Biomarkers for lysosomal storage diseases

Abstract

Methods for assessing lysosomal dysfunction and diagnosing certain lysosomal storage diseases are provided. The methods use CD63 as a biomarker and can be used for diagnosing and monitoring disease states, as well as for monitoring and / or adjusting therapeutic interventions for such conditions. Assays and kits for use in the methods are also provided.

Description

[Technical Field]

[0001] The present disclosure relates to biomarkers for lysosomal storage diseases. The present disclosure provides methods useful for diagnosing and monitoring certain lysosomal storage diseases, as well as for monitoring and / or adjusting therapeutic interventions for such conditions. [Background technology]

[0002] Lysosomal storage disorders (LSDs) are a group of genetic disorders that include Fabry disease (FD), Gaucher disease (GD), and mucopolysaccharidoses (MPS). These disorders primarily involve the dysfunction of lysosomal hydrolases, resulting in impaired substrate degradation. Disruption of lysosomal function can lead to the accumulation of undegraded substrates in endosomes and lysosomes, ultimately impairing cellular function. Although each LSD typically results from mutations in different genes and results in defects in enzyme activity or protein function, all LSDs share a common biochemical feature: the accumulation of substrates within lysosomes.

[0003] Although lysosomal proteins are ubiquitously distributed, the accumulation of undegraded substrates in patients with lysosomal storage disorders is usually restricted to cells, tissues, and organs with high substrate turnover. The accumulation of primary storage materials can trigger a cascade of secondary disruptions to other biochemical and cellular functions, resulting in severe pathology in lysosomal storage disorders.

[0004] The degree and severity of LSDs typically depend on the type and amount of substrate accumulated, but almost all disorders are progressive. Many clinical similarities are observed between and within groups. Common clinical features of many LSDs include bone abnormalities, organomegaly, central nervous system dysfunction, and coarse hair and facial features. Many patients with lysosomal storage disorders die in infancy or childhood, and those who survive into adulthood often have a shortened lifespan and significant morbidity.

[0005] Although individual LSDs are classified as rare diseases, their prevalence, when considered as a group of disorders, is substantial and represents a significant health issue. A limited number of studies have investigated the incidence of LSDs, defined as the total number of diagnosed cases within a given time period divided by the total number of live births within that same period. One of the main problems associated with obtaining accurate epidemiological data for these individually rare disorders is that most countries have numerous diagnostic centers, further complicating the problem of collecting and correlating diagnoses. The estimated prevalence of LSDs worldwide is approximately 1 in 7,500 live births. The true prevalence is likely higher due to misdiagnosed or undiagnosed cases.

[0006] Gaucher disease (GD) is an inherited metabolic disorder caused by mutations in the GBA gene, resulting in glucocerebrosidase (GCase) deficiency. Accumulation of glucosylceramide (GL1), the primary storage substrate in macrophage lysosomes, affects cells of the reticuloendothelial system, including the liver, spleen, and bone marrow. It is the most common LSD, with a prevalence of approximately 1 in 40,000 in the general population. In Ashkenazi Jewish populations, the prevalence has historically been as high as 1 in 1,000.

[0007] Fabry disease (FD) is the second most common LSD after Gaucher disease. It is an X-linked lysosomal storage disorder characterized by deficient activity of the enzyme α-galactosidase A (α-Gal), encoded by the GLA gene. The enzyme deficiency leads to the progressive intracellular accumulation of glycosphingolipids, primarily globotriaosylceramide (GL3), in various cell types and tissues, including the kidney, heart, liver, spleen, and skin, as well as the peripheral and central nervous systems.

[0008] Mucopolysaccharidoses (collectively "MPS") are inherited autosomal recessive disorders (except for MPS type II, which is X-linked) in which mucopolysaccharides—also known as glycosaminoglycans (GAGs)—accumulate in connective tissue and other tissues throughout the body, including the skin, cartilage, cornea, liver, spleen, and vascular tissue. Severe symptoms manifest in preschool children with developmental delay, short stature, recurrent ear and respiratory infections, hepatosplenomegaly, and coarsened facies. Over time, children develop hearing loss, atrial valve disease, airway obstruction, skeletal contractures, and distinctive facial features with macrocephaly, thick eyebrows, enlarged gums, macroglossia, and thickened lips and nasal alae. Intelligence is impaired, and patients degenerate as the disease progresses. IQ is below 70 in 61% of untreated patients and borderline in 25% at age 2–3 years. Other complications include corneal opacity, carpal tunnel syndrome, hydrocephalus, glaucoma, cardiac arrhythmias, cervical spine instability, and spinal cord compression. If untreated, life expectancy is 20 or 30 years.

[0009] Currently, there are no cures for LSDs, and no approved treatments for many of these conditions. A particular challenge is developing effective therapies for treating the CNS symptoms common in LSDs. For LSDs for which treatment is available, the quality of life for these patients has improved significantly, especially for those whose disease is diagnosed and treated early.

[0010] Existing treatments for LSDs, where available, typically involve either enzyme replacement therapy (ERT), in which an active version of the defective enzyme is administered to the patient to compensate for the reduced or abnormal activity of the patient's own enzyme, or substrate suppression therapy (SRT), in which a modulator of an enzyme involved in a defective lysosomal pathway alters the flux of substrates through the pathway, reducing the levels of the problematic substrate. In either case, regular and repeated administration of the therapy is required. Approximately 70% of approved therapies involve ERT. Approved treatments for FD are based on ERT using recombinant α-Gal, i.e., agalsidase beta (Fabrazyme® - Sanofi, EU approved in 2001, US approved in 2003) and agalsidase alfa (Replagal® - Takeda, EU approved in 2001). FD patients can also be treated with the small molecule chaperone migalastat (Galafold® - Amicus Therapeutics, EU approved in 2016), if they have the appropriate GLA mutation. Treatments for GD include ERT, such as imiglucerase (Cerezyme® - Sanofi, EU approved 1997), and SRT, such as eliglustat (Cerdelga® - Sanofi, EU approved 2015) and miglustat (Zavesca® - Janssen, EU approved 2002). Treatment for MPS may involve ERT using alpha-L-iduronidase (Aldurazyme® - Sanofi, EU approved for MPS I in 2003). Other active agents are being evaluated for the treatment of lysosomal storage diseases; one such agent is benglustat, (3S)-1-azabicyclo[2.2.2]octan-3-yl N-{2-[2-(4-fluorophenyl)-1,3-thiazol-4-yl]propan-2-yl}carbamate, which is in phase 2 clinical trials as an SRT treatment for FD.

[0011] Before treatment for LSDs can begin, the condition must be diagnosed. Given the relative rarity of LSDs, diagnostic methods should aim for high levels of sensitivity and specificity. Historically, diagnosis has been based on multiple factors, including family history, clinical symptoms, and biopsy, but this can miss early (presymptomatic) disease, which is particularly problematic in the case of severe pediatric LSDs. More recently, genetic screening and enzyme or substrate assays have been developed to investigate some LSDs. However, such tests are typically highly specific for each LSD or subtype of LSD, and no broad diagnostic assays for a common group of LSDs utilizing a single biomarker exist. Multiplex screening assays have been proposed, but they have not yet been widely adopted (see, for example, International Patent Publication No. WO 2004 / 088322, which measures levels of multiple lysosomal protein markers, such as LAMP-1, saposin C, α-glucosidase, and α-iduronidase, in newborn blood spots).

[0012] Until the early 2000s, the canonical biomarker for FD was globotriaosylceramide (GL3), the major accumulation substrate resulting from α-Gal inactivation. Increasing uncertainty about the role of GL3 in disease pathology and the lack of correlation between GL3 and α-Gal activity or between GL3 and disease symptoms led to a need for more robust diagnostic and prognostic biomarkers. α-Gal activity itself was thought to have diagnostic potential in male patients, but its diagnostic sensitivity in female patients was low. In the late 2000s, globotriaosylsphingosine (lyso-GL3), the deacylated form of GL3, was investigated as a biomarker for FD and was found to have superior sensitivity compared to GL3 and α-Gal activity. Lyso-GL3 is now an important diagnostic biomarker and is often used to complement full genetic analysis in determining FD cases. It is also useful for monitoring disease progression and treatment.

[0013] Using a similar rationale, biomarker-based diagnostic tests for GD have been developed. This disease is characterized by the accumulation of glucosylceramide (GL1) and its deacylated form, glucosylsphingosine (lyso-GL1), in the body due to a deficiency in GCase. Lyso-GL1 levels, β-glucosidase activity, and gene GBA sequencing are currently the most common diagnostic and prognostic indicators for GD. In the case of lyso-GL1, several studies suggest that its pathological involvement correlates with disease burden and clinical severity. The proteins chitotriosidase and CCL18 have also been identified as biomarkers for GD, but are not commonly used in clinical practice.

[0014] Mucopolysaccharidosis type I (MPS I), also known as Hurler syndrome, is characterized by a deficiency in the enzyme alpha-L-iduronidase (IDUA), which leads to the accumulation of its catabolic substrates, dermatan sulfate and heparan sulfate, in lysosomes. In most clinical settings, the first step in diagnosing MPS I is IDUA enzyme activity. If enzyme activity is decreased, a second-stage test for mucopolysaccharidosis is performed on blood or blood spots to check for elevated dermatan sulfate and heparan sulfate. Along with diagnosis, dermatan sulfate and heparan sulfate levels are the standard for monitoring treatment. Similar assays for specific GAG levels exist and can be used to diagnose and / or monitor other mucopolysaccharidoses.

[0015] However, for many LSDs, there are no clinically validated biomarkers for diagnosis or monitoring of treatment progress. Therefore, there is an urgent need to develop biomarkers for the early detection of common LSDs, as well as new and improved methods for characterizing and monitoring specific LSDs. Summary of the Invention [Means for solving the problem]

[0016] This application demonstrates that the cell surface glycoprotein CD63 can be used as a biomarker for diagnosing and / or monitoring multiple LSDs. Thus, CD63 may serve as a common biomarker for LSD pathogenesis. Not only does CD63 function as a biomarker for disease progression by itself, but measuring CD63 levels can also be used in conjunction with other clinical measures to diagnose and / or monitor specific disease states.

[0017] CD63 was first detected as a marker of platelet activation, but its exact function is unknown. It is localized to the membranes of melanosomes and platelet dense bodies. Some cells, such as activated basophils and proliferating mast cells, are enriched in CD63, and CD63 is often used in cell biology as a marker for multivesicular bodies. It is also used as a marker for extracellular vesicles released from either multivesicular bodies or the plasma membrane. CD63 can also be used as a cellular marker, for example, to quantify platelet size, number, or volume (see, e.g., International Publication No. 2004 / 088322, cited above). CD63 is heavily glycosylated, which may protect it from lysosomal enzyme degradation. When the structural gene and cDNA for CD63 were first isolated and sequenced, it was found to be identical to ME491, an antigen associated with early melanoma cells. CD63 also appears to be identical to granulophysin, a protein associated with platelet dense granules. Due to its known uses, there are many commercial kits available for its detection and quantification in biological samples, which are typically based on ELISAs using specific anti-CD63 antibodies.

[0018] Without wishing to be bound by theory, based on the observations described in this example, it is believed that CD63 may function as a circulating biomarker for multiple lysosomal storage diseases, particularly those that share the same glycosphingolipid pathway. Furthermore, because CD63 levels in patients may be more stable over time than levels of conventional biomarkers for LSDs (e.g., GD), CD63 represents a particularly advantageous biomarker for monitoring certain LSDs.

[0019] Thus, a first aspect provides a method of diagnosing a subject as suffering from or at risk of suffering from a lysosomal storage disease (LSD), the method comprising measuring the level of CD63 in a sample from the subject, wherein CD63 is the only biomarker used in the method.

[0020] In embodiments, the subject is diagnosed with or at risk of developing a lysosomal storage disorder, including Fabry disease, Gaucher disease, MPS I, MPS II, and MPS III.

[0021] Another aspect provides a method for detecting or diagnosing lysosomal dysfunction in a subject, the method comprising measuring the level of CD63 in a sample from the subject, wherein CD63 is the only biomarker used in the method.

[0022] Another aspect provides a method for detecting or diagnosing abnormal glycosphingolipid processing in a subject, the method comprising measuring the level of CD63 in a sample from the subject, wherein CD63 is the only biomarker used in the method.

[0023] Another aspect provides a method of generating quantitative data about a subject, the method comprising determining a level of a single biomarker in a sample from the subject, wherein the biomarker is CD63.

[0024] In embodiments, the subject has not been previously diagnosed with an LSD and / or has not been evaluated for risk factors associated with lysosomal dysfunction or abnormal glycosphingolipid processing. In embodiments, the sample comprises (e.g., consists of) a blood fraction selected from plasma and serum.

[0025] In embodiments, a subject is diagnosed as having or at risk of having a lysosomal storage disease, or is diagnosed as having or at risk of having a lysosomal storage disease, if the level of CD63 is greater than a control value, where the control value is measured as the CD63 level at an earlier time point from the same subject, or in a sample obtained from one or more healthy subjects.

[0026] Another aspect provides the use of CD63 as a biomarker in diagnosing a lysosomal storage disease in a subject, in detecting or diagnosing lysosomal dysfunction in a subject, or in detecting or diagnosing abnormal glycosphingolipid processing in a subject, wherein CD63 is used as the sole biomarker in said detection or diagnosis.

[0027] Another aspect provides a method of diagnosing Fabry disease in a subject suspected of being at risk of having Fabry disease, the method comprising measuring the level of CD63 in a sample from the subject.

[0028] In embodiments, a subject is suspected of being at risk for Fabry disease as a result of exhibiting one or more of the following: a family history of Fabry disease, fatigue, pain, lens or corneal opacities, vortex keratopathy, angiokeratoma, dyspnea, palpitations, edema, renal disease, myocardial dysfunction, cardiac conduction abnormalities with shortened PR intervals, cardiac arrhythmias, dizziness, headache, crossed diplopia, dysarthria, hemiataxia, transient ischemic attack, premature stroke, and dementia. In embodiments, a subject is diagnosed with Fabry disease if the level of CD63 measured in a sample from the subject is greater than a control value, where the control value is determined as the CD63 level in a sample taken from one or more healthy subjects. In embodiments, a subject is diagnosed with Fabry disease if the level of CD63 measured in a sample from the subject is at least 100% greater than the control value.

[0029] Another aspect provides a method of generating quantitative data about a subject, the method comprising (e.g., consisting of) determining a level of CD63 in a sample from a subject, the subject having or suspected of having Fabry disease.

[0030] Another aspect provides the use of CD63 as a biomarker in the diagnosis of Fabry disease in a subject suspected of being at risk of suffering from Fabry disease.

[0031] Another aspect provides the use of CD63 as a biomarker to improve methods of diagnosing Fabry disease in a subject, optionally wherein CD63 is used as a biomarker together with GL3, lyso-GL3, and / or α-Gal activity.

[0032] Another embodiment provides a method of treating a subject diagnosed with Fabry disease by the methods defined below, wherein the treatment comprises administering to the subject one or more therapeutic treatments for Fabry disease.

[0033] Another aspect provides a method of treating Fabry disease in a patient in need thereof, the patient having a higher than normal plasma CD63 level, the method comprising administering to the subject an effective amount of a therapeutic treatment for Fabry disease.

[0034] Another aspect provides a method for generating quantitative data for a subject, the method comprising: (a) determining a level of CD63 in a sample from the subject; (b) determining whether the level of CD63 is greater than a control value, where the control value is measured as the CD63 level in a sample taken from one or more healthy subjects; and (c) applying one or more therapeutic treatments to the subject if the level of CD63 in the sample is greater than the control value.

[0035] In embodiments, the one or more therapeutic treatments include (e.g., consist of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy. In embodiments, the treatment includes administering benglustat or migalastat, e.g., benglustat, to the subject. In other embodiments, the treatment includes administering recombinant α-galactosidase, e.g., agalsidase beta, to the subject.

[0036] Another aspect provides a therapeutic agent for the treatment of Fabry disease in a subject, the subject having been diagnosed with Fabry disease by the previously defined methods.

[0037] In embodiments, the one or more therapeutic treatments include (e.g., consist of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy. In embodiments, the treatment includes administering benglustat or migalastat, e.g., benglustat, to the subject. In other embodiments, the treatment includes administering recombinant α-galactosidase, e.g., agalsidase beta, to the subject.

[0038] Another aspect provides a method of monitoring the progression of Fabry disease in a subject diagnosed with Fabry disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is taken from the subject after the first sample is taken from the subject; (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample; and (d) determining that the subject's Fabry disease has become more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample; determining that the subject's Fabry disease has not progressed if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample; and determining that the subject's Fabry disease is in remission if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0039] Another aspect provides a method for generating quantitative data for a subject diagnosed with Fabry disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; and (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

[0040] In embodiments, the sample is a blood sample, for example a plasma sample.

[0041] Another aspect provides a method for monitoring the progress of treatment for Fabry disease in a subject diagnosed with Fabry disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering therapeutic treatment for Fabry disease to the subject; (c) measuring the level of CD63 in a second sample from the subject, the second sample being taken from the subject after the therapeutic treatment has been administered; and (d) determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0042] Another aspect provides a method of treating or preventing the onset or progression of Fabry disease in a subject assessed to be at risk for Fabry disease, the method comprising: (a) obtaining a first biological sample from the subject and analyzing the sample for CD63 concentration; (b) initiating a course of therapeutic treatment for Fabry disease in the subject if the CD63 concentration is above the control value; and, optionally, (c) obtaining a second biological sample from the subject after treating the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0043] Another aspect provides a method for adjusting the dosage of a therapeutic treatment for Fabry disease in a subject receiving said therapeutic treatment, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is taken from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample.

[0044] Another aspect provides a method for generating quantitative data for a subject having Fabry disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; and (b) determining the level of CD63 in a second sample from the subject after the subject has been administered a therapeutic treatment for Fabry disease.

[0045] In embodiments, the dosage of the therapeutic treatment should be increased if the level of CD63 determined in step (b) is substantially the same as or greater than the level of CD63 determined in step (a).

[0046] In embodiments, the therapeutic treatment comprises (e.g., consists of) substrate inhibition therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment comprises administering benglustat or migalastat, e.g., benglustat, to the subject. In other embodiments, the treatment comprises administering recombinant α-galactosidase, e.g., agalsidase beta, to the subject.

[0047] In embodiments, the second or subsequent sample is taken from the subject at least 8 weeks after the start of therapeutic treatment.

[0048] Another aspect provides the use of CD63 as a biomarker for monitoring the progression of Fabry disease in a subject diagnosed with Fabry disease, for monitoring the progress of a treatment for Fabry disease, or for adjusting the dosage of a therapeutic treatment for Fabry disease in a subject diagnosed with Fabry disease.

[0049] Another aspect provides a method of diagnosing Gaucher disease in a subject suspected of being at risk for Gaucher disease, the method comprising measuring the level of CD63 in a sample from the subject.

[0050] In embodiments, a subject is suspected of being at risk for Gaucher disease as a result of exhibiting one or more of the following: a family history of Gaucher disease, hepatomegaly and splenomegaly, pain, osteoporosis, skin pigmentation, pancytopenia, neurological symptoms, and Parkinson's disease. In embodiments, a subject is diagnosed with Gaucher disease if the level of CD63 measured in a sample from the subject is greater than a control value, where the control value is measured as the CD63 level in a sample taken from one or more healthy subjects. In embodiments, a subject is diagnosed with Gaucher disease if the level of CD63 measured in a sample from the subject is at least about 100% greater than the control value.

[0051] Another aspect provides a method for generating quantitative data for a subject, the method comprising (e.g., consisting of) determining a level of CD63 in a sample from a subject, the subject having or suspected of having Gaucher disease.

[0052] Another aspect provides the use of CD63 as a biomarker in the diagnosis of Gaucher disease in a subject suspected of being at risk for Gaucher disease.

[0053] Another aspect provides the use of CD63 as a biomarker to improve a method of diagnosing Gaucher disease in a subject, optionally wherein CD63 is used as a biomarker together with GL1, lyso-GL1, and / or β-glucosidase (GCas) activity.

[0054] Another aspect provides a method of treating a subject diagnosed with Gaucher disease by the methods previously defined, wherein the treatment comprises administering to the subject one or more therapeutic treatments for Gaucher disease.

[0055] Another aspect provides a method of treating Gaucher disease in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level, the method comprising administering to the subject an effective amount of a therapeutic treatment for Gaucher disease.

[0056] Another aspect provides a method for generating quantitative data for a subject, the method comprising: (a) determining a level of CD63 in a sample from the subject; (b) determining whether the level of CD63 is greater than a control value, where the control value is measured as the CD63 level in a sample taken from one or more healthy subjects; and (c) applying one or more therapeutic treatments to the subject if the level of CD63 in the sample is greater than the control value.

[0057] In embodiments, the one or more therapeutic treatments include (e.g., consist of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy. In embodiments, the treatment includes administering benglustat, eliglustat, or miglustat to the subject. In embodiments, the treatment includes administering recombinant glucocerebrosidase, e.g., imiglucerase, to the subject.

[0058] Another aspect provides a therapeutic agent for the treatment of Gaucher disease in a subject, wherein the subject has been diagnosed with Gaucher disease by the previously defined methods.

[0059] In embodiments, the therapeutic agent is a therapeutic treatment as previously defined.

[0060] Another aspect provides a method of monitoring the progression of Gaucher disease in a subject diagnosed with Gaucher disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, where the second sample is taken from the subject after the first sample is taken from the subject; (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample; and (d) determining that the subject's Gaucher disease has become more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample; determining that the subject's Gaucher disease has not progressed if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample; and determining that the subject's Gaucher disease is in remission if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0061] Another aspect provides a method for generating quantitative data for a subject diagnosed with Gaucher disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; and (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

[0062] In embodiments, the sample is a blood sample, for example a plasma sample.

[0063] Another aspect provides a method for monitoring the progress of treatment for Gaucher disease in a subject diagnosed with Gaucher disease, the method comprising: (A) measuring the level of CD63 in a first sample from the subject; (b) administering a therapeutic treatment for Gaucher disease to the subject; (c) measuring the level of CD63 in a second sample, the second sample being taken from the subject after the therapeutic treatment has been administered; and (d) determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0064] Another aspect provides a method for treating or preventing the onset or progression of Gaucher disease in a subject assessed to be at risk for Gaucher disease, the method comprising: (a) obtaining a first biological sample from the subject and analyzing the sample for CD63 concentration; (b) initiating a course of therapeutic treatment for Gaucher disease in the subject if the CD63 concentration is above the control value; and, optionally, (c) after treating the subject, obtaining a second biological sample from the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0065] Another aspect provides a method for adjusting the dosage of a therapeutic treatment for Gaucher disease in a subject receiving said therapeutic treatment, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, where the second sample is taken from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample.

[0066] Another aspect provides a method for generating quantitative data about a subject with Gaucher disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; and (b) determining the level of CD63 in a second sample from the subject after the subject has been administered a therapeutic treatment for Gaucher disease.

[0067] In embodiments, the dosage of the therapeutic treatment should be increased if the CD63 level determined in step (b) is substantially the same as or greater than the CD63 level determined in step (a). In embodiments, the therapeutic treatment comprises (e.g., consists of) substrate inhibition therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment comprises administering benglustat, eliglustat, or miglustat to the subject. In embodiments, the treatment comprises administering recombinant glucocerebrosidase, e.g., imiglucerase, to the subject. In embodiments, the second or subsequent sample is taken from the subject at least 8 weeks after the start of the therapeutic treatment.

[0068] Another aspect provides the use of CD63 as a biomarker for monitoring the progression of Gaucher disease in a subject diagnosed with Gaucher disease, or for monitoring the progress of a treatment for Gaucher disease in a subject diagnosed with Gaucher disease, or for adjusting the dosage of a therapeutic treatment for Gaucher disease.

[0069] Another aspect provides a method of diagnosing MPS in a subject suspected of being at risk of suffering from MPS, the method comprising measuring the level of CD63 in a sample from the subject.

[0070] In embodiments, a subject is suspected of being at risk for MPS as a result of exhibiting one or more of the following: a family history of MPS, macrocephaly, hearing loss, corneal opacities, malocclusion, rigidity, hip dysplasia, clawed hands, joint laxity, valve thickening, left ventricular hypertrophy, recurrent respiratory infections, obstructive airway disease, hepatomegaly / splenomegaly, umbilical / inguinal hernia, growth retardation, ventricular dilation, enlarged perivascular spaces, hyperactive or aggressive behavior, leukocyte dysgranulogenesis, hydrops fetalis, and proteinuria. In embodiments, a subject is diagnosed with MPS if the level of CD63 measured in a sample from the subject is greater than a control value, where the control value is determined as the CD63 level in a sample taken from one or more healthy subjects. In embodiments, a subject is diagnosed with MPS if the level of CD63 measured in a sample from the subject is at least 100% greater than the control value.

[0071] Another aspect provides a method of generating quantitative data about a subject, the method comprising (e.g., consisting of) determining a level of CD63 in a sample from a subject, the subject having or suspected of having MPS.

[0072] Another aspect provides the use of CD63 as a biomarker in the diagnosis of MPS in subjects suspected of being at risk of suffering from MPS.

[0073] Another aspect provides the use of CD63 as a biomarker to improve the diagnosis of MPS in a subject, optionally wherein CD63 is used as a biomarker together with one or more glycosaminoglycans (GAGs) or glycans.

[0074] Another embodiment provides a method of treating a subject diagnosed with MPS by the methods previously defined, wherein the treatment comprises administering to the subject one or more therapeutic treatments for MPS.

[0075] Another aspect provides a method of treating MPS in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level, the method comprising administering to the subject an effective amount of a therapeutic treatment for MPS.

[0076] Another aspect provides a method for generating quantitative data for a subject, the method comprising: (a) determining a level of CD63 in a sample from the subject; (b) determining whether the level of CD63 is greater than a control value, where the control value is measured as the CD63 level in a sample taken from one or more healthy subjects; and (c) applying one or more therapeutic treatments to the subject if the level of CD63 in the sample is greater than the control value.

[0077] In embodiments, the one or more therapeutic treatments comprise (e.g., consist of) enzyme replacement therapy, gene therapy, and / or hematopoietic stem cell transplantation. In embodiments, the treatment comprises (e.g., consists of) enzyme replacement therapy with α-L-iduronidase, iduronidase-2-sulfatase, or heparan-N-sulfatase.

[0078] Another embodiment provides a therapeutic agent for the treatment of MPS in a subject, wherein the subject has been diagnosed with MPS by the previously defined methods.

[0079] In embodiments, the therapeutic agent is a therapeutic treatment as previously defined.

[0080] Another aspect provides a method for monitoring the progression of MPS in a subject diagnosed with MPS, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, where the second sample is obtained from the subject after the first sample is obtained from the subject; (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample; and (d) determining that the subject's MPS has become more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample; determining that the subject's MPS has not progressed if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample; and determining that the subject's MPS is in remission if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0081] Another aspect provides a method of generating quantitative data for a subject diagnosed with MPS, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; and (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

[0082] In embodiments, the sample is a blood sample, for example a plasma sample.

[0083] Another embodiment provides a method for monitoring the progress of treatment for MPS in a subject diagnosed with MPS, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering a therapeutic treatment for MPS to the subject; (c) measuring the level of CD63 in a second sample from the subject, the second sample being taken from the subject after the therapeutic treatment has been administered; and (d) determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0084] Another aspect provides a method of treating or preventing the onset or progression of MPS in a subject assessed to be at risk for MPS, the method comprising: (a) obtaining a first biological sample from the subject and analyzing the sample for CD63 concentration; (b) initiating a course of therapeutic treatment for MPS in the subject if the CD63 concentration is above a control value; and, optionally, (c) after treating the subject, obtaining a second biological sample from the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0085] Another aspect provides a method for adjusting the dosage of a therapeutic treatment for MPS in a subject receiving said therapeutic treatment, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is taken from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample.

[0086] Another aspect provides a method for generating quantitative data about a subject having MPS, the method comprising: (a) determining the level of CD63 in a first sample from the subject; and (b) determining the level of CD63 in a second sample from the subject after administering a therapeutic treatment for MPS to the subject.

[0087] In embodiments, the dosage of the therapeutic treatment should be increased if the CD63 level determined in step (b) is substantially the same as or greater than the CD63 level determined in step (a). In embodiments, the therapeutic treatment comprises (e.g., consists of) enzyme replacement therapy, gene therapy, and / or hematopoietic stem cell transplantation. In embodiments, the treatment comprises (e.g., consists of) enzyme replacement therapy with α-L-iduronidase, iduronidase-2-sulfatase, or heparan-N-sulfatase. In embodiments, the second or subsequent sample is taken from the subject at least 8 weeks after the start of the therapeutic treatment.

[0088] Another aspect provides the use of CD63 as a biomarker for monitoring the progression of MPS in a subject diagnosed with MPS, or for monitoring the progress of a treatment for MPS in a subject diagnosed with MPS, or for adjusting the dosage of a therapeutic treatment for MPS.

[0089] Another aspect provides a kit for detecting or diagnosing a particular lysosomal storage disease in a subject, the kit comprising: (a) means for detecting CD63 in a sample from the subject; and (b) means for detecting one or more biomarkers of the lysosomal storage disease in the sample from the subject.

[0090] In embodiments, the particular lysosomal storage disease is Fabry disease, and the kit comprises (a) means for detecting CD63 in a sample from a subject, and (b) means for detecting one or more biomarkers of Fabry disease in the sample (e.g., means for detecting GL3 and / or lyso-GL3 in the sample).

[0091] In other embodiments, the particular lysosomal storage disease is Gaucher disease, and the kit comprises (a) means for detecting CD63 in a sample from the subject; and (b) means for detecting one or more biomarkers of Gaucher disease in the sample (e.g., means for detecting lyso-GL1 in the sample).

[0092] In yet other embodiments, the lysosomal storage disease is MPS, and the kit comprises (a) means for detecting CD63 in a sample from the subject, and (b) means for detecting one or more biomarkers of MPS in the sample. In embodiments, (i) the lysosomal storage disease is MPS I, and the kit comprises, in part, (b) means for detecting dermatan sulfate and optionally heparan sulfate in the sample, (ii) the lysosomal storage disease is MPS II, and the kit comprises, in part, (b) means for detecting dermatan sulfate and heparan sulfate in the sample, or (iii) the lysosomal storage disease is MPS III, and the kit comprises, in part, (b) means for detecting heparan sulfate in the sample.

[0093] In embodiments, the means for detecting CD63 comprises at least one anti-CD63 antibody.

[0094] Additional features and advantages of the compounds, compositions, and methods disclosed herein will be apparent from the following detailed description. [Brief explanation of the drawings]

[0095] [Figure 1] Figure 1 shows biomarker response to benglustat treatment in a clinical trial over time in patients with Fabry disease compared to healthy controls. The biomarkers analyzed are CD63 (Figure 1A) - displayed as "linear NPX," i.e., normalized protein expression; GL3 (Figure 1B); and lyso-GL3 (Figure 1C). All biomarkers demonstrate a response to treatment. CD63 and lyso-GL3 do not return to control levels over the study period. GL3 shows no separation between baseline and control. [Figure 2]Estimated marginal mean (log2) values of CD63 (Figure 2A), GL3 (Figure 2B), and lyso-GL3 (Figure 2C) are shown for benglustat treatment in Fabry disease, measured over time from baseline to 156 weeks. Horizontal lines above the curves indicate statistical significance, and asterisks indicate the confidence of significance (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). The first significant decrease in CD63 occurs by week 26. [Figure 3] Figure 3 shows intraclass correlation coefficient (ICC) data (10,000 permutations) for measured biomarkers from the Fabry ACT / LTS benglustat Phase II clinical trial. The ICC (Figure 3A) is a measure of inter-patient variability, whereas the ICC residual (Figure 3C) is a measure of intra-patient variability. The distribution intercepts of the ICC are shown in Figure 3B. Each plot shows, from left to right: log(CD63); log(GL3); and log(lyso-GL3). CD63 and lyso-GL3 have comparable intra-subject variability, but both are much lower than GL3. [Figure 4] Probability density functions comparing the distribution of CD63 values (log2) in healthy subjects (left) and diseased and treated Gaucher patients (right) are shown, with sample metadata shown below in Table 2. There is a statistically significant separation between the two groups, with CD63 being upregulated in diseased patients (Wilcoxon test: p=8.89e-10). [Figure 5A]Pairwise spaghetti plots are shown to visualize intra-patient variability of CD63 (Figure 5A), lyso-GL1 (LGL1; Figure 5B), and chitotriosidase (CHITO; Figure 5C) in patients with Gaucher disease. Intra-subject variability is visualized by plotting biomarker changes between adjacent time points, i.e., changes compared to previous measurements. All patients included in the plots have been treated for Gaucher disease for several years and represent biomarker steady state during treatment. Results for each patient are displayed as separate lines (displayed with different line styles). Note that data were not collected for all patients at each time point. Lyso-GL1 and CHITO appear to be more variable than CD63, which is confirmed in Figure 6. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A-C] Correlations between CD63 and other biomarkers are shown. Figures 6A-6C show intraclass correlation coefficient (ICC) data (10,000 permutations) for biomarkers measured from the Gaucher disease cohort outlined in Table 2 below. The ICC (Figure 6A) is a measure of interpatient variability, whereas the ICC residuals (Figure 6C) are a measure of intrapatient variability. The ICC distribution intercepts are shown in Figure 6B. Each plot shows, from left to right: log(CD63); log(lyso-GL1); and log(CHITO). Note that CD63 has significantly lower intrapatient variability than either lyso-GL1 or CHITO. Figure 6D illustrates the correlation in another way: data points show CD63 levels in samples from Gaucher disease patients versus lyso-GL1 levels (light gray circles), CCL18 levels (black triangles), and chitotriosidase activity (dark gray squares). Lines of best fit are shown for lyso-GL1 (lower line, light gray) with r = 0.65 and p < 0.0001; CCL18 (middle line, black) with r = 0.63 and p = 0.0119; and chitotriosidase (upper line, dark gray) with r = 0.78 and p < 0.0001. [Figure 6D] Same as above. [Figure 7]A probability density plot comparing the distribution of CD63 values (log2) in healthy subjects and diseased MPS patients (Figure 7A) is shown, with all sample metadata listed in Table 3 below. The shaded area on the right side of the plot contains data from all three MPS subtypes. The curves within the shaded area on the right side represent the distributions of the different MPS subtypes (indicated by arrows). The statistical difference between the healthy and MPS distributions is shown in Figure 8C. A comparison of CD63 values between male and female MPS disease subjects is also shown (Figure 7B). At α = 0.05, the two distributions are not statistically different (p = 0.0746). [Figure 8A] Results of the Fabry (Figure 8A), Gaucher (Figure 8B), and MPS (Figure 8C) studies analyzed separately for male and female patients (and controls) are shown. P values are provided for each plot. The only instance of p>0.05 is for the female Fabry patient. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9] Figure 1 shows Olink® proteome profiling of a sample from a male adult Fabry disease patient (baseline) compared to a healthy control. Proteins with at least a 1.5-fold change and p<0.05 were considered significant and are shown in grey. CD63 is circled. [Figure 10] Box plots of CD63 levels in CSF samples from healthy controls (leftmost plots) compared with MPS I (middle plots) and MPS II (rightmost plots) patients are shown. [Figure 11A] Figure 11A shows CD63 levels in CSF samples from healthy controls compared to patients with type 3 Gaucher disease (GD3). GD3 samples were collected at three time points: baseline, 26 weeks after treatment with benglustat and imiglucerase, and 52 weeks after treatment. Mean levels are elevated in GD3 patients at baseline compared to healthy controls and decrease over time (Figure 11A). This trend is also seen within individual patients (Figure 11B). [Figure 11B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0096] Specific embodiments of the present disclosure will now be described with reference to preparations and schemes, but it should be understood that such embodiments are illustrative only and merely illustrate a few of the many possible specific embodiments that may represent applications of the principles of the present disclosure. Various changes and modifications will be apparent to those skilled in the art upon consideration of the benefit of this disclosure and are deemed to be within the spirit and scope of the present disclosure as further defined in the appended claims.

[0097] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods, devices, and materials are described herein. All technical and patent publications mentioned herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0098] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Michael R. Green and Joseph Sambrook, Molecular Cloning (4 thed.,Cold Spring Harbor Laboratory Press 2012);the series Ausubel et al.eds.(2007)Current Protocols in Molecular Biology;the series Methods in Enzymology(Academic Press,Inc.,N.Y.);MacPherson et al.,(1991)PCR 1:A Practical Approach(IRL Press at Oxford University Press);MacPherson et al.,(1995)PCR 2:A Practical Approach;Harlow and Lane eds.(1999)Antibodies,A Laboratory Manual;Freshney(2005)Culture of Animal Cells:A Manual of Basic Technique,5 thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0099] All numerical designations (e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges) are approximations that are varied (+) or (-) by increments of, for example, 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0100] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells (including mixtures thereof). Unless otherwise specified or clear from the context, the term "or" as used herein is understood to be inclusive. The term "comprising" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0101] As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements, but do not exclude other elements.

[0102] "Consisting essentially of," when used to define compositions and methods, shall mean excluding other elements of essential importance for the purpose described. Thus, a composition consisting essentially of elements as defined herein will not exclude trace contaminants from isolation and purification methods, pharmaceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, etc.

[0103] "Consisting of" shall mean the exclusion of other ingredients beyond trace elements, as well as the exclusion of substantial method steps for administering the disclosed compositions or process steps for producing the compositions or achieving the intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure. The use of the term "comprising" herein is intended to encompass and disclose corresponding statements in which the term "comprising" is replaced with "consisting essentially of" or "consisting of."

[0104] "Subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, primates, and humans. In embodiments, mammals include horses, dogs, and cats. In embodiments, a mammal is a human.

[0105] "Administering" is defined herein as a means of providing a subject with an agent or a composition containing the agent in a manner that brings the agent into contact with (e.g., present within) the body of a subject. Such administration can be by any route, including, but not limited to, oral administration, transdermal (e.g., vaginal, rectal, or oral mucosal) administration, administration by injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, to the CNS), or administration by inhalation (e.g., oral or nasal). Administration can also involve providing a substance or composition to a portion of the surface of the subject's body, for example, by topical administration to the skin. Pharmaceuticals are, of course, administered in a form appropriate for each administration route.

[0106] "Treating" or "treatment" of a disease includes: (1) preventing the disease, i.e., preventing clinical symptoms of the disease from developing in a patient who may be susceptible to the disease but who has not yet experienced or manifested symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and / or (3) palliating the disease, i.e., causing regression of the disease or its clinical symptoms.

[0107] In particular, the term "suffering from" (or "having") with respect to a lysosomal storage disease refers to a patient or individual who has developed at least some of the pathology characteristic of the disease and / or exhibits one or more symptoms of the disease (e.g., one or more biomarkers characteristic of the disease), regardless of whether they have actually been diagnosed with the disease. A patient may be said to be "at risk for" such a disease if they have the potential to be affected by the disease (e.g., have a history of the disease in their family lineage or have a genetic mutation associated with the disease) but have not yet developed all or some of the pathology characteristic of the disease.

[0108] An "effective amount" or "therapeutically effective amount" is an amount sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or dosages. Such delivery depends on many variables, including the period for which individual dosage units are used, the bioavailability of the therapeutic agent, the route of administration, and the like. However, the specific dosage level of a therapeutic agent for any particular subject will depend on a variety of factors (e.g., the activity of the specific compound employed; the subject's age, weight, general health, sex, and diet; the time of administration; the rate of excretion; the drug combination; and the severity of the particular disorder being treated; and the administration form). Treatment dosages may generally be titrated to optimize safety and efficacy. Typically, dose-effect relationships from in vitro and / or in vivo studies can provide useful guidance initially regarding appropriate dosages for patient administration. Generally, one will desire to administer an amount of compound effective to achieve serum levels commensurate with concentrations found to be effective in vitro. These considerations, as well as effective formulations and administration procedures, are known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat (e.g., ameliorate) one or more symptoms associated with a lysosomal storage disease. For example, oral administration may require a total daily dose of between 0.1 mg and 1000 mg of active agent. The total daily dose may be administered in single or divided doses and, at the physician's discretion, may fall outside of the typical ranges given herein.

[0109] When a compound such as eliglustat or benglustat is referred to herein, it includes the compound itself and its pharmaceutically acceptable salts. For example, a reference to "eliglustat" includes eliglustat hemitartrate, and a reference to "benglustat" includes benglustat malate. Benglustat is (S)-1-azabicyclo[2.2.2]octan-3-yl-N-[2-[2-(4-fluorophenyl)-1,3-thiazol-4-yl]propan-2-yl]carbamate. Eliglustat is N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-hydroxy-3-pyrrolidin-1-ylpropan-2-yl]octanamide.

[0110] If desired, any embodiment (e.g., method) provided herein can be combined with any one or more of the other embodiments (e.g., methods) provided herein.

[0111] The following abbreviations are used herein: α-Gal α-galactosidase A BCA Bicinchoninic Acid BL Baseline BSA Bovine serum albumin CCL18 chemokine (CC motif) ligand 18 cDNA complementary DNA CHITO Chitotriosidase CNS central nervous system CS Chondroitin Sulfate CSF cerebrospinal fluid CV confidence score DNA deoxyribonucleic acid DS Dermatan Sulfate ELISA enzyme-linked immunosorbent assay ERT enzyme replacement therapy ESI electrospray ionization FACS fluorescence-assisted cell sorting FD Fabry disease GAG glycosaminoglycans GBA glucocerebrosidase gene GCase glucocerebrosidase (also known as acid β-glucosidase) GD Gaucher disease GD3 Type 3 Gaucher disease GI gastrointestinal GL1 Glucosylceramide GL3 Globotriaosylceramide GM1 monosialotetrahexosylganglioside GM2 monosialotrihexosylganglioside GM3 monosialodihexosylganglioside HS Heparan sulfate HSCT hematopoietic stem cell transplantation ICC Intraclass Correlation Coefficient IDUA α-L-iduronidase IQ intelligence quotient KS keratan sulfate LCMS Liquid Chromatography Mass Spectrometry LC-MS / MS Liquid Chromatography Tandem Mass Spectrometry LSD Lysosomal storage disorder Lyso-GL1 glucosylsphingosine (also called LGL1) Lyso-GL3 Globotriaosylsphingosine MPS mucopolysaccharidosis (or mucopolysaccharidoses) NCL neuronal ceroid lipofuscinosis(es) NPX normalized protein expression PEA proximity extension assay PBS Phosphate-buffered saline PCR polymerase chain reaction RNA ribonucleic acid SD standard deviation SEM Standard error of the mean SRT substrate suppression therapy Tris Tris(hydroxymethyl)aminomethane UPLC Ultra High Performance Liquid Chromatography WK week

[0112] Methods for diagnosing or detecting lysosomal storage diseases generally Provided herein is a method for diagnosing a subject as suffering from or at risk of suffering from a lysosomal storage disease, comprising measuring the level of CD63 in a sample from the subject. This method has the advantage of being able to detect irregularities in the glycosphingolipid pathway that are common to many LSDs. Thus, the method can provide a general diagnosis of lysosomal storage disorders, i.e., the diagnosis need not be limited to the assessment of a single disease state, but provides a more holistic picture of lysosomal function and glycosphingolipid processing. Accordingly, a related aspect provides a method for detecting or diagnosing lysosomal dysfunction in a subject, comprising measuring the level of CD63 in a sample from the subject. Another related aspect provides a method for detecting or diagnosing abnormal glycosphingolipid processing in a subject, comprising measuring the level of CD63 in a sample from the subject. In embodiments, CD63 is the only biomarker used in the method.

[0113] Other related aspects provide for the use of CD63 as a biomarker in diagnosing a lysosomal storage disease in a subject. Related aspects provide for the use of CD63 as a biomarker in detecting or diagnosing lysosomal dysfunction in a subject. Related aspects provide for the use of CD63 as a biomarker in detecting or diagnosing abnormal glycosphingolipid processing in a subject. In embodiments, CD63 is used as the sole biomarker in such detection and / or diagnosis.

[0114] Alternatively, these aspects provide a method for generating quantitative data about a subject, the method comprising determining a level of a biomarker in a sample from the subject, wherein the biomarker is CD63. In embodiments, the method comprises determining the level of a single biomarker, namely, CD63.

[0115] Although the subject being evaluated may be identified as at risk for or suspected of having an LSD, for example, by considering family history or clinical observation, it is contemplated that the method will be performed on subjects for whom risk factors for lysosomal diseases or dysfunctions have not yet been evaluated. Thus, in embodiments, the subject has not previously been diagnosed with an LSD and / or has not been evaluated for risk factors associated with lysosomal dysfunction or abnormal glycosphingolipid processing. Such risk factors include, but are not limited to, subjects with parents who have a genetic mutation known to cause an LSD, such as, for example, Ashkenazi Jewish, Finnish, Asian, or Dutch parents, or related parents.

[0116] As noted above, the present methods can provide a general diagnosis of lysosomal storage disorders. Without wishing to be bound by theory, the methods may be used to treat a variety of conditions, including Fabry disease, Krabbe disease, Gaucher disease (e.g., types 1, 2, and 3), Niemann-Pick disease (e.g., types A, B, and C), metachromatic leukodystrophy, Farber disease, Krabbe disease, galactosialidosis, Schindler disease, GM1 gangliosidosis, GM2 gangliosidosis (e.g., AB variant, Sandhoff disease, and Tay-Sachs disease), GM3 gangliosidosis, lysosomal acid lipase deficiency, Wolman disease, cholesterol ester storage disease, multiple sulfatase deficiency, Pompe disease, Danon disease, Salla disease, alpha-mannosidosis, beta-mannosidosis, aspartylglucosaminuria, fucosidosis, MPS I (e.g., Hurler syndrome, Scheie syndrome, and Hurler-Scheie syndrome), MPS II (e.g., Hunter syndrome), MPS III (e.g., Hunter syndrome), MPS IV ... MPS III (e.g., Sanfilippo syndrome types A, B, C, and D), MPS IV (e.g., Morquio syndrome types A and B), MPS VI (e.g., Maroteaux-Lamy syndrome), MPS VII (e.g., Sly syndrome), MPS It is hypothesized that the diagnostic methods may be suitable for diagnosing a broad range of diseases, including lysosomal storage diseases, including type IX (e.g., hyaluronidase deficiency), mucolipidoses (e.g., sialidosis, inclusion cell disease, pseudo-Hurler polydystrophy / phosphotransferase deficiency, and mucolipidin 1 deficiency), and neuronal ceroid lipofuscinoses (e.g., Santavori-Hartzia disease / pediatric NCL, Jansky-Bielschotsky disease / late-onset pediatric NCL, Batten-Spielmeyer-Voigt disease / juvenile NCL, Kuchs disease / adult NCL, Finnish variant / type 5, late-onset pediatric variant / type 6, type 7, Nordic epilepsy / Turkish late-onset pediatric / type 8, German / Serbian late-onset pediatric / type 9, and congenital cathepsin D deficiency). Thus, in embodiments, a subject is diagnosed with or at risk of developing a lysosomal storage disease selected from the aforementioned conditions.In other embodiments, the subject is diagnosed with Fabry disease, Krabbe disease, Gaucher disease (e.g., types 1, 2, and 3), metachromatic leukodystrophy, Farber disease, Krabbe disease, galactosialidosis, Schindler disease, GM1 gangliosidosis, GM2 gangliosidosis (e.g., AB variant, Sandhoff disease, and Tay-Sachs disease), GM3 gangliosidosis, lysosomal acid lipase deficiency, Wolman disease, cholesterol ester storage disease, multiple sulfatase deficiency, Pompe disease (e.g., infantile-onset Pompe disease), Danon disease, Salla disease, alpha-mannosidosis, beta-mannosidosis, aspartylglucosaminuria, fucosidosis, MPS I (e.g., Hurler syndrome, Scheie syndrome, and Hurler-Scheie syndrome), MPS II (e.g., Hunter syndrome), MPS III (e.g., urinary tract infection), or a combination of these. MPS III (e.g., Sanfilippo syndrome types A, B, C, and D), MPS IV (e.g., Morquio syndrome types A and B), MPS VI (e.g., Maroteaux-Lamy syndrome), MPS VII (e.g., Sly syndrome), MPS The patient has or is at risk of having a lysosomal storage disease selected from type IX (e.g., hyaluronidase deficiency), mucolipidoses (e.g., sialidosis, inclusion cell disease, pseudo-Hurler polydystrophy / phosphotransferase deficiency, and mucolipidin 1 deficiency), and neuronal ceroid lipofuscinoses (e.g., Santavori-Hartzia disease / pediatric NCL, Jansky-Bielschotsky disease / late-onset pediatric NCL, Batten-Spielmeyer-Voigt disease / juvenile NCL, Kuchs disease / adult NCL, Finnish variant / type 5, late-onset pediatric variant / type 6, type 7, Nordic epilepsy / Turkish late-onset pediatric / type 8, German / Serbian late-onset pediatric / type 9, and congenital cathepsin D deficiency). In yet other embodiments, the subject is diagnosed with or at risk of having Fabry disease, Gaucher disease, MPS I, MPS II, and MPS III. In embodiments, the Gaucher disease is Gaucher disease type 1, type 2, or type 3 (e.g., type 3).

[0117] The sample from a subject in which CD63 is measured or determined can be any sample containing lysosomes, exosomes, and / or other cellular fractions in which CD63 may accumulate after impairment of lysosomal function. The sample can consist essentially of one sample type (e.g., one tissue or liquid type) or can consist of multiple sample types (e.g., several tissue and / or liquid types). Sample types can be selected from, for example, blood, blood fractions, urine, cerebrospinal fluid, saliva, lymphatic fluid, skin tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Most conveniently, the sample is of a type that can be obtained by a relatively non-invasive method. Thus, in embodiments, the sample from a subject comprises (e.g., consists of) blood, blood fractions, and / or urine. In certain embodiments, the sample from a subject comprises (e.g., consists of) a blood fraction selected from plasma and serum. In other embodiments, the sample from a subject comprises (e.g., consists of) CSF.

[0118] When diagnosing lysosomal storage diseases or detecting lysosomal dysfunction or abnormal glycosphingolipid processing, the level of CD63 in a sample is typically compared to a control value to determine whether it is within a normal (e.g., healthy) range or outside the normal range. Determining the normal range value for any given sample type is within the capabilities of those skilled in the art. As described in detail herein, detection and quantification of CD63 in a sample is typically performed using an immunoassay such as an ELISA, although other methods (e.g., as described herein) may also be used. There are several commercially available sources for suitable ELISA kits, such as those mentioned below and in the Examples.

[0119] In an embodiment, the method includes measuring the level of CD63 in a sample from the subject (i.e., the test sample level) and comparing the level to a control value; if the test sample level is greater than the control value, it is considered to be outside the normal range. In one embodiment, the control value is a baseline CD63 level previously measured in the same subject (i.e., a previous sample from the subject), e.g., a baseline CD63 level measured at least one month before obtaining the test sample from the subject, e.g., a baseline CD63 level measured at least 2, 3, 6, 9, 12, 18, or 24 months before obtaining the test sample from the subject. The control value may correspond to the CD63 level measured in a single previous sample from the subject, or may be the average of values from multiple previous samples. In an alternative embodiment, the control value is the CD63 level in a sample taken from a healthy subject (or the average value for a cohort of healthy subjects), i.e., from subjects who are not affected by and not at risk of developing a lysosomal storage disease as described above. In an embodiment, the healthy subject is matched to the subject being evaluated, e.g., by age and / or gender. In certain embodiments, the control value corresponds to the level of CD63 in the same type of sample being assessed in the method, e.g., a sample obtained, processed, and / or stored in the same manner as the test sample. For example, when the method utilizes a plasma sample, the control value typically corresponds to the level of CD63 in a control plasma sample, e.g., a previous plasma sample taken from the same subject or a plasma sample obtained from one or more healthy subjects.

[0120] In embodiments where the control value for CD63 concentration is a point value (or average, e.g., mean value), the CD63 level in the test sample is considered to be outside the normal range if it is numerically greater than the control value. For example, it may be at least about 5% greater than the control value, e.g., at least about 7.5%, 10%, 12.5%, 15%, 20% greater than the control value. It may also be 25%, 30%, 40%, 50%, 75%, or 100% greater. In particular, it may be at least about 125% greater than the control value, e.g., at least about 150%, 175%, 200%, 250%, 300%, 350%, 400%, or 500% greater than the control value. In embodiments, the CD63 level in the test sample is about 200% to about 700% greater than the control value (e.g., about 300% to about 500% greater, or about 350% to about 450% greater).

[0121] In embodiments where the control value for CD63 concentration is, for example, a range of values explained by some variation around the mean, the CD63 level in the test sample is considered to be outside the normal range if it is more than about 1 standard error above the mean, e.g., more than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, or 3.0 standard errors above the mean. In other embodiments (e.g., where the patient is female and / or suspected of having or being at risk for Fabry disease), the CD63 level in the test sample is considered to be outside the normal range if it is more than about 0.25 standard errors above the mean, e.g., more than about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 standard errors above the mean.

[0122] Thus, in embodiments, there is provided a method of diagnosing a subject as having or at risk of having a lysosomal storage disease, the method comprising: - measuring the level of CD63 in a test sample (e.g., a plasma sample) from the subject; - comparing the level measured in the test sample with a control value, where the control value is measured as the CD63 level in a sample (e.g., a plasma sample) taken from the same subject, at an earlier time point, or from one or more healthy subjects; - diagnosing the subject as suffering from or at risk of suffering from a lysosomal storage disease if the level measured in the test sample is greater than the control value.

[0123] In another embodiment, a method for detecting or diagnosing lysosomal dysfunction in a subject is provided, the method comprising: - measuring the level of CD63 in a test sample (e.g., a plasma sample) from the subject; - comparing the level measured in the test sample with a control value, where the control value is measured as the CD63 level in a sample (e.g., a plasma sample) taken from the same subject, at an earlier time point, or from one or more healthy subjects; - detecting or diagnosing lysosomal dysfunction in the subject if the level measured in the test sample is greater than the control value.

[0124] In another embodiment, a method for detecting or diagnosing aberrant glycosphingolipid processing in a subject is provided, the method comprising: - measuring the level of CD63 in a test sample (e.g., a plasma sample) from the subject; - comparing the level measured in the test sample with a control value, where the control value is measured as the CD63 level in a sample (e.g., a plasma sample) taken from the same subject, at an earlier time point, or from one or more healthy subjects; - detecting or diagnosing abnormal glycosphingolipid processing in the subject if the level measured in the test sample is greater than the control value.

[0125] The above embodiments may optionally include obtaining a test sample from a subject, for example, taking a blood sample, and optionally separating the blood sample into its fractions (e.g., a plasma sample).

[0126] Methods for diagnosing, treating, and monitoring certain disease conditions As detailed above, CD63 is not only useful for the detection and diagnosis of lysosomal dysfunction and abnormal glycosphingolipid processing, but can also be used as a biomarker in the diagnosis, monitoring, and therapeutic treatment of certain lysosomal storage diseases.

[0127] Thus, in one embodiment, a method for diagnosing a particular lysosomal storage disease (e.g., a condition defined herein) in a subject is provided, the method comprising measuring the level of CD63 in a sample from the subject. A related embodiment provides the use of CD63 as a biomarker in diagnosing a particular lysosomal storage disease in a subject. Viewed alternatively, these embodiments provide methods for generating quantitative data about a subject who may be suspected of being at risk for a particular lysosomal storage disease, the method comprising determining the level of CD63 in a sample from the subject. A related embodiment provides the use of CD63 as a biomarker to improve methods for diagnosing a particular lysosomal storage disease in a subject.

[0128] It will be understood that when the methods are directed to a specific condition, these embodiments typically rely on additional measurements other than CD63 levels in a test sample from a subject. For example, the methods may be performed on a subject already suspected of being at risk for a particular LSD and / or may use other biomarkers characteristic of the condition in question. A subject may be considered at risk for a disease through the identification of certain risk factors, such as a family history of the disease, genetic testing, analysis of other characteristic biomarkers, or clinical symptoms. In this case, the CD63 level in a sample from a subject is typically measured after the risk factors have been assessed, thereby already considering the subject at risk for the disease, and the measurement of CD63 serves to confirm the diagnosis of lysosomal dysfunction and thus the disease in the subject. Alternatively, the measurement of CD63 levels in a sample from a subject may be used together with one or more other characteristic measurements of the disease, for example, as part of a panel of biomarkers used for diagnostic purposes. In this case, CD63 measurement can help improve diagnostic accuracy, e.g., by reducing the rate of false positives and / or increasing the rate of true positives in the population of test subjects (compared to corresponding diagnostic methods in which CD63 levels are not taken into account).

[0129] This diagnosis of a specific lysosomal storage disease in a subject can be used to prescribe a therapeutic treatment for the disease. Thus, in one embodiment, a method is provided for treating a subject diagnosed with a specific lysosomal storage disease by the previously defined method, wherein the treatment comprises administering one or more therapeutic treatments for the disease to the subject. A related embodiment provides a therapeutic agent for treating a specific lysosomal storage disease in a subject, wherein the subject has been diagnosed with the disease by the previously defined method. Also provided is a method for generating quantitative data for a subject, the method comprising (e.g., consisting of) (a) determining the level of CD63 in a sample from the subject; (b) determining whether the CD63 level is greater than a control value (e.g., the control value is measured as the CD63 level in a sample taken from one or more healthy subjects); and (c) administering one or more therapeutic treatments for the specific lysosomal storage disease to the subject if the CD63 level in the sample is greater than the control value. In embodiments, the therapeutic treatment (e.g., the administered agent) comprises (e.g., consists of) a type of therapy generally known to treat the disease in question, such as substrate inhibition therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy.

[0130] CD63 levels can also be used to monitor the progression of a particular lysosomal storage disease, for example, to monitor the natural time course of the disease (e.g., to assess the appropriate time to initiate therapeutic intervention) or to monitor the effect of therapeutic intervention on the disease (e.g., as a guide for dosage adjustments, to assess the efficacy of treatment, etc.).

[0131] Thus, in one aspect, a method is provided for monitoring the progression of a particular lysosomal storage disease in a subject diagnosed with the disease, the method comprising comparing the level of CD63 in a first sample from the subject with the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the first sample was obtained from the subject. Typically, the method will determine that the disease is becoming more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample, or that the subject's disease is not progressing if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample, or that the disease is in remission if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. A related aspect provides the use of CD63 as a biomarker for monitoring the progression of a particular lysosomal storage disease in a subject diagnosed with the disease. Viewed another way, these embodiments provide a method for generating quantitative data about a subject diagnosed with a particular lysosomal storage disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; and (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

[0132] Another aspect provides a method for monitoring the progress of treatment for a specific lysosomal storage disease in a subject diagnosed with the disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering to the subject a therapeutic treatment (e.g., a treatment defined herein) for the specific lysosomal storage disease; and (c) measuring the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the therapeutic treatment. Typically, the method further comprises step (d) of determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. In embodiments, the dosage of the therapeutic treatment should be increased if the level of CD63 determined in step (c) is substantially the same as or greater than the level of CD63 determined in step (a). A related aspect provides the use of CD63 as a biomarker for monitoring the progress of treatment for a specific lysosomal storage disease in a subject diagnosed with the disease. Viewed another way, these aspects provide a method for generating quantitative data for a subject having a particular lysosomal storage disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; and (b) determining the level of CD63 in a second sample from the subject after administering a therapeutic treatment for the disease to the subject. In embodiments, the dosage of the therapeutic treatment should be increased if the level of CD63 determined in step (b) is substantially the same as or greater than the level of CD63 determined in step (a).

[0133] Another aspect provides a method of treating a lysosomal storage disease in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level, e.g., a level at least about 0.25 standard deviations (e.g., at least about 0.5, 0.75, 1, or 1.5 standard deviations) below the mean value of a population of healthy subjects. In embodiments, the method of treatment comprises administering to the patient an effective amount of a therapeutic treatment capable of treating the lysosomal storage disease. In embodiments, the therapeutic treatment is substrate inhibition therapy and / or enzyme replacement therapy (e.g., as described herein) capable of treating the lysosomal storage disease.

[0134] Normal plasma levels can be determined, for example, by methods described herein (such as the Olink® assay described in detail in the Examples). In embodiments, the healthy population of subjects is a population with a similar genetic background, e.g., a population from the same genetic and / or geographic location as the patient. In embodiments, the lysosomal storage disease is Fabry disease, Gaucher disease (e.g., Gaucher disease type 1, 2, or 3), or MPS (e.g., MPS type I, II, or III).

[0135] A further aspect provides a method of treating a lysosomal storage disease in a subject diagnosed as being at risk for the disease, wherein the patient has a plasma CD63 level that is higher than a control level in a sample previously collected from the subject, e.g., at least about 10% higher (e.g., at least about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher). In embodiments, the treatment method comprises administering to the subject an effective amount of substrate reduction therapy or enzyme replacement therapy (e.g., as described herein) capable of treating the lysosomal storage disease.

[0136] In embodiments, the lysosomal storage disease is Fabry disease (e.g., the subject is male), Gaucher disease (e.g., Gaucher disease type 1, 2, or 3), or MPS (e.g., MPS type I, II, or III). In embodiments, the plasma control level is from a sample taken from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level. In embodiments, the control value is derived from measuring the CD63 level in a single sample taken from the subject. In other embodiments, the control value is derived from measuring the level of CD63 in multiple samples (e.g., 2, 3, 4, 6, 8, or more samples) obtained from the subject.

[0137] Another aspect provides a method of treating or preventing the onset or progression of a lysosomal storage disease in a subject assessed to be at risk for the disease, the method comprising: (a) obtaining a first biological sample (e.g., blood or a blood fraction such as plasma) from the subject and analyzing the sample for CD63 concentration; (b) if the CD63 concentration is above a control value, initiating a course of therapeutic treatment in the subject (e.g., a therapeutically effective amount of substrate reduction therapy or enzyme replacement therapy (e.g., as described herein) capable of treating the lysosomal storage disease); and, optionally, (c) after treating the subject, obtaining a second biological sample (e.g., of the same type as the first biological sample) from the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0138] In embodiments, the control value is about 10% higher (e.g., about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher) than the control level in a sample previously collected from the same subject. In embodiments, the control level is derived from measuring the CD63 level in a single sample collected from the subject. In other embodiments, the control value is derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8, or more samples) obtained from the subject. In embodiments, the plasma control level is from a sample collected from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level.

[0139] A further aspect provides a method of reducing CD63 levels in a patient (e.g., in the patient's blood) suffering from a lysosomal storage disease, the method comprising administering an effective amount of substrate reduction therapy or enzyme replacement therapy (e.g., as described herein). In embodiments, the lysosomal storage disease is Fabry disease, Gaucher disease (e.g., Gaucher disease type 1, 2, or 3), or MPS (e.g., MPS type I, II, or III). In embodiments, the reduction in CD63 is a reduction in plasma CD63 levels.

[0140] It will be understood that various embodiments of the methods described above may be applied to the above method (e.g., with respect to the selection of sample types for the test sample and control sample). In certain embodiments, the particular lysosomal storage disease is selected from the conditions described above.

[0141] These methods are further illustrated below for Fabry disease, Gaucher disease and MPS, which are also the subject of this example.

[0142] Fabry disease Fabry disease (FD) is a lysosomal storage disorder characterized by defective activity of α-Gal, encoded by the GLA gene. The enzyme deficiency leads to the progressive intracellular accumulation of glycosphingolipids, primarily GL3, in various cell types and tissues, including the kidney, heart, liver, spleen, and skin, as well as the peripheral and central nervous systems. Symptoms presented by FD patients vary widely and depend on the severity and stage of the condition. Symptoms typically begin in childhood or adolescence and can include: acrotactile dysesthesias (severe pain in the extremities, e.g., a burning sensation in the arms or legs that worsens with exercise and hot weather), lens and corneal opacities, angiokeratoma, edema, abdominal pain, impaired circulation and increased risk of heart attack or stroke, cardiac hypertrophy, progressive kidney damage (e.g., leading to kidney failure), decreased sweating, fever, and gastrointestinal problems. Renal, cardiac, and cerebrovascular symptoms tend to characterize the later stages of the disease and are a significant cause of morbidity. Fabry disease is an X-linked lipid storage disorder; boys have a 50% chance of inheriting the disorder from their mothers, and girls have a 50% chance of being carriers. Mild disease is more common in women, but affected women can occasionally present with severe symptoms similar to those seen in affected men. Historically, diagnosis was based on clinical symptoms and family history, followed by presumptive assays of α-Gal activity in leukocytes or plasma and / or detection of GL3 in tissue biopsies; diagnosis could be confirmed by molecular genetic analysis (Breunig et al., Kidney International (2003) 63(84):S181-S185). More recently, lyso-GL3 has been used as a diagnostic biomarker (Maruyama et al., Genet. Med. (2019) 21(1):44-52; for other diagnostics, see also Levstek et al., Genes (Basel) (2020) 11(9):1091-1109). Treatments for FD include ERT using recombinant α-Gal (e.g., agalsidase beta and agalsidase alpha), small molecule chaperone therapy (e.g., migalastat), and substrate reduction therapy (e.g., benglustat).Other therapeutic interventions are also under investigation (see, e.g., Oder et al., Cardiovasc Diagn Ther. (2021) 11(2):683-695; and also Lenders et al., Drugs (2021) 81(6):635-645).

[0143] As shown in the accompanying examples, CD63 levels are increased in samples from Fabry disease patients compared to matched healthy control subjects. In particular, CD63 has now been observed to be upregulated in the plasma of Fabry patients, CD63 levels decrease linearly with treatment (both SRT and ERT), and the response of CD63 to treatment (SRT with benglustat) is comparable in its variability to the standard Fabry disease biomarkers GL3 and lyso-GL3. Thus, the identification of CD63 as a biomarker for FD represents an important advance in the diagnosis and monitoring of this disease.

[0144] Thus, in one aspect, there is provided a method of diagnosing Fabry disease in a subject suspected of being at risk of having Fabry disease, the method comprising measuring the level of CD63 in a sample from the subject. A related aspect provides the use of CD63 as a biomarker in the diagnosis of Fabry disease in a subject suspected of being at risk of having Fabry disease. Viewed alternatively, these aspects provide methods of generating quantitative data about a subject, the method comprising determining the level of CD63 in a sample from the subject, the subject being suspected of having (or having) Fabry disease.

[0145] In embodiments, a subject is considered at risk for Fabry disease through family history of the disease, genetic testing, analysis of other characteristic biomarkers, clinical symptoms, and identification of specific risk factors, such as those described herein. In embodiments, the specific risk factors are selected from one or more of clinical symptoms, family history, genetic testing, enzyme activity, and glycosphingolipid levels. Clinical symptoms include one or more of fatigue, pain (e.g., acrotactile paresthesia or abdominal pain), lens or corneal opacities, vortex keratopathy, angiokeratoma, dyspnea, palpitations, edema, renal disease (e.g., proteinuria, microscopic hematuria, or lipiduria, or end-stage renal disease), myocardial dysfunction (e.g., concentric or asymmetric left ventricular hypertrophy, or heart failure), cardiac conduction abnormalities with shortened PR interval, cardiac arrhythmias, dizziness, headache, crossed diplopia, dysarthria, hemiataxia, transient ischemic attack, early-onset stroke, and dementia. Family history may include the medical history of ancestors and / or close relatives (e.g., aunts, uncles, cousins, etc.; see Laney et al., J Genet Couns. (2008) 17(1):79-83). Genetic testing may involve sequencing part or all of the GLA gene and comparing it to known disease-causing variants (see, e.g., "Fabry Disease: Perspectives from 5 Years of FOS," Oxford PharmaGenesis (2006), Ed. Mehta, Beck and Sunder-Plassmann; Chapter 33). The patient's mutational status may allow for a diagnosis of Fabry disease with a high degree of confidence, for example, in the case of some mutations that result in premature termination of enzyme translation. However, in other cases, it may not be possible to completely correlate variant GLA with Fabry disease. Enzyme activity can be assessed, for example, by testing the activity of α-Gal in whole blood or blood spot samples (see, e.g., https: / / www.discoverfabry.com / hcp / diagnosing-fabry; and Nakao et al., Kidney Int. (2003) 64(3):801-807).Glycosphingolipid levels can be assessed by measuring the concentration of GL3 and / or lyso-GL3 in a sample, such as a whole blood or plasma sample (see, e.g., Maruyama et al., 2019 (supra)).

[0146] In embodiments, the control value is a baseline CD63 level previously measured in the same subject (i.e., a previous sample from the subject), e.g., a baseline CD63 level measured at least 1 month before obtaining the test sample from the subject, e.g., at least 2, 3, 6, 9, 12, 18, or 24 months before obtaining the test sample from the subject. The control value may correspond to the CD63 level measured in a single previous sample from the subject, or may be the average of values from multiple previous samples. In alternative embodiments, the control value is the CD63 level in a sample taken from one or more healthy subjects (e.g., the average value of a cohort of healthy subjects). In embodiments, the healthy subject is matched to the subject being evaluated, e.g., by age and / or gender. The healthy subject is generally matched to the subject being evaluated by gender. Typically, the control value corresponds to the level of CD63 in the same type of sample being evaluated in the method, e.g., a sample obtained, processed, and / or stored in the same manner as the test sample. For example, if the method utilizes a plasma sample, the control value typically corresponds to the level of CD63 in a control plasma sample, e.g., a previous plasma sample taken from the same subject or a plasma sample obtained from one or more healthy subjects.

[0147] In embodiments where the control value for CD63 concentration is a point value (or average, e.g., mean), the CD63 level in a test sample is considered to be outside the normal range if it is at least about 5% greater than the control value, e.g., at least about 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, or 100% greater than the control value. In some embodiments, the CD63 level in a test sample is considered to be outside the normal range if it is at least about 2-fold greater than the control value, e.g., at least about 3, 4, 5, 6, 8, or 10-fold greater than the control value. In embodiments, the CD63 level in a test sample is about 3 to about 8-fold greater than the control value, e.g., about 4 to about 7-fold greater than the control value, or about 5 to about 6-fold greater than the control value. In embodiments, the CD63 level in a test sample is about 5-fold greater than the control value, or about 6-fold greater than the control value. In embodiments where the control value for CD63 concentration is a range of values, e.g., explained by some variation around the mean value, the CD63 level in the test sample is considered to be outside the normal range if it is more than about 1 standard error above the mean value, e.g., more than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, or 3.0 standard errors above the mean value.

[0148] In embodiments, CD63 is used as the only biomarker in the methods of the present disclosure.

[0149] Alternatively, measurement of CD63 levels in a sample from a subject is used together with one or more other characteristic measures of the disease, for example, as part of a panel of biomarkers used for diagnostic purposes. This aspect provides a method for improving the diagnosis of Fabry disease in a subject, characterized in that CD63 is used as a biomarker. A related aspect provides the use of CD63 as a biomarker to improve methods for diagnosing Fabry disease in a subject. In embodiments, CD63 is used as a biomarker together with GL3, lyso-GL3, and / or α-Gal activity, optionally together with one or more additional biomarkers. In embodiments, CD63 is used as a biomarker together with lyso-GL3, optionally together with one or more additional biomarkers. In embodiments, the other biomarkers used (i.e., other than CD63) do not include any of the following: α-iduronidase, α-glucosidase, saposin C, LAMP-1, LAMP-2, β-glucosidase, α-galactosidase A, iduronate-2-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, saposin B, heparan-N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA:glucosamine N-acetyltransferase enzymes, N-acetylglucosamine 6-sulfatase, β-galactosidase, β-glucuronidase, aspartylglucosaminidase, acid lipase, β-hexosaminidase A, β-hexosaminidase B, GM2-activator, acid ceramidase, α-L-fucosidase, α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferase g-subunit, palmitoyl protein thioesterase, tripeptidyl peptidase I, cathepsin K, α-galactosidase B, sialic acid transporter, CD45 leukocyte common biomolecule, or LIMP II.

[0150] In embodiments, the use of CD63 as a biomarker reduces the rate of false positives and / or increases the rate of true positives compared to a corresponding diagnosis in which CD63 levels are not measured.

[0151] Another embodiment provides a method of treating a subject diagnosed with Fabry disease by a method defined herein, wherein the treatment comprises administering one or more therapeutic treatments for Fabry disease to the subject. A related embodiment provides a therapeutic agent for treating Fabry disease in a subject, wherein the subject has been diagnosed with Fabry disease by a method defined herein. Also provided is a method for generating quantitative data about a subject, the method comprising (e.g., consisting of) (a) determining the level of CD63 in a sample from the subject, (b) determining whether the level of CD63 is greater than a control value (e.g., the control value measured as the CD63 level in a sample taken from one or more healthy subjects), and (c) administering one or more therapeutic treatments for Fabry disease to the subject if the level of CD63 in the sample is greater than the control value.

[0152] In embodiments, the therapeutic treatment or agent comprises (e.g., consists of) substrate reduction therapy (e.g., benglustat), chaperone therapy (e.g., migalastat), enzyme replacement therapy (e.g., agalsidase alfa or agalsidase beta), and / or gene therapy (e.g., using the GLA gene or an active fragment thereof). In embodiments, the therapeutic treatment comprises (consists of) administering benglustat or migalastat (e.g., benglustat) to the subject. In other embodiments, the treatment comprises administering recombinant α-galactosidase (e.g., agalsidase beta) to the subject.

[0153] A further aspect provides a method of monitoring the progression of Fabry disease in a subject diagnosed with Fabry disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the first sample is obtained from the subject; and (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample. Typically, the method further comprises: (d) determining that the disease is becoming more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample; determining that the subject's disease is not progressing if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample; and determining that the disease is in remission if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. Related embodiments provide for the use of CD63 as a biomarker for monitoring the progression of Fabry disease in subjects diagnosed with Fabry disease. Viewed alternatively, these embodiments provide methods for generating quantitative data about subjects diagnosed with Fabry disease, the methods comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; and (c) comparing the level of CD63 determined in step (a) with the level determined in step (b). The method may comprise a further step (d) in which disease progression is assessed based on the comparison made in step (c).

[0154] The sample may be of the type mentioned herein. In embodiments, the sample contains lysosomes and / or exosomes. In embodiments, the sample is selected from blood, blood fractions, urine, cerebrospinal fluid, saliva, lymph, skin tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Most conveniently, the sample from the subject comprises (e.g., consists of) blood (e.g., whole blood), blood fractions, and / or urine. In embodiments, the sample from the subject comprises (e.g., consists of) a blood fraction selected from plasma and serum. In embodiments, the sample is a blood sample, e.g., a plasma sample. In embodiments, the sample comprises (e.g., consists of) CSF.

[0155] In embodiments, the first and second (or subsequent) samples are obtained from the subject at least about one day apart, e.g., at least about two, three, or four days, or at least about one, two, or four weeks apart. In other embodiments, the first and second (or subsequent) samples are obtained from the subject at least about eight weeks apart, e.g., at least about nine, ten, twelve, fifteen, or twenty weeks apart.

[0156] In embodiments, the method includes successive measurements of one or more further samples taken from the subject, for example at the intervals specified above.

[0157] Another aspect provides a method of monitoring the progress of treatment for Fabry disease in a subject diagnosed with Fabry disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering therapeutic treatment for Fabry disease to the subject; and (c) measuring the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the therapeutic treatment has been administered. Typically, the method further comprises step (d) of determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. In embodiments, the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment should be increased if the level of CD63 determined in step (c) is substantially the same as or greater than the level of CD63 determined in step (a). A related aspect provides the use of CD63 as a biomarker for monitoring the progress of treatment for Fabry disease in a subject diagnosed with Fabry disease. Viewed alternatively, these aspects provide a method of generating quantitative data for a subject with Fabry disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; and (b) determining the level of CD63 in a second sample from the subject after administering a therapeutic treatment for Fabry disease to the subject. In embodiments, the dosage of the therapeutic treatment should be increased if the level of CD63 determined in step (b) is substantially the same as or greater than the level of CD63 determined in step (a).

[0158] In embodiments, the treatment for Fabry disease is as described herein. Accordingly, the treatment may include (e.g., consist of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment includes administering benglustat or migalastat (e.g., benglustat) to the subject. In other embodiments, the treatment includes administering recombinant alpha-galactosidase (e.g., agalsidase beta) to the subject.

[0159] The second sample is typically collected a period of time after the therapeutic treatment. In embodiments, the second sample is collected from the subject at least about 1 week after the therapeutic treatment (e.g., after the start of the therapeutic treatment), for example, at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the therapeutic treatment. In one embodiment, the second sample is collected from the subject at least about 8 weeks after the start of the therapeutic treatment.

[0160] In embodiments, the method includes serial measurements of one or more additional samples taken from the subject, e.g., at intervals of about one week, e.g., about every 2, 4, 6, 8, 10, or 12 weeks. This serial measurement can be particularly useful when the dosage of therapy is adjusted based on changes in CD63 levels in the subject.

[0161] Another aspect provides a method of treating Fabry disease in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level above the mean for a population of healthy subjects (e.g., a level of at least about 0.25 standard deviations, e.g., at least about 0.5, 0.75, 1, or 1.5 standard deviations), the method comprising administering to the patient an effective amount of a therapeutic treatment for Fabry disease. In an embodiment, the healthy population of subjects is a population with a similar genetic background, e.g., a population from the same genetic and / or geographic location as the patient.

[0162] A further aspect provides a method of treating Fabry disease in a subject diagnosed as being at risk for developing the disease, wherein the patient has a plasma CD63 level that is higher than a control level in a sample previously collected from the subject (e.g., at least about 10% higher than the plasma control level, e.g., at least about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher than the plasma control level), the method comprising administering to the patient an effective amount of a therapeutic treatment for Fabry disease (e.g., as described herein). In embodiments, the plasma control level is from a sample collected from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level. In embodiments, the control value is derived from measuring CD63 levels in a single sample collected from the subject. In other embodiments, the control value is derived from measuring the level of CD63 in multiple samples (eg, 2, 3, 4, 6, 8, or more samples) obtained from the subject.

[0163] A further aspect provides a method of reducing CD63 levels in a patient (e.g., in the patient's blood) suffering from Fabry disease, the method comprising administering to the patient an effective amount of a therapeutic treatment (e.g., substrate reduction therapy or enzyme replacement therapy as described herein). In an embodiment, the reduction in CD63 is a reduction in plasma CD63 levels.

[0164] Another aspect provides a method of treating or preventing the onset or progression of Fabry disease in a subject assessed to be at risk of developing Fabry disease, the method comprising: (a) obtaining a first biological sample (e.g., blood or a blood fraction such as plasma) from the subject and analyzing the sample for CD63 concentration; (b) initiating a course of therapeutic treatment in the subject (e.g., a therapeutically effective amount of substrate reduction therapy or enzyme replacement therapy, such as those described herein) if the CD63 concentration is above a control value; and, optionally, (c) after treating the subject, obtaining a second biological sample (e.g., of the same type as the first biological sample) from the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0165] In embodiments, the control value is about 10% higher (e.g., about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher) than the control level in a sample previously collected from the same subject. In embodiments, the control level is derived from measuring the CD63 level in a single sample collected from the subject. In other embodiments, the control value is derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8, or more samples) obtained from the subject. In embodiments, the plasma control level is from a sample collected from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level.

[0166] In embodiments, the treatment for Fabry disease is as described herein. Accordingly, the treatment may include (e.g., consist of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment includes administering benglustat or migalastat (e.g., benglustat) to the subject. In other embodiments, the treatment includes administering recombinant alpha-galactosidase (e.g., agalsidase beta) to the subject.

[0167] The second sample is typically collected a period of time after the therapeutic treatment. In embodiments, the second sample is collected from the subject at least about 1 week after the therapeutic treatment (e.g., after the start of the therapeutic treatment), for example, at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the therapeutic treatment. In one embodiment, the second sample is collected from the subject at least about 8 weeks after the start of the therapeutic treatment.

[0168] In embodiments, the method includes serial measurements of one or more additional samples taken from the subject, e.g., at intervals of about one week, e.g., about every 2, 4, 6, 8, 10, or 12 weeks. This serial measurement can be particularly useful when the dosage of therapy is adjusted based on changes in CD63 levels in the subject.

[0169] Another aspect provides a method of adjusting the dosage of a therapeutic treatment for Fabry disease in a subject receiving said therapeutic treatment, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, where the second sample is obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample. Typically, adjusting the dosage in step (c) comprises maintaining the dosage of the therapeutic treatment if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, and increasing the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment if the level of CD63 in the second sample is substantially the same as or greater than the level of CD63 in the first sample. In embodiments, if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment is reduced. A related aspect provides the use of CD63 as a biomarker for adjusting the dosage of a therapeutic treatment for Fabry disease in a subject receiving such therapeutic treatment.

[0170] In embodiments, the subject is a male subject. In other embodiments, the subject is a female subject.

[0171] Gaucher disease Gaucher disease (GD) is an inherited metabolic disorder caused by mutations in the GBA gene, which results in GCase (also known as acid β-glucosidase) deficiency and the associated accumulation of GL1 and lyso-GL1 in macrophage lysosomes. This affects cells of the reticuloendothelial system, including the liver, spleen, and bone marrow, and can result in splenic and renal enlargement, renal insufficiency, skeletal damage, bone lesions, or severe neurological impairment. GD is classified into three major types: type 1 GD, the most common type, primarily affects adults with a mean age of 28 years at diagnosis; type 2 GD, an acute neuropathic form typically affecting infants between 3 and 6 months of age; and type 3 GD, a chronic neuropathic form with a slower and milder onset than type 2. Gaucher disease type 3 (GD3) is characterized by progressive encephalopathy and systemic symptoms similar to type 1. It is caused by mutations in the GBA gene (1q21) and is characterized by prominent central nervous system (CNS) involvement, which poses significant challenges for diagnosis and monitoring.

[0172] Lyso-GL1 levels, β-glucosidase activity, and gene GBA sequencing are currently the most common diagnostic and prognostic indicators for GD. In the case of lyso-GL1, several studies have suggested that its pathological involvement correlates with disease burden and clinical severity. Chitotriosidase and CCL18 have also been identified as biomarkers for Gaucher disease, but are not commonly used in clinics (see, e.g., Rolfs et al., PLoS ONE (2013) 8(11): e79732).

[0173] As shown in the accompanying examples, CD63 levels are elevated in Gaucher disease patients and show much lower variability in treated patients than standard biomarkers of GD, such as lyso-GL1 or chitotriosidase. Thus, CD63 represents a valuable biomarker for use in diagnosing and monitoring GD.

[0174] Thus, in one aspect, a method of diagnosing Gaucher disease in a patient suspected of being at risk for Gaucher disease is provided, the method comprising measuring the level of CD63 in a sample from the subject. A related aspect provides the use of CD63 as a biomarker in the diagnosis of Gaucher disease in a subject suspected of being at risk for Gaucher disease. Viewed alternatively, these aspects provide a method for generating quantitative data about a subject, the method comprising determining the level of CD63 in a sample from the subject, the subject being suspected of having (or suffering from) Gaucher disease. In an embodiment, the Gaucher disease is type 1 GD, type 2 GD, or type 3 GD. In an embodiment, the Gaucher disease is type 3 GD.

[0175] In embodiments, a subject is considered at risk for Gaucher disease (e.g., type 1 GD) through family history of the disease, genetic testing, analysis of other characteristic biomarkers, clinical symptoms, etc., as well as the identification of specific risk factors, such as those described herein. In embodiments, the specific risk factors are selected from one or more of clinical symptoms, family history, genetic testing, enzyme activity, and glycosphingolipid levels. Clinical symptoms include one or more of hepatomegaly and splenomegaly, pain (particularly severe pain in the joints, e.g., hips and / or knees), osteoporosis, skin pigmentation, pancytopenia (e.g., causing anemia, neutropenia, leukopenia, and / or thrombocytopenia, accompanied by an increased risk of infection and bleeding), and neurological symptoms (e.g., impaired olfactory and cognitive function, particularly in type 1 GD; seizures, hypertension, intellectual disability, and apnea, particularly in type 2 GD; and myoclonic dementia and ocular apraxia, particularly in type 3 GD), and Parkinson's disease. Family history may include a history of Gaucher disease in ancestors and / or close relatives (e.g., aunts, uncles, cousins, etc.). Genetic testing may involve sequencing part or all of the GBA gene and comparing it to known disease-causing variants (see, e.g., Riboldi et al., Cells (2019) 8:364-380). The patient's mutation status may allow a diagnosis of Gaucher disease to be made with high confidence, for example, in the case of mutations known to be associated with severe disease. However, in other cases, it may not be possible to completely correlate variant GBA with Gaucher disease. Enzyme activity may be assessed, for example, by testing the activity of GCase in whole blood or blood spot samples (see, e.g., Miyamoto et al., Intern Med. (2021) 60:699-707). Glycosphingolipids can be assessed by measuring the concentration of GL1 and / or lyso-GL1 in a sample, e.g., a whole blood or plasma sample (see, e.g., Rolfs et al., 2013, supra).

[0176] In embodiments, the control value is a baseline CD63 level previously measured in the same subject (i.e., a previous sample from the subject), e.g., a baseline CD63 level measured at least 1 month before obtaining the test sample from the subject, e.g., at least 2, 3, 6, 9, 12, 18, or 24 months before obtaining the test sample from the subject. The control value may correspond to the CD63 level measured in a single previous sample from the subject, or may be the average of values from multiple previous samples. In alternative embodiments, the control value is the CD63 level in a sample taken from one or more healthy subjects (e.g., the average value of a cohort of healthy subjects). In embodiments, the healthy subjects are matched to the subject being evaluated, e.g., by age and / or sex. Typically, the control value corresponds to the level of CD63 in a sample of the same type as that being evaluated in the method, e.g., a sample obtained, processed, and / or stored in the same manner as the test sample. For example, if the method utilizes a plasma sample, the control value typically corresponds to the level of CD63 in a control plasma sample, e.g., a previous plasma sample taken from the same subject or a plasma sample obtained from one or more healthy subjects.

[0177] In embodiments where the control value for CD63 concentration is a point value (or average, e.g., mean), the CD63 level in a test sample is considered to be outside the normal range if it is at least about 5% greater than the control value, e.g., at least about 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, or 100% greater than the control value. In some embodiments, the CD63 level in a test sample is considered to be outside the normal range if it is at least about 2-fold greater than the control value, e.g., at least about 3, 4, 5, 6, 8, or 10-fold greater than the control value. In embodiments, the CD63 level in a test sample is about 3 to about 8-fold greater than the control value, e.g., about 4 to about 6-fold greater than the control value, or about 4 to about 5-fold greater than the control value. In embodiments, the CD63 level in a test sample is about 4-fold greater than the control value, or about 5-fold greater than the control value. In embodiments where the control value for CD63 concentration is a range of values, e.g., explained by some variation around the mean value, the CD63 level in the test sample is considered to be outside the normal range if it is more than about 1 standard error above the mean value, e.g., more than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, or 3.0 standard errors above the mean value.

[0178] In embodiments, CD63 is used as the only biomarker in the methods of the present disclosure.

[0179] Alternatively, measurement of CD63 levels in a sample from a subject is used together with one or more other characteristic measurements of the disease, e.g., as part of a panel of biomarkers used for diagnostic purposes. This aspect provides a method for improving the diagnosis of Gaucher disease in a subject, characterized in that CD63 is used as a biomarker. A related aspect provides the use of CD63 as a biomarker to improve the method for diagnosing Gaucher disease in a subject. In embodiments, the Gaucher disease is type 1 Gaucher disease, type 2 Gaucher disease, or type 3 Gaucher disease. In embodiments, CD63 is used as a biomarker together with GL1, lyso-GL1, CCL18, and / or chitotriosidase, optionally together with one or more additional biomarkers. In embodiments, the other biomarkers used (i.e., other than CD63) do not include any of the following: α-iduronidase, α-glucosidase, saposin C, LAMP-1, LAMP-2, β-glucosidase, α-galactosidase A, iduronate-2-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, saposin B, heparan-N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA:glucosamine N-acetyltransferase enzymes, N-acetylglucosamine 6-sulfatase, β-galactosidase, β-glucuronidase, aspartylglucosaminidase, acid lipase, β-hexosaminidase A, β-hexosaminidase B, GM2-activator, acid ceramidase, α-L-fucosidase, α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferase g-subunit, palmitoyl protein thioesterase, tripeptidyl peptidase I, cathepsin K, α-galactosidase B, sialic acid transporter, CD45 leukocyte common biomolecule, or LIMP II.

[0180] In embodiments, the use of CD63 as a biomarker reduces the rate of false positives and / or increases the rate of true positives compared to a corresponding diagnosis in which CD63 levels are not measured.

[0181] Another aspect provides a method of treating a subject diagnosed with Gaucher disease by a method defined herein, wherein the treatment comprises administering to the subject one or more therapeutic treatments for Gaucher disease. A related aspect provides a therapeutic agent for treating Gaucher disease in a subject, wherein the subject has been diagnosed with Gaucher disease by a method defined herein. Also provided is a method for generating quantitative data about a subject, the method comprising (e.g., consisting of) (a) determining the level of CD63 in a sample from the subject, (b) determining whether the level of CD63 is greater than a control value (e.g., the control value measured as the CD63 level in a sample taken from one or more healthy subjects), and (c) administering to the subject one or more therapeutic treatments for Gaucher disease if the level of CD63 in the sample is greater than the control value. In an embodiment, the Gaucher disease is type 1 GD, type 2 GD, or type 3 GD.

[0182] In embodiments, the therapeutic treatment or medicament comprises (e.g., consists of) substrate reduction therapy (e.g., eliglustat, benglustat, or miglustat), chaperone therapy, enzyme replacement therapy (e.g., imiglucerase, velaglucerase, or taliglucerase), and / or gene therapy (e.g., using the GBA gene). In embodiments, the therapeutic treatment comprises (e.g., consists of) administering eliglustat to the subject. In embodiments, the therapeutic treatment comprises (e.g., consists of) administering benglustat to the subject. In other embodiments, the treatment comprises administering recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the therapeutic treatment comprises (e.g., consists of) administering benglustat and recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the Gaucher disease is type 1 GD and the therapeutic treatment comprises (e.g., consists of) administering eliglustat to the subject. In embodiments, the Gaucher disease is type 2 or type 3 GD and the therapeutic treatment comprises (e.g., consists of) administering benglustat to the subject. In embodiments, the Gaucher disease is type 3 GD and the therapeutic treatment comprises (e.g., consists of) administering benglustat and recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the Gaucher disease is type 3 GD and the therapeutic treatment comprises (e.g., consists of) administering benglustat to the subject.

[0183] A further aspect provides a method for monitoring the progression of Gaucher disease in a subject diagnosed with Gaucher disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the first sample is obtained from the subject; and (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample. Typically, the method further comprises: (d) determining that the disease has become more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample; determining that the subject's disease has not progressed if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample; and determining that the disease has remitted if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. A related aspect provides the use of CD63 as a biomarker for monitoring the progression of Gaucher disease in a subject diagnosed with Gaucher disease. Viewed alternatively, these aspects provide a method for generating quantitative data about a subject diagnosed with Gaucher disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; and (c) comparing the level of CD63 determined in step (a) with the level determined in step (b). The method may comprise a further step (d) in which disease progression is assessed based on the comparison made in step (c). In embodiments, the Gaucher disease is type 1 GD, type 2 GD, or type 3 GD.

[0184] The sample may be of the type mentioned herein. In embodiments, the sample contains lysosomes and / or exosomes. In embodiments, the sample is selected from blood, blood fractions, urine, cerebrospinal fluid, saliva, lymph, skin tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Conveniently, the sample from the subject comprises (e.g., consists of) blood (e.g., whole blood), blood fractions, and / or urine. In embodiments, the sample from the subject comprises (e.g., consists of) a blood fraction selected from plasma and serum. In embodiments, the sample is a blood sample, e.g., a plasma sample. In embodiments, the sample is a CSF sample. In embodiments, the sample does not contain platelets.

[0185] In embodiments, the first and second (or subsequent) samples are obtained from the subject at least about one day apart, e.g., at least about two, three, or four days, or at least about one, two, or four weeks apart. In other embodiments, the first and second (or subsequent) samples are obtained from the subject at least about eight weeks apart, e.g., at least about nine, ten, twelve, fifteen, or twenty weeks apart.

[0186] In embodiments, the method includes successive measurements of one or more further samples taken from the subject, for example at the intervals specified above.

[0187] Another aspect provides a method for monitoring the progress of treatment for Gaucher disease in a subject diagnosed with Gaucher disease, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering a therapeutic treatment for Gaucher disease to the subject; and (c) measuring the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the therapeutic treatment. Typically, the method further comprises step (d) of determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. In embodiments, the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment should be increased if the level of CD63 determined in step (c) is substantially the same as or greater than the level of CD63 determined in step (a). A related aspect provides the use of CD63 as a biomarker for monitoring the progress of treatment for Gaucher disease in a subject diagnosed with Gaucher disease. Viewed another way, these aspects provide a method for generating quantitative data for a subject with Gaucher disease, the method comprising: (a) determining the level of CD63 in a first sample from the subject; and (b) determining the level of CD63 in a second sample from the subject after administering a therapeutic treatment for Gaucher disease to the subject. In embodiments, the dosage of the therapeutic treatment should be increased if the level of CD63 determined in step (b) is substantially the same as or greater than the level of CD63 determined in step (a). In embodiments, the Gaucher disease is type 1 GD, type 2 GD, or type 3 GD. In embodiments, the Gaucher disease is type 3 GD, and the sample comprises (e.g., consists of) CSF.

[0188] In embodiments, the treatment for Gaucher disease is as described herein. Accordingly, the treatment may include (e.g., consist of) substrate reduction therapy, enzyme replacement therapy, and / or gene therapy. In embodiments, the treatment includes administering eliglustat or miglustat (e.g., eliglustat) to the subject. In other embodiments, the treatment includes administering recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the method includes monitoring a change from a first treatment (e.g., ERT with imiglucerase) to a second treatment (e.g., SRT with eliglustat), e.g., checking whether the patient improves or remains stable after the change.

[0189] The second sample is typically collected a period of time after the therapeutic treatment. In embodiments, the second sample is collected from the subject at least about 1 week after the therapeutic treatment (e.g., after the start of the therapeutic treatment), for example, at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the therapeutic treatment. In one embodiment, the second sample is collected from the subject at least 8 weeks after the start of the therapeutic treatment.

[0190] In embodiments, the method includes serial measurements of one or more additional samples taken from the subject, e.g., at intervals of about every week, e.g., about every 2, 4, 6, 8, 10, or 12 weeks. This serial measurement can be particularly useful when the dosage of therapy is being adjusted based on changes in CD63 levels in the subject.

[0191] Another aspect provides a method of treating Gaucher disease in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level above the mean for a population of healthy subjects (e.g., a level of at least about 0.25 standard deviations, e.g., at least about 0.5, 0.75, 1, or 1.5 standard deviations), the method comprising administering to the patient an effective amount of a therapeutic treatment for Gaucher disease. In an embodiment, the healthy population of subjects is a population with a similar genetic background, e.g., a population from the same genetic and / or geographic location as the patient.

[0192] A further aspect provides a method of treating Gaucher disease in a subject diagnosed as being at risk for developing the disease, wherein the patient has a plasma CD63 level that is higher than a control level in a sample previously collected from the subject (e.g., a level that is at least about 10% higher, e.g., at least about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher than the plasma control level), the method comprising administering to the subject an effective amount of a therapeutic treatment for Gaucher disease (e.g., as described herein). In embodiments, the plasma control level is from a sample collected from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level. In embodiments, the control value is derived from measuring CD63 levels in a single sample collected from the subject. In other embodiments, the control value is derived from measuring the level of CD63 in multiple samples (eg, 2, 3, 4, 6, 8, or more samples) obtained from the subject.

[0193] A further aspect is a method of reducing CD63 levels in a patient (e.g., in the patient's blood) suffering from Gaucher disease, the method comprising administering to the patient an effective amount of a therapeutic treatment (e.g., substrate reduction therapy or enzyme replacement therapy as described herein). In an embodiment, the reduction in CD63 is a reduction in plasma CD63 levels.

[0194] Another aspect provides a method of treating or preventing the onset or progression of Gaucher disease in a subject assessed to be at risk for Gaucher disease, the method comprising: (a) obtaining a first biological sample (e.g., blood or a blood fraction such as plasma) from the subject and analyzing the sample for CD63 concentration; (b) initiating a course of therapeutic treatment in the subject (e.g., a therapeutically effective amount of substrate reduction therapy and / or enzyme replacement therapy, such as those described herein) if the CD63 concentration is above a control value; and, optionally, (c) after treating the subject, obtaining a second biological sample (e.g., of the same type as the first biological sample) from the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0195] In embodiments, the control value is about 10% higher (e.g., about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher) than the control level in a sample previously collected from the same subject. In embodiments, the control level is derived from measuring the CD63 level in a single sample collected from the subject. In other embodiments, the control value is derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8, or more samples) obtained from the subject. In embodiments, the plasma control level is from a sample collected from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level.

[0196] In embodiments, the treatment for Gaucher disease is as described herein. Accordingly, the treatment may include (e.g., consist of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy. In embodiments, the treatment includes administering benglustat, eliglustat, or miglustat (e.g., benglustat or eliglustat) to the subject. In other embodiments, the treatment includes administering recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the therapeutic treatment includes (e.g., consists of) administering benglustat and recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the Gaucher disease is type 1 GD, and the therapeutic treatment includes (e.g., consists of) administering eliglustat to the subject. In embodiments, the Gaucher disease is type 2 or type 3 GD, and the therapeutic treatment includes (e.g., consists of) administering benglustat to the subject. In embodiments, the Gaucher disease is type 3 GD and the therapeutic treatment comprises (e.g., consists of) administering benglustat and recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the Gaucher disease is type 3 GD and the therapeutic treatment comprises (e.g., consists of) administering benglustat to the subject.

[0197] The second sample is typically collected a period of time after the therapeutic treatment. In embodiments, the second sample is collected from the subject at least about 1 week after the therapeutic treatment (e.g., after the start of the therapeutic treatment), for example, at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the therapeutic treatment. In one embodiment, the second sample is collected from the subject at least about 8 weeks after the start of the therapeutic treatment.

[0198] In embodiments, the method includes serial measurements of one or more additional samples taken from the subject, e.g., at intervals of about one week, e.g., about every 2, 4, 6, 8, 10, or 12 weeks. This serial measurement can be particularly useful when the dosage of therapy is adjusted based on changes in CD63 levels in the subject.

[0199] Another aspect provides a method for adjusting the dosage of a therapeutic treatment for Gaucher disease in a subject receiving said therapeutic treatment, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, where the second sample is obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample. Typically, adjusting the dosage in step (c) comprises maintaining the dosage of the therapeutic treatment if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, and increasing the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment if the level of CD63 in the second sample is substantially the same as or greater than the level of CD63 in the first sample. In embodiments, if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment is reduced. A related aspect provides the use of CD63 as a biomarker for adjusting the dosage of a therapeutic treatment for Gaucher disease in a subject receiving said therapeutic treatment.

[0200] Mucopolysaccharidoses Mucopolysaccharidoses are genetic disorders in which GAGs accumulate in connective and other tissues throughout the body as a result of a deficiency in the lysosomal enzymes that break them down. Symptoms vary depending on the severity of the disease but typically result from damage to bone, connective tissue, and organs (e.g., dysplasia, joint stiffness, and hepatomegaly / splenomegaly), as well as compression of spinal nerves or nerve roots (e.g., pain, motor dysfunction, and other peripheral nervous system complications). Seven clinical types of MPS have currently been identified, with multiple subtypes classified according to the affected genes (see, e.g., Celik et al., Diagnostics (2021) 11(2):273-311). Current treatment options include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT), but early diagnosis and initiation of treatment are usually essential to have any significant impact on disease progression. The general characteristics of MPS are shown in the following table (adapted from Celik et al., supra, where HS is heparan sulfate, DS is dermatan sulfate, CS is chondroitin sulfate, e.g., chondroitin-4-sulfate or chondroitin-6-sulfate, and KS is keratan sulfate):

[0201] [Table 1]

[0202] Clinical diagnosis of MPS has historically been performed by measuring total GAG levels in urine. However, this method is difficult to collect and store, making it impractical and expensive for screening urine from newborns. Spot tests for urinary GAG levels have been developed based on dye staining, but these suffer from reliability and sensitivity issues, especially in the first few days after birth. These dye-based methods are not generally applicable to blood spot samples and cannot distinguish MPS types or reliably predict disease severity (see, e.g., Tomatsu et al., Mol Genet Metab. (2013) 110(0):42-53). For example, affinity binding methods (ELISAs) have been developed that can detect specific GAGs, such as keratin sulfate. While such methods can be used to assess GAG levels in blood samples, they are not suitable for simultaneous analysis of multiple GAGs (see, e.g., Khan et al., Mol Genet Metab. (2020) 130(2):101-109). More recently, mass spectrometry methods (e.g., LC-MS / MS) have been developed to detect and quantify multiple GAGs in blood samples (see, e.g., Khaledi et al., Anal Chem. (2020) 92(17):11721-11727), but these methods are not suitable for diagnosing all MPS types in blood samples.

[0203] Thus, there remains a need to improve methods for diagnosing MPS and to provide new methods for monitoring disease progression and treatment effectiveness. As shown in the accompanying examples, CD63 levels are elevated in MPS patients and can be used as a measure for detecting and monitoring MPS in these subjects.

[0204] Thus, in one aspect, a method of diagnosing MPS in a subject suspected of being at risk for MPS is provided, the method comprising measuring the level of CD63 in a sample from the subject. A related aspect provides the use of CD63 as a biomarker in the diagnosis of MPS in a subject suspected of being at risk for MPS. Viewed alternatively, these aspects provide a method for generating quantitative data about a subject, the method comprising determining the level of CD63 in a sample from the subject, the subject being suspected of having (or suffering from) MPS. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is selected from MPS I and MPS II. In embodiments, the MPS is MPS I (e.g., IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0205] In embodiments, a subject is considered at risk for developing MPS through the identification of certain risk factors, such as a family history of the disease, genetic testing, analysis of other characteristic biomarkers, clinical symptoms, etc. In embodiments, the certain risk factors are selected from one or more of clinical symptoms, family history, genetic testing, enzyme activity, and GAG levels. Clinical manifestations may include one or more symptoms affecting the head and neck (e.g., macrocephaly, hearing loss, corneal opacities, and malocclusion), the joints and skeleton (e.g., stiffness, hip dysplasia, clawed hands, joint laxity), the cardiovascular system (e.g., valvular hypertrophy and left ventricular hypertrophy), the airway (e.g., recurrent respiratory infections and obstructive airway disease), the abdomen (e.g., hepatomegaly / splenomegaly and umbilical / inguinal hernia), as well as neurological symptoms (e.g., growth retardation, ventricular dilation, enlarged perivascular spaces, and hyperactive or aggressive behavior), and other symptoms (e.g., dysgranulogenesis in white blood cells, hydrops fetalis, and proteinuria). Many of these signs and symptoms are common to different types of MPS, and patients presenting with a single symptom typically cannot be diagnosed with a high degree of certainty on that basis. Developmental delay and hip symptoms may be most relevant for screening programs aimed at early diagnosis of these conditions (for details on the differential diagnosis of MPS, see, e.g., Kubaski et al., Diagnostics (Basel). (2020) 10(3):172). Family history may include a history of MPS among ancestors and / or close relatives (e.g., aunts, uncles, cousins, etc.). Genetic testing may involve sequencing part or all of the relevant gene (e.g., as shown in the table above) and comparing it to known disease-causing variants (see, e.g., Kubaski et al., 2020, supra). Depending on the patient's mutation status, a diagnosis of MPS can be made with a high degree of confidence, although it may not be possible to fully correlate the variant gene with MPS.Enzyme activity can be assessed, for example, by testing the activity of the missing enzyme in a whole blood sample or blood spot sample (see, e.g., Filocamo et al., Italian Journal of Pediatrics (2018) 44(S2):129; and Lehman et al., Rheumatology (2011) 50(S5):v41-48). GAG levels can be assessed by measuring the concentration of glycans in a sample, such as whole blood, plasma, serum, or urine (see, e.g., Khan et al., 2020 (supra)).

[0206] In embodiments, the control value is a baseline CD63 level previously measured in the same subject (i.e., a previous sample from the subject), e.g., a baseline CD63 level measured at least 1 month before obtaining the test sample from the subject, e.g., at least 2, 3, 6, 9, 12, 18, or 24 months before obtaining the test sample from the subject. The control value may correspond to the CD63 level measured in a single previous sample from the subject, or may be the average of values from multiple previous samples. In alternative embodiments, the control value is the CD63 level in a sample taken from one or more healthy subjects (e.g., the average value of a cohort of healthy subjects). In embodiments, the healthy subjects are matched to the subject being evaluated, e.g., by age and / or sex. Typically, the control value corresponds to the level of CD63 in a sample of the same type as that being evaluated in the method, e.g., a sample obtained, processed, and / or stored in the same manner as the test sample. For example, if the method utilizes a plasma sample, the control value typically corresponds to the level of CD63 in a control plasma sample, e.g., a previous plasma sample taken from the same subject or a plasma sample obtained from one or more healthy subjects.

[0207] In embodiments where the control value for CD63 concentration is a point value (or average, e.g., mean), the CD63 level in a test sample is considered to be outside the normal range if it is at least about 5% greater than the control value, e.g., at least about 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, or 100% greater than the control value. In some embodiments, the CD63 level in a test sample is considered to be outside the normal range if it is at least about 2-fold greater than the control value, e.g., at least about 3, 4, 5, 6, 8, or 10-fold greater than the control value. In embodiments, the CD63 level in a test sample is about 3 to about 8-fold greater than the control value, e.g., about 3 to about 6-fold greater than the control value, or about 4 to about 5-fold greater than the control value. In embodiments, the CD63 level in a test sample is about 4-fold greater than the control value, or about 5-fold greater than the control value. In embodiments where the control value for CD63 concentration is a range of values, e.g., explained by some variation around the mean value, the CD63 level in the test sample is considered to be outside the normal range if it is more than about 1 standard error above the mean value, e.g., more than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, or 3.0 standard errors above the mean value.

[0208] In embodiments, CD63 is used as the only biomarker in the methods of the present disclosure.

[0209] Alternatively, measurement of CD63 levels in a sample from a subject is used together with one or more other characteristic measurements of the disease, for example, as part of a panel of biomarkers used for diagnostic purposes. This aspect provides a method for improving the diagnosis of MPS in a subject, characterized in that CD63 is used as a biomarker. A related aspect provides the use of CD63 as a biomarker to improve the method for diagnosing MPS in a subject. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., type IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., type IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).In embodiments, the other biomarkers used (i.e., other than CD63) do not include any of the following: α-iduronidase, α-glucosidase, saposin C, LAMP-1, LAMP-2, β-glucosidase, α-galactosidase A, iduronate-2-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, saposin B, heparan-N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA:glucosamine N-acetyltransferase enzymes, N-acetylglucosamine 6-sulfatase, β-galactosidase, β-glucuronidase, aspartylglucosaminidase, acid lipase, β-hexosaminidase A, β-hexosaminidase B, GM2-activator, acid ceramidase, α-L-fucosidase, α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferase g-subunit, palmitoyl protein thioesterase, tripeptidyl peptidase I, cathepsin K, α-galactosidase B, sialic acid transporter, CD45 leukocyte common biomolecule, or LIMP II.

[0210] In embodiments, the use of CD63 as a biomarker reduces the rate of false positives and / or increases the rate of true positives compared to a corresponding diagnosis in which CD63 levels are not measured.

[0211] Another aspect provides a method of treating a subject diagnosed with MPS by the methods defined herein, wherein the treatment comprises administering one or more therapeutic treatments for MPS to the subject. A related aspect provides a therapeutic agent for treating MPS in a subject, wherein the subject has been diagnosed with MPS by the methods defined herein. Also provided is a method for generating quantitative data about a subject, the method comprising (e.g., consisting of) (a) determining the level of CD63 in a sample from the subject, (b) determining whether the level of CD63 is greater than a control value (e.g., the control value measured as the CD63 level in a sample taken from one or more healthy subjects), and (c) administering one or more therapeutic treatments for MPS to the subject if the level of CD63 in the sample is greater than the control value. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., IH, IS, or IH / S type). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., type IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0212] In embodiments, the therapeutic treatment or medicament comprises (e.g., consists of) enzyme replacement therapy, gene therapy, and / or hematopoietic stem cell transplantation. In embodiments, the MPS is MPS I, MPS II, MPS IV, MPS VI, or MPS VII, and the therapeutic treatment comprises (e.g., consists of) enzyme replacement therapy and / or hematopoietic stem cell transplantation. In embodiments, the therapeutic treatment or medicament comprises (e.g., consists of) enzyme replacement therapy with α-L-iduronidase, iduronidase-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase, α-glucosaminidase, acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-6-sulfate sulfatase, β-galactosidase, N-acetylglucosamine-4-sulfatase, β-glucuronidase, or hyaluronidase. In embodiments, the MPS is MPS I, and the therapeutic treatment comprises (e.g., consists of) administering to the subject recombinant α-L-iduronidase (e.g., laronidase). In other embodiments, the MPS is MPS II, and the therapeutic treatment comprises (e.g., consists of) administering to the subject recombinant iduronidase-2-sulfatase (e.g., idursulfase). In other embodiments, the MPS is MPS IV A, and the therapeutic treatment comprises (e.g., consists of) administering to the subject recombinant N-acetylgalactosamine-6-sulfatase (e.g., elosulfase alfa); the MPS is MPS VI, and the therapeutic treatment comprises (e.g., consists of) administering to the subject recombinant N-acetylglucosamine-4-sulfatase (e.g., galsulfase); or the MPS is MPS VII, and the therapeutic treatment comprises (e.g., consists of) administering to the subject recombinant β-glucuronidase (e.g., bestronidase alfa).

[0213] A further embodiment provides a method for monitoring the progression of MPS in a subject diagnosed with MPS, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the first sample was obtained from the subject; and (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample. Typically, the method further comprises: (d) determining that the disease is becoming more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample; determining that the subject's disease is not progressing if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample; and determining that the disease is in remission if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. A related aspect provides the use of CD63 as a biomarker for monitoring the progression of MPS in a subject diagnosed with MPS. Viewed alternatively, these aspects provide a method for generating quantitative data for a subject diagnosed with MPS, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; and (c) comparing the level of CD63 determined in step (a) with the level determined in step (b). The method may comprise a further step (d) in which disease progression is assessed based on the comparison made in step (c). In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0214] The sample may be of the type mentioned herein. In embodiments, the sample contains lysosomes and / or exosomes. In embodiments, the sample is selected from blood, blood fractions, urine, cerebrospinal fluid, saliva, lymph, skin tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Most conveniently, the sample from the subject comprises (e.g., consists of) blood, blood fractions, and / or urine. In embodiments, the sample from the subject comprises (e.g., consists of) a blood fraction selected from plasma and serum. In embodiments, the sample is a blood sample, e.g., a plasma sample. In other embodiments, the sample comprises (e.g., consists of) urine. In other embodiments, the sample comprises (e.g., consists of) CSF. In embodiments, the sample does not comprise fibroblasts.

[0215] In embodiments, the first and second (or subsequent) samples are obtained from the subject at least about one day apart, e.g., at least about two, three, or four days, or at least about one, two, or four weeks apart. In other embodiments, the first and second (or subsequent) samples are obtained from the subject at least about eight weeks apart, e.g., at least about nine, ten, twelve, fifteen, or twenty weeks apart.

[0216] In embodiments, the method includes successive measurements of one or more further samples taken from the subject, for example at the intervals specified above.

[0217] Another aspect provides a method for monitoring the progress of treatment for MPS in a subject diagnosed with MPS, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering a therapeutic treatment for MPS to the subject; and (c) measuring the level of CD63 in a second sample from the subject, the second sample being obtained from the subject after the therapeutic treatment. Typically, the method further comprises step (d) of determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample. In embodiments, the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment should be increased if the level of CD63 determined in step (c) is substantially the same as or greater than the level of CD63 determined in step (a). A related aspect provides the use of CD63 as a biomarker for monitoring the progress of treatment for MPS in a subject diagnosed with MPS. Viewed another way, these aspects provide a method for generating quantitative data about a subject with MPS, the method comprising: (a) determining the level of CD63 in a first sample from the subject; and (b) determining the level of CD63 in a second sample from the subject after administering a therapeutic treatment for MPS to the subject. In embodiments, the dosage of the therapeutic treatment should be increased if the level of CD63 determined in step (b) is substantially the same as or greater than the level of CD63 determined in step (a). In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., type IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., type IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., type IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0218] In embodiments, the treatment for MPS is as described herein. Accordingly, the treatment may include (e.g., consist of) enzyme replacement therapy, gene therapy, or hematopoietic stem cell transplantation. In embodiments, the MPS is MPS I, MPS II, MPS IV, MPS VI, or MPS VII, and the treatment includes (e.g., consists of) enzyme replacement therapy and / or hematopoietic stem cell transplantation. In embodiments, the therapeutic treatment or medicament comprises (e.g., consists of) enzyme replacement therapy with α-L-iduronidase, iduronidase-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase, α-glucosaminidase, acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-6-sulfate sulfatase, β-galactosidase, N-acetylglucosamine-4-sulfatase, β-glucuronidase, or hyaluronidase. In embodiments, the MPS is MPS I, and the treatment comprises (e.g., consists of) administering recombinant α-L-iduronidase (e.g., laronidase) to the subject. In other embodiments, the MPS is MPS II and the treatment comprises (e.g., consists of) administering to the subject a recombinant iduronidase-2-sulfatase (e.g., idursulfase). In other embodiments, the MPS is MPS IV A and the treatment comprises (e.g., consists of) administering to the subject a recombinant N-acetylgalactosamine-6-sulfatase (e.g., elosulfase alfa); the MPS is MPS VI and the treatment comprises (e.g., consists of) administering to the subject a recombinant N-acetylglucosamine-4-sulfatase (e.g., galsulfase); or the MPS is MPS VII and the treatment comprises (e.g., consists of) administering to the subject a recombinant β-glucuronidase (e.g., bestronidase alfa).

[0219] The second sample is typically collected a period of time after the therapeutic treatment. In embodiments, the second sample is collected from the subject at least about one week after the therapeutic treatment (e.g., after initiation), for example, at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the therapeutic treatment. In one embodiment, the second sample is collected from the subject at least about 8 weeks after the initiation of the therapeutic treatment.

[0220] In embodiments, the method includes serial measurements of one or more additional samples taken from the subject, e.g., at intervals of about every week, e.g., about every 2, 4, 6, 8, 10, or 12 weeks. This serial measurement can be particularly useful when the dosage of therapy is being adjusted based on changes in CD63 levels in the subject.

[0221] Another aspect provides a method of treating MPS in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level above the mean for a population of healthy subjects (e.g., a level of at least about 0.25 standard deviations, e.g., at least about 0.5, 0.75, 1, or 1.5 standard deviations), the method comprising administering to the patient an effective amount of a therapeutic treatment for MPS. In embodiments, the healthy population of subjects is a population with a similar genetic background, e.g., a population in the same genetic and / or geographic location as the patient. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., type IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., type IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., type IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0222] A further aspect is a method of treating MPS in a subject diagnosed as being at risk for developing the disease, wherein the patient has a plasma CD63 level that is higher than a control level in a sample previously collected from the subject (e.g., at least about 10% higher than the plasma control level, e.g., at least about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher), the method comprising administering to the subject an effective amount of a therapeutic treatment for MPS (e.g., as described herein). In embodiments, the plasma control level is from a sample collected from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level. In embodiments, the control value is derived from measuring CD63 levels in a single sample collected from the subject. In other embodiments, the control value is derived from measuring CD63 levels in multiple samples (e.g., 2, 3, 4, 6, 8, or more samples) obtained from the subject. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., type IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., type IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0223] A further aspect provides a method of reducing CD63 levels in a patient (e.g., in the patient's blood) suffering from MPS, the method comprising administering to the patient an effective amount of a therapeutic treatment (e.g., enzyme replacement therapy as described herein). In embodiments, the reduction in CD63 is a reduction in plasma CD63 levels. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0224] Another aspect provides a method of treating or preventing the onset or progression of MPS in a subject assessed to be at risk for MPS, the method comprising: (a) obtaining a first biological sample (e.g., blood or a blood fraction such as plasma) from the subject and analyzing the sample for CD63 concentration; (b) initiating a course of therapeutic treatment in the subject (e.g., a therapeutically effective amount of enzyme replacement therapy, such as those described herein) if the CD63 concentration is above a control value; and, optionally, (c) obtaining a second biological sample (e.g., of the same type as the first biological sample) from the subject after treating the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., type IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0225] In embodiments, the control value is about 10% higher (e.g., about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher) than the control level in a sample previously collected from the same subject. In embodiments, the control level is derived from measuring the CD63 level in a single sample collected from the subject. In other embodiments, the control value is derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8, or more samples) obtained from the subject. In embodiments, the plasma control level is from a sample collected from the subject about 1 to about 52 weeks (e.g., 4 to 32 weeks, or 12 to 26 weeks, e.g., about 12, 16, 22, 26, or 30 weeks) prior to measuring the plasma CD63 level.

[0226] In embodiments, the treatment for MPS is as described herein. Accordingly, the treatment may include (e.g., consist of) enzyme replacement therapy, gene therapy, and / or hematopoietic stem cell transplantation. In embodiments, the treatment includes (e.g., consists of) enzyme replacement therapy with α-L-iduronidase, iduronidase-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase, α-glucosaminidase, acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-6-sulfate sulfatase, β-galactosidase, N-acetylglucosamine-4-sulfatase, β-glucuronidase, or hyaluronidase.

[0227] The second sample is typically collected a period of time after the therapeutic treatment. In embodiments, the second sample is collected from the subject at least about 1 week after the therapeutic treatment (e.g., after the start of the therapeutic treatment), for example, at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the therapeutic treatment. In one embodiment, the second sample is collected from the subject at least about 8 weeks after the start of the therapeutic treatment.

[0228] In embodiments, the method includes serial measurements of one or more additional samples taken from the subject, e.g., at intervals of about one week, e.g., about every 2, 4, 6, 8, 10, or 12 weeks. This serial measurement can be particularly useful when the dosage of therapy is adjusted based on changes in CD63 levels in the subject.

[0229] Another aspect provides a method for adjusting the dosage of a therapeutic treatment for MPS in a subject receiving said therapeutic treatment, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, where the second sample is obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample. Typically, adjusting the dosage in step (c) comprises maintaining the dosage of the therapeutic treatment if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, and increasing the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment if the level of CD63 in the second sample is substantially the same as or greater than the level of CD63 in the first sample. In embodiments, if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, the dosage (e.g., amount and / or frequency of administration) of the therapeutic treatment is reduced. A related aspect provides the use of CD63 as a biomarker for adjusting the dosage of a therapeutic treatment for MPS in a subject receiving said therapeutic treatment. In embodiments, the MPS is selected from MPS I, MPS II, and MPS III. In embodiments, the MPS is MPS I (e.g., type IH, IS, or IH / S). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., type IIIA, IIIB, IIIC, or IIID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (e.g., MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (e.g., MPS IIIA).

[0230] Assay Assays that can be used to measure and quantify CD63 levels in biological samples are known. Specific examples of suitable assays are provided in the Examples below. Generally speaking, the assays rely on having a molecule (e.g., an antibody) that specifically binds to CD63, and a molecule that can itself be detected and / or quantified directly or via another binding partner (e.g., using a labeled universal antibody).

[0231] Thus, in embodiments, detection and / or quantification of CD63 is performed by immunoassays (e.g., ELISA, microfluidic ELISA, or bead-based high-sensitivity ELISA, or proximity extension assay), mass spectrometry, microchips, biophysical assays (e.g., surface plasmon resonance using an anti-CD63 capture antibody, such as Biocore®), nanoneedle bioarrays (e.g., from Nanomosaic®), or nucleic acid-binding aptamers (e.g., from Somalogic®). Exemplary embodiments utilize ELISAs, such as indirect or sandwich ELISAs, in which a primary or capture antibody specifically binds to CD63 (e.g., a mouse antibody that binds to human CD63) and a secondary or detection antibody binds to the first antibody (e.g., a goat anti-mouse IgG) or to a different portion of CD63 and is labeled for detection and / or quantification. The primary or capture antibody is typically a monoclonal antibody capable of reacting with CD63 from one species or more than one species. Such antibodies are widely available from commercial sources (e.g., Invitrogen®, AbCam®, etc.). Furthermore, the sequence of CD63 is well known, and one of skill in the art can produce monoclonal antibodies to CD63 using standard techniques (see also "Leukocyte and Stromal Cell Molecules: The CD Markers", Wiley; Ed. Zola et al., 2007, pp. 150). Another exemplary embodiment utilizes cell sorting techniques, e.g., FACS, using fluorescently labeled antibodies to CD63, CD8, and / or CD81.

[0232] An alternative exemplary embodiment utilizes a proximity extension assay, using a matched pair of CD63-binding antibodies, each of which is labeled with a unique oligonucleotide such that the oligonucleotide hybridizes when the antibodies bind to their target. The annealed products are then amplified by PCR and detected (e.g., in a multiplexed format), typically in a high-throughput fluidic chip system.

[0233] A sample for use according to the present method can be, for example, purified and / or separated from other (e.g., non-CD63-containing) components in the sample. The sample can also be enriched for a component of interest, such as exosomes. Methods for enriching and / or purifying exosomes and other CD63-containing components are known in the art and / or described herein. As an example, a sample can be enriched for exosomes by ultracentrifugation and / or antibody capture methods. Ultracentrifugation of plasma, serum, urine, or cell culture samples can include, for example, centrifugation at 100,000 g to pellet exosomes and resuspension in an appropriate buffer. Affinity capture methods can, for example, use antibody-coated beads to capture targets on exosome membranes, followed by recovery of the beads by centrifugation or use of a magnet.

[0234] kit A further aspect provides a kit for (e.g., suitable for) detecting or diagnosing lysosomal dysfunction or aberrant glycosphingolipid processing in a sample from a subject, the kit comprising means for detecting CD63 and, optionally, means for detecting one or more additional biomarkers of lysosomal dysfunction. In embodiments, the means for detecting CD63 comprises an anti-CD63 antibody (e.g., a monoclonal antibody). In embodiments, the means for detecting CD63 comprises a pair of anti-CD63 antibodies, each of which is linked to a unique oligonucleotide, such that the unique oligonucleotide can hybridize when the antibody pair binds to CD63.

[0235] Another aspect provides a kit for (e.g., suitable for) monitoring the progression of a particular lysosomal storage disease or the response of a particular lysosomal storage disease to treatment in a subject, the kit comprising a means for detecting CD63 and, optionally, a means for detecting one or more additional biomarkers of lysosomal dysfunction. In embodiments, the means for detecting CD63 comprises an anti-CD63 antibody (e.g., a monoclonal antibody). In embodiments, the means for detecting CD63 comprises a pair of anti-CD63 antibodies, each of which is linked to a unique oligonucleotide, such that the unique oligonucleotide can hybridize when the antibody pair binds to CD63.

[0236] Yet another aspect provides a kit for (e.g., suitable for) detecting or diagnosing a particular lysosomal storage disease in a subject, the kit comprising: (a) means for detecting CD63 in a sample from the subject; and (b) means for detecting one or more biomarkers of the lysosomal storage disease in the sample from the subject. In embodiments, the means for detecting CD63 comprises an anti-CD63 antibody (e.g., a monoclonal antibody). In embodiments, the means for detecting CD63 comprises a pair of anti-CD63 antibodies, each linked to a unique oligonucleotide, such that the unique oligonucleotide can hybridize when the antibody pair binds to CD63.

[0237] In embodiments, the lysosomal storage disease is Fabry disease, and the kit comprises (a) means for detecting CD63 in a sample from the subject (e.g., an anti-CD63 antibody, or a matched pair of anti-CD63 antibodies, each linked to a unique oligonucleotide); and (b) means for detecting one or more biomarkers of Fabry disease in the sample (e.g., means for detecting GL3 and / or lyso-GL3 in the sample).

[0238] In embodiments, the lysosomal storage disease is Gaucher disease, and the kit comprises (a) means for detecting CD63 in a sample from the subject (e.g., an anti-CD63 antibody, or a matched pair of anti-CD63 antibodies, each linked to a unique oligonucleotide); and (b) means for detecting one or more biomarkers of Gaucher disease in the sample (e.g., means for detecting lyso-GL1 in the sample).

[0239] In embodiments, the lysosomal storage disease is an MPS (e.g., MPS I, MPS II, or MPS III), and the kit comprises (a) means for detecting CD63 in a sample from a subject (e.g., an anti-CD63 antibody, or a matched pair of anti-CD63 antibodies, each linked to a unique oligonucleotide); and (b) means for detecting one or more biomarkers of that MPS in the sample, e.g., means for detecting dermatan sulfate and optionally heparan sulfate in the sample (e.g., in the case of MPS I), means for detecting dermatan sulfate and heparan sulfate in the sample (e.g., in the case of MPS II), or means for detecting heparan sulfate in the sample (e.g., in the case of MPS III).

[0240] Kits of the present disclosure may further include instructions for use of the kit in one or more of the methods described herein, e.g., one or more methods for detecting and / or diagnosing lysosomal storage diseases, lysosomal dysfunction, or aberrant glycosphingolipid processing in a biological sample.

[0241] Having generally described herein, the following non-limiting examples are provided to further illustrate the present disclosure. [Example]

[0242] Experimental protocol Protein assays CD63 was measured by Olink® profiling as described below using the Olink® Target96 Neuro-Exploratory (Olink Proteomics AB, Uppsala, Sweden) according to the manufacturer's instructions.

[0243] Chitotriosidase activity was assayed according to the method described by Schoonhoven, et al., Clin Chim Acta. (2007) 381(2):136-139, with minor modifications. Briefly, 5 μL of serum sample was mixed with 100 μL of 26 μM 4-methylumbelliferyl-β-DN,N',N"-triacetylchitotrioside (Sigma, M5639) in 0.1 M / 0.2 M citrate-phosphate buffer. The mixture was incubated at 37°C for 15 min. To stop the reaction, glycine-sodium hydroxide buffer (210 μL of 0.5 M Gly-NaOH, pH 10.6) was added. Fluorescence was measured at 360 nm excitation and 455 nm emission using a plate reader (Biotek®). Chitotriosidase activity could also be assayed according to the method described by Adelino et al., JIMD Rep. (2013) 9:85-91 and was reported in nmol / h / ml.

[0244] CCL18 levels were measured by sandwich ELISA according to the method described in Boot, et al., Blood (2004) 103(1):33-39.

[0245] Protein detection can be performed using Olink® (Olink Proteomics AB, Uppsala, Sweden) profiling according to the manufacturer's recommended protocol (for exemplary methods, see, e.g., Assarsson et al., PLoS ONE (2014) 9(4):e95192; and Jabbari et al., Journal of Neurology, Neurosurgery & Psychiatry (2019) 90:768-773). The proximity extension assay (PEA) technology used in the Olink® protocol has been well described (Assarsson et al., 2014, supra) and allows for the simultaneous analysis of 92 specimens per panel, using 1 μL of each sample. Briefly, pairs of oligonucleotide-labeled antibody probes bind to their target proteins, and when the two probes are brought into close proximity, the oligonucleotides hybridize in pairs. Addition of DNA polymerase results in a proximity-dependent DNA polymerase event, generating a unique PCR target sequence. The resulting DNA sequences can then be detected and quantified using a microfluidic real-time PCR instrument (Biomark HD, Fluidigm). Internal extension and interplate controls are then used to quality control and normalize the data, adjusting for intra- and inter-run variability. The final assay readout is presented as protein relative quantification (NPX), an arbitrary unit on the log2-scale where higher values correspond to higher protein expression. All assay validation data (e.g., detection limits, intra- and inter-assay precision data, etc.), experimental protocols, and data processing are available on the manufacturer's website (www.olink.com).

[0246] The PEA assay can be calibrated to quantify protein levels, e.g., CD63. Typically, this involves preparing samples of known concentration (e.g., by titration in the buffer defined above) and generating an absolute concentration standard curve. To facilitate correct folding and / or post-translational modifications (e.g., glycosylation), recombinant human CD63 produced in a mammalian expression system is typically obtained (e.g., from a commercial supplier), although the protein can also be expressed in baculovirus or Escherichia coli (E. coli) systems using known techniques. Concentration can be determined using well-known methods, for example, using a bicinchoninic acid (BCA) assay with a BSA sample of known concentration. Concentration can also be determined by measuring the total amino acid concentration after protein hydrolysis. A suitable range of decreasing protein concentrations is selected to be within the dynamic range of the Olink® assay. For example, 3-fold dilutions can be used to obtain samples with protein concentrations of 1, 3, 9, 12, 36, 108, 324, and 972 pg / ml. A 0 pg / ml sample (i.e., simply buffer) is used as a blank to determine the background. Once calibrated using samples of known CD63 concentration, the assay is used to test human patient samples in the same assay run. Human patient samples are typically run undiluted, but can be diluted with a known amount of buffer whose protein levels fall outside the range of the calibration curve. The absolute concentration of CD63 in the test sample can be calculated by reference to the calibration curve—by comparing Olink® NPX values (e.g., average of triplicate measurements)—by applying a linear fit to the calibration sample values versus pg / ml, or by curve fitting, such as a four-parameter nonlinear curve fit, as needed, to obtain a good curve fit. If the NPX value of the test sample is within the dynamic range of the calibration curve, e.g., higher than the 0 pg / ml blank level with acceptable variability (e.g., within 20% CV), the absolute concentration can be accurately determined by reference to the fitted calibration curve. In this way, the PEA assay can provide absolute quantification of protein levels (in pg / ml).

[0247] For the measurement of protein levels in CSF, the following protocol was used: Biomarker expression in CSF was measured using four Olink® Explore panels (cardiometabolic, inflammatory, neurological, and oncological) as described, for example, by Wik et al. ("Proximity Extension Assay in Combination with NextGeneration Sequencing for High-throughput Proteome-wide Analysis, 2021, Mol Cell Proteomics 20, 100168). Briefly, 10 μL of CSF samples and Olink controls were placed in a 384-well sample source plate and then transferred to a Mosquito and Dragonfly liquid handler (both SPT The samples were diluted to 1:10, 1:100, 1:1000, and 1:100,000 in a 384-well sample dilution plate using a PCR probe (from Labtech). For the immunoreaction, these diluted samples were mixed with Olink® probes (DNA oligo-conjugated antibodies) in a 384-well incubation plate using a Mosquito handler and incubated at 4°C for 16 to 24 hours. The next day, the reagent mixture for the first PCR amplification step (PCR1 step) was added to the immunoreaction mixture using a Dragonfly handler, and the PCR1 reaction for the pre-amplification step was performed using a ProFlex PCR system (Applied Biosciences). The PCR1 product was then transferred to an epMotion The PCR2 products were pooled using a 5075 liquid handler (Eppendorf), and then the same handler was used to add reagents for the second PCR amplification step (PCR2 step), and the PCR2 reaction for amplification and sample indexing were performed using the ProFlex system. The PCR2 products were pooled using an epMotion handler to create four libraries for each Olink® Explore panel, which were then purified using magnetic beads and their QC was performed using a Bioanalyzer 2100 (Agilent).Four libraries were analyzed by next-generation sequencing using a NovaSeq 6000 (Illumina). Samples with more than 10% missing data were excluded from the analysis. No imputation was performed for missing data.

[0248] Alternative methods may be used to measure levels of proteins such as CD63, including immunoassays using antibodies to CD63 (e.g., widely available ELISAs, microfluidic ELISAs such as Protein Simple Ella, or sensitive ELISAs based on SIMOA Quanterix), biophysical methods using anti-CD63 antibodies to capture CD63 (e.g., Biacore surface plasmon resonance), and nanoneedles (e.g., Nanomosaic) or nucleic acid-binding aptamers (e.g., Somalogic). Plasma, serum, cell, and tissue samples for conventional ELISA assays are typically diluted 4-fold or more in sample dilution buffer to obtain acceptable assay performance (dilution linearity).

[0249] Assays for glycosphingolipids Glycosphingolipids (lyso-GL1, GL3, and lyso-GL3) were measured by ESI-LC-MS / MS according to the method described by Murugesan et al., Am J Hematol. (2016) 91(11):1082-1089. Briefly, 20 μL of plasma aliquots were added to 1 mL of chloroform:methanol (2:3) in an Eppendorf tube and then centrifuged. The supernatant was removed and extracted with chloroform (220 μL) and water (520 μL) by mixing and centrifugation. The upper phase was re-extracted with chloroform and added to the lower phase. The combined samples were dried, resuspended in 100 μL of methanol:water (9:1), and injected into the LC-MS / MS system for tandem mass spectrometry. Separation of glycosphingolipids and other matrix components was achieved using UPLC under gradient conditions with two mobile phases: 0.1% formic acid in water; and 0.1% formic acid in acetonitrile. Mass spectrometry (MS) was performed, typically with parent (M+H + The assay was performed in selected ion monitoring mode using m / z 462.5 > 282.4 for lyso-GL1. The assay was performed using known glycosphingolipid standards (lyso-GL1, lyso-GL3, and dimethylpsychosine as an internal standard for GL3, respectively). 13 Calibration was performed using C6-labeled lyso-GL3 (GelbChem) and C17-GL3 (Matreya LLC, Cat. No. 1523).

[0250] Sample preparation Preparation of plasma and serum samples was performed according to standard protocols. Cell samples were prepared by mixing / agitation. Tissue samples were solubilized by homogenization in a standard buffer (e.g., PBS, optionally containing a mild detergent such as 0.1% TWEEN 20 and / or a carrier protein such as 1% BSA (which may improve stability and / or reproducibility); or 50 mM Tris / HCl (pH 7.5)). To aid solubilization, additional detergent, e.g., up to 1% Triton, was typically added to the tissue sample at a higher concentration. Plasma and serum samples were typically run undiluted in the Olink® assay. Preparation of cell and tissue samples involves the addition of buffer to a final protein concentration within the dynamic range of the Olink® assay.

[0251] The sample to be tested can be enriched for exosomes using, for example, FACS as described herein. Alternative methods for exosome enrichment or purification include ultracentrifugation and antibody capture methods, both of which are known and commercially available. Standard ultracentrifugation for the preparation of exosomes from plasma / serum, urine, or cell cultures involves, for example, centrifugation at 100,000 g for 1 hour in a typical floor-standing ultracentrifuge using a typical rotor and buckets that hold 10 ml to 50 ml of sample, or centrifugation for 5 minutes in a benchtop ultracentrifuge using 0.5 ml or 1.5 ml sample tubes. Exosomes are pelleted to the bottom of the tube and resuspended in an appropriate buffer. Commercially available kits (e.g., from System Biosciences Inc.) are available for antibody affinity capture of exosomes and exosome surface / membrane proteins. This method typically uses antibody-coated beads to capture exosome targets, followed by bead recovery by centrifugation or use of a magnet.

[0252] Analysis of patient and control samples for biomarkers of LSD Plasma from Fabry disease patients and normal controls Plasma from patients with FD was obtained from three different cohorts—hereafter referred to as (i) ACT / LTS, (ii) Cohort 1, and (iii) Cohort 2.

[0253] ACT / LTS samples were obtained from the Sanofi-benglustat Phase II study. This study was calibrated with male patients only. Demographics are shown in Table 1 below. The Sanofi-benglustat Phase II study is a longitudinal study, and patient plasma was collected at six set intervals: baseline (week 0), 12 weeks, 26 weeks, 52 weeks, 104.2 weeks, and 156.4 weeks. Some patients had missing time points. All patients were treated with benglustat (substrate reduction therapy - SRT) during this study.

[0254] Cohort 1 is a collection of plasma samples provided by Dr. Gavin Oudit at the University of Alberta. Patient demographic information is provided in Table 1 below. Briefly, these plasma samples were from a mix of male and female patients ranging in age from 25 to 68 years. They were non-serialized samples (e.g., one sample per patient), and the plasma samples were a mix of treated (enzyme replacement therapy - ERT) and untreated Fabry patients, as well as healthy control subjects.

[0255] Cohort 2 is a collection of plasma samples provided by Dr. Michael Mauer at the University of Minnesota. Patient demographic information is provided in Table 1 below. Briefly, these plasma samples were from a mix of male and female patients ranging in age from 4 to 59 years. They were mostly non-serial samples (e.g., <3 samples per patient). There were some serial samples with and without baseline. Plasma samples were from a mix of treated (enzyme replacement therapy - ERT) and untreated Fabry disease patients.

[0256] Healthy controls for the Fabry disease plasma samples were mostly from commercial sources (Sanguine Bioscience and BioIVT). These plasma samples were age- and sex-matched to the Fabry disease cohort, and their demographic information is provided in Table 1 below. Additional healthy controls were provided along with the Cohort 1 samples.

[0257] [Table 2]

[0258] Sera from Gaucher disease patients and normal controls Samples from the Gaucher disease cohort were obtained from Yale University (Prof. Pram Mistry). Patient demographic information is provided in Table 2 below. Briefly, the Gaucher disease cohort consisted of 89 samples from 21 patients, both male and female. These samples spanned a large age range, with a mean age of 50.5 years. Only two of the patients had baseline samples, while 15 patients had longitudinal data. The majority of the longitudinal data were from samples obtained several years after initial treatment. Most patients were treated with ERT, although some patients were switched to SRT at some point during treatment.

[0259] Healthy control samples were obtained from commercial (BioIVT) and internal (Genzyme donor program samples, non-clinical and internal clinical trials) sources. Patient demographic information is provided in Table 2 below. Briefly, there were 43 samples from 43 healthy donors and no longitudinal healthy control samples. The mean age of healthy control patients for the Gaucher disease cohort was 45 years, with a broad age range similar to that of the affected samples.

[0260] [Table 3]

[0261] Plasma from MPS patients and normal controls Patient demographic information for the MPS cohort (samples provided by Dr. Giugliani, University of Porto Alegre) is provided below in Table 3. Briefly, all patients were young, with a mean age of approximately 6 years. There were 28 samples for 28 patients, and no longitudinal samples were obtained for this cohort. The samples were from a mix of male and female subjects. Furthermore, none of the patients were receiving treatment, so they were all baseline disease samples. Patient disease represented an approximately equal distribution of MPS I, MPS II, and MPS IIIA.

[0262] Demographic information for patients in the healthy subject cohort (samples from BioIVT) is provided below in Table 3. Patients in the healthy control cohort were age-matched to the diseased patients, and all were pediatric samples with an average age of approximately 6 years. In total, there were 19 healthy control samples from 19 patients, which were a mix of both male and female donors.

[0263] [Table 4]

[0264] CSF from patients with MPS and Gaucher disease (type 3) Although serum biomarkers can provide insight into systemic changes, analysis of cerebrospinal fluid (CSF) biomarkers may provide a direct and accurate assessment of neural processes occurring within the CNS. Therefore, we investigated the potential of CD63 as a CSF biomarker for detecting MPS, detecting GD3, and assessing treatment response in GD3.

[0265] To ensure consistency and reliability, CSF samples were meticulously collected from consenting patients at each center according to center-specific protocols. To minimize batch effects, samples were randomized during plating based on center, sex, and disease status. Olink proteomics methods (described above) were used to profile changes in protein biomarkers within the CSF samples.

[0266] Patient demographic information for the MPS cohort is shown in Table 4 below. Because our data suggested no significant age-related differences in CD63, healthy adult CSF samples served as reference controls to allow for comparative analysis. Controls were not age-matched (pediatric CSF control samples were not readily available).

[0267] [Table 5]

[0268] Patient samples for the GD3 study were collected as part of the Sanofi-sponsored LEAP trial, and informed patient consent was obtained (see, e.g., Schifffmann et al., Brain (2023) 146(2):461-474 for details). Samples from 11 adult patients were obtained by lumbar puncture at baseline and at weeks 26 and 52 when the patients were receiving benglustat and imiglucerase combination treatment (Cerezyme®, Sanofi, Cambridge USA). CSF samples from healthy adults were used as controls. Changes in protein biomarkers in the CSF samples were analyzed using the Olink proteomics method (described above).

[0269] Results and Conclusions Assay results performed on Fabry disease samples demonstrate that CD63 is significantly upregulated in the plasma of untreated Fabry disease patients ("Baseline" in the longitudinal study shown in Figure 1A). Upon treatment, CD63 levels decline toward healthy levels over a 30-month period (Figure 1A; "Linear NPX"). A response to treatment is observed after 12 weeks, and the subsequent decline in CD63 levels remains consistent over several years (Figure 2; showing a linear decline in estimated marginal mean (log2) values of CD63 from 12 to 156.4 weeks). The CD63 response to benglustat treatment is comparable to the standard Fabry disease biomarkers GL3 and lyso-GL3 in terms of their interpatient variability (Figure 3A). Intrapatient variability is much lower for CD63 and lyso-GL3 than for GL3 (Figure 3C). When analyzed separately, the CD63 response appears to be greater in male Fabry patients than in female patients (Fig. 8A).

[0270] Analysis of samples from the Gaucher disease cohort demonstrates that CD63 is also significantly upregulated in the plasma of Gaucher disease patients compared with healthy controls (Figure 4). Samples from male Gaucher disease patients appear to have little difference in CD63 levels compared with female Gaucher disease patients (Figure 8B). The day-to-day variability of CD63 levels in Gaucher disease patients receiving long-term treatment (Figure 5A) is lower than that of other established biomarkers, such as lyso-GL1 (Figure 5B) or chitotriosidase (Figure 5C). This low level of intrapatient variability is highly significant (Figure 6C; p<0.002), establishing CD63 as an exceptional biomarker for monitoring the status and progression of LSD, especially compared with the known biomarkers lyso-GL1 and chitotriosidase. Furthermore, CD63 levels correlate with the levels of known biomarkers for Gaucher disease (Figure 6D).

[0271] CD63 levels are also significantly elevated in MPS patients (Figure 7A) and in both males and females (Figure 8C). Although there appears to be no statistically significant difference between the mean CD63 levels in male and female patients, the different distribution profiles may suggest some differences in subtypes within each population (Figure 7B, p>0.05; see also Figure 8C). This observation further supports the use of CD63 as a general biomarker for detecting and / or monitoring LSDs.

[0272] Olink® profiling of samples from male adult Fabry patients showed that CD63 was the most significantly upregulated protein compared to healthy controls (FIG. 9).

[0273] Analysis of data collected from MPS CSF samples revealed compelling results for CD63. Figure 10 (box plot) shows a substantial increase in CD63 levels among MPS patients (NPX values = -3.186 and -3.037 for MPS I and MPS II, respectively) compared to the control group (NPX value = -6.402). The p-values for the differences between MPS I / MPS II and healthy controls are all <0.001. These findings strongly support the feasibility and clinical utility of CD63 as a biomarker for MPS in CSF.

[0274] The GD3 samples measured over time also showed a significant increase in CD63 levels in the CSF at baseline compared with the mean of healthy controls (Figure 11A), as well as a decrease over time with treatment. The same trend was observed in individual patients (Figure 11B). Notably, there was a substantial increase in CD63 levels in baseline GD3 patients compared with healthy controls (Figure 11A), with NPX values of -6.1 and -4.3 for controls and GD3 patients, respectively. Even without considering disease severity, statistical analysis showed high significance (p-value of 0.0036), demonstrating a clear difference between the two groups. Furthermore, treatment with benglustat, an investigational brain-penetrating glucosylceramide synthase inhibitor, consistently resulted in a decrease in CD63 levels, indicating the efficacy of treatment. Furthermore, changes in CD63 showed a positive correlation with changes in lysoGL-1 in the CSF, further supporting the potential of CD63 as a diagnostic marker for GD3 in the CSF.

[0275] These findings highlight the potential utility of CD63 as a CSF biomarker for the detection and monitoring of GD3. Assessing CD63 levels in the CSF can provide valuable insight into GD3 disease progression and treatment response.

[0276] The above results indicate that CD63 is a useful biomarker for assessing the pathology of lysosomal storage diseases in general, and in particular for LSDs involving the lysosomal glycosphingolipid pathway (for which no single biomarker is currently available). The ready availability of simple assays for quantifying and monitoring CD63, such as ELISA, and its demonstrated use in blood samples, make CD63 a particularly attractive biomarker in this context.

[0277] Additionally, when a feature or aspect is described in terms of a Markush group, one skilled in the art will recognize that such feature or aspect is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0278] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

Claims

1. 1. A method for diagnosing a subject as suffering from or at risk of suffering from a lysosomal storage disease (LSD), the method comprising measuring the level of CD63 in a sample from the subject, wherein CD63 is the only biomarker used in the method.

2. 10. The method of claim 1, wherein the subject is diagnosed with or at risk for a lysosomal storage disorder, including Fabry disease, Gaucher disease, MPS I, MPS II, and MPS III.

3. A method for detecting or diagnosing lysosomal dysfunction in a subject, said method comprising measuring the level of CD63 in a sample from said subject, wherein CD63 is the only biomarker used in said method.

4. A method for detecting or diagnosing abnormal glycosphingolipid processing in a subject, said method comprising measuring the level of CD63 in a sample from said subject, wherein CD63 is the only biomarker used in said method.

5. 1. A method of generating quantitative data about a subject, said method comprising determining the level of a single biomarker in a sample from said subject, said biomarker being CD63.

6. 6. The method of any one of claims 1 to 5, wherein the subject has not been previously diagnosed with an LSD and / or has not been evaluated for risk factors associated with lysosomal dysfunction or aberrant glycosphingolipid processing.

7. The method of any one of claims 1 to 6, wherein the sample comprises (e.g. consists of) a blood fraction selected from plasma and serum.

8. 8. The method of any one of claims 1 to 7, wherein the subject is diagnosed as suffering from or at risk of suffering from a lysosomal storage disease, or as having a lysosomal storage defect or abnormal glycosphingolipid processing, if the level of CD63 is greater than a control value, wherein the control value is measured as the CD63 level in a sample taken from the same subject at an earlier time point, or from one or more healthy subjects.

9. Use of CD63 as a biomarker in diagnosing a lysosomal storage disease in a subject, detecting or diagnosing lysosomal dysfunction in a subject, or detecting or diagnosing abnormal glycosphingolipid processing in a subject, wherein CD63 is used as the only biomarker in said detection or diagnosis.

10. 1. A method for diagnosing Fabry disease in a subject suspected of being at risk of having Fabry disease, said method comprising measuring the level of CD63 in a sample from said subject.

11. 11. The method of claim 10, wherein the subject is suspected of being at risk for developing Fabry disease as a result of exhibiting one or more of the following: a family history of Fabry disease, fatigue, pain, lens or corneal opacities, vortex keratopathy, angiokeratoma, dyspnea, palpitations, edema, renal disease, myocardial dysfunction, cardiac conduction abnormalities with shortened PR interval, cardiac arrhythmias, dizziness, headache, crossed diplopia, dysarthria, hemiataxia, transient ischemic attack, early-onset stroke, and dementia.

12. The method of claim 10 or 11, wherein the subject is diagnosed with Fabry disease if the level of CD63 in the sample from the subject is greater than a control value, the control value being measured as the CD63 level in a sample taken from one or more healthy subjects.

13. 13. The method of claim 12, wherein the subject is diagnosed with Fabry disease if the level of CD63 measured in the sample from the subject is at least about 100% greater than the control value.

14. 1. A method for generating quantitative data about a subject, the method comprising (e.g. consisting of) determining the level of CD63 in a sample from the subject, the subject having or suspected of having Fabry disease.

15. The use of CD63 as a biomarker in the diagnosis of Fabry disease in subjects suspected of being at risk of having Fabry disease.

16. Use of CD63 as a biomarker to improve the diagnosis of Fabry disease in a subject, optionally wherein CD63 is used as a biomarker in parallel with GL3, lyso-GL3, and / or α-Gal activity.

17. 14. A method of treating a subject diagnosed with Fabry disease by the method of any one of claims 10 to 13, wherein said treating comprises administering to said subject one or more therapeutic treatments for Fabry disease.

18. 1. A method of treating Fabry disease in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level, the method comprising administering to the subject an effective amount of a therapeutic treatment for Fabry disease.

19. 1. A method for generating quantitative data about a subject, the method comprising: (a) determining the level of CD63 in a sample from said subject; (b) determining whether the level of CD63 is greater than a control value, wherein the control value is measured as the CD63 level in a sample taken from one or more healthy subjects; (c) administering to the subject one or more therapeutic treatments for Fabry disease if the level of CD63 in the sample is greater than the control value.

20. 20. The method of any one of claims 17 to 19, wherein the one or more therapeutic treatments comprise (e.g. consist of) substrate inhibition therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy.

21. 21. The method of claim 20, wherein the treatment comprises administering benglustat or migalastat, e.g., benglustat, to the subject.

22. 22. The method of claim 20 or 21, wherein the treatment comprises administering to the subject a recombinant alpha-galactosidase, such as agalsidase beta.

23. A therapeutic agent for the treatment of Fabry disease in a subject, wherein the subject has been diagnosed as having Fabry disease by the method of any one of claims 10 to 13.

24. The method of claim 23, wherein the therapeutic agent is a therapeutic treatment according to any one of claims 20 to 22.

25. 1. A method for monitoring the progression of Fabry disease in a subject diagnosed with Fabry disease, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after the first sample is obtained from the subject; and (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample; (d) determining that the subject's Fabry disease has become more severe if the level of CD63 in the first sample is greater than the level of CD63 in the first sample, determining that the subject's Fabry disease has not progressed if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample, and determining that the subject's Fabry disease is in remission if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

26. 1. A method for generating quantitative data for a subject diagnosed with Fabry disease, said method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

27. 27. The method of claim 25 or 26, wherein the sample is a blood sample, such as a plasma sample.

28. 1. A method for monitoring the progress of treatment for Fabry disease in a subject diagnosed with Fabry disease, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering to said subject a therapeutic treatment for Fabry disease; (c) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after the therapeutic treatment has been administered; and (d) determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

29. 1. A method of treating or preventing the onset or progression of Fabry disease in a subject assessed to be at risk of suffering from Fabry disease, said method comprising: (a) obtaining a first biological sample from the subject and analyzing said sample for CD63 concentration; (b) initiating a course of therapeutic treatment for Fabry disease in said subject if said CD63 concentration is above a control value; and optionally, (c) obtaining a second biological sample from the subject after treating the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; (d) adjusting said therapeutic treatment based on said observed change in CD63 levels.

30. 1. A method for adjusting the dosage of a therapeutic treatment for Fabry disease in a subject receiving said therapeutic treatment, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample.

31. 1. A method for generating quantitative data for a subject with Fabry disease, said method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a second sample from the subject after administering to the subject a therapeutic treatment for Fabry disease.

32. 32. The method of claim 31 , wherein the dosage of the therapeutic treatment should be increased if the CD63 level determined in step (b) is substantially the same as or greater than the CD63 level determined in step (a).

33. 33. The method of any one of claims 28 to 32, wherein the therapeutic treatment comprises (e.g. consists of) substrate inhibition therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy.

34. 34. The method of claim 33, wherein the treatment comprises administering benglustat or migalastat, e.g., benglustat, to the subject.

35. 34. The method of claim 33, wherein the treatment comprises administering to the subject a recombinant alpha-galactosidase, e.g., agalsidase beta.

36. 36. The method of any one of claims 25 to 35, wherein the second or subsequent sample is taken from the subject at least 8 weeks after initiation of the therapeutic treatment.

37. Use of CD63 as a biomarker for monitoring the progression of Fabry disease in a subject diagnosed with Fabry disease, or for monitoring the progress of treatment for Fabry disease in a subject diagnosed with Fabry disease, or for adjusting the dosage of therapeutic treatment for Fabry disease.

38. 1. A method for diagnosing Gaucher disease in a subject suspected of being at risk of suffering from Gaucher disease, said method comprising measuring the level of CD63 in a sample from said subject.

39. 39. The method of claim 38, wherein the subject is suspected of being at risk for Gaucher disease as a result of exhibiting one or more of the following: a family history of Gaucher disease, hepatomegaly and splenomegaly, pain, osteoporosis, skin pigmentation, pancytopenia, neurological symptoms, and Parkinson's disease.

40. 40. The method of claim 38 or 39, wherein the subject is diagnosed with Gaucher disease if the level of CD63 measured in the sample from the subject is greater than a control value, the control value being measured as the CD63 level in a sample taken from one or more healthy subjects.

41. 41. The method of claim 40, wherein the subject is diagnosed with Gaucher disease when the level of CD63 measured in the sample from the subject is at least about 100% greater than the control value.

42. 1. A method for generating quantitative data about a subject, the method comprising (e.g., consisting of) determining the level of CD63 in a sample from the subject, the subject having or suspected of having Gaucher disease.

43. The use of CD63 as a biomarker in the diagnosis of Gaucher disease in subjects suspected of being at risk for Gaucher disease.

44. 1. Use of CD63 as a biomarker to improve a method of diagnosing Gaucher disease in a subject, optionally wherein CD63 is used as a biomarker in parallel with GL1, lyso-GL1, and / or β-galactosidase (GCase) activity.

45. 42. A method of treating a subject diagnosed with Gaucher disease by the method of any one of claims 38 to 41, wherein said treating comprises administering to said subject one or more therapeutic treatments for Gaucher disease.

46. 1. A method of treating Gaucher disease in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level, the method comprising administering to the subject an effective amount of a therapeutic treatment for Gaucher disease.

47. 1. A method for generating quantitative data about a subject, the method comprising: (a) determining the level of CD63 in a sample from said subject; (b) determining whether the level of CD63 is greater than a control value, wherein the control value is measured as the CD63 level in a sample taken from one or more healthy subjects; (c) administering to the subject one or more therapeutic treatments for Gaucher disease if the level of CD63 in the sample is greater than the control value.

48. 48. The method of any one of claims 45 to 47, wherein the one or more therapeutic treatments comprise (e.g., consist of) substrate inhibition therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy.

49. 49. The method of claim 48, wherein the treatment comprises administering to the subject benglustat, eliglustat, or miglustat.

50. 50. The method of claim 48 or 49, wherein the treatment comprises administering a recombinant glucocerebrosidase, e.g., imiglucerase, to the subject.

51. A therapeutic agent for the treatment of Gaucher disease in a subject, wherein the subject has been diagnosed with Gaucher disease by the method of any one of claims 38 to 41.

52. The method of claim 51, wherein the therapeutic agent is a therapeutic treatment according to any one of claims 48 to 50.

53. 1. A method for monitoring the progression of Gaucher disease in a subject diagnosed with Gaucher disease, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after the first sample is obtained from the subject; and (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample; (d) determining that the subject's Gaucher disease has become more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample, determining that the subject's Gaucher disease has not progressed if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample, and determining that the subject's Gaucher disease has remitted if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

54. 1. A method for generating quantitative data for a subject diagnosed with Gaucher disease, said method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

55. 55. The method of claim 53 or 54, wherein the sample is a blood sample, such as a plasma sample.

56. 1. A method for monitoring the progress of treatment for Gaucher disease in a subject diagnosed with Gaucher disease, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering to said subject a therapeutic treatment for Gaucher disease; (c) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after the therapeutic treatment has been administered; and (d) determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

57. 1. A method for treating or preventing the onset or progression of Gaucher disease in a subject assessed to be at risk for Gaucher disease, said method comprising: (a) obtaining a first biological sample from the subject and analyzing said sample for CD63 concentration; (b) initiating a course of therapeutic treatment for Gaucher disease in said subject if said CD63 concentration is above a control value; and optionally, (c) obtaining a second biological sample from the subject after treating the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; (d) adjusting said therapeutic treatment based on said observed change in CD63 levels.

58. 1. A method for adjusting the dosage of a therapeutic treatment for Gaucher disease in a subject receiving said therapeutic treatment, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample.

59. 1. A method for generating quantitative data for a subject with Gaucher disease, said method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a second sample from the subject after administering to the subject a therapeutic treatment for Gaucher disease.

60. 60. The method of claim 59, wherein the dosage of the therapeutic treatment should be increased if the CD63 level determined in step (b) is substantially the same as or greater than the CD63 level determined in step (a).

61. 61. The method of any one of claims 56 to 60, wherein the therapeutic treatment comprises (e.g. consists of) substrate inhibition therapy, chaperone therapy, enzyme replacement therapy, or gene therapy.

62. 62. The method of claim 61, wherein the treatment comprises administering to the subject benglustat, eliglustat, or miglustat.

63. 62. The method of claim 61, wherein the treatment comprises administering a recombinant glucocerebrosidase, e.g., imiglucerase, to the subject.

64. 64. The method of any one of claims 53-63, wherein the second or subsequent sample is taken from the subject at least 8 weeks after initiation of the therapeutic treatment.

65. Use of CD63 as a biomarker for monitoring the progression of Gaucher disease in a subject diagnosed with Gaucher disease, or for monitoring the progress of treatment for Gaucher disease in a subject diagnosed with Gaucher disease, or for adjusting the dosage of therapeutic treatment for Gaucher disease.

66. A method for diagnosing MPS in a subject suspected of being at risk of suffering from MPS, said method comprising measuring the level of CD63 in a sample from said subject.

67. 67. The method of claim 66, wherein the subject is suspected of being at risk for MPS as a result of exhibiting one or more of the following: family history of MPS, macrocephaly, hearing loss, corneal opacities, malocclusion, rigidity, hip dysplasia, clawed hands, joint laxity, valvular hypertrophy, left ventricular hypertrophy, recurrent respiratory infections, obstructive airway disease, hepatomegaly / splenomegaly, umbilical / inguinal hernia, growth retardation, ventricular dilation, enlarged perivascular spaces, hyperactive or aggressive behavior, dysgranulogenesis in white blood cells, fetal hydrops, and proteinuria.

68. The method of claim 66 or 67, wherein the subject is diagnosed with MPS if the level of CD63 measured in the sample from the subject is greater than a control value, the control value being measured as the CD63 level in a sample taken from one or more healthy subjects.

69. 69. The method of claim 68, wherein the subject is diagnosed with MPS if the level of CD63 measured in the sample from the subject is at least about 100% greater than the control value.

70. A method for generating quantitative data about a subject, the method comprising (e.g., consisting of) determining the level of CD63 in a sample from the subject, the subject having or suspected of having MPS.

71. The use of CD63 as a biomarker in the diagnosis of MPS in subjects suspected of being at risk for MPS.

72. Use of CD63 as a biomarker to improve methods of diagnosing MPS in a subject, optionally wherein CD63 is used as a biomarker in parallel with one or more glycosaminoglycans (GAGs) or glycans.

73. 14. A method of treating a subject diagnosed with MPS by the method of any one of claims 10 to 13, wherein said treating comprises administering to said subject one or more therapeutic treatments for MPS.

74. 1. A method of treating MPS in a patient in need thereof, wherein the patient has a higher than normal plasma CD63 level, the method comprising administering to the subject an effective amount of a therapeutic treatment for MPS.

75. 1. A method for generating quantitative data about a subject, the method comprising: (a) determining the level of CD63 in a sample from said subject; (b) determining whether the level of CD63 is greater than a control value, wherein the control value is measured as the CD63 level in a sample taken from one or more healthy subjects; (c) administering to the subject one or more therapeutic treatments for MPS if the level of CD63 in the sample is greater than the control value.

76. 76. The method of any one of claims 73 to 75, wherein the one or more therapeutic treatments comprise (e.g. consist of) enzyme replacement therapy, gene therapy, and / or hematopoietic stem cell transplantation.

77. 77. The method of claim 76, wherein the treatment comprises (e.g., consists of) enzyme replacement therapy with α-L-iduronidase, iduronidase-2-sulfatase, or heparan-N-sulfatase.

78. A therapeutic agent for the treatment of MPS in a subject, wherein the subject has been diagnosed as having MPS by the method of any one of claims 66 to 69.

79. 79. The method of claim 78, wherein the therapeutic agent is a therapeutic treatment according to claim 76 or 77.

80. 1. A method for monitoring the progression of MPS in a subject diagnosed with MPS, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after the first sample is obtained from the subject; and (c) comparing the level of CD63 in the first sample with the level of CD63 in the second sample; (d) determining that the subject's MPS has become more severe if the level of CD63 in the second sample is greater than the level of CD63 in the first sample, determining that the subject's MPS has not progressed if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample, and determining that the subject's MPS has improved if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

81. 1. A method for generating quantitative data for a subject diagnosed with MPS, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a subsequent sample from the subject; (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

82. 82. The method of claim 80 or 81, wherein the sample is a blood sample, such as a plasma sample.

83. 1. A method for monitoring the progress of treatment of MPS in a subject diagnosed with MPS, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering to said subject a therapeutic treatment for MPS; (c) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after the therapeutic treatment has been administered; and (d) determining that the treatment is successful if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

84. 1. A method for treating or preventing the onset or progression of MPS in a subject assessed to be at risk for developing MPS, said method comprising: (a) obtaining a first biological sample from the subject and analyzing said sample for CD63 concentration; (b) initiating a course of therapeutic treatment for MPS in said subject if said CD63 concentration is above a control value; and optionally, (c) obtaining a second biological sample from the subject after treating the subject and analyzing the sample for CD63 concentration to determine a change in CD63 levels; (d) adjusting said therapeutic treatment based on said observed change in CD63 levels.

85. 1. A method for adjusting the dosage of a therapeutic treatment for MPS in a subject receiving said therapeutic treatment, said method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dosage of the therapeutic treatment based on the difference between the level of CD63 in the first sample and the level of CD63 in the second sample.

86. 1. A method for generating quantitative data for a subject with MPS, the method comprising: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in a second sample from the subject after administering to the subject a therapeutic treatment for MPS.

87. 87. The method of claim 86, wherein the dosage of the therapeutic treatment should be increased if the CD63 level determined in step (b) is substantially the same as or greater than the CD63 level determined in step (a).

88. 88. The method of any one of claims 83 to 87, wherein said therapeutic treatment comprises (e.g. consists of) enzyme replacement therapy, gene therapy, and / or hematopoietic stem cell transplantation.

89. 89. The method of claim 88, wherein the treatment comprises (e.g., consists of) enzyme replacement therapy with α-L-iduronidase, iduronidase-2-sulfatase, or heparan-N-sulfatase.

90. 90. The method of any one of claims 80-89, wherein the second or subsequent sample is taken from the subject at least 8 weeks after initiation of the therapeutic treatment.

91. Use of CD63 as a biomarker for monitoring the progression of MPS in a subject diagnosed with MPS, or for monitoring the progress of treatment for MPS in a subject diagnosed with MPS, or for adjusting the dosage of therapeutic treatment for MPS.

92. A kit for detecting or diagnosing a specific lysosomal storage disease in a subject, the kit comprising: (a) means for detecting CD63 in a sample from the subject; and (b) means for detecting one or more biomarkers of the lysosomal storage disease in a sample from the subject.

93. 93. The kit of claim 92, wherein the specific lysosomal storage disease is Fabry disease, and the kit comprises: (a) means for detecting CD63 in a sample from the subject; and (b) means for detecting one or more biomarkers of Fabry disease in the sample (e.g., means for detecting GL3 and / or lyso-GL3 in the sample).

94. 93. The kit of claim 92, wherein the specific lysosomal storage disease is Gaucher disease, and the kit comprises: (a) means for detecting CD63 in a sample from the subject; and (b) means for detecting one or more biomarkers of Gaucher disease in the sample (e.g., means for detecting lyso-GL1 in the sample).

95. 93. The kit of claim 92, wherein the lysosomal storage disease is MPS, and the kit comprises: (a) means for detecting CD63 in a sample from the subject; and (b) means for detecting one or more biomarkers of MPS in the sample.

96. 96. The kit of claim 95, wherein (i) the lysosomal storage disease is MPS I and the kit comprises, in part, (b) means for detecting dermatan sulfate and optionally heparan sulfate in the sample; (ii) the lysosomal storage disease is MPS II and the kit comprises, in part, (b) means for detecting dermatan sulfate and heparan sulfate in the sample; or (iii) the lysosomal storage disease is MPS III and the kit comprises, in part, (b) means for detecting heparan sulfate in the sample.

97. The kit of any one of claims 92 to 96, wherein the means for detecting CD63 comprises at least one anti-CD63 antibody.

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