Biomarkers for lysosomal storage disease

Abstract

Methods for assessing lysosomal dysfunction and for diagnosing specific lysosomal storage diseases are provided. The methods employ CD63 as a biomarker and can be used to diagnose and monitor conditions, as well as to monitor and / or modulate therapeutic interventions against 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 that can be used to diagnose and monitor specific lysosomal storage diseases, as well as for monitoring and / or modulating therapeutic interventions for such disorders. Background Art

[0002] Lysosomal storage disorders (LSDs) are a group of genetic diseases that include Fabry disease (FD), Gaucher disease (GD), and mucopolysaccharidoses (MPS). These disorders primarily involve dysfunction of lysosomal hydrolases, resulting in impaired substrate degradation. Disruption of lysosomal function may lead to the accumulation of one or more undegraded substrates in endosomes and lysosomes, ultimately impairing cellular function. Although each LSD is typically caused by mutations in a different gene (and therefore lack of enzyme activity or protein function), all LSDs share common biochemical features in that they result in the accumulation of substrates within lysosomes.

[0003] Although lysosomal proteins are ubiquitously distributed, the accumulation of one or more undegraded substrates in LSD patients is often restricted to those cells, tissues, and organs with high substrate turnover rates. The accumulation of a primary storage material may result in a cascade of secondary disruptions to other biochemical and cellular functions, leading to the severe pathology of lysosomal storage disorders.

[0004] The extent and severity of LSDs typically depend on the type and amount of substrate that accumulates, but nearly all disorders are progressive. Many clinical similarities are observed between and within groups. Common clinical features of many LSDs include skeletal 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 typically have shortened lifespans and significant morbidity.

[0005] Individual LSDs are classified as rare diseases, but when considered as a group of disorders, their prevalence is significant and they represent an important health problem. A limited number of studies have investigated the incidence of LSDs, which is defined as the total number of cases diagnosed in a certain period of time divided by the total number of live births in the same period of time. One of the main problems associated with obtaining accurate epidemiological data on these individual rare disorders is that in most countries there are many diagnostic centres, complicating the problem of collecting and linking diagnoses. The combined estimated prevalence of LSDs worldwide is approximately 1 in 7,500 live births. The true prevalence is likely to be greater due to misdiagnosed or undiagnosed cases.

[0006] Gaucher disease (GD) is an inherited metabolic disorder caused by mutations in the GBA gene, which results in a deficiency of the enzyme glucocerebrosidase (GC enzyme). Accumulation of glucosylceramide (GL1), the major storage substance in the lysosomes of macrophages, affects cells of the reticuloendothelial system, which includes 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 the Ashkenazi Jewish population, 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 insufficient activity of the enzyme alpha-galactosidase A (α-Gal), encoded by the GLA gene. The enzyme deficiency leads to progressive intracellular accumulation of glycosphingolipids, most notably globotriaosylceramide (GL3), in multiple cell types and tissues, including the kidney, heart, liver, spleen, and skin, as well as in the peripheral and central nervous systems.

[0008] Mucopolysaccharidoses (collectively referred to as "MPS") are inherited autosomal recessive disorders (except 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 (such as skin, cartilage, cornea, liver, spleen, and vascular tissue). Severe manifestations are seen in preschool children with developmental delay, short stature, repeated ear and respiratory infections, hepatosplenomegaly, and coarse facial features. Over time, children develop hearing loss, heart valve disease, airway obstruction, skeletal contractures, and a unique facial appearance with macrocephaly, thick eyebrows, gingival hypertrophy, macroglossia, and thickened lips and nose wings. Intellectual impairment, and patients experience regression as the disease progresses. In 61% of untreated patients, IQ <70, and 25% are borderline at 2-3 years. Other complications include corneal clouding, carpal tunnel syndrome, hydrocephalus, glaucoma, cardiac arrhythmias, cervical spine instability, and spinal cord compression. Untreated life expectancy is twenty or thirty years.

[0009] Currently, there is no cure for any LSD, and there are no approved treatments for many of these conditions. A particular challenge is developing effective therapies to treat the CNS manifestations that are so common in LSDs. For treatable LSDs, the quality of life of these patients, especially those whose disease is diagnosed and treated at an early stage, is significantly improved.

[0010] Existing treatments for LSDs, where they are available, typically involve enzyme replacement therapy (ERT) (in which an active form of the missing enzyme is administered to the patient to compensate for the reduced or abnormal activity of the patient's own enzyme) or substrate reduction therapy (SRT) (in which modulators of enzymes involved in the defective lysosomal pathway are administered to alter the flux of substrates through the pathway and reduce 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. Authorized treatments for FD are based on the use of recombinant α-Gal (i.e., recombinant alglucosidase beta ( -Sanofi, approved in the EU in 2001 and in the US in 2003) and recombinant alglucosidase alfa ( -Takeda, EU approved in 2001))). If FD patients have a compliant GLA mutation, they can also be treated with the small molecule chaperone migalastat ( -Amicus Therapeutics, EU approved in 2016) for treatment. Treatments for GD include ERT such as imiglucerase ( -Sanofi, approved by the EU in 1997) and SRT such as ilrustat ( -Sanofi, EU approved in 2015) and milusostat ( -Janssen, EU approved 2002). Treatment for MPS may involve the use of α-L-iduronidase ( -Sanofi, EU approved in 2003 for MPS I). Other active agents are being evaluated for the treatment of lysosomal storage diseases; one such agent is venglustat, (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 a Phase 2 clinical trial as an SRT treatment for FD.

[0011] Before starting LSD treatment, it is necessary to diagnose the condition. In view of the relative rarity of LSD, the method of diagnosis should aim at a high level of sensitivity and specificity. Historically, diagnosis will be based on a variety of factors (including family history, clinical manifestations and biopsy), although this may miss early (pre-symptomatic) disease; this is particularly problematic in the case of severe childhood LSD. Recently, genetic screening and enzyme or substrate assays have been developed to study some LSDs. However, such tests are typically highly specific for each LSD or even for each subtype of LSD, and there is generally no extensive diagnostic assay using a single biomarker for the LSD group. Multiple screening assays have been proposed, but these have not yet been widely adopted (see, for example, International Patent Publication WO 2004 / 088322, which measures the levels of multiple lysosomal protein markers (such as LAMP-1, saponin C, α-glucosidase and α-iduronidase) in neonatal blood spots).

[0012] The typical biomarker of FD that has been used until the early 21st century is globotriaosylceramide (GL3) - the main accumulation substrate caused by α-Gal inactivity. The 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 manifestation have increased the demand for more robust diagnostic and prognostic biomarkers. α-Gal activity itself is considered to have diagnostic potential in male patients, but the diagnostic sensitivity in female patients is poor. In the late 21st century, the deacylated form of GL3, globotriaosylsphingosine (lyso-GL3), was studied as a biomarker for FD and found to have excellent sensitivity compared to GL3 and α-Gal activity. Lyso-GL3 is now an important diagnostic biomarker and is often used to supplement complete genetic analysis in the determination of FD cases. It can also be used to monitor disease progression and treatment.

[0013] Similar principles can be used to develop diagnostic tests using biomarkers for GD. This disease is characterized by the accumulation of glucosylceramide (GL1) and its deacylated form glucosylsphingosine (lyso-GL1) in the body due to the deficiency of GC enzyme. 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, some studies have shown that its pathological involvement is associated 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 of the α-L-iduronidase (IDUA) enzyme, which results in the accumulation of its catabolism substrates dermatan sulfate and heparan sulfate in the lysosomes. Most commonly in the clinical setting, the first level of MPS I diagnosis is IDUA enzyme activity. If the enzyme activity is reduced, a second level test for mucopolysaccharidosis is performed on the blood or blood spots to look for elevated dermatan sulfate and heparan sulfate. Along with diagnosis, dermatan sulfate and heparan sulfate levels are the standard for treatment monitoring. Similar assays that observe the levels of specific GAGs also exist, which 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 treatment progress. Thus, there is an urgent need to develop biomarkers for early detection of LSDs in general, and to develop new and improved methods for characterizing and monitoring specific LSDs.

[0016] This application demonstrates that the cell surface glycoprotein CD63 can be used as a biomarker for diagnosing and / or monitoring a variety of LSDs. Therefore, CD63 can serve as a common biomarker for the pathogenesis of LSDs. Not only can CD63 itself function as a biomarker for disease progression, but the measurement of CD63 levels can also be used in conjunction with other clinical measurements to diagnose and / or monitor specific conditions.

[0017] CD63 is a protein that is first detected as a marker of platelet activation, although its exact function is unknown. It is localized to the membranes of melanosomes and platelet dense bodies. Some cells are rich in CD63, such as activated basophils and proliferated mast cells, and CD63 is often used as a marker of multivesicular bodies in cell biology. It is also used as a marker of extracellular vesicles released from multivesicular bodies or plasma membranes. CD63 can also be used as a cell marker, for example, to quantify platelet size, quantity or volume (see, for example, WO 2004 / 088322 above). CD63 is highly glycosylated, which may protect it from lysosomal enzyme degradation. When the structural gene and cDNA of CD63 were first isolated and sequenced, it was found that it was identical to ME491 (antigen associated with early melanoma cells). CD63 also appears to be identical to dense granule membrane protein (granulophysin) (protein associated with platelet dense bodies). Due to its known uses, there are many commercial kits that can be used for detection and quantification in biological samples. These are typically based on ELISA using specific anti-CD63 antibodies.

[0018] Based on the observations described in the Examples herein, although not wishing to be bound by theory, it is hypothesized that CD63 may serve as a circulating biomarker for a variety of lysosomal storage diseases, particularly those that share the same glycosphingolipid pathway. In addition, because the levels of CD63 in patients may be more stable over time than the levels of conventional biomarkers for LSDs (e.g., for GD), CD63 represents a particularly advantageous biomarker for monitoring specific LSDs. Summary of the invention

[0019] Thus, a first aspect provides a method of diagnosing a subject suffering from or at risk of developing 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 employed in the method.

[0020] In embodiments, the subject is diagnosed as having or being at risk for a lysosomal storage disease, including Fabry disease, Gaucher disease, MPS Type I, MPS Type II, and MPS Type III.

[0021] Another aspect provides a method of 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 employed in the method.

[0022] Another aspect provides a method of detecting or diagnosing aberrant 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 employed in the method.

[0023] Another aspect provides a method for generating quantitative data for a subject, the method comprising determining the 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 a LSD and / or has not been assessed 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, the subject is diagnosed as having or being at risk for a lysosomal storage disease or as having a lysosomal dysfunction or abnormal glycosphingolipid processing if the level of CD63 is greater than a control value, wherein the level of CD63 in a sample taken from the same subject or from one or more healthy subjects at an earlier time point is measured as the control value.

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

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

[0028] In embodiments, the subject is suspected of being at risk for Fabry disease due to the presence of one or more of the following: family history of Fabry disease, fatigue, pain, lens or corneal opacities, vortex keratopathy, angiokeratoma, shortness of breath, palpitations, edema, kidney disease, myocardial dysfunction, conduction abnormalities with shortened PR interval, arrhythmias, vertigo, headache, diplopia, dysarthria, hemiataxia, transient ischemic attack, early-onset stroke, and dementia. In embodiments, the subject is diagnosed as having Fabry disease if the level of CD63 measured in a sample from the subject is greater than a control value, wherein the level of CD63 in a sample obtained from one or more healthy subjects is measured as the control value. In embodiments, the subject is diagnosed as having Fabry disease if the level of CD63 measured in a sample from the subject is at least about 100% higher than the control value.

[0029] Another aspect provides a method for generating quantitative data from a subject, wherein the method comprises (e.g., consists of) determining the level of CD63 in a sample from the subject, wherein the subject has Fabry disease or is suspected of having Fabry disease.

[0030] Another aspect provides the use of CD63 as a biomarker in diagnosing Fabry disease in a subject suspected of being at risk for developing 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 aspect provides a method of treating a subject who has been diagnosed as having Fabry disease by a method as defined above, said treatment comprising administering to said 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, wherein the patient has higher than normal plasma CD63 levels, the method comprising administering to the subject an effective amount of a therapeutic treatment for Fabry disease.

[0034] On the other hand, a method for generating quantitative data for a subject is provided, wherein the method comprises: (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, wherein the level of CD63 in a sample obtained from one or more healthy subjects is measured as the control value; and (c) if the level of CD63 in the sample is greater than the control value, applying one or more therapeutic treatments for Fabry disease to the subject.

[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 to the subject venglustat or migalastat, e.g., venglustat. In other embodiments, the treatment includes administering to the subject a recombinant α-galactosidase, e.g., a recombinant alglucosidase β.

[0036] Another aspect provides a therapeutic agent for use in treating Fabry disease in a subject, wherein the subject has been diagnosed as having Fabry disease by a method as defined above.

[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 to the subject venglustat or migalastat, e.g., venglustat. In other embodiments, the treatment includes administering to the subject a recombinant α-galactosidase, e.g., a recombinant alglucosidase β.

[0038] On the other hand, a method for monitoring the progression of Fabry disease in a subject diagnosed with Fabry disease is provided, 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 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 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 has become less severe if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0039] On the other hand, a method for generating quantitative data of a subject diagnosed with Fabry disease is provided, the method comprising the steps of: (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, such as a plasma sample.

[0041] On the other hand, a method for monitoring the progress of a treatment for Fabry disease in a subject diagnosed with Fabry disease is provided, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering a therapeutic treatment for Fabry disease to the subject; (c) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after administration of the therapeutic treatment; 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] On the other hand, a method for treating or preventing the development or progression of Fabry disease in a subject assessed to be at risk for developing Fabry disease is provided, the method comprising the steps of: (a) obtaining a first biological sample from the subject and analyzing the CD63 concentration of the sample; (b) if the CD63 concentration is above a control value, initiating a course of therapeutic treatment for Fabry disease in the subject, and optionally: (c) after treating the subject, obtaining a second biological sample from the subject and analyzing the CD63 concentration of the sample to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0043] On the other hand, a method for adjusting the dose of a therapeutic treatment for Fabry disease in a subject is provided, 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 obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dose 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] On the other hand, a method for generating quantitative data of a subject having Fabry disease is provided, the method comprising the following steps: (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 a therapeutic treatment for Fabry disease to the subject.

[0045] In embodiments, 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), the dose of said therapeutic treatment will be increased.

[0046] In embodiments, the therapeutic treatment comprises (e.g., consists of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment comprises administering to the subject venglustat or migalastat, e.g., venglustat. In other embodiments, the treatment comprises administering to the subject a recombinant α-galactosidase, e.g., a recombinant alglucosidase β.

[0047] In embodiments, said second or subsequent sample is obtained from said subject at least 8 weeks after initiation of said 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, or for monitoring the progress of a treatment for Fabry disease, or for adjusting the dose 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 developing Gaucher disease, the method comprising measuring the level of CD63 in a sample from the subject.

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

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

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

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

[0054] Another aspect provides a method of treating a subject who has been diagnosed as having Gaucher disease by a method as defined above, said treatment comprising administering to said subject one or more therapeutic treatments for Gaucher disease.

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

[0056] On the other hand, a method for generating quantitative data for a subject is provided, wherein the method comprises: (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, wherein the level of CD63 in a sample obtained from one or more healthy subjects is measured as the control value; and (c) if the level of CD63 in the sample is greater than the control value, applying one or more therapeutic treatments for Gaucher disease to the subject.

[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 to the subject venglustat, ilukastat, or milukastat. In embodiments, the treatment includes administering to the subject a recombinant glucocerebrosidase, such as imiglucerase.

[0058] Another aspect provides a therapeutic agent for use in treating Gaucher disease in a subject, wherein the subject has been diagnosed as having Gaucher disease by a method as defined above.

[0059] In an embodiment, the therapeutic agent is a therapeutic treatment as defined above.

[0060] On the other hand, a method for monitoring the progression of Gaucher disease in a subject diagnosed with Gaucher disease is provided, 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 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 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 become less severe if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0061] On the other hand, a method for generating quantitative data of a subject diagnosed with Gaucher disease is provided, the method comprising the following steps: (a) determining the level of CD63 in a first sample from the subject; (b) determining the level of CD63 in subsequent samples 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, such as a plasma sample.

[0063] On the other hand, a method for monitoring the progress of a treatment for Gaucher disease in a subject diagnosed with Gaucher disease is provided, 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 from the subject, wherein the second sample is obtained from the subject after administration of the therapeutic treatment; 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] On the other hand, a method for treating or preventing the development or progression of Gaucher disease in a subject assessed to be at risk for developing Gaucher disease is provided, the method comprising the steps of: (a) obtaining a first biological sample from the subject and analyzing the CD63 concentration of the sample; (b) if the CD63 concentration is above a control value, initiating a therapeutic treatment course for Gaucher disease in the subject, and optionally: (c) after treating the subject, obtaining a second biological sample from the subject and analyzing the CD63 concentration of the sample to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0065] On the other hand, a method for adjusting the dose of a therapeutic treatment for Gaucher disease in a subject receiving the therapeutic treatment is provided, 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 obtained from the subject after administering one or more doses of the therapeutic treatment; and (c) adjusting the dose 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] On the other hand, a method for generating quantitative data of a subject having Gaucher disease is provided, the method comprising the following steps: (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 a therapeutic treatment for Gaucher disease to the subject.

[0067] In embodiments, if the CD63 level determined in step (b) is substantially the same as or greater than the CD63 level determined in step (a), the dose of the therapeutic treatment will be increased. In embodiments, the therapeutic treatment comprises (e.g., consisting of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment comprises administering venglustat, ilukastat, or milukastat to the subject. In embodiments, the treatment comprises administering a recombinant glucocerebrosidase, such as imiglucerase, to the subject. In embodiments, the second or subsequent sample is obtained from the subject at least 8 weeks after initiation 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, or for adjusting the dose of a therapeutic treatment for Gaucher disease in a subject diagnosed with Gaucher disease.

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

[0070] In embodiments, the subject is suspected of being at risk for MPS due to the presence of one or more of the following: family history of MPS, macrocephaly, hearing loss, corneal opacity, abnormal dentition, rigidity, hip dysplasia, claw hand, joint laxity, valve thickening, left ventricular hypertrophy, recurrent respiratory tract infections, obstructive airway disease, hepatomegaly / splenomegaly, umbilical / inguinal hernia, developmental delay, ventriculomegaly, enlarged perivascular spaces, hyperactive or aggressive behavior, abnormal leukocyte granules, fetal hydrops, and proteinuria. In embodiments, the subject is diagnosed as having MPS if the level of CD63 measured in a sample from the subject is greater than a control value, wherein the level of CD63 in a sample taken from one or more healthy subjects is measured as the control value. In embodiments, the subject is diagnosed as having MPS if the level of CD63 measured in a sample from the subject is at least about 100% higher than the control value.

[0071] Another aspect provides a method for generating quantitative data from a subject, wherein the method comprises (eg, consists of) determining the level of CD63 in a sample from the subject, wherein the subject has MPS or is suspected of having MPS.

[0072] Another aspect provides the use of CD63 as a biomarker in diagnosing MPS in a subject suspected of being at risk for developing MPS.

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

[0074] Another aspect provides a method of treating a subject who has been diagnosed as having MPS by a method as defined above, said treatment comprising administering to said 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 higher than normal plasma CD63 levels, 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, wherein the method comprises: (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, wherein the level of CD63 in a sample taken from one or more healthy subjects is measured as the control value; and (c) if the level of CD63 in the sample is greater than the control value, applying one or more therapeutic treatments for MPS to the subject.

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

[0078] Another aspect provides a therapeutic agent for use in treating MPS in a subject, wherein the subject has been diagnosed as having MPS by a method as defined above.

[0079] In an embodiment, the therapeutic agent is a therapeutic treatment as defined above.

[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, wherein 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 has become less severe if the level of CD63 in the second sample is lower than the level of CD63 in the first sample.

[0081] On the other hand, a method for generating quantitative data of a subject diagnosed with MPS is provided, the method comprising the steps of: (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, such as a plasma sample.

[0083] Another aspect provides a method for monitoring the progress of a 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, wherein the second sample is obtained from the subject after administration of the therapeutic treatment; 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] On the other hand, a method for treating or preventing the development or progression of MPS in a subject assessed to be at risk for MPS is provided, the method comprising the steps of: (a) obtaining a first biological sample from the subject and analyzing the CD63 concentration of the sample; (b) if the CD63 concentration is above a control value, initiating a therapeutic treatment course for MPS in the subject, and optionally: (c) after treating the subject, obtaining a second biological sample from the subject and analyzing the CD63 concentration of the sample to determine a change in CD63 levels; and (d) adjusting the therapeutic treatment based on the observed change in CD63 levels.

[0085] In another aspect, a method for adjusting the dose of a therapeutic treatment for MPS in a subject receiving the therapeutic treatment is provided, 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 obtained from the subject after administration of one or more doses of the therapeutic treatment; and (c) adjusting the dose of the therapeutic treatment based on a difference between the level of CD63 in the first sample and the level of CD63 in the second sample.

[0086] On the other hand, a method for generating quantitative data of a subject having MPS is provided, the method comprising the following steps: (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 a therapeutic treatment for MPS to the subject.

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

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

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

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

[0092] In yet other embodiments, the lysosomal storage disease is MPS, and the kit comprises: (a) a means for detecting CD63 in a sample from the subject; and (b) a 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) a 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) a 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) a means for detecting heparan sulfate in the sample.

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

[0094] Other features and advantages of the compounds, compositions, and methods disclosed herein will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 Shown is the response of biomarkers to venglustat treatment in a clinical trial over time in patients with Fabry disease compared to healthy controls. The biomarkers analyzed were: CD63 ( Figure 1A) - labeled "Linear NPX", i.e. normalized protein expression; GL3 ( Figure 1 B); and lyso-GL3( Figure 1 C). All biomarkers showed response to treatment. CD63 and lyso-GL3 did not return to control levels during the trial period. There was no separation of GL3 between baseline and control.

[0096] Figure 2 CD63 ( Figure 2 A) GL3( Figure 2 B) and lyso-GL3( Figure 2 C) Estimated marginal mean (log2) values, longitudinally showing from baseline to 156 weeks of venglustat treatment for Fabry disease. The horizontal line above the curve indicates a statistically significant difference, and the star indicates the confidence of the significant difference (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). The first significant decrease in CD63 occurred at week 26.

[0097] Figure 3 Intraclass correlation coefficient (ICC) data (10,000 permutations) of biomarkers measured from the Fabry ACT / LTS venglustat Phase II clinical trial are shown. ICC ( Figure 3 A) is a measure of variability between patients, while the ICC residual ( Figure 3 C) is a measure of intra-patient variability. The ICC distribution intercept is shown in Figure 3 In B. Each graph shows from left to right: log2(CD63); log2(GL3); and log2(lyso-GL3). CD63 and lyso-GL3 have comparable within-subject variability, but both are much lower than GL3.

[0098] Figure 4 The probability density function comparing the distribution of CD63 values ​​(log2) for healthy subjects (left) and diseased, treated Gaucher disease patients (right) is shown; sample metadata is listed below in Table 2. There is a statistically significant separation between the two groups, and CD63 is upregulated in disease patients (Wilcoxon test: p=8.89e -10 ).

[0099] FIG5 shows paired spaghetti plots to visualize CD63 ( Figure 5A ), lyso-GL1(LGL1; Figure 5B ) and chitotriosidase (CHITO; Figure 5C) within the patient variability. The intra-subject variability is visualized by plotting the biomarker changes (i.e., changes compared to the previous measurement) between adjacent time points. All patients included in the figure have received treatment for Gaucher disease for several years, representing the biomarker steady state during treatment. The results for each patient are shown in a separate line (and in a different line style). Note that data for all patients are not collected at each time point. Lyso-GL1 and CHITO appear to have greater variability than CD63, and this is confirmed in Figure 6.

[0100] Fig. 6 shows the correlation between CD63 and other biomarkers. Fig. 6A to Fig. 6C show the intraclass correlation coefficient (ICC) data (10,000 permutations) of the biomarkers measured from the Gaucher disease cohort (cohort) summarized in Table 2 below. ICC (Fig. 6A) is a measure of variability between patients, and ICC residual (Fig. 6C) is a measure of intra-patient variability. The ICC distribution intercept is shown in Fig. 6B. Each figure shows from left to right: log2 (CD63); log2 (lyso-GL1); and log2 (CHITO). Note that the intra-patient variability of CD63 is significantly lower than that of lyso-GL1 or CHITO. Fig.6D Correlation is presented in another way: data points show CD63 levels in samples from patients with Gaucher disease, versus lyso-GL1 levels (light grey circles), CCL18 levels (black triangles), and chitotriosidase activity (dark grey squares). The line of best fit is shown, with r=0.65 and p<0.0001 for lyso-GL1 (lower line, light grey); r=0.63 and p=0.0119 for CCL18 (middle line, black), and r=0.78 and p<0.0001 for chitotriosidase (upper line, dark grey).

[0101] Figure 7 A probability density plot comparing the distribution of CD63 values ​​(log2) of healthy subjects and patients with MPS disease is shown ( Figure 7 A); all sample metadata are listed in Table 3 below. The shaded area on the right side of the figure contains data from all three MPS subtypes. The curves within the shaded area on the right represent the distribution of different MPS subtypes (indicated by arrows). The statistical differences between the healthy distribution and the MPS distribution are shown in Figure 8C Also shown is a comparison of CD63 values ​​between male and female MPS disease subjects ( Figure 7 B) At α = 0.05, the two distributions were not statistically different (p = 0.0746).

[0102] FIG8 shows the Fabry disease ( Fig. 8A )、Gaucher disease( Figure 8B ) and MPS( Figure 8C ) study. P values ​​are provided in each figure. The only example with p>0.05 was a female patient with Fabry disease.

[0103] Fig. 9 Shown are samples from male adult Fabry disease patients (baseline) compared to healthy controls. Proteomic profiling. Proteins with at least 1.5-fold change and p<0.05 were considered significant and are shown in grey. CD63 is circled.

[0104] Fig.10 Shown are box plots of CD63 levels in CSF samples from healthy controls (leftmost panel) compared to MPS I (middle panel) and MPS II (rightmost panel) patients.

[0105] Figure 11 shows CD63 levels in CSF samples from healthy controls compared to patients with Gaucher disease type 3 (GD3); GD3 samples were collected at three time points: baseline, 26 weeks and 52 weeks of treatment with venglustat and imiglucerase. Mean levels in GD3 patients were elevated at baseline compared to healthy controls and decreased over time ( Fig.11A This trend was also seen in individual patients ( Fig. 11B ). DETAILED DESCRIPTION

[0106] Although specific embodiments of the present disclosure will now be described with reference to preparations and schemes, it should be understood that such embodiments are by way of example only and are only a few of the many possible specific embodiments that can represent the application of the principles of the present disclosure. Various changes and modifications will be apparent to those skilled in the art, given the benefit of the present disclosure, and are deemed to be within the spirit and scope of the present disclosure as further defined in the appended claims.

[0107] definition

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. Although any method and material similar to or equivalent to those described herein can be used in the implementation or testing of the present disclosure, exemplary methods, equipment and materials are now described. All technologies and patents cited herein are openly incorporated herein by reference in their entirety. Any content of this paper should not be construed as admitting that the present disclosure is not entitled to such disclosure content earlier than the prior public text.

[0109] Unless otherwise indicated, the present disclosure will employ 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 (4th ed., Cold Spring Harbor Laboratory Press 2012); Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., NY); 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, 5th ed.; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acids Hybridization; Anderson (1999) Nucleic AcidHybridization; Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor) Laboratory); Makrides editor (2003) GeneTransfer and Expression in Mammalian Cells;Mayer and Walker, editors (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al., editors (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (editor) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press). ;

[0110] All numerical designations, such as pH, temperature, time, concentration, molecular weight, etc. (including ranges), are approximate values ​​(where appropriate) varying in increments of, for example, 0.1 or 1.0 ((+) or (-)). It should be understood that, although not always expressly stated, all numerical representations are preceded by the term "about". It should also be understood that, although not always expressly stated, the reagents described herein are exemplary only and that equivalents thereof are known in the art.

[0111] Unless the context clearly indicates otherwise, as used in the specification and claims, the singular forms "a", "an", and "the" include plural referents. For example, the term "cell" includes a plurality of cells, including mixtures thereof. Unless otherwise specified or obvious from the context, as used herein, the term "or" should be understood to be inclusive. The term "including" is used herein to mean and is used interchangeably with the phrase "including but not limited to".

[0112] As used herein, the terms "comprising" or "comprises" are intended to indicate that compositions and methods include the listed elements, but do not exclude other elements.

[0113] When used to define compositions and methods, "consisting essentially of shall mean excluding other elements of any significance for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, etc.

[0114] "Consisting of shall mean excluding more than trace elements and substantial method steps for applying the compositions of the present disclosure, or for producing the compositions or achieving the desired results. Embodiments defined by each of these transitional terms are within the scope of the present disclosure. The use of the term "comprising" herein is intended to encompass and disclose corresponding statements in which the term "comprising" is replaced by "consisting essentially of" or "consisting of."

[0115] "Subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, rodents, rats, rabbits, monkeys, cattle, sheep, pigs, dogs, cats, livestock, sports animals, pets, horses, primates, and humans. In embodiments, the mammals include horses, dogs, and cats. In embodiments, the mammal is a human.

[0116] "Administering" is defined herein as a means of providing a medicament or a composition containing the medicament to a subject in a manner that causes the medicament to contact the subject's body (e.g., in vivo). Such administration can be by any route, including but not limited to oral, percutaneous (e.g., vaginal, rectal or oral mucosa), by injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, into the CNS), or by inhalation (e.g., oral or nasal). Administration can also involve providing a substance or composition to a portion of the subject's body surface, such as by topical application to the skin. Of course, the pharmaceutical preparation is given in a form suitable for each route of administration.

[0117] "Treating" or "treatment" of a disease includes: (1) preventing the disease from occurring in a patient who may be susceptible to the disease but does not yet experience or exhibit symptoms of the disease, i.e., causing clinical 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) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

[0118] In particular, the term "suffering from" (or "suffering from") in relation to a lysosomal storage disease refers to a patient or individual who has developed at least some of the characteristic pathology of the disease and / or is exhibiting one or more signs of the disease (e.g., one or more biomarkers characteristic of the disease), regardless of whether they have actually been diagnosed with the disease. In cases where a patient is susceptible to a disease (e.g., has a history in their family pedigree or due to the presence of a genetic mutation associated with the disease), but they have not yet developed all or some of the characteristic pathology of the disease, they may be referred to as being "at risk for developing the disease."

[0119] An "effective amount" or "therapeutically effective amount" is an amount sufficient to achieve a beneficial or desired result. The effective amount can be administered in one or more administrations, applications or dosages. Such delivery depends on many variables, including the time period of using a single dosage unit, the bioavailability of the therapeutic agent, the route of administration, etc. However, it should be understood that the specific dosage level of the therapeutic agent for any particular subject depends on a variety of factors, including, for example, the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, the time of administration, the excretion rate, the drug combination, and the severity and form of administration of the specific disorder being treated. The therapeutic dose can usually be adjusted stepwise to optimize safety and efficacy. Typically, the dose-effect relationship from in vitro and / or in vivo testing can initially provide useful guidance on the appropriate dose for patient administration. Generally, people will wish to administer a compound that effectively reaches an amount of serum levels commensurate with the concentration found to be effective in vitro. These considerations and effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutically effective amount" is an amount sufficient to treat (e.g., improve) one or more symptoms associated with lysosomal storage diseases. For example, oral administration may require a total daily dose of 0.1 mg to 1000 mg of active agent. The total daily dose may be administered in a single or divided dose and may exceed the typical range given herein at the discretion of the physician.

[0120] When compounds such as elukastat or venglustat are mentioned herein, these compounds include the compounds themselves as well as pharmaceutically acceptable salts thereof. For example, reference to "elukastat" includes elukastat hemi-tartrate, and reference to "venglustat" includes venglustat malate. Venglustat is (S)-1-azabicyclo[2.2.2]octan-3-yl-N-[2-[2-(4-fluorophenyl)-1,3-thiazol-4-yl]propan-2-yl]carbamate. Ilustat is N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxaphenyl-6-yl)-1-hydroxy-3-pyrrolidin-1-ylpropan-2-yl]octanamide.

[0121] Where appropriate, any embodiment (eg, method) provided herein may be combined with any one or more other embodiments (eg, methods) provided herein.

[0122] The following abbreviations are used in this article:

[0123] α-Gal α-galactosidase A

[0124] BCA Bicinchoninic Acid

[0125] BL Baseline

[0126] BSA bovine serum albumin

[0127] CCL18 Chemokine (CC motif) ligand 18

[0128] cDNA complementary DNA

[0129] CHITO Chitotriosidase

[0130] CNS Central Nervous System

[0131] CS Chondroitin Sulfate

[0132] CSF Cerebrospinal fluid

[0133] CV Confidence

[0134] DNA deoxyribonucleic acid

[0135] DS Dermatan Sulfate

[0136] ELISA enzyme-linked immunosorbent assay

[0137] ERT enzyme replacement therapy

[0138] ESI Electrospray ionization

[0139] FACS fluorescence-assisted cell sorting

[0140] FD Fabry disease

[0141] GAG Glycosaminoglycan

[0142] GBA glucocerebrosidase gene

[0143] GC enzyme glucocerebrosidase (also called acid beta-glucosidase)

[0144] GD Gaucher disease

[0145] GD3 Gaucher disease type 3

[0146] GI

[0147] GL1 Glucosylceramide

[0148] GL3 Globulin triacylceramide

[0149] GM1 Monosialotetrahexosylganglioside

[0150] GM2 Monosialotriahexosylganglioside

[0151] GM3 monosialodihexosylganglioside

[0152] HS Heparan Sulfate

[0153] HSCT Hematopoietic stem cell transplantation

[0154] ICC intraclass correlation coefficient

[0155] IDUA α-L-iduronidase

[0156] IQ

[0157] KS Keratan Sulfate

[0158] LCMS Liquid chromatography mass spectrometry

[0159] LC-MS / MS Liquid chromatography tandem mass spectrometry

[0160] LSD Lysosomal Storage Disorders

[0161] lyso-GL1 glucosylceramide (also known as LGL1)

[0162] lyso-GL3 spherical triacylsphingosine

[0163] MPS mucopolysaccharidosis (or mucopolysaccharidoses)

[0164] NCL Neuronal ceroid lipofuscinosis

[0165] NPX normalized protein expression

[0166] PEA proximity extension assay

[0167] PBS Phosphate buffered saline

[0168] PCR polymerase chain reaction

[0169] RNA Ribonucleic acid

[0170] SD Standard deviation

[0171] SEM Standard error of the mean

[0172] SRT Substrate Reduction Therapy

[0173] Tris(hydroxymethyl)aminomethane

[0174] UPLC Ultra-Performance Liquid Chromatography

[0175] WK Zhou

[0176] Methods for diagnosing or testing lysosomal storage disorders are usually

[0177] Provided herein is a method for diagnosing a subject suffering from or at risk of suffering from a lysosomal storage disease, the method comprising measuring the level of CD63 in a sample from the subject. The advantage of this method is that it is able to detect irregularities in the glycosphingolipid pathway common to many LSDs. Therefore, the method is able to provide a general diagnosis of lysosomal storage disorders, that is, the diagnosis does not have to be limited to the assessment of a single disease state, but rather provides a more comprehensive picture of lysosomal function and glycosphingolipid processing. Therefore, a related 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. Another related 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. In an embodiment, CD63 is the only biomarker used in the above method.

[0178] Other related aspects provide for the use of CD63 as a biomarker in the diagnosis of 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 the detection and / or diagnosis.

[0179] Viewed in another way, these aspects provide a method for generating quantitative data of a subject, the method comprising determining the 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 (i.e., CD63).

[0180] Although the subject to be evaluated may have been identified as being at risk for or suspected of having a LSD (e.g., by consideration of family history or clinical observation), it is contemplated that the methods of the invention will be performed on subjects who have not yet been evaluated for risk factors for a lysosomal disease or dysfunction. Thus, in embodiments, the subject has not been previously diagnosed as having a 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 a LSD; including parents of Ashkenazi Jewish, Finnish, Asian, or Dutch descent; or parents who are related to each other.

[0181] As mentioned above, the method of the present invention can provide a general diagnosis of lysosomal storage disorders. Without wishing to be bound by theory, it is assumed that the method can be suitable for diagnosing a large class of diseases, including Fabry disease, Krabb disease, Gaucher disease (e.g., Type 1, Type 2 and Type 3), Niemann-Pick disease (e.g., Type A, Type B and Type C), metachromatic leukodystrophy, Farber disease, Krabb disease, galactosialidase storage disease, 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, disease), α-mannosidosis, β-mannosidosis, asparaginyl glucosamineuria, fucosidosis, MPS I (e.g., Hurler syndrome, Scheie syndrome, and Hurler-Scheie syndrome), MPS II (e.g., Hunter syndrome), 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 Type IX (e.g., hyaluronidase deficiency), mucolipidosis (e.g., sialidosis, inclusion body cell disease, pseudo-Hurler polydystrophy / phosphotransferase deficiency, and mucolipin 1 deficiency), and neuronal ceroid lipofuscinosis (e.g., Santavuori-Haltia disease / infantile NCL, Jansky-Bielschowsky disease / late infantile NCL, Batten-Spielmeyer-Vogt disease / juvenile NCL, Kufs disease / adult NCL, Finnish variant / type 5, late infantile variant / type 6, type 7, Northern epilepsy type / Turkish late infantile / type 8, German / Serbian late infantile / type 9, and congenital cathepsin D deficiency).Thus, in embodiments, the subject is diagnosed as having or being at risk for developing a lysosomal storage disease selected from the above-mentioned conditions. In other embodiments, the subject is diagnosed with or is at risk for a lysosomal storage disease selected from the group consisting of Fabry disease, Krabbe disease, Gaucher disease (e.g., type 1, type 2, and type 3), metachromatic leukodystrophy, Farber disease, Krabbe disease, galactosialidase storage disease, 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, Sala disease, alpha-mannosidosis, beta-mannosidosis, asparaginyl glucosamineuria, fucosidosis, MPS I (e.g., Hurler syndrome, Scheherez syndrome, and Hurler-Schwein syndrome), MPS MPS II (e.g., Hunter syndrome), 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., Maroto-Lami syndrome), MPS VII (e.g., Slater syndrome), MPS In some embodiments, the subject is diagnosed with or at risk for a lysosomal storage disease selected from the group consisting of Fabry disease, Gaucher disease, MPS type I, MPS type II, and MPS type III. In embodiments, the Gaucher disease is Gaucher disease type 1, type 2, or type 3 (eg, type 3).

[0182] The sample from the subject determining or measuring CD63 can be any sample containing lysosomes, exosomes and / or other cell fractions that CD63 may accumulate after lysosomal function is impaired. The sample can be essentially composed of one sample type (e.g., one tissue or fluid type), or it can be composed of multiple sample types (e.g., several tissues and / or fluid types). The sample type can be, for example, selected from blood, blood fractions, urine, cerebrospinal fluid, sputum, lymph, dermal tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Most conveniently, the sample is a type that can be obtained in a relatively non-invasive manner. Therefore, in an embodiment, the sample from the subject includes (e.g., consisting of) blood, blood fractions and / or urine. In a specific embodiment, the sample from the subject includes (e.g., consisting of) a blood fraction selected from plasma and serum. In other embodiments, the sample from the subject includes (e.g., consisting of) CSF.

[0183] In the diagnosis of lysosomal storage diseases or the detection of lysosomal dysfunction or abnormal glycosphingolipid processing, the level of CD63 in the sample is typically compared to a control value to determine whether it is within the normal (e.g., healthy) range or outside the normal range. The determination of the normal range 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 ELISA (although other methods (e.g., as described herein) may also be employed). There are multiple commercial sources for suitable ELISA kits, such as those mentioned below and in the Examples.

[0184] In embodiments, the method includes measuring the CD63 level (i.e., test sample level) in a sample from the subject, and comparing the level to a control value, wherein if the test sample level is greater than the control value, the test sample level is considered to be outside the normal range. In one embodiment, the control value is a baseline CD63 level, which was previously measured in the same subject (i.e., in an earlier sample from the subject), such as at least 1 month before obtaining the test sample from the subject (e.g., at least 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months before obtaining the test sample from the subject). The control value can represent the CD63 level measured in a single earlier sample from the subject, or it can be the average value of the values ​​from multiple earlier samples. In alternative embodiments, the control value is the CD63 level (or the average value of a group of healthy subjects in the same period) in a sample obtained from a healthy subject (i.e., a subject who does not suffer from and has no risk of suffering from lysosomal storage disease), such as those listed above. In embodiments, healthy subjects are matched to the subjects being evaluated, for example, by age and / or sex. In specific embodiments, the control value represents the level of CD63 in the same type of sample (e.g., a sample that has been obtained, processed and / or stored in the same manner as the test sample) evaluated in the methods of the invention. For example, where the methods of the invention utilize plasma samples, the control value typically represents the level of CD63 in a control plasma sample (e.g., an earlier plasma sample obtained from the same subject or a plasma sample obtained from one or more healthy subjects).

[0185] In embodiments where the control value for CD63 concentration is a point value (or an average value, such as an average value), if the CD63 level in the test sample is numerically greater than the control value, the CD63 level in the test sample is considered to be outside the normal range. For example, it can be at least about 5% greater than the control value, such as at least about 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, or 100% greater than the control value. In particular, it can be at least about 125% greater than the control value, such as 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%, or about 350% to about 450%).

[0186] In embodiments where the control value for CD63 concentration is a range of values ​​(e.g., described by some variability around a mean), the CD63 level in the test sample is considered to be outside the normal range if the CD63 level in the test sample is greater than the mean by more than about 1 standard error, e.g., greater 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. In other embodiments (e.g., where the patient is female and / or suspected of having or at risk for Fabry disease), the CD63 level in the test sample is considered to be outside the normal range if the CD63 level in the test sample is greater than the mean by more than about 0.25 standard errors, e.g., greater than the mean by more than about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 standard errors.

[0187] Thus, in an embodiment, a method of diagnosing a subject as having or being at risk of developing a lysosomal storage disease is provided, the method comprising:

[0188] - measuring the level of CD63 in a test sample (e.g., a plasma sample) from the subject;

[0189] - comparing the level measured in the test sample to a control value, wherein the control value is measured as the level of CD63 in a sample (e.g., a plasma sample) taken at an earlier time point from the same subject or from one or more healthy subjects; and

[0190] - If the level measured in the test sample is greater than the control value, the subject is diagnosed as having or being at risk of developing a lysosomal storage disease.

[0191] In other embodiments, a method of detecting or diagnosing lysosomal dysfunction in a subject is provided, the method comprising:

[0192] - measuring the level of CD63 in a test sample (e.g., a plasma sample) from the subject;

[0193] - comparing the level measured in the test sample to a control value, wherein the control value is measured as the level of CD63 in a sample (e.g., a plasma sample) taken at an earlier time point from the same subject or from one or more healthy subjects; and

[0194] - If the level measured in the test sample is greater than the control value, a lysosomal dysfunction is detected or diagnosed in said subject.

[0195] In other embodiments, a method of detecting or diagnosing abnormal glycosphingolipid processing in a subject is provided, the method comprising:

[0196] - measuring the level of CD63 in a test sample (e.g., a plasma sample) from the subject;

[0197] - comparing the level measured in the test sample to a control value, wherein the control value is measured as the level of CD63 in a sample (e.g., a plasma sample) taken at an earlier time point from the same subject or from one or more healthy subjects; and

[0198] - If the level measured in the test sample is greater than the control value, abnormal glycosphingolipid processing is detected or diagnosed in the subject.

[0199] The above embodiments may optionally include the step of obtaining a test sample from a subject, for example, taking a blood sample and optionally separating the blood sample into its fractions (eg, a plasma sample).

[0200] Methods for diagnosing, treating and monitoring specific conditions

[0201] As detailed above, CD63 can be used not only to detect and diagnose lysosomal dysfunction and aberrant glycosphingolipid processing, but can also be used as a biomarker for the diagnosis, monitoring and therapeutic treatment of specific lysosomal storage diseases.

[0202] Thus, in one aspect, a method for diagnosing a specific lysosomal storage disease (e.g., a condition as defined herein) in a subject is provided, the method comprising measuring the level of CD63 in a sample from the subject. Related aspects provide the use of CD63 as a biomarker in diagnosing a specific lysosomal storage disease in a subject. Viewed another way, these aspects provide a method for generating quantitative data of a subject who may be suspected of being at risk for a specific lysosomal storage disease, the method comprising determining the level of CD63 in a sample from the subject. Related aspects provide the use of CD63 as a biomarker to improve methods of diagnosing a specific lysosomal storage disease in a subject.

[0203] It will be appreciated that where the methods of the invention are directed to a particular condition, these aspects will typically rely on additional measurements in addition to CD63 levels in a test sample from a subject. For example, the methods may be performed on subjects who are already suspected of being at risk for a particular LSD, and / or they may employ other biomarkers that are characteristic of the condition in question. A subject may be considered at risk for the disease by identifying specific risk factors, such as family history of the disease, genetic testing, analysis of other characteristic biomarkers, clinical symptoms, and the like. In this case, CD63 levels are typically measured in a sample from a subject after assessing the risk factors, such that the subject is already considered at risk for the disease; here, the role of CD63 measurement is to confirm the diagnosis of lysosomal dysfunction in the subject and thereby confirm the disease. 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 biomarker panel for diagnostic purposes). In this case, CD63 measurement is used to improve the accuracy of the diagnosis, for example, to reduce the proportion of false positives and / or increase the proportion of true positives in a population of test subjects (compared to a corresponding diagnostic method in which CD63 levels are not taken into account).

[0204] Such diagnosis of a specific lysosomal storage disease in a subject can be used to guide therapeutic treatment for the disease. Thus, in one aspect, a method of treating a subject who has been diagnosed with a specific lysosomal storage disease by a method as defined above is provided, the treatment comprising administering to the subject one or more therapeutic treatments for the disease. A related aspect provides a therapeutic agent for treating a specific lysosomal storage disease in a subject, wherein the subject has been diagnosed with the disease by a method as defined above. A method for generating quantitative data for a subject is also provided, wherein the method comprises (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., a control value measured as the level of CD63 in a sample taken from one or more healthy subjects); and (c) if the level of CD63 in the sample is greater than the control value, one or more therapeutic treatments for a specific lysosomal storage disease are applied to the subject. In embodiments, the therapeutic treatment (e.g., the agent to be administered) comprises (e.g., consists of) the types of therapies generally known to treat the disease in question, such as substrate reduction therapy, chaperone therapy, enzyme replacement therapy, and / or gene therapy.

[0205] CD63 levels can also be used to monitor the progression of specific lysosomal storage diseases, such as monitoring the natural time course of the disease (e.g., assessing the correct time to initiate therapeutic intervention) or monitoring the effect of therapeutic intervention on the disease (e.g., assessing the efficacy of treatment, as a guide for dose adjustments, etc.).

[0206] Thus, in one aspect, a method for monitoring the progression of a subject diagnosed with a particular lysosomal storage disease is provided, the method comprising comparing the level of CD63 in a first sample from the subject to 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. Typically, the method will determine that if the level of CD63 in the second sample is greater than the level of CD63 in the first sample, the disease has become more severe, or if the level of CD63 in the second sample is substantially the same as the level of CD63 in the first sample, the subject's disease has not progressed, or if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, the disease has become less severe. Related aspects provide the use of CD63 as a biomarker for monitoring the progression of the disease in a subject diagnosed with a particular lysosomal storage disease. Viewed another way, these aspects provide a method for generating quantitative data for a subject diagnosed with a particular lysosomal storage disease, the method comprising the steps of: (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) to the level determined in step (b).

[0207] Another aspect provides a method for monitoring the progress of a 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 a therapeutic treatment for the specific lysosomal storage disease (e.g., a treatment as defined herein) to the subject; and (c) measuring the level of CD63 in a second sample from the subject, wherein the second sample is obtained from the subject after the administration of the therapeutic treatment. Typically, the method further comprises a step (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. In an embodiment, 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), the dose of the therapeutic treatment will be increased. A related aspect provides the use of CD63 as a biomarker for monitoring the progress of a treatment for the disease in a subject diagnosed with a specific lysosomal storage 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 the steps of: (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, 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), the dose of the therapeutic treatment will be increased.

[0208] 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 that is at least about 0.25 standard deviations (such as at least about 0.5, 0.75, 1, or 1.5 standard deviations) higher than the mean for 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 a substrate reduction therapy and / or an enzyme replacement therapy (e.g., as described herein) capable of treating the lysosomal storage disease.

[0209] This can be accomplished, for example, by methods as described herein (such as described in detail in the Examples). In embodiments, the healthy subject population is a population with a similar genetic background, e.g., a population with the same ancestry and / or geographic location as the patient. In embodiments, the lysosomal storage disease is Fabry disease, Gaucher disease (e.g., Gaucher disease type 1, type 2, or type 3), or MPS (e.g., MPS type I, type II, or type III).

[0210] Another aspect provides a method of treating a lysosomal storage disease in a subject diagnosed as being at risk for the disease, wherein the patient's plasma CD63 level is higher than a control level in a sample previously taken from the subject, e.g., at least about 10% higher than the plasma control level (such as at least about 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher than the plasma control level). In embodiments, the method of treatment comprises administering to the subject an effective amount of a substrate reduction therapy or enzyme replacement therapy capable of treating the lysosomal storage disease (e.g., as described herein).

[0211] In embodiments, the lysosomal storage disease is Fabry disease (e.g., wherein the subject is male), Gaucher disease (e.g., Gaucher disease type 1, type 2, or type 3), or MPS (e.g., MPS type I, type II, or type III). In embodiments, the plasma control level is from a sample drawn from the subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks, or 30 weeks) prior to measuring the plasma CD63 level. In embodiments, the control value is derived by measuring the CD63 level in a single sample drawn from the subject. In other embodiments, the control value is an average value derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8, or more samples) drawn from the subject.

[0212] In another aspect, a method for treating or preventing the development or progression of a lysosomal storage disease in a subject assessed to be at risk for the disease is provided, the method comprising the steps of: (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 for the subject (e.g., a therapeutically effective amount of a substrate reduction therapy or an 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.

[0213] In embodiments, the control value is about 10% higher than the control level in a sample previously taken from the same subject (e.g., about 20%, 30%, 40%, 50%, 60%, 80% or 100% higher than the control level). In embodiments, the control level is derived by measuring the CD63 level in a single sample extracted from a subject. In other embodiments, the control value is the average value derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8 or more samples) extracted from a subject. In embodiments, the plasma control level is from a sample extracted from a subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks or 30 weeks) prior to measuring the plasma CD63 level.

[0214] Another aspect provides a method of reducing CD63 levels in a patient with a lysosomal storage disease (e.g., in the patient's blood), the method comprising administering an effective amount of a substrate reduction therapy or an 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, type 2, or type 3), or MPS (e.g., MPS type I, type II, or type III). In embodiments, the reduction in CD63 is a reduction in plasma CD63 levels.

[0215] It should be understood that various embodiments of the methods described above can be applied to the above methods (eg, such as the selection of sample types for test and control samples). In certain embodiments, the specific lysosomal storage disease is selected from the disorders listed above.

[0216] These methods are further described below for Fabry disease, Gaucher disease, and MPS, which are also the subject of embodiments of the present invention.

[0217] Fabry disease

[0218] Fabry disease (FD) is a lysosomal storage disorder characterized by insufficient α-Gal activity encoded by the GLA gene. Enzyme deficiency leads to progressive intracellular accumulation of glycosphingolipids (mostly GL3) in multiple cell types and tissues (including kidneys, heart, liver, spleen and skin) as well as in the peripheral and central nervous systems. The symptoms experienced by patients with FD are highly variable and depend on the severity and stage of the condition. Symptoms usually begin in childhood or adolescence and may include: paresthesia of the extremities (severe pain in the extremities, e.g., burning sensation in the arms and legs, aggravated by exercise and hot weather), opacities of the lens and cornea, angiokeratoma, edema, abdominal pain, impaired blood circulation, and increased risk of heart attack or stroke, enlarged heart, progressive renal damage (e.g., leading to renal failure) and reduced sweating, fever and gastrointestinal difficulties. Renal, cardiac and cerebrovascular symptoms tend to characterize the late stages of the disease and are important causes of morbidity. Fabry disease is an X-linked lipid storage disease, with boys having a 50% chance of inheriting the disorder from their mothers, and daughters having a 50% chance of becoming carriers. Mild forms of the disease are common in women, although affected women may occasionally experience severe symptoms similar to those of men with the disorder. Historically, a diagnosis can be made on an assumed basis by considering clinical symptoms and family history, then determining α-Gal activity in leukocytes or plasma and / or detecting GL3 in tissue biopsies; the diagnosis can then be confirmed by molecular genetic analysis (Breunig et al., Kidney International (2003) 63 (84): S181-S185). 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., recombinant alglucosidase β and recombinant alglucosidase α), small molecule chaperone therapy (e.g., migalastat), and substrate reduction therapy (e.g., venglustat). Other therapeutic interventions are also under investigation (see, e.g., Oder et al., Cardiovasc Diagn Ther. (2021) 11(2): 683-695; and Lenders et al., Drugs (2021) 81(6): 635-645).

[0219] As shown in the accompanying examples, CD63 levels are increased in samples from Fabry disease patients compared to matched healthy control subjects. In particular, it has now been observed that CD63 is upregulated in the plasma of Fabry disease patients; CD63 levels decrease in a linear manner (with both SRT and ERT); CD63 response to treatment (with SRT of venglustat) is comparable in variability to the classic Fabry biomarkers GL3 and lyso-GL3. Therefore, the identification of CD63 as a biomarker for FD represents an important advance in the diagnosis and monitoring of this disease.

[0220] Thus, in one aspect, a method for diagnosing Fabry disease in a subject suspected of being at risk for Fabry disease is provided, the method comprising measuring the level of CD63 in a sample from the subject. Related aspects provide the use of CD63 as a biomarker in diagnosing Fabry disease in a subject suspected of being at risk for Fabry disease. Viewed in another way, these aspects provide a method for generating quantitative data for a subject, the method comprising determining the level of CD63 in a sample from a subject, wherein the subject is suspected of having (or having) Fabry disease.

[0221] In embodiments, by identifying specific risk factors, such as family history of the disease, genetic testing, analysis of other characteristic biomarkers, clinical symptoms, etc., and those described herein, it can be considered that the subject is at risk for Fabry disease. 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 may include one or more of the following: fatigue, pain (e.g., paresthesia of the extremities or abdominal pain), lens or corneal opacities, vortex keratopathy, angiokeratoma, shortness of breath, palpitations, edema, kidney disease (e.g., proteinuria, microscopic hematuria or fat urine, or end-stage renal disease), myocardial dysfunction (e.g., concentric or asymmetric left ventricular hypertrophy, or heart failure), conduction abnormalities with shortened PR intervals, arrhythmias, vertigo, headaches, diplopia, dysarthria, hemiataxia, transient ischemic attacks, early-onset strokes, and dementia. Family history may include a history of Fabry disease among ancestors and / or close relatives (e.g., aunts, uncles, cousins, etc.; see, e.g., 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 with known pathogenic variants (see, e.g., "Fabry Disease: Perspectives from 5 Years of FOS", Oxford Pharma Genesis (2006), ed. Mehta, Beck and Sunder-Plassmann; Chapter 33). For example, in the case of some mutations that lead to premature termination of enzyme translation, the mutation status of the patient can allow a diagnosis of Fabry disease with high confidence. However, in other cases, variant GLA may not be absolutely associated with Fabry disease. Enzyme activity can be assessed by testing the activity of α-Gal in, for example, whole blood or blood spot samples (see, for example, 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 GL3 and / or lyso-GL3 concentrations in samples (e.g., whole blood or plasma samples) (see, for example, Maruyama et al., 2019, supra).

[0222] In embodiments, the control value is a baseline CD63 level that was previously measured in the same subject (i.e., in an earlier sample from the subject), such as at least 1 month before the test sample was obtained from the subject (e.g., at least 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months before the test sample was obtained from the subject). The control value may represent the CD63 level measured in a single earlier sample from the subject, or it may be the average of the values ​​from multiple earlier samples. In alternative embodiments, the control value is the CD63 level in a sample obtained from one or more healthy subjects (e.g., the average of a cohort of healthy subjects at the same time). In embodiments, healthy subjects are matched with the subjects being evaluated, for example, by age and / or gender. Healthy subjects are usually matched with the subjects being evaluated by gender. Typically, the control value represents the CD63 level in the same type of sample (e.g., a sample that has been obtained, processed, and / or stored in the same manner as the test sample) evaluated in the methods of the present invention. For example, where the methods of the invention utilize plasma samples, the control value typically represents the level of CD63 in a control plasma sample (eg, an earlier plasma sample taken from the same subject or a plasma sample obtained from one or more healthy subjects).

[0223] In embodiments where the control value for CD63 concentration is a point value (or average value, e.g., an average value), if the CD63 level in the test sample 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%, then the CD63 level in the test sample is considered to be outside the normal range. In some embodiments, if the CD63 level in the test sample is at least about 2 times greater than the control value, e.g., at least about 3, 4, 5, 6, 8, or 10 times the control value, then the CD63 level in the test sample is considered to be outside the normal range. In embodiments, the CD63 level in the test sample is about 3 to about 8 times the control value, e.g., about 4 to about 7 times the control value, or about 5 to about 6 times the control value. In embodiments, the CD63 level in the test sample is about 5 times the control value or about 6 times the control value. In embodiments where the control value for CD63 concentration is a range of values ​​(e.g., described by some variability around the mean), the CD63 level in the test sample is considered outside the normal range if the CD63 level in the test sample is greater than about 1 standard error of the mean, e.g., greater 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.

[0224] In embodiments, CD63 is used as the sole biomarker in the methods of the disclosure.

[0225] Alternatively, the measurement of CD63 levels in a sample from a subject is used together with one or more other characteristic measures of the disease (e.g., as part of a biomarker panel 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 the method of diagnosing Fabry disease in a subject. In an embodiment, CD63 is used as a biomarker with GL3, lyso-GL3 and / or α-Gal activity, optionally together with one or more other biomarkers. In an embodiment, CD63 is used as a biomarker with lyso-GL3, optionally together with one or more other biomarkers. In an embodiment, the other biomarkers used (i.e., in addition to CD63) do not include any of the following: α-iduronidase, α-glucosidase, saposin C (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, saponin B, heparan-N-sulfatase, α-N-acetylglucosamine glycosidase, acetyl-CoA:glucosamine N-acetyltransferase, N-acetylglucosamine 6-sulfatase, β-galactosidase, β-glucuronidase, asparaginyl glucosaminidase, acid lipase, β-hexosaminidase A, β-hexosaminidase B, GM2-promoter, acid ceramidase, α-L-fucosidase, α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferase g-subunit, palmitoyl-protein thioesterase, tripeptidyl peptidase I, cathespsin K, α-galactosidase B, sialic acid transporter, CD45 leukocyte common biomolecule or LIMP II.

[0226] In embodiments, use of CD63 as a biomarker reduces the proportion of false positives and / or increases the proportion of true positives compared to a corresponding diagnostic that does not measure CD63 levels.

[0227] On the other hand, a method for treating a subject who has been diagnosed with Fabry disease by a method as defined herein is provided, the treatment comprising administering one or more therapeutic treatments for Fabry disease to the subject. A related aspect provides a therapeutic agent for treating Fabry disease in a subject, wherein the subject has been diagnosed with Fabry disease by a method as defined herein. A method for generating quantitative data for a subject is also provided, wherein the method comprises (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., a control value of the level of CD63 measured as a sample taken from one or more healthy subjects); and (c) if the level of CD63 in the sample is greater than the control value, one or more therapeutic treatments for Fabry disease are applied to the subject.

[0228] In embodiments, the therapeutic treatment or agent comprises (e.g., consists of) substrate reduction therapy (e.g., venglustat), chaperone therapy (e.g., migalastat), enzyme replacement therapy (e.g., recombinant alglucosidase alpha or recombinant alglucosidase beta), and / or gene therapy (e.g., using a GLA gene or an active fragment thereof). In embodiments, the therapeutic treatment comprises (e.g., consists of) administering venglustat or migalastat (e.g., venglustat) to the subject. In other embodiments, the treatment comprises administering a recombinant α-galactosidase (e.g., recombinant alglucosidase beta) to the subject.

[0229] Another aspect provides a method for 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 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 the step (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 become less severe 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 Fabry disease in a subject diagnosed with Fabry disease. Viewed in another way, these aspects provide a method for generating quantitative data of a subject diagnosed with Fabry disease, the method comprising the steps of: (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 include an additional step (d), wherein the progression of the disease is assessed based on the comparison made in step (c).

[0230] The sample can 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, sputum, lymph, dermal tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Most conveniently, the sample from the subject includes (e.g., consisting of) blood (e.g., whole blood), blood fractions and / or urine. In embodiments, the sample from the subject includes (e.g., consisting of) blood fractions selected from plasma and serum. In embodiments, the sample is a blood sample, such as a plasma sample. In embodiments, the sample includes (e.g., consisting of) CSF.

[0231] In embodiments, the first and second (or subsequent) samples are obtained from the subject at intervals of at least about 1 day, e.g., at least about 2, 3, or 4 days, or at least about 1, 2, or 4 weeks. In other embodiments, the first and second (or subsequent) samples are obtained from the subject at intervals of at least about 8 weeks, e.g., at least about 9, 10, 12, 15, or 20 weeks.

[0232] In embodiments, the method comprises ongoing measurements of one or more additional samples taken from the subject, eg, at the intervals specified above.

[0233] On the other hand, a method for monitoring the progress of a treatment for Fabry disease in a subject diagnosed with Fabry disease is provided, the method comprising: (a) measuring the level of CD63 in a first sample from the subject; (b) administering a therapeutic treatment for Fabry disease to the subject; and (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 is administered. Typically, the method further comprises a step (d): if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, the treatment is determined to be successful. In an embodiment, if the level of CD63 determined in step (c) is substantially the same as the level of CD63 determined in step (a) or is greater than the level of CD63 determined in step (a), the dose (e.g., the amount and / or frequency of administration) of the therapeutic treatment will be increased. A related aspect provides the use of CD63 as a biomarker for monitoring the progress of a treatment for Fabry disease in a subject diagnosed with Fabry disease. Viewed another way, these aspects provide a method for generating quantitative data of a subject with Fabry disease, the method comprising the steps of: (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, 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), the dose of the therapeutic treatment will be increased.

[0234] In embodiments, the treatment for Fabry disease is as described herein. Thus, the treatment may include (e.g., consisting of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment includes administering venglustat or migalastat (e.g., venglustat) to the subject. In other embodiments, the treatment includes administering recombinant alpha-galactosidase (e.g., recombinant alglucosidase β) to the subject.

[0235] The second sample is typically obtained after a period of time has passed since the administration of the therapeutic treatment. In embodiments, the second sample is obtained from the subject at least about 1 week after the administration of the therapeutic treatment (e.g., after initiation) (e.g., at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the administration of the therapeutic treatment). In one embodiment, the second sample is obtained from the subject at least about 8 weeks after the initiation of the therapeutic treatment.

[0236] In embodiments, the method comprises continuous measurement of one or more additional samples taken from the subject, for example at intervals of about every about 1 week, such as about every 2, 4, 6, 8, 10, or 12 weeks. Such ongoing measurements may be particularly valuable in cases where the dosage of a therapy is adjusted based on changes in CD63 levels in a subject.

[0237] On the other hand, a method for treating Fabry disease in a patient in need thereof is provided, wherein the patient has a higher than normal plasma CD63 level (e.g., at least about 0.25 standard deviations, such as a level of at least about 0.5, 0.75, 1, or 1.5 standard deviations) that is higher than the mean of a healthy population of subjects, the method comprising administering to the patient an effective amount of a therapeutic treatment for Fabry disease. In embodiments, the healthy subject population is a population with a similar genetic background, e.g., a population with the same ancestry and / or geographic location as the patient.

[0238] On the other hand, a method for treating Fabry disease in a subject diagnosed as being at risk of developing the disease is provided, wherein the patient has a plasma CD63 level that is higher than a control level in a sample previously taken from the subject (e.g., at least about 10% higher than the plasma control level, such as at least about 20%, 30%, 40%, 50%, 60%, 80% or 100%), the method comprising administering to the subject an effective amount of a therapeutic treatment for Fabry disease (e.g., as described above). In embodiments, the plasma control level is from a sample extracted from the subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks or 30 weeks) prior to measuring the plasma CD63 level. In embodiments, the control value is derived by measuring the CD63 level in a single sample extracted from the subject. In other embodiments, the control value is an average derived from measuring CD63 levels in multiple samples (eg, 2, 3, 4, 6, 8, or more samples) taken from the subject.

[0239] Another aspect provides a method of reducing CD63 levels in a patient with Fabry disease (e.g., in the patient's blood), the method comprising administering to the patient an effective amount of a therapeutic treatment (e.g., a substrate reduction therapy or an enzyme replacement therapy as described herein). In embodiments, the reduction in CD63 is a reduction in plasma CD63 levels.

[0240] On the other hand, a method for treating or preventing the development or progression of Fabry disease in a subject assessed to be at risk for developing Fabry disease is provided, the method comprising the steps of: (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 for the subject (e.g., a therapeutically effective amount of substrate reduction therapy or enzyme replacement therapy, such as those described herein), 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.

[0241] In embodiments, the control value is about 10% higher than the control level in a sample previously taken from the same subject (e.g., about 20%, 30%, 40%, 50%, 60%, 80% or 100% higher than the control level). In embodiments, the control level is derived by measuring the CD63 level in a single sample extracted from a subject. In other embodiments, the control value is the average value derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8 or more samples) extracted from a subject. In embodiments, the plasma control level is from a sample extracted from a subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks or 30 weeks) prior to measuring the plasma CD63 level.

[0242] In embodiments, the treatment for Fabry disease is as described herein. Thus, the treatment may include (e.g., consisting of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy, or gene therapy. In embodiments, the treatment includes administering venglustat or migalastat (e.g., venglustat) to the subject. In other embodiments, the treatment includes administering recombinant alpha-galactosidase (e.g., recombinant alglucosidase β) to the subject.

[0243] The second sample is typically obtained after a period of time has passed since the administration of the therapeutic treatment. In embodiments, the second sample is obtained from the subject at least about 1 week after the administration of the therapeutic treatment (e.g., after initiation) (e.g., at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the administration of the therapeutic treatment). In one embodiment, the second sample is obtained from the subject at least about 8 weeks after the initiation of the therapeutic treatment.

[0244] In embodiments, the method comprises continuous measurement of one or more additional samples taken from the subject, for example at intervals of about every about 1 week, such as about every 2, 4, 6, 8, 10, or 12 weeks. Such ongoing measurements may be particularly valuable in cases where the dosage of a therapy is adjusted based on changes in CD63 levels in a subject.

[0245] On the other hand, a method for adjusting the dose of the therapeutic treatment of a subject receiving therapeutic treatment for Fabry disease is provided, the method comprising: (a) measuring the CD63 level in a first sample from the subject; (b) measuring the CD63 level in a second sample from the subject, wherein the second sample is obtained from the subject after administering one or more doses of the therapeutic treatment; and (c) adjusting the dose of the therapeutic treatment based on the difference between the CD63 level in the first sample and the CD63 level in the second sample. Typically, the dose adjustment in step (c) includes: if the CD63 level in the second sample is lower than the CD63 level in the first sample, then maintaining the dose of the therapeutic treatment, and if the CD63 level in the second sample is substantially the same as the CD63 level in the first sample or greater than the CD63 level in the first sample, then increasing the dose (e.g., the amount and / or frequency of administration) of the therapeutic treatment. In an embodiment, if the CD63 level in the second sample is lower than the CD63 level in the first sample, then reducing the dose (e.g., the amount and / or frequency of administration) of the therapeutic treatment. Related aspects provide the use of CD63 as a biomarker for adjusting the dosage of a therapeutic treatment for Fabry disease in a subject receiving said therapeutic treatment.

[0246] In embodiments, the subject is a male subject. In other embodiments, the subject is a female subject.

[0247] Gaucher disease

[0248] Gaucher disease (GD) is an inherited metabolic disorder caused by mutations in the GBA gene, resulting in a deficiency of the GC enzyme (also known as acid beta-glucosidase) and an associated accumulation of GL1 and lyso-GL1 in the lysosomes of macrophages; this affects cells of the reticuloendothelial system (including the liver, spleen, and bone marrow), which may lead to enlargement of the spleen and liver, liver dysfunction, skeletal disorders, bone lesions, or severe neurological complications. The forms of GD are grouped into three main types - type 1 GD is a non-neurological form that is the most common type and primarily affects adults, with a mean age at diagnosis of approximately 28 years; type 2 GD is an acute neurological form that typically affects infants in the first 3-6 months of life; and type 3 is a chronic neurological form with a later and slower onset than type 2. Gaucher disease type 3 (GD3) is characterized by progressive encephalopathy and systemic manifestations similar to type 1. It is caused by mutations in the GBA gene (1q21) and is characterized by significant central nervous system (CNS) involvement, which presents significant challenges for diagnosis and monitoring.

[0249] Lyso-GL1 level, β-glucosidase activity and gene GBA sequencing are currently the most common GD diagnostic and prognostic indicators. In the case of lyso-GL1, some studies have shown that its pathological involvement is associated with disease burden and clinical severity. Chitotriosidase and CCL18 have also been identified as biomarkers for Gaucher disease, but are not commonly used in clinical practice (see, for example, Rolfs et al., PLoS ONE (2013) 8 (11): e79732).

[0250] As shown in the accompanying examples, CD63 levels are elevated in Gaucher disease patients and show much lower variability in treated patients compared to typical biomarkers of GD, such as lyso-GL1 or chitotriosidase. Therefore, CD63 represents a valuable biomarker for use in diagnosing and monitoring GD.

[0251] Thus, in one aspect, a method for diagnosing Gaucher disease in a subject suspected of being at risk for developing Gaucher disease is provided, the method comprising measuring the level of CD63 in a sample from the subject. Related aspects provide the use of CD63 as a biomarker in diagnosing Gaucher disease in a subject suspected of being at risk for developing Gaucher disease. Viewed another way, these aspects provide a method for generating quantitative data for a subject, the method comprising determining the level of CD63 in a sample from a subject, wherein the subject is suspected of having (or having) Gaucher disease. 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.

[0252] In embodiments, a subject may be considered at risk for Gaucher disease (e.g., type 1 GD) by identifying specific risk factors, such as family history of the disease, genetic testing, analysis of other characteristic biomarkers, clinical symptoms, and the like, as well 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 may include one or more of the following: hepatomegaly and splenomegaly, pain (especially severe pain in joints (e.g., hips and / or knees)), osteoporosis, skin pigmentation, pancytopenia (e.g., leading to anemia, neutropenia, leukopenia, and / or thrombocytopenia, with increased risk of infection and bleeding), and neurological symptoms (e.g., impaired olfaction and cognition, especially in type 1 GD; convulsions, hypertonia, intellectual disability, and apnea, especially in type 2 GD; and myoclonus, convulsions, dementia, and ocular muscle apraxia, especially in type 3 GD) and Parkinsonism. Family history may include a history of Gaucher disease between ancestors and / or close relatives (e.g., aunts, uncles, cousins, etc.). Genetic testing may involve sequencing some or all of the GBA gene and comparing it with known pathogenic variants (see, e.g., Riboldi et al., Cells (2019) 8: 364-380). For example, in the case of mutations known to be associated with severe disease, the patient's mutation status may allow for a diagnosis of Gaucher disease with high confidence. However, in other cases, variant GBA may not be absolutely associated with Gaucher disease. Enzyme activity can be assessed by testing the activity of GC enzymes, for example, in whole blood or blood spot samples (see, e.g., Miyamoto et al., Intern Med. (2021) 60: 699-707). Glycosphingolipid levels can be assessed by measuring GL1 and / or lyso-GL1 concentrations in samples (e.g., whole blood or plasma samples) (see, e.g., Rolfs et al., 2013, supra).

[0253] In embodiments, the control value is a baseline CD63 level that was previously measured in the same subject (i.e., in an earlier sample from the subject), e.g., at least 1 month before the test sample was obtained from the subject (e.g., at least 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months before the test sample was obtained from the subject). The control value may represent the CD63 level measured in a single earlier sample from the subject, or it may be the average of the values ​​from multiple earlier samples. In alternative embodiments, the control value is the CD63 level in a sample obtained from one or more healthy subjects (e.g., the average of a cohort of healthy subjects at the same time). In embodiments, healthy subjects are matched to the subjects being evaluated, e.g., by age and / or gender. Typically, the control value represents the CD63 level in the same type of sample evaluated in the methods of the present invention (e.g., a sample that has been obtained, processed, and / or stored in the same manner as the test sample). For example, where the methods of the invention utilize plasma samples, the control value typically represents the level of CD63 in a control plasma sample (eg, an earlier plasma sample taken from the same subject or a plasma sample obtained from one or more healthy subjects).

[0254] In embodiments where the control value for CD63 concentration is a point value (or average value, e.g., an average value), if the CD63 level in the test sample 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%, then the CD63 level in the test sample is considered to be outside the normal range. In some embodiments, if the CD63 level in the test sample is at least about 2 times greater than the control value, e.g., at least about 3, 4, 5, 6, 8, or 10 times the control value, then the CD63 level in the test sample is considered to be outside the normal range. In embodiments, the CD63 level in the test sample is about 3 to about 8 times the control value, e.g., about 4 to about 6 times the control value, or about 4 to about 5 times the control value. In embodiments, the CD63 level in the test sample is about 4 times the control value or about 5 times the control value. In embodiments where the control value for CD63 concentration is a range of values ​​(e.g., described by some variability around the mean), the CD63 level in the test sample is considered outside the normal range if the CD63 level in the test sample is greater than about 1 standard error of the mean, e.g., greater 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.

[0255] In embodiments, CD63 is used as the sole biomarker in the methods of the disclosure.

[0256] Alternatively, the measurement of CD63 levels in a sample from a subject is used together with one or more other characteristic measures of the disease (e.g., as part of a biomarker panel 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. Related aspects provide the use of CD63 as a biomarker to improve the method of diagnosing Gaucher disease in a subject. In an embodiment, the Gaucher disease is type 1 GD, type 2 GD, or type 3 GD. In an embodiment, CD63 is used as a biomarker together with GL1, lyso-GL1, CCL18 and / or chitotriosidase, optionally together with one or more other biomarkers. In an embodiment, the other biomarkers used (i.e., in addition to CD63) do not include any of the following: α-iduronidase, α-glucosidase, saposin C (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, saponin B, heparan-N-sulfatase, α-N-acetylglucosamine glycosidase, acetyl-CoA:glucosamine N-acetyltransferase, N-acetylglucosamine 6-sulfatase, β-galactosidase, β-glucuronidase, asparaginyl glucosaminidase, acid lipase, β-hexosaminidase A, β-hexosaminidase B, GM2-promoter, acid ceramidase, α-L-fucosidase, α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferase g-subunit, palmitoyl-protein thioesterase, tripeptidyl peptidase I, cathespsin K, α-galactosidase B, sialic acid transporter, CD45 leukocyte common biomolecule or LIMP II.

[0257] In embodiments, use of CD63 as a biomarker reduces the proportion of false positives and / or increases the proportion of true positives compared to a corresponding diagnostic that does not measure CD63 levels.

[0258] On the other hand, a method for treating a subject who has been diagnosed with Gaucher disease by a method as defined herein is provided, the treatment comprising 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 as defined herein. A method for generating quantitative data for a subject is also provided, wherein the method comprises (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., a control value of the level of CD63 measured as a sample taken from one or more healthy subjects); and (c) if the level of CD63 in the sample is greater than the control value, one or more therapeutic treatments for Gaucher disease are applied to the subject. In an embodiment, the Gaucher disease is type 1 GD, type 2 GD, or type 3 GD.

[0259] In embodiments, the therapeutic treatment or agent comprises (e.g., consists of) substrate reduction therapy (e.g., ilukastat, venglustat, or milustat), chaperone therapy, enzyme replacement therapy (e.g., imiglucerase, velaglucerase, or taliglucerase), and / or gene therapy (e.g., using a GBA gene). In embodiments, the therapeutic treatment comprises (e.g., consists of) administering ilukastat to a subject. In embodiments, the therapeutic treatment comprises (e.g., consists of) administering venglustat to a subject. In other embodiments, the treatment comprises administering recombinant glucocerebrosidase (e.g., imiglucerase) to a subject. In embodiments, the therapeutic treatment comprises (e.g., consists of) administering venglustat and recombinant glucocerebrosidase (e.g., imiglucerase) to a subject. In embodiments, the Gaucher disease is type 1 GD, and the therapeutic treatment comprises (e.g., consists of) administering ilukastat to a subject. In embodiments, the Gaucher disease is type 2 or type 3 GD, and the therapeutic treatment comprises (e.g., consists of) administering venglustat to the subject. In embodiments, the Gaucher disease is type 3 GD, and the therapeutic treatment comprises (e.g., consists of) administering venglustat and a 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 venglustat to the subject.

[0260] Another 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, 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. Typically, the method further comprises the step (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 become less severe 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 another way, these aspects provide a method for generating quantitative data of a subject diagnosed with Gaucher disease, the method comprising the steps of: (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 include an additional step (d), wherein the progression of the disease 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.

[0261] The sample can 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, sputum, lymph, dermal tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Conveniently, the sample from the subject includes (e.g., consisting of) blood (e.g., whole blood), blood fractions and / or urine. In embodiments, the sample from the subject includes (e.g., consisting of) blood fractions selected from plasma and serum. In embodiments, the sample is a blood sample, such as a plasma sample. In embodiments, the sample is a CSF sample. In embodiments, the sample does not include platelets.

[0262] In embodiments, the first and second (or subsequent) samples are obtained from the subject at intervals of at least about 1 day, e.g., at least about 2, 3, or 4 days, or at least about 1, 2, or 4 weeks. In other embodiments, the first and second (or subsequent) samples are obtained from the subject at intervals of at least about 8 weeks, e.g., at least about 9, 10, 12, 15, or 20 weeks.

[0263] In embodiments, the method comprises ongoing measurements of one or more additional samples taken from the subject, eg, at the intervals specified above.

[0264] On the other hand, a method for monitoring the progress of treatment for Gaucher disease in a subject diagnosed with Gaucher disease is provided, 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, wherein the second sample is obtained from the subject after the therapeutic treatment is administered. Typically, the method further comprises a step (d): if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, the treatment is determined to be successful. In an embodiment, if the level of CD63 determined in step (c) is substantially the same as the level of CD63 determined in step (a) or is greater than the level of CD63 determined in step (a), the dose (e.g., the amount and / or frequency of administration) of the therapeutic treatment will be increased. 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 the steps of: (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, 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), the dose of the therapeutic treatment will be increased. 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.

[0265] In embodiments, the treatment for Gaucher disease is as described herein. Therefore, the treatment may include (e.g., consisting of) substrate reduction therapy, enzyme replacement therapy and / or gene therapy. In embodiments, the treatment includes administering ilukastat or milukastat (e.g., ilukastat) to the subject. In other embodiments, the treatment includes administering recombinant glucocerebrosidase (e.g., imiglucerase) to the subject. In embodiments, the method monitors changes from a first treatment (such as ERT, e.g., with imiglucerase) to a second treatment (such as SRT, e.g., with ilukastat), e.g., to check that the patient improves or remains stable after the change.

[0266] The second sample is typically obtained after a period of time has passed since the administration of the therapeutic treatment. In embodiments, the second sample is obtained from the subject at least about 1 week after the administration of the therapeutic treatment (e.g., after initiation) (e.g., at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the administration of the therapeutic treatment). In one embodiment, the second sample is obtained from the subject at least 8 weeks after the initiation of the therapeutic treatment.

[0267] In embodiments, the method comprises continuous measurement of one or more additional samples taken from the subject, for example at intervals of about every 1 week, such as about every 2, 4, 6, 8, 10, or 12 weeks. Such ongoing measurements may be particularly valuable in cases where the dosage of a therapy is adjusted based on changes in CD63 levels in a subject.

[0268] On the other hand, a method of treating Gaucher disease in a patient in need thereof is provided, wherein the patient has a higher than normal plasma CD63 level (e.g., at least about 0.25 standard deviations, such as a level of at least about 0.5, 0.75, 1, or 1.5 standard deviations) above the mean of a healthy population of subjects, the method comprising administering to the patient an effective amount of a therapeutic treatment for Gaucher disease. In embodiments, the population of healthy subjects is a population of similar genetic background, e.g., a population of the same ancestry and / or geographic location as the patient.

[0269] On the other hand, a method for treating Gaucher disease in a subject diagnosed as being at risk for the disease is provided, wherein the patient has a plasma CD63 level that is higher than a control level in a sample previously taken from the subject (e.g., at least about 10% higher than the plasma control level, such as at least about 20%, 30%, 40%, 50%, 60%, 80% or 100%), the method comprising administering to the subject an effective amount of a therapeutic treatment for Gaucher disease (e.g., as described above). In embodiments, the plasma control level is from a sample extracted from the subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks or 30 weeks) prior to measuring the plasma CD63 level. In embodiments, the control value is derived by measuring the CD63 level in a single sample extracted from the subject. In other embodiments, the control value is an average derived from measuring CD63 levels in multiple samples (eg, 2, 3, 4, 6, 8, or more samples) taken from the subject.

[0270] Another aspect provides a method of reducing CD63 levels in a patient with Gaucher disease (e.g., in the patient's blood), the method comprising the step of administering to the patient an effective amount of a therapeutic treatment (e.g., a substrate reduction therapy or an enzyme replacement therapy as described herein). In embodiments, the reduction in CD63 is a reduction in plasma CD63 levels.

[0271] On the other hand, a method for treating or preventing the development or progression of Gaucher disease in a subject assessed to be at risk for developing Gaucher disease is provided, the method comprising the steps of: (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 for the subject (e.g., a therapeutically effective amount of substrate reduction therapy and / or enzyme replacement therapy, such as those described herein), 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.

[0272] In embodiments, the control value is about 10% higher than the control level in a sample previously taken from the same subject (e.g., about 20%, 30%, 40%, 50%, 60%, 80% or 100% higher than the control level). In embodiments, the control level is derived by measuring the CD63 level in a single sample extracted from a subject. In other embodiments, the control value is the average value derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8 or more samples) extracted from a subject. In embodiments, the plasma control level is from a sample extracted from a subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks or 30 weeks) prior to measuring the plasma CD63 level.

[0273] In embodiments, the treatment for Gaucher disease is as described herein. Therefore, the treatment may include (e.g., consisting of) substrate reduction therapy, chaperone therapy, enzyme replacement therapy and / or gene therapy. In embodiments, the treatment includes administering venglustat, ilukastat or milustat (e.g., venglustat or ilukastat) 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., consisting of) administering venglustat 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., consisting of) administering ilukastat to the subject. In embodiments, the Gaucher disease is type 2 or type 3 GD, and the therapeutic treatment includes (e.g., consisting of) administering venglustat to the subject. In embodiments, the Gaucher disease is type 3 GD, and the therapeutic treatment comprises (e.g., consists of) administering venglustat and a 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 venglustat to the subject.

[0274] The second sample is typically obtained after a period of time has passed since the administration of the therapeutic treatment. In embodiments, the second sample is obtained from the subject at least about 1 week after the administration of the therapeutic treatment (e.g., after initiation) (e.g., at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the administration of the therapeutic treatment). In one embodiment, the second sample is obtained from the subject at least about 8 weeks after the initiation of the therapeutic treatment.

[0275] In embodiments, the method comprises continuous measurement of one or more additional samples taken from the subject, for example at intervals of about every about 1 week, such as about every 2, 4, 6, 8, 10, or 12 weeks. Such ongoing measurements may be particularly valuable in cases where the dosage of a therapy is adjusted based on changes in CD63 levels in a subject.

[0276] On the other hand, a method for adjusting the dose of a therapeutic treatment for Gaucher disease in a subject receiving therapeutic treatment is provided, the method comprising: (a) measuring the CD63 level in a first sample from the subject; (b) measuring the CD63 level in a second sample from the subject, wherein the second sample is obtained from the subject after administering one or more doses of the therapeutic treatment; and (c) adjusting the dose of the therapeutic treatment based on the difference between the CD63 level in the first sample and the CD63 level in the second sample. Typically, the dose adjustment in step (c) comprises: if the CD63 level in the second sample is lower than the CD63 level in the first sample, then maintaining the dose of the therapeutic treatment, and if the CD63 level in the second sample is substantially the same as or greater than the CD63 level in the first sample, then increasing the dose (e.g., amount and / or frequency) of the therapeutic treatment. In an embodiment, if the CD63 level in the second sample is lower than the CD63 level in the first sample, then reducing the dose (e.g., amount and / or frequency) of the therapeutic treatment. Related aspects provide 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.

[0277] Mucopolysaccharidosis

[0278] Mucopolysaccharidosis is a hereditary disorder in which GAGs accumulate in connective tissue and other tissues throughout the body due to a lack of lysosomal enzymes that degrade GAGs. Symptoms vary depending on the severity of the disease, but are typically caused by damage to bones, connective tissue, and organs (e.g., dysplasia, joint stiffness, and hepatomegaly / splenomegaly) and compression of nerves or spinal nerve roots (e.g., pain, impaired motor function, and other complications of the peripheral nervous system). There are currently seven identified MPS clinical types, as well as 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 treatment initiation are usually crucial to have any significant impact on disease progression. The general characteristics of MPS are indicated in the following table (adapted from Celik et al., supra, wherein: HS is heparan sulfate; DS is dermatan sulfate; CS is chondroitin sulfate, such as 4- or 6- chondroitin sulfate; and KS is keratan sulfate):

[0279] Historically, the clinical diagnosis of MPS is carried out by measuring the total GAG level in urine. However, due to the difficulty in collection and storage, this method is impractical and costly for screening neonatal urine. A spot test of urine GAG ​​levels based on dye staining has been developed, but this test has reliability and sensitivity problems, especially in the first few days after birth. These dye-based methods are generally not suitable for blood spot samples, and cannot distinguish MPS types or predict the severity of the disease with great reliability (see, for example, Tomatsu et al., Mol Genet Metab. (2013) 110 (0): 42-53). An affinity binding method (ELISA) that can detect specific GAGs (such as keratan sulfate) has been developed. Such methods can be used to assess the GAG ​​levels in blood samples, but are not suitable for simultaneous analysis of multiple GAGs (see, for example, Khan et al., Mol Genet Metab. (2020) 130 (2): 101-109). 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 the diagnosis of all types of MPS in blood samples.

[0280] Therefore, there remains a need for improved methods for MPS diagnosis and for providing new methods for monitoring disease progression and therapeutic efficacy.As shown in the appended examples, CD63 levels are elevated in MPS patients and can be used as a measure for detecting and monitoring MPS in these subjects.

[0281] Thus, in one aspect, a method for diagnosing MPS in a subject suspected of being at risk of MPS is provided, the method comprising measuring the level of CD63 in a sample from the subject. Related aspects provide the use of CD63 as a biomarker in diagnosing MPS in a subject suspected of being at risk of MPS. Viewed in another way, these aspects provide a method for generating quantitative data for a subject, the method comprising determining the level of CD63 in a sample from a subject, wherein the subject is suspected of having (or having) 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 type). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., IIIA, IIIB, IIIC, or HID type). 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 (eg, MPS IIIA).

[0282] In embodiments, a subject may be considered at risk for MPS by identifying specific risk factors, such as family history of the disease, genetic testing, analysis of other characteristic biomarkers, clinical symptoms, etc. In embodiments, the specific risk factors are selected from one or more of clinical symptoms, family history, genetic testing, enzyme activity, and GAG levels. Clinical symptoms may include one or more symptoms affecting the head and neck (e.g., macrocephaly, hearing loss, corneal opacity, and abnormal dentition), joints and bones (e.g., stiffness, hip dysplasia, claw hands, and joint laxity), cardiovascular system (e.g., valve thickening and left ventricular hypertrophy), airway (e.g., recurrent respiratory tract infections and obstructive airway disease), abdomen (e.g., hepatomegaly / splenomegaly and umbilical / inguinal hernia), as well as neurological symptoms (e.g., developmental delay, ventriculomegaly, enlarged perivascular spaces, and hyperactive or aggressive behavior), and other symptoms (e.g., abnormal leukocyte granules, fetal hydrops, and proteinuria). Many of these signs and symptoms are common to different types of MPS, and patients presenting isolated symptoms cannot typically be diagnosed with high certainty on this basis alone. Developmental delay and osteoarticular manifestations may be most relevant to screening programs for early diagnosis of these conditions (for more details on MPS differential diagnosis, see, for example, Kubaski et al., Diagnostics (Basel). (2020) 10 (3): 172). Family history may include a history of MPS between ancestors and / or close relatives (e.g., aunts, uncles, cousins, etc.). Genetic testing may involve sequencing some or all of the relevant genes (e.g., as shown in the table above) and comparing them with known pathogenic variants (see, for example, Kubaski et al., 2020, supra). The mutation status of the patient can allow the diagnosis of MPS with high confidence, however, it may not be possible to absolutely associate the variant gene with MPS. Enzyme activity can be assessed by testing the activity of the missing enzyme in, for example, whole blood or blood spot samples (see, for example, 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 (e.g., whole blood, plasma, serum, or urine sample) (see, for example, Khan et al., 2020, supra).

[0283] In embodiments, the control value is a baseline CD63 level that was previously measured in the same subject (i.e., in an earlier sample from the subject), e.g., at least 1 month before the test sample was obtained from the subject (e.g., at least 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months before the test sample was obtained from the subject). The control value may represent the CD63 level measured in a single earlier sample from the subject, or it may be the average of the values ​​from multiple earlier samples. In alternative embodiments, the control value is the CD63 level in a sample obtained from one or more healthy subjects (e.g., the average of a cohort of healthy subjects at the same time). In embodiments, healthy subjects are matched to the subjects being evaluated, e.g., by age and / or gender. Typically, the control value represents the CD63 level in the same type of sample evaluated in the methods of the present invention (e.g., a sample that has been obtained, processed, and / or stored in the same manner as the test sample). For example, where the methods of the invention utilize plasma samples, the control value typically represents the level of CD63 in a control plasma sample (eg, an earlier plasma sample taken from the same subject or a plasma sample obtained from one or more healthy subjects).

[0284] In embodiments where the control value for CD63 concentration is a point value (or average value, e.g., an average value), if the CD63 level in the test sample 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%, then the CD63 level in the test sample is considered to be outside the normal range. In some embodiments, if the CD63 level in the test sample is at least about 2 times greater than the control value, e.g., at least about 3, 4, 5, 6, 8, or 10 times the control value, then the CD63 level in the test sample is considered to be outside the normal range. In embodiments, the CD63 level in the test sample is about 3 to about 8 times the control value, e.g., about 3 to about 6 times the control value, or about 4 to about 5 times the control value. In embodiments, the CD63 level in the test sample is about 4 times the control value or about 5 times the control value. In embodiments where the control value for CD63 concentration is a range of values ​​(e.g., described by some variability around the mean), the CD63 level in the test sample is considered outside the normal range if the CD63 level in the test sample is greater than about 1 standard error of the mean, e.g., greater 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.

[0285] In embodiments, CD63 is used as the sole biomarker in the methods of the disclosure.

[0286] Alternatively, the measurement of CD63 levels in a sample from a subject is used together with one or more other characteristic measures of the disease (e.g., as part of a biomarker panel 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. Related aspects provide the use of CD63 as a biomarker to improve the method of 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., IH, IS, or IH / S type). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., IIIA, IIIB, IIIC, or HID type). 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., in addition to CD63) do not include any of the following: alpha-iduronidase, alpha-glucosidase, saponin C, LAMP-1, LAMP-2, beta-glucosidase, alpha-galactosidase A, iduronate-2-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, saponin B, heparan-N-sulfatase, alpha-N-acetylglucosamine glycosidase, acetyl-CoA:glucosamine N-acetyltransferase, N-acetylg ... Glycosamine 6-sulfatase, β-galactosidase, β-glucuronidase, asparaginyl glucosaminidase, acid lipase, β-hexosaminidase A, β-hexosaminidase B, GM2-promoter, acid ceramidase, α-L-fucosidase, α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferase g-subunit, palmitoyl-protein thioesterase, tripeptidyl peptidase I, cathespsin K, α-galactosidase B, sialic acid transporter, CD45 leukocyte common biomolecule or LIMP II.

[0287] In embodiments, use of CD63 as a biomarker reduces the proportion of false positives and / or increases the proportion of true positives compared to a corresponding diagnostic that does not measure CD63 levels.

[0288] Another aspect provides a method of treating a subject who has been diagnosed with MPS by a method as defined herein, the treatment comprising administering to the subject one or more therapeutic treatments for MPS. A related aspect provides a therapeutic agent for treating MPS in a subject, wherein the subject has been diagnosed with MPS by a method as defined herein. A method for generating quantitative data for a subject is also provided, wherein the method comprises (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., a control value measured as the level of CD63 in a sample taken from one or more healthy subjects); and (c) if the level of CD63 in the sample is greater than the control value, applying one or more therapeutic treatments for MPS to 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., 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 HID). 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).

[0289] In embodiments, the therapeutic treatment or medicament includes (e.g., consisting 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 includes (e.g., consisting of) enzyme replacement therapy and / or hematopoietic stem cell transplantation. In embodiments, the therapeutic treatment or medicament includes (e.g., consisting of) enzyme replacement therapy and / or hematopoietic stem cell transplantation: α-L-iduronidase, iduronidase-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase, α-glucosaminidase, acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-6-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 a 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 a recombinant iduronidase-2-sulfatase (e.g., idursulfatase). In other embodiments, the MPS is MPS IVA and the therapeutic 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 therapeutic 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 therapeutic treatment comprises (e.g., consists of) administering to the subject a recombinant β-glucuronidase (e.g., vestronidase alfa).

[0290] 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, 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. Typically, the method further comprises the step (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 become less severe 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 another way, these aspects provide a method for generating quantitative data for a subject diagnosed with MPS, the method comprising the steps of: (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) to the level determined in step (b). The method may include an additional step (d), wherein the progression of the disease is assessed based on the comparison performed 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., 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 HID). 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).

[0291] The sample can be 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, sputum, lymph, dermal tissue, kidney tissue, heart tissue, spleen tissue, bone marrow, etc. Most conveniently, the sample from the subject includes (e.g., consisting of) blood, blood fractions and / or urine. In embodiments, the sample from the subject includes (e.g., consisting of) blood fractions selected from plasma and serum. In embodiments, the sample is a blood sample, such as a plasma sample. In other embodiments, the sample includes (e.g., consisting of) urine. In other embodiments, the sample includes (e.g., consisting of) CSF. In embodiments, the sample does not include fibroblasts.

[0292] In embodiments, the first and second (or subsequent) samples are obtained from the subject at intervals of at least about 1 day, e.g., at least about 2, 3, or 4 days, or at least about 1, 2, or 4 weeks. In other embodiments, the first and second (or subsequent) samples are obtained from the subject at intervals of at least about 8 weeks, e.g., at least about 9, 10, 12, 15, or 20 weeks.

[0293] In embodiments, the method comprises ongoing measurements of one or more additional samples taken from the subject, eg, at the intervals specified above.

[0294] Another aspect provides a method for monitoring the progress of a 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, wherein the second sample is obtained from the subject after the administration of the therapeutic treatment. Typically, the method further comprises a step (d): if the level of CD63 in the second sample is lower than the level of CD63 in the first sample, determining that the treatment is successful. In an embodiment, 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), the dose (e.g., amount and / or frequency of administration) of the therapeutic treatment will be increased. A related aspect provides the use of CD63 as a biomarker for monitoring the progress of a treatment for MPS in a subject diagnosed with MPS. Viewed in another way, these aspects provide a method for generating quantitative data of a subject with MPS, the method comprising the steps of: (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, 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), the dose of the therapeutic treatment will be increased. 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 HID). 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 (eg, MPS IIIA).

[0295] In embodiments, the treatment for MPS is as described herein.Therefore, the treatment may include (for example, consisting 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 (for example, consisting of) enzyme replacement therapy and / or hematopoietic stem cell transplantation.In embodiments, the therapeutic treatment or medicament includes (for example, consisting of) the following enzyme replacement therapy: α-L-iduronidase, iduronidase-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase, α-glucosamine, acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-6-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 to the subject a recombinant α-L-iduronidase (e.g., laronidase). 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., iduronidase). In other embodiments, the MPS is MPS IVA, and the treatment comprises (e.g., consists of) administering to the subject a recombinant N-acetylgalactosamine-6-sulfatase (e.g., elosulfatase α); 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., galactase); or the MPS is MPS VII, and the treatment comprises (e.g., consists of) administering to the subject a recombinant β-glucuronidase (e.g., vicrinidase α).

[0296] The second sample is typically obtained after a period of time has passed since the administration of the therapeutic treatment. In embodiments, the second sample is obtained from the subject at least about 1 week after the administration of the therapeutic treatment (e.g., after initiation) (e.g., at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the administration of the therapeutic treatment). In one embodiment, the second sample is obtained from the subject at least about 8 weeks after the initiation of the therapeutic treatment.

[0297] In embodiments, the method comprises continuous measurement of one or more additional samples taken from the subject, for example at intervals of about every 1 week, such as about every 2, 4, 6, 8, 10, or 12 weeks. Such ongoing measurements may be particularly valuable in cases where the dosage of a therapy is adjusted based on changes in CD63 levels in a subject.

[0298] On the other hand, a method of treating MPS in a patient in need thereof is provided, wherein the patient has a higher than normal plasma CD63 level (e.g., a level of at least about 0.25 standard deviations, such as at least about 0.5, 0.75, 1, or 1.5 standard deviations) that is higher than the average of a healthy population of subjects, the method comprising administering to the patient an effective amount of a therapeutic treatment for MPS. In embodiments, the healthy subject population is a population with a similar genetic background, for example, a population with the same ancestry 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., IH, IS, or IH / S type). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., IIIA, IIIB, IIIC, or HID type). 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).

[0299] On the other hand, a method of treating the disease in a subject diagnosed as being at risk of developing MPS is provided, wherein the patient has a plasma CD63 level that is higher than a control level in a sample previously taken from the subject (e.g., at least about 10% higher than the plasma control level, such as at least about 20%, 30%, 40%, 50%, 60%, 80% or 100%), the method comprising administering to the subject an effective amount of a therapeutic treatment for MPS (e.g., as described above). In an embodiment, the plasma control level is from a sample extracted from the subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks or 30 weeks) before measuring the plasma CD63 level. In an embodiment, the control value is derived by measuring the CD63 level in a single sample extracted from the subject. In other embodiments, the control value is an average value derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8 or more samples) extracted 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 HID). 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).

[0300] On the other hand, a method for reducing the level of CD63 in a patient with MPS (e.g., in the blood of the patient) is provided, the method comprising the step of administering to the patient an effective amount of a therapeutic treatment (e.g., an enzyme replacement therapy as described herein). In embodiments, the reduction of 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 type). In other embodiments, the MPS is MPS II. In other embodiments, the MPS is MPS III (e.g., IIIA, IIIB, IIIC, or HID type). 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).

[0301] Another aspect provides a method for treating or preventing the development or progression of MPS in a subject assessed to be at risk for MPS, the method comprising the steps of: (a) obtaining a first biological sample (e.g., blood or a blood fraction such as plasma) from the subject and analyzing the CD63 concentration of the sample; (b) if the CD63 concentration is above a control value, initiating a course of therapeutic treatment for the subject (e.g., a therapeutically effective amount of enzyme replacement therapy, such as those described herein), 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 CD63 concentration of the sample to determine a change in the CD63 level; and (d) adjusting the therapeutic treatment based on the observed change in the CD63 level. 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 HID). In embodiments, the MPS is not MPS I. In embodiments, the MPS is not MPS III (eg, MPS IIIA). In embodiments, the MPS is not MPS I or MPS III (eg, MPS IIIA).

[0302] In embodiments, the control value is about 10% higher than the control level in a sample previously taken from the same subject (e.g., about 20%, 30%, 40%, 50%, 60%, 80% or 100% higher than the control level). In embodiments, the control level is derived by measuring the CD63 level in a single sample extracted from a subject. In other embodiments, the control value is the average value derived from measuring the CD63 level in multiple samples (e.g., 2, 3, 4, 6, 8 or more samples) extracted from a subject. In embodiments, the plasma control level is from a sample extracted from a subject between about 1 and about 52 weeks (e.g., between 4 and 32 weeks, or between 12 and 26 weeks, such as about 12 weeks, 16 weeks, 22 weeks, 26 weeks or 30 weeks) prior to measuring the plasma CD63 level.

[0303] In embodiments, the treatment for MPS is as described herein.Therefore, the treatment may include (for example, consisting of) enzyme replacement therapy, gene therapy and / or hematopoietic stem cell transplantation.In embodiments, the treatment includes (for example, consisting of) using the following enzyme replacement therapy: α-L-iduronidase, iduronidase-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase, α-glucosaminidase, acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-6-sulfatase, β-galactosidase, N-acetylglucosamine-4-sulfatase, β-glucuronidase or hyaluronidase.

[0304] The second sample is typically obtained after a period of time has passed since the administration of the therapeutic treatment. In embodiments, the second sample is obtained from the subject at least about 1 week after the administration of the therapeutic treatment (e.g., after initiation) (e.g., at least about 2, 3, 4, 6, 8, 10, 12, 15, or 20 weeks after the administration of the therapeutic treatment). In one embodiment, the second sample is obtained from the subject at least about 8 weeks after the initiation of the therapeutic treatment.

[0305] In embodiments, the method comprises continuous measurement of one or more additional samples taken from the subject, for example at intervals of about every about 1 week, such as about every 2, 4, 6, 8, 10, or 12 weeks. Such ongoing measurements may be particularly valuable in cases where the dosage of a therapy is adjusted based on changes in CD63 levels in a subject.

[0306] On the other hand, a method for adjusting the dose of a therapeutic treatment for MPS in a subject receiving therapeutic treatment is provided, the method comprising: (a) measuring the CD63 level in a first sample from the subject; (b) measuring the CD63 level in a second sample from the subject, wherein the second sample is taken from the subject after administering one or more doses of the therapeutic treatment; and (c) adjusting the dose of the therapeutic treatment based on the difference between the CD63 level in the first sample and the CD63 level in the second sample. Typically, the dose adjustment in step (c) comprises: if the CD63 level in the second sample is lower than the CD63 level in the first sample, then maintaining the dose of the therapeutic treatment, and if the CD63 level in the second sample is substantially the same as or greater than the CD63 level in the first sample, then increasing the dose (e.g., amount and / or frequency) of the therapeutic treatment. In an embodiment, if the CD63 level in the second sample is lower than the CD63 level in the first sample, then reducing the dose (e.g., amount and / or frequency) of the therapeutic treatment. Related aspects provide for the use of CD63 as a biomarker for adjusting the dose of a therapeutic treatment for MPS in a subject receiving the 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 HID). 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).

[0307] Determination

[0308] Assays that can be used to measure and quantify CD63 levels in biological samples are known. Specific examples of suitable assays are given in the Examples below. In general, the assays rely on molecules (e.g., antibodies) that have specific binding to CD63 and which themselves can be detected and / or quantified directly or via another binding partner (e.g., using a labeled universal antibody).

[0309] Thus, in embodiments, CD63 detection and / or quantification is performed by immunoassay (e.g., ELISA, microfluidic ELISA, or bead-based high sensitivity ELISA, or proximity extension assay), mass spectrometry, microchip, biophysical assay (e.g., surface plasmon resonance using an anti-CD63 capture antibody, such as from ), nanoneedle bioarrays (e.g., from ) or nucleic acid binding aptamers (e.g., from ). Exemplary embodiments employ an ELISA, such as an indirect or sandwich ELISA, 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., 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 that can react with CD63 from one species or from more than one species. Such antibodies can be obtained from commercial sources (e.g., In addition, the sequence of CD63 is well known, and the skilled person can generate monoclonal antibodies against CD63 using standard techniques (see also "Leukocyte and Stromal Cell Molecules: The CD Markers", Wiley; ed. Zola et al., 2007, p. 150). Another exemplary embodiment employs cell sorting techniques, such as FACS using fluorescently labeled antibodies against CD63, CD8 and / or CD81.

[0310] An alternative exemplary embodiment employs a proximity extension assay using matched pairs of antibodies that bind CD63, wherein each of the matched pairs of antibodies is labeled with a unique oligonucleotide such that when the antibodies bind to their targets, the oligonucleotides hybridize. The annealed products are then amplified by PCR and detected (e.g., in a multiplexed manner), typically in a high-throughput fluidic chip system.

[0311] The sample for use according to the method of the invention can be purified, for example, and / or separated from other (e.g., non-CD63-containing) components in the sample. The target component of the sample, such as exosomes, can also be concentrated. Methods for concentrating and / or purifying exosomes and other CD63-containing components are known in the art and / or described herein. For example, the exosomes of the sample can be enriched by ultracentrifugation and / or antibody capture methods. Ultracentrifugation of plasma, serum, urine or cell culture samples can include, for example, centrifugation at 100,000g to precipitate the exosomes and resuspend them in an appropriate buffer. Affinity capture methods can, for example, use antibody-coated beads to capture exosome membrane targets, and then recover the beads by centrifugation or using a magnet.

[0312] Reagent test kit

[0313] Another aspect provides a kit for (e.g., suitable for) detecting or diagnosing lysosomal dysfunction or abnormal glycosphingolipid processing in a sample from a subject, the kit comprising a device for detecting CD63 and optionally a device for detecting one or more other biomarkers of lysosomal dysfunction. In an embodiment, the device for detecting CD63 comprises an anti-CD63 antibody (e.g., a monoclonal antibody). In an embodiment, the device for detecting CD63 comprises an anti-CD63 antibody pair, each of which is linked to a unique oligonucleotide so that when the antibody pair binds to CD63, the unique oligonucleotide can hybridize.

[0314] Another aspect provides a kit for (e.g., suitable for) monitoring the progression of a particular lysosomal storage disease or monitoring the response of a particular lysosomal storage disease to treatment of a subject, the kit comprising a device for detecting CD63 and optionally a device for detecting one or more other biomarkers of lysosomal dysfunction. In embodiments, the device for detecting CD63 comprises an anti-CD63 antibody (e.g., a monoclonal antibody). In embodiments, the device for detecting CD63 comprises an anti-CD63 antibody pair, each of which is linked to a unique oligonucleotide such that the unique oligonucleotide can hybridize when the antibody pair binds to CD63.

[0315] 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) a device for detecting CD63 in a sample from the subject; and (b) a device for detecting one or more biomarkers of the lysosomal storage disease in a sample from the subject. In embodiments, the device for detecting CD63 comprises an anti-CD63 antibody (e.g., a monoclonal antibody). In embodiments, the device for detecting CD63 comprises an anti-CD63 antibody pair, each of which is linked to a unique oligonucleotide such that the unique oligonucleotide can hybridize when the antibody pair binds to CD63.

[0316] In an embodiment, the lysosomal storage disease is Fabry disease, and the kit comprises: (a) a device for detecting CD63 in a sample from a subject (e.g., an anti-CD63 antibody or a matched pair of anti-CD63 antibodies, each of which is linked to a unique oligonucleotide); and (b) a device for detecting one or more biomarkers for Fabry disease in the sample (e.g., a device for detecting GL3 and / or lyso-GL3 in the sample).

[0317] In an embodiment, the lysosomal storage disease is Gaucher disease, and the kit comprises: (a) a device for detecting CD63 in a sample from a subject (e.g., an anti-CD63 antibody or a matched pair of anti-CD63 antibodies, each of which is linked to a unique oligonucleotide); and (b) a device for detecting one or more biomarkers for Gaucher disease in the sample (e.g., a device for detecting lyso-GL1 in the sample).

[0318] In embodiments, the lysosomal storage disease is MPS (e.g., MPS I, MPS II, or MPS III), and the kit comprises: (a) a device for detecting CD63 in a sample from a subject (e.g., an anti-CD63 antibody or a matched pair of anti-CD63 antibodies, each of which is linked to a unique oligonucleotide); and (b) a device for detecting one or more biomarkers of MPS in the sample, such as a device for detecting dermatan sulfate and, optionally, heparan sulfate in a sample (e.g., in the case of MPS I), a device for detecting dermatan sulfate and heparan sulfate in a sample (e.g., in the case of MPS II), or a device for detecting heparan sulfate in a sample (e.g., in the case of MPS III).

[0319] The kits of the present disclosure may further comprise instructions for use of the kit in one or more of the methods described herein (e.g., in one or more methods for detecting and / or diagnosing a lysosomal storage disease, lysosomal dysfunction, or aberrant glycosphingolipid processing in a biological sample).

[0320] Having generally described herein, the following non-limiting examples are provided to further illustrate the disclosure. Example

[0321] Experimental protocol

[0322] Protein determination

[0323] According to the manufacturer's instructions, Spectral analysis (described below) uses CD63 was measured by the Target96 Neuro-Exploratory panel (Olink Proteomics AB, Uppsala, Sweden).

[0324] Chitotriosidase activity was determined according to the method described in Schoonhoven et al., Clin Chim Acta. (2007) 381(2): 136-139, with slight modifications. Briefly, 5 μL of serum sample was mixed with 100 μL of 26 μM 4-methylumbelliferyl-β-DN, N′, N″-triacetyl chitotriosidase (Sigma, M5639) in 0.1 M / 0.2 M citrate-phosphate buffer. This mixture was incubated at 37° C. for 15 min. To stop the reaction, glycine-sodium hydroxide buffer (210 μL 0.5 M Gly-NaOH, pH 10.6) was added. Plate reader was used. Fluorescence was measured at 360 nm excitation and 455 nm emission. Chitotriosidase activity can also be determined according to the method described by Adelino et al., JIMD Rep. (2013) 9:85-91 and reported in nmol / hr / ml.

[0325] CCL18 levels were measured by sandwich ELISA according to the method described in Boot et al., Blood (2004) 103(1):33-39.

[0326] Can be used (Olink Proteomics AB, Uppsala, Sweden) Spectral analysis Protein detection was performed 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) technique of the scheme has been well described (Assarsson et al., 2014, supra), and can simultaneously analyze each group of 92 analytes to be analyzed using 1 μL of each sample. In short, oligonucleotide-labeled antibody probes bind to their target proteins, and if the two probes are brought into close proximity, the oligonucleotides will hybridize in pairs. Adding DNA polymerase will result in proximity-dependent DNA polymerization events, producing unique PCR target sequences. Subsequently, a microfluidic real-time PCR instrument (Biomark HD, Fluidigm) can be used to detect and quantify the resulting DNA sequence. The data is then quality controlled and normalized using internal extension controls and inter-plate controls to adjust for intra-run variations and inter-run variations. The final assay readout is presented in normalized protein performance (NPX) values, which are arbitrary units on a log2 scale, where high values ​​correspond to higher protein performance. All assay validation data (e.g., detection limits, intra-assay precision data, and inter-assay precision data, etc.), experimental protocols, and data processing can be obtained on the manufacturer's website (www.olink.com).

[0327] The PEA assay can be calibrated to quantify protein levels, such as the protein levels of CD63. Typically, this involves preparing a sample of known concentration (e.g., by titration dilution in a buffer as defined above) and generating an absolute concentration calibration curve. Although proteins can be expressed in baculovirus or E. coli systems using known techniques, recombinant human CD63 typically obtained (e.g., from a commercial supplier) has been produced in a mammalian expression system to promote correct folding and / or post-translational modification (e.g., glycosylation). The concentration can be determined using well-known methods (e.g., using a bicinchoninic acid (BCA) assay with a BSA sample of known concentration). The concentration can also be determined by measuring the total amino acid concentration after protein hydrolysis. In An appropriate range of gradually decreasing protein concentrations is selected within the dynamic range of the assay. For example, a 3-fold dilution can be used to generate samples with protein concentrations of 1, 3, 9, 12, 36, 108, 324, and 972 pg / ml. A 0 pg / ml sample (i.e., just buffer) is used as a blank to determine background. Once calibration is performed using samples with known CD63 concentrations, the assay is used to test human patient samples in the same assay experiment. Human patient samples are typically run without dilution, but can be diluted with a known amount of buffer in the event that the protein level exceeds the range of the calibration curve. The absolute concentration of CD63 in the test sample can be determined by reference to the calibration curve - by comparison NPX values ​​(e.g., mean values ​​of triplicate determinations) - are calculated by fitting the calibration sample values ​​to a linear fit for pg / ml or by a curve fit such as a 4-parameter nonlinear curve fit (as appropriate) to give a good curve fit. Assuming the test sample NPX values ​​are within the dynamic range of the calibration curve - e.g., levels above the 0 pg / ml blank, with acceptable variability (e.g., within 20% CV) - the absolute concentration is accurately determined by reference to the fitted calibration curve. In this manner, the PEA assay can provide absolute quantification of protein levels (in pg / ml).

[0328] To measure protein levels in CSF, the following protocol was used: four The exploratory panels (cardiometabolic, inflammatory, neurological, and oncological) measured biomarker expression in CSF as described, for example, by Wik et al. (“Proximity Extension Assay in Combination with Next Generation Sequencing for High-throughput Proteome-wide Analysis, 2021, Mol Cell Proteomics 20, 100168). Briefly, 10 μL of CSF sample and Olink control were placed in a 384-well sample source plate and then diluted 1:10, 1:100, 1:1000, and 1:100,000 in a 384-well sample diluent plate using Mosquito and Dragonfly liquid handlers (both from SPT Labtech). For immune responses, these diluted samples were mixed with The probes (DNA oligo-conjugated antibodies) were mixed in a 384-well culture plate and incubated at 4°C for 16-24 hours. The next day, the reagent mixture for the first PCR amplification step (PCR1 step) was added to the immunoreaction mixture using a Dragonfly processor, and the PCR1 reaction for the pre-amplification step was performed using the ProFlex PCR system (Applied Biosciences). The PCR1 products were pooled using an epMotion 5075 liquid handler (Eppendorf), and then the reagents for the second PCR amplification step (PCR2 step) were added using the same handler, and the PCR2 reaction for amplification and sample indexing was performed using the ProFlex system. The PCR2 products were pooled using the epMotion processor to obtain a total of 100 samples for each. Four libraries were created for the discovery group, and the products were then purified using magnetic beads and their QC was performed using a Bioanalyzer 2100 (Agilent). The four libraries were analyzed by next-generation sequencing using a NovaSeq 6000 (Illumina). Analytes with more than 10% missing data were excluded from the analysis. No missing data imputation was performed.

[0329] Alternative methods can be used to measure protein levels (such as CD63), including, for example, immunoassays using antibodies to CD63 (e.g., widely commercially available ELISA, microfluidic ELISA such as Protein Simple Ella, or bead-based high-sensitivity ELISA such as 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 traditional ELISA assays are typically diluted 4-fold or more in sample dilution buffer to give acceptable assay performance (dilution linearity).

[0330] Determination of glycosphingolipids

[0331] According to Murugesan et al., Am J Hematol. (2016) 91 (11): 1082-1089, glycosphingolipids (lyso-GL1, GL3 and lyso-GL3) were measured by ESI-LC-MS / MS. In short, 20 μL plasma aliquots were added to 1 mL chloroform in an Eppendorf tube: methanol (2: 3), mixed and then centrifuged. The supernatant was taken out 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 methanol: water (9: 1), and injected into the LC-MS / MS system for tandem mass spectrometry. UPLC was used to achieve separation of glycosphingolipids and other matrix components under gradient conditions with two mobile phases (0.1% formic acid in water; and 0.1% formic acid in acetonitrile). In selected ion monitoring mode, typically using the precursor (M+H + ) ion transitions (e.g., for lyso-GL1, m / z 462.5>282.4) were used for mass spectrometry (MS). Known glycosphingolipid standards (dimethylsphingosine, 13 The assay was calibrated with C6-labeled lyso-GL3 (GelbChem) and C17-GL3 (Matreya LLC, catalog number 1523) as internal standards for lyso-GL1, lyso-GL3, and GL3, respectively.

[0332] Sample preparation

[0333] Plasma and serum sample preparation followed standard protocols. Cell samples were prepared by mixing / agitation. Tissue samples were dissolved by homogenization in a standard buffer (e.g., PBS; or 50 mM Tris / HCl pH 7.5, optionally containing a mild detergent such as 0.1% TWEEN20 and / or a carrier protein such as 1% BSA, which may improve stability and / or reproducibility). Additional detergent (e.g., up to 1% Triton) was typically added to tissue samples at higher concentrations to account for dissolution. Plasma and serum samples were typically prepared at The assay is run undiluted. Preparation of cell and tissue samples involves adding buffer to Final protein concentration within the dynamic range of the assay.

[0334] Exosomes of the sample to be tested can be enriched, for example, using FACS as described herein. Alternative methods for exosome enrichment or purification include ultracentrifugation and antibody capture methods, both of which are known and readily available. Standard ultracentrifugation for preparing exosomes from plasma / serum, urine or cell culture will include, for example, centrifugation at 100,000 g for 1 hour in a typical floor-standing ultracentrifuge using a typical rotor and a bucket accommodating 10ml-50ml samples, or centrifugation in a desktop ultracentrifuge for 5 minutes using 0.5ml or 1.5ml sample tubes. Exosomes are precipitated to the bottom of the tube and resuspended in an appropriate buffer. Commercial kits for antibody affinity capture of exosomes and exosome surface / membrane proteins are available (e.g., from System Biosciences Inc.). The method typically uses antibody-coated beads to capture exosome targets, and then recovers the beads by centrifugation or using a magnet.

[0335] Analysis of LSD biomarkers in patient and control samples

[0336] Plasma from Fabry disease patients and normal controls

[0337] Plasma from FD patients was obtained from 3 different cohorts, referred to below as (i) ACT / LTS, (ii) Cohort 1, and (iii) Cohort 2.

[0338] ACT / LTS samples were obtained from the Sanofi–venglustat Phase II trial. This trial consisted of male patients only. Their demographic information is given in Table 1 below. The Sanofi–venglustat Phase II trial was a longitudinal study and plasma was collected from patients at the following 6 set intervals: baseline (0 weeks), 12 weeks, 26 weeks, 52 weeks, 104.2 weeks, and 156.4 weeks. Some patients were missing time points. During this trial, all patients were treated with venglustat (substrate reduction therapy-SRT).

[0339] Cohort 1 is a collection of plasma samples provided by Dr. Gavin Oudit of the University of Alberta. Patient demographics are given in Table 1 below. In short, these plasma samples are from a mixture of male and female patients in the age range of 25 to 68 years old. They are non-longitudinal samples (e.g., 1 sample per patient), and the plasma samples are a mixture of treated Fabry disease patients (enzyme replacement therapy-ERT), untreated Fabry disease patients, and healthy control subjects.

[0340] Cohort 2 is a collection of plasma samples provided by Dr. Michael Mauer of the University of Minnesota. Patient demographics are given in Table 1 below. In short, these plasma samples are from a mixture of male and female patients in the age range of 4 to 59 years old. They are mostly non-longitudinal samples (e.g., <3 samples per patient). There are some longitudinal patients with baseline, and some longitudinal patients without baseline. Plasma samples are from a mixture of treated (enzyme replacement therapy-ERT) and untreated Fabry disease patients.

[0341] Healthy controls for Fabry disease plasma samples were primarily obtained from commercial sources (Sanguine Bioscience and BioIVT). These plasma samples were age and sex matched to the Fabry cohort, and their demographics are given in Table 1 below. Cohort 1 samples provided additional healthy controls.

[0342] Table 1: Patient Demographics of the Fabry Disease Cohort

[0343] Sera from Gaucher disease patients and normal controls

[0344] The samples from the Gaucher disease cohort were from Yale University (Professor Pram Mistry). The patient demographics are given in Table 2 below. In short, the Gaucher disease cohort consisted of 89 samples from 21 patients (both male and female). These samples spanned a wide age range, with an average age of 50.5 years. Only 2 of the patients had baseline samples, but 15 patients had longitudinal data. Most of the longitudinal data were from samples obtained several years after the initial treatment. Most patients were treated with ERT, but some patients were changed to SRT at a certain point in the treatment process.

[0345] Healthy control samples were from commercial (BioIVT) and internal (Genzyme donor program samples, nonclinical and internal clinical studies) sources. Patient demographics are given in Table 2 below. Briefly, there were 43 samples from 43 healthy donors; there were no longitudinal healthy control samples. The mean age of healthy control patients in the Gaucher disease cohort was 45 years, and the age range covered a wide range similar to that of the diseased samples.

[0346] Table 2: Patient Demographics of the Gaucher Disease Cohort Cohort Gaucher disease samples Control samples Number of samples (n) 89 43 Age (average) 50.5 45 Age (range) 5 to 83 24 to 76 Male patient count 8 14 Female patient count 13 29 Untreated sample (baseline) 2 Processed samples 87 ERT 20 SRT 5 Vertical with baseline 2 Vertical without baseline 13

[0347] Plasma from MPS patients and normal controls

[0348] The patient demographics of the MPS cohort (samples provided by Dr. Giugliani - Univ PortoAlegre) are given in Table 3 below. In short, the patients were all young, with an average age of about 6 years. There were 28 samples from 28 patients; there were no longitudinal samples in this cohort. The samples were from a mix of male and female subjects. In addition, no patients were receiving treatment, so these were all baseline disease samples. The patient disease covered MPS I, MPS II, and MPS IIIA, with almost equal representation.

[0349] The patient demographics in the healthy control cohort (samples from BioIVT) are given in Table 3 below. The patients in the healthy control cohort were age matched to the disease patients; they were all pediatric samples with an average age of approximately 6 years. There were a total of 19 healthy control samples from 19 patients (a mix of both male and female donors).

[0350] Table 3: Patient Demographics of the MPS Cohort Cohort MPS Samples Control samples Number of samples (n) 28 19 Age (average) 5.9 6.3 Age (range) 0.75 to 26 1 to 12 Male patient count 16 8 Female patient count 12 11 Untreated sample (baseline) 28 Processed samples 0

[0351] CSF from patients with MPS and Gaucher disease (type 3)

[0352] While serum biomarkers can provide insight into systemic changes, analysis of cerebrospinal fluid (CSF) biomarkers may provide a direct and more accurate assessment of neurological processes occurring within the CNS. Therefore, the potential of CD63 as a CSF biomarker for detecting MPS and for detecting GD3 and evaluating treatment response in GD3 was investigated.

[0353] CSF samples were carefully collected from consenting patients at each center according to center-specific protocols to ensure consistency and reliability. To minimize batch effects, samples were randomly assigned based on center, gender, and disease status during plating. The Olink proteomics method (above) was used to profile the changes in protein biomarkers in CSF samples.

[0354] Patient demographics for the MPS cohort are given below in Table 4. As our data showed no significant age-related differences in CD63, healthy adult CSF samples served as reference controls, allowing for comparative analysis. The control group was not age matched (pediatric CSF control samples were not readily available).

[0355] Table 4: Patient Demographics of the MPS / CSF Cohort Group(n) Gender (male / female) Age (years) Comparison (40) 19 / 21 20-60 MPS I(17) 11 / 6 2-15、19、29、30 MPS II(11) 11 / 0 2-16、21、29

[0356] Patient samples for the GD3 study were collected as part of the Sanofi-sponsored LEAP trial with informed consent (see, e.g., Schiffmann et al., Brain (2023) 146(2):461-474 for details). During the combination treatment with 11 IL-67 inhibitors (Sanofi, Cambridge, USA), samples from 11 adult patients were obtained by lumbar puncture at baseline and at weeks 26 and 52. CSF samples from healthy adults were used as controls. The Olink proteomics method (above) was used to analyze changes in protein biomarkers in CSF samples.

[0357] Results and Conclusions

[0358] Results from the assay of Fabry disease samples showed that CD63 was significantly upregulated in the plasma of untreated Fabry disease patients ( Figure 1 The “baseline” in the longitudinal study is shown in A). While on treatment, CD63 levels decreased over a 30-month period toward healthy levels ( Figure 1 A; "Linear NPX"). Response to treatment was observed after 12 weeks, and subsequent reductions in CD63 levels were consistent over several years ( Figure 2 ; shows a linear decrease in the estimated marginal mean (log2) values ​​of CD63 from week 12 to week 156.4). The response of CD63 to venglustat treatment was comparable to the classic Fabry disease biomarkers GL3 and lyso-GL3 in terms of its interpatient variability ( Figure 3 A). When analyzed separately, the intra-patient variability of CD63 and lyso-GL3 was much lower than that of GL3 ( Figure 3 C), CD63 responses appear to be greater in male Fabry disease patients than in female patients ( Fig. 8A ).

[0359] Analysis of samples from a Gaucher disease cohort showed that CD63 was also significantly upregulated in the plasma of Gaucher disease patients compared with healthy controls ( Figure 4 There appeared to be little difference in CD63 levels in samples from male Gaucher patients compared to female Gaucher patients ( Figure 8B Day-to-day variability of CD63 levels in patients with Gaucher disease receiving long-term treatment ( Figure 5A ) is lower than other established biomarkers (such as lyso-GL1 ( Figure 5B ) or chitotriosidase ( Figure 5C)). This low level of intra-patient variability was highly significant (Figure 6C; p<0.002) and establishes CD63 as a unique biomarker for monitoring LSD status and progression, especially when compared with the known biomarkers lyso-GL1 and chitotriosidase. Furthermore, CD63 levels correlated with levels of known biomarkers for Gaucher disease ( Fig.6D ).

[0360] MPS patients Figure 7 A) (both male and female ( Figure 8C )) also had significantly elevated CD63 levels. Although the different profiles of the distribution may suggest some differences in the subtypes of each population, there did not appear to be a statistically significant difference between the mean CD63 levels of male and female patients ( Figure 7 B, p>0.05; see also Figure 8C ). This observation further supports the use of CD63 as a universal biomarker for the detection and / or monitoring of LSDs.

[0361] Compared with healthy controls, samples from male adult Fabry disease patients Profiling analysis showed that CD63 was the most significantly upregulated protein ( Fig. 9 ).

[0362] Analysis of data collected from MPS CSF samples revealed compelling results in the case of CD63. Fig.10 (Box plot) shows that CD63 levels were significantly elevated in MPS patients (NPX values ​​for MPS I and MPS II = -3.186 and -3.037, respectively) compared to controls (NPX value = -6.402). The p values ​​for the differences between MPS I / MPS II and healthy references were all <0.001. These findings strongly support the feasibility and clinical utility of CD63 as a biomarker for MPS in CSF.

[0363] Longitudinal GD3 samples also showed that CD63 levels in CSF at baseline were significantly elevated compared with the mean values ​​of healthy controls ( Fig.11A ), and decreased with treatment duration. The same trend was observed in individual patients ( Fig. 11B In particular, CD63 levels were significantly elevated in GD3 patients at baseline compared with healthy controls ( Fig.11A), NPX values ​​for control patients and GD3 patients were -6.1 and -4.3, respectively. Even without considering the severity of the disease, the statistical analysis showed a high significance (p value of 0.0036), indicating a clear difference between the two groups. In addition, treatment with venglustat, an investigational brain-penetrating glucosylceramide synthase inhibitor, consistently resulted in a decrease in CD63 levels, indicating the effectiveness of the treatment. In addition, the data on changes in CD63 positively correlated with changes in lysoGL-1 in CSF, further supporting the potential of CD63 as a diagnostic marker for GD3 in CSF.

[0364] These findings highlight the potential use of CD63 as a CSF biomarker for the detection and monitoring of GD3. By evaluating CD63 levels in CSF, we can gain valuable insights into GD3 disease progression and treatment response.

[0365] The results discussed above suggest that CD63 is a circulating biomarker that can be used to assess the pathogenesis of lysosomal storage diseases in general and LSDs involving the lysosomal glycosphingolipid pathway in particular (for which no single biomarker is currently available). The ready availability of simple assays (such as ELISA) for quantifying and monitoring CD63 and its demonstrated use in blood samples make CD63 a particularly attractive biomarker in this context.

[0366] In addition, where features or aspects are described in terms of Markush groups, those skilled in the art will recognize that these features or aspects are also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0367] 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 case of conflict, the present specification, including definitions, will control.

Claims

1. A method of diagnosing a subject as having or being at risk for developing 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 employed in the method.

2. The method of claim 1, wherein the subject is diagnosed as having or being at risk for developing a lysosomal storage disease, comprising Fabry disease, Gaucher disease, MPS type I, MPS type II, and MPS type III.

3. A method of 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 employed in the method.

4. 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 employed in the method.

5. A method for generating quantitative data from a subject, the method comprising determining the level of a single biomarker in a sample from the subject, wherein the biomarker is CD63.

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

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

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

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

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

11. The method of claim 10, wherein the subject is suspected of being at risk for Fabry disease due to the presence of one or more of the following: family history of Fabry disease, fatigue, pain, lens or corneal opacities, vortex keratopathy, angiokeratoma, shortness of breath, palpitations, edema, kidney disease, myocardial dysfunction, conduction abnormalities with shortened PR interval, arrhythmia, vertigo, headache, diplopia, dysarthria, hemiataxia, transient ischemic attack, early-onset stroke, and dementia.

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

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

14. A method for generating quantitative data from a subject, wherein the method comprises (e.g., consists of) determining the level of CD63 in a sample from the subject, wherein the subject has Fabry disease or is suspected of having Fabry disease.

15. Use of CD63 as a biomarker for diagnosing Fabry disease in a subject suspected of being at risk of developing Fabry disease.

16. 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.

17. A method of treating a subject who has been diagnosed with Fabry disease by a method according to any one of claims 10-13, said treatment comprising administering to said subject one or more therapeutic treatments for Fabry disease.

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

19. A method for generating quantitative data of a subject, wherein the method comprises: (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, wherein the level of CD63 in a sample taken from one or more healthy subjects is measured as the control value; 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.

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

21. The method of claim 20, wherein the treatment comprises administering venglustat or migalastat, such as venglustat, to the subject.

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

23. A therapeutic agent for treating Fabry disease in a subject, wherein the subject has been diagnosed as having Fabry disease by the method according to 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 20-22.

25. A method for 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 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 Fabry disease has become more severe if the CD63 level in the second sample is greater than the CD63 level in the first sample, determining that the subject's Fabry disease has not progressed if the CD63 level in the second sample is substantially the same as the CD63 level in the first sample, and determining that the subject's Fabry disease has become less severe if the CD63 level in the second sample is lower than the CD63 level in the first sample.

26. A method for generating quantitative data of a subject diagnosed with Fabry disease, the method comprising the steps of: (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; as well as (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

27. A method according to claim 25 or claim 26, wherein the sample is a blood sample, such as a plasma sample.

28. A method for monitoring the progress of a 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 to the 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 administration of the therapeutic treatment; as well as (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. A method of treating or preventing the development or progression of Fabry disease in a subject assessed to be at risk for developing Fabry disease, the method comprising the steps of: (a) obtaining a first biological sample from the subject, and analyzing the CD63 concentration of the sample; (b) if the CD63 concentration is above the control value, initiating a course of therapeutic treatment for Fabry disease on the subject, 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 said therapeutic treatment based on observed changes in said CD63 levels.

30. A method for adjusting the dose of a therapeutic treatment for Fabry disease in a subject receiving the 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 obtained from the subject after administration of one or more doses of the therapeutic treatment; as well as (c) adjusting the dose 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. A method for generating quantitative data of a subject suffering from Fabry disease, the method comprising the steps of: (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 a therapeutic treatment for Fabry disease to the subject.

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

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

34. The method of claim 33, wherein the treatment comprises administering venglustat or migalastat, such as venglustat, to the subject.

35. The method of claim 33, wherein the treatment comprises administering to the subject a recombinant alpha-galactosidase, such as recombinant alglucosidase beta.

36. The method of any one of claims 25 to 35, wherein the second or subsequent sample is obtained 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 as having Fabry disease, or for monitoring the progress of a treatment for Fabry disease, or for adjusting the dose of a therapeutic treatment for Fabry disease in a subject diagnosed as having Fabry disease.

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

39. The method of claim 38, wherein the subject is suspected to be at risk for Gaucher disease due to the presence of one or more of the following: family history of Gaucher disease, hepatomegaly and splenomegaly, pain, osteoporosis, skin pigmentation, pancytopenia, neurological symptoms, and Parkinsonism.

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

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

42. A method for generating quantitative data from a subject, wherein the method comprises (e.g., consists of) determining the level of CD63 in a sample from the subject, wherein the subject has Gaucher disease or is suspected of having Gaucher disease.

43. Use of CD63 as a biomarker for diagnosing Gaucher disease in a subject suspected of being at risk for developing Gaucher disease.

44. Use of CD63 as a biomarker to improve methods of diagnosing Gaucher disease in a subject, optionally wherein CD63 is used as a biomarker together with GL1, lyso-GL1 and / or beta-glucosidase (GC enzyme) activity.

45. A method of treating a subject who has been diagnosed with Gaucher disease by a method according to any one of claims 38-41, said treatment comprising administering to said subject one or more therapeutic treatments for Gaucher disease.

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

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

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

49. The method of claim 48, wherein the treatment comprises administering venglustat, elukastat, or milukastat to the subject.

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

51. A therapeutic agent for treating Gaucher disease in a subject, wherein the subject has been diagnosed as having Gaucher disease by the method according to any one of claims 38-41.

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

53. 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, wherein 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 Gaucher disease has become more severe if the CD63 level in the second sample is greater than the CD63 level in the first sample, determining that the subject's Gaucher disease has not progressed if the CD63 level in the second sample is substantially the same as the CD63 level in the first sample, and determining that the subject's Gaucher disease has become less severe if the CD63 level in the second sample is lower than the CD63 level in the first sample.

54. A method for generating quantitative data of a subject diagnosed with Gaucher disease, the method comprising the steps of: (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; as well as (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

55. A method according to claim 53 or claim 54, wherein the sample is a blood sample, such as a plasma sample.

56. A method for monitoring the progress of a 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 to the 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 administration of the therapeutic treatment; as well as (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. A method of treating or preventing the development or progression of Gaucher disease in a subject assessed to be at risk for developing Gaucher disease, the method comprising the steps of: (a) obtaining a first biological sample from the subject, and analyzing the CD63 concentration of the sample; (b) if the CD63 concentration is above the control value, initiating a course of therapeutic treatment for Gaucher disease on the subject, 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 said therapeutic treatment based on observed changes in said CD63 levels.

58. A method for adjusting the dose of a therapeutic treatment for Gaucher disease in a subject receiving the 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 obtained from the subject after administration of one or more doses of the therapeutic treatment; as well as (c) adjusting the dose 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. A method for generating quantitative data of a subject suffering from Gaucher disease, the method comprising the steps of: (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 a therapeutic treatment for Gaucher disease to the subject.

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

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

62. The method of claim 61, wherein the treatment comprises administering venglustat, elukastat, or milukastat to the subject.

63. The method of claim 61, wherein the treatment comprises administering to the subject a recombinant glucocerebrosidase, such as imiglucerase.

64. The method of any one of claims 53 to 63, wherein the second or subsequent sample is obtained 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 a treatment for Gaucher disease, or for adjusting the dose of a therapeutic treatment for Gaucher disease in a subject diagnosed with Gaucher disease.

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

67. The method of claim 66, wherein the subject is suspected to be at risk for MPS due to the presence of one or more of the following: family history of MPS, macrocephaly, hearing loss, corneal opacities, abnormal dentition, rigidity, hip dysplasia, claw hands, joint laxity, valvular thickening, left ventricular hypertrophy, recurrent respiratory tract infections, obstructive airway disease, hepatomegaly / splenomegaly, umbilical / inguinal hernia, developmental delay, ventriculomegaly, enlarged perivascular spaces, hyperactive or aggressive behavior, abnormal leukocyte granules, fetal hydrops, and proteinuria.

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

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

70. A method for generating quantitative data from a subject, wherein the method comprises (e.g., consists of) determining the level of CD63 in a sample from the subject, wherein the subject has MPS or is suspected of having MPS.

71. Use of CD63 as a biomarker for diagnosing MPS in a subject suspected of being at risk of developing 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 together with one or more glycosaminoglycans (GAGs) or glycans.

73. A method of treating a subject who has been diagnosed as having MPS by a method according to any one of claims 10-13, said treatment comprising administering to said subject one or more therapeutic treatments for MPS.

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

75. A method for generating quantitative data of a subject, wherein the method comprises: (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, wherein the level of CD63 in a sample taken from one or more healthy subjects is measured as the control value; 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.

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

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

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

80. 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, wherein 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 CD63 level in the second sample is greater than the CD63 level in the first sample, determining that the subject's MPS has not progressed if the CD63 level in the second sample is substantially the same as the CD63 level in the first sample, and determining that the subject's MPS has become less severe if the CD63 level in the second sample is lower than the CD63 level in the first sample.

81. A method for generating quantitative data of a subject diagnosed with MPS, the method comprising the steps of: (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; as well as (c) comparing the level of CD63 determined in step (a) with the level determined in step (b).

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

83. A method for monitoring the progress of a 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 to the 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 administration of the therapeutic treatment; as well as (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. A method of treating or preventing the development or progression of MPS in a subject assessed to be at risk for developing MPS, the method comprising the steps of: (a) obtaining a first biological sample from the subject, and analyzing the CD63 concentration of the sample; (b) if the CD63 concentration is above the control value, initiating a course of therapeutic treatment for MPS on the subject, 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 said therapeutic treatment based on observed changes in said CD63 levels.

85. A method for adjusting the dosage of a therapeutic treatment for MPS in a subject receiving the 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 obtained from the subject after administration of one or more doses of the therapeutic treatment; as well as (c) adjusting the dose 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. A method for generating quantitative data of a subject suffering from MPS, the method comprising the steps of: (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 a therapeutic treatment for MPS to the subject.

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

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

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

90. The method of any one of claims 80 to 89, wherein the second or subsequent sample is obtained 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 as having MPS, or for monitoring the progress of a treatment directed against MPS, or for adjusting the dose of a therapeutic treatment directed against MPS in a subject diagnosed as having MPS.

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

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

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

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

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