WO2025193499A1 - Compositions and methods to potentiate enzyme therapies for treating homocystinuria and homocysteine remethylation disorders - Google Patents

Abstract

Embodiments of the present disclosure generally relate to compositions and methods for treating diseases such as homocystinuria and homocysteine remethylation disorders in a patient. In an embodiment, a composition for potentiating a Cystathionine beta-synthase (CBS) enzyme replacement therapy in a patient undergoing treatment for homocystinuria or homocysteine remethylation disorders is provided. The composition includes a sulfur- containing reducing agent. In another embodiment, a combination therapy for treating homocystinuria or homocysteine remethylation disorders in a patient is provided. The combination therapy includes a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof. The combination therapy further includes a sulfur-containing reducing agent. Methods for treatment and methods for adjusting an amount of a metabolite in a patient are also described herein.

Description

COMPOSITIONS AND METHODS TO POTENTIATE ENZYME THERAPIES FOR TREATING HOMOCYSTINURIA AND HOMOCYSTEINE REMETHYLATION DISORDERS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63/564,451, filed on March 12, 2024, which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] This application contains references to amino acid and nucleic acid sequences which have been submitted as the sequence listing text file entitled “SEQ ID NOs 1-6”, file size 8.60 KiloBytes (KB), created January 10, 2024, which is hereby incorporated by reference in its entirety.

FIELD

[0003] Embodiments of the present disclosure generally relate to compositions and methods for treating diseases such as homocystinuria and homocysteine remethylation disorders in a patient.

BACKGROUND

[0004] Cystathionine beta-synthase (CBS) is the first enzyme in the transsulfuration pathway, catalyzing the condensation of serine and homocysteine (Hey) to cystathionine (Cth), which is then hydrolyzed to cysteine (Cys) by Cystathionine Gamma Lyase (CGL). The biological transsulfuration system seems to largely lack redundancy of components, making this system prone to mutational perturbations. For example, the CBS enzyme is the only component that can process homocysteine to cystathionine. A limited system redundancy partly exists as homocysteine, the first metabolite that funnels into the pathway, can alternatively be converted to methionine through the remethylation pathway, thus relieving the homocysteine load. In addition, cysteine, a downstream product, can be obtained directly from diet. Nevertheless, these pathways are limited in their capacity to maintain normal levels of metabolites, and lack of CBS function has detrimental consequences for human patients if left untreated. Inactivation of CBS results in cystathionine beta-synthase-defi cient homocystinuria (CBSDH), more commonly referred to as classical homocystinuria. Limited therapeutic options exist to treat CBSDH and conventional treatment options reduce homocysteine but do not tend to normalize Cth or Cys and thus these treatment options are inadequate for providing robust and effective treatment options. More effective treatment strategies for individuals with homocystinuria are needed.

[0005] There is a need for new and improved compositions and methods for treating diseases such as homocystinuria.

SUMMARY

[0006] Embodiments of the present disclosure generally relate to compositions and methods for treating diseases such as homocystinuria and homocysteine remethylation disorders in a patient. Compositions of the present disclosure include a sulfur-containing reducing agent, for example, a thiol-based reductant. The inventor discovered that sulfur- containing reducing agents can serve to scavenge for and preferably bind to a highly reactive homocysteine or can replace homocysteine bound to protein sulfhydryl (cysteine) residues, thus resulting in a decreased homocysteine binding to plasma proteins. Liberated homocysteine can form mixed disulfides with the reducing agents, which consequently can increase elimination of homocysteine from plasma. Furthermore, liberated homocysteine not forming disulfides can be a substrate for cystathionine beta-synthase-based enzyme replacement therapies and thus can increase efficacy of such treatments and enzymatic elimination of homocysteine from plasma. Such compositions can be utilized to treat diseases such as homocystinuria and homocysteine remethylation disorders.

[0007] In an embodiment, a composition for potentiating a cystathionine beta-synthase (CBS) enzyme replacement therapy in a patient undergoing treatment for homocystinuria or homocysteine remethylation disorders is provided. The composition includes a sulfur- containing reducing agent.

[0008] In another embodiment, a composition for increasing efficacy of a CBS enzyme replacement therapy is provided. The composition includes a sulfur-containing reducing agent. [0009] In another embodiment, a composition for potentiating, sensitizing, and/or amplifying a CBS enzyme replacement therapy in a patient is provided. The composition includes a sulfur-containing reducing agent.

[0010] In another embodiment, a method for increasing efficacy of a CBS enzyme replacement therapy in a patient is provided. The method includes administering to the patient a therapy comprising a composition that includes a sulfur-containing reducing agent. The method further includes administering to the patient a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof. [0011] In another embodiment, a combination therapy for treating homocystinuria or homocysteine remethylation disorders in a patient is provided. The combination therapy includes a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof. The combination therapy further includes a sulfur-containing reducing agent.

[0012] In another embodiment, a combination therapy for decreasing homocysteine levels in a patient is provided. The combination therapy includes a composition comprising a sulfur- containing reducing agent. The combination therapy further includes a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof.

[0013] In another embodiment, a method for potentiating enzyme therapy for treating homocystinuria or homocysteine remethylation disorders in a patient is provided. The method includes administering to the patient a CBS polypeptide, the CBS mutant polypeptide, the CBS variant, or combinations thereof. The method further includes administering to the patient a sulfur-containing reducing agent. The sulfur-containing reducing agent can be administered before and/or after the CBS polypeptide, the CBS mutant polypeptide, the CBS variant, or combinations thereof.

[0014] In another embodiment, a method of treatment is provided. The method of treatment includes administering to the patient a combination therapy described herein, wherein the combination therapy is administered as a single joint shot or dose (for example, by the same route); or the combination therapy is administered separately by administering the composition comprising a sulfur-containing reducing agent orally and administering the CBS polypeptide, a CBS mutant polypeptide, or combinations thereof parenterally (subcutaneously).

[0015] In another embodiment, a method for potentiating enzyme therapy for treating homocystinuria or homocysteine remethylation disorders in a patient is provided. The method includes administering to the patient a combination therapy described herein, wherein the combination therapy is administered as a single joint shot or dose (for example, by the same route); or the combination therapy is administered separately by administering the composition comprising a sulfur-containing reducing agent orally and administering the CBS polypeptide, a CBS mutant polypeptide, or combinations thereof parenterally (subcutaneously).

[0016] In another embodiment, a method of adjusting an amount of a metabolite in a patient is provided. The method includes administering to a patient a composition comprising a sulfur- containing reducing agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, can be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, and the exemplary embodiments can admit to other equally effective embodiments.

[0018] FIG. 1A is a gel showing results of SDS-PAGE analysis for example modified cystathionine beta synthase (CBS) proteins according to at least one embodiment of the present disclosure.

[0019] FIG. IB shows non-limiting data for the specific activity of example modified CBS proteins according to at least one embodiment of the present disclosure.

[0020] FIG. 2 is a graphical representation of an animal study design according to at least one embodiment of the present disclosure.

[0021] FIG. 3 A shows non-limiting data for the effect of sulfur-containing reducing agents alone and in combination with modified CBS proteins on plasma total homocysteine (tHcy) levels in I278T mice according to at least one embodiment of the present disclosure.

[0022] FIG. 3B shows non-limiting data for the effect of sulfur-containing reducing agents alone and in combination with modified CBS proteins on plasma total cysteine (tCys) levels in I278T mice according to at least one embodiment of the present disclosure.

[0023] FIG. 3C shows non-limiting data for the effect of sulfur-containing reducing agents alone and in combination with modified CBS proteins on plasma cystathionine (Cth) levels in I278T mice according to at least one embodiment of the present disclosure.

[0024] FIG. 3D shows non-limiting data for the effect of sulfur-containing reducing agents alone and in combination with modified CBS proteins on plasma methionine (Met) levels in I278T mice according to at least one embodiment of the present disclosure.

[0025] FIG. 4 shows non-limiting data for the distribution of plasma Hey fractions during evaluation of sulfur-containing reducing agents in I278T mice according to at least one embodiment of the present disclosure. The plots in FIG. 4 display relative proportions of protein-bound Hey fractions and protein-unbound Hey fractions of plasma tHcy pool (representing 100%) at baseline (D10), after treatment with sulfur-containing reducing agents (D17) and after co-administration of PEG-CBS and sulfur-containing reducing agents (D31) in I278T cohorts designated as PBS (control), NAC, MESNA and dcCYS. Numbers within and above the bars corresponds to average Hey concentrations in protein-bound/unbound fractions and plasma tHcy, respectively. Numerical percentage with arrow designates a change in relative proportion of protein-bound versus protein-unbound plasma Hey fractions with a significance of the change (ns - non-significant, */**/*** = p<0.05/0.01/0.001).

[0026] Figures included herein illustrate various embodiments of the disclosure. It is contemplated that elements and features of one embodiment can be beneficially incorporated in other embodiments without further recitation.

DETAILED DESCRIPTION

[0027] Embodiments of the present disclosure generally relate to compositions and methods for treating diseases such as homocystinuria and homocysteine remethylation disorders in a patient. As described herein, the CBS enzyme catalyzes the condensation of serine and Hey to cystathionine, which is then hydrolyzed by cysteine. Along the transsulfuration pathway, the CBS enzyme is the only component that can process Hey to cystathionine. A limited system redundancy partly exists as homocysteine can be converted to methionine through the remethylation pathway. However, this pathway has limited capacity to maintain normal levels of metabolites in plasma. In patients that have poor functioning of the CBS enzyme, the consequences are dire. One consequence is CBSDH (or classical homocystinuria). Conventional treatment options do not effectively normalize the levels of Hey, Cth, Cys, or combinations thereof in a patient. More effective treatment strategies for patients presenting with homocystinuria or homocysteine remethylation disorders.

[0028] To this end, the inventor discovered that sulfur-containing reducing agents (for example, a thiol-based reductant) helps maintain proper levels of various metabolites in plasma of patients undergoing treatment for homocystinuria or homocysteine remethylation disorders. The sulfur-containing reducing agent can scavenge for and preferably bind to a highly reactive homocysteine (Hey), thus resulting in a decreased Hey binding to plasma proteins, increased retention of cysteine (Cys) and consequently increased elimination of Hey from plasma. Compositions that include such a sulfur-containing reducing agent can be utilized to treat diseases such as homocystinuria and homocysteine remethylation disorders.

[0029] In at least one embodiment is provided a composition for increasing efficacy of a cystathionine beta-synthase (CBS) enzyme replacement therapy that includes a sulfur- containing reducing agent. By increasing the efficacy of the CBS enzyme replacement therapy, the composition can be used to potentiate treatment, for example, by decreasing homocysteine levels closer to a normal level, by decreasing the dose of the enzyme replacement therapy, or combinations thereof.

[0030] In some embodiments, a combined therapy of enzyme replacement therapy (ERT) and sulfur-containing reducing agents is provided. Here, the increased availability of non- protein-bound Hey can improve the efficacy of the ERT. The ERT can include human CBS, or a modified version thereof (for example, human truncated CBS (htCBS), yeast truncated CBS, or a variant thereof), to reduce serum homocysteine (Hey) concentrations, and restore levels of its downstream metabolites such as cystathionine and cysteine. The combined therapy can be utilized for treatment of diseases such as homocystinuria and homocysteine remethylation disorders.

[0031] The combination of sulfur-containing reducing agents (for example, thiol-based reductants) and ERT can achieve a greater reduction in homocysteine than conventional methods. While not wishing to be bound by any theory, it is believed that the sulfur-containing reducing agents can free up homocysteine by breaking bonds that homocysteine forms and the ERT can then metabolize a greater amount of homocysteine, leading to a greater reduction in homocysteine levels.

[0032] As used herein, and unless specified to the contrary or the context clearly indicates otherwise, “CBS” refers to cystathionine beta synthase, “Hey” refers to homocysteine, “HCU” refers to homocystinuria, “tHcy” refers to total homocysteine, “tCys” refers to total cysteine, “Cth” refers to cystathionine, “Met” refers to methionine, “NHS” refers to N- hydroxy succinimide, and “PEG” refers to polyethylene glycol.

[0033] The use of headings is for purposes of convenience only and does not limit the scope of the present disclosure. Embodiments described herein can be combined with other embodiments.

[0034] Embodiments of the present disclosure generally relate to compositions that include sulfur-containing reducing agents. The term sulfur-containing reducing agent may be used interchangeably with the term sulfur-containing reductant. When the sulfur-containing reducing agent includes a thiol (-SH) functional group, the sulfur-containing reducing agent may be referred to as a thiol-based reductant. As used herein, a “composition” can include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof. Compositions of the present disclosure can be prepared by any suitable mixing process. [0035] Compositions described herein can be utilized for the treatment of diseases such as homocystinuria and homocysteine remethylation disorders in a patient. For treatment of diseases, sulfur-containing reducing agents of the present disclosure can be used, for example, to potentiate, sensitize, amplify, increase efficacy, etc., of a CBS enzyme replacement therapy. The sulfur-containing reducing agent can change an amount of homocysteine, cysteine, cystathionine, methionine, or combinations thereof in plasma of the patient. The sulfur- containing reducing agent can change a relative proportion of protein-bound homocysteine versus protein-unbound homocysteine in plasma of the patient. For example, the sulfur- containing reducing agent can decrease homocysteine binding to proteins in plasma of the patient.

[0036] Any suitable sulfur-containing reducing agent can be utilized. The sulfur-containing reducing agent can include a sulfur-containing functional group such as a thiol (-SH), a disulfide (-S-S-), a sulfonic acid (-SO3H), a sulfonate (-SO3 ), or combinations thereof. The sulfur-containing reducing agent may include one or more sulfur-containing functional groups. Illustrative, but non-limiting, examples of sulfur-containing reducing agent can include N- acetylcysteine (NAC; CAS No.: 616-91-1; structure la), 2-mercaptoethanesulfonic acid (CAS No.: 3375-50-6; structure lb), cysteamine (dcCys; CAS No.: 60-23-1, structure 1c), a salt thereof, or combinations thereof. Example salts include sodium 2-mercaptoethanesulfonate (MESNA), hydrochloride salts of cysteamine, hydrobromide salts of cysteamine. The structures of N-acetylcysteine (la), 2-mercaptoethanesulfonic acid (lb), and cysteamine (1c) are shown below:

[0037] Embodiments of the present disclosure also generally relate to pharmaceutical compositions. The pharmaceutical compositions can be used for the treatment of diseases such as homocystinuria and homocysteine remethylation disorders in a patient. [0038] Pharmaceutical compositions of the present disclosure include a sulfur-containing reducing agent. Pharmaceutical compositions of the present disclosure can further include a pharmaceutically acceptable carrier or excipient.

[0039] Pharmaceutical compositions described herein can be formulated with any suitable pharmaceutically acceptable carrier or diluents as well as any other suitable adjuvant or excipient in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19^th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. In one embodiment, the pharmaceutical compositions can include a combination of multiple (for example, two or more) CBS mutant polypeptides.

[0040] A pharmaceutically acceptable carrier can include any suitable solvent, dispersion medium, coating, antibacterial agent, antifungal agent, isotonic delaying agent, and absorption delaying agent, and the like that are physiologically compatible. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion). Depending on the route of administration, the active compound, for example, CBS mutant polypeptide, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound. The potentiating ingredient, for example, sulfur-containing reducing agent, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0041] Pharmaceutically acceptable salts can be used. Such salts are those that retain the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, S. M. Berge, et al., J. Pharm. Sci., 1977, 66, 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, combinations thereof, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine, combinations thereof, among others. The term “biological activity” refers to any biological activity typically attributed to a nucleic acid or protein by those skilled in the art. Examples of biological activities are enzymatic activity, ability to dimerize, fold or bind another protein or nucleic acid molecule, etc.

[0042] In some embodiments, the carrier or excipient for use with the composition disclosed herein can include, but is not limited to, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, surfactant polyoxyethylene-sorbitan monooleate, or combinations thereof.

[0043] Actual dosage levels of the active ingredients in compositions and pharmaceutical compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level can depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0044] Embodiments of the present disclosure also generally relate to combination therapies for the treatment of diseases such as homocystinuria and homocysteine remethylation disorders in a patient. The combination therapy may include (i) a sulfur-containing reducing agent or composition thereof described herein, and (ii) an enzyme replacement therapy. The enzyme replacement therapy can include any suitable CBS polypeptide described herein such as a CBS polypeptide, a CBS mutant polypeptide, a CBS variant, or combinations thereof. In some examples, the CBS mutant polypeptide can be PEGylated.

[0045] Embodiments of the present disclosure also generally relate to methods for the treatment of diseases such as homocystinuria and homocysteine remethylation disorders in a patient. Methods for the treatment of diseases may include administering to the patient a composition described herein, a pharmaceutical composition described herein, a combination therapy described herein, or combinations thereof. [0046] Methods described herein can include administering to a patient an amount of a sulfur-containing reducing agent or composition thereof described herein. The amount can be a single amount or an amount administered over a selected number of days.

[0047] A method of treating homocystinuria or a homocysteine remethylation disorder in a patient can include administering to the patient an effective amount of a combination therapy comprising (i) a sulfur-containing reducing agent or composition thereof, and (ii) optionally an enzyme replacement therapy. The enzyme replacement therapy can include any suitable CBS polypeptide described herein such as a CBS polypeptide, a CBS mutant polypeptide, a CBS variant, or combinations thereof. The enzyme replacement therapy can include an (isolated) CBS polypeptide, an (isolated) CBS mutant polypeptide described herein, or combinations thereof.

[0048] A combination therapy of the present disclosure can include any suitable dosing regimen. For example, combination therapies can include comprises a dosing regimen comprising:

[0049] (a) administering a sulfur-containing reducing agent on days 1, 2, 3, 4, 5, 15, 16,

17, 18, and/or 19 of a first 19-day cycle; and

[0050] (b) optionally, administering the enzyme replacement therapy (CBS or mutant construct thereof) on day 14, 15, 16, 17, and/or 18 of a first 19-day cycle.

[0051] The sulfur-containing reducing agent can be administered in any suitable amount such as from about 50 mg/kg to about 2,000 mg/kg, such as from about 100 mg/kg to about 1,500 mg/kg, such as from about 200 mg/kg to about 1,000 mg/kg, such as from about 400 mg/kg to about 800 mg/kg, or about 200 mg/kg, or about 1,000 mg/kg, though other values are contemplated. These amounts can be given at each treatment day or be a total amount administered over the 19-day cycle.

[0052] The CBS (or mutant construct thereof) can be administered in any suitable amount such as from about 1 mg/kg to about 15 mg/kg, such as from about 3 mg/kg to about 12 mg/kg, such as from about 5 mg/kg to about 10 mg/kg, such as about 8 mg/kg, though other values are contemplated. These amounts can be given at each treatment day or be a total amount administered over the 19-day cycle.

[0053] Administration can be performed by any suitable method such as intravenously, such as an intravenous (IV) infusion. [0054] “Coding sequence” refers to that portion of a nucleic acid (for example, a gene) that encodes (i) an mRNA that is translated into an amino acid sequence of a protein; or (ii) a functional RNA, such as an interfering RNA or antisense molecule.

[0055] “Recombinant,” when used with reference to, for example, a cell, nucleic acid, polypeptide, expression cassette or vector, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto but produced or derived from synthetic materials. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell (i.e., “exogenous nucleic acids”) or express native genes that are otherwise expressed at a different level, typically, under-expressed or not expressed at all.

[0056] Recombinant techniques can include, for example, use of a recombinant nucleic acid such as a cDNA encoding a protein or an antisense sequence, for insertion into an expression system, such as an expression vector; the resultant construct is introduced into a cell, and the cell expresses the nucleic acid, and the protein, if appropriate. Recombinant techniques also encompass the ligation of nucleic acids to coding or promoter sequences from different sources into one expression cassette or vector for expression of a fusion protein, constitutive expression of a protein, or inducible expression of a protein.

[0057] The terms “subject”, “individual”, or “patient” are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, nonhuman primates, rodents such as rats or mice, and to domestic animals such as dogs and cats, among other animals.

[0058] Compositions, combination therapies, methods, and treatments described herein are not limited to human diseases, but are also applicable to other mammals such as nonhuman primates, rodents such as rats or mice, and to domestic animals such as dogs and cats, among other animals.

[0059] “Associated” refers to coincidence with the development or manifestation of a disease, condition, or phenotype. Association can be due to, but is not limited to, genes responsible for housekeeping functions whose alteration can provide the foundation for a variety of diseases and conditions, those that are part of a pathway that is involved in a specific disease, condition, or phenotype and those that indirectly contribute to the manifestation of a disease, condition, or phenotype. [0060] “Physiological conditions” or “physiological solution” refers to an aqueous environment having an ionic strength, pH, and temperature substantially similar to conditions in an intact mammalian cell or in a tissue space or organ of a living mammal. Typically, physiological conditions comprise an aqueous solution having about 150 mM NaCl, pH 6.5- 7.6, and a temperature of approximately 22-37°C. Generally, physiological conditions are suitable binding conditions for intermolecular association of biological macromolecules. For example, physiological conditions of 150 mM NaCl, pH 7.4, at 37 °C are generally suitable.

[0061] “Pharmaceutically acceptable excipient or carrier” refers to an excipient that can optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient. In particular, in the present instance, such refers to an excipient that can be taken into the mammalian subject’s body in association with an active compound (for example, a PEGylated CBS or variant thereof) with no significant adverse toxicological effects to the subject.

[0062] The term “excipient” or “vehicle” as used herein refers to any substance, not itself a therapeutic agent, used as a carrier for delivery of a therapeutic agent and suitable for administration to a patient, for example a mammal or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition into a discrete article such as a capsule or tablet suitable for oral administration. Excipients and vehicles include any such materials known in the art, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is nontoxic and which does not interact with other components of the composition in a deleterious manner. Administration can refer to oral administration, inhalation, enteral administration, feeding, or inoculation by intravenous injection. The excipients can include standard pharmaceutical excipients, and can also include any components that can be used to prepare foods and beverages for human and/or animal consumption, feed or bait formulations or other foodstuffs. [0063] The ERT and the sulfur-containing reducing agents can induce a desired biological or pharmacological effect, which can include, but is not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing an infection, (2) alleviating a condition caused by a disease, for example, alleviating pain or inflammation caused as a result of disease, and/or (3) either alleviating, reducing, or completely eliminating the disease from the organism. The effect can be local, such as providing for a local anesthetic effect, or it can be systemic. [0064] The terms “pharmacologically effective amount” or “therapeutically effective amount” refer to a non-toxic, but sufficient amount of the active agent (or composition containing the active agent) to provide the desired level in the bloodstream or at the site of action (for example, intracellularly) in the patient to be treated, and/or to provide a desired physiological, biophysical, biochemical, pharmacological or therapeutic response, such as amelioration of the manifestations of homocystinuria. The exact amount required will vary from patient to patient, and will depend on numerous factors, such as the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (for example, the number of doses administered per day), as well as patient considerations, such as species, age, and general condition of the patient, the severity of the condition being treated, additional drugs being taken by the patient, mode of administration, and the like. These factors and considerations can be determined by one skilled in the art. An appropriate “effective” amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0065] For example, a therapeutically effective amount of a sulfur-containing reducing agent can refer to an amount of the sulfur-containing reducing agent that changes or adjusts an amount or level of a metabolite (for example, homocysteine, cysteine, cystathionine, methionine, or combinations thereof) in plasma of a patient. In the case of thiols, the therapeutically effective amount can refer to an amount that changes the redox status of the thiol metabolites, for example, change the balance in the pools of free, disulfide-bound, and protein-bound fractions. As another example, a therapeutically effective amount of a sulfur- containing reducing agent (or a composition that includes sulfur-containing reducing agent) can refer to an amount of the sulfur-containing reducing agent (or the composition that includes sulfur-containing reducing agent) to potentiate, sensitize, amplify, increase efficacy, etc. of a CBS enzyme replacement therapy .

[0066] The term “nucleic acid” can be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA. The DNA can be double-stranded or single-stranded, and if single-stranded can be the coding strand or the non-coding (anti-sense, complementary) strand. The nucleic acid may be in the form of a nucleic acid sequence such as those nucleic acid sequences described herein.

[0067] As used herein, a “variant” is a nucleic acid, protein, or polypeptide which is not identical to, but has significant homology (for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) over the entire length of the wild type nucleic acid or amino acid sequence, as exemplified by sequences in the public sequence databases, such as GenBank. As used herein, a “protein, polypeptide or peptide fragment thereof’ refers to the full-length protein or a portion of it having an amino acid sequence usually at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length, although dipeptides, tripeptides and tetrapeptides are also contemplated and encompassed by the present disclosure. [0068] As used herein, a “mutant” is a mutated protein designed or engineered to alter properties or functions relating to glycosylation, protein stabilization, and/or ligand binding.

[0069] As used herein, the terms “native” or “wild-type” relative to a given cell, polypeptide, nucleic acid, trait or phenotype, refers to the form in which that is typically found in nature.

[0070] As used herein, the terms “protein,” “polypeptide,” “oligopeptide” and “peptide” have their conventional meaning and are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (for example, glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Furthermore, the polypeptides described herein are not limited to a specific length. Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide can be an entire protein, or a subsequence thereof. Polypeptides can also refer to amino acid subsequences comprising epitopes, for example, antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.

[0071] “Position corresponding to” refers to a position of interest (for example, base number or residue number) in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein. Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99%. The position of interest is then given the number assigned in the reference nucleic acid molecule. For example, if a particular polymorphism in Gene-X occurs at nucleotide 173 of SEQ ID No. X, to identify the corresponding nucleotide in another allele or isolate, the sequences are aligned and then the position that lines up with 173 is identified. Because various alleles can be of different length, the position designate 173 may not be nucleotide 173, but instead is at a position that “corresponds” to the position in the reference sequence.

[0072] “Percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions (for example, gaps) as compared to the reference sequence (which does not comprise additions or deletions) for alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as “seeds” for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0073] While all of the above mentioned algorithms and programs are suitable for a determination of sequence alignment and % sequence identity, for purposes of the disclosure herein, determination of % sequence identity will typically be performed using the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.

[0074] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of embodiments of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for.

Examples

[0075] Various sulfur-containing reducing agents were evaluated for their effect on, for example, plasma homocysteine (Hey) and cysteine (Cys) levels as well as their individual chemical forms (for example, free, disulfide, protein-bound) in a mouse model of homocystinuria (HCU). Various example modified cystathionine beta synthase (CBS) proteins were constructed and investigated as example enzyme replacement therapies. A combined therapy of enzyme replacement therapy (ERT) and sulfur-containing reducing agents in HCU mouse model was also investigated to achieve, for example, a synergistic effect and normalization of plasma Hey levels.

[0076] Homocysteine, cysteine, cystathionine, and methionine are found in tissues, plasma, and other fluids of a patient as well. Plasma is the compartment which is typically assessed in patients and where enzyme therapy distributes and acts on these compounds. Accordingly, embodiments described herein apply to plasma and can apply to tissues of the patient and other fluids of the patient.

[0077] The following constructs were prepared: (1) human truncated CBS construct with C-terminal 6xHis tag (C-htCBS v2) and (2) yeast truncated CBS construct (C-SCCBSA345- 507). Both constructs were prepared in pET28 vector, subcloned into E. coli BL21, expressed, and purified using a two-column protocol (capture TALON cobalt IMAC column and polishing DEAE column). PEGylation was performed using NHS-activated PEG of various lengths. Additional details regarding preparation of the construction and PEGylation are further described below.

[0078] A mouse model for CBS deficiency (I278T mice) was used for evaluation. Here, the most common mutated CBS allele is 833T>C (I278T), which is associated with pyridoxineresponsive homocystinuria.

1. Experimental Procedures l.A. Construction of human truncated CBS with C-terminal 6xHis Tag (C-htCBS v2)

[0079] Compared to an annotated sequence of human CBS (uniprot# P35520), this construct lacks amino acid regions 2-39 and 398-551 and contains permanent C-terminal 6xHis tag. The coding sequence was codon optimized for expression in a bacterial host. l.A.l, Generation of the construct

[0080] Full length codon optimized human CBS gene was synthesized by Genscript and supplied in pUC57 vector (as described in J. Clin. Invest. 2016;126(6):2372-2384). The desired construct lacking residues 2-39 and 398-551 (pET28-C-hCBSOPTdl-39&397-551) was created as follows. The pET28 vector was obtained from Novagen, digested with Ncol and Xhol, dephosphorylated with CIP/SAP, gel extracted and purified, and served as a vector for ligation. The insert was prepared by PCR amplification of the desired region using the following primers and conditions:

[0081] pET28-C-htCBSv2 (d2-39&d398-551) [0082] Ta=58-63°C, elongation 1 min sec - 1090 bp PCR amplicon, 1076 bp after cleavage

[0083] 891 CT AG CCATGG AACCGCTGTGGATTCGTCCGG (FWD, Ncol site,

Tm=67.8°C) (SEQ ID NO: 5)

[0084] 872 CTAG CTCGAG GAAGCCTTTTTGCAGCATCCAACGGT (REV, Xhol site, Tm=67.6°C) (SEQ ID NO: 6)

[0085] The 891FWD+872REV PCR amplicon was kit purified, cleaved using Ncol and Xhol (NEB), gel separated and extracted, purified, and served as an insert for ligation. The vector and the insert were ligated using T4 DNA ligase-based ligation kit (NEB) and transformed into E. coli XL- 10. Multiple colonies were picked, plasmids were purified using Qiagen mini kit, and DNA sequence was verified (Eurofins genomics).

LA, 2, Expression and purification

[0086] The construct was transformed into Escherichia coli BL21 and its authenticity was confirmed by DNA sequencing. The purification of C-htCBS v2, which carries a permanent 6xHis affinity tag at the C-terminus, follows a modified procedure of T. Majtan et al., “Folding and activity of mutant cystathionine betasynthase depends on the position and nature of the purification tag: Characterization of the R266K CBS mutant,” Protein Expr Purif 82 (2012): 317-324. After the first immobilized metal affinity chromatography step (TALON column; Clontech) and subsequent desalting on a Sephadex G-25 (GE Healthcare) column, the sample was loaded onto a DEAE Sepharose (GE Healthcare) column equilibrated in the DEAE loading buffer (15 mM potassium phosphate, pH 7.2, 1 mM EDTA, 1 mM DTT, 10% ethylene glycol). The bound CBS was washed with 2 column volumes of the DEAE loading buffer followed by 5 column volumes of the DEAE wash buffer (50 mM potassium phosphate pH 7.2, 1 mM EDTA, 1 mM DTT, 10% ethylene glycol). The enzyme was then eluted with 150 mM potassium phosphate in the DEAE loading/wash buffer. The enzyme was buffer exchanged into the final storage buffer (20 mM HEPES pH 7.4, 1 mM TCEP) on a Sephadex G-25 column and subsequently concentrated using an ultrafiltration device (Amicon) equipped with an YM- 30 (Millipore) membrane. Finally, the enzyme was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0087] The C-terminal human truncated CBS construct (C-htCBS v2) has an amino acid sequence as set forth in SEQ ID NO: 1, and is shown in Table 2. C-htCBS v2 has a nucleotide sequence as set forth in SEQ ID NO. 2, and is shown in Table 2. The nucleotide sequence set forth in SEQ ID NO: 2 includes the terminal STOP codon.

Table 2: C-htCBS v2

l.B. Construction of yeast truncated CBS construct (C-SCCBSA345-507) [0088] This yeast truncated CBS construct was prepared using non-human CBS, specifically from yeast. Unlike human CBS, yeast CBS (uniprot# P32582) does not contain heme, has higher specific activity, and its activity is not regulated by S-adenosylmethionine. Similar to the human construct described above, the C-terminal regulatory domain (residues 346-507) was removed, extra glycine was added after initial methionine, and permanently attached the C-terminal 6xHis tag to facilitate purification. The construct was prepared in pET28 vector as described above and transformed into E. coli BL21. Its authenticity was confirmed by DNA sequencing. The purification of the yeast truncated CBS was performed as described in T. Majtan et al. (2014) “Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases,” PLOS ONE 9(8): el05290.

[0089] The yeast truncated CBS construct (C-SCCBSA345-507) has an amino acid sequence as set forth in SEQ ID NO: 3, and is shown in Table 3. C-htCBS v2 has a nucleotide sequence as set forth in SEQ ID NO. 4, and is shown in Table 3. The nucleotide sequence set forth in SEQ ID NO: 4 includes the terminal STOP codon.

Table 3: C-SCCBSA345-507 l.C. PEGylation

[0090] Activated PEG molecules with either maleimide or NHS ester coupling group characterized by the absence of impurities, such as diols, and narrow molecular weight distributions were purchased from NOF (Japan). PEGylation with maleimide or NHS ester PEG was carried out in 100 mM potassium phosphate pH 6.5 and 50 mM sodium phosphate pH 7.2, respectively, by adding PEG dissolved in Milli-Q water in the desired molar ratio (typically CBS subunit/PEG = 1 : 10) to a final protein concentration of 5 mg/mL. The reaction was carried out at 4°C overnight. Subsequently, the reaction mixture was diluted twice with Milli-Q water, buffer exchanged into Gibco l x PBS (Thermo Fisher Scientific, MA, USA), and concentrated using Labscale TFF system with Pellicon XL 50 Biomax 100 cartridge (poly(ether sulfone) 100 kDa MWCO membrane). The final PEG-CBS conjugates were filter sterilized (Millipore’s 0.2 pm PVDF membrane filter), aliquoted, flash frozen in liquid nitrogen, and stored at -80°C.

[0091] A molar ratio of PEG molecules to the CBS (or mutant construct thereof) was 20: 1 for the 5 kDa NHS ester-activated PEG, 15: 1 for the 10 kDa NHS ester-activated PEG, and 10: 1 for the 20 kDa NHS ester-activated PEG. l.D. Enzyme (CBS) activity assay

[0092] CBS activity was determined by a radioisotope assay using [¹⁴C(U)] L-serine as the labeled substrate. Briefly, a purified enzyme (420 ng) was assayed in a 100 mL reaction mixture for 30 min at 37°C. The reaction mixture contained 100 mM Tris-HCl pH 8.6, 10 mM L-serine, 0.2 mM Pyridoxal 5 '-phosphate (PLP), 0.3 pCi L-[¹⁴C(U)]-serine, and 0.5 mg/mL bovine serum albumin (BSA). The reaction was performed in the presence or absence of AdoMet in a final concentration of 0.3 mM. The reaction mixture with enzyme was incubated at 37°C for 5 minutes and the reaction was initiated by addition of L-homocysteine to a final concentration of 10 mM. The reaction was terminated by an immediate cooling of the mixture in ice water and the labeled product was separated from the substrates by paper chromatography. Spots corresponding to cystathionine (Cth) were cut-out and radioactivity was determined by using a scintillation counter. l.E. SDS-PAGE

[0093] Denatured proteins were separated by SDS-PAGE using a 9% separating gel with a 4% stacking gel. Native samples were separated in 4-15% polyacrylamide gradient precast gels (Mini-PROTEAN TGX, Bio-Rad). For visualization, the denatured gels were stained with Simple Blue (Invitrogen). Western blot analysis of crude cell lysates under denaturing or native conditions was then performed as described in T. Majtan et al., “Rescue of cystathionine betasynthase (CBS) mutants with chemical chaperones: purification and characterization of eight CBS mutant enzymes”, The Journal of Biological Chemistry 285 (2010): 15866-15873. l.E. Determination of metabolite concentrations

[0094] Plasma metabolites (for example, homocysteine, cystathionine and cysteine) were determined by stable-isotope-dilution gas chromatography mass spectrometry as described in H. Allen et al., “Serum betaine, N,N-dimethylglycine and N-methylglycine levels in patients with cobalamin and folate deficiency and related inborn errors of metabolism”, Metabolism 42 (1993), 1448-1460. Metabolites in tissues, which homogenized in the presence of 5mM DTT and deproteinized with 4 volumes of 0.1M perchloric acid, were determined using stable- isotope-dilution liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) as described in E. Arning et al., “Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in plasma using liquid chromatography-electrospray tandem mass spectrometry”, Methods Mol. Biol. 1378 (2016) 255-262 and S.C. Lai et al., “The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system”, FASEB J. 27 (2013), 2468-2475. l.E. Animal protocols

[0095] All animal procedures were approved by the University of Colorado — Denver IACUC, which is an AAALAC Accredited (#00235), Public Health Service Assured (#A 3269- 01) and USDA Licensed (#84-R-0059) Institution. A breeding pair of heterozygous transgenic I278T mice on the C57BL6 CBS knockout background was provided by Fox Chase Cancer Center, Philadelphia, PA, USA. Mice were propagated and genotyped at our facility, as described elsewhere (Wang et al., 2005. Hum. Mol. Genet. 14, 2201-2208). Breeding pairs were maintained on water containing 25 mM zinc chloride to induce transgene expression and thus rescue the homozygous I278T pups from neonatal death. After weaning at 21 days of age, mice switched to a regular water supply and were maintained on extruded standard diet 2920X (Envigo, CA, USA). A single-use lancet for submandibular bleeding was used for blood collection into Capiject T-MLHG lithium heparin (12.5 IU) tubes with gel (Terumo Medical Corporation, NJ, USA). Tubes were then centrifuged at 1200 G for 10 min, followed by collection of plasma to 1.5 mL tubes and storage at -80° C.

[0096] Genotyping: As the heterozygous CBS +/- females were used for breeding with homozygous CBS -I- males, all pups were analyzed for CBS knockout homozygosity by qPCR. Tail biopsies were processed by the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). DNA quality was monitored by NanoDrop 1000 (Thermo Scientific, DE, USA). 20 ng samples of DNA were run single-plex in triplicate using Applied Biosystems’ (CA, USA) Gene Expression master mix (Item #4369016). Amplification was performed on Applied Biosystems’ 7500 Fast Instrument using the standard curve method. Applied Biosystems’ Tert (Item #4458366) or Tfrc (Item #4458368) copy number reference assays were used as the homozygous one copy calibrator. Applied Biosystems’ assay Mr00299300 was used to detect the Neo gene. The presence of the I278T transgene was not routinely verified as all animals were expected to carry at least a single copy of the transgene as described elsewhere (Wang et al., 2005. Hum. Mol. Genet. 14, 2201-2208).

2, Non-limiting Examples

2.A. Generation of modified CBS proteins

[0097] The following CBS proteins were generated as described above: C-htCBS v2; and C-SCCBSA345-507. C-htCBS v2 is a human truncated CBS sequence having sequences removed from both the N- and C-terminal ends and a permanently attached C-terminal 6xHis tag to facilitate purification. C-SCCBSA345-507 is a yeast truncated CBS carrying permanently attached C-terminal 6xHis tag. PEG-modified versions of these CBS proteins were also investigated to determine the effect on size and activity.

[0098] FIG. 1 A shows an SDS-PAGE of the CBS proteins and CBS proteins modified with 5 kDA, 10 kDA, and 20 kDA NHS-activated PEGs. The results indicated that the PEGylation resulted in modification of C-htCBS v2 and C-SCCBSA345-507 protein subunits. The PEGylation did result in the presence of variably modified species, most likely determined by the number of PEG moi eties covalently attached to one or more protein lysine residues. While not wishing to be bound by any theory, it is believed that the presence of the smaller (for example, less PEGylated) species does not substantially impact both pharmacokinetics and pharmacodynamics of PEG-CBS. In addition, it is believed that shorter PEGs, such as 5 kDa or 10 kDa NHS PEGs, may provide technological advantages as the viscosity of such PEG- CBS species is much lower than the 20 kDa NHS PEGs.

[0099] FIG. IB indicates that PEGylation does not significantly impair catalytic activity of the modified enzymes. C-SCCBSA345-507 is about 50% more active than C-htCBS v2. C- htCBS v2 and C-SCCBSA345-507 were further investigated with in vivo studies.

2.B. Safety profile of sulfur-containing reducing agents

[0100] To evaluate safety profile after intraperitoneal (IP) injection of the selected sulfur- containing reducing agent (NAC, MESNA, dcCYS), I278T mice (n = 6 per each cohort) were injected with an initial dose of 200 mg/kg/day (daily dose split into two IP injections: 6AM and 6PM) for a period of 5 days and monitored their health for any unusual symptoms and signs, such as lethargy, shaking, hyperactivity, aggressivity, unexpected loss/gain of weight, etc. No unusual symptoms and signs were observed. Therefore after two weeks a higher dose of 500 mg/kg/day were tested following a similar protocol. The I278T mice receiving 500 mg/kg/day dcCYS were observed to be less active, had problems to maintain body temperature, and one mouse died. The other two compounds (NAC and MESNA) were tolerated well, therefore after additional two weeks, the higher doses of 1,000 mg/kg/day were evaluated. This higher dose was well-tolerated by I278T mice. Based on these results, NAC and MESNA were evaluated at the dose of 1,000 mg/kg/day, while dcCYS were administered at 5* lower dose (200 mg/kg/day) in order to prevent its apparent toxicity in I278T mice.

2.C. Animal study design

[0101] Once the highest safe doses of sulfur-containing reducing agents in I278T mice were obtained, these values were utilized to design and execute an animal study to assess the efficacy of sulfur-containing reducing agents affecting dynamic balance of plasma Hey forms by themselves as well as in combination with enzyme therapy (for example, a PEG-CBS described herein). This approach allowed minimization of the use of experimental animals and to monitor and compare changes upon sulfur-containing reducing agent and PEG-CBS administration in the same animals during the course of the study. [0102] FIG. 2 shows a graphical representation of the study design. The PEG-CBS utilized for the study is the yeast truncated CBS construct (C-SCCBSA345-507) PEGylated with 20 kDa NHS-activated PEG.

[0103] Three cohorts each representing one selected sulfur-containing reducing agent (NAC, MESNA and dcCYS) and control PBS-injected cohort of I278T mice (n=6-8 per group) were transferred from a complete standard rodent chow 2920X and set up on amino acid- defined diet TD.170063 containing 0.4% methionine and 0.35% cystine. After a 12-day acclimation to the new diet, PBS (vehicle) or sulfur-containing reducing agent dissolved in PBS (1000 mg/kg/day NAC or MESNA; 200 mg/kg/day dcCYS) was administered daily for five days intraperitoneally (IP) in two doses each day (executed at 7AM and 7PM). After 10 days recovery period back to initial metabolite levels, a similar regimen was executed again now accompanied with administration of PEG-CBS to assess synergistic effect of sulfur- containing reducing agents and enzyme therapy for HCU. Treatment with PEG-CBS herein preceded administration of sulfur-containing reducing agents by one day just to have an initial efficacious CBS activity in plasma before the first dose of sulfur-containing reducing agents. The PEG-CBS was administered at dose of 8 mg/kg/day in a single subcutaneous (SC) injection each day at 9AM for a period of 5 days (for example, until PEG-CBS reaches steady-state plasma levels in mice. Blood samples were collected on day 1 (DI) (normal diet basal levels), D10 (amino-acid defined diet basal levels), D14 & D17 (initial & steady-state efficacy of sulfur-containing reducing agents alone), D24 (recovery of basal levels) and D28 & D31 (initial & steady-state efficacy of sulfur-containing reducing agents together with PEG-CBS). (D = Day).

2.D. Impact of sulfur-containing reducing agents — with or without PEG-CBS — on plasma sulfur amino acid metabolites

[0104] FIGS. 3A-3D show non-limiting data for the evaluation of thiol-based reductants in I278T mice. The plots in FIGS. 3A-3D are based on four I278T mouse cohorts (n = 6-8 each) were acclimated on amino acid-defined diet containing 0.4% Met and received vehicle (PBS), NAC, MESNA, or dcCYS alone and after a washout period received the same together with PEG-CBS as described in detail in the main text. Hexagons by the x-axis denote administration days for PBS or reductants, while stars by the x-axis designate days of ERT (PEG-CBS) coadministration. (FIG. 3A) Plasma tHcy, (FIG. 3B) plasma tCys, (FIG. 3C) plasma Cth, (FIG. 3D) plasma Met. Asterisks denote significance (*/**/*** = p<0.05/0.01/0.001). [0105] FIG. 3A shows the effect of sulfur-containing reducing agents alone and in combination with PEG-CBS on plasma total homocysteine (tHcy) levels. After 5 days of treatment, a decrease of about 27.5% and about 26.3% was observed after treatment with NAC and dcCYS, respectively (p<0.01, day 10 (DIO) vs day 17 (DI 7)), while treatment with MESNA showed a -10.9% drop in plasma tHcy levels (p=ns). Co-administration of PEG-CBS resulted in substantially decreased plasma tHcy levels in all cohorts. While PEG-CBS administration resulted in tHcy decrease by about 91.4% and about 89.8% in PBS- and dcCYS- injected I278T mice, respectively (p<0.001, day 24 (D24) vs day 31 (D31)), mice receiving NAC and MESNA showed even bigger drop in plasma tHcy levels after co-administration of PEG-CBS by about 95.1% and about 95.8%, respectively (p<0.001). Specifically, tHcy concentration dropped to about 43 pM and about 49 pM in PBS and dcCYS cohorts, respectively, while NAC and MESNA cohorts showed on average plasma tHcy of about 25 pM and about 23 pM, respectively (p<0.01). Taken together, administration of NAC and dcCYS alone resulted in a significant decrease of tHcy levels. On the other hand, NAC and MESNA acted in synergy with PEG-CBS and jointly achieved reduction of plasma tHcy by >95%, which almost equaled to normalization on a background of unrestricted normal Met intake.

[0106] FIG. 3B shows the effect of sulfur-containing reducing agents alone and in combination with PEG-CBS on plasma total cysteine (tCys) levels. After 5 days of treatment, an increase of about 13.9% increase was observed after treatment with NAC (p<0.01, day 10 (D10) vs day 17 (DI 7)), while treatment with dcCYS showed a -5.6% increase in plasma tCys levels with MESNA and PBS cohorts showing no difference (p=ns). Co-administration of PEG-CBS resulted in a substantial increase of plasma tCys levels in all cohorts. While PEG- CBS administration resulted in normalization of tCys in PBS-injected cohort (294 pM, p<0.001, day 24 (D24) vs day 31 (D31)), elevation of plasma tCys was less profound in cohorts receiving sulfur-containing reducing agent. Specifically, plasma tCys increased to about 242 pM and about 232 pM in NAC- and MESNA-treated mice (p<0.001), respectively, while dcCYS increased tCys only to about 176 pM (p<0.05). Taken together, only NAC alone was able to slightly increase low tCys levels observed in I278T mice. However, all studied sulfur- containing reducing agents reduced efficacy of PEG-CBS to improve plasma tCys levels under the conditions tested. While co-administration of NAC and MESNA with PEG-CBS still resulted in a lower-limit normalization compared to PEG-CBS alone (PBS cohort), dcCYS apparently interfered with plasma tCys normalization under the conditions tested. [0107] FIG. 3C shows the effect of sulfur-containing reducing agents alone and in combination with PEG-CBS on plasma cystathionine (Cth) levels. The sulfur-containing reducing agents alone did not change plasma Cth levels. A marked elevation of plasma Cth levels was observed only after co-administration of PEG-CBS. Interestingly, I278T mice receiving PBS or dcCYS showed significantly higher plasma Cth levels compared to those on NAC and MESNA (p<0.05). Specifically, plasma Cth concentration increased to about 74.1 pM and about 88.3 pM in PBS and dcCYS cohorts, respectively (p<0.001, day 24 (D24) vs day 31 (D31)), while the mice on NAC and MESNA achieved plasma Cth levels of about 44.7 pM and about 48.7 pM, respectively (p<0.001). Taken together, plasma Cth levels increased only with administration of PEG-CBS as a marker of PEG-CBS activity and efficacy. Interestingly, production of Cth and its plasma accumulation was much lower in NAC- and MESNA-treated cohorts compared to PBS-injected mice and dcCYS-injected mice, which correlated with the difference in plasma tHcy and partially tCys levels (FIGS. 3A and 3B).

[0108] FIG. 3D shows the effect of sulfur-containing reducing agents alone and in combination with PEG-CBS on plasma methionine (Met) levels. Under the conditions tested, sulfur-containing reducing agents alone did not show a significant effect on plasma Met levels despite apparent downward trend. Co-administration of PEG-CBS resulted in a significant decrease of plasma Met concentrations in all cohorts by about 29.9%, about 35.5%, about 45.5% and about 67.8% to about 66 pM, about 50 pM, about 52 pM, and about 41 pM plasma Met in cohorts injected with PBS, NAC, MESNA and dcCYS, respectively (p<0.05 for PBS and p<0.01 for the remaining cohorts), which essentially resulted in plasma Met normalization (about 50 pM in WT mice on the same diet). Taken together, and under the conditions tested, only PEG-CBS alone or in combination with sulfur-containing reducing agents resulted in a significant decrease and essentially normalization of plasma Met concentration in I278T mice.

2.E. Impact of sulfur-containing reducing agents on Plasma Hey fractions

[0109] FIG. 4 shows relative proportions of protein-bound Hey fractions and protein- unbound Hey fractions from tHcy pools when the mice got acclimated to amino acid-defined diet (day 10 (D10)), after 5 days of treatment with sulfur-containing reducing agents (day 17 (D17)) and at steady state levels of PEG-CBS when co-administered with sulfur-containing reducing agents (day 31 (D31)). Both tHcy and protein-unbound pools were determined experimentally, while protein-unbound fraction was calculated as a difference between tHcy and protein-bound Hey. The PBS injections did not significantly change Hey fractionation in plasma. However, administration of sulfur-containing reducing agents resulted in a significant increase of protein-unbound fraction confirming the hypothesis that sulfur-containing reducing agents can shift the balance towards protein-unbound Hey. Specifically, administration of NAC and MESNA resulted in about 46% and about 39% increase of protein-unbound Hey at DI 7, respectively (p<0.001), while administration of dcCYS yielded much smaller response of about 21% (p<0.05). Interestingly, when co-administered with PEG-CBS, which by itself resulted in >90% decreased of tHcy (FIG. 3A), distribution of Hey fractions has been restored and essentially returned to pre-treatment levels. Taken together, and under the conditions tested, sulfur-containing reducing agents, such as NAC and MESNA, markedly increased proportion of protein-unbound Hey fraction. Despite normalization of Hey distribution after coadministration of PEG-CBS and sulfur-containing reducing agents under the conditions tested, the impact of NAC and MESNA has apparently resulted in potentiation or synergistic effect with PEG-CBS as plasma tHcy dropped to about 24 pM levels compared to about 46 pM concentration for PBS- and dcCYS-injected I278T cohorts (see also FIG. 3A).

[0110] The presented experimental data support the idea that sulfur-containing reducing agents affect the proportion of protein-bound and protein-unbound Hey fractions within plasma tHcy pool. Treatment with NAC and MESNA resulted in a substantial (about 40%) increase of protein-unbound plasma Hey fraction. Further, co-administration of PEG-CBS, with NAC and MESNA resulted in a significantly lower plasma tHcy levels compared to those in I278T mice injected with vehicle (PBS) or dcCYS. This improvement of plasma tHcy levels (essentially almost normalization) by NAC and MESNA together with PEG-CBS was accompanied by a lesser normalization of plasma tCys concentrations and lower production/accumulation of Cth, a product of PEG-CBS, in circulation.

[OHl] The results clearly show the synergistic effect of the modified CBS proteins with the sulfur-containing reducing agents. That is, the results clearly showed that the result was not merely additive and is of a significant practical advantage by showing, at least, a significant improvement in, for example, plasma tHcy levels.

[0112] Overall, the data presented herein indicates that sulfur-containing reducing agents displayed clear potential to decrease plasma tHcy levels by themselves or in combination with novel enzyme therapy for HCU.

Embodiments Listing

[0113] The present disclosure provides, among others, the following embodiments, each of which can be considered as optionally including any alternate embodiments: [0114] Embodiment 1. A composition for increasing efficacy of a CBS enzyme replacement therapy, the composition comprising: a sulfur-containing reducing agent.

[0115] Embodiment 2. A composition for potentiating, sensitizing, and/or amplifying a CBS enzyme replacement therapy in a patient, the composition comprising: a sulfur-containing reducing agent.

[0116] Embodiment 3. The composition of Embodiment 1 or Embodiment 2, wherein the composition decreases homocysteine levels in a patient, decreases a dose of the enzyme replacement therapy, or combinations thereof.

[0117] Embodiment 4. The composition of any one of Embodiments 1-3, wherein the sulfur-containing reducing agent comprises a thiol (-SH), a disulfide (-S-S-), a sulfonic acid (-SO3H), a sulfonate (-SO3 ), a salt thereof, or combinations thereof.

[0118] Embodiment 5. The composition of any one of Embodiments 1-4, wherein the sulfur-containing reducing agent comprises a hydrochloride salt, a hydrobromide salt, a sodium salt, a potassium salt, a lithium salt, or combinations thereof

[0119] Embodiment 6. The composition of any one of Embodiments 1-5, wherein the sulfur-containing reducing agent comprises N-acetylcysteine (NAC), 2- mercaptoethanesulfonic acid, sodium 2-mercaptoethanesulfonate, cysteamine, cysteamine hydrochloride, cysteamine hydrobromide, a salt thereof, or combinations thereof.

[0120] Embodiment 7. A method for increasing efficacy of a CBS enzyme replacement therapy in a patient, the method comprising: administering to the patient a therapy comprising the composition of any one of Embodiments 1-6; and administering to the patient a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof.

[0121] Embodiment 8. The method of Embodiment 7, wherein the composition of any one of Embodiments 1-6 is co-administered with the CBS polypeptide, the CBS mutant polypeptide, or combinations thereof.

[0122] Embodiment 9. The method of Embodiment 7 or Embodiment 8, wherein the composition of any one of Embodiments 1-6 is administered to the patient prior to and/or after the CBS polypeptide, the CBS mutant polypeptide, or combinations thereof.

[0123] Embodiment 10. A combination therapy for decreasing homocysteine levels in a patient, the combination therapy comprising: a composition comprising a sulfur-containing reducing agent; and a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof. [0124] Embodiment 11. The combination therapy of Embodiment 10, wherein the CBS mutant polypeptide comprises a human truncated CBS polypeptide or a yeast truncated polypeptide.

[0125] Embodiment 12. The combination therapy of Embodiment 10 or Embodiment 11, wherein the CBS mutant polypeptide comprises an amino acid sequence as set forth in one or more of SEQ ID NOs: 1 or 3, or an amino acid sequence having at least 80%, or 85%, or 90%, or 95% sequence identity to one or more of SEQ ID NOs: 1 or 3.

[0126] Embodiment 13. The combination therapy of any one of Embodiments 10-12, wherein the CBS mutant polypeptide is PEGylated and a C-terminal regulatory region of the CBS mutant polypeptide is truncated.

[0127] Embodiment 14. The combination therapy of any one of Embodiments 10-13, wherein the CBS mutant polypeptide is PEGylated with a polyethylene glycol having a molecular weight that is from about 2 kDa to about 40 kDa.

[0128] Embodiment 15. The combination therapy of any one of Embodiments 10-14, wherein the CBS mutant polypeptide is PEGylated with a polyethylene glycol that is branched. [0129] Embodiment 16. The combination therapy of any one of Embodiments 10-15, wherein the CBS mutant polypeptide is PEGylated with a 5 kDa NHS ester-activated PEG, a 10 kDa NHS ester-activated PEG, or a 20 kDa NHS ester-activated PEG.

[0130] Embodiment 17. A method for treating homocystinuria in a patient, the method comprising: administering to the patient the combination therapy of any one of Embodiments 10-16, wherein: the combination therapy is administered as a single joint shot or dose (for example, by the same route); or the combination therapy is administered separately by administering the composition comprising a sulfur-containing reducing agent orally and administering the CBS polypeptide, a CBS mutant polypeptide, or combinations thereof parenterally (subcutaneously).

[0131] Embodiment 18. The method of Embodiment 17, wherein the composition comprising the sulfur-containing reducing agent is administered to the patient prior to the CBS polypeptide, the CBS mutant polypeptide, or combinations thereof. [0132] Embodiment 19. A method of adjusting an amount of a metabolite in a patient, the method comprising: administering to a patient a composition comprising a sulfur-containing reducing agent.

[0133] Embodiment 20. The method of Embodiment 19, further comprising: administering to the patient a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof.

[0134] Embodiment 21. The method of Embodiment 20, wherein: the CBS mutant polypeptide is PEGylated; a C-terminal regulatory region of the CBS mutant polypeptide is truncated; a N-terminal region of the CBS polypeptide is truncated; or combinations thereof.

[0135] Embodiment 22. The method of Embodiment 20 or Embodiment 21, wherein the composition comprising the sulfur-containing reducing agent is administered to the patient before, during, or after the CBS mutant polypeptide.

[0136] Embodiment 23. The method of any one of Embodiments 19-22, wherein the metabolite comprises homocysteine, cysteine, cystathionine, methionine, or combinations thereof.

[0137] Embodiment 24. A composition for potentiating a CBS enzyme replacement therapy in a patient undergoing treatment for homocystinuria or homocysteine remethylation disorders, the composition comprising a sulfur-containing reducing agent.

[0138] Embodiment 25. The composition of Embodiment 24, wherein the sulfur- containing reducing agent changes an amount of homocysteine, cysteine, cystathionine, methionine, or combinations thereof in plasma of the patient.

[0139] Embodiment 26. The composition of Embodiment 24 or 25, wherein the sulfur- containing reducing agent changes a relative proportion of protein-bound homocysteine versus protein-unbound homocysteine in plasma of the patient.

[0140] Embodiment 27. The composition of any one of Embodiments 24-26, wherein the sulfur-containing reducing agent decreases homocysteine binding to proteins in plasma of the patient.

[0141] Embodiment 28. The composition of any one of Embodiments 24-27, wherein the sulfur-containing reducing agent comprises a thiol (-SH), a disulfide (-S-S-), a sulfonic acid (-SO3H), a sulfonate (-SO3 ), a salt thereof, or combinations thereof.

[0142] Embodiment 29. The composition of any one of Embodiments 24-28, wherein the sulfur-containing reducing agent comprises a hydrochloride salt, a hydrobromide salt, a sodium salt, a potassium salt, a lithium salt, or combinations thereof. [0143] Embodiment 30. The composition of any one of Embodiments 24-29, wherein the sulfur-containing reducing agent comprises N-acetylcysteine, 2-mercaptoethanesulfonic acid, sodium 2-mercaptoethanesulfonate, cysteamine, cysteamine hydrochloride, cysteamine hydrobromide, a salt thereof, or combinations thereof.

[0144] Embodiment 31. A combination therapy for treating homocystinuria or homocysteine remethylation disorders in a patient, the combination therapy comprising: a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof; and a sulfur-containing reducing agent.

[0145] Embodiment 32. The combination therapy of Embodiment 31, wherein the sulfur- containing reducing agent changes a relative proportion of protein-bound homocysteine versus protein-unbound homocysteine in plasma of the patient.

[0146] Embodiment 33. The combination therapy of Embodiment 31 or Embodiment 32, wherein the sulfur-containing reducing agent decreases homocysteine levels in plasma of the patient.

[0147] Embodiment 34. The combination therapy of any one of Embodiments 31-33, wherein the sulfur-containing reducing agent comprises a thiol (-SH), a disulfide (-S-S-), a sulfonic acid (-SO3H), a sulfonate (-SO3 ), a salt thereof, or combinations thereof.

[0148] Embodiment 35. The combination therapy of any one of Embodiments 31-34, wherein the sulfur-containing reducing agent comprises N-acetylcysteine, 2- mercaptoethanesulfonic acid, sodium 2-mercaptoethanesulfonate, cysteamine, cysteamine hydrochloride, cysteamine hydrobromide, a salt thereof, or combinations thereof.

[0149] Embodiment 36. The combination therapy of any one of Embodiments 31-35, wherein the CBS mutant polypeptide comprises a human truncated CBS polypeptide or a yeast truncated polypeptide.

[0150] Embodiment 37. The combination therapy of any one of Embodiments 31-36, wherein the CBS mutant polypeptide comprises an amino acid sequence as set forth in one or more of SEQ ID NOs: 1 or 3.

[0151] Embodiment 38. The combination therapy of any one of Embodiments 31-37, wherein the CBS mutant polypeptide is PEGylated and a C-terminal regulatory region of the CBS mutant polypeptide is truncated.

[0152] Embodiment 39. The combination therapy of any one of Embodiments 31-38, wherein the CBS mutant polypeptide is PEGylated with a polyethylene glycol having a molecular weight that is from about 2 kDa to about 40 kDa. [0153] Embodiment 40. The combination therapy of any one of Embodiments 31-39, wherein the CBS mutant polypeptide is PEGylated with a polyethylene glycol that is branched. [0154] Embodiment 41. The combination therapy of any one of Embodiments 31-40, wherein the CBS mutant polypeptide is PEGylated with a 5 kDa NHS ester-activated PEG, a 10 kDa NHS ester-activated PEG, or a 20 kDa NHS ester-activated PEG.

[0155] Embodiment 42. A method for potentiating enzyme therapy for treating homocystinuria or homocysteine remethylation disorders in a patient, the method comprising: administering to the patient a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof; and administering to the patient a sulfur-containing reducing agent or a therapeutically effective amount of a sulfur-containing reducing agent.

[0156] Embodiment 43. The method of Embodiment 42, wherein the sulfur-containing reducing agent or the therapeutically effective amount of the sulfur-containing reducing agent changes an amount of homocysteine, cysteine, cystathionine, methionine, or combinations thereof in plasma of the patient.

[0157] As is apparent from the foregoing general description and the specific embodiments, while forms of the embodiments have been illustrated and described, various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, an element or a group of elements is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, element, or elements and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.

[0158] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit can be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit can be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit can be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value can serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0159] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, embodiments comprising “a sulfur-containing reducing agent” includes embodiments comprising one, two, or more sulfur-containing reducing agents, unless specified to the contrary or the context clearly indicates only one sulfur-containing reducing agent is included.

[0160] As used herein, reference to chemical compound without specifying a particular isomer (such as butanol) expressly discloses all isomers (such as n-butanol, iso-butanol, secbutanol, and tert-butanol). For example, reference to a chemical compound having 4 carbon atoms expressly discloses all isomers thereof. When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomer, diastereomer, and enantiomer of the compound described individually or in any combination. [0161] All patents, patent applications, patent publications, scientific articles and the like, cited or identified in this application are hereby incorporated by reference in their entirety in order to describe more fully the state of the art to which the present application pertains.

[0162] While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure can be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

Claims What is claimed is:

1. A composition for potentiating a CBS enzyme replacement therapy in a patient undergoing treatment for homocystinuria or homocysteine remethylation disorders, the composition comprising a sulfur-containing reducing agent.

2. The composition of claim 1, wherein the sulfur-containing reducing agent changes an amount of homocysteine, cysteine, cystathionine, methionine, or combinations thereof in plasma of the patient.

3. The composition of claim 1, wherein the sulfur-containing reducing agent changes a relative proportion of protein-bound homocysteine versus protein-unbound homocysteine in plasma of the patient.

4. The composition of claim 1, wherein the sulfur-containing reducing agent decreases homocysteine binding to proteins in plasma of the patient.

5. The composition of claim 1, wherein the sulfur-containing reducing agent comprises a thiol (-SH), a disulfide (-S-S-), a sulfonic acid (-SO3H), a sulfonate (-SO3 ), a salt thereof, or combinations thereof.

6. The composition of claim 1, wherein the sulfur-containing reducing agent comprises a hydrochloride salt, a hydrobromide salt, a sodium salt, a potassium salt, a lithium salt, or combinations thereof.

7. The composition of claim 1, wherein the sulfur-containing reducing agent comprises N-acetylcysteine, 2-mercaptoethanesulfonic acid, sodium 2-mercaptoethanesulfonate, cysteamine, cysteamine hydrochloride, cysteamine hydrobromide, a salt thereof, or combinations thereof.

8. A combination therapy for treating homocystinuria or homocysteine remethylation disorders in a patient, the combination therapy comprising: a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof; and a sulfur-containing reducing agent.

9. The combination therapy of claim 8, wherein the sulfur-containing reducing agent changes a relative proportion of protein-bound homocysteine versus protein-unbound homocysteine in plasma of the patient.

10. The combination therapy of claim 8, wherein the sulfur-containing reducing agent decreases homocysteine levels in plasma of the patient.

11. The combination therapy of claim 8, wherein the sulfur-containing reducing agent comprises a thiol (-SH), a disulfide (-S-S-), a sulfonic acid (-SO3H), a sulfonate (-SO3 ), a salt thereof, or combinations thereof.

12. The combination therapy of claim 8, wherein the sulfur-containing reducing agent comprises N-acetylcysteine, 2-mercaptoethanesulfonic acid, sodium 2- mercaptoethanesulfonate, cysteamine, cysteamine hydrochloride, cysteamine hydrobromide, a salt thereof, or combinations thereof.

13. The combination therapy of claim 8, wherein the CBS mutant polypeptide comprises a human truncated CBS polypeptide or a yeast truncated polypeptide.

14. The combination therapy of claim 8, wherein the CBS mutant polypeptide comprises an amino acid sequence as set forth in one or more of SEQ ID NOs: 1 or 3.

15. The combination therapy of claim 8, wherein the CB S mutant polypeptide is PEGylated and a C-terminal regulatory region of the CBS mutant polypeptide is truncated.

16. The combination therapy of claim 8, wherein the CB S mutant polypeptide is PEGylated with a polyethylene glycol having a molecular weight that is from about 2 kDa to about 40 kDa.

17. The combination therapy of claim 8, wherein the CB S mutant polypeptide is PEGylated with a polyethylene glycol that is branched.

18. The combination therapy of claim 8, wherein the CBS mutant polypeptide is PEGylated with a 5 kDa NHS ester-activated PEG, a 10 kDa NHS ester-activated PEG, or a 20 kDa NHS ester-activated PEG.

19. A method for potentiating enzyme therapy for treating homocystinuria or homocysteine remethylation disorders in a patient, the method comprising: administering to the patient a CBS polypeptide, a CBS mutant polypeptide, or combinations thereof; and administering to the patient a sulfur-containing reducing agent.

20. The method of claim 19, wherein the sulfur-containing reducing agent changes an amount of homocysteine, cysteine, cystathionine, methionine, or combinations thereof in plasma of the patient.