WO2012082830A1

WO2012082830A1 - Serum biomarkers for hibm

Info

Publication number: WO2012082830A1
Application number: PCT/US2011/064791
Authority: WO
Inventors: Daniel Darvish; Yadira Valles-Ayoub; Emil D. Kakkis
Original assignee: Hibm Research Group, Inc.
Priority date: 2010-12-14
Filing date: 2011-12-14
Publication date: 2012-06-21

Abstract

The present invention relates to methods of diagnosing/ monitoring and assessing sialic acid deficiencies such as Hereditary Inclusion Body Myopathy (HIBM) and to methods of predicting/determining responsiveness to treatment.

Description

SERUM BIOMARKERS FOR HIBM

TECHNICAL FIELD

[0001] The present invention relates to serum biomarkers and methods of using same for assessing sialylation in serum for evaluating and managing sialic acid deficiencies such as Hereditary Inclusion Body Myopathy (HIBM).

BACKGROUND

[0002] Sialic acid is the only sugar that contains a net negative charge and is typically found on terminating branches of N-glycans, O-glycans, and glycosphingolipids (gangliosides) (and occasionally capping side chains of GPI anchors). The sialic acid modification of cell surface molecules is crucial for many biological phenomena including protein structure and stability, regulation of cell adhesion, and signal transduction. Sialic acid deficiency disorders such as

Hereditary Inclusion Body Myopathy (HIBM or HIBM type 2), Nonaka myopathy, and Distal Myopathy with Rimmed Vacuoles (DMRV) are clinical diseases resulting from a reduction in sialic acid production.

[0003] HIBM is a rare autosomal recessive neuromuscular disorder case by a specific biosynthetic defect in the sialic acid synthesis pathway. Eisenberg et al., Nat. Genet. 29:83-87 (2001). The disease manifests between the ages of 20 to 40 with foot drop and slowly progressive muscle weakness and atrophy. Patients may suffer difficulties walking with foot drop, gripping and using their hands, and normal body functions like swallowing. Histologically, it is associated with muscle fiber

degeneration and formation of vacuoles containing 15-18 nm tubulofilaments that immunoreact like β-amyloid, ubiquitin, prion protein and other amyloid-related proteins. Askanas et al., Curr Opin Rheumatol. 10:530-542 (1998). Both the progressive weakness and histological changes initially spare the quadriceps and certain other muscles of the face. However, the disease is relentlessly progressive with patients becoming incapacitated and wheelchair-confined within two to three decades. There are no treatments currently available.

[0004] Studies of an Iranian- Jewish genetic isolate mapped the mutation associated with HIBM to chromosome 9pl2-13. Argov et al., Neurology 60: 1519-1523 (2003). The causative mutations were identified for HIBM in the gene GNE, which encodes the bifunctional enzyme UDP-N- acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK). Eisenberg et al., Nat. Genet. 29:83-87 (2001). DMRV is a Japanese variant, allelic to HIBM. Nishino et al., Neurology 59: 1689-1693 (2002). [0005] The biosynthesis steps and feedback regulation of GNE/MNK is depicted in Figure 1. The production of sialic acid on glycoconjugates requires the conversion of N-acetylglucosamine (conjugated to its carrier nucleotide sugar UDP) to sialic acid. The sialic acid subsequently enters the nucleus where it is conjugated with its nucleotide sugar carrier CMP to make CMP-sialic acid, which is used as a donor sugar for glycosylation reactions in the cell. CMP-sialic acid is a known regulator of GNE/MNK activity. Jay et al., Gene Reg. & Sys. Biol. 3: 181-190 (2009). Patients with HIBM have a deficiency in the production of sialic acid via the rate controlling enzyme GNE/MNK, which conducts the first two steps of this sequence: 1) epimerization of the glucosamine moiety to mannosamine with release of UDP, and 2) phosphorylation of the N-acetylmannosamine. The mutations causing HIBM occur in the regions encoding either the epimerase domain (GNE) or the kinase domain (MNK). Nearly twenty GNE mutations have been reported in HIBM patients from different ethnic backgrounds with founder effects among the Iranian Jews and Japanese. Broccolini et al., Hum. Mutat. 23:632 (2004). Most are missense mutations and result in decreased enzyme GNE activity and underproduction of sialic acid. Sparks et al., Glycobiology 15(11): 1102-10 (2005);

Penner et al., Biochemistry 45:2968-2977 (2006).

[0006] Knock-out of the GNE/MNK gene in mice is lethal as no sialic acid is incompatible with life, but knock-in introduction of human mutant forms of GNE/MNK have allowed the production of mouse models with human disease features. In the DMRV-HIBM mouse model in which Gne- deficient mice transgenically express the human GNE gene with D176V mutation Gne

hGN£D176V-Tg), these mice show hyposialylation in various organs in addition to the characteristic features of muscle atrophy, weakness and degeneration, and amyloid deposition. In these mice, hyposialylation is documented from birth, yet the mice develop muscle symptoms only several weeks later, including decreased twitch force production in isolated muscles starting at 10 weeks of age and impairment of motor performance from 20 weeks of age onward. Muscle atrophy and weakness were, however, reduced or prevented after treatment with administration of a sialic acid metabolite, N-acetylmannosamine (ManNAc), sialic acid, or sialyl-lactose, in water. Malicdan et al., Nat.

Medicine 15(6):690-695 (2009). All three sialic acid metabolites tested showed similar treatment effects. In another mouse model of HIBM in which knockin mice harbor the M712T Gne mutation, mice homozygous for the M712T Gne mutation died within 72 hours after birth, but lacked a muscle phenotype. Galeano et al., /. Clin. Investigation 117(6) 1585-1594 (2007). Homozygous mice, however, did have severe glomerular hematuria and podocytopathy, including effacement of the podocyte foot processes and segmental splitting of the glomerular basement membrane (GBM). Administration of ManNAc in water to mutant mice improved survival, improved renal histology including less flattened and fused podocyte foot processes, increased sialylation of renal

podocalyxin, and increased sialylation of brain PSA-NCAM. Galeano et al., /. Clin. Investigation 117(6): 1585-1594 (2007).

[0007] In individuals with DMRV, there is a 25% reduction of sialic acid in muscle tissue;

however, there is no difference in sialic acid content in sera between DMRV individuals and normal control individuals. See Noguchi et al. JBC 279(12): 11402-11407 (2004). Noguchi et al. reason that sialic acids are predominantly produced in the liver and transferred to synthesized glycloproteins, which are then released into the blood plasma. Free sialic acid in the plasma is derived from desialylation of these glycoproteins. GNE is expressed in the liver in large amounts; therefore, the reduction in enzymatic acid by mutations may not significantly affect the synthesis of sialic acid in the liver of DMRV patients, and sialic acid is present at concentrations comparable with normal blood levels. In contrast, Noguchi et al. reason that in DMRV skeletal muscles, the sialic acid contents are reduced. The reduced enzymatic activities along with weak expression of GNE protein are probably responsible for the more serious reduction in sialic acid synthesis in muscle tissue compared with plasma. Noguchi et al., JBC 279(12): 11402-11407, 11406 (2004).

[0008] One sialic acid containing glycoprotein, Neural Cell Adhesion Molecule (NCAM) has been shown to play an important role in cell to cell interactions not only in brain, but also in muscle. Normally NCAM is hypersialylated with as many as 10 sialic acid residues per oligosaccharride chain in a structure referred to as poly sialic acid (PSA). PSA-NCAM is a component of the cell surface membrane of myoblasts in the muscle. It has been shown that HIBM patients have a form of NCAM on the surface of the muscle that is hyposialyated with reduced or completely absent sialic acid residues. Broccolini et al., Neurology 75 265-272 (2010). This has been confirmed in HIBM knock-in mice by showing these mice also produce NCAM that is hyposialylated. Gagiannis et al., Glycoconjugate Journal 24 125-130 (2007).

[0009] The current assessment of HIBM patients requires the use of a muscle biopsy and the assessment of sialylation of muscle bound glycoproteins such as NCAM. See, for example, Ricci, E. et al., Neurology, 66(5), 755-8 (2006); Broccolini, A. et al., Neurology, 75(3), 265-272 (2010);

Tajima, Y. et al., The American Journal of Pathology, 166(4), 1121-1130 (2005); Nemunaitis, G. et al., / Gene Med, 12(5), 403-12 (2010). Muscle biopsies cannot be assessed regularly, are difficult to quantify and cannot be used reliably for regular management or drug development studies. [0010] Given the problems associated with current methods for diagnosing HIBM and determining responsiveness to and/or monitoring treatment of HIBM patients, there is a need for methods which allow easy detection of hyposialylated NCAM.

[0011] All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

[0012] Disclosed herein are: methods of determining whether an individual suffers from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual; methods of assessing disease severity in an individual suffering from a sialic acid deficiency based upon sialylation state of one or more glycoproteins in a blood sample from the individual; methods of treating an individual suffering from a sialic acid deficiency comprising administering an effective amount of a therapeutic agent to the individual, wherein treatment is based upon sialylation state of one or more glycoproteins in a blood sample from the individual; methods of identifying an individual suffering from a sialic acid deficiency as suitable or not suitable for treatment with a therapeutic agent, comprising detecting sialylation state of one or more

glycoproteins in a blood sample from the individual, wherein the sialylation state indicates that the individual is suitable or not suitable for treatment; methods of treating an individual suffering from a sialic acid deficiency comprising: (a) selecting an individual based upon sialylation state of one or more glycoproteins in a blood sample from the individual; and (b) administering to the selected individual an effective amount of a therapeutic agent; methods of monitoring responsiveness or lack of responsiveness to treatment with a therapeutic agent in an individual suffering from a sialic acid deficiency comprising detecting sialylation state one or more glycoproteins in a blood sample from the individual, wherein responsiveness is indicated by sialylation state of one or more glycoproteins.

[0013] Also disclosed herein are: methods of identifying an individual suffering from a sialic acid deficiency as suitable or not suitable to continue treatment with a therapeutic agent based upon sialylation state of one or more glycoproteins in a blood sample from the individual; methods of aiding diagnosis of a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual; methods of aiding assessment of disease severity in an individual suffering from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual; methods of aiding assessment of whether an individual suffering from a sialic acid deficiency is suitable or not suitable for treatment with a therapeutic agent comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual, wherein the sialylation state indicates that the individual is suitable or not suitable for treatment; methods of aiding monitoring responsiveness or lack of responsiveness to treatment with a therapeutic agent in an individual suffering from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual, wherein responsiveness is indicated by sialylation state of one or more glycoproteins; and methods of aiding assessment of whether an individual suffering from a sialic acid deficiency is suitable or not suitable to continue treatment with a therapeutic agent comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual.

BRIEF DESCRIPTION OF FIGURES

[0014] Figure 1 provides a diagram of intracellular sialic acid metabolism.

[0015] Figure 2A and B show soluble sialylated and non-sialylated NCAM from serum samples detected by a monoclonal anti-NCAM antibody. Figure 2A: Standards (Lane 1), normal human serum from two different people (Lane 5 and 6), a HIBM patient serum before treatment showing a weak upper and middle bands but the same lower bands (Lane 7). Figure 2B: Standards (Lane 1 and 9), Normal human serum from three (Lanes 2-4), a HIBM patient serum showing abnormal upper band (Lane 5), serum from a patient with myopathy but not HIBM (Lane 6), serum from a HIBM patient on ManNAc therapy for 2 yrs (Lane 7), normal human serum treated with sialidase showing that the upper and middle bands disappear if sialic acid is removed (Lane 8).

[0016] Figure 3 shows soluble sialylated and non-sialylated NCAM from serum samples detected by a monoclonal anti-NCAM antibody. Standard size ladder 20-250kD (lanes 1 and 10, Precision Plus Protein™ WesternC™ Standards, Biorad, CA), two different HIBM patients (Lanes 2-3), two different patients suffering from non-GNE related myopathy (Lanes 4-5), normal human sera from two control individuals (Lanes 6-7), same normal sera as used in Lanes 6-7 following treatment with sialidase showing that upper and middle bands are lighter if sialic acid is removed (Lanes 8-9).

DETAILED DESCRIPTION

[0017] The present application provides diagnostic, prognostic and therapeutic methods, which are based, at least in part, on determination of sialylation state of one or more glycoproteins in a biological fluid sample. In particular, the inventors have discovered that polysialylated and hyposialylated versions of soluble glycoproteins such as NCAM present in a biological fluid sample can be used as a marker for a sialylation deficiency. The sialylation state of serum NCAM reflects the muscle disease state of hyposialylation and can thus be used to monitor the severity and course of the disease such as HIBM in previously diagnosed patients, as well as to monitor the effects of a therapy for these patients.

General Techniques

[0018] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al , 1989); Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Handbook of Experimental Immunology (D.M. Weir & C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); The

Immunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Technique s(Greg T. Hermanson, ed., Academic Press, 1996); and Methods of Immunological Analysis (R. Masseyeff, W.H. Albert, and N.A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993).

Definitions

[0019] The terms "treating" and "treatment" as used herein refer to an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing the severity and/or frequency one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), delay or slowing the progression of the disease, ameliorating the disease state, increasing production of sialic acid, the sialylation precursor CMP-sialic acid (e.g. , increasing intracellular production of sialic acid) and restoring the level of sialylation in muscle and other proteins, decreasing the dose of one or more other medications required to treat the disease, and/or increasing the quality of life.

[0020] "Prophylaxis," "prophylactic treatment," "or preventive treatment" refers to prevention of the occurrence of one or more symptoms and/or their underlying cause, for example, prevention of a disease or condition in a patient susceptible to developing a disease or condition (e.g., at a higher risk, as a result of genetic predisposition, environmental factors, predisposing diseases or disorders, or the like). Prophylaxis includes HIBM myopathy in which chronic disease changes in the muscles are irreversible and for which animal model data suggests treatment benefit in prophylaxis.

[0021] As used herein, "delaying" the progression of the disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.

[0022] As used herein, an "at risk" individual is an individual who is at risk of developing a sialic acid deficiency. An individual "at risk" may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein. "At risk" denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of a sialic acid deficiency, which are described herein. An individual having one or more of these risk factors has a higher probability of developing a sialic acid deficiency than an individual without these risk factor(s).

[0023] The term "effective amount" refers to the amount of a pharmaceutical formulation including one or more compounds in the sialic acid biosynthetic pathway that provides the one or more compounds in the sialic acid biosynthetic pathway in a sufficient amount to render a desired treatment outcome. An effective amount may be comprised within one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint.

[0024] A "therapeutically effective amount" refers to an amount of a pharmaceutical formulation including one or more compounds in the sialic acid biosynthetic pathway sufficient to produce a desired therapeutic outcome (e.g., reduction of severity of a disease or condition). A

"prophylactically effective amount" refers to an amount of a pharmaceutical formulation including one or more compounds in the sialic acid biosynthetic pathway sufficient to prevent or reduce severity of a future disease or condition when administered to an individual who is susceptible and/or who may develop a disease or condition.

[0025] The term "derivative" as used herein includes derivatives, analogs, prodrugs, and unnatural precursors.

[0026] "Soluble" as used herein in relation to the glycoproteins provided herein means capable of dissolving under normal physiological conditions in a biological fluid such as one or more of blood, serum, plasma, urine and cerebrospinal fluid without substantial binding to body tissue.

[0027] As used herein, "a biological fluid sample" refers to a blood, serum, or plasma, cerebrospinal fluid or urine sample which contains a molecule which is to be characterized and/or identified, for example, based on physical, biochemical, chemical physiological, and/or genetic characteristics. A biological fluid sample does not encompass a tissue biopsy sample.

[0028] "Predicting" or "prediction" is used herein to refer to the likelihood that an individual is likely to respond either favorably or unfavorably to a treatment regimen.

[0029] As used herein, "at the time of starting treatment" or "baseline" refers to the time period at or prior to the first exposure to the treatment.

[0030] As used herein, "based upon" includes assessing, determining, or measuring the individual' s characteristics as described herein (and preferably selecting an individual suitable for receiving treatment).

[0031] When a marker is "used as a basis" for selection, assessing, measuring, or determining method of treatment and/or prevention as described herein, the marker is measured before and/or during treatment, and the values obtained are used by a clinician in assessing any of the following: (a) probable or likely suitability of an individual to initially receive treatment(s); (b) probable or likely unsuitability of an individual to initially receive treatment(s); (c) responsiveness to treatment; (d) probable or likely suitability of an individual to continue to receive treatment(s); (e) probable or likely unsuitability of an individual to continue to receive treatment(s); or (f) predicting likelihood of clinical benefits. As would be well understood by one in the art, an evaluation of an individual's health-related quality of life in a clinical setting is a clear indication that this parameter was used as a basis for initiating, continuing, and/or ceasing administration of the treatments described herein.

[0032] A method of "aiding assessment" as used herein refers to methods that assist in making a clinical determination and may or may not be conclusive with respect to the assessment.

[0033] The term "disorder" or "disease" used interchangeably herein, refers to any alteration in the state of the body or one of its organs and/or tissues, interrupting or disturbing the performance of organ function and/or tissue function (e.g., causes organ dysfunction) and/or causing a symptom such as discomfort, dysfunction, distress, or even death to a subject afflicted with the disease.

[0034] The term "individual" refers to a mammal. Preferably, the individual is a human.

[0035] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0036] It is understood that aspect and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments. [0037] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0038] As is apparent to one skilled in the art, an individual assessed, selected for, and/or receiving treatment may be an individual in need of assessment, selection for and/or receiving treatment.

Methods of Use

[0039] In some embodiments of the methods described below, the levels of glycoprotein expression are unchanged between diseased individual and a healthy individual. However, in a diseased individual the extent of sialylation of the glycoprotein is reduced as compared with that of the healthy individual. Thus, in these embodiments, the methods measure the extent of protein sialylation. In other embodiments, the protein expression in a diseased individual is reduced as compared with that of a healthy individual. Thus, in these embodiments, the methods measure the extent of protein expression.

[0040] Provided herein are methods of diagnosing and/or assessing disease severity of a sialic acid deficiency in an individual by detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual.

[0041] Specifically, provided herein are methods of identifying an individual as suitable or not suitable for treatment, selecting an individual for treatment and/or monitoring responsiveness or lack of responsiveness to treatment in an individual, based on sialylation state of one or more

glycoproteins in a biological fluid sample from the individual. Provided herein are also methods of treating an individual suffering from a sialic acid deficiency and/or identifying an individual as suitable or not suitable to continue treatment with a therapeutic agent based on sialylation state of one or more glycoproteins in a biological fluid sample from the individual.

[0042] Provided herein are methods of determining whether an individual suffers from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual. In some embodiments, the biological fluid sample is a blood sample {e.g., serum or plasma sample). In some embodiments, the glycoprotein is Neural Cell Adhesion Molecule (NCAM). In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM). In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual suffers from a sialic acid deficiency.

[0043] Further provided herein are methods of assessing disease severity in an individual suffering from a sialic acid deficiency based upon sialylation state of one or more glycoproteins in a biological fluid sample from the individual. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation.

[0044] Also provided herein is a method of treating an individual suffering from a sialic acid deficiency comprising administering an effective amount of a therapeutic agent to the individual, wherein treatment is based upon sialylation state of one or more glycoproteins in a biological fluid sample from the individual. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is suitable for treatment with a therapeutic agent. In some embodiments, the sialylation state is non-hyposialylation and non-hyposialylation indicates that the individual is not suitable for treatment with a therapeutic agent. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0045] Further provided herein is a method of identifying an individual suffering from a sialic acid deficiency as suitable or not suitable for treatment with a therapeutic agent, comprising detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual, wherein the sialylation state indicates that the individual is suitable or not suitable for treatment. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is suitable for treatment with a therapeutic agent. In some embodiments, the sialylation state is non-hyposialylation and non-hyposialylation indicates that the individual is not suitable for treatment with a therapeutic agent. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0046] Provided herein is also a method of treating an individual suffering from a sialic acid deficiency comprising: (a) selecting an individual based upon sialylation state of one or more glycoproteins in a biological fluid sample from the individual; and (b) administering to the selected individual an effective amount of a therapeutic agent. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is selected for treatment with a therapeutic agent. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0047] Provided herein is also a method of monitoring responsiveness or lack of responsiveness to treatment with a therapeutic agent in an individual suffering from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual, wherein responsiveness is indicated by sialylation state of one or more glycoproteins. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is nonresponsive to treatment. In some embodiments, the sialylation state is non- hyposialylation and non-hyposialylation indicates that the individual is responsive to treatment. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0048] Provided herein is also a method of identifying an individual suffering from a sialic acid deficiency as suitable or not suitable to continue treatment with a therapeutic agent based upon sialylation state of one or more glycoproteins in a biological fluid sample from the individual In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is not suitable to continue treatment. In some embodiments, the sialylation state is non- hyposialylation and non-hyposialylation indicates that the individual is suitable to continue treatment. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0049] Further provided herein is also a method of aiding diagnosis of a sialic acid deficiency in an individual comprising detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is Neural Cell Adhesion Molecule (NCAM). In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM).

[0050] Provided herein is also a method of aiding assessment of disease severity in an individual suffering from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is Neural Cell Adhesion Molecule (NCAM). In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM).

[0051] Provided herein is also a method of aiding assessment of whether an individual suffering from a sialic acid deficiency is suitable or not suitable for treatment with a therapeutic agent, comprising detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual, wherein the sialylation state indicates that the individual is suitable or not suitable for treatment. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is suitable for treatment with a therapeutic agent. In some embodiments, the sialylation state is non-hyposialylation and non-hyposialylation indicates that the individual is not suitable for treatment with a therapeutic agent. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0052] Provided herein is also a method of aiding monitoring responsiveness or lack of responsiveness to treatment with a therapeutic agent in an individual suffering from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual, wherein responsiveness is indicated by sialylation state of one or more glycoproteins. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and

hyposialylation indicates that the individual is nonresponsive to treatment. In some embodiments, the sialylation state is non-hyposialylation and non-hyposialylation indicates that the individual is responsive to treatment. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0053] Provided herein is also a method of aiding assessment of whether an individual suffering from a sialic acid deficiency is suitable or not suitable to continue treatment with a therapeutic agent comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is NCAM. In some embodiments, the sialic acid deficiency is HIBM. In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is not suitable to continue treatment. In some embodiments, the sialylation state is non-hyposialylation and non-hyposialylation indicates that the individual is suitable to continue treatment. In some embodiments, the therapeutic agent comprises a compound in the sialic acid synthesis pathway or derivative thereof.

[0054] In some embodiments, the individual is suitable for treatment or suitably treated. When the individual is suitable for treatment or suitably treated, the individual is likely to exhibit one or more desirable clinical or non-clinical outcome as compared to individuals having the same disease and receiving the same treatment but possessing a different sialylation state of one or more glycoproteins. A desirable clinical or non-clinical outcome includes but is not limited to increased production of sialic acid, restored level of sialylation in muscle and other proteins, increased muscle function, increased muscle strength (e.g., muscle strength of the quadriceps), increased muscle tensile force, improved muscle movement, improved limb movement, muscle growth, increased muscle stamina, decrease in muscle fatigability, decrease in muscle atrophy, decrease in neuronal atrophy, increase in pulmonary function, reduction in proteinuria (e.g., lower amounts of protein in the urine), reduction in hematuria (e.g., lower amounts of red blood cells in the urine) increased activity, stable disease (e.g., preventing or delaying the worsening of the disease), and/or increase or elongation of overall survival. The clinical outcome(s) will then be considered, and a decision as to whether the patient is suitable for the therapy will be made accordingly, taking into account the patient's specific situation and the relevance of the clinical or non-clinical outcomes.

[0055] In some embodiments, the individual is "likely to respond" or "responsive." When an individual is likely to respond or responsive to treatment, the individual is likely to experience an improvement or positive clinical or non-clinical response selected from, but not limited to increased production of sialic acid, restored level of sialylation in muscle and other proteins, increased muscle function, increased muscle strength (e.g., muscle strength of the quadriceps), increased muscle tensile force, improved muscle movement, improved limb movement, muscle growth, increased muscle stamina, decrease in muscle fatigability, decrease in muscle atrophy, decrease in neuronal atrophy, increase in pulmonary function, reduction in proteinuria (e.g., lower amounts of protein in the urine), reduction in hematuria (e.g., lower amounts of red blood cells in the urine) increased activity, stable disease (e.g., preventing or delaying the worsening of the disease), and/or increase or elongation of overall survival.

[0056] In some embodiments in any of the methods provided herein, the sialylation state of the glycoprotein is hyposialylation. In some embodiments, hyposialylation encompasses a sialylation state characterized by a lower level of one or more forms of sialylated glycoprotein in a biological fluid sample compared to a reference. In some embodiments in any of the methods provided herein, the sialylation state of one or more glycoproteins is non-hyposialylation. In some embodiments, non- hyposialylation encompasses a sialylation state characterized by an about equal or higher level of one or more forms of sialylated glycoprotein in a biological sample compared to a reference.

[0057] In some embodiments, the reference is the level of unsialylated glycoprotein within the same biological fluid sample. In some embodiments, the reference is the level of one or more sialylated glycoprotein in a biological fluid sample of the same individual before and/or after treatment. In some embodiments, the reference is the median level of one or more forms of sialylated glycoprotein in a biological fluid sample for a given patient population. In some embodiments the patient population is a group of healthy individuals. In some embodiments, the patient population is a group of individuals diagnosed with a sialic acid deficiency.

[0058] In some embodiments, when the sialylation state is hyposialylation, the lower level of one or more forms of sialylated glycoprotein in a biological fluid sample is about any of 1.25X, 1.5X, 1.75X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 25X, 50X, or 100X lower than a reference. In some embodiments, the lower level of one or more forms of sialylated glycoprotein in a biological fluid sample is between about any of 1.25X and 100X, 1.25X and 50X, 1.5X and 100X, 1.5X and 50X, 2X and 100X, 2X and 50X, 1.25X and 10X, 1.5X and 10X, 2X and 10X lower than a reference.

[0059] In some embodiments, the lower level of one or more forms of sialylated glycoprotein in a biological fluid sample is a decrease of at least about any of 20%, 25%, 30%, 35% or 40% compared to a reference.

[0060] In some embodiments, when the sialylation state is non-hyposialylation, the equal or higher level of one or more forms of sialylated glycoprotein in a biological fluid sample is about any of IX, 1.25X, 1.5X, 1.75X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 25X, 50X, or 100X higher than a reference. In some embodiments, the equal or higher level of one or more forms of sialylated glycoprotein in a biological fluid sample is between about any of IX and 100X, l.X and 50X, 1.25X and 100X, 1.25X and 50X, 1.5 and 100X, 1.5 and 50X, 2X and 100X, 2X and 50X, IX and 10X, 1.25X and 10X, 1.5X and 10X, 2X and 10X higher than a reference.

[0061] In some embodiments, the equal or higher level of one or more forms of sialylated glycoprotein in a biological fluid sample is an increase of at least about any of 20%, 25%, 30%, 35% or 40% compared to a reference.

[0062] In some embodiments, the levels of one or more forms of sialylated glycoprotein in a biological fluid sample compared to a reference are expressed at statistically significant differential levels. In some embodiments, statistical significance is determined at a p-value of 0.1 or less, 0.05 or less, or 0.01 or less. In some embodiments, the p-value is between about any of 0.01 and 0.05 or 0.01 and 0.1. In some embodiments, the p-values are corrected for multiple comparisons. In some embodiments, multiple comparisons are corrected for using Bonferroni correction. In some embodiments, p-values are determined using permutation approaches, which are well known to those in the art. Permutation tests include randomization tests, re -randomization tests, exact tests, the jackknife, the bootstrap and other resampling schemes. In some embodiments, the threshold criterion comprises a correlation value. In some embodiments, the correlation value is r. In some

embodiments, r is greater than or equal to about any of 0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30 or 0.25.

[0063] In some embodiments, the measured levels of one or more forms of sialylated glycoprotein in a biological fluid sample are normalized. For example, the levels are normalized against a protein in the biological fluid sample, the level of which does not change (or does not change significantly) among different samples.

[0064] Sialylation state of a glycoprotein can be determined by methods known in the art. In the first step of the determination methods, the glycoprotein in the sample is isolated and purified.

Isolation and purification methods are known in the art, for example, SDS PAGE, size exclusion chromatography, affinity resin or beads, filteration/isolation columns, various centrifugation methods to separate fractions, and the like. In some embodiments, the sialylation state of a glycoprotein is determined by SDS PAGE and Western Blot using an antibody to the glycoprotein capable of detecting sialylated and unsialylated glycoprotein as described for Neural Cell Adhesion Molecule (NCAM) in the Examples disclosed herein.

[0065] In some embodiments in any of the methods provided herein, the biological fluid sample is blood, serum, plasma, cerebrospinal fluid or urine. In some embodiments, the biological fluid sample is blood. In some embodiments, the biological fluid sample is serum. In some embodiments, the biological fluid sample is plasma.

[0066] In some embodiments in any of the methods provided herein, the sialic acid deficiency is a myopathy associated with sialic acid deficiency. In some embodiments, the myopathy associated with sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM), Nonaka myopathy, and/or Distal Myopathy with Rimmed Vacuoles (DMRV). In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM). Glycoproteins

[0067] Glycoproteins of which sialylation status is evaluated and used in the methods provided herein include but are not limited to Neural Cell Adhesion Molecule (NCAM), alpha-dystroglycan (alpha-DG), beta-dystroglycan (beta-DG), sarcoglycans, PC-1, biglycan, podocalyxin, gangliosides, sialylated ion channels, cytokines, and immune globulins. In general, sialylation status of all enzymes and proteins that may spill into blood upon compromising muscle cell membrane integrity can be measured using the methods disclosed herein.

[0068] In some embodiments of the methods provided herein, the glycoprotein is a soluble glycoprotein. In some embodiments, the glycoprotein is a transmembrane or GPI-anchored glycoprotein which has been released from the cell membrane into a biological fluid. In some embodiments, the glycoprotein is a secreted glycoprotein.

[0069] In some embodiments of the methods provided herein, the glycoprotein is Neural Cell Adhesion Molecule (NCAM). NCAM is a heterogeneous glycoprotein of the Immunoglobulin(Ig) superfamily which mediates cell-cell and cell-substratum interactions. There are numerous alternatively-spliced NCAM mRNAs produced, giving a wide diversity of NCAM isoforms. Reyes AA et al., Mol Cell Biol. 11 (3): 1654-61 (1991). Transcription, translation and post-translational modifications of NCAM are regulated in a developmental and cell-specific fashion. In mammalian brain, the glycosylphosphatidylinositol (GPI) -anchored 125-kDa isoform and transmembrane 140- and 160-kDa isoforms are primarily expressed. In muscle, three isoforms have been described: a GPI-anchored 125-kDa isoform and transmembrane 140- and 155-kDa isoforms. Walsh et al., Development 105:803-811 (1989). A secreted NCAM isoforms which includes an in-frame stop codon and thus prematurely terminates the coding sequence, generating a truncated N-CAM polypeptide has also been reported. Gower et al., Cell 55:955-964 (1988). Furthermore, intact transmembrane isoforms of NCAM can be released from the plasma membrane and present in cerebrospinal fluid. Olsen et al., Biochem J 295:833-840.

[0070] In some embodiments of the methods provided herein, the glycoprotein is any soluble NCAM isoform present in a biological fluid of an individual. In some embodiments, the glycoprotein is a GPI-anchored or transmembrane isoforms of NCAM which has been released from the plasma membrane into a biological fluid. In some embodiments, the glycoprotein is the 120 and 140 kD NCAM identified by monoclonal Ab NCAM (RNL-1): sc-53007 obtained from Santa Cruz Biotech, CA. In other embodiments, the NCAM glycoprotein is any isoform of NCAM, the sialylation amount of which is detectable by antibodies, lectins, or other molecules that allow detection of sialic acid residues.

[0071] In some embodiments, the glycoprotein is alpha-dystroglycan (a-DG). a-DG is an essential component of the dystrophin-glycoprotein complex. Michele et al. Nature 418, 417-422 (2002); Michele et al. J Biol Chem 278, 15457-15460 (2003). a-DG is heavily glycosylated with O-mannosyl glycans (mannose-N- acetylglucosamine-galactose-sialic acid) linked to a serine or threonine; these glycans are critical for a-DG's interactions with laminin and other extracellular ligands. In some embodiments, the glycoprotein is soluble a-DG. In some embodiments, the glycoprotein is a-DG released from the plasma membrane into a biological fluid. In other embodiments, the a-DG is any isoform or molecular parts of a-DG, the sialylation amount of which is detectable by antibodies, lectins, or other molecules that allow detection of sialic acid residues.

Therapeutic Agents and Pharmaceutical Formulations

[0072] Therapeutic agents for use in any of the methods described herein include but are not limited to compounds in the sialic acid biosynthetic pathway or derivative thereof and nucleic acids encoding wildtype GNE coding for UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase or homologues thereof with at least 80% sequence identity.

[0073] Compounds in the sialic acid biosynthetic pathway or derivative thereof include, but are not limited to, mannosamine, N-acetyl mannosamine (ManNAc), ManNac-6-phosphate (ManNAc-6- P), UDP-GlcNAc, N-acetylneuraminic acid (NeuAc), NeuAc-9-phosphate (NeuAc-9-P), sialic acid {i.e., 5-N-acetylneuraminic acid), CMP-sialic acid, and/or derivatives thereof.

[0074] In some embodiments, the one or more compounds in the sialic acid biosynthetic pathway or derivative thereof include ManNAc or a derivative thereof. Structures of such ManNAc and derivatives thereof include, but are not limited to, those defined by the formula below:

wherein: Ri, R3, R4, or R5 is hydrogen, lower alkanoyl, carboxylate or lower alkyl; and R₂ is lower alkyl, lower alkanoylalkyl, lower alkyl alkanoyloxy.

[0075] The term lower alkyl refers to (C]-C₆)alkyl. A lower alkyl includes methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, (C3-C₆)cycloalkyl {e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), (C3-C6)cycloalkyl(Ci-C6)alkyl (e.g., cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2- cyclopentylethyl, or 2-cyclohexylethyl), (Ci-C₆)alkoxy (e.g. , methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy) (C2-C6)alkenyl (e.g. , vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 ,-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl), (C2-C₆)alkynyl (e.g. , ethynyl, 1-propynyl, 2-propynyl, 1 -butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4- pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl), (Ci-C6)alkanoyl (e.g., acetyl, propanoyl or butanoyl), halo(Ci-C6)alkyl (e.g. , iodomethyl, bromomethyl, chloromethyl,

fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl), hydroxy(Ci-C6)alkyl (e.g., hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2- hydroxypropyl, 3-hydroxypropyl, 1 -hydroxy butyl, 4-hydroxybutyl, 1-hydroxypentyl, 5- hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl), (Ci-C6)alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl), (C]-C6)alkylthio (e.g., methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio), and/or (C2-C6)alkanoyloxy (e.g. , acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy).

[0076] In some embodiments, R₂ is methyl, and Ri, R3, R4, and R5 is hydrogen. In some embodiments, the ManNAc or derivative thereof is N-acetyl mannosamine (ManNAc). In some embodiments, the ManNAc or derivative thereof is N-levulinoylmannosamine (ManLev) or N- azidoacetylmannosamine (ManNAz).

[0077] In some embodiments, the one or more compounds in the sialic acid biosynthetic pathway or derivative thereof include sialic acid or a derivative thereof. In some embodiments, the sialic acid or derivative thereof is sialic acid. In some embodiments, the sialic acid or derivative thereof is a sialic acid analog such as N-levulinoyl sialic acid (SiaLev) or N-azidoacetyl sialic acid (SiaNAz). In some embodiments, the sialic acid or derivative thereof is bound as a glycoconjugate. In some embodiments, the sialic acid or derivative thereof is an unnatural precursor such as sialylactose. In some embodiments the sialic acid or derivative thereof is conjugated to an immunoglobulin.

[0078] In some embodiments of any of the methods described herein, the therapeutic agent is formulated for various forms of administration by any of the methods well known to the

pharmaceutical arts. See, for example, WO 2008/150477 and US 200902981 12, which are incorporated herein in their entirety. The therapeutic agent may be administered, for example, at a dose of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 200 to 400 mg/kg, at least about 1 mg/kg to about 25 to 200 mg/kg, at least about 1 mg/kg to about 1000 mg/kg, at least about 200 mg/kg to about 1000 mg/kg, at least about 400 mg/kg to about 1000 mg/kg, or at least about 600 mg/kg to about 1000 mg/kg of body weight, although other dosages may provide beneficial results. The amount administered will vary depending on various factors including, but not limited to the disease, the weight, the physical condition, the health, the age of the mammal, whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.

Formulations

[0079] Various pharmaceutical formulations comprising one or more therapeutic agents may be used in any of the methods described herein. In particular, provided herein are pharmaceutical formulations comprising one or more therapeutic agents (e.g., those described herein) and a pharmaceutically acceptable carrier, diluent, and/or excipient. Examples of suitable carriers, excipients, and diluents include, but are not limited to, sugars, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum such as xanthan gum, guar gum, or gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyethylene glycols, polyvinylpyrrolidone, phospholipics, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, mineral oil, lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents, disintegrating agents, antioxidants, surfactants, and/or flavoring agents.

[0080] Pharmaceutical formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch,

microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0081] The pharmaceutical formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.

[0082] In some embodiments, when the therapeutic agent is a nucleic acid, the therapeutic agent may be used and delivered to a system in connection with an appropriate delivery vehicle (such as a liposome or lipid nanoparticle).

[0083] For topical administration, the pharmaceutical formulation may be a cream, milk, gel, dispersion, or microemulsions, lotion thickened to a greater or lesser extent, impregnated pad, ointment or stick, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap.

[0084] The pharmaceutical formulation may be a food supplement or incorporated into food or drink item such as a nutritional bar, snack bar, cookie, candy, cereal, pudding, ice cream, frozen confectionary, chewing gum, drink mix, soda pop, liquid supplement, sauce, salad dressing, gravy, jelly, jam, spread, margarine, peanut butter, nut spread, frosting, and the like. In essence, can be used in any food, composition or supplement in which sugar is employed. Hence, the therapeutic agent and/or derivatives thereof can be used as a partial or full substitute for sugar.

[0085] Such food supplements, drinks and food items can include any other food ingredient including, for example, flour, oil, cream, butter, sugar, salt, spices and the like. In addition, the food supplements, drinks and food items can include vitamins and nutrients commonly found in other nutritional supplements.

Routes of Administration

[0086] Various routes of administration may be used in any of the methods described herein. In some embodiments of any of the methods described herein, the therapeutic agent can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular, intraperitoneal, intraarticular, intraarterial, intrasynovial, or infusion techniques), rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.

[0087] Administration of the therapeutic agents in accordance may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the therapeutic agent may be essentially continuous over a pre-selected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.

Kits and/or Articles of Manufacture

[0088] Provided herein are diagnostic, prognostic and therapeutic methods, which are based, at least in part, on determination of sialylation state of one or more glycoproteins in a biological fluid sample. In some embodiments, the methods use an agent to detect sialylated and unsialylated glycoprotein(s). In some embodiments, the methods use one or more antibody to detect sialylated and unsialylated glycoprotein(s). In some embodiments, the methods encompass administration of a therapeutic agent. Accordingly, the invention provides kits and/or articles of manufacture for performing these methods as well as instructions for carrying out the methods of this invention such as collecting a biological fluid sample, performing the screen, and/or analyzing the results, and/or administration of an effective amount of a therapeutic agent as defined herein. These can be used alone or in combination with other suitable therapy.

[0089] Provided herein are kits and/or articles of manufacture comprising packaging material and at least one vial comprising an agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein the agent is used to determine sialylation state of one or more glycoproteins in a biological fluid sample. Also provided herein are kits and/or articles of manufacture comprising packaging material and at least one vial comprising a therapeutic agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent. Further provided herein are kits and/or articles of manufacture comprising packaging material and at least one vial comprising an agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein the agent is used to determine sialylation state of one or more glycoproteins in a biological fluid sample and at least one vial comprising a therapeutic agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent. [0090] Provided herein are kits and/or articles of manufacture for use in diagnosing and/or assessing disease severity of a sialic acid deficiency in an individual by detecting sialylation state of one or more glycoproteins in a biological fluid sample from the individual. The kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable agent to determine sialylation state of one or more glycoproteins in a biological fluid sample, and instructions for use thereof. In some embodiments, the kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable antibody to determine sialylation state of one or more glycoproteins, and instructions for use thereof. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is Neural Cell Adhesion Molecule (NCAM). In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM). In some embodiments, the sialylation state is hyposialylation.

[0091] Further provided herein are kits and/or articles of manufacture for use in treating an individual suffering from a sialic acid deficiency, identifying an individual as suitable or not suitable for treatment and/or selecting an individual for treatment based on sialylation state of one or more glycoproteins in a biological fluid sample from the individual. The kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable agent to determine sialylation state of one or more glycoproteins, one or more therapeutic agent and instructions for use thereof. In some embodiments, the kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable antibody, one or more therapeutic agent and instructions for use thereof. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is Neural Cell Adhesion Molecule (NCAM). In some embodiments, the sialic acid deficiency is Hereditary

Inclusion Body Myopathy (HIBM). In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is suitable or selected for treatment. In some

embodiments, the sialylation state is non-hyposialylation and non-hyposialylation indicates that the individual is not suitable for treatment.

[0092] Provided herein are also kits and/or articles of manufacture for use in monitoring responsiveness or lack of responsiveness to treatment in an individual and/or identifying an individual as suitable or not suitable to continue treatment with a therapeutic agent based on sialylation state of one or more glycoproteins in a biological fluid sample from the individual. The kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable agent to determine sialylation state of one or more glycoproteins, one or more therapeutic agent and instructions for use thereof. In some embodiments, the kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable antibody, one or more therapeutic agent and instructions for use thereof. In some embodiments, the biological fluid sample is a blood sample (e.g., serum sample). In some embodiments, the glycoprotein is Neural Cell Adhesion Molecule (NCAM). In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM). In some embodiments, the sialylation state is hyposialylation and hyposialylation indicates that the individual is non-responsive or not suitable to continue treatment. In some embodiments, the sialylation state is non- hyposialylation and non-hyposialylation indicates that the individual is responsive to treatment or suitable to continue treatment.

[0093] The kits and/or articles of manufacture can include all or some of the positive controls, negative controls, reagents, and antibodies described herein for determining the sialylation state of one or more glycoproteins in a biological fluid sample.

[0094] As amenable, these suggested kit and/or article of manufacture components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit and/or article of manufacture components may be provided in solution or as a liquid dispersion or the like.

EXAMPLES

Example 1: Detection of soluble sialylated and unsialylated NCAM in serum

[0095] Serum samples from normal individuals, a HIBM patient before treatment, a patient suffering from myopathy but not HIBM, and a HIBM patient on ManNAc therapy for 2 years were purified to reduce serum proteins. The samples were subjected to SDS PAGE and stained with a monoclonal antibody to NCAM (Figure 2A and B). As a control, serum samples were prepared as described above and treated with sialidase to remove sialic acid.

[0096] Some NCAM in serum is sialylated and NCAM sialylation varies in disease and treatment (Figure 2). The upper and middle bands detected by western blot with the monoclonal antibody to NCAM are due to sialic acid containing forms, since they disappear after treatment with sialidase (Figure 2B, Lane 8). These bands are fuzzy and decreased in HIBM patients (Figure 2A, Lane 7; Figure 2B, Lane 5), and improve after treatment (Figure 2B, Lane 7). They are not abnormal in patients with muscle disease but not HIBM (Figure 2B, Lane 6). Example 2: Diagnosis ofHIBM

[0097] Serum samples of individuals at risk of developing HIBM and individuals experiencing symptoms of myopathy are prepared as described in Example 1. Polysialylated and unsialylated NCAM is assessed as described in Example 1. Individuals identified as being at risk of the disease based on the results of sialylation detection tests are further tested for clinical muscle wasting based on age of onset and patterns typical of HIBM and undergo genetic testing to confirm the presence of the disease. The level of sialylation of NCAM may also decrease with advancing age irrespective of HIBM, and detection of NCAM sialylation levels may also serve as a diagnostic or predictive marker of ageing or various disorders associated with ageing.

Example 3: Determining responsiveness to treatment in HIBM patient

[0098] Individuals diagnosed with HIBM are treated with ManNAc or by other means of increasing cellular sialic acid pools (e.g. other molecules or gene therapy) and responsiveness to treatment is assessed in serum samples of these individuals by detecting polysialylated and unsialylated NCAM as described in Example 1.

[0099] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

CLAIMS We claim:

1. A method of determining whether an individual suffers from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual.

2. A method of assessing disease severity in an individual suffering from a sialic acid deficiency based upon sialylation state of one or more glycoproteins in a blood sample from the individual.

3. A method of treating an individual suffering from a sialic acid deficiency comprising administering an effective amount of a therapeutic agent to the individual, wherein treatment is based upon sialylation state of one or more glycoproteins in a blood sample from the individual.

4. A method of identifying an individual suffering from a sialic acid deficiency as suitable or not suitable for treatment with a therapeutic agent, comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual, wherein the sialylation state indicates that the individual is suitable or not suitable for treatment.

5. A method of treating an individual suffering from a sialic acid deficiency comprising: (a) selecting an individual based upon sialylation state of one or more glycoproteins in a blood sample from the individual; and (b) administering to the selected individual an effective amount of a therapeutic agent.

6. A method of monitoring responsiveness or lack of responsiveness to treatment with a therapeutic agent in an individual suffering from a sialic acid deficiency comprising detecting sialylation state one or more glycoproteins in a blood sample from the individual, wherein responsiveness is indicated by sialylation state of one or more glycoproteins.

7. A method of identifying an individual suffering from a sialic acid deficiency as suitable or not suitable to continue treatment with a therapeutic agent based upon sialylation state of one or more glycoproteins in a blood sample from the individual.

8. A method of aiding diagnosis of a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual.

9. A method of aiding assessment of disease severity in an individual suffering from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual.

10. A method of aiding assessment of whether an individual suffering from a sialic acid deficiency is suitable or not suitable for treatment with a therapeutic agent comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual, wherein the sialylation state indicates that the individual is suitable or not suitable for treatment.

11. A method of aiding monitoring responsiveness or lack of responsiveness to treatment with a therapeutic agent in an individual suffering from a sialic acid deficiency comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual, wherein responsiveness is indicated by sialylation state of one or more glycoproteins.

12. A method of aiding assessment of whether an individual suffering from a sialic acid deficiency is suitable or not suitable to continue treatment with a therapeutic agent comprising detecting sialylation state of one or more glycoproteins in a blood sample from the individual.

13. The method of any one of claims 3-5 and 10, wherein the sialylation state is hyposialylation and hyposialylation indicates that a) the individual is suitable for treatment or b) the individual is selected for treatment.

14. The method of any one of claims 3-4 and 10, wherein the sialylation state is non- hyposialylation and non-hyposialylation indicates that a) the individual is not suitable for treatment or b) the individual is not selected for treatment.

15. The method of any one of claims 6-7 and 11-12, wherein the sialylation state is

hyposialylation and hyposialylation indicates that a) the individual is nonresponsive or b) the individual is not suitable for continuing treatment.

16. The method of any one of claims 6-7 and 11-12, wherein the sialylation state is non- hyposialylation and non-hyposialylation indicates that a) the individual is responsive or b) the individual is suitable for continuing treatment.

17. The method of any one of claims 3-7 and 10-16, wherein the therapeutic agent comprises one or more compounds in the sialic acid biosynthetic pathway or derivative thereof.

18. The method of claim 17, wherein the therapeutic agent comprises N-acetyl-mannosamine or a derivative thereof.

19. The method of claim 17, wherein the therapeutic agent comprises sialic acid.

20. The method of any one of claims 1-19, wherein the sialic acid deficiency is a myopathy associated with a sialic acid deficiency.

21. The method of claim 20, wherein the myopathy associated with a sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM), Nonaka myopathy and/or Distal Myopathy with Rimmed Vacuoles (DMRV).

22. The method of any one of claims 1-21, wherein the blood sample is a plasma sample.

23. The method of claim any one of claims 1-21, wherein the blood sample is a serum sample.

24. The method of any one of claims 1-23, wherein the glycoprotein is Neural Cell Adhesion Molecule (NCAM)

25. The method of claim 24, wherein NCAM is a GPI anchored or transmembrane NCAM isoforms released from a muscle cell.

26. The method of claim 24, wherein NCAM is the 120, 140 kD NCAM identified by monoclonal Ab NCAM (RNL-1): sc-53007.