with sulfite accumulation usually present symptoms such as intractable seizures, metabolic acidosis, intracranial hemorrhage, exaggerated startle reactions, and feeding difficulties. Neurological damage is severe and rapidly progressive as a result of accumulation of toxic levels of sulfite and its metabolites in the brain. Death commonly occurs in the neonatal period, and patients who survive that period usually develop encephalopathy and psychomotor retardation.

[0005] Sulfite oxidase deficiency is a rare autosomal inherited disease caused by mutations in the sulfite oxidase gene, resulting in reduced enzyme activity.

Individuals affected with sulfite oxidase deficiency most commonly present in the neonatal period with intractable seizures, characteristic dysmorphic features, and profound mental retardation.

[00063 Most cases diagnosed as sulfite oxidase deficiency, however, are

actually caused by a deficiency in the molybdenum-containing pterin cofactor, an essential cofactor in the reaction catalyzed by sulfite oxidase. Molybdenum cofactor deficiency ( oCD) is a rare autosomal recessive disorder characterized by the loss of activity of all molybdenum-dependent enzymes, namely sulfite oxidase, xanthine oxidase, aldehyde oxidases and the mitochondria! amidoxide-reducing enzyme (mARC), due to mutations in various molybdenum cofactor biosynthesis genes, including MOCS1 (Type A disorder, observed in about two thirds of patients with the MoCD disorder), MOCS2 (Type B disorder), and gephyrin gene (GPHN) mutations (Type C disorder).

[0007] While substantially reduced sulfite oxidase activity can be fatal, even slightly impaired sulfite oxidase activity may be detrimental to human health.

Accumulation of sulfite and its metabolites can trigger allergic reactions. Symptoms of sulfite sensitivity include asthma, urticaria, angioedema, abdominal pain, nausea, diarrhea, seizures, and anaphylactic shock.

[0008] There is currently no cure for all cases of sulfite oxidase deficiency and all types of MoCD.

SUMMARY

[0009] This disclosure provides a solution to these issues by providing a

pharmaceutical composition comprising a mammalian oxygen-reactive sulfite oxidase variant. In certain embodiments, the mammalian sulfite oxidase variant is one that lacks the heme domain. In certain embodiments, the mammalian sulfite oxidase variant is PEGylated. In certain embodiments, the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSO_A«VATV (SEQ ID NO: 10) and human sulfite oxidase variant HSC APTV (SEQ ID NO: 11).

[0010] In another aspect, a pharmaceutical composition is provided

comprising PEGylated sulfite oxidase. In certain embodiments, the PEGylated sulfite oxidase is a plant sulfite oxidase, or a variant (i.e., a mutant) thereof, or a PEGylated vertebrate sulfite oxidase, or a variant thereof.

[ 0011] In another aspect, a method is provided of treating a sulfite oxidase deficiency, an excess sulfite accumulation, or reduced sulfite oxidase activity resulting from molybdenum cofactor deficiency in a patient, the method comprising administering a pharmaceutical composition disclosed herein or a pharmaceutical composition comprising sulfite oxidase to the patient. In certain embodiments, the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSOAKVATV (SEQ ID NO: 10) and human sulfite oxidase variant

(SEQ ID NO: 1 1 ).

[ 0012 ] In another aspect, a method is provided of reducing sulfite level in a subject, the method comprising administering a pharmaceutical composition disclosed herein or a pharmaceutical composition comprising sulfite oxidase to the subject. In certain embodiments, the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSOAKVATV (SEQ ID NO: 10) and human sulfite oxidase variant HSO_AKVAPTV (SEQ ID NO: 1 1 ).

[0013] In a further aspect, a PEGylated mammalian oxygen-reactive sulfite oxidase variant is provided, in particular a PEGylated oxygen-reactive sulfite oxidase variant wherein the mammalian sulfite oxidase variant lacks part or all of the heme domain; more specifically, wherein the variant is at least one of human sulfite oxidase variant HSOAKVATV (SEQ ID NO: 10) and human sulfite oxidase variant HSO_FIKVAPW (SEQ ID NO: 11 ).

[0014] Numerous other aspects are provided in accordance with these and other aspects of the disclosure. Other features and aspects of the present disclosure will become more fully apparent from the following drawings, detailed description, and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure (FIG.) 1. The heme domain impacts hydrogen peroxide formation in mammalian SO.

[0016] Sulfite-dependent formation of H₂0₂ by murine sulfite oxidase (MSO)

(SEQ ID NO: 13), human sulfite oxidase (HSO) (SEQ ID NO: 9), plant sulfite oxidase (PSO), and their variants, in the presence (filled circles) and absence (open circles) of 0.5 μ catalase is shown in FIG. 1. H₂0₂ formation is shown after 30 min incubation of 1 μΜ wt MSO (SEQ ID NO: 13) (A), wt HSO (SEQ ID NO: 9) (B), PSO (C), MSOi_heme (SEQ ID NO: 14) (D), HSC TV (SEQ ID NO: 10) (E) and HSOA VAPTV (SEQ ID NO: 11 ) (F). Linear regression curves were determined for the activity without catalase (SEQ ID NO: 6) (Slopes: C: y = 0.96x -1.30, D: y = 0.72x +0.92, E: y = 0.41x +0.72, F: y = 0.54x +0.11). All experiments were repeated at least three times (n=3) and results are meansi standard deviation.

[0017] FIG. 2. Heme deletion enables oxygen reactivity of mammalian

SO.

[0018] Influence of heme domain deletion in mammalian SO on sulfite:

ferricyanide activity and H₂0₂ production was investigated in murine and human SO, (A, B) Michaelis-Menten plot of initial ferricyanide: sulfite velocities of murine (SEQ ID NO: 13) (A) and human (SEQ ID NO: 9) (B) heme domain-deleted SO (SOMO)- SO activity was measured at an enzyme concentration of 500 nM (based on Moco saturation) and the kinetic parameters were determined according to Michaelis-Menten fitting (A: _m 87 ± 7 μΜ; Jr_cat 211 ± 6 min^"1, B: Km 46 ± 6 μΜ; k_mt 10⁹ ± 4 min^"1). (C, D) H₂0₂ formation by 1 μΜ murine (SEQ ID NO: 13) (C) and human (SEQ ID NO: 9) (D) SO_MO in the absence (open circles) and presence (filled circles) of 0.5 μΜ catalase (SEQ ID NO: 8) and linear regression curves were determined for the activity without catalase (SEQ ID NO: 8) (Slopes: C: y = 0.68x - 4.04, D: y = 0.71 x -3.43). AH experiments were repeated at least three times (n=3) and results are means± standard deviation.

[0019 ] FIG. 3. Oxygen consumption by p!ant and mammalian SO

variants. [0020] Oxygen consumption rates of PSO and mammalian SO variants were measured using an oxygraph instrument (A) Kinetic determination of the oxygen concentration in the oxygraph with 0.25 μΜ PSO following the addition of 60 μΜ sulfite {arrow). (B) Initial velocities of oxygen consumption by 250 nM of either PSO, MSO (SEQ ID NO: 13), MSO_Aheme (SEQ ID NO: 14), MSO_Mo{SEQ ID NO: 16), HSO (SEQ ID NO: 9), HSO_AKVAT_V (SEQ ID NO: 10), HSOM_VA_PTV (SEQ ID NO: 11) or HSOM_O (SEQ ID NO: 4) following the addition of 60 μΜ sulfite. All experiments were repeated at least three times (n=3) and results are means± standard deviation. (Student's t-test, **P < 0.01 ; ***P < 0.001 ).

[0021] FIG.4. SO expression and activity are low in mouse brain in

comparison to liver and kidney.

[0022] SO expression and activity were investigated in liver, kidney and brain extracts from three different adult mice. (A) Crude extracts (50 g) were separated by a 10% SDS-PAGE and subsequently analyzed by western blot using an anti-SO antibody (upper panel) and an anti-actin antibody (lower panel). (B) SO activity in liver, kidney and brain extracts was determined using the sulfite: cytochrome c assay. SO activity was determined in triplicates using at least three different biological samples (n=3) and results are means! standard deviation.

[0023 ] FIG. 5. PEGylation induces changes in stability and

ollgomerization of SO proteins.

[ 0024] (A) PEGylation of PSO increases its molecular weight in a time- dependent manner. PSO was PEGylated with a 4.2 kDa (kilodalton) branched PEG and aliquots were taken after 0-30 min. (B) PSO was PEGylated with a branched 4.2 kDa and linear 5 kDa PEG. 10 and 20 vg of non-modified and PEGylated PSO were separated by a 12 % SDS-PAGE. (C) Size exclusion chromatography of non- modified and PEGylated (by 0.5 and 5 kDa PEGs) PSO (C) and HSO_Mo (SEQ ID NO: 4) (D) using a HR10/30 Superdex 200 column.

[0025] FIG. 6. PEGylation of SO retains catalytic activity and oxygen reactivity. [0026] PSO and HSO_Mo were PEGylated with a linear 0.5 or 5 kDa PEG and the influence of PEGylation on catalytic activity (A-D) and H₂0₂ formation (E-H) was investigated. Similarly to PSO, substrate inhibition fitting was used for the determination of the kinetic parameters of the PEGylated plant proteins (A: 0.5 kDa PEG and B, 5 kDa PEG), while ichaelis-Menten fitting was used for PEGylated HSOM_O ((SEQ ID NO: 4) C: 0.5 kDa PEG and D, 5 kDa PEG). Activities of the plant and mammalian SOs were measured at concentration of 50 nM and 500 n , respectively and the determined kinetic parameters are summarized in Table 3. (F- H) Suifite-dependent H₂0₂ formation using 1 μΜ of PSO (E, F) or HSO_Mo (SEQ ID NO: 4) (G, H) either PEGylated with 0.5 kDa PEG (E, G) or with 5 kDa PEG (F, H). H₂0₂ quantifications were performed with (open circles) and without (closed circles) addition of catalase (0.5 μΜ) and linear regression curves were determined for the activity without catalase (SEQ ID NO: 6) (Slopes. E: y = 0.93x -1.38, F: y = 0.94x - 1.03, G: y = 0.78x -4.72, H: y = 0.74x -4.60). All experiments were repeated at least three times (n=3) and results are means ± standard deviation.

[0027] FIG. 7. SO prevents suifite-dependent hydrogen peroxide toxicity in HEK cells.

[0028] The ability of plant and human SO to generate H₂0₂ in cultures of HEK cells was evaluated as a function of cell survival using the MTT assay. Ceil viability was investigated in the presence of increasing concentrations of sulfite in the absence of PSO and catalase (SEQ ID NO: 6) (A), with PSO (B) and with PSO and catalase (SEQ ID NO: 6) (C). (D, E) Cell viability was investigated at 0.5 mM sulfite in the absence of protein and in the presence of 0.5 μΜ PSO; PEG-PSO; HSO_MO (SEQ ID NO: 4); PEG-HSO^ (SEQ ID NO: 4) and full length HSO either in the absence of catalase (D) or in the presence of 0.5 μΜ catalase (SEQ ID NO: 8) (E). All experiments were repeated at least three times (n=3) and results are means± standard deviation. (Student's t-test, **P < 0.01 , ***P < 0.001 , ns: not significant).

[002 ] FIG. 8: impact of heme binding and hinge deletions on the folding of mammalian SO.

[00303 (A-C) Disruption of heme binding in MSO (SEQ ID NO: 13) did not impact the folding of the protein. 20 pg of purified wt MSO (SEQ ID NO: 13) and

MSO_Aheme (SEQ ID NO: 14) were separated on a 12 % SDS-PAGE (A) and subsequently analyzed by circular dichrotsm (CD) spectroscopy (B) and differences in the overall secondary structural elements as predicted by CD spectroscopy are presented in (C). (D-E) Tether deletions within the tether connecting the Mo and heme domains in human SO did not disrupt the basic folding of the protein.

Expression and purification of wt HSO (SEQ ID NO: 9), HSC ATV (SEQ ID NO: 10) and HSC APTV (SEQ ID NO: 11) (20 pg) were analyzed by 12 % SDS-PAGE (D) and impact of the tether deletion on folding of HSO was assessed by CD spectroscopy (E) and the resulting distribution of secondary structural elements is depicted in (F). All CD spectroscopy measurements were conducted at a protein concentration of 0.2 mg/ml in a potassium phosphate buffer pH 8.0. CD

spectroscopy was performed using a quartz cuvette with 0.1 cm path length and a total volume of 300 μΙ. Data were collected between 180 nm and 260 nm under a continuous nitrogen flow. Data collection was performed using the JASCO Spectra Manager software (Version 1.53.01, Build 1.0) in Spectrum measurement mode and final data analysis and deconvolution of far-UV CD spectra was conducted with CDNN (Version 2.1).

[0031] FIG. i: Impact of hinge deletions on activity and oxygen reactivity of human SO.

[ 0032 3 (A) The influence of deletions within the linker region between the oco and heme domain was investigated by monitoring the sulfite: cytochrome c activity of wild-type (wt) HSO (SEQ ID NO: 9) and its deletion variants HSO_AKVATV (SEQ ID NO: 10) and HSOAKVAPTV (SEQ ID NO: 11) using 10 n enzyme. The catalytic turnover of the different variants was determined according to ichaelis- Menten fitting (wt: lf_cat 15 ± 0.6 s^'\ HSO_A VAW: feat 5 ±0.4 s^"\ HSO_AKVAPTV: feat 1 ± 0.06 s^"1). (B) Ferricyanide: sulfite activity of PSO. PSO activity was measured at an enzyme concentration of 50 nM and due to the strong inhibition observed at high substrate concentrations, the kinetic parameters were determined using a substrate inhibition fitting model, which resulted in K_m 23 ± 5 μΜ; k_cA 511 ± 41 min^"1 and K_\ 680 ± 140 μΜ. (C) Non-enzymatic reaction of 50 μΜ H₂0₂ with increasing concentrations of sulfite (0 to 50 μΜ) and the corresponding linear regression curve is highlighted in the panel (slope, y = 0.923x +52.60). All experiments were repeated at least three times (n=3) and error bars indicate standard deviation. [0033 ] FIG. 10: Structure of the NHS-PEG molecules used in the study,

[0034] The structure of the 0.5 kDa linear PEG corresponds to a methyl- PEOs-NHS ester with a molecular weight of 509.54 Da and a spacer arm length of

30.8A. (B) The linear 5 kDa PEG used in this study had an average molecular weight of 5 kDa. The length of the sequence was not exactly determined due to its polydisperisty (n). (C) Structure of the branched 4.2 kDa PEG corresponding to a 6- arm branched methyl-NHS-ester PEG with a formula of

and a molecular weight of 4212.12 Da.

[0035] FIG. 11: PEGyfation of SO increases is molecular weight without loss of Moco.

[0036] The PEGylation of PSO, MSO_Mo (SEQ ID NO: 16) and HSO_Mo (SEQ ID

NO: 4) were performed using two linear PEG molecules (0.5 and 5 kDa) and impact on MW and cofactor saturation were investigated. (A) Analysis of non-modified and PEGylated SO proteins by a 12 % SDS-PAGE showing the increase in molecular weight (MW) of the PEGylated proteins. (B) Moco saturation of non-modified and PEGylated proteins was measured by HPLC Form A analysis. Triplicate samples were used in panel B and the experiment was repeated at least three times.

Symbols indicate mean ± standard deviation. (n=3).

[0037] FIG. 12: Size exclusion chromatography of non-modified and

PEGylated murine SO_MO.

[0038] Size exclusion chromatography of non-modified and PEGylated (by 0.5 and 5 kDa PEGs) MSO_MO(SEG ID NO: 16). Proteins were PEGylated at room temperature (25 °C) and analyzed by size exclusion chromatography using a HR10/30 Superdex 200 column.

[0039] FIG. 13: Impact of heme domain deletion and PEGylation on

stability of human SO.

[0040] Human wt SO (wt HSO) (SEQ ID NO: 9), the human heme-deletion variant (HSO_Mo). and HSO_Mo (SEQ ID NO: 4) being PEGylated with a linear 0.5 kDa PEG (0.5 PEG-HSO_MO) or with a linear 5 kDa PEG (5 PEG-HSO_MO) were analyzed by CD spectroscopy (A) and the resulting distribution of structural elements is highlighted in (B). All proteins were analyzed at the same concentration of 0.2 mg/ml and under the same set-up used for the CD spectroscopy analysis of the tether deletion variants of HSO. The stability of the human SO wt, deletion and PEGylated variants was assessed at room temperature (25 °C) by measuring either the sutfiterferri cyanide activity (C) or by measuring cofactor saturation of the proteins through Form A analysis (D) over a time period of 10 hours. SO activity was measured at a protein concentration of 0.5 μΜ and a sulfite concentration of 500 μΜ. All experiments in C and D were repeated at least three times (n=3) and error bars indicate standard deviations.

[0041] FIG. 14: PEGylation of murine SO_MO preserves catalytic activity and oxygen reactivity.

[00 2] Murine SO_Mo was PEGylated with either linear 0.5 or 5 kDa PEGs and the influence of PEGylation on catalytic activity (A, B) and H₂0₂ formation (C, D) was investigated. (A, B) ichaelis- enten plots of ferricyanide:sulfite activity of

PEGylated MSO_Mo with 0.5 kDa (A) and 5 kDa (B). SO activities were measured at a concentration of 500 nM and the kinetic parameters were determined according to Michaelis-Menten fitting (A: K_m 149 ± 11 μΜ; 303 ± 7 min^"1, B: K_m 58 ± 5 μΜ; Jc_cat 175 ± 4 min^"1). H₂0₂ formation was measured after sulfite oxidation using 1 μΜ of PEGylated MSO_Mo (SEQ ID NO: 16) with 0.5 kDa (C) and 5 kDa (D) in the presence (open circles) and absence (filled circles) of purified catalase PSO (0.5 μΜ) and linear regression curves were determined for the activity in the absence of catalase (Slopes- C: y = 0.70x -4,02, D: y= 0.73x -2.58). All experiments were repeated at least three times (n=3) and error bars indicate ±standard deviation.

[00 3] FSG. 15: Hydrogen peroxide-dependent toxicity in the absence and presence of purified catalase.

[00443 Cell viability was investigated in HE cells using the TT assay either in the presence of increasing concentrations of H₂0₂ alone (A) or after

preincubation with 0.5 μΜ purified catalase (B). Ail experiments were repeated at least three times (n=3) and error bars indicate istandard deviation. DETAILED DESCRIPTION

[0045] As used herein, the word "a" or "plurality" before a noun represents one or more of the particular noun. For example, the phrase "a mammalian cell" represents "one or more mammalian cells" or "at least one mammalian cell."

[0046] AH references cited herein, including patent applications and

publications, are hereby incorporated by reference in their entirety. [00 7] Some of the abbreviations used are: SO -- sulfite oxidase; Moco - molybdenum cofactor; PSO - plant sulfite oxidase; HSO_MO - human sulfite oxidase molybdenum domain; MSO_MO— murine sulfite oxidase molybdenum domain; IET - intramolecular electron transfer; PEG - polyethylene glycol; MoCD - molybdenum cofactor deficiency; SOD - sulfite oxidase deficiency. "Sulfite" is also spelled as "sulphite" in the literature; or herein.

[00 8] In mammals, sulfite oxidase is mainly found in the liver, where it

catalyzes the oxidation of sulfite, generated throughout the catabolism of cysteine. Molybdenum (Mo) is bound to the pterin-based molybdenum cofactor (Moco) of sulfite oxidase. Animal sulfite oxidase is a dimeric enzyme, harboring a cytochrome b₅-type heme domain in addition to the pterin-based molybdenum cofactor domain. The catalytic cycle of animal sulfite oxidase involves electron transfer from sulfite to pterin-based molybdenum cofactor, followed by two electron transfer steps via the cytochrome b₅ domain to the terminal electron acceptor cytochrome c. The orthologue plant sulfite oxidase (PSO) lacks the heme domain and thereby constitutes the simplest eukaryotic Mo-enzyme. As a result, electrons derived from sulfite oxidation are directly passed to molecular oxygen, a process generating hydrogen peroxide (H₂0₂) in subsequent steps, which, in the presence of the enzyme cataiase, is further converted into water and oxygen. Mammalian and plant SO are localized in different cellular compartments catalyzing the oxidation of sulfite by coupling electron transfer either to mitochondrial respiration or peroxidation, respectively. As a result, mammalian SO requires a heme domain- mediating electron transfer to cytochrome c, while PSO consists only of a single catalytic domain, which passes electrons directly to molecular oxygen. Neither vertebrate nor bacterial forms of SO were reported to react with oxygen at any appreciable rates, while PSO uses molecular oxygen as electron acceptor for sulfite oxidation and consequently produces H₂0₂. In contrast, with mammalian SOs, H₂0₂ formation is very low. Administration of PSO to animals would likely result in undesirable inflammatory or allergic reactions.

[00 9] The reaction mechanism of sulfite oxidase can be divided into a

reductive and an oxidative half-reaction. In the reductive half-reaction, sulfite binds at the Mo^vl center and is oxidized to sulfate by the transfer of two electrons to the molybdenum center yielding the reduced Mo^ species. According to the respective reduction potentials, one electron is transferred via IET to the heme domain creating a paramagnetic Mo^v intermediate state, which can be detected using electron paramagnetic resonance (EPR) spectroscopy. The oxidative half-reaction is initiated with the transfer of one electron from heme to the final electron acceptor cytochrome c. Then, the second electron can leave the Mo center by a second IET step via heme to a second cytochrome c yielding the fully oxidized form of the enzyme. The crystal structure of plant sulfite oxidase from Arabidopsis thaliana depicted the dose resemblance of the Mo domain between animal and plant SOs, suggesting a similar reductive half-reaction during the catalytic cycle of sulfite oxidation for animal and plant sulfite oxidases. However, the absence of the heme domain in plant sulfite oxidase implicates a different oxidative half-reaction than in animal sulfite oxidase. In fact, it has been shown that the Mo domain of plant sulfite oxidase reacts directly with oxygen, leading to the formation of superoxide ions as the immediate product of the oxidative half-reaction, which is spontaneously d is mutated to H₂0₂. The plant sulfite oxidase reaction with oxygen is unique as none of the vertebrate Mo-enzymes characterized so far are able to react with significant rates with oxygen (i.e., are not oxygen reactive). Furthermore, plant and animal sulfite oxidase share structurally very similar Mo center and residues important for substrate binding are highly conserved.

[0050] Defects in any step of the biosynthesis of pterin-based molybdenum cofactor lead to pterin-based molybdenum cofactor deficiency (MoCD), a rare inherited metabolic disorder resulting in the loss of activity of all Mo-enzymes and affected patients usually die in early childhood. Mutations in the SUOX gene result in sulfite oxidase deficiency (SOD). Both deficiencies are characterized by a severe neurodegenerative phenotype resulting from the accumulation of sulfite and other toxic metabolites such as S-sulfocysteine. MoCD can be grouped into three types according to the underlying genetic defect. Type A deficiency affects two-thirds of all patients and is caused by mutations in the MOCS1 gene. Type B patients accumulate the first Moco intermediate cyclic pyranopterin monophosphate (cPMP) due to defects in the MOCS2 gene. Type C deficiency affects the GPHN gene.

[0051] Until recently, no effective therapy was available for MoCD and death in early childhood was the usual outcome. The ability to purify the first pterin-based molybdenum cofactor intermediate cPMP [Santamaria-Araujo, J. A., Fischer, B., Otte, T., Nimtz, M., Mendel, R. R., Wray. V. and Schwarz, G. (2004) J Biol Chem. 279, 15994-15999] was the starting point for MoCD type A treatment, and since 2010, several cases that received cPMP treatment have been reported for MoCD type A patients. Treated patients were exposed to repetitive intravenous injections of cPMP, which resulted in the restoration of Mo-enzyme activities and the normalization of typical disease biomarkers. However, cPMP is the only reported stable pterin-based molybdenum cofactor intermediate and similar therapies for MoCD type B and C are not feasible.

[0052] In one aspect, a pharmaceutical composition is provided comprising a mammalian oxygen-reactive sulfite oxidase variant. In certain embodiments, the mammalian oxygen-reactive sulfite oxidase variant is a mammalian sulfite oxidase variant lacking the heme domain function, caused by partial or complete deletion, mutational change, or altering the linking peptide between the Moco and heme domain. In certain embodiments, the mammalian oxygen-reactive sulfite oxidase variant is a mammalian sulfite oxidase variant that lacks a functional heme domain, A mammalian sulfite oxidase includes, for example, sulfite oxidase from at least one of human, mouse, monkey, rat, etc. In certain embodiments, the mammalian oxygen-reactive sulfite oxidase variant is PEGylated. In certain embodiments, the pharmaceutical composition further comprises a catalase. In certain embodiments, the pharmaceutical composition may further comprise a hydrogen peroxide

reducing agent or sequester. Mammalian SO variants are able to transfer electrons from sulfite to oxygen, but only with reasonable rates in the absence of efficient heme reduction. Heme-deleted mammalian SO variants can catalyze the formation of H₂0₂. In certain embodiments, oxygen-reactive mammalian SO variants preserve their catalytic activity. In certain embodiments, the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSO_AKVATV (SEQ ID NO: 10) and human sulfite oxidase variant HSC APTV (SEQ ID NO: 11 ). [0053] A mammalian oxygen-reactive sulfite oxidase variant is a variant that, unlike its wild type counterpart, readily reacts directly with oxygen, leading to the formation of superoxide ions as the immediate product of the oxidative half- reaction, which is spontaneously dismutated to H₂0₂, H₂0₂ (which can be present in a stoichiometric or near stoichiometric amount) is thus produced in this reaction, in an amount greater than the amount produced by its wild-type counterpart. A

mammalian oxygen-reactive sulfite oxidase variant includes a SO variant that has the heme domain completely deleted or the heme domain mutated at a heme- coordinating residue or the hinge region (the surface exposed tether peptide connecting the Mo and heme domains) deleted by 5 or six residues, and an SO variant with one or more point mutations in the heme domain and/or in the hinge region, in particular one to 20, preferably 1 to 10, more preferably one to 5 point mutations; and a SO variant with a non-functional or low functioning heme domain: such variants have impaired, reduced, or abolished heme domain-mediated electron transfer (IET) to cytochrome c. Some examples of a human oxygen- reactive sulfite oxidase variants include HSO_AKVATV (SEQ ID NO: 10), (residues 108-110, 112-113 of SEQ ID NO: 9 deleted or residues 86-88, 90-91 of SEQ ID NO: 2 deleted; and HSO_AKVAPTV (SEQ ID NO: 11), (residues 108-113 of SEQ ID NO: 9 deleted or residues 86-91 of SEQ ID NO: 2 deleted). The human SO hinge region is at amino acid residues 105-115 of SEQ ID NO: 9 or at amino acid residues 86-91 of SEQ ID NO: 2.

[0054] The mammalian sulfite oxidase can be from any mammalian species that has a sulfite oxidase. In certain embodiments, the mammalian sulfite oxidase is from mouse, human, rat, or monkey. In certain embodiments, the mammalian sulfite oxidase is from a human.

[0055] The cataiase can be from any source that has a catalase, including from any mammal that has a catalase. In certain embodiments, the mammalian catalase is from mouse, human, rat, or monkey. The catalase can be wild-type or a variant that retains or shows altered function. The catalase can be from the same species as the sulfite oxidase to be administered to a patient, or from different species.

[0056] In another aspect, a pharmaceutical composition is provided

comprising PEGylated sulfite oxidase. In certain embodiments, the sulfite oxidase is a plant sulfite oxidase, or a variant thereof, or a vertebrate sulfite oxidase, or a variant thereof. The pharmaceutical composition may further comprise catalase or a hydrogen peroxide reducing agent or sequester, i.e. a potvdentate (multiple bonded) ligand which is able to form two or more separate coordinate bonds to a single central metal atom.

[0057 ] In yet another aspect, a method of treating a sulfite oxidase deficiency, an excess sulfite accumulation (due to any reason), or reduced sulfite oxidase activity resulting from pterin-based molybdenum cofactor deficiency in a patient is provided, comprising administering an effective amount of a pharmaceutical composition comprising sulfite oxidase to a patient in need thereof. In certain embodiments, the pharmaceutical composition is a pharmaceutical composition described herein. In certain embodiments, the method further comprises administering a pharmaceutical composition comprising a catalase to said patient. The catalase and the sulfite oxidase, or a variant thereof, can be in the same or in a different pharmaceutical composition. In certain embodiments, the method further comprises administering a hydrogen peroxide reducing agent or a sequester.

Hydrogen peroxide is a byproduct of oxygen-reactive sulfite oxidase activity and such reducing agents and sequester can prevent or reduce hydrogen peroxide accumulation.

[0058] In yet another aspect, a method of reducing sulfite level in a patient is provided, comprising administering an effective amount of a pharmaceutical composition comprising sulfite oxidase to a patient in need thereof. In certain embodiments, the pharmaceutical composition is a pharmaceutical composition as disclosed herein. In certain embodiments, the method further comprises administering a pharmaceutical composition comprising a catalase to said patient. The catalase and the sulfite oxidase, or a variant thereof, can be in the same or in a different pharmaceutical composition. In certain embodiments, the method further comprises administering a hydrogen peroxide reducing agent or sequester.

Hydrogen peroxide is a byproduct of sulfite oxidase activity and reducing agents and sequester can prevent or reduce hydrogen peroxide accumulation.

[0059] Impaired sulfite oxidation is the major cause of neuronal cell death in MoCD and SOD. Furthermore, the majority of sulfite in MoCD and SOD originates from peripheral tissue. Sulfite is primarily generated in the liver and kidneys and is transported to the brain via the vascular system. Consequently, the methods and compositions for enzyme replacement therapies of the present disclosure targeting sulfite removal from the blood are effective in preventing sulfite toxicity. The disclosed mammalian SO variants (e.g., the mammalian oxygen-reactive SO variants) can function outside the mitochondria by transferring electrons to oxygen.

[0060] Vertebrate sulfite oxidase includes sulfite oxidase from any vertebrate source that has a sulfite oxidase, such as mammalian, human, bovine, and murine sulfite oxidase. Mammalian sulfite oxidase includes sulfite oxidase from any mammalian source, such as human and mice. Plant sulfite oxidase includes sulfite oxidase from any source, including Arabidopsis sulfite oxidase as described in Eilers et al., (2001) J. Biol, Chem 276, 46989-46994, Chlamydamonas reanhardtii sulfite oxidase as described in Merchant, SS et al., (2007) Science 318(5848) 245- 50, Aspergillus fumigatus sulfite oxidase as described in Nierman WC et al., (2005) Nature 438(7071 ) 1151-8, and others available on GENBANK^®. Reference herein to "sulfite oxidase" includes: sulfite oxidase that is a hybrid between one or more species; a sulfite oxidase that is an enzymatically active fragment; and a sulfite oxidase that is part of an enzymatically active fusion protein, fused with an appropriate other protein(s), peptide(s), or polypeptide(s). "Enzymatic activity" means an activity to catalyse sulfite oxidation, i.e. activity in transferring electrons to oxygen, resulting in H₂0₂, and is measured by sulfite:ferricyanide assay as decribed below.

[0061] Standard method for the determination of sulfite oxidase activity in the full-length animal enzyme: Sulfite oxidase activity of full-length sulfite oxidase can determined use the sulfitexytochrome c-dependent activity assay, which is based on the sulfite-dependent reduction of cytochrome c monitored at 560 rim. Sulfite-dependent reduction of cytochrome c results in increase of cytochrome c absorption at 560 nm. Sulfite oxidase variants with either defects in the catalytic molybdenum domain or the heme domain or the electron transfer between domains will show reduced or absent sulfitexytochrome c activity.

[0062] Alternative method for the determination of partial sulfite oxidase activity at the molybdenum site: Sulfite oxidase activity of full-length sulfite oxidase as well as sulfite oxidase molybdenum domain or variants with impaired electron transfer can be monitored using the sulfite:ferricyanide activity assay, which is based on the sulfite-dependent reduction of ferricyanide monitored at 420 nm. Sulfite-dependent reduction of ferricyanide results in the reduction of ferricyanide absorption at 420 nm. Sulfite oxidase variants with defects in the catalytic moSybdenum domain will show reduced or absent su!fite:ferricyanide activity, while variants with a defective heme domain or defect in the electron transfer will remain active in this assay.

[0063 ] Reference herein to "sulfite oxidase variant" or "sulfite oxidase mutant" includes a sulfite oxidase variant having one or more, in particular one to 20, preferably one to 10, more preferably one to 5 amino acid substitutions, deletions, or additions provided that the sulfite oxidase is active in transferring electrons to oxygen, resulting in H₂0₂. Reference herein to "sulfite oxidase variant" or "sulfite oxidase mutant" includes a sulfite oxidase variant that is a hybrid between one or more species; an enzymatically active fragment; a part of an enzymatically active fusion protein, fused with an appropriate other protein(s) peptide(s), or

polypeptide(s); and cova!entiy or non-covalently modified with an agent, such as PEG.

[0064] Reference herein to "catalase" includes a catalase variant having one or more amino acid substitutions, deletions or additions provided that the catalase is active in catalyzing the reaction of H₂0₂ to water and oxygen determined by a colorimetric method, Catalase activity can be determined by Hydrogen peroxide quantification, which is based on the quantification of the complex formation between xylenol orange and ferric ions, which is produced by the peroxide- dependent oxidation of ferrous iron. The method can be performed using a commercial kit (National Diagnostics) and detection can be carried out

coiorimetrica!ly following the protocol of the manufacturer. Quantification of the product can be carried out after an incubation time of 30 min at room temperature (25 °C) by measuring the absorption at 560 nm. Reference herein to "catalase" includes a catalase from any source, including from human or mouse. Reference herein to "catalase" includes a catalase that is a hybrid between one or more species; a catalase that is an enzymatically active fragment, wherein catalytic activity refers to the before-mentioned activity, namely catalyzing the reaction of H₂0₂ to water; a part of an enzymatically active fusion protein, fused with an appropriate other protein(s) peptide(s), or polypeptide(s); and covalently or non- covalently modified with an agent, such as PEG.

[0065] Proteins are obtained by standard techniques well known in the art, such as by recombinant DNA technology, expression and purification. Mutants are also obtained by standard techniques well known in the art, such as by

recombinant DNA technology. Purification of proteins are also known in the art.

[0066] The sulfite oxidase and the catalase can be further modified covaiently, or non-covalently, by methods known in the art. For example, the proteins may be PEGylated.

[0067] As used herein, "PEG" refers to polyethylene glycol, a water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art. The number of ethylene glycol units in PEG is approximated for the molecular mass described in Daltons. For example, if two PEG molecules are attached to a linker where each PEG molecule has the same molecular mass of 10 kDa, then the 20 total molecular mass of PEG on the linker is about 20 kDa. The molecular masses of the PEG attached to the linker can also be different, e.g., of two molecules on a linker one PEG molecule can be 5 kDa and one PEG molecule can be 15 kDa.

[0068] Covalent modification of proteins with polyethylene glycol (PEG) is known to be a useful method to extend the circulating half-lives of proteins in the body. Other known advantages of PEGylation are an increase of solubility and a decrease in protein immunogenicity. A method for the PEGylation of proteins is the use of PEG activated with amino-reactive reagents like N-hydroxysuccinimkJe (NHS). Any method for the PEGylation of proteins can be used.

[0069] In certain embodiments, a PEG terminates on one end with hydroxy or methoxy (methoxy PEG, mPEG) and is, on the other end, covalently attached to a linker moiety via an ether oxygen bond. The PEG polymer is either linear or branched. Useful PEG reagents are, e.g., available from Nektar Therapeutics. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. Branched PEGs usually have from 2 to 8 arms and are described in, for example, EP-A 0 473 084 and U.S. Pat. No. 5,932,462.

[0070] In certain embodiments, the composition comprises 2-12 PEG

molecules per monomer, 4-10 PEG molecules per monomer, 4, 6, or 8 PEG molecules per monomer or any other number of PEG molecules per monomer as identified as active. In a certain embodiment, the composition comprises methyl PEG8-NHS ester, linear NHS activated PEG with 5kDa in size (polydisperse), linear activated PEG with 10 kDA in size (polydisperse) or branched NHS-activated PEG with 4.2 kDa in size (mortodisperse). In another embodiment the number of PEG molecules attached to sulfite oxidase is 4 moiecu!es per monomer (branched PEG) or 8 molecules per monomer (5 kDa PEG). In certain embodiments, all of the surface exposed lysine of a sulfite oxidase protein or variant SO protein is

PEGy!ated.

[0071] The production of PEGylated sulfite oxidase can be carried out at pH 4-10, pH 6, 7, 8, or 9 using techniques known to persons skilled in the art.

[0072] To achieve maximum PEGylation using methyl-PEG8-NHS ester a higher pH may be used. Use of pH 9 attached 12.5 PEGs. PEGylation at lower pH may avoid pH induced denaturatton of sulfite oxidase. To introduce more PEG groups the sulfite oxidase-amino acid sequence may be varied to include more surface lysine residues, for example by site-directed mutagenesis.

[0073] Any suitable pharmaceutical compositions and formulations, as well as suitable methods for formulating and suitable routes and suitable sites of administration, are within the scope of this disclosure, and are known in the art.

Also, any suitable dosage(s) and frequency of administration are contemplated.

[0074] The pharmaceutical compositions can include a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are

physiologically compatible (see e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing Company, edited by Oslo et al. (e.g. pp. 1435- 1712). The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge et al. (1977) J Pharm Sci. 66:1-19).

[0075] In certain embodiments, the protein compositions can be stabilized and formulated as a solution, microemulsion, dispersion, liposome, lyophilized (freeze- dried) powder, or other ordered structure suitable for stable storage at high

concentration. Sterile injectable solutions can be prepared by incorporating an enzyme in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by sterile filtration.

Generally, dispersions are prepared by incorporating an enzyme into a sterile vehicle that contains a basic dispersion medium. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drytng that yield a powder of a an enzyme plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin.

[0076] The terms "treating™ and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or

underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and improvement or remediation of damage. Thus, for example, the present method of "treating" a disorder encompasses both prevention of the disorder in a predisposed individual and treatment of the disorder in a clinically symptomatic individual. "Treating™ as used herein covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human, and includes; inhibiting the condition, i.e., arresting its development; or relieving or ameliorating the effects of the condition, i.e., causing regression of the effects of the condition.

[0077 ] "Prophylaxis" or "prophylactic" or "preventative" therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it.

[0078] The composition may be administered by any suitable route, such as orally, topically, or parenterally, parenterally being particularly preferred.

[0079] The compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of

administration. The route can be, e.g., intravenous ("IV") injection or infusion, subcutaneous ("SC") injection, intraperitoneal ("IP") injection, pulmonary delivery such as by intrapulmonary injection, intraocular injection, intraarticular injection, or intramuscular ("IM") injection. The composition can be administered by intra-hepatic injection.

[0080] The term parenteral as used herein includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, subconjunctival, intracavity, transdermal and subcutaneous injection, aerosol for administration to lungs or nasal cavity or administration by infusion by, for example, osmotic pump.

Preparations for parenteral administration include, for example, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors, and inert gases and the like.

[0081] A suitable dose can depend on a variety of factors including, e.g., the age, gender, and weight of a subject to be treated. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the disease and/or the extent of the sulfite accumulation. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will depend upon the judgment of the treating medical practitioner (e.g., doctor or nurse).

[0082 ] The treatment may involve administration of one or more other agents.

Such agents can provide supplemental, additional, or enhanced function for sulfite oxidase. Such agents include, without limitation, mo!ybdate, a composition of a cofactor required for sulfite oxidase activity, cyclic pyranopterin monophosphate (cP P), molybdopterin precursor Z and derivatives (see U.S. Patent No. 7,504,095 and WO2012/112922), molybdopterin or molybdenum cofactor, catalase, IV fluids, or cytochrome C. Such agents can be formulated with the composition or may be administered simultaneously or sequentially to a subject being treated.

[0083] The term "subject" is used interchangeably with the term "patient" and includes adults, neonates, and infants, including, for example, those aged less than

6 weeks at the time of diagnosis and aged less than 8 weeks at the start of treatment. A neonate is considered to be a baby from birth to 4 weeks and an infant is considered a baby under 12 months old. The subject may also be aged less than one week at the time of diagnosis and aged less than one week at the start of treatment. The subject may also be older. A "subject," as used herein, can be a human. A "patient" is used herein interchangeably with a "subject." In certain embodiments, the patient (or the subject) is a human patient (or human subject).

[0084] The term "an effective amount" or "a therapeutically effective amount" is a dosage that is sufficient to reduce sulfite levels and/or S-sulfocysteine levels in a subject. Generally, a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition in the subject. The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. The term "an effective amount" or "a therapeutically effective amount" can also be a dosage that is sufficient to elicit a desired medical outcome, such as improved symptoms of MoCD or SOD in a patient, and improved survival of the patient, by any amount of time, including one day. These symptoms can be monitored and tested for using methods known in the art. [0085] In certain embodiments, a therapeutic treatment includes a series of doses, which will usually be administered concurrently with the monitoring of clinical endpoints with the dosage levels adjusted as needed to achieve the desired clinical outcome,

[0086] Appropriate dosages for administering the composition may range from

100 pg/kg body weight to 100 mg/kg body weight, with a 0.2 mg to 100 mg, from

0.55 mg to 85 mg, from 1 mg to 70 mg, or from 2 mg to 60 mg. The compositions can be administered in one dose, or at intervals such as once daily, once every second day, once weekly, and once monthly or for a substantial part or the whole of the lifetime of the patient. Dosage schedules can be adjusted depending on the half-life of the composition, or the severity of the patient's condition. In certain embodiments, the compositions are administered as a bolus dose or continuous infusion, to maximize the circulating levels of the composition of the seventh aspect for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose. A typical dosage regime can involve daily to weekly administration of a composition to a subject at a dose of 10-640 pg/kg, including a dose of 10, 50, 100, 150, 160, 200, 250, 300, 320, 350, 400, 450, 480, 500, 550, 600, or 640 pg/kg.

[0087] Safety and tolerability assessments may be made during the dosage regime. A physical examination including head circumference, neurological examination and imaging (including EEG and MRI), ECG, vital signs (heart rate, non-invasive blood pressure, respiratory rate, and temperature), adverse events, blood gas analysis, blood chemistry (including urea, electrolytes, creatinine, uric acid, and liver function tests), hematology, and urinalysis (including creatinine), s- sulfocysteine [SSC], S-sulfonated transthyretin, homocysteine, cystine, thiosulfate, and dipstick testing for sulfite in urine. Methods for these assessments are known in the art.

[0088] Methods for assessing enzyme activity are known in the art. For

example, sulfite oxidase activity can be determined by the sulfite:ferricyanide assay. For another example, laser flash photolysis can be used to measure the IET rate constants where the heme is present. Methods for measuring oxygen consumption are also known in the art. For example, the oxygen consumption of SO can be determined by using an oxygraph instrument, which allows direct determination of sulfrte:oxygen activity.

[00893 All of the proteins and mutants may be constructed, produced, and purified by standard techniques, such as by recombinant DNA technology.

[0090] Examples

[0091] The following examples are set forth for purposes of illustration only and are not to be construed as limiting the scope of the claims in any manner.

[0092 ] Example 1. Purification of Plant Sulfite Oxidase

[0093] Recombinant hexa-histidine-tagged Arabidopsis sulfite oxidase ("hexa- histidine" disclosed as SEQ ID NO: 7) was made and purified from E. colt. Plant sulfite oxidase (PSO) was expressed as described in Eilers et a!., 2001, J. Biol, Chem. 276, 48989-46994 using the plasmid pQE80-AtPSO. The protein was purified using its C-terminal histidine tagged by metal ion chelate chromatography and ion exchange chromatography (see Schrader et ai., (2003) Structure 11 , 1251- 1263).

Example 2. PEGyiation of PSO

[0095] PSO was PEGylated in order to protect PSO from rapid clearance and degradation and to avoid an immunogenic response to the plant protein. Four different N- hydroxysuccinimide-(NHS) activated PEG molecules were used to modify PSO by PEGyiation: Methyl-PEG8-NHS ester; Linear NHS-activated PEG with 5 kDa in size (polydisperse); Linear NHS-activated PEG with 10 kDa in size (polydisperse); and Branched NHS-activated PEG with 4.2 kDa in size

(monodisperse).

[0096] The PEGyiation efficiency was investigated for the Methyl-PEG8-NHS ester at different pH conditions showing highest PEGyiation with pH 9 (12.5 PEGs attached). To avoid unspecific pH-induced denaturation of PSO pH 8 was chosen for the following PEGyiation reactions. [0097] The PEGylated proteins were de-salted and analyzed by SDS-

PAGE, size exclusion chromatography, and mass spectrometry. The number of attached PEG molecules as determined by ALDI-TOF were; branched PEG: 4 molecules per monomer and 5 kDa PEG: 8 molecules per monomer. Note that the use of SDS-PAGE and size exclusion chromatography cannot accurately determine the number of added PEGs, but does illustrate that the protein still runs as a single band and at a larger, PEGylated size.

[0098] Activity of PEGylated PSO

[0099] To probe if PEGylation affected the catalytic properties of PSO, steady state kinetics of the methyl PE-8 NHS ester (0.5 kDa) modified form of PSO generated at different pH values were performed. The results showed that

PEGylation results in no significant change of the catalytic parameters K_m and k_cat,

[00100] Preliminary animal studies in MOCS1 -deficient mice

[00101] To probe whether PEG-PSO is able to avoid sulfite toxicity in oco-deficient MOCS1 -/- mice, PEG-PSO was injected into these animals. The following preliminary experiments were performed:

[00102] Single Intra-hepatic injection of 5 kDa PEG-PSO (different amounts)

administered only once starting right after birth:

[00103] Table 1.

5 kDa PEG-PSO (per animal) Number of animals Life span (day)

3218pmol 140 pg 2 Mocsl-I- 15 ± 1

322 pmol 14 pg 2 Mocsl-I- 16.5 ± 1.5

32 pmol 1.4 pg 2 Mocsl-I- 11.5 ± 0.5

7.5

0 0 Mocs1-l-

3218 pmol 140 pg 4 Mocs1+l+ >100

PSO (per animal)

3218pmol 140 pg 2 Mocsl-I- 11.75 ± 0.4 [00104] This experiment shows that a single injection of PEG- PSO can significantly extend life span in a murine model as compared to untreated Mocs1-f- mice, which lived an average 7.5 days (Schwarz et al. 2004 Hum. Mol. Genet. 13:1249-1255), All PEG-PSO treated animals showed prior to death no signs of sulfite-induced neurotoxicity. Post-mortem dissection confirmed a gastrointestinal inflammation. As wild type animals treated with PEG-PSO showed no effect (at least up to 100 days), a toxicity of PEG-PSO protein can be excluded. Non- PEGylated PSO did not extend the life span with statistical significance.

[00105] Examples 3-10.

[00106] in these examples, when using either a murine SO variant with

deficient heme binding, or two human SO variants with hinge truncations impairing heme domain mobility, or heme-deficient murine and human variants, reducing or abolishing IET between the Mo and heme domains enabled oxygen reactivity of mammalian SOs. The highest levels of oxygen reactivity were found for the heme- deficient SOM_O variants as well as MSC ne (SEQ ID NO: 14), while intermediate activities were found for the hinge deletion variants of HSO (SEQ ID NO: 9). In particular, HSO_AKVATV (SEQ ID NO: 10) has been shown in previous studies to exhibit an almost 100-fold reduction in IET rate, which in contrast resulted in only three-fold decreased sulfite ytochrome c activity (FIG. 9A). An additional deletion of Pro¹¹¹ (conserved residue in animal SO) [residue number 111 of SEQ ID NO: 9 and residue number 89 of SEQ ID NO: 2] further reduced the steady state activity in HSOAKVAPTV (SEQ ID NO: 11), suggesting a further diminished IET, which correlated well with the increased reactivity towards oxygen.

[00107] PEGylated mammalian SO_MO variants appear as homogenous dimeric proteins in contrast to the non-modified proteins, which showed a high degree of oligomeric heterogeneity, probably due to the lack of heme domain and subsequent exposure of hydrophobic surface patches. PEGylated SO proteins showed only minor changes in their activity as demonstrated by su!fate:ferricyanide steady state kinetics. More importantly, H₂0₂ production and thereby oxygen reactivity was preserved for all PEGylated proteins. [00108] Example 3. Electron transfer between Moco and heme determines

SO reactivity towards oxygen

[00109] SO from chicken ( Galtus gallus) and plant (Arabidopsis thaliana) share 47 % sequence identity despite the lack of the heme domain in PSO. However, only PSO has been reported to react with oxygen, a process involving the formation of superoxide ions, which are further dismutated to H₂0₂.

[00110] H₂0₂ production was determined as a function of sulfite oxidation in both animal and plant SOs. For this purpose, a colorimetric method that quantifies all organic peroxides including superoxide ions and H₂0₂ was used and the exclusive production of H₂0₂was probed by the addition of recombinantly expressed and purified human catalase. Sulfite-dependent H₂0₂ production (for 30 min) of wt murine ( SO) and human SO (HSO) was determined and compared to that of PSO. H₂0₂ formation was low in the presence of MSO, showing 15 μΜ H₂0₂ formation with 75 μΜ sulfite (FIG. 1A) while HSO did not lead to any significant H₂0₂ production (< 2 μΜ, FIG. 1B). In contrast, when using PSO, H₂0₂ formation correlated with the increase in sulfite in a linear manner suggesting a stoichiometric turn over (FIG. 1C). In all experiments, no signal was detected upon the addition of catalase (FIG. 1 , open circles), suggesting that under these experimental conditions only H₂0₂ is formed and detected.

[00111] A heme-defictent murine SO (M_so) variant (MSO^^) (SEQ ID NO: 14) was generated by replacing both heme-coordinating histidines to alanines (H119A, H144A), thus resulting in a loss of heme binding [Kiein, J. M. and Schwarz, G. (2012) Cofactor-dependent maturation of mammalian sulfite oxidase links two mitochondrial import pathways. Journal of Cell Science, 125, 4676-4885].

Disruption of heme binding did not result in any perturbation of the folding of MSO, as assessed by circular dichroism (CD) spectroscopy (FIG. 8 A-C). In contrast to wt MSO (SEQ ID NO: 13), MSO_Aheme (SEQ ID NO: 14) showed a linear sulfite- dependent production of H₂0₂ resulting in more than 50 μΜ H₂0₂ formed out of 75 μΜ sulfite (FIG. 1D), demonstrating that in the absence of a functional heme domain, the reactivity of MSO towards oxygen is favored.

[00112] The impact of deletions of the tether connecting the Mo and heme

domains on oxygen reactivity of human SO (HSO) (SEQ ID NO: 9) was investigated. In mammalian SO, 1ET between the Mo and heme domains is essentia! to complete the catalytic cycle. The 1ET process was extensively investigated in HSO using laser flash photolysis, which enables the measurement of the IET rate constants between both redox centers in HSO and identified the importance of the tether linking Mo and heme during the IET process [Johnson- Winters, K., Tollin, G. and Enemark, J. H. (2010) Biochemistry 49, 7242-7254], Accordingly, deletion of five residues within the tether in the

variant resulted in a strong decrease in the IET rate constant (467 ± 19 s^"1 in wt HSO and 5.59 ± 0.03 s^'1 in HSO_AKVATV) due to restricted domain mobility [Johnson-Winters, K., Tollin, G. and Enemark, J. H. (2010) Biochemistry 49, 7242-7254].

[00113] Therefore, the same HSO deletion variant (HSOA VATV) and a second deletion variant (HSOAKVAPTV) were generated by additionally deleting a proline residue (Pro¹¹¹) within the tether between the Mo and heme domains [Johnson- Winters, K., Tollin, G. and Enemark, J. H. (2010) Biochemistry 49, 7242-7254]. Similar to MSOi_heme, both HSO deletion variants did not display structural changes in comparison to wt HSO as assessed by CD spectroscopy (FIG. 8 D-F). However, both tether deletions in HSO resulted in a dramatic decrease in sulfite:cytochrome c activity as documented by 3-fold and 15-fold decrease in Jc_cal for HSO&KVAW and HSOAKVAPTV, respectively, as compared to wt HSO (FIG. 9 A). When using

HSOA VATV, which is not deficient in heme but shows a reduced IET rate constant [Johnson-Winters, K., Tollin, G. and Enemark, J. H. (2010) Biochemistry 49, 7242- 7254] between Moco and heme, again a linear H₂0₂ production was observed (FIG. 1E). However, the molar ratio of H₂0₂ formed per sulfite dropped from 0.72 to 0.41 as compared to MSO_AHEME (FIG. 1D). The additional deletion of Pro111 in

HSOAKVAPTV further reduced sulfite:cytochrome c activity (FIG. 9A), while the rate of H₂0₂ formation per mole sulfite increased to 0.54 (FIG. 1F).

[00114] Example 4. Mammalian SO molybdenum domains show oxygen reactivity similar to PSO

[00115] To structurally mimic PSO, bacterial expression constructs of the Moco and dimerization domains of murine (MSO_Mo) and human SO (HSO_Mo)

corresponding to the C-terminal 378 residues of both mammalian proteins were generated, recombinantly expressed, and purified. First, these mammalian SO_MO variants were characterized by steady state kinetics using the sulfite: ferri cyanide assay (FIG.2A-B) and compared their activities to that of PSO (FIG. 9B).

Mammalian SO_Mo variants revealed two major differences to PSO. First, SO_Mo variants showed no inhibition at high substrate concentration, which was in contrast to PSO. Second, the determined Jc_cat values for MSO_MO (SEQ ID NO: 16) and HSO_MO (SEQ ID NO; 4) were 2.4 and 4.7-fold lower, respectively, as compared to PSO (FIG. 2 A-B and FSG. 9B).

[00116] Sulfite-dependent H₂0₂ formation was demonstrated for both

mammalian SO_Mo variants, confirming their ability to use molecular oxygen as electron acceptor (FIG. 2 C-D). In contrast to PSO, which showed a nearly stoichiometric (0.98, FIG. iC) correlation between H₂0₂ formation and sulfite concentration (FIG. 1C), H₂0₂ formation mediated by MSO_Mo and HSO_Mo showed a molar ratio between H₂0₂ and sulfite of 0.68 and 0.71 , respectively (Fig. 2 C-D). Knowing that H₂0₂ is able to oxidize sulfite non-enzymatically [Hansen, R., Lang, C, Riebeseel, E., Lindigkeit, R., Gessler, A., Rennenberg, H. and Mendel, R. R. (2006) J Biol Chem. 281, 6884-6888] (FIG. 9C), it is proposed that the reduced catalytic activity of the SO heme-deleted variants measured by sulfite.ferricyanide assay (FIG.2A, B) favored the non-catalytic sulfite oxidation by H₂0₂ (compare FIG. 1D-F), thus leading to an overall reduction in H₂0₂ accumulation. Additionally, H₂0₂ was almost not detected at low sulfite concentrations (below 10 μΜ) using the mammalian SO_Mo variants (FIG. 2 C-D). This is due to the fact that under aerobic conditions and low concentrations, sulfite is susceptible to air oxidation, which combined with the non-catalytic sulfite oxidation mediated by H₂0_2l might explain the detection limit of H₂0₂ at low sulfite concentrations.

[00117] Example 5. Reduced IET increases oxygen consumption in

mammalian SO

[00118] Next, the oxygen consumption of SO variants was determined by using an oxygraph instrument, which allows direct determination of sulfite:oxygen activity. Addition of 60 μΜ sulfite to 0.25 μΜ PSO induced a rapid decrease (within 5 seconds (s)) in the oxygen concentration from 190 μΜ to 124 μΜ, which correlated well with the amount of added sulfite, thus suggesting a stoichiometric conversion of sulfite into H₂0₂ (FIG. 3A), The resulting velocity of 14 μΜ s ¹ (Fig. 3B) converts to a rate of 56 s^'1, which is comparable to the rates reported previously [Hemann, C, Hood, B. L, Fulton, M., Hansch, R., Schwa rz, G., Mendel, R. R., Kirk, M. L. and Hiile, R. (2005) J Am Chem Soc. 127, 16567-16577]. However, under these assay conditions, inhibition at substrate concentrations higher than 80 μΜ was observed for the PSO-cataiyzed reaction (FIG. 9B). Therefore, under saturating sulfite concentrations, a direct comparison of the oxygen consumption rates of plant and mammalian SO variants is not feasible.

[001193 Oxygen consumption rates were measured for all mammalian SO

variants (FIG. 3B). The activity of MSO and HSO remained very low (1-2 μΜ s^"1) supporting the previous results of H₂0₂ formation (FIG. 3B). Comparison of MSO with its heme-deficient and domain variants or HSO with its deletion and domain variants, showed an increase in oxygen consumption, which correlates with the respective decrease in lET between Moco and heme in the HSO deletion variants (measured by sulfitexytochrome c activity). Consequently, oxygen reactivity was highest when the heme domain was completely absent (using heme-deficient or SO_Mo domain variants, FIG. 3B).

[00120] Accordingly, the absence of the heme cofactor or a reduced IET results in a strong increase in oxygen reactivity of mammalian SO, thus suggesting that oxygen is competing with the heme cofactor for the electrons derived from sulfite oxidation.

[00121] Example 8. Mouse brain lacks capacity for su!fite oxidation

[00122] Given that SO is the most important Mo-enzyme in mammals and that both SOD and fvloCD are mainly characterized by sulfite-induced neuronal cell death. The extent to which sulfite is oxidized in the mouse brain was determined.

[00123] SO expression levels and SO activities were determined in mouse liver, kidney and brain crude protein extracts. SO protein was not detectable in the brain while liver and kidney showed strong expression (FIG. 4A). Furthermore, SO activity was nearly undetectable in the brain, while liver and kidney revealed high activities (FIG. 4B) as expected from the SO expression levels (FIG. 4A). Knowing that liver is characterized by a high capacity for sulfur amino acid catabolism

[Stipanuk, M. H., Ueki, I., Dominy, J. E., Jr., Simmons, C. R. and Hirschberger, L. L. (2003) Amino Acids. 37, 55-63], it is not surprising that sulfite is primarily generated in the liver [Stipanuk, M. H., Ueki, I., Dominy, J. E., Jr., Simmons, C. R. and Hirschberger, L. L. (2009) Amino Acids 37, 55-63]. Accordingly, a treatment option for MoCD and SOD could rely in removing sulfite from the circulation, which would be favorable in preventing sulfite-induced brain damage.

[00124] Example 7. PEGylatlon of SO induces changes in stability and oligomerizatton

[00125] Up to now, a replacement therapy for MoCD and SOD using

mammalian SO is not feasible due to the requirement for mammalian SO translocation into mitochondria where its native electron acceptor, oxidized cytochrome c, is localized. The ability of mammalian SO_Mo domain variants to use oxygen as electron acceptor offers the possibility for the development of an enzyme replacement therapy towards MoCD and SOD, in which SO variants can use dissolved oxygen in blood for sulfite oxidation.

[00126] To increase the stability of SO_Mo proteins and to suppress potential immune responses, protein PEGyiation was investigated as a modification method.

[001273 For PEGyiation, N-hydroxysuccinimide (NHS) activated PEG

molecules were used that were covalently coupled to surface-exposed lysine residues of different SO variants. This method is based on the reaction of activated PEG esters with primary amino groups of the protein, which lead to the formation of stable amide bonds between lysine residues and PEG molecules [Roberts, M. J., Bentley, M. D. and Harris, J. M. (2002) Adv Drug Deliv Rev. 54, 459-476]. Thus, the number of PEG molecules attached on the surface of the protein is directly proportional to the number and the accessibility of surface exposed lysine residues. As a result, the molecular weight ( W) of the modified protein increases according to the number and MW of the attached PEG molecules.

[00128] First, PSO was PEGylated and parameters such as incubation time and structure of PEG molecules on the efficacy of modification were investigated (FIG. 5 A and B). When using a 4.2 kDa PEG molecule, approximately 50 % of PSO was PEGylated within 2 minutes (min), as depicted by a shift in MW and within 30 min, PEGyiation was nearly completed (FIG. 5A). Next, two PEG molecules differing in size and structure were used to PEGylate PSO: a branched (4.2 kDa) and a linear PEG (5 kDa) (FIG. 10). SDS-PAGE analysis showed an increase in MW of all PEGylated proteins demonstrating that the PEG molecules were covalently bound (FIG. 5B). The exact MW of the PEGylated proteins could not be determined by SDS-PAGE, as they displayed a heterogeneous distribution pattern in the gel (branched PEG) or the apparent MW exceeded the resolution range of the gel (linear PEG). Therefore, mass spectrometry was applied and the number of added PEG molecules was determined based on the apparent mass increase. Four and eight PEGs were found coupled to PSO by using the branched or linear PEG, respectively (Table 2),

[00129] Due to the higher coupling efficiency of PSO with the linear 5 kDa PEG, MSO_Mo and HSO_Mo were PEGylated using two sizes of linear PEGs (0.5 and 5 kDa, FIG. 10). Modified proteins were separated from excess PEG molecules by size exclusion chromatography (FIG. 5 C and D). SDS-PAGE analysis showed again a shift in the MW of all PEGylated proteins (FIG. 11 A). PEGylation with the 5 kDa PEG resulted in proteins displaying a heterogeneous band pattern, which was due to the polydisperse nature of the PEG used. When using the monodisperse 0.5 kDa PEG, modified proteins displayed a single sharp band within the SDS-PAGE (FIG. 11 A). Furthermore, PEGylation of SO proteins did not change their oco content (FIG. 1 B).

[00130] Size exclusion chromatography confirmed the corresponding increase in MW of PEGylated PSO (FIG. 5C). Size exclusion chromatography of non- modified PSO showed a single peak corresponding to a PSO monomer as described before [Eilers et al. (2001) J. Biol. Chem. 278, 46989-46994] and PEGylation resulted in a clear reduction of the elution volume indicating an increase in MW, which was greater when using 5 kDa PEG molecules in comparison to 0.5 kDa PEG (FIG. 5C). However the increase in MW of the

PEGylated proteins did not correlate with the MW and number of added PEG molecules as previously determined by mass spectrometry (see Table 2). In fact, PEG is a highly soluble amphiphilic polyether diol that can be linear or branched. The increase in MW of PEGylated proteins is mainly due to the large hydrodynamic volume of the PEG and not only due to its MW as depicted by differences in the MW determined for PEGylated PSO by SDS-PAGE and mass spectrometry. PSO modified with a 5 kDa linear PEG showed in SDS-PAGE an apparent MW of 170 kDa, while by mass spectrometry the determined MW was 85 kDa, which corresponds to the addition of eight PEG molecules per monomer. Furthermore, the number of attached PEG molecules to the protein largely depended on the chemistry of the PEG molecule used, as depicted by the differences between PSO PEGylated with either the branched or the linear PEG (see Table 2). The number of added PEG molecules was twice as much when using the linear 5 kDa PEG as compared to the branched one, while both PEG molecules had a similar MW. This observation can be explained by steric hindrance resulting from the large surface occupied by branched PEGs as compared to linear PEGs. In addition, all

PEGylated proteins eluted differently to globular proteins on size exclusion chromatography, resulting in earlier elution volumes, which did not correlate with the increase in MW. Therefore the unequivocal determination of the oligomeric state or the apparent MW of PEGylated proteins was not possible using size exclusion chromatography, as this method is based on the elution volume of standard globular proteins,

[00131] Table 2. Mass spectrometric identification of the molecular weight of PEGylated PSO

Molecular Number of added

Protein

weight (kDa) PEG molecules

PSO 44 -

Branched

PEG-PSO (5 63 4

kDa)

Linear PEG-

85 8

PSO (5 kDa)

[00132] Human SO_Mo (HSO½₀) displayed a heterogeneous elution profile

depicted by the presence of three peaks corresponding to monomers, dimers and high molecular oligomers (FIG. 5D), However, PEGylation of HSO_Mo variants resulted in the formation of a single homogenous peak in the size exclusion chromatogram (FIG. 5D) and similar results were obtained using the murine SO_Mo variant (FIG. 12). Dimerizatiori of Mo-enzymes is expected to follow the

incorporation of Moco [Mendel, R. R. and Schwarz, G, {2011} Coordin. Chem. Rev. 255, 1145-1158] and it has been shown that mammalian SO dimerization and maturation is independent of heme binding [Klein, J, M. and Schwarz, G. (2012) Journal of Ceil Science. 125, 4876-4885], Neither deletion of the heme domain in the HSOMO variant nor PEGylation of HSO_Mo resulted in major structural changes in comparison to wt HSO as assessed by CD spectroscopy (FIG. 13 A and B).

Furthermore, stability of wild type (wt) HSO, HSO_Mo and PEGylated HSO_Mo proteins were not affected as Moco saturation as well as sutfite:cytochrome c activities remained stable over a time period of 10 h (FIG. 13 C and D). Thus, it appears that the impact of HSO_Mo PEGylation observed in size exclusion chromatography does not rely on an increased stability but is rather due to an increased solubility due to the amphiphilic character of the PEG molecules used.

[00133] Example 8. PEGylation of SO preserves Is catalytic activity

[00134] Following PEGylation, a change or loss in catalytic activity may occur, if the attached PEG molecules hinder substrate-binding, conformational flexibility and/or catalysis. Therefore, first sulfite: ferricyanide steady state kinetics of

PEGylated PSO, MSO_Mo and HSO_Mo were determined. PEGylation of SO proteins did not result in major changes in the corresponding kinetic parameters (FIG. 6 A- D, Table 3 and FIG. 14 A and B). PEGylation of PSO with 5 kDa PEG did not alter its catalytic turn over (k of 483 min^"1 versus 511 min^"1), whereas a slight increase in Jc_cat was observed when the 0.5 kDa PEG was used (Jr_catof 650 min^"1 versus 511 min^"1). On the other hand, an approximately two-fold increase in K_m was determined for both PEGylated PSO proteins as compared to native PSO. See Table 3.

[00135] Table 3. Kinetic parameters of non-modified and PEGyiated SO proteins

PSO 23 ± 5 511 ± 41 22 ± 8 0.68 ± 0.14

PEG-PSO (0.5 kDa) 68 ± 6 651 ± 29 9.5 ± 4.7 1 .38 ± 0.18

PEG-PSO (5 kDa) 63 ± 7 483 ± 28 7.7 ± 3.5 2.42 ± 0.60

MSOMO 67 ± 7 211 ± 6 3.2 ± 0.8

PEG-MSOMO (0.5 kDa) 149 ± 11 303 ± 7 2 ± 0.7

PEG-MSOMO (5 kDa) 58 ± 5 176 ± 4 3 ± 0.8

HSOMO 46 ± 6 109 ± 4 2.4 ± 0.6

PEG-HSOMO (0.5 kDa) 102 ± 7 111 ± 2 1.1 ± 0.3

PEG-HSOMO (5 kDa) 110 ± 5 134 ± 2 1.2 ± 0.4

[00136]

¹ Kinetic data resulting from the activities of plant SOs were fitted using a substrate inhibition mode! and an additional kinetic parameter K, was calculated.

[00137] Influence of PEGylation on the activity of mammalian SO_Mo proteins was similar to that observed for PSO, resulting in an approximately two-fold increase in K_m with either similar or even increased fc_cat values as compared to those of the non-modified enzymes (FIG. 6 C and D, Table 3 and FIG, 14 A and B). Furthermore, PEGylation of PSO and mammalian SO_Mo variants did not alter their ability to generate H₂0₂. All PEGyiated SO proteins showed similar levels of H₂0₂ production as compared to non-modified proteins (FIG. 6 E-H and FIG. 14 C and D). Interestingly, also here a very low H₂0₂ concentration was detected at low sulfite concentrations (below 10 μΜ) for the PEGyiated proteins, which was more visible using mammalian SO_Mo variants, attesting for a higher contribution of the non-catalytic sulfite oxidation mediated by H₂0₂ in mammalian SOM_O variants in comparison to PSO (FIG. 6 E-H and FIG. 14 C-D).

[00138] Example §, SO is able to catalyze sulfite oxidation using dissol ed oxygen in cell culture [00139] Previous examples (examples 3-8) showed that mammalian SO variants are able to react with oxygen in vitro. Furthermore, it was shown that PEGylation as a masking method, consisting of shielding the protein by the cova!ent attachment of PEG molecules to the surface-exposed lysine residues of the protein, did not cause loss of activity. The capacity of both plant and human SOs to use oxygen as an electron acceptor, thus generating H₂0₂ were evaluated in a cell-based assay. For this purpose, human embryonic kidney cells (HEK 293) were exposed to SO-dependent H₂0₂ toxicity. Cell viability was determined using the MTT (3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide) cell

proliferation assay. Already very low amounts of H₂0₂-induced toxicity in HEK cells, which was prevented if purified human cata!ase was co-incubated with the cells exposed to H202 (FIG. 15 A and B).

[00140] Next, PSO-mediated H₂0₂ generation was probed. Exposing HEK cells only to sulfite concentrations up to 0.5 m was not toxic, given that those cells express functional SO (FIG. 7A). However, when HEK cells were co-incubated with 0.1 mM sulfite (and higher concentrations) and PSO, a significant cell death with only 20 % cell viability was measured (FIG. 7B). PSO-mediated toxicity was prevented when purified catalase was added (FIG. 7C), being in line with the PSO- dependent formation of H₂0₂ in the presence of sulfite. Based on these results, 0.5 mM sulfite was used and the sulfite-dependent H₂0₂ formation of non-modified and PEGylated PSO and HSO_Mo were investigated as well as full length HSO. Similar to PSO, HSOMO as well as the PEGylated proteins were equally effective in inducing cell death resulting in 20 % cell survival as compared to control (FIG. 7D), and toxicity was again prevented if purified catalase was added (FIG. 7E). In contrast, full length HSO was not able to induce cell toxicity, which again confirms its inability to accept oxygen as a suitable co-substrate (FIG. 7D).

[001 13 Example 10, Molecular Biological Methods and Materials Used for Examples 3-9.

[00142 ] Expression constructs for human and murine SO molybdenum domain (HSOMO and MSO_Mo) were generated by cloning the coding sequence for the dimerization and oco domains (HSO, GEN BANK® accession number

AY056018.1 , residue 1 10 to 488; MSO, GENBANK* accession number

BC027197.1 , residues 168 to 546) into pQE80L (QIAGEN, Valencia CA) using Sail and Hindi!! restriction sites. The same restriction sites were used for cloning of HSO WT. HSO deletion variants HSOAKVATV and HSC AFTV were generated by fusion PGR and cloned into the pQE80L vector using Sac! and Sal! restriction sites. MSO (wt) and MSO_Aheme were generated as previously described [Klein, J. M. and Schwa rz, G. (2012) Journal of Cell Science 125, 4876-4885]. For catalase expression, the coding sequence of human catalase (GenBank^® accession number BC110398.1 ) was PCR-cloned into pQE80L using Sail and Hindll! restriction sites. For recombinant expression of PSO, the previously described rAt-SO construct was used [Eilers, T„ Schwarz, G., Brinkmann, H., Witt, C, Richter, T., Nieder, J., Koch, B., Hille, R., Hansch, R. and Mendel, R. R. (2001) J Biol Chem. 276, 46989-46994].

[00143] Protein Expression and Purification

[00144] All SO proteins were expressed in E. co/ TP 1000 [Temple, C. A., Graf, T. N. and Rajagopalan, K. V. (2000) Archives of biochemistry and biophysics. 383, 281-287] as previously described [Belaidi, A. A. and Schwarz, G. (2013)

Biochemical Journal 450, 149-157]. The strain is available from Tracy Palmer, Head of Division of Molecular Microbiology, School of Life Sciences, University of Dundee (http://www.lifesci.dundee.ac.uk groups/tracy_palmer/moco.html). Human catalase was expressed in £ colt BL21 (DE3). The strain is available under catalog No. C2530H from New England Biolabs Inc.JExpression was induced with 0.1 mM isopropyl β-thiogalactoside at OD600 = 0.1 and continued for 15 h at 30°C. All His- tagged proteins were purified by nickel nitrilotriacetic acid affinity as recommended (QIAGEN, Valencia CA). For plant SO, a second purification step consisting of an anion exchange chromatography was performed as previously described

[Schrader, N.» Fischer, K., Theis, K., Mendel, R. R., Schwarz, G. and Kisker, C. (2003) Structure 11 , 1251-1263.]. All purified proteins were exchanged into the same buffer (20 mM Tris HCI pH 8.0, 50 mM NaCI) and stored at -80 °C.

[00145] Determination of Moco saturation

[00146] Moco saturation was determined by denaturing 500 pmol of protein using acid iodine oxidation and alkaline phosphatase treatment resulting in the formation of the stable Moco oxidation product FormA-dephospho, which was further quantified using HPLC reverse phase chromatography as described [Klein, E. L, Belaidi, A. A., Raitsimring, A. M., Davis, A. C, Kramer, T„ Astashkin, A. V., Neese, F,, Schwa rz, G. and Enemark, J. H. (2014) inorganic Chemistry 53, 961- 971].

[00147] Determination of SO activity

[001 8] For PSO, HSOM_O and MSO_Mo, activities were either measured using the sulfite: fern cyanide or sulfite ytochrome c assay. Sulfite: ferricyanide activity was measured by monitoring the reduction of ferricyanide (Potassium hexacyanoferrate: Fe{CN)_s) at 420 nm (ε420=1020 M^"1 cm ) [Cohen, H. J. and Fridovich. i. (1971 ) J Biol Chem. 246, 359-366]. The assay included the following components: 140 pi of 100 mM Tris/acetate pH 8, 20 μΙ of protein solution, 20 μΙ of 4 mM Fe(CN)₆ and the reaction was started by the addition of 20 μΐ of sodium sulfite (varying

concentrations). Activities were measured at an enzyme concentration of 50 n and 500 nM for the plant and mammalian SO proteins, respectively.

[001 9] Sulfite: cytochrome c activity was determined for HSO and deletion variants HSO_AKVATV and HSOiKVAPrv b monitoring the absorption change of cytochrome c at 550 nm (ε550= 19630 M^"1 cm^"1) [Feng, C, Wilson, H. L, Hurley, J. K., Hazzard, J. T., Toll in, G., Rajagopalan, K. V. and Enemark, J. H. (2003) J Biol

Chem. 278, 2913-2920]. Briefly, equal protein concentrations (10 nM) were incubated in a 200 μΐ final volume with a mixture containing: 50 mM Tris/acetate pH 8; sodium sulfite (varying concentrations) and the reaction was started by adding 12 μΙ of cytochrome c (10 mg/ml). SO activity in crude protein extracts was

determined in a similar way with the following modifications: 50 pg of protein crude extract were used and the assay buffer mixture contained 50 mM Tris/acetate pH 8; 0.2 mM deoxycholic acid; 0.1 mM potassium cyanide and 0.5 mM sodium sulfite. All activities were measured at room temperature (25 °C) using a 96 well-plate reader (BioTeK, Germany).

[00150] Hydrogen peroxide quantification

[00151] The assay is based on the formation of a complex between xylenol orange and ferric ions, which is produced by the peroxide-dependent oxidation of ferrous iron. The method was performed using a commercial kit (National

Diagnostics, Atlanta GA) and detection was carried out colorimetrically following the protocol of the manufacturer. Quantification was carried out after an incubation time of 30 min at room temperature (25 °C) by measuring the absorption at 560 nm using a 96 well-plate reader (BioTeK, Germany).

[00152] Oxygraph measurement

[00153] Oxygen consumption was measured using an Oroboros Oxygraph 2k Instrument (Oroboros Instruments GmbH, Austria). First, a 2 ml solution containing

100 mM Tris pH 8.0 and 250 nM purified enzyme was introduced into the Oxygraph chamber and maintained at 37 °C for approx. 15 min to equilibrate oxygen concentration in the chamber. The reaction was started by addition of 60 μΜ sulfite and the slopes corresponding to the linear change in oxygen concentration of the plots were used to calculate the oxygen consumption rates. Data were analyzed using Datl_ab4 software (version 4.3).

[00154] Western blotting

[00155] Western blots were performed from mouse crude extracts of liver, kidney and brain derived from three different animals. Protein concentration was determined by Bradford and 50 pg of each crude extract was separated by a 10% SDS-PAGE. Primary antibodies used were anti-suffite oxidase (Eurogentec, San Diego, CA) and anti-actin (Santa Cruz, Dallas TX). Secondary antibodies coupled to horseradish peroxidase (Abeam, Cambridge, UK) were visualized using Super Signal West Pico Chemiluminescent Substrate (ThermoFisher Scientific, Waltham MA) and analysis of the protein bands was performed with the Chemiluminescence DeVision HQ2 camera system and the GEL-PRO ANALYZER software (Decon Science Tec, Hohengandern, Germany).

[00156] PEGylation reaction

[00157 ] For PEGylation of plant and mammalian SO proteins the N-

Hydroxysuccinimide (NHS) method was used [Roberts, M. J., Bentley, M. D. and Harris, J. M. (2002) Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev. 54, 459-478]. Three different PEG reagents (mPEG NHS ester, Celares, Berlin, Germany) being different in size and chemistry were applied (for details see FIG. 10). Briefly, the SO proteins were exchanged into phosphate buffer saline (PBS) and concentrated to 5-10 mg/ml. PEG reagent was added to the protein solution in a 20-fold molar excess and the solution was incubated for 30 min at room temperature (25 °C). Finally, excess of non-reacted PEG was removed by size exclusion chromatography using a HR16/30 Superdex 200 column (GE

Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

[00158] Ceil viability

[00159] Cell viability studies were conducted in human embryonic kidney cells (HEK 293) using the MTT (3-(4 5-dimethy!thiazo!-2-yl)-2 5-diphenyltetrazolium bromide) assay (Promega, Germany). Briefly, 80 μΙ of HEK cell suspension (containing 2 * 10⁴ cells) were dispensed into each well of a 96-well tissue culture plate and incubated overnight at 37 °C in a humidified, 5 % C0₂ atmosphere. Next, 10 μ! of SO and/or catalase proteins were added to each well in a final

concentration of 0.5 μΜ and allowed to incubate for 30 min at 37 °C. Following, 10 pi sulfite (varying concentrations) was added to each well and incubated for 15 hours at 37 °C in a humidified, 5 % C0₂ atmosphere. H₂0₂ toxicity was also investigated in the absence and presence of catalase using a concentration range of 0 to 0.5 mM enzyme. Finally, cell viability was evaluated using the MTT dye according to the supplier protocol (Promega, Germany) and absorption at 570 nm (reference 650 nm) was recorded using a well plate reader (TECAN, Germany).

[00160] Example 11. Murine and rat models

[00161] The well-established Mocsl animal model of fvloCD provides a starting point for a proof of concept study. Given that MoCD result also in the loss of xanthine oxidase, MoCD mice could potentially suffer from kidney failure. Studies will be conducted in animals that are whole body or organ-specific SO knock outs, which would on the one hand enable the study of isolated SO deficiency and on the other hand could serve as model for the development of the enzyme substitution therapy. In both models, PEGylated SO_Mo are applied by intravenous application and are reacted with oxygen, thus Iowering the levels of circulating sulfite. Knowing the toxicity of H₂0₂, it is important to ensure low levels of H₂0₂ by either co- application of catalase or by using a dosing scheme that allows sulfite-dependent non-enzymatic clearance of H₂0_?. [00162] Likewise, a sulfite oxidase-deficient rat mode! system can also be used to study sulfite-dependent H₂0₂ formation. See, e.g., Gunnison et a!. Fd. Cosmet. Toxicol. Vol. 19, pp. 209-220 (1981). Alternatively, a sulfite oxidase-deficient whole body or organ-specific knock-out mode! could be used in mice.

[00163] Example 12. HEK cells as model system

[00164] HEK cells were used as a cellular model system to study sulfite- dependent H₂0₂ formation. While sulfite (up to 500 μΜ) is well tolerated, SO- dependent H₂0₂ formation severely impacted cell survival. By using this assay it was confirmed that mammalian heme-deleted SO variants catalyzed sulfite oxidation by using oxygen from the culture medium as electron acceptor. A

catalase was required to efficiently remove produced H₂0₂. Alternatively, in the presence of low amounts of SO, the clearance rate of sulfite would be low, which in turn would enable the non-enzymatic oxidation of sulfite by H₂0₂, formed in the first case.

[00165] Other Embodiments

[00166] The foregoing description discloses only exemplary embodiments.

[00167] It is to be understood that while the detailed description describes certain embodiments, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Thus, while only certain features have been illustrated and described, many modifications and changes will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the present disclosure.

[00168] Table 4 DMA and Amino acid sequences

[00163] Human sulfite oxidase: HSO [00170] Homo sapiens sulfite oxidase (SUOX) gene

[00171] GenBank^® accession number: AY056018.1

[001723 HSO DNA sequence (without mitochondrial targeting sequence)

GAGTCAACACACATATACACTAAGGAGGAAGTGAGTTCCGACACCAGCCCTGAGACTG

GGATCTGGGTGACTCTGGGCTCTGAGGTCTTTGATGTCACAGAATTTGTGGACCTACAT

CCAGGGGGGCCTTCAAAGCTGATGCTAGCAGCTGGGGGTCCCCTAGAGCCCTTCTGG GCCCTCTATGCTGTTCACAACCAGTCCCATGTGCGTGAGTTACTGGCTCAGTACAAGAT TGGGGAGCTGAATCCTGAAGACAAGGTAGCCCCCACCGTGGAGACCTCTGACCCTTAT GCTGATGATCCTGTACGTCACCCAGCCCTGAAGGTCAACAGCCAGCGGCCCTTTAATG CAGAGCCTCCCCCTGAGCTGCTGACAGAAAACTACATCACACCCAACCCTATCTTCTTC ACCCGGAACCATCTGCCTGTACCTAACCTGGATCCAGACACCTATCGCTTACACGTAGT AGGAGCACCTGGGGGTCAGTCACTGTCTCTTTCCCTGGATGACTTGCACAACTTTCCCA GGTACGAGATCACAGTCACTCTGCAGTGTGCCGGCAACCGACGCTCTGAGATGACTCA GGTCAAAGAAGTAAAAGGTCTGGAGTGGAGAACAGGAGCCATCAGCACTGCACGCTGG GCTGGGGCACGGCTCTGTGATGTGTTAGCCCAGGCTGGCCACCAACTCTGTGAAACTG AGGCCCACGTCTGCTTTGAGGGACTGGACTCAGACCCTACTGGGACTGCCTATGGAGC ATCCATCCCTCTGGCTCGGGCCATGGACCCTGAAGCTGAGGTCCTGCTGGCATATGAG ATGAATGGGCAGCCTCTGCCACGTGACCACGGCTTCCCTGTGCGTGTGGTGGTTCCTG GAGTGGTGGGTGCCCGCCATGTCAAATGGCTGGGCAGAGTGAGTGTGCAGCCAGAGG AAAGTTACAGCCACTGGCAACGGCGGGATTACAAAGGCTTCTCTCCATCTGTGGACTGG GAGACTGTAGATTTTGACTCTGCTCCATCCATTCAGGAACTTCCTGTCCAGTCCGCCAT CACAGAGCCCCGGGATGGAGAGACTGTAGAATCAGGGGAGGTGACCATCAAGGGCTAT GCATGGAGTGGTGGTGGCAGGGCTGTGATCCGGGTGGATGTGTCTCTGGATGGGGGC CTAACCTGGCAGGTGGCTAAGCTGGATGGAGAGGAACAGCGCCCCAGGAAGGCCTGG GCATGGCGTCTGTGGCAGTTGAAAGCCCCTGTGCCAGCTGGACAAAAGGAACTGAACA TTGTTTGTAAGGCTGTGGATGATGGTTACAATGTGCAGCCAGACACCGTGGCCCCAATC TGGAACCTGCGAGGTGTTCTCAGCAATGCCTGGCATCGTGTCCATGTCTATGTCTCCCC ATGA (SEQ ID NO: 1)

[00173] HSO Amino acid sequence (without mitochondrial targeting

sequence) ESTHIYTKEEVSSHTSPETGIWVTLGSEVFDVTEFVDLHPGGPSKLMLAAGGPLEPFWALYA VHNQSHVRELLAQYK!GELNPEDKVAPTVETSDPYADDPVRHPALKVNSQRPFNAEPPPEL LTENYITPNPIFFTRNHLPVPNLDPDTYRLHWGAPGGQSLSLSLDDLHNFPRYEITVTLQCA GNRRSEMTQVKEVKGLEWRTGAISTARWAGARLCDVLAQAGHQLCETEAHVCFEGLDSD PTGTAYGAS I PLARAM DPEAEVLLAYEM NGQ PLPRDHG FPVRVWPG WGARH VKWLG V SVQPEESYSHWQRRDYKGFSPSVDWETVDFDSAPSIQELPVQSAITEPRDGETVESGEVT! KGYAWSGGGRAVIRVDVSLDGGLTWQVAKLDGEEQRPRKAWAWRLWQLKAPVPAGQKE LNIVCKAVDDGYNVQPDTVAPIWNLRGVLSNAWHRVHVYVSP (SEQ ID NO: 2) [00174] DNA sequence of wild type (WT) HSO (with mitochondria!

targeting sequence)

ATGGGGACCCTATTAGGTCTCGGTGCAGTGTTGGCCTATCAGGACCATCGGTGTAGGG CTGCTCAGGAGTCAACACACATATACACTAAGGAGGAAGTGAGTTCCCACACCAGCCCT GAGACTGGGATCTGGGTGACTCTGGGCTCTGAGGTCTTTGATGTCACAGAATTTGTGGA CCTACATCCAGGGGGGCCTTCAAAGCTGATGCTAGCAGCTGGGGGTCCCCTAGAGCCC TTCTGGGCCCTCTATGCTGTTCACAACCAGTCCCATGTGCGTGAGTTACTGGCTCAGTA CAAGATTGGGGAGCTGAATCCTGAAGACAAGGTAGCCCCCACCGTGGAGACCTCTGAC CCTTATGCTGATGATCCTGTACGTCACCCAGCCCTGAAGGTCAACAGCCAGCGGCCCTT TAATGCAGAGCCTCCCCCTGAGCTGCTGACAGAAAACTACATCACACCCAACCCTATCT TCTTCACCCGGAACCATCTGCCTGTACCTAACCTGGATCCAGACACCTATCGCTTACAC GTAGTAGGAGCACCTGGGGGTCAGTCACTGTCTCTTTCCCTGGATGACTTGCACAACTT TCCCAGGTACGAGATCACAGTCACTCTGCAGTGTGCCGGCAACCGACGCTCTGAGATG ACTCAGGTCAAAGAAGTAAAAGGTCTGGAGTGGAGAACAGGAGCCATCAGCACTGCAC GCTGGGCTGGGGCACGGCTCTGTGATGTGTTAGCCCAGGCTGGCCACCAACTCTGTGA AACTGAGGCCCACGTCTGCTTTGAGGGACTGGACTCAGACCCTACTGGGACTGCCTAT GGAGCATCCATCCCTCTGGCTCGGGCCATGGACCCTGAAGCTGAGGTCCTGCTGGCAT ATGAGATGAATGGGCAGCCTCTGCCACGTGACCACGGCTTCCCTGTGCGTGTGGTGGT TCCTGGAGTGGTGGGTGCCCGCCATGTCAAATGGCTGGGCAGAGTGAGTGTGCAGCC AGAGGAAAGTTACAGCCACTGGCAACGGCGGGATTACAAAGGCTTCTCTCCATCTGTG GACTGGGAGACTGTAGATTTTGACTCTGCTCCATCCATTCAGGAACTTCCTGTCCAGTC CGCCATCACAGAGCCCCGGGATGGAGAGACTGTAGAATCAGGGGAGGTGACCATCAA GGGCTATGCATGGAGTGGTGGTGGCAGGGCTGTGATCCGGGTGGATGTGTCTCTGGAT GGGGGCCTAACCTGGCAGGTGGCTAAGCTGGATGGAGAGGAACAGCGCCCCAGGAAG GCCTGGGCATGGCGTCTGTGGCAGTTGAAAGCCCCTGTGCCAGCTGGACAAAAGGAAC TGAACATTGTTTGTAAGGCTGTGGATGATGGTTACAATGTGCAGCCAGACACCGTGGCC CCAATCTGGAACCTGCGAGGTGTTCTCAGCAATGCCTGGCATCGTGTCCATGTCTATGT

CTCCCCATGA (SEQ ID NO: 8)

[00175] Amino acid sequence of wild type (WT) HSO (with mitochondrial targeting sequence)

MGTLLGLGAVLAYQDH CRAAQ ESTH I YTKEEVSS HTSPETG IWVTLGS EVFDVTEFVDLH P GGPSKLMLAAGGPLEPFWALYAVHNQSHVRELLAQYKIGELNPEDKVAPTVETSDPYADDP VRHPALKVNSQRPFNAEPPPELLTENYITPNPIFFTRNHLPVPNLDPDTYRLHWGAPGGQS LSLSLDDLHNFPRYEITVTLQCAGNRRSEMTQVKEVKGLEWRTGAISTARWAGARLCDVLA QAGHQLCETEAHVCFEGLDSDPTGTAYGASIPLARAMDPEAEVLLAYEMNGQPLPRDHGF PVRWVPGWGARHVKWLGRVSVQPEESYSHWQRRDYKGFSPSVDWETVDFDSAPSIQE LPVQSAITEPRDGETVESGEVTIKGYAWSGGGRAVIRVDVSLDGGLTWQVAKLDGEEQRP RKAWAWRLWQLKAPVPAGQKELNtVCKAVDDGYNVQPDTVAP!WNLRGVLSNAWHRVHV YVSP (SEQ ID NO: 9)

[00176] Amino acid sequences of HSO variants

[00177] HSOAKVATV (Deletion of the amino acid residues: 108, 109, 110, 112, 113 of WT HSO)

MGTLLGLGAVLAYQDHRCRAAQESTHIYTKEEVSSHTSPETGIWVTLGSEVFDVTEFVDLHP

GGPSKL LAAGGPLEPFWALYAVHNQSHVRELLAQYKIGELNPEDPETSDPYADDPVRHP

ALKVNSQRPFNAEPPPELLTENYITPNPIFFTRNHLPVPNLDPDTYRLHWGAPGGQSLSLSL DDLHNFPRYEITVTLQCAGNRRSEMTQVKEVKGLEWRTGAISTARWAGARLCDVLAQAGH QLCETEAHVCFEGLDSDPTGTAYGASIPLARA DPEAEVLLAYE NGQPLPRDHGFPVRW VPGWGARHVKWLGRVSVQPEESYSHWQRRDYKGFSPSVDWETVDFDSAPSIQELPVQS AITEPRDGETVESGEVTIKGYAWSGGGRAVIRVDVSLDGGLTWQVAKLDGEEQRPRKAWA WRLWQLKAPVPAGQKELNIVCKAVDDGYNVQPDTVAPIWNLRGVLSNAWHRVHVYVSP

(SEQ ID NO: 10)

[00178] HSOA VAPTV (Deletion of the amino acid residues: 108, 109, 110, 111, 112, 113 of WT HSO) MGTLLGLGAVLAYQDHRCRAAQESTHIYTKEEVSSHTSPETGIWVTLGSEVFDVTEFVDLHP GGPSKLMLAAGGPLEPFWALYAVHNQSHVRELLAQYKIGELNPEDETSDPYADDPVRHPAL KVNSQRPFNAEPPPELLTENYITPNPiFFTRNHLPVPNLDPDTYRLHWGAPGGQSLSLSLD DLHNFPRYEITVTLQCAGNRRSE TQVKEVKGLEWRTGAISTARWAGARLCDVLAQAGHQ LCETEAHVCFEGLDSDPTGTAYGASIPLARAMDPEAEVLLAYEMNGQPLPRDHGFPVRVW PGWGARHVKWLGRVSVQPEESYSHWQRRDYKGFSPSVDWETVDFDSAPSIQELPVQSA! TEPRDGETVESGEVTIKGYAWSGGGRAVIRVDVSLDGGLTWQVAKLDGEEQRPRKAWAW RLWQLKAPVPAGQKELNIVCKAVDDGYNVQPDTVAPIWNLRGVLSNAWHRVHVYVSP

(SEQ !D NO: 11 )

[00179] For the generation of bacterial expression constructs of WT HSO, HSOAKVATV, and HSOAKVAPTV, the DNA sequences encoding the first 22 amino acid residues corresponding to a putative trans-membrane domain were deleted in the final constructs.

[00180] Human sulfite oxidase Molybdenum domain: HSO o

[00181] HSOMO DNA sequence

GCCCCCACCGTGGAGACCTCTGACCCTTATGCTGATGATCCTGTACGTCACCCAGCCC TGAAGGTCAACAGCCAGCGGCCCTTTAATGCAGAGCCTCCCCCTGAGCTGCTGACAGA AAACTACATCACACCCAACCCTATCTTCTTCACCCGGAACCATCTGCCTGTACCTAACCT GGATCCAGACACCTATCGCTTACACGTAGTAGGAGCACCTGGGGGTCAGTCACTGTCT CTTTCCCTGGATGACTTGCACAACTTTCCCAGGTACGAGATCACAGTCACTCTGCAGTG TGCCGGCAACCGACGCTCTGAGATGACTCAGGTCAAAGAAGTAAAAGGTCTGGAGTGG AGAACAGGAGCCATCAGCACTGCACGCTGGGCTGGGGCACGGCTCTGTGATGTGTTAG CCCAGGCTGGCCACCAACTCTGTGAAACTGAGGCCCACGTCTGCTTTGAGGGACTGGA CTCAGACCCTACTGGGACTGCCTATGGAGCATCCATCCCTCTGGCTCGGGCCATGGAC CCTGAAGCTGAGGTCCTGCTGGCATATGAGATGAATGGGCAGCCTCTGCCACGTGACC ACGGCTTCCCTGTGCGTGTGGTGGTTCCTGGAGTGGTGGGTGCCCGCCATGTCAAATG GCTGGGCAGAGTGAGTGTGCAGCCAGAGGAAAGTTACAGCCACTGGCAACGGCGGGA TTACAAAGGCTTCTCTCCATCTGTGGACTGGGAGACTGTAGATTTTGACTCTGCTCCATC CATTCAGGAACTTCCTGTCCAGTCCGCCATCACAGAGCCCCGGGATGGAGAGACTGTA GAATCAGGGGAGGTGACCATCAAGGGCTATGCATGGAGTGGTGGTGGCAGGGCTGTG ATCCGGGTGGATGTGTCTCTGGATGGGGGCCTAACCTGGCAGGTGGCTAAGCTGGATG GAGAGGAACAGCGCCCCAGGAAGGCCTGGGCATGGCGTCTGTGGCAGTTGAAAGCCC CTGTGCCAGCTGGACAAAAGGAACTGAACATTGTTTGTAAGGCTGTGGATGATGGTTAC AATGTGCAGCCAGACACCGTGGCCCCAATCTGGAACCTGCGAGGTGTTCTCAGCAATG CCTGGCATCGTGTCCATGTCTATGTCTCCCCATGA (SEQ ID NO; 3)

[00182] HSOmo Amino acid sequence APTVETSDPYADDPVRHPALKVNSQRPFNAEPPPELLTENYITPNPIFFTRNHLPVPNLDPDT YRLHWGAPGGQSLSLSLDDLHNFPRYEITVTLQCAGNRRSEMTQVKEVKGLEWRTGAIST ARWAGARLCDVLAQAGHQLCETEAHVCFEGLDSDPTGTAYGASIPLARAMDPEAEVLLAYE MNGQPLPRDHGFPVRVWPGWGARHVKWLGRVSVQPEESYSHWQRRDYKGFSPSVDW ETVDFDSAPSIQELPVQSAITEPRDGETVESGEVTIKGYAWSGGGRAVIRVDVSLDGGLTW QVAKLDGEEQRPRKAWAWRLWQLKAPVPAGQKELNIVCKAVDDGYNVQPDTVAPIWNLR GVLSNAWHRVHVYVSP (SEQ ID NO: 4)

[00183] Human cataiase

[00184] Human cataiase DNA sequence

ATGGCTGACAGCCGGGATCCCGCCAGCGACCAGATGCAGCACTGGAAGGAGCAGCGG GCCGCGCAGAAAGCTGATGTCCTGACCACTGGAGCTGGTAACCCAGTAGGAGACAAAC TTMTGrrATTACAGTAGGGCCCCGTGGGCCCCTTCTTGTTCAGGATGTGGTTTTCACT GATGAAATGGCTCATTTTGACCGAGAGAGAATTCCTGAGAGAGTTGTGCATGCTAAAGG AGCAGGGGCCTTTGGCTACTTTGAGGTCACACATGACATTACCAAATACTCCAAGGCAA AGGTATTTGAGCATATTGGAAAGAAGACTCCCATCGCAGTTCGGTTCTCCACTGTTGCT GGAGAATCGGGTTCAGCTGACACAGTTCGGGACCCTCGTGGGTTTGCAGTGAAATTTTA CACAGAAGATGGTAACTGGGATCTCGTTGGAAATAACACCCCCATTTTCTTCATCAGGG ATCCCATATTGTTTCCATCTTTTATCCACAGCCAAAAGAGAAATCCTCAGACACATCTGA AGGATCCGGACATGGTCTGGGACTTCTGGAGCCTACGTCCTGAGTCTCTGCATCAGGT TTCTTTC7TGTTCAGTGATCGGGGGATTCCAGATGGACATCGCCACATGAATGGATATG GATCACATAC7TTCAAGCTGGTTAATGCAAATGGGGAGGCAGTTTATTGCAAATTCCATT ATAAGACTGACCAGGGCATCAAAAACCTTTCTG7TGAAGATGCGGCGAGACTTTCCCAG GAAGATCCTGACTATGGCATCXX3GGATCTTTTTAACGCCATTGCCACAGGAAAGTACCC CTCCTGGACTTTTTACATCCAGGTCATGACATTTAATCAGGC^GAAACTmCCATTTAAT CCATTCGATCTCACCAAGGTTTGGCCTCACAAGGACTACCCTCTCATCCCAGTTGGTAA ACTGGTCTTAMCCGGAATCCAGTTAATTACTTTGCTGAGGTTGAACAGATAGCCTTCGA CCCAAGCAACATGCCACCTGGCATTGAGGCCAGTCCTGACAAAATGCTTCAGGGCCGC

C 1 1 1 I ! GCCTATCCTGACACTCACCGCCATCGCCTGGGACCCAATTATCTTCATATACCT GTGAACTGTCCCTACCGTGCTCGAGTGGCCAACTACCAGCGTGACGGCCCGATGTGCA TGCAGGACAATCAGGGTGGTGCTCCAAATTACTACCCCAACAGCTTTGGTGCTCCGGAA CAACAGCCTTCTGCCCTGGAGCACAGCATCCAATATTCTGGAGAAGTGCGGAGATTCAA CACTGCCAATGATGATAACGTTACTCAGGTGCGGGCATTCTATGTGAACGTGCTGAATG

AG G AACAG AGG AAACGTCTGTGTG AG AACATTG CCGG CCACCTG AAG GATG CACAAAT TTTCATCCAGAAGAAAGCGGTCAAGAACTTCACTGAGGTCCACCCTGACTACGGGAGCC ACATCCAGGCTCTTCTGGACAAGTACAATGCTGAGAAGCCTAAGAATGCGATTCACACC TTTGTGCAGTCCGGATCTCACTTGGCGGCAAGGGAGAAGGCAAATCTGTGA (SEQ ID

NO: 5)

[00185] Human catalase Amino acid sequence

MADSRDPASDQ QHWKEQRAAQKADVLTTGAGNPVGDKLNVITVGPRGPLLVQDVVFTD EMAHFDRERiPERWHAKGAGAFGYFEVTHDUKYSKAKVFEHIGKKTPIAVRFSTVAGESG SADTVRDPRGFAVKFYTEDGNWDLVGNNTP!FFIRDPiLFPSFIHSQKRNPQTHLKDPDMVW DFWSLRPESLHQVSFLFSDRGIPDGHRHMNGYGSHTFKLVNANGEAVYCKFHYKTDQG!K NLSVEDAARLSQEDPDYGIRDLFNAIATGKYPSWTFYIQV TFNQAETFPFNPFDLTKVWPH KDYPLIPVGKLVLNRNPVNYFAEVEQIAFDPSN PPGIEASPDKMLQGRLFAYPDTHRHRLG PNYLHIPVNCPYRARVANYQRDGP CMQDNQGGAPNYYPNSFGAPEQQPSALEHS!QYS GEVRRFNTANDDNVTQVRAFYVNVLNEEQRKRLCENIAGHLKDAQIFIQKKAVKNFTEVHPD YGSHIQALLDKYNAEKPKNAIHTFVQSGSHLAAREKANL (SEQ ID NO: 6)

[00186] Murine sulfite oxidase: (MSO)

[00187] Mus muscuius sylfite oxidase (SUOX) gene

[00188] GENBANK^® accession number: BC027197.1

[00189] DNA sequence of wild type (WT) MSO

ATGCTGCTGCAGCTATACAGATCCGTGGTTGTGAGGCTTCCACAGGCCATCAGAGTCAA GTCAACCCCCTTGAGGCTCTGCATTCAAGCATGCTCCACAAATGATTCACTTGAGCCCC AGCATCCCAGCCTTACCTTTTCTGATGATAACTCAAGGACTCGGAGATGGAAAGTCATG GGGACCCTGTTAGGCCTGGGTGTGGTGCTGGTCTACCATGAGCATCGGTGTAGGGCTT CTCAGGAGTCACCACGGATGTACTCTAAAGAGGATGTGCGTTCTCACAACAACCCTAAA ACTGGAGTCTGGGTAACTCTAGGCTCTGAGGTCTTCGATGTCACAAAATTTGTGGACCT GCATCCAGGAGGACCATCAAAACTGATGCTAGCAGCTGGAGGTCCCCTAGAACCCTTC TGGGCCCTCTATGCTGTGCACAACCAGCCCCATGTACGTGAGTTACTGGCCGAGTATAA GATTGGGGAACTGAACCCCGAAGATAGCATGTCCCCCTCCGTGGAAGCCTCTGACCCT TATGCTGATGATCCTATTCGTCATCCAGCCCTGAGGATTAATAGCCAGCGCCCCTTTAAT GCAGAGCCTCCTCCTGAACTGCTAACTGAAGGCTACATCACACCAAATCCTATTTTCTTC ACCCGTAACCATCTGCCTGTACCTAACCTGGACCCACACACCTATCGCTTACATGTAGT AGGGGCACCTGGAGGTCAGTCACTGTCTCTGTCCTTGGATGATTTGCATAAGTTTCCCA

AACATGAGGTCACTGTCACTCTGCAGTGTGCTGGTAACCGGCGCTCCGAAATGAGTAA GGTCAAGGAAGTGAAAGGTCTGGAATGGAGAACAGGGGCGATCAGCACAGCACGCTG GGCTGGGGCCCGGCTCTGTGATGTGTTAGCCCAGGCTGGTCACCGACTCTGTGACTCT GAGGCCCATGTCTGTTTTGAAGGACTGGATTCAGACCCCACTGGAACTGCCTATGGAG GATCGATCCCTCTGGCTCGGGCCATGGATCCTGAAGCCGAGGTCCTCCTGGCTTATGA AATGAATGGTCAGCCTCTACCTCGTGACCATGGCTTCCCTGTACGGGTGGTGGTTCCTG GTGTAGTAGGTGGCCGTCATGTCAAATGGCTCGGCAGAGTGAGTGTGGAATCAGAGGA GAGTTATAGTCACTGGCAGAGGCGGGATTACAAAGGCTTTTCTCCATCTGTGGACTGGG ACACGGTAAACTTTGACCTAGCTCCATCAATTCAGGAACTACCTATCCAGTCAGCTATCA CAGAACCTCAAGATGGGGCCATTGTAGAGTCAGGCGAGGTGACTATCAAGGGCTATGC ATGGAGTGGTGGTGGCAGGGCTGTGATTCGAGTGGATGTGTCTGTGGATGGGGGACTA ACCTGGCAGGAAGCTGAGCTAGAGGGAGAGGAAGAGTGTCCCAGGAAGGCCTGGGGT TGGCGAATATGGCAGTTGAAAGCTCAGGTGCCGGCTGAGCAAAAGGAATTGAACATCAT TTGCAAAGCTGTAGATGACAGTTACAATGTGCAGCCAGACACTGTAGCCCCAATCTGGA ACCTTCGGGGCGTACTCAGCAATGCCTGGCACCGTGTCCATGTTCAGGTGGTCCCATG

A (SEQ ID NO: 12)

[00130] Amino acid sequence of wild type (WT) MSO

MLLQLYRSVWRLPQAIRVKSTPLRLCIQACSTNDSLEPQHPSLTFSDDNSRTRRWKVMGT LLGLGWLWHEHRCRASQESPR YSKEDVRSHNNPKTGVWVTLGSEVFDVTKFVDLHPG GPSKLMLAAGGPLEPFWALYAVHNQPHVRELLAEYKIGELNPEDSMSPSVEASDPYADDPI RHPALRINSQRPFNAEPPPELLTEGYITPNPIFFTRNHLPVPNLDPHTYRLHWGAPGGQSLS LSLDDLHKFPKHEVTVTLQCAGNRRSE SKVKEVKGLEWRTGAISTARWAGARLCDVLAQ AGHRLCDSEAHVCFEGLDSDPTGTAYGASIPLARA DPEAEVLLAYEMNGQPLPRDHGFP VRVWPGWGARHVKWLGRVSVESEESYSHWQRRDYKGFSPSVDWDTVNFDLAPSIQEL PIQSAITQPQDGAIVESGEVTIKGYAWSGGGRAVIRVDVSVDGGLTWQEAELEGEEQCPRK AWAWRIWQLKAQVPAEQKELNIICKAVDDSYNVQPDTVAP1WNLRGVLSNAWHRVHVQW P (SEQ ID NO: 13)

[00191] Amino acid sequence of MSO variants

[00192] MSOa_he_me The amino acid residues number: 119 and 144 (both

histidines) of wt MSO have been exchanged to alanines) MLLQLYRSVWRLPQAIRVKSTPLRLCIQACSTNDSLEPQHPSLTFSDDNSRTRRWKVMGT LLGLGWLVYHEHRCRASQESPRMYSKEDVRSHNNPKTGVWVTLGSEVFDVTKFVDLAPG GPSKL LAAGGPLEPFWALYAVANQPHVRELLAEYKIGELNPEDSMSPSVEASDPYADDPI RHPALRINSQRPFNAEPPPELLTEGYITPNPIFFTRNHLPVPNLDPHTYRLHWGAPGGQSLS LSLDDLHKFPKHEVTVTLQCAGNRRSEMSKVKEVKGLEWRTGAiSTARWAGARLCDVLAQ AGHRLCDSEAHVCFEGLDSDPTGTAYGASIPLARAMDPEAEVLLAYEMNGQPLPRDHGFP VRVWPGWGARHVKWLGRVSVESEESYSHWQRRDYKGFSPSVDWDTVNFDLAPSIQEL PIQSAITQPQDGAIVESGEVT!KGYAWSGGGRAVIRVDVSVDGGLTWQEAELEGEEQCPRK AWAWRlWQLKAQVPAEQKELNIICKAVDDSYNVQPDTVAPiWNLRGVLSNAWHRVHVQW P (SEQ ID NO: 14)

[00193 ] For the generation of bacterial expression constructs of wt MSO and MSO&heme. the first 80 amino acid residues corresponding to mitochondrial targeting peptide and a putative trans-membrane domain were deleted in the final constructs.

[00194] Murine sulfite oxidase Molybdenum domain: MSOM_O

[ 00195] DNA sequence of MSO_Mo(DNA sequence coding for the amino acid residues: 168 to 546 of wt MSO)

TCCCCCTCCGTGGAAGCCTCTGACCCTTATGCTGATGATCCTATTCGTCATCCAGCCCT GAGGATTAATAGCCAGCGCCCCTTTAATGCAGAGCCTCCTCCTGAACTGCTAACTGAAG GCTACATCACACCAAATCCTATTTTCTTCACCCGTAACCATCTGCCTGTACCTAACCTGG ACCCACACACCTATCGCTTACATGTAGTAGGGGCACCTGGAGGTCAGTCACTGTCTCTG TCCTTGGATGATTTGCATAAGTTTCCCAAACATGAGGTCACTGTCACTCTGCAGTGTGCT GGTAACCGGCGCTCCGAAATGAGTAAGGTCAAGGAAGTGAAAGGTCTGGAATGGAGAA CAGGGGCCATCAGCACAGCACGCTGGGCTGGGGCCCGGCTCTGTGATGTGTTAGCCC AGGCTGGTCACCGACTCTGTGACTCTGAGGCCCATGTCTGTTTTGAAGGACTGGATTCA GACCCCACTGGAACTGCCTATGGAGCATCGATCCCTCTGGCTCGGGCCATGGATCCTG AAGCCGAGGTCCTCCTGGCTTATGAAATGAATGGTCAGCCTCTACCTCGTGACCATGGC TTCCCTGTACGGGTGGTGGTTCCTGGTGTAGTAGGTGCCCGTCATGTCAAATGGCTCG GCAGAGTGAGTGTGGAATCAGAGGAGAGTTATAGTCACTGGCAGAGGCGGGATTACAA AGGCTTTTCTCCATCTGTGGACTGGGACACGGTAAACTTTGACCTAGCTCCATCAATTCA GGAACTACCTATCCAGTCAGCTATCACACAACCTCAAGATGGGGCCATTGTAGAGTCAG GCGAGGTGACTATCAAGGGCTATGCATGGAGTGGTGGTGGCAGGGCTGTGATTCGAGT GGATGTGTCTGTGGATGGGGGACTAACCTGGCAGGAAGCTGAGCTAGAGGGAGAGGA ACAGTGTCCCAGGAAGGCCTGGGCTTGGCGAATATGGCAGTTGAAAGCTCAGGTGCCG GCTGAGCAAAAGGAATTGAACATCATTTGCAAAGCTGTAGATGACAGTTACAATGTGCA GCCAGACACTGTAGCCCCAATCTGGAACCTTCGGGGCGTACTCAGCAATGCCTGGCAC CGTGTCCATGTTCAGGTGGTCCCATGA (SEQ ID NO: 15)

[00196] Amino acid sequence of MSO_mo

SPSVEASDPYADDPIRHPALRINSQRPFNAEPPPELLTEGYITPNPIFFTRNHLPVPNLDPHT YRLHWGAPGGQSLSLSLDDLHKFPKHEVTVTLQCAGNRRSE SKVKEVKGLEWRTGAIST AR AGARLCDVLAQAGHRLCDSEAHVCFEGLDSDPTGTAYGASIPLARAMDPEAEVLLAY EMNGQPLPRDHGFPVRVWPGWGARHVKWLGRVSVESEESYSHWQRRDYKGFSPSVD WDTVNFDLAPSIQELP!QSAtTQPQDGAIVESGEVTIKGYAWSGGGRAVIRVDVSVDGGLTW QEAELEGEEQCPRKAWAWRIWQLKAQVPAEQKELNIICKAVDDSYNVQPDTVAPIWNLRG VLSNAWHRVHVQWP (SEQ ID NO: 16)

Claims

Claims

A pharmaceutical composition comprising a mammalian oxygen-reactive sulfite oxidase variant.

The pharmaceutical composition of claim 1 , wherein the mammalian oxygen-reactive sulfite oxidase variant is a mammalian sulfite oxidase variant that lacks part or all of the heme domain; more specifically, wherein the variant is at least one of human sulfite oxidase variant HSO_A<VATV (SEQ ID NO: 10) and human sulfite oxidase variant HSC _PTV (SEQ ID NO: 11).

The pharmaceutical composition of any of the preceding claims, wherein the mammalian oxygen-reactive sulfite oxidase variant is a mammalian sulfite oxidase variant that has a non-functional heme domain.

The pharmaceutical composition of any of the preceding claims, wherein the sulfite oxidase variant is PEGyiated.

The pharmaceutical composition of any of the preceding claims, further comprising a catalase.

The pharmaceutical composition of any of the preceding claims, wherein the mammalian oxygen-reactive sulfite oxidase variant is a human oxygen-reactive sulfite oxidase variant.

A method of treating a sulfite oxidase deficiency, an excess sulfite accumulation, or reduced sulfite oxidase activity resulting from pterin-based molybdenum cofactor deficiency in a patient, comprising administering an effective amount of a

pharmaceutical composition comprising a mammalian oxygen-reactive sulfite oxidase variant to a patient in need thereof.

The method of claim 7, wherein the mammalian oxygen-reactive sulfite oxidase variant is a human oxygen-reactive sulfite oxidase variant; more specifically, wherein the variant is at least one of human sulfite oxidase variant HSC _ATV (SEQ ID NO: 10) and human sulfite oxidase variant HSO_&KV_APTV (SEQ ID NO: 11).

9. The method of claim 7 or 8, wherein the sulfite oxidase is PEGylated.

10. The method of any one of claims 7-9, further comprising administering a

pharmaceutical composition comprising a catalase to said patient, wherein the catalase and the sulfite oxidase variant are in the same or in a different

pharmaceutical composition.

11. A method of reducing sulfite level in a patient, comprising administering an effective amount of a pharmaceuticai composition comprising a mammalian oxygen-reactive sulfite oxidase variant to a patient in need thereof; more specifically, wherein the variant is at least one of human sulfite oxidase variant HSC VATV (SEQ ID NO: 10) and human sulfite oxidase variant HSO_&KVAPTV (SEQ ID NO; 1 1 ).

12. The method of claim 11 , wherein the sulfite oxidase is PEGylated.

13. The method of any one of claims 11 and 12, further comprising administering a pharmaceutical composition comprising a catalase to said patient, wherein the catalase and the sulfite oxidase variant are in the same or in a different

pharmaceutical composition.

14. A PEGylated mammalian oxygen-reactive sulfite oxidase variant.

15. The PEGylated mammalian oxygen-reactive sulfite oxidase variant of claim 14, wherein the mammalian sulfite oxidase variant lacks part or all of the heme domain; more specifically, wherein the variant is at least one of human sulfite oxidase variant HSOAKVATV (SEQ ID NO: 10) and human sulfite oxidase variant HSO VAPTV (SEQ ID NO: 11 ).