Methods for treating congenital disorders of glycosylation

Abstract

Embodiments of the disclosure include methods and compositions for treating one or more congenital disorders of glycosylation (CDG), such as Phosphomannomutase-2 (PMM2) deficiency (or PMM2-CDG). In specific embodiments, one or more GSK3 inhibitors are utilized as a therapeutic agent for an individual with PMM2-CDG. In certain embodiments, the individual may also be provided one or more aldose reductase inhibitors and / or one or more carbonic anhydrase inhibitors.

Description

METHODS FOR TREATING CONGENITAL DISORDERS OF GLYCOSYLATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 551,882, filed February 9, 2024, which is incorporated by reference herein in its entiretyBACKGROUNDI. Field

[0002] This disclosure relates at least to the field of compounds and pharmaceutical compositions for increasing glycogen and glycosylation and treating congenital disorders of glycosylation. In certain embodiments, this disclosure relates at least to the fields of biochemistry, cell biology, molecular biology, metabolism, and medicine.II. Background

[0003] Congenital disorders of glycosylation (CDG) include more than 160 inborn errors of metabolism that affect protein and lipid-linked glycosylation. CDGs are typically classified as Types I (CDG-1) and II (CDG-II).

[0004] Phosphomannomutase-2 (PMM2) deficiency (or PMM2-CDG, previously known as CDG-Ia) is the most common disorder of glycosylation with more than 1000 patients reported worldwide. It is an autosomal recessive disease caused by mutations in the PMM2 gene, which encodes for phosphomannomutase-2. The phosphomannomutase-2 enzyme catalyzes the conversion of mannose 6-phosphate into mannose 1 -phosphate, which in turn leads to the synthesis of GDP-mannose: an essential sugar nucleotide involved in protein and lipid glycosylation. Insufficient levels of GDP-mannose lead to hypoglycosylation. PMM2 deficiency also affects intracellular metabolic balance, including alterations in glucose flux and polyol metabolism. Thus, resolving the glycosylation defect, and restoring metabolic balance, may all or singly contribute to a therapeutic effect in the disease.

[0005] The clinical presentation of PMM2-CDG varies widely and is characterized by a multisystem phenotype that includes neurologic involvement (ataxia, speech delay, and seizures), global developmental delay, intellectual disability, coagulation abnormalities, and liverabnormalities. There is no cure for PMM2-CDG and treatment is mainly supportive and symptomatic, focusing on the management of individual symptoms as they arise.SUMMARY

[0006] Embodiments of the present disclosure provide compositions and methods for increasing glycosylation, increasing glycogen, and / or restoring metabolic balance in an individual in need thereof. In particular embodiments, the disclosure provides methods for treating congenital disorders of glycosylation. In specific embodiments, the present disclosure provides compositions and methods for increasing glycosylation, increasing glycogen, and restoring metabolic balance in an individual in need thereof, such as by administering to the individual a therapeutically effective amount of a compound having a structure in FIG. 1 .

[0007] Embodiments of the disclosure include methods of treating a congenital disorder of glycosylation (CDG) or a congenital disorder of deglycosylation (CDDG) in an individual, comprising the step of administering to the individual a therapeutically effective amount of one or more glycogen synthase kinase 3 (GSK3) inhibitors. In some embodiments, the individual has a functional defect in Phosphomannomutase-2 (PMM2). The individual may be a fetus, newborn, infant, child, adolescent, or adult. In specific embodiments, the GSK3 inhibitor inhibits GSK-3a, GSK-3 , or both. In specific embodiments, the GSK3 inhibitor is Tideglusib, CCG 50014, or a combination thereof. In some embodiments, the method further comprises the step of administering to the individual a therapeutically effective amount of one or more aldose reductase inhibitors and / or one or more carbonic anhydrase inhibitors. In some embodiments, an individual has been tested for one or more mutations in the PMM2 gene and determined to have at least one mutations in one or both PMMS genes. In certain embodiments, one or both biological parents of the individual are determined to be, or are known to be, a carrier of a mutation (or defect) in the PMM2 gene. Some methods encompassed herein may further comprise the step of analyzing for a mutation in the PMM2 gene from a sample from the individual. The analyzing step may or may not be part of routine testing for the individual or may or may not prompted by a family history of CGD. Some methods encompassed herein may further comprise the step of testing a sample from one or both biological parents of the individual for a mutation in the PMM2 gene. In some embodiments, the analyzing step is part of routine testing for one or both biological parents or is prompted by a family history of CDG in one or both biological parents. Testing a sample for amutation or defect includes but is not limited to sequencing or assaying with one or more nucleic acid probes or primers.

[0008] Embodiments of the disclosure may comprise pharmaceutical compositions comprising one or more GSK3 inhibitors, and one or both of (a) one or more aldose reductase inhibitors; and (b) one or more carbonic anhydrase inhibitors. In some embodiments, the composition comprises one or more aldose reductase inhibitors but not one or more carbonic anhydrase inhibitors. In some embodiments, the composition comprises one or more carbonic anhydrase inhibitors but not one or more aldose reductase inhibitors. In some embodiments, the composition comprises 1, 2, 3, 4, or 5 different GSK3 inhibitors and / or aldose reductase inhibitors and / or different carbonic anhydrase inhibitors. Furthermore, it is contemplated that one or more specific aldose reductase inhibitor(s) or specific carbonic anhydrase inhibitors may be excluded in aspects of the disclosure.

[0009] Embodiments of the disclosure include kits comprising one or more GSK3 inhibitors, and one or both of (a) one or more aldose reductase inhibitors; and (b) one or more carbonic anhydrase inhibitors. In some embodiments, the kit comprises 1, 2, 3, 4, 5, or more aldose reductase inhibitors but not one or more carbonic anhydrase inhibitors. In some embodiments, the kit comprises one or more carbonic anhydrase inhibitors but not 1, 2, 3, 4, 5, or more aldose reductase inhibitors.

[0010] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the subject matter of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the subject matter of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The subject matter of the disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] FIG. 1 illustrates examples of compounds of the disclosure. Compound 1, tideglusib, is a non-ATP competitive GSK-3p inhibitor. Compound 2, CCG 50014, is a tool compound andan inhibitor of Regulator of G-protein Signaling protein type 4s, (RGS4) and RGS8; structural similarity to tideglusib indicates that in specific embodiments it is also a GSK-3p inhibitor.

[0013] FIG. 2 illustrates the growth defect of haploid (FIG. 2A) and heterozygous diploid SEC53 patient alleles (FIG. 2B) in a 24-hour luminescent-based growth assay. Wild Type (“WT”); SEC53 haploid mutants (“V108K” “F164S” “R148H”); SEC53 compound heterozygous mutant V108K / F164S (“V108K / F164S”).

[0014] FIG. 3 illustrates the Z-score distribution of heterozygous diploid mutant cells treated with 20 pM of compounds compared to DMSO controls. Mean -score for each group is shown. Compound 1 was the most potent rescuer of mutant yeast growth, followed by Compound 2. compound-treated SEC53 mutant (“VI 08K / F164S (compounds)”); DMSO SEC53 mutant; DMSO wild type (“WT (DMSO)”); Compound 1 (“tideglusib”); Compound 2 (“CCG 5014”).

[0015] FIG. 4 illustrates the glycosylation defects and metabolic imbalance in PMM2-CDG patient fibroblast (FIG. 4A) and brain organoid (FIG. 4B) models.

[0016] FIG. 5 illustrates an example of restoring metabolic balance, glycogen levels, and glycosylation in pathways in an individual.

[0017] FIGS. 6A-6D concern the role of Tideglusib in glycogen synthesis and glycosylation remodeling in PMM2 deficient hCOs upon Tideglusib treatment. 6A) Schematic showing decreased glycogen storage in PMM2-CDG and the potential of Tideglusib in increasing glycogen storage. 6B) Volcano plot depicting the differentially expressed glycopeptides in vehicle- treated PMM2-CDG in comparison to control vehicle- treated hCOs. X-axis is log2 fold-change (PMM2- CDG Vehicle / Control Vehicle). 6C) Volcano plot depicting the differentially expressed glycopeptides in PMM2-CDG treated with 1 uM Tideglusib in comparison to PMM2-CDG vehicle-treated hCOs. X-axis is log2 fold-change (PMM2-CDG 1 / zM Tideglusib / PMM2-CDG Vehicle). 6D) Volcano plot depicting the differentially expressed glycopeptides in PMM2-CDG treated with 10 gM Tideglusib in comparison to PMM2-CDG vehicle-treated hCOs. X-axis is log2 fold-change (PMM2-CDG 10 / zM Tideglusib / PMM2-CDG Vehicle). 6B-6D) Y-axis is the negative logarithm of p-value from a t test. The horizontal dashed red line represents the cutoff for significance (<0.05). Some of the changing glycopeptides are marked in red circles and glycoproteins’ names, glycosylation sites, and glycan structures are drawn.

[0018] FIGS. 7A-7C concern rdemodeling of Metabolites in PMM2 deficient hCOs upon Tideglusib Treatment. 7A) Volcano plot depicting the differentially expressed metabolites invehicle-treated PMM2-CDG in comparison to control vehicle-treated hCOs. X-axis is log2 foldchange (PMM2-CDG Vehicle / Control Vehicle). 7B) Volcano plot depicting the differentially expressed metabolites in PMM2-CDG treated with 10 qM Tideglusib in comparison to PMM2- CDG vehicle-treated hCOs. X-axis is log2 fold-change (PMM2-CDG 10 qM Tideglusib / PMM2- CDG Vehicle). ). 7A-7B) Y-axis is the negative logarithm of p-value from a t test. The horizontal dashed black line represents the cutoff for significance (<0.05). The vertical dashed black line represents an FC of 1.3. Metabolites that are significant and with an FC equal to or greater than 1.3 are labeled with their name. 7C) PLS-DA plot of treated vs vehicle PMM2 deficient and control hCOs.DETAILED DESCRIPTION

[0019] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0020] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0021] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0022] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0023] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0024] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0025] The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0026] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g, water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g, antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0027] The term “subject,” as used herein, may be used interchangeably with the term “individual” and generally refers to an organism having, or that is suspected of having, or that is at risk for having, CDG or CDDG. The subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens),household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject may be undergoing or having undergone treatment. The subject may be asymptomatic. The subject may be healthy individuals but that are desirous of prevention of CDG or CDDG. The “subject” or "individual", as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. An individual may comprise any age of a human or non- human animal and therefore includes both adult and juveniles (i.e., children) and infants and includes in utero individuals. It is not intended that the term connote a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.

[0028] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e g., CDG or CDDG. Treatment can involve optionally either the reduction or amelioration of one or more symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. Treating may mean alleviation of at least one symptom of the disease or condition.

[0029] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.* * * *I. General Embodiments

[0030] Embodiments of the disclosure include methods in which a phosphomannomutase-2 (PMM2) deficiency is treated in an individual in need thereof. Specific embodiments include methods in which a disorder of glycosylation is treated in an individual in need thereof, and the disorder may be congenital. In specific embodiments, there are methods of treating an individual having insufficient conversion of mannose 6-phosphate into mannose 1 -phosphate. In certain embodiments, there are methods of treating an individual having insufficient levels of GDP- mannose. In some embodiments, there are methods of increasing glycogen and / or glycosylationin an individual having lower than normal levels of glycogen and / or glycosylation. In some embodiments, there are methods of treating an individual having hypoglycosylation. Certain embodiments include methods of treating an individual having an intracellular metabolic imbalance, examples of which include alterations in glucose flux and / or polyol metabolism. In specific embodiments, there are methods of treating an individual having under-glycosylated glycoproteins, under-glycosylated lysosomal enzymes, and / or under-glycosylated serum proteins. In certain embodiments, there are methods of treating a disorder of N-glycosylation. In some embodiments, there are methods of treating an enzyme deficiency or other malfunction somewhere along the N-glycosylation pathway or other glycosylation pathways. Embodiments of the disclosure include methods of treating an individual having a medical condition including neurologic involvement (ataxia, speech delay, and seizures), global developmental delay, intellectual disability, coagulation abnormalities, and / or liver abnormalities, as examples.

[0031] Embodiments of the disclosure include methods of treating PMM2-CDG, and the CDG may be because of a defect of oligosaccharide assembly and transfer (type 1). PMM2 deficiency disrupts the metabolic equilibrium, resulting in alterations across multiple metabolic pathways related to sugar processing and utilization. The clinical presentation of PMM2-CDG may be characterized by neurological symptoms, developmental delays, failure to thrive, and / or multiorgan involvement.

[0032] Provided herein are methods for increasing glycosylation and / or increasing glycogen in an individual in need thereof comprising administering a therapeutically effective amount of a compound as described herein, or a combination of the compounds described herein, or a composition as described herein.

[0033] In some embodiments, increasing glycosylation provides increased levels of glycosylated glycoproteins, glycosylated lysosomal enzymes, and / or glycosylated serum proteins as compared to levels of the respective glycoproteins, lysosomal enzymes, or serum proteins prior to administration. Measurement of glycosylated glycoproteins, glycosylated lysosomal enzymes, and / or glycosylated serum proteins are methods known in the art. See, e. ., Carchon et al, Clinical Chemistry, 50: 1, 101-111 (2004).

[0034] In some embodiments, increasing glycogen levels provides increased levels of glycosylated glycoproteins, glycosylated lysosomal enzymes, or glycosylated serum proteins as compared to levels of respective glycoproteins, lysosomal enzymes, or serum proteins prior toadmini strati on. Measurement of glycosylated glycoproteins, lysosomal enzymes, or serum proteins are methods known in the art. See, e.g., Carchon el al, Clinical Chemistry, 50: 1, 101-111 (2004).

[0035] Provided herein are methods for treating a condition or disorder mediated, at least in part, by PMM2 (including PMM2 having one or more mutations that affect the enzyme activity) in an individual in need thereof comprising administering a therapeutically effective amount of a compound as described herein, or a combination of the compounds described herein, or a composition as described herein.

[0036] In some embodiments, the condition or disorder mediated, at least in part, by PMM2, is a congenital disorder of glycosylation. In some embodiments, the congenital disorder of glycosylation is a Type I disorder (e.g., la, lb, Ic, Id, le, If, Ih, li, Ij, Ik, IL, Im, In, Io, Ip, Iq, Ir, DPM2-CDG, TUSC3-CDG, MAGT1-CDG, DHDDS- CDG, and l / IIx). In some embodiments, the congenital disorder of glycosylation is a Type II disorder (e g. Ila, lib, lie, lid, He, Ilf, Ilg, Ilh, Hi, Ilj, HL, ATP6V0A2-CDG, MAN1B1-CDG, and ST3GAL3-CDG). In some embodiments, the congenital disorder of glycosylation is not classified as Type I or Type II.

[0037] Provided herein are methods for treating a congenital disorder of glycosylation in a patient in need thereof comprising administering a therapeutically effective amount of a compound as described herein, or a combination of the compounds described herein, or a composition as described herein. In some embodiments, a method for treating a congenital disorder of glycosylation in a patient in need thereof comprises administering a therapeutically effective amount of one or both compounds of FIG. 1 (including Compound 1) or a pharmaceutically acceptable salt thereof. In some embodiments, a method for treating a congenital disorder of glycosylation in a patient in need thereof comprises administering a therapeutically effective amount of one or more GSK3 inhibitors, or a pharmaceutically acceptable salt thereof.

[0038] In some embodiments, the congenital disorder of glycosylation is a Type I disorder. In some embodiments, the congenital disorder of glycosylation is a Type II disorder. In some embodiments, the congenital disorder of glycosylation is not classified as Type I or Type II. In some embodiments, the congenital disorder of glycosylation is PMM2-CDG.

[0039] Provided herein are methods for treating PMM2-CDG in a patient in need thereof comprising administering a therapeutically effective amount of a compound as described herein, or a combination of the compounds described herein, or a composition as described herein.

[0040] It is contemplated herein that, in some embodiments, a compound described herein may be useful for treating congenital disorders of deglycosylation (CDDG). In some embodiments, a compound described herein may be useful for treating NGLY1 -related congenital disorder of deglycosylation (NGLY1-CDDG) (the NGLY1 gene encodes PNGase, also known as peptide:N- glycanase or N-glycanase 1). In some embodiments, a compound encompassed herein may be useful for treating MAN2C1 -related congenital disorder of deglycosylation MAN2C1-CDDG (the MAN2C1 gene encodes mannosidase, alpha, class 2C, member 1).

[0041] In any of the embodiments described herein, a patient is administered one or more of the compounds described herein, The one or more compounds can be administered simultaneously or sequentially. When administered simultaneously, the compounds may or may not be in the same formulation. Whether administered simultaneously or sequentially, the routes of administration may or may not be the same for the compounds. Any order of administration of delivery of multiple compounds may be utilized, in specific embodiments.

[0042] In any of the embodiments described herein, a patient is administered a pharmaceutical composition that comprises one or more of the compounds encompassed herein.

[0043] In any of the embodiments described herein, the patient is administered a therapeutically effective amount of a therapeutic agent useful for a medical condition, including useful for increasing glycogen, and in specific embodiments the therapeutic agent comprises one or more glycogen synthase kinase 3 (GSK-3) inhibitors.

[0044] Any method encompassed herein may include one or more steps, and these steps may be utilized in a variety of combinations depending on the method. In particular embodiments of the disclosure, one or more of the following steps may be utilized: administering or delivering to an individual a therapeutically effective amount of a compound to treat CDG or CDDG; administering or delivering to an individual a therapeutically effective amount of multiple compounds to treat CDG or CDDG, including 2, 3, 4, 5, or more different compounds; administering or delivering to an individual a therapeutically effective amount of a GSK-3 inhibitor; administering or delivering to an individual a therapeutically effective amount of multiple GSK-3 inhibitors, including 2, 3, 4, 5, or more different GSK-3 inhibitors; administering or delivering to an individual a therapeutically effective amount of Tideglusib; administering or delivering to an individual a therapeutically effective amount of Tideglusib and one or more additional GSK-3 inhibitors; administering or delivering to an individual a therapeuticallyeffective amount of Tideglusib and a therapeutically effective amount of CCG 50014; administering or delivering to an individual a therapeutically effective amount of Tideglusib and a therapeutically effective amount of 1, 2, 3, 4, 5, or more aldose reductase inhibitors; administering or delivering to an individual a therapeutically effective amount of Tideglusib and a therapeutically effective amount of 1, 2, 3, 4, 5, or more carbonic anhydrase inhibitors; administering or delivering to an individual a therapeutically effective amount of Tideglusib and a therapeutically effective amount of 1, 2, 3, 4, 5, or more aldose reductase inhibitors and a therapeutically effective amount of 1, 2, 3, 4, 5, or more carbonic anhydrase inhibitors; administering or delivering to an individual a therapeutically effective amount of CCG 50014; administering or delivering to an individual a therapeutically effective amount of CCG 50014 and one or more additional GSK-3 inhibitors; administering or delivering to an individual a therapeutically effective amount of CCG 50014 and a therapeutically effective amount of 1, 2, 3, 4, 5, or more aldose reductase inhibitors; administering or delivering to an individual a therapeutically effective amount of CCG 50014 and a therapeutically effective amount of 1, 2, 3, 4, 5, or more carbonic anhydrase inhibitors; administering or delivering to an individual a therapeutically effective amount of CCG 50014 and a therapeutically effective amount of 1, 2, 3, 4, 5, or more aldose reductase inhibitors and a therapeutically effective amount of 1, 2, 3, 4, 5, or more carbonic anhydrase inhibitors; analyzing a sample from an individual for a defect in the PMM2 gene; analyzing a sample from a pregnant mother for a defect in the PMM2 gene; analyzing a sample from a biological father for a defect in the PMM2 gene; analyzing amniotic fluid and / or blood from a pregnant mother for a defect in the PMM2 gene; analyzing a sample from an in utero fetus for a defect in the PMM2 gene; analyzing a sample from an individual known to have CDG or suspected of having CDG for a defect that would directly or indirectly cause CDG; ascertaining a family history for one or more symptoms of CDG; diagnosing an individual for CDG; subjecting an individual to routine testing for a defect in the PMM2 gene; subjecting a male adult, female adult, in utero fetus, infant, child, or adolescent for routine testing for CDG; subjecting a male adult, female adult, in utero fetus, infant, child, or adolescent for routine testing for a defect in the PMM2 gene; and so forth. It is specifically contemplated that in any composition or method, Tideglusib and / or CCG 50015 is excluded.IL PMM2-CDG and Treatment Thereof: Glycosylation defects and MetabolicImbalance

[0045] In particular embodiments, the present disclosure concerns treatment of PMM2-CDG or other CDGs - including Type I, Type II, and those that cannot be classified into these types. Other CDGs include N-linked type I forms of CDG (such as PMM2-CDG, MPI-CDG, GMPPA- CDG, and PGM1-CDG); N-linked type II forms of CDG (such as MAN1GB1-CDG and MGAT2- CDG); O-linked forms of CDG (such as EXT1 / EXT2-CDG, B4GALT7-CDG and B3GALTL- CDG); CDG due to defects in multiple glycosylation pathways (such as DPMI -CDG and DOLK- CDG); CDG due to GPI-anchor synthesis defects (such as PIGN-CDG or PIGV-CDG), and CDG due toglycosphingolipid synthesis defects (such as ST3GAL5-CDG or B4GALNT1-CDG).

[0046] PMM2-CDG leads to insufficient levels of the nucleotide sugar GDP -mannose, resulting in under-glycosylation of proteins / lipids. As illustrated in FIG. 4, the disorder leads to metabolic imbalance on several axes. Glucose flux is shifted away from GDP -mannose synthesis and glycosylation, toward polyol metabolism (Radenkovic et al., Cell Rep Med 4, 101056, 2023). Glycogen levels are depleted in PMM2 brain organoid models resulting in defects in glycosylation and energy metabolism (manuscript under review). Stored primarily in astrocytes, glycogen serves as a critical energy reserve in the brain, providing neurons with lactate as a vital energy substrate (Belanger et al, Cell Metab 14, 724-738, 201 l).It is also a reservoir for glucosamine used to produce nucleotide sugar UDP-GlcNAc for glycosylation (Sun et al. CellMetab. 33, 1404-1417, 2021). Abnormal glycogen metabolism impairs N-glycosylation in the brain and contributes to disease (e.g., Glycogen Storage Diseases) (Sun etal. CellMetab. 33, 1404-1417, 2021).

[0047] In particular embodiments, metabolic balance and glycosylation may be restored in individuals with PMM2-CDG, such as by aldose reductase inhibition using epalrestat that rewires glucose metabolism. In such cases, glucose is shuttled away from polyol production towards GDP- mannose production and glycosylation (FIG. 5).

[0048] In specific embodiments, there are methods of preventing a congenital disorder of glycosylation, including at least PMM2-CDG of any kind. As one example, a biological female individual and a biological male individual may be aware of each having a mutation in PMM2 and desire to procreate. They may be aware because of routine screening, having a family history of a CDG (including PMM2-CDG), both, and so forth. Following conception, the pregnant mother, the fetus, or both may be administered one or more compounds encompassed herein. The fetus may be administered one or more compounds encompassed herein in utero. In some cases, the pregnant mother and the fetus in utero are administered the same therapeutic compound(s),whereas in other cases the pregnant mother and the fetus in utero are administered a different therapeutic compound, or different combinations of therapeutic compounds. In particular embodiments, one or more symptoms of PMM2-CDG are prevented from occurring following administration of one or more compounds encompassed herein to an affected individual and / or the biological mother pregnant with the affected individual. In particular embodiments, one or more symptoms of PMM2-CDG are delayed in onset following administration of one or more compounds encompassed herein to an affected individual and / or the biological mother pregnant with the affected individual. In particular embodiments, one or more symptoms of PMM2-CDG are reduced in severity following administration of one or more compounds encompassed herein to an affected individual and / or the biological mother pregnant with the affected individual. Such symptoms include at least a neurological symptom (e.g., ataxia, speech delay, and seizures), a global developmental delay, an intellectual disability, coagulation abnormalities, and / or liver abnormalities.

[0049] An individual at risk for having PMM2-CDG, including an individual having at least one mutation in the PMM2 gene, may be administered one or more compounds encompassed herein.

[0050] In some embodiments, there are methods of treating, preventing, delaying the onset of one or more symptoms, or reducing the severity of one or more symptoms of a congenital disorder of deglycosylation (CDDG), including CDDG1 or CDDG2. In CDDG1, the mutation is in the NGLY1 gene, whereas in CDDG2, the mutation is in the MAN2C1 gene.III. Glycogen synthase kinase (GSK3) and Inhibitors Thereof

[0051] Embodiments of the disclosure include methods for treatment or prevention or reduction in severity of one or more symptoms and / or delay in onset of one or more symptoms of a congenital disorder of glycosylation. In particular embodiments, such methods utilize one or more Glycogen synthase kinase (GSK3) inhibitors. GSK3 is a Ser / Thr kinase with -100 substrates; it is involved in glycogen metabolism, cell signaling, insulin metabolism, gene expression, and protein synthesis (Beurel et al. Pharmacol Ther., 148: 114-31, 2015).

[0052] GSK3 exists in two isozymes encoded by two homologous genes GSK-3a (GSK3A) and GSK-30 (GSK3B). The isoforms have shared and distinct roles / substrates; both isoforms are involved in glycogen synthesis regulation but are tissue-type specific (Wang, Li, Di; Med Res Rev42(2):946-982 2022); GSK-3P implicated in diabetes (Teli and Gajjar. BioorgMed Chem Volume 92, 2023).

[0053] Glycogen synthase (GS) is involved in conversion of glucose to glycogen (UDP-Glc —> glycogen), and phosphorylation of GS by GSK3 reduces glycogen synthesis. There are several GSK3 and GSK3 isoform-specific inhibitors in clinical development (Wang, 2022; Teli and Gajjar, 2023).

[0054] In any medical condition encompassed, there may be methods of treating or preventing or delaying the onset and / or severity of at least one symptom by providing to the individual a therapeutically effective amount of one or more GSK3 inhibitors to the individual. In specific embodiments, the GSK3 inhibitor(s) comprises Tideglusib (also called NP-12, NP031112, or 4- Benzyl-2-(naphthalen-l-yl)-l, 2, 4-thiadiazolidine-3, 5-dione, AMO-02, for example) and / or CCG 50014 (FIG. 1). Tideglusib is an orally bioavailable small molecule inhibitor of GSK-3P in clinical development for Myotonic Dystrophy ALS, Progressive Supranuclear Palsy, Alzheimer’ s disease, and Autism Spectrum Disorder (Saraswati etal. EurJMed Chem 144:843-858, 2018). In specific embodiments, GSK-3P inhibition by tideglusib may increase glycogen, which in turn would increase glycosylation and restore metabolic balance in PMM2-CDG individuals.In addition to Tideglusib and / or CCG 50014, or as alternatives, one may utilize one or more of the following GSK3 inhibitors: Enzastaurin; one or more maleimide derivatives, such as 3-(7- azaindolyl)-4-arylmaleimides, SB-216763, SB-415286, BIP-135, indolylmaleimides, heteroaryl- maleimides, and / or macrocyclic azaindolylmaleimides; isonicotinamides, pyrazolopyrimidines; pyrazolopyridines; imidazopyridine; the pyrrolopyrimidine TWS119; the oxindolypyridine AZD1080, SAR502250; CHIR98014; CHIR98023; CHIR990218023, the triazolopyridine JGK- 263; 7-hydroxy-lH-benzoimidazole; a lH-indazole-3-carboxamide; AF3581; an amino thiazole, such as AR-A014418, VP2.51, and / or VP2.54; pyrazine analogs, such as AZD2858; 1,3,4- oxadiazole derivates MMBO and / or TCS2002; an oxazole-carboxamide derivate, such as PF- 04802367; BRD0705 and / or BRD3731 ; a thiadiazolidinone, such as belonging to the class of non- ATP competitive inhibitors and including the small heterocyclic TDZD inhibitor family; a uinolone analog VP0.7, VP3.35, and / or SC100, having been synthesized based on the heterocyclic thiadiazolidinone core; a chloromethyl thienyl ketone; a halomethyl phenyl ketone; lithium; small peptides L803mts and / or L807mts; a thiazolidinedione, such as TDZD-8; iminothiadiazoles, such as 5-imino-l,2,4-thiadizoles, VP 1.14, and / or VP 1.16; or a combination thereof.IV. Combination Therapy

[0055] In any of the embodiments described herein, an individual may be administered one or more therapeutic agents in addition to being administered one or more GSK3 inhibitors. The different compounds may be administered at substantially the same time or at different times. When administered at different times, the duration between administrations may be about 1-60 minutes and any range derivable therein, about 1-24 hours and any range derivable therein, about 1-7 days and any range derivable therein, 1-4 weeks and any range derivable therein, 1-12 months and any range derivable therein, 1-10 years and any range derivable therein, and so forth. The routes of administration may or may not be the same for the GSK3 inhibitor compared to another one or more therapeutic agents. When administered at substantially the same time, the compounds may or may not be administered in the same formulation and / or by the same route.

[0056] In specific embodiments, the individual is further administered a therapeutically effective amount of one or more therapeutic agents that increase glycosylation, such as one or more aldose reductase inhibitors e.g., epalrestat). One or more aldose reductase inhibitors may include any compound from WO 2020 / 040831, which is incorporated by reference herein in its entirety. Examples of aldose reductase inhibitors include, but are not limited to, alrestatin, benurestat, epalrestat, fidarestat, imirestat, lidorestat, minalrestat, ponalrestat, ranirestat, risarestat, sorbinil, tolrestat, zenarestat, and / or zopolrestat. Any of these may be included (alone or in combination) or excluded in compositions or methods described herein.

[0057] In any of the embodiments described herein, an individual is further administered a therapeutically effective amount of another therapeutic agent, such as wherein the therapeutic agent is one or more carbonic anhydrase inhibitors (e.g., acetazolamide). Examples of carbonic anhydrase inhibitors include, but are not limited to, at least acetazolamide, dorzolamide, methazolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, indisulam, and a combination thereof. Any of these may be included (alone or in combination) or excluded in compositions or methods described herein.V. Yeast Avatar Generation, Screening Platform, and Yeast Avatar Characterization

[0058] To facilitate high-throughput screening for compounds that may treat PMM2-CDG, a yeast avatar of a particular patient was generated. The patient is a compound heterozygote, havingone maternal -derived pathogenic PMM2 variant and a different paternal -derived pathogenic PMM2 variant. The yeast version of PMM2 is called SEC53. As shown in the protein sequence alignment below, the amino acid order is offset by +7 in yeast, so position 157 in human PMM2 is position 164 in yeast SEC53; position 101 in human PMM2 is position 108 in yeast SEC53.

[0059] The patient has aF157S mutation and a N101K mutation. TheF157S mutation replaces an evolutionarily conserved phenylalanine with a serine residue and was predicted to be pathogenic in the genetic testing report. The N101K mutation was also predicted to be pathogenic but it is not evolutionarily conserved between humans and yeast. However, replacing either human asparagine 101 or yeast valine 108 with a lysine residue is predicted to be deleterious. Both mutations are in in the cap domain, which is required for dimer formation and proper active site stabilization. Modeling indicates that both variants of the patient are indirectly interacting with each other.

[0060] The PMM2 variants of the patient were initially assessed as VI 08K and F 164S haploid yeast avatars so their residual function could be assessed separately. Then the two haploid avatars were combined to generate a diploid compound heterozygote that matched the exact genotype of the patient. Each haploid variant avatar had a growth defect as severe as another variant that was considered a functional null having no detectable enzymatic activity (SEC53 null mutant (R148H)). The diploid compound heterozygous avatar also had a severe growth defect. The growth defect was measured by a luminescence-based growth assay (BacTiter-Glo) compared to WT yeast. The diploid mutant V108K / F164S was screened with a TargetMol® library for compounds that rescue growth.VI. Administration of Therapeutic Compositions

[0061] The therapy provided herein may comprise administration of one or a combination of therapeutic agents. The therapies may be administered in any suitable manner known in the art. The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, intrauterinely, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0062] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0063] Any therapeutic agent or combination of agents in a composition encompassed by the disclosure may be administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,I I I, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547,548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg.

[0064] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg / kg to 2000 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000, 2000 pg / kg, mg / kg, pg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0065] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 0.001 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0066] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0067] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0068] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5,6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0069] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0070] The active compounds can be formulated for parenteral administration, e.g., formulated for inj ection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0071] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0072] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0073] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity 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. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Inmany cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0074] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile inj ectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0075] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0076] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above..

[0077] In certain aspects, the compositions or agents for use in the methods are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as the uterus or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0078] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0079] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of nonlimiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0080] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0081] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0082] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0083] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0084] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0085] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0086] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0087] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g.,alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.Examples

[0088] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1GENERAL METHODS

[0089] Embodiments of general methods are provided herein.Strains and plasmids

[0090] All strains used herein are in the S288c background. Strains were grown in Synthetic Complete (“SC”) or SC drop out media (US Biological) + 2% dextrose at 30°C unless otherwise noted. Standard procedures were followed for yeast transformation and strain generation. Previously published SEC53 rescue plasmid (pPLl) generated by cloning the SEC53 promoter, open reading frame, and terminator sequences into the episomal pRS316 containing the URA3 selectable marker was used in the study (Lao et al., 2019). SEC53 variants were generated by cloning the pACTl promoter, SEC53 gene, and CYC1 terminator sequences into an integrating plasmid containing the LEU2 selectable marker. Overlapping primers bearing requisite mutations were used to amplify SEC53. Plasmids were generated by Gibson assembly. Strains were generated in house using standard yeast transformation.Growth assay

[0091] Cells from overnight cultures were resuspended in SD-leu+FOA media to ODeoo = 0.4, then serial diluted into 100 pL SD-leu +FOA media in 96-well plates at 10-1, 10-2, 10-3, 10-4 and10 -5. Plates were incubated at 30°C for 24 hours. 50 pL of yeast cell suspension was transferred to a 96-well plate into with 50 pL of BacTiter-Glo (Promega), briefly vortexed and incubated for 5 minutes. Luminescence readings were measured by a plate reader (Tecan M1000 pro). 5- floroortic acid was purchased from US Biological and was used at a final concentration of 1 mg / mL.Drug Screen

[0092] 200 nL of compounds or DMSO were dispensed into 384-well plates using the Echo acoustic dispenser (Beckman Coulter Labcyte Echo 650) to achieve a final concentration of 20 pM. Cells from cultures were resuspended in SD-leu+FOA media to ODeoo = 0.0025 and 25uL of yeast cell suspensions were dispensed into the 384-well plates containing compounds or DMSO with a EL406 automated dispenser (Biotek). Plates were covered and incubated at 30°C for 24 hours. 25 pL of BacTiter-Glo was dispensed into the plates. Plates were briefly vortexed and then incubated for 10 minutes prior to reading (Envision Plate reader).EXAMPLE 2YEAST MUTANT MODELS OF PMM2 DISEASEModeling PMM2 patient alleles in yeast SEC53

[0093] Mammalian PMM2 and the yeast homolog, SEC53 share 55% identity at the aminoacid level. Two PMM2 disease-causing alleles (N101K and F157S) were generated. PMM2 N101K and F157S correspond to SEC53 V108K and F164S, respectively. N101 and F157 are in the cap domain of PMM2, which is required for dimer formation and stabilization of the active site. Existing data on the N101K variant is limited. Based on structural modeling (AlphaFold), N101 is located within the same region of the dimerization interface of PMM2 as E93. The E93K patient allele in yeast SEC53 (E100K) results in a severe growth-defect (Lao etal., 2019), therefore the N101K variant is similarly predicted to result in a severe loss-of-function in PMM2. F157S has been reported in several PMM2-CDG patients and has low or no detectable enzyme activity in patients.SEC53 patient alleles cause a severe growth defect

[0094] To overcome the complication that SEC53 is an essential gene, a wild type SEC53 copy was placed on a E1RA3 plasmid that we can conditionally remove by growing cells in 5- fluoroorotic acid (5-FOA) as described in Lao et al. 2019. The phenotype of each variant is revealed when the wild type URA3 containing plasmid is counter- selected in media containing 5- FOA.

[0095] Haploid V108K and F164S mutants exhibit a severe growth defect that is comparable to the R148H null allele (R141H in human PMM2), which has no detectable enzyme activity in patients (FIG. 2A). MostPMM2-CDG patients have compound heterozygous mutations, therefore a heterozygous diploid of V108K and F164S was generated. Similarly, the diploid V108K / F164S mutant exhibits a severe growth defect (FIG. 2B).EXAMPLE 3EXAMPLES OF COMPOUNDS FOR PMM2Drug screen in a yeast model of PMM2-CDG identifies chemical modifiers

[0096] The SEC53 V108K / F164S heterozygous diploid was advanced to a high-throughput drug screen. The 8,387 compound TargetMol® library collection comprising FDA-approved drugs, drugs in various stages of clinical development, bioactive tool compounds, and natural products was used for screening at 20 pM. Each strain was screened in singlicate and in 384-well plates, with each plate containing 32 wells of the negative control (no drugs) and 32 wells of the positive controls (wild type cells), each in 0.72% DMSO.

[0097] The positive and negative controls exhibited a distinct separation of Z-scores, enabling the identification of compounds which rescued growth of the mutant strain. Using a Z-score cutoff of 0.2, 18 compounds were identified that rescued the growth of the mutant strain. The most potent compound (Compound 1) was the GSK-3P inhibitor tideglusib (Z-score = 1.08), which rescued mutant growth by 2.2-fold (FIG. 3). The second-most potent compound, Compound 2 (Z- score = 0.4), shares structural similarities with Compound 1, suggesting its potential as a GSK-30 inhibitor.

[0098] In particular embodiments, Compound 1 increases glycosylation, or reverses any other determinantal effects caused by the mutations in SEC53, by increasing glycogen levels in yeast. It is expected that Compound 1 will reverse or alleviate symptoms of PMM2-CDG, caused by defects in the orthologous human PMM2 gene.EXAMPLE 4

[0099] Examples of Material and Methods

[0100] Reprogramming of PMM2 deficient fibroblast to induced pluripotent stem cells.

[0101] The Sendai virus Cytotune 2.0 kit was used by the Mayo Clinic core to generate iPSCs from control and patient fibroblast. The iPSCs were cultured at 37 °C and 5% CO2 on Geltrex™ coated plates and fed mTeSRTM plus media with 1% Anti -Anti.

[0102] All iPSC’s passed assessment for pluripotency markers and ability to differentiate into all 3 germ layers. Mycoplasma testing was performed and was negative for all cell lines.

[0103] Treatment and Differentiation of PMM2 deficient cortical organoids

[0104] iPSCs cells were cultured in a 10 cm plate to a confluency of 80-90% in mTESR plus media with 1% AA. The cells were washed with 5 ml of DPBS and 5 ml of Acutase was added. The cells were incubated for 5-7 min to allow for cell detachment and moved to a 50 ml canonical tube. The plate was washed with 5 ml DMEM-F12 and that was also added to the 50 ml canonical tube. The cells were counted, spun down at 200 g for 5 min, and resuspended in mTESR plus media with 1% AA and 10 uM Rock Inhibitor. 2.5 million cells were added per AggreWell, spun down at 200 g for 5 min and placed in incubator for 24 hours. After 24 hours, spheroids were moved into ultra-low attachment plates with 12 ml of the mTESR with 1% AA and 10 uM Rock Inhibitor (day 0). After 48 hours, the media was changed to Neural Induction Media (NIM) composed of 77.5 % DMEM-F12, 20% Knockout Serum (KSR), 1% Non-Essential Amino Acid, 0.5% Glutamax,0.1 mM 2-Mercaptaenthanol, 5 pM Dorsomorphin, 10 pM SB-431542, and 20ng / mL FGF2 The organoids are fed with NIM media every day until day 6. On day 6, the media is changed to Neural Media (NM) composed with Neurobasal A media, 2% B27 supplement without vitamin A, 1% Glutamax, and 1% AA with the addition of 20 ng / mL FGF2, and 20 ng / mLEGF. The organoids were fed the NM media+ EGF+FGF2 every day through day 15 and every other day through day 24. At day 25, the base NM media remained the same, but the factors were changed to 20 ng / ml BDNF, and 20 ng / ml NT-3 and this media was fed every other day to the organoids until day 42. At day 43, all factors were removed, and the human cortical brain organoids (hCOs) were only fed NIM. At Day 44, the hCOs were treated with 10 M DMSO, 10 pM Tideglusib, and 1 pM Tideglusib. Every 3 days, fresh media with drug or vehicle was added to the hCOs. Treatment was stopped on day 58.

[0105] Organoid Lysis and Protein Digestion

[0106] At day 58, four hCOs each from PMM2 cell lines (11367-CDG, 11368-CDG, 11547- CDG) and from control lines (CTRL-1651, CTRL-8399, and CTRL-8856.1) with 10 pM DMSO, 10 pM Tideglusib, and 1 pM Tideglusib were collected and washed 3 times with DPBS, flash frozen in dry ice, and kept in -80 °C till time of assay. The samples were lysed using Bioruptor sonication device in 8M urea (in 100 mM TEABC) with 1% protease inhibitor cocktail (Thermo Scientific). Protein amount was quantified in the organoid lysates using BCA colormetric assay as per the manufacturer’s instructions (Thermo Scientific). Equal quantity of protein from both PMM2-CDG and controls were first reduced using 10 mM TCEP for 30 minutes at 55°C on a thermomixer, then alkylated with 40 mM iodoacetamide for 30 minutes in the dark at room temperature. The proteins were then digested with 1 :20 w / w (protein: trypsin) ratio of trypsin (Worthington, USA) at 37°C overnight with mild shaking on thermomixer. Resulting peptides were desalted using C18 cartridges and labeled with tandem mass tags (TMT) (Thermo Fisher Scientific, USA) as per the manufacturer’s protocol.

[0107] Liquid chromatography tandem mass spectrometry (LC-MS / MS)

[0108] LC-MS / MS analysis of fractionated peptides and glycopeptides both proteomics and glycoproteomics, respectively was conducted as previously described 12, 13 with some modifications. Briefly, 12 concatenated fractions from bRPLC and 12 concatenated fractions from SEC were analyzed on an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) equipped with Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific Inc.). The peptides / glycopeptides were first trapped on a trap column (100 mm x 2 cm, Acclaim PepMaplOO Nano-Trap, Thermo Fisher Scientific) at a flow rate of 20 pl / min. LC separation was performed on an analytical column (Easy Spray 75 pm x 50 cm, Cl 8 2 pm, 100 A) at a flow rate of 300 nl / min for 150 min using a linear gradient of 0.1% formic acid in water (solvent A) and 0. 1% formic acidin acetonitrile (solvent B). All experiments were performed in a data-dependent acquisition (DDA) mode at an isolation window of 0.7 m / z. Precursor ions were acquired at a resolution of 120,000 (at m / z 200) and fragment ions at a resolution of 30,000 (at m / z 200). Data acquisition was performed with option of “lock mass” (m / z 441.12002) for all data. Precursor ions were acquired in the Orbitrap mass analyzer in m / z range of 350-1,700 for proteomics and 350-2,000 for N- glycoproteomics. Precursor fragmentation was conducted using normalized higher-energy collisional dissociation (HCD) method of 34 for proteomics and normalized stepped HCD at 15, 25 and 40% for N-glycoproteomics.

[0109] Data Analysis for Proteomics and Glycoproteomics

[0110] The proteomics data were searched using Sequest search engine in Proteome Discoverer 2.5 against the human Uniprot protein database. The N-glycoproteomics data using the publicly available software pGlyco version 3.0 with an in-built N-glycan database for identifying glycans and human Uniprot protein database for identifying peptide sequences. Two missed cleavages were allowed for both proteomics and glycoproteomics analysis. Error tolerance for precursor and fragment ions were set to 10 ppm and 0.02 Da, respectively, for proteomics and 10 ppm and 20 ppm, respectively, for glycoproteomics. Cysteine carbamidomethylation was set as fixed modification and, oxidation of methionine as a variable modification. False discovery rate (FDR) was set to 1% at the peptide-spectrum matches (PSMs), peptide, protein and glycopeptides levels. For proteomics, quantitation of peptides across PMM2-CDG and control hCOs was done using TMT reporter ion intensities using “reporter ion quantifier” node. To quantify glycopeptides, reporter ion quantification was performed for glycoproteomics raw files in Proteome Discoverer and glycopeptide IDs obtained from pGlyco were matched with quantitation data on a scan-to- scan basis (MS / MS).

[0111] Organoid Preparation for Metabolomics

[0112] At day 58, four hCOs each from PMM2 cell lines (11367-CDG, 11368-CDG, 11547- CDG) and from control lines (CTRL-1651, CTRL-8399, and CTRL-8856.1) with 10 / / M DMSO, 10 M Tideglusib, and 1 pM Tideglusib were collected and washed 3 times with 0.9 % NaCl, flash frozen in dry ice, and kept in -80 °C till time of assay. At time of assay, hCOs were weighed. 350 pL of ice-cold extraction buffer (80 % MeOH, Internal Standards) and a few sonication beads were added to the sample and placed in Bioruptor sonication device for lysing. After lysing, the samples were stored overnight at -80 °C and subsequently centrifuged at 15,000 rpm for 20 minutes at 4°C. 100 ul of the supernatant was collected and 35 ul of dd H2O and 800 ul of 100% chloroform was added to it. The sample were then vortexed and placed at 4 °C overnight. The next day, the polar phase (top layer) was collected and sent for LC / MS.

[0113] LC / MS analysis for Metabolites

[0114] LC / MS analysis on metabolites was conducted. A Cl 8 ion-pairing liquid chromatography column was used to separate 10 ul of the sample and subsequently placed in Thermo Fisher Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer in the negative ion mode (full scan 70-1050 m / z, resolution 140,000 at 200 m / z, AGC at 3e6, 512 ms ion fill time) to further resolve the metabolites. The ESI was set to capillary temperature at 350 °C, 50 sheet gas flow rate, S-lens RF level of 60, spray voltage of 4 kV, and auxiliary gas flow rate 15. The m / z ratio and elution time using in house metabolite standard library and El-Maven v0.12.0 / Polly TM Labeled LC-MS Workflow20 was used to identify the metabolites. The El-Maven v0.12.0 / Polly TM Labeled LC-MS Workflow20 was also used to correct for naturally occurring carbon isotopes. The abundance of the metabolites was normalized to internal standards and the weight of the hCOs. Relative abundance was established by utilizing control hCOs as reference and absolute quantification was not performed.

[0115] Glycosylation Remodeling in PMM deficient hCOs upon Tideglusib Treatment

[0116] The glycoproteomics analysis identified 1,668 glycopeptides in hCOs. In terms of baseline comparison between vehicle treated PMM2 deficient and control hCOs, dysregulations in glycosylation of glycopeptide associated with WNT signaling (LRP1), ECM to cell connections (LRP1, NEGRI, ITAV, THY1, NCAM1, & LSAMP), neuronal activity (DPP6), neuronal migration (TSN3, NCAM1,& ITAV), and axon guidance(LSAMP & NCAM1) could be seen (FIG 6B). Upon treatment with 1 / / M Tideglusib, none of the glycopeptides for PMM2-CDG hCOs were significantly hypoglycosylated but instead shifted towards a hyperglycosylated state (FIG. 6C). This included glycopeptides associated with WNT signaling (LRP1), ECM to cell connections (NCAM1, NFASC, & LAMB1), neuronal migration (NCAM1 & LAMB1), and axon guidance (NCAM1 & NFASC). Upon treatment with 10 [ M Tideglusib, a similar trend of protein hyperglycosylation (FIG. 6D) associated with WNT signaling (WNT8B), ECM component synthesis (CHPF2), ECM to cell connections (NEGRI & NCAM1), neuronal migration (TSN3 & NCAM1), and axon guidance (NCAM1) could be seen.

[0117] Metabolic Remodeling in PMM2 deficient hCOs upon Tideglusib Treatment

[0118] In terms of baseline comparison between vehicle treated PMM2 deficient and control hCOs, PMM2 deficient hCOs show a reduction in metabolites (FIG. 7A) associated with polyol pathway (sorbitol & ribitol), glycolysis (fructose 1,6-bisphosphate), pentose phosphate pathway (D-Sedoheptulose), and glycosylation pathway (N- Acetylneuraminic acid). Upon treatment with 10 M Tideglusib, an increase in metabolites (FIG. 7B) for the polyol pathway (sorbitol & ribitol), glycolysis (fructose 1,6-bisphosphate & ATP), and nucleotide synthesis (CTP & UTP) can be seen. Furthermore, UDP-glucose, a precursor to glycogen synthesis, was also upregulated upon 10 pM Tideglusib treatment (FIG. 7B). Partial Least Squares Discriminant Analysis showed separation between PMM2 deficient Vehicle and 10 pM Tideglusib treated groups, indicating significant differences (FIG. 7C).* * *

[0119] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0120] Belanger et al, Cell Metab, 14, 724-738, 2011.

[0121] Beurel et al. Pharmacol Ther, 148: 114-31, 2015

[0122] Carchon et l., Clinical Chemistry, 50: 1, 101-111, 2004.

[0123] Lao etal.G3, 9, 413-423, 2019.

[0124] Radenkovic et al., Cell Rep Med 4, 101056, 2023.

[0125] Saraswati et al. Eur J Med Chem 144:843-858, 2018.

[0126] Sun et al. CellMetab. 33, 1404-1417, 2021.

[0127] Teli and Gajjar. BioorgMed Chem Volume 92, 2023.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a congenital disorder of glycosylation (CDG) or a congenital disorder of deglycosylation (CDDG) in an individual, comprising the step of administering to the individual a therapeutically effective amount of one or more glycogen synthase kinase 3 (GSK3) inhibitors.

2. The method of claim 1, wherein the individual has CDG.

3. The method of claim 1 or 2, wherein the individual has a functional defect in Phosphomannomutase-2 (PMM2).

4. The method of any one of the preceding claims, wherein the individual is a fetus, newborn, infant, child, adolescent, or adult.

5. The method of any one of the preceding claims, wherein the GSK3 inhibitor inhibits GSK- 3a, GSK-3P, or both.

6. The method of any one of the preceding claims, wherein the GSK3 inhibitor is Tideglusib, CCG 50014, or a combination thereof.

7. The method of any one of the preceding claims, further comprising the step of administering to the individual a therapeutically effective amount of one or more aldose reductase inhibitors and / or one or more carbonic anhydrase inhibitors.

8. The method of any one of the preceding claims, wherein one or both biological parents are determined to be, or are known to be, a carrier of a defect in the PMM2 gene.

9. The method of any one of the preceding claims, further comprising the step of analyzing from a sample from the individual for a defect in the PMM2 gene.

10. The method of claim 9, wherein the analyzing is part of routine testing for the individual or is prompted by a family history of CGD.

11. The method of any one of the preceding claims, further comprising the step of analyzing a sample from one or both biological parents of the individual for a defect in the PMM2 gene.

12. The method of claim 11, wherein the analyzing is part of routine testing for one or both biological parents or is prompted by a family history of CDG in one or both biological parents13. A pharmaceutical composition comprising one or more GSK3 inhibitors, and one or both of (a) one or more aldose reductase inhibitors; and (b) one or more carbonic anhydrase inhibitors.

14. The composition of claim 13, wherein the composition comprises one or more aldose reductase inhibitors but not one or more carbonic anhydrase inhibitors.

15. The composition of claim 13, wherein the composition comprises one or more carbonic anhydrase inhibitors but not one or more aldose reductase inhibitors.

16. A kit comprising one or more GSK3 inhibitors, and one or both of (a) one or more aldose reductase inhibitors; and (b) one or more carbonic anhydrase inhibitors.

17. The kit of claim 16, wherein the kit comprises one or more aldose reductase inhibitors but not one or more carbonic anhydrase inhibitors.

18. The kit of claim 16, wherein the kit comprises one or more carbonic anhydrase inhibitors but not one or more aldose reductase inhibitors

Images