Field of the Invention

The invention relates to a method of detecting an increased or decreased volume and/or pH of the late endosomal/lysosomal (Le/Lys) system in cell sample(s). The invention also concerns methods for diagnosing, treating and monitoring Le/Lys disorders, as well as methods of screening for compounds capable of increasing or decreasing the volume and/or pH of the Le/Lys system. Such compounds can be used to treat Le/Lys disorders.

Background to the Invention

Late endosomal/lysosomal (Le/Lys) disorders represent about 50 distinct genetic diseases. They are have a birth prevalence of about 1 : 7,700 and are caused by the absence or impaired function of specific late endosomal or lysosomal enzymes. They are characterized by an increased volume and/or pH of the acidic compartment of the endolysosomal system, namely the late endosom.es and lysosomes. Their pathology affects many of the body' s systems, but mainly the nervous system. Mental retardation is a common symptom. Such disorders are generally severely progressive and unremitting. They tend to present in the first few years of life and the severe progression results in frequent hospitalization. If left untreated, patients often die in their mid-teens.

The efficacy of treatment and prevention of irreversible damage to the nervous system is dependent on an early diagnosis of the Le/Lys disorder. However, current diagnostic approaches for such disorders are limited. Early screening for Le/Lys disorders often involves measuring late endosomal or lysosomal enzyme expression and/or activity (Meikle et al., MoI. Genet. Metab., 2006; 88: 307 to 314). However, different Le/Lys disorders result from the impairment or absence of different enzymes. This makes it difficult to rapidly screen newborns for all Le/Lys disorders. Diagnosis is usually carried out on the basis of the particular type(s) of lipid or non-lipid stored in the Le/Lys system.

Current therapeutic approaches for Le/Lys disorders are also limited. There are few, if any, curative treatments and many of the therapeutic options merely improve quality of life. Some Le/Lys disorders have been responsive to bone marrow transplantation or enzyme replacement therapy. Some benefit has also been reported in a clinical trial using an inhibitor of GSL biosynthesis: the irnino sugar drug, miglustat (Patterson et al, Rev Neurol (separata) 2006; 43: 8). However, there are currently no non-specific treatments that benefit all Le/Lys disorders. There is therefore a need to develop improved diagnostic assays for and treatments of Le/Lys disorders.

Summary of the Invention The invention provides a novel assay which is capable of rapidly detecting an increased or decreased volume and/or pH of the Le/Lys system of cells. The assay allows multiple samples of cells to be simultaneously screened for an increased or a decreased volume and/or pH of the Le/Lys system. The non-specific nature of the assay allows the detection of an increased or decreased volume and/or pH of the Le/Lys system irrespective of its cause. The assay of the invention therefore provides a rapid screen for any and all Le/Lys disorders. The screen can be carried out on a large number of samples, and hence patients, at the same time.

The assay of the invention also allows the rapid identification and investigation of compounds that affect the volume and/or pH of the Le/Lys system. Such compounds have the potential to be used to treat Le/Lys disorders. The assay of the invention also allows the progression and/or treatment of Le/Lys disorders to be easily monitored in one or more patients.

The assay of the invention utilises a marker for the volume and/or pH of the Le/Lys system. The inventors have surprisingly shown that such a marker can be used to rapidly and simultaneously measure the volume and/or pH of the Le/Lys system in more than one sample of cells. The assay of the invention is a multiplex assay. The inventors have also surprisingly shown that the assay of the invention has a small standard of error. For instance, the volume and pH of the Le/Lys system in wild- type cells remains in a tight range even between different repetitions of the assay and between different individuals having different ages and ethnicity. The assay of the invention is also very sensitive and so may be used to detect small changes in the volume and/or pH of the Le/Lys system.

Accordingly, in a first embodiment, the present invention provides a method for detecting an increased or decreased volume and/or pH of the late endosomal/lysosomal (Le/Lys) system in two or more cell samples, the method comprising:

(a) providing two or more test samples comprising cells;

(b) incubating each test sample with a marker for the volume and/or pH of the Le/Lys system in the cells; (c) concurrently measuring in each test sample the signal from the marker;

(d) comparing the signal measured in each test sample with a control value obtained using a control sample of cells exhibiting no increase or decrease in the volume and/or pH of the Le/Lys system and thereby determining whether or not the cells in each test sample display an increased or decreased volume and/or pH of the Le/Lys system; wherein at least two of the test samples are different from one another, wherein an increased signal in a test sample compared with the control value indicates that the test sample contains cells displaying an increased volume and/or pH of the Le/Lys system and wherein a decreased signal in a test sample compared with the control value indicates that the test sample contains cells displaying a decreased volume and/or pH of the Le/Lys system.

In a second embodiment, the invention provides a method for detecting an increased or decreased volume and/or pH of the Le/Lys system in at least one cell sample, the method comprising: (a) providing at least one test sample comprising cells;

(b) providing at least one control sample comprising cells exhibiting no increase or decrease in the volume and/or pH of the Le/Lys system;

(c) incubating each test sample and each control sample with a marker for the volume and/or pH of the Le/Lys system in the cells; (d) concurrently measuring in each test sample and each control sample the signal from the marker;

(e) comparing the signal measured in each test sample with a control value obtained using the control sample(s) and thereby determining whether or not the cells in each test sample display an increased or decreased volume and/or pH of the Le/Lys system; wherein an increased signal in a test sample compared with the control value indicates that the test sample contains cells displaying an increased volume and/or pH of the Le/Lys system and wherein a decreased signal in a test sample compared with the control value indicates that the test sample contains cells displaying a decreased volume and/or pH of the Le/Lys system. The invention further provides: - a method for diagnosing a late endosomal/lysosomal (Le/Lys) disorder in two or more patients, the method comprising carrying out a method of the first embodiment using a test sample from each patient and thereby determining whether or not each patient has a Le/Lys disorder, wherein an increased or decreased volume and/or pH of the Le/Lys system in a test sample indicates that the patient has a Le/Lys disorder; - a method for diagnosing a Le/Lys disorder in a patient, the method comprising carrying out a method of the second embodiment using a test sample from the patient and thereby determining whether or not the patient has a Le/Lys disorder, wherein an increased or decreased volume and/or pH of the Le/Lys system in a sample indicates that the patient has a Le/Lys disorder; a method for screening two or more compounds for their ability to increase or decrease the volume and/or pH of the Le/Lys system, the method comprising:

(a) providing two or more test samples comprising cells;

(b) incubating each test sample with one of the two or more compounds; (c) incubating each test sample with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells;

(c) concurrently measuring in each test sample the signal from the marker;

(d) comparing the signal measured in each test sample with a control value obtained using a control sample of cells which has not been incubated with any of the two or more compounds and thereby determining whether or not any of the two or more compounds are able to increase or decrease the volume and/or pH of the Le/Lys system; wherein an increased signal in a test sample compared with the control value identifies the compound used to treat the test sample as being able to increase the volume and/or pH of the Le/Lys system; and wherein a decreased signal in a test sample compared with the control value identifies the compound used to treat the test sample as being able to decrease the volume and/or pH of the Le/Lys system; a method for screening a compound for its ability to increase or decrease the volume and/or pH of the Le/Lys system, the method comprising: (a) providing two samples comprising cells;

(b) incubating only one of the samples with the compound;

(c) incubating both samples with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells;

(c) concurrently measuring in each sample the signal from the marker; (d) comparing the signal measured in the sample incubated with the compound with the signal measured in the sample of cells which has not been incubated with the compound and thereby determining whether or not the compound is able to increase or decrease the volume and/or pH of the Le/Lys system; wherein an increased signal in the sample incubated with the compound compared with the sample which has not been incubated with the compound identifies the compound as being able to increase the volume and/or pH of the Le/Lys system; and wherein a decreased signal in the sample incubated with the compound compared with the sample which has not been incubated with the compound identifies the compound as being able to decrease the volume and/or pH of the Le/Lys system; a compound capable of increasing or decreasing the volume and/or pH of the Le/Lys system identified using a method of the invention; a compound of the invention for use in increasing or decreasing the volume and/or pH of the Le/Lys system of a cell; a compound of the invention for use in treating or preventing a Le/Lys disorder; a method of treating or preventing a Le/Lys disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of a compound of the invention; an in vitro method of increasing or decreasing the volume and/or pH of the Le/Lys system of a cell, the method comprising administering to the cell an effective amount of a compound of the invention; - a compound of the invention for use in treating or preventing an age-related disorder; a method of treating or preventing an age-related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of a compound of the invention; a method for monitoring the progressive effect of a compound that is capable of increasing or decreasing the volume and/or pH of the Le/Lys system, the method comprising:

(a) providing two or more test samples comprising cells;

(b) incubating each test sample with the compound for a different amount of time within a range of times;

(c) incubating each test sample with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells;

(c) concurrently measuring in each test sample the signal from the marker;

(d) plotting the signal obtained from each test sample against the corresponding time for which the sample was incubated with the compound and thereby monitoring the progressive effect of the compound on the volume and/or pH of the Le/Lys system; wherein the signal at each time point provides an indication of the volume and/or pH of the Le/Lys system of the cells; a method for monitoring the progression of a Le/Lys disorder or an age-related disorder in a patient, the method comprising: (a) providing two or more test samples comprising cells each taken from the patient at different times;

(b) incubating each test sample with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells; (c) concurrently measuring in each test sample the signal from the marker;

(d) plotting the signal obtained from each sample against the time at which sample was taken from the patient and thereby monitoring the progression of the disorder in the patient; wherein the signal at each time point provides an indication of the volume and/or pH of the Le/Lys system of the patient; - a method for determining the effectiveness of treatment of a Le/Lys disorder or an age-related disorder in two or more patients, the method comprising:

(a) providing a test sample comprising cells taken from each of the patients after treatment has begun;

(b) incubating each test sample with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells;

(c) concurrently measuring in each test sample the signal from the marker;

(d) comparing the signal measured in each test sample with a control value obtained using a control sample of cells taken from the same patient before the treatment was begun and thereby determining whether or not the treatment is effective; wherein a decreased signal in a test sample compared with the control value or no significant difference between the signal in a test sample and the control value indicates that the treatment is effective in the patient; a method for determining the effectiveness of a treatment of a Le/Lys disorder or an age- related disorder in a patient, the method comprising: (a) providing a control sample comprising cells taken from the patient before the treatment was begun;

(a) providing a test sample comprising cells taken from the patient after the treatment has begun;

(c) incubating the test sample and control sample with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells;

(d) concurrently measuring the signal provided by the marker in the test sample and in the control sample; (d) comparing the signal in the test sample with the signal in the control sample and thereby determining whether or not the treatment is effective; wherein a decreased signal in the test sample compared with the control sample or no significant difference between the signal in the test sample and the signal in the control sample indicates that the treatment is effective in the patient; a method for monitoring the continued effectiveness of treatment of a Le/Lys disorder or an age-related disorder in two or more patients, the method comprising:

(a) providing a test sample comprising cells from each of the patients;

(b) incubating each test sample with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells;

(c) concurrently measuring in each test sample the signal from the marker;

(d) comparing the signal measured in each test sample with a control value obtained using a control sample of cells taken from the same patient earlier in the treatment and thereby determining whether or not the treatment is continuing to be effective; wherein a decreased signal in a test sample compared with the control value or no significant difference in the signal in a test sample and the control value indicates that the treatment is continuing to be effective in the patient; a method for monitoring the continued effectiveness of a treatment of Le/Lys disorder or an age-related disorder in a patient, the method comprising: (a) providing two or more test samples comprising cells each taken from the patient at different times;

(b) incubating each test sample with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells;

(c) concurrently measuring in each test sample the signal from the marker; (d) plotting the signal obtained from each test sample against the time at which the sample was taken from the patient and thereby monitoring the continued effectiveness of the treatment; wherein a decreased signal in a test sample compared with the signal in a test sample taken earlier during the treatment or no significant difference between the signal in a test sample and the signal in a test sample taken earlier during the treatment indicates that the treatment is continuing to be effective in the patient; and a kit for carrying out a method of the invention, which kit comprises a marker that is capable of measuring the volume and/or pH of the Le/Lys system in cells and a flat plate comprising two or more wells. Brief Description of the Figures

Fig. 1 shows the results of an experiment to determine the requirements for linear LysoTracker® fluorescence intensity. A, using a fixed cell number of 50,000 cells (so that each well in the 96 well plate has a confluent cell monolayer), the concentration of LysoTracker® was adjusted from 5OnM to 50OnM and fluorescence intensity recorded with a BMG Labtech fluorimeter (n=5, graph is a representative experiment). B ₅ having determined optimal LysoTracker® concentration (20OnM), the number of cells was adjusted from 1,000 to 100,000 to determine linear - saturated labeling. LysoTracker® fluorescence is non-linear at low and high concentrations indicating over and sub optimal loading. At a cell number of 40,000, fluorescence remains linear but not saturated allowing further elevation (for storage cells), n=5. The graph is a representative experiment.

Fig. 2 shows the results of an experiment to determine the specificity of sub-cellular LysoTracker® compartment loading. A, RAW macrophages were incubated initially with 0.5mg/ml Rhodamine-dextran for 12h followed by 12h pulse to label lysosomes or 15min followed by 30min pulse with 5mg/ml Rhodamine-dextran to label late endosomes. Cells were then incubated with 20OnM LysoTracker® and visualised using a Zeiss Axioplan fluorescence microscope equipped with charge coupled device digital camera and Axiovision software. 89% overlap (merge) was observed between LysoTracker® and lysosomal dextran and 78% overlap was observed between LysoTracker® and late endosomal dextran. Smller, non-merged red punctate staining was indicative of early endosomes which, because of their higher pH, do not colocalise with LysoTracker® (n=2). B, To confirm that LysoTracker® only labels acidic endosomal compartments, RAW macrophages (50,000 cells) were grown on 96 well plates, labelled with LysoTracker® (lOmin, 20OnM, room temperature loading) and then the acidic stores were disrupted with a variety of drugs. All treatments reduced LysoTracker® fluorescence to background levels (~5000 arbitrary fluorescence units), n = 8. Fig. 3 shows the results of an experiment to determine the method by which LysoTracker® accumulates in acidic endosomes. A, Chinese hamster ovary (CHO) cells were grown overnight in 96 well plates (40,000 cells per well) in the presence or absence of 20OmM sucrose to induce swelling of acidic compartments (via absorption of anions). An elevation of ~ 47% in LysoTracker® staining was observed in cells incubated with sucrose compared to untreated controls, which was indicative of increased LysoTracker® loading into these organelles. In both untreated and treated cells,

LysoTracker® fluorescence was reduced down to equal levels following incubation with GPN to disrupt acidic endosome stability (n=4). B, to confirm that sucrose induced lysosomal swelling, LysoTracker® stained CHO cells treated with or without 20OmM sucrose overnight were observed by fluorescence microscopy. Enlarged vesicles were clearly present (n=2). These data indicated that sucrose-induced swelling of acidic endosomes leads to enhanced uptake of LysoTracker® and a specific increase in fluorescence within that compartment. Fig. 4 shows the results of an experiment to determine the sensitivity of the 96 well based

LysoTracker® assay. A, RAW macrophages grown in 96 well plates (40,000 cells per well) were incubated with 2mg/ml Ul 8666A for the indicated times prior to lysosomal labeling with LysoTracker® (lOmin, 20OnM room temperature), n=6. B ₃ biochemical evaluation of lipid storage, sphingosine and glycosphingolipids were extracted from cells harvested at each time point and analysed by HPLC. Cholesterol was analysed by Molecular Probes Amplex Red cholesterol assay kit, n=4.

Fig. 5 shows the results of an experiment confirming that the 96 well plate LysoTracker® assay can detect storage and non-storage patient cells. A, human patient fibroblasts (obtained from the Coriell Cell Repository) were grown in 96 well plates (40,000 cells per well) prior to staining with LysoTracker® green (lOmin, 20OnM, room temperature) fluorescence was measured using a BMG labtech fluorimeter, n=6. B, confirmation by fluorescence microscopy of the extent of LysoTracker® fluorescence in a select group of patient fibroblasts. No increase is seen in Gaucher cells as with the plate assay, whereas Tay-Sachs and Sandhoff cells are increasingly brighter respectively correlating with the fluorimeter data in A. Fig. 6 shows the results of an experiment confirming that the 96 well plate LysoTracker® assay can be used for monitoring therapeutic improvement. A, human patient fibroblasts (obtained from the Coriell Cell Repository) were grown initially in the presence of the indicated drug for 48h followed by a further 24h with drug in 96 well plates (40,000 cells per well) prior to staining with LysoTracker® green (lOmin, 20OnM, room temperature). A statistically significant reduction in lysosomal NPCl staining was seen with NB-DNJ, does reduces NPCl lipid storage at 72h as previously reported biochemically. A far greater reduction in storage was seen with ISP-I as indicated by reduction in cholesterol storage, which does not occur with NB-DNJ (see inset). The assay is sufficiently sensitive to detect the reduction in GSL storage alone caused by NB-DNJ treatment. B, curcumin treatment reduces lipid storage in NPCl cells with a dramatic reduction over 24h, which can be accurately Recorded using the LysoTracker® 96 well assay.

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Fig. 7 shows the results of an experiment confirming that the 96 well plate LysoTracker® assay can be used as a high throughput screening system for agents that induce or correct lysosomal dysfunction. A panel of 13 novel natural products was screened and four were found to be potentially beneficial in treating NPCl cells.

Fig. 8 shows binding of human B-lymphoblasts to streptavidin-biotin-CD19 coated 96 well microplates followed by LysoTracker® red staining. Fig. 9 shows the chemical structures of LysoTracker®. A is LysoTracker® blue. B is

LysoTracker® green. C is LysoTracker® red.

Fig. 10 shows increased LysoTracker® incorporation into Danon disease (Lampl deficiency; column C) and cystic fibrosis (CFTR deficiency, abnormal lysosomal pH; column B) human patient fibroblasts compared to normal human control fibroblasts (column A). This indicates the presence of lysosomal storage in fibroblasts of patients with both Danon disease and cystic fibrosis. The Y axis is fluorescence intensity.

Fig. 11 shows chloroquine-induced phospholipidosis increases LysoTracker® signal intensity. The Y axis is fluorescence intensity. The X axis is treatment time: column A concerns control cells (i.e. cells that are not treated with chloroquine), column B concerns cells treated with chloroquine for 10 minutes and column C concerns cells treated with chloroquine for 24 hours. Treatment of human fibroblast cells with chloroquine for 10 minutes after LysoTracker® staining reduces the LysoTracker® signal (deacidifies the lysosomes; column B). This effect in time induces lysosomal storage which after 24 hours can be measured as an increase in LysoTracker® staining (column C) compared to untreated control cells (column A).

Detailed Description of the Invention

It is to be understood that different applications of the disclosed methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a marker" includes "markers", reference to "a sample" includes two or more such samples, reference to "a disorder" includes two or more such disorders, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Methods of detecting an increased or decreased volume and/or pH of the Le/Lvs system

The present invention provides methods for detecting an increased or decreased volume and/or pH of the late endosomal/lysosomal (Le/Lys) system. The methods are preferably carried out in vitro.

In a first embodiment, the method is carried out using two or more test cell samples that are different from one another. A cell sample is a sample comprising cells. All the test samples are incubated with a marker for the volume and/or pH of the Le/Lys system in the cells. The incubating may be carried out in any suitable volume. Typical volumes of the samples range from about lOμl to about 1ml, preferably from about 50μl to about 500μl, more preferably from about lOOμl to about 200μl. Typically, the length of time for which the cells are incubated with the marker is from about 5 minutes to about 50 hours, for example from about 10 minutes to about 40 hours, from about 20 minutes to about 30 hours, from about 30 minutes to about 20 hours, from about 45 minutes to about 12 hours, from about 1 hour to about 6 hours, preferably from about 10 minutes to about 2 hours. The cells may be incubated overnight.

The cells may be incubated with the marker at any suitable temperature. The suitable temperature is typically in the same range as the normal body temperature of the human or animal from which the cells are derived. Typically, the incubation is carried out at a fixed temperature between about 4 ⁰ C and about 38 ⁰ C, preferably from about 20 ⁰ C to about 25 ⁰ C, more preferably at 20 ⁰ C.

The signal from the marker is concurrently or simultaneously measured in each test sample. The marker is typically washed off the cells before the signal is measured. However, if the method is carried out using a small volume or sample size, the marker does not have to be washed off the cells. Suitable washes will be known in the art. The signal measured in each test sample is compared with a control value.

An increased signal in a test sample compared with the control value indicates that the test sample contains cells displaying an increased volume and/or pH of the Le/Lys system. An increased signal in a test sample compared with the control value preferably indicates that the test sample contains cells displaying an increased volume and pH of the Le/Lys system. The volume of the Le/Lys system may be increased because of increased storage of lipid or non-lipid in the Le/Lys system or because of swelling of the Le/Lys system.

A decreased signal in a test sample compared with the control value indicates that the test sample contains cells displaying a decreased volume and/or pH of the Le/Lys system. A decreased signal in a test sample compared with the control value preferably indicates that the test sample contains cells displaying a decreased volume and pH of the Le/Lys system. The volume of the Le/Lys system may be decreased because of decreased storage of lipid or non-lipid in the Le/Lys system or because of shrinkage of the Le/Lys system.

In the first embodiment, the method may be carried out using any number of test samples as long as it is greater than or equal to 2. For instance, the method may be carried out using 5, 10, 15, 20, 30, 40, 50 , 100, 150, 200 or more test samples. The method is preferably carried out using 6, 12, 24,

48, 96 or 384 or 1526 samples.. This allows high-throughput screening of cells.

The cells are preferably captured or immobilized on a surface. Any method of immobilizing or capturing the cells can be used. The cells may be immobilized or captured on the surface using Fc receptors, capture antibodies, avidimbiotin, lectins, polymers or any other capture chemicals. The cells may be cells, such as fibroblasts, that adhere to the surface.

The samples are typically present in wells. The samples are preferably present in the wells of a flat plate. The samples are more preferably present in the wells of a standard 96 or 384 well plate.

Such plates are commercially available Fisher scientific, VWR suppliers, Nunc, Starstedt or Falcon.

The cells are preferably immobilized or captured on a surface of one or more, preferably all, of the wells. Any method of immobilizing or capturing the cells can be used. The cells may be immobilized or captured on a surface of the well(s) by coating the surface with Fc receptors, capture antibodies, avidin:biotin, lectins, polymers or any other capture chemicals.

The wells typically have a capacity of from about 25μl to about 250μl, from about 30μl to about 200μl, from about 40μl to about 150μl or from about 50 to lOOμl. In the first embodiment, the two or more test samples may be different from each other in any way. For instance, they may contain different compounds or culture media. The two or more test samples are preferably derived from different sources, such as from different patients. The two or more test samples are more preferably derived from different patients. The two or more test samples preferably contain the same type of cells. Cell types are discussed in more detail below. In the first embodiment, the control value is obtained using a control sample of cells exhibiting no increase or decrease in the volume and/or pH of the Le/Lys system. Such cells are well known to a person skilled in the art. The control value is obtained by incubating the control sample with the marker for the volume and/or pH of the Le/Lys system in the cells and measuring the signal from the marker. The control value may be derived from more than one control samples as described below for the second embodiment. In order to allow an effective comparison, the control value has the same units as the signal from the test sample with which it is being compared. A person skilled in the art is capable of obtaining such a value. In the first embodiment, the control value is typically obtained separately from the method of the invention. For instance, the control value may be obtained beforehand and recorded, for instance on a computer. The control value is typically used for comparison purposes in the first embodiment. The control value may be used for multiple repetitions of the method of the first embodiment. The control value is preferably obtained under the same conditions, such as type of marker, concentration of marker, cell number and cell type, under which the method of the first embodiment is carried out.

In a second embodiment, the method is carried out using at least one test cell sample and at least one control cell sample. The control sample comprises cells exhibiting no increase or decrease in the volume and/or pH of the Le/Lys system. All the samples (i.e. each test sample and each control sample) are incubated with a marker for the volume and/or pH of the Le/Lys system in the cells as described above. The signal from the marker is concurrently or simultaneously measured in each test sample and each control sample and the signal measured in each test sample is compared with a control value obtained using the control sample(s). An increased signal in a test sample compared with the control value indicates that the test sample contains cells displaying an increased volume and/or pH of the Le/Lys system. A decreased signal in a test sample compared with the control value indicates that the test sample contains cells displaying a decreased volume and/or pH of the Le/Lys system.

The test samples and control samples are typically present in wells as described above.

In the second embodiment, the method may be carried out using any number of test samples. For instance, the method may be carried out using 1, 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200 or more test samples. The method is preferably carried out using 95 or 383 test samples. If such an embodiment is carried out using a standard 96 or 384 well plate, the remaining well is used for the control sample,

In the second embodiment, the method may be carried out using any number of control samples. For instance, the method may be carried out using 1, 2, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200 or more control samples.

In the second embodiment, multiple test samples may be identical or different from each other. They may be different from each other in any way. For instance, they may contain different test compounds or culture media. Multiple test samples are preferably derived from different sources. Multiple test samples are more preferably derived from different patients. Multiple test samples preferably contain the same type of cells. The test sample(s) and control sample(s) preferably comprise the same type of cells.

In the second embodiment, the control value is obtained while carrying out the method of the invention. This ensures that the control value is obtained under the same conditions, such as type of marker, concentration of marker, cell number and cell type, as the signal from the test sample(s). The control value may be derived from more than one control sample. For instance, the control value may be the arithmetic mean of the signal obtained from each control sample. The control value may be recorded, for instance on a computer, and used in a method of the first embodiment. In order to allow an effective comparison, the control value has the same units as the signal from the test sample(s) with which it is being compared.

The methods of the two embodiments have various advantages. The methods are capable of rapidly detecting an increased or decreased volume and/or pH of the Le/Lys system in multiple samples. This allows for high throughput screening of multiple samples. As discussed above, up to 384 samples can be run simultaneously. It is estimated that 384 samples can be assayed in a 30 minute period. As will be apparent from the discussion below, the multiplex aspect of the methods means that they have a variety of important uses.

The methods are highly sensitive. They allow the detection of small increases or decreases in the volume and/or pH of the Le/Lys system. The methods are cheap and simple to use. They involve the use of commercially-available markers and reading equipment, such as a fluorescence/absorbance flat plate reader. This is discussed in more detail below. They do not involve the use of large and expensive pieces of equipment, such as a fluorescent-activated cell sorting (FACS) machine, confocal microscope or mass spectrometer. They do not require an expert user to use and maintain the equipment.

Sample and cells

The sample(s) used in the methods of the two embodiments may be any suitable sample comprising cells. The sample can be obtained from or extracted from any eukaryotic organism, such an animals, plant, fungi and protests. The sample can be a cell or cells derived from a tissue sample, such as lung, brain, liver, skin, bone marrow or nails, or a fluid sample comprising cells, such as blood. The sample is preferably derived from blood and is processed as described below before being used in the methods. The sample is preferably derived from a newborn, infantile, juvenile or adult blood sample. The sample is most preferably derived from a newborn blood spot. Typically, the sample is human in origin, but alternatively it may be non-human. For instance, the sample can be from animals such as from commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets such as cats, dogs, rodents or lagomorphs. The sample can also be from other organisms, such as insects. The sample can be from a human or non-human animal undergoing treatment for a Le/Lys disorder.

The sample is typically processed prior to being used in the invention, for example by centrifugation, by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells, or by lysis of unwanted cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be stored prior to use in the methods, preferably below -70 ⁰ C, such as down to -200 ⁰ C.

Cells extracted from organisms can be cultured before being used in the methods. This allows equal numbers of adherent cells to be present in each sample being assayed. Alternatively, if the cells are immobilized or captured, the cells, such as fresh blood cells, can be counted before plating. The importance equal number of adherent cells is discussed below. Techniques for culturing cells are well known to a person skilled in the art. The cells are typically cultured under standard conditions of 37 ⁰ C, 5% CO ₂ in medium supplemented with serum.

The cells may be cultured in any suitable flask or vessel and then be transferred to wells. The cells are typically cultured in wells. The cells are preferably cultured in a flat plate comprising two or more wells, such as a standard 96 or 384 well plate. Incubating the cells with the marker typically involves replacing the culture medium in each well with a suitable solution comprising the marker. Suitable solutions are well known to a person skilled in the art.

As mentioned above, the samples are typically present or cultured in wells, such as those of a flat plate. The wells within which the samples are contained or cultured may be modified to facilitate the methods of the two embodiments. For instance, the wells may be modified to facilitate culture of the cells, for instance by including a growth matrix. The wells are preferably modified to allow attachment of the cells to the wells or to allow immobilization of the cells onto a surface of the wells. This facilitates replacement of any fluid in the sample, such as culture medium, with a solution containing the marker. It also avoids the need to isolate the cells from other cells or molecules in the sample, for instance using centrifugation of filtration. If the cells are immobilized or captured on the wells, for instance by antibody capture or the use of adherent cells such as fibroblasts, they can be easily separated from any impurities in the sample by simply replacing any fluid in the sample and washing the cells as appropriate. This allows the assay to be carried out quickly after a sample has been obtained or extracted from an organism. One or more surfaces of the wells are more preferably coated with Fc receptors, capture antibodies, avidin:biotin, lectins, polymers or any other capture chemicals that bind to the cells and immobilize or capture them on the well or the surface(s). AIl eukaryotic cells comprise have a late endosomal/lysosomal (Le/Lys) system. The cells are alive. Any suitable cells may be used in the two embodiments. The cells can be lung, brain, liver, skin, bone marrow, nail or blood cells. The cells are preferably fibroblasts or B lymphocyte cells. This is advantageous because it is quick and easy to take a skin biopsy or blood from an organism and isolate fibroblasts or B lymphocyte cells respectively,

Each test sample preferably comprises the same type of cells, such as fibroblasts or B lymphocyte cells. This allows each test sample to be compared with a single control value. Similarly, each control sample preferably comprises the same type of cells as each test sample. This allows an effective comparison between the signal from the test sample(s) and the control value. Furthermore, if used as described below, each calibration sample preferably comprises the same type of cells as each test sample and each control sample. This ensures that the method is calibrated for the type of cells upon which it will be carried out.

Marker The sample(s) are incubated with a marker for the pH and/or volume of the Le/Lys system in the cells. The marker is preferably capable of measuring the volume and pH of the Le/Lys system.

Any marker that is capable of measuring the volume and/or pH of the Le/Lys system may be used. A marker is capable of measuring the volume and/or pH of the Le/Lys system if it provides a signal that varies depending on the volume and/or pH of the Le/Lys system. It is preferred that the signal increases as the volume and/or pH of the Le/Lys system increases and decreases as the volume and/or pH of the Le/Lys system decreases. It is even more preferred that the signal displays a linear relationship with the volume and/or pH of the Le/Lys system.

For a particular number of cells, the marker is typically capable of saturation at a particular concentration. Saturation means that the signal from the marker will not increase any further even though its concentration is increased. For a particular concentration of marker, the relationship between cell number and the signal generated by the marker typically displays at least some linearity.

This linear relationship allows the methods to function efficiently. Calibration of the number of cells and the concentration of marker is discussed in more detail below.

Suitable markers include, but are not limited to, those that are reactive to protonation and other compounds in the Le/Lys system, such as lysines. The marker is preferably capable of permeating the cell membrane of the cells and is primarily retained in the Le/Lys system. The signal provided by the marker may be any signal that can be measured. The marker may comprise a fluorescent molecule, radioisotope, e.g. ¹²⁵ 1, ³⁵ S, enzyme, antigen, antibody or linker, such as biotin. The marker preferably comprises a fluorescent molecule.

The marker is more preferably any of the following: - N-(3-((2,4-dimtrophenyl)amino)propyl)-N-(3-arninopropyl)meth ylamine, dihydrochloride

(DAMP), which can be detected using anti-DAMP antibodies and is commercially- available from Invitrogen;

Monodansylcadaverine, which is an fluorescent marker; a LysoTracker®, which is a fluorescent marker and is commercially-available from both Invitrogen and also Lonza; pH indicators such as Lysosensor® (Invitrogen); Fluorescent dextrans, which are indicator of lysosomal loading; 4-nitro-7-piperazino-2, 1 ,3-benzoxadiazole (ΝBD-PZ), which is a fluorescent marker; Acridine Orange, which is a fluorescent marker; and - cellular expression constructs of lysosomal proteins, such as Lamp, expressing pH-fluoro, a fluorescent pH indicator.

The marker is most preferably a LysoTracker®. The LysoTracker® may be blue, blue-white, yellow, green or red. The chemical structures of LysoTracker® blue, green and red are shown in Fig. 9.

Calibration

The marker may be used at any concentration that allows it to function correctly. A person skilled in the art is able to determine an effective concentration of the marker. The marker is typically not used at a concentration that can induce alteration is the Le/Lys system. In both the first and second embodiments, the concentration of the marker has preferably been calibrated. Such calibration does not need to be done every time that the methods are carried out. For instance, calibration can be done once and the methods may be carried several times using the particular marker at the calibrated concentration. In a preferred embodiment, the methods further comprise calibrating the concentration of the marker. Calibration of the concentration of the marker ensures that the concentration is at or approaching saturation. The marker is at saturation if its signal does not increase any further as its concentration is increased. The marker is approaching saturation if it gives a maximum signal in a cell known to display an increased volume and/or pH of the Le/Lys system. A strong signal is achieved from the marker if it is used at a concentration that is at or approaching saturation. This ensures that even small changes in the volume and/or pH of the Le/Lys system can be detected.

The concentration of the marker is preferably calibrated using multiple, such as 1, 2, 5, 6, 10, 15 or more, calibration samples of cells. Each calibration sample comprises approximately the same number of cells. In particular, each calibration sample comprises a number of cells that differs from a fixed number by less than 5%, such as less than 3%, less than 2% or less than 1%. This can be achieved by culturing the cells before calibration is carried out. The fixed number is typically chosen on the basis of the number needed to form a confluent monolayer in the well within which the calibration sample is contained.

Each calibration sample is incubated with a different concentration of the marker within a range of concentrations. A person skilled in the art will be able to determine a suitable range of concentrations. For instance, a suitable range of concentrations for LysoTracker® is from 0 to 60OnM. The signal from the marker in each calibration sample is measured. This allows the determination of the relationship between signal and marker concentration. For instance, the relationship can be determined simply by plotting the signal from the marker against the concentration of the marker. A concentration of the marker which provides a signal which is at or approaching saturation of the marker is used in the methods of the first and second embodiments.

Any number of cells may be present in each test or control sample. A person skilled in the art is able to determine an effective number of cells. The number is typically chosen on the basis of the number needed to form a confluent monolayer in the well within which the sample is contained. The numbers will differ between different cell types. Generally from about 10 ⁴ to about 10 ⁷ cells are present in each test or control sample. For example, from about 1.5 x 10 ⁴ to 7 x 10 ⁴ cells may be present in the sample. Preferably, about 2.0 x 10 ⁴ cells may be present in each sample. In both the first and second embodiments, the number of cells in each test sample and each control sample has preferably been calibrated. Such calibration does not need to be done every time that the methods are carried. For instance, calibration can be done once and the methods may be carried several times using the calibrated number of cells. In a preferred embodiment, the methods further comprise calibrating the number of cells. Calibration of the number of cells is intended to result in the signal provided by the marker falling within a linear relationship between the number of cells and signal provided by the marker. This ensures that even small increases or decreases in the volume and/or pH of the Le/Lys system can be detected by the marker. Calibration of the number of cells therefore results in a sensitive assay. The number of cells is preferably calibrated using several, such as 1, 2, 5, 6, 10, 15 or more, calibration samples of cells. Each calibration sample comprises a different number of cells within a range of numbers. This can be achieved by culturing the cells in each sample for different times before calibration is carried out. A person skilled in the art will be able to determine a suitable range of number of cells. For instance, if using a standard 96 well plate, a suitable range of number of cells 0 to 100,000 cells per well.

Each calibration sample is incubated with the marker. Each calibration sample is incubated with approximately the same concentration of marker. A person skilled in the art will be able to determine a suitable concentration. The concentration is preferably the concentration calibrated as described above. The signal from the marker in each calibration sample is measured. This allows the determination of the relationship between signal and cell number. For instance, the relationship between the two can be determined by plotting the signal from the marker against the number of cells in each sample. A number of cells which falls within a linear relationship between signal and cell number is used in the methods of the first and second embodiments. A person skilled in the art will be able to determine a linear relationship. For instance, such a relationship may be determined using a standard computer-based graphical program. A signal falls within a linear relationship between signal and cell number as long as it forms part of the linear relationship. The signal is typically towards the middle of the linear relationship. For instance, if the linear relationship runs between a signal of about 9,000 and about 17,000, a number of cells is chosen that results in a signal of about 13,000. This means that the signal can increase in a linear fashion as the volume and/or pH of the Le/Lys system increases without saturating.

In both the first and second embodiments, the concentration of the marker and the number of cells have more preferably been calibrated. In the most preferred embodiment, the methods further comprise calibrating the concentration of the marker and the number of cells.

Measuring the signal

The signal from the marker can be read and measured using any method known in the art. The signal is preferably read and measured using a flat plate reader. Suitable readers are commercially- available from BMG Labtech or Spectramax.

Diagnosis

The present invention also provides methods for diagnosing a Le/Lys disorder, such as a Le/Lys lipid storage disorder, in one or more patients. These diagnostic methods are based on the assays described above. These diagnostic methods have several advantages. First, they allow large numbers of patients to be screened quickly. The classical assay for diagnosis of Niemann-Pick type C (NPCl), a Le/Lys disorder, is a skin biopsy followed by elevated staining of cellular cholesterol levels. However, staining for cellular cholesterol levels takes much longer than the diagnostic methods of the present invention and cannot screen as many patients simultaneously. Second, the diagnostic methods of the present invention allow the identification of Le/Lys disorders irrespective of the mechanism by which the relevant disorder alters Le/Lys function. The diagnostic methods are therefore advantageous because they are capable of detecting any disorder that of the Le/Lys system irrespective of its cause. In other words, the diagnostic methods are for diagnosing any or all Le/Lys disorders. This allows a large number of patients to be rapidly screened for the presence of any Le/Lys disorder or dysfunction. The methods will not necessarily diagnose which disorder a particular patient has. However, it will allow more focussed tests to be carried out to determine the particular Le/Lys disorder, such as Le/Lys lipid storage disorder or Le/Lys trafficking disorder, in the patient. The diagnostic methods are therefore typically followed by more specific tests involving measuring enzyme expression and/or activity or determining the lipid stored in the Le/Lys system. Methods of doing this are known in the art.

A Le/Lys disorder is any disorder that involves dysfunction or disruption in the Le/Lys system. A Le/Lys disorder involves an increased or decreased volume and/or pH of the Le/Lys system. Even certain types of specific cancer, such as invasive bladder cancer (Daugaard et al. Cancer Research, 2007) can be detected using the methods of the invention.

The Le/Lys disorder is preferably a Le/Lys storage disorder. A Le/Lys storage disorder is any disorder that involves an increased volume and/or pH of the Le/Lys system. The Le/Lys storage disorder preferably involves an increased volume and pH of the Le/Lys system. The Le/Lys storage disorder may involve increased storage of lipid or non-lipid. The Le/Lys lipid storage disorder may be a primary lysosomal hydrolase defect, a post-translational processing defect of lysosomal enzymes, a trafficking defect for lysosomal enzymes, a defect in lysosomal enzyme protection, a defect in soluble non-enzymatic lysosomal proteins, a transmembrane (non-enzyme) protein defect or an unclassified defect.

Primary lysosomal hydrolase defects include, but are not limited to, Gaucher disease (Glucosylceramidase defect), GMl gangliosidosis (GMl-β-galactosidase defect), Tay-Sachs disease (β -Hexosaminidase A defect), Sandhoff disease (β -Hexosaminidase A+B defect), Fabry disease (α - Galactosidase A defect), Krabbe disease (β-Galactosyl ceramidase defect), Niemann-Pick disease Type A and B (Sphingomyelinase defect), Metachromatic leukodystrophy (Arylsulphatase A defect), MPS IH (Hurler syndrome; α-Iduronidase defect), MPS IS (Scheie syndrome; α-Iduronidase defect), MPS II (Hunter syndrome; Iduronate sulphatase defect), MPS IIIA (Sanfϊlippo A syndrome; Heparan sulphamidase defect), MPS HIB (Sanfilippo B syndrome; Acetyl α-glucosaminidase defect), MPS IIIC (Sanfilippo C syndrome; Acetyl CoA: α-glucosaminide N-acetyltransferase defect), MPS HID

(Sanfilippo D syndrome; N-Acetyl glucosamine-6-sulphatase defect), MPS IV A (Morquio A disease; Acetyl galactosamine-6-sulphatase defect), MPS IVB (Morquio B disease; β-Galactosidase defect), MPS V (redesignated MPS IS), MPS VI (Maroteaux Lamy Syndrome; Acetyl galactosamine-4- sulphatase (Arylsulphatase B) defect), MPS VII (Sly Syndrome; β-Glucuronidase defect), MPS IX (Hyaluronidase defect), Wolman disease (WD; Acid lipase defect), Farber disease (Acid ceramidase defect), Cholesteryl ester storage disease (Acid lipase defect), Pompe disease (Type II; α 1,4- Glucosidase defect), Aspartylglucosaminuria (Glycosylasparaginase defect), Fucosidosis (α-Fucosidase defect), α-Mannosidosis (α-Mannosidase defect), β-Mannosidosis (β-Mannosidase defect), Schindler disease (N-Acetylgalactosaminidase defect), Sialidosis (α-Neuraminidase defect), Infantile neuronal ceroid lipofuscinoses (CLNl; Palmitoyl protein thioesterase defect) and Late infantile neuronal ceroid lipofuscinoses (CLN2; Carboxypeptidase defect).

Post-translational processing defects of lysosomal enzymes include, but are not limited to, Mucosulphatidosis (MSD; Multiple sulphatase defect).

Trafficking defects for lysosomal enzymes include, but are not limited to, Mucolipidosis type II (I-cell disease; N- Acetyl glucosamine phosphoryl transferase defect), Mucolipidosis type IIIA (pseudo- Hurler polydystrophy; N- Acetyl glucosamine phosphoryl transferase defect) and Mucolipidosis type IIIC.

Defects in lysosomal enzyme protection include, but are not limited to, Galactosialidosis (Protective protein cathepsin A (PPCA) defect), β-galactosidase defects and neuraminidase defects. Defects in soluble non-enzymatic lysosomal proteins include, but are not limited to, GM2 activator protein deficiency (Variant AB), Sphingolipid activator protein (SAP) deficiency and Neuronal ceroid lipofuscinoses (NCL) (CLN5).

Transmembrane (non-enzyme) protein defects include, but are not limited to, Danon disease (Lysosome-associated membrane protein 2 (LAMP2) defect), Niemann-Pick Type C (NPCl and NPC2 defect), Cystinosis (Cystinosin defect), Infantile free sialic acid storage disease (ISSD; Sialin defect), Salla disease (free sialic acid storage; Sialin defect), Juvenile neuronal ceroid lipofuscinoses (CLN3, Batten disease), Neuronal ceroid lipofuscinoses (NCL) (CLN6 and CLN8) and Mucolipidosis type IV (Mucolipin defect).

Unclassified defects include, but are not limited to, Neuronal ceroid lipofuscinoses (NCL) (CLN4 and CLN7).

The Le/Lys lipid storage disorder is preferably any of Niemann-Pick type C (NPCl), NPC2, Smith-Lemli-Opitz Syndrome (SLOS), an inborn error of cholesterol synthesis, Tangier disease, Pelizaeus-Merzbacher disease, the Neuronal Ceroid Lipofuscinoses, primary glycosphingolipidoses (i.e. Gaucher, Fabry, GMl, GM2 gangliosidoses, Krabbe and MLD), Farber disease and Multiple Sulphatase Deficiency.

The Le/Lys disorder is preferably a Le/Lys trafficking disorder. A Le/Lys trafficking disorder is any disorder that involves defective Le/Lys recycling and/or transport. The Le/Lys trafficking disorder may be any of Mucolipidosis type IV, Mucolipidosis type II, cystic fibrosis, Alzheimer's and Huntington's disease.

There are additional Le/Lys disorders which are caused by deficiency in a lysosomal protein of unknown function or which are not lipid storage disorders or trafficking disorders. These include Danon's disease (Lamp deficiency), Chediak-Higashi syndrome and Cystinosis.

The Le/Lys disorder is preferably drug-induced. The drug-induced Le/Lys disorder is preferably drug-induced phospholipidosis. Drug-induced phospholipidosis can be induced by a variety of drugs including, but not limited to, the anti-malarial chloroquine, the anti-breast cancer drug tamoxifen and cocaine. The Le/Lys disorder is preferably a pathogenic infection that is capable of blocking lysosome- phagosome fusion via induction of a NPC like cellular phenotype. Mycobacterium tuberculosis (Mtb) successfully survives within cells of the innate immune system, including macrophages and monocytes. Multiple mechanisms are involved in the intracellular survival of Mtb, including defective acidification of the phagosome and inhibition of phosphatidylinositol dependent trafficking pathways, via secretion of inositol-like lipids by the mycobacteria. Bacilli interact with cell surface complement receptors and are ingested into phagosomes that mature but do not fuse with lysosomes. Binding to the complement receptor is normally followed by the stimulation of phago-lysosome fusion. However, in M. tuberculosis infected macrophages, this is substantially reduced. Other pathogenic infections which are capable of blocking phagosome-lysosome fusion include infection by Salmonella, Brucella, Coxiella, Chlamydia and Anaplasma phagocytophilum bacteria.

The ability of lysosomes to fuse with late endosomes relies upon calcium release, specifically from the late endosomal/lysosomal compartment itself. When insufficient calcium is released from acidic stores there is a complete block in late endosome-lysosome fusion. A severe human disease results from calcium deficiency in acidic the acidic compartment, the lysosomal storage disease termed NPC. NPC is unusual as it is caused by mutations in two genes, NPCl or NPC2, that function as part of the same cellular pathway. However, the precise mechanistic link between these two genes remains unknown and the functional roles of these proteins remains enigmatic. NPCl encodes a multimembrane spanning protein of the limiting membrane of the late endosome/lysosome where as NPC2 is a soluble cholesterol binding protein of the lysosome.

When NPCl is inactivated, sphingosine is the first lipid to be stored, suggesting that NPCl plays a role in the transport of sphingosine from the lysosome, where it is normally generated as part of sphingolipid catabolism. Elevated sphingosine in turn causes a defect in calcium entry into acidic stores resulting in greatly reduced calcium release from this compartment. This then prevents late endosome-lysosome fusion (LE/Lys fusion), which is a calcium dependent process, and causes the secondary accumulation of lipids (cholesterol, sphingomyelin and glycosphingolipids) that are cargos in transit through the late endocytic pathway. Other secondary consequences of inhibiting NPCl function include defective endocytosis and failure to clear autophagic vacuoles. It has been shown that the NPC1/NPC2 cellular pathway is targeted by pathogenic mycobacteria to promote their survival in late endosomes.

The term "Niemann-Pick type C disease like cellular phenotype" as used herein, means a cellular phenotype which includes: (a) abnormal cholesterol metabolism and trafficking; (b) abnormal sphingolipid storage and trafficking; and (c) defective endocytosis. Typically, the abnormal sphingolipid storage (b) involves the majority of sphingolipids in the cell being present at abnormally elevated levels. Typically, (c) comprises defective endocytosis of substantially all biomolecules in the endocytic pathway, including lipids and biomolcules other than lipids, for instance proteins.

Pathogenic infections which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype include infections by any pathogen that that secretes a molecule (for instance a lipid or a secondary amine) that can inhibit NPCl function or any pathogen that prevents

NPCl from getting to the compartment in which it normally functions (for instance by inhibition of late endosome acidification). Typically, intracellular cholesterol storage would also be observed in combination with either of these phenotypes.

Pathogenic infections which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype include, but are not limited to, infections caused by the following types of bacteria: Mycobacteria, Salmonella, Brucella, Coxiella, Chlamydia and Anaplasma phagocytophilum.

In one embodiment, the pathogenic infection is an infection caused by Mycobacteria (a mycobacterial infection). Typically, the pathogenic infection is an infectious disease, such as infectious disease tuberculosis.

The mycobacterium which most commonly causes tuberculosis in humans is Mycobacterium tuberculosis. However, other mycobacteria, such as Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannetti, and Mycobacterium microtti, also cause tuberculosis (although these species are less common in humans). Thus, in one embodiment, the pathogenic infection is an infection caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannetti and/or Mycobacterium microtti.

In another embodiment, the pathogenic infection is one caused by Salmonella, Brucella, Coxiella or Chlamydia. Thus, for instance, the infection may be one caused by Salmonella enterica,

Brucella abortus, Coxiella burnetii or Chlamydia trachomatis.

In another embodiment, the pathogenic infection is one caused by Anaplasma phagocytophilum. Anaplasma phagocytophilum is a bacterium that lives inside white blood cells, and causes the disease human granulocytic anaplasmosis (HGA). Accordingly, in one embodiment, the Le/Lys disorder is human granulocytic anaplasmosis.

In one embodiment, the method is for diagnosing a late endosomal/lysosomal (Le/Lys) disorder in two or more patients. This method is based on the method of the first embodiment described above.

The method involves detecting an increased or decreased volume and/or pH of the Le/Lys system in a sample from each patient. An increased or decreased volume and/or pH of the Le/Lys system in a sample indicates that the patient has a Le/Lys disorder. An increased volume and/or pH of the Le/Lys system in a sample preferably indicates that the patient has a Le/Lys storage disorder, such as a Le/Lys lipid storage disorder.

In another embodiment, the method is for diagnosing a Le/Lys disorder in a single patient. This method is based on the method of the second embodiment described above. The method involves detecting an increased or decreased volume and/or pH of the Le/Lys system in a sample from the patient. An increased or decreased volume and/or pH of the Le/Lys system in the sample indicates that the patient has a Le/Lys disorder. An increased volume and/or pH of the Le/Lys system in a sample preferably indicates that the patient has a Le/Lys storage disorder, such as a Le/Lys lipid storage disorder. In both diagnostic methods, any of the methods of the first and second embodiments described above may be used. In both diagnostic methods, the concentration of the marker and/or the number of cells has preferably been calibrated as described above. The patient(s) are preferably human. The patient(s) can be newborns, infants, juveniles or adults. The patient(s) are preferably newborn children. The method is therefore particularly useful for the rapid screening of large numbers of newborns for a Le/Lys disorder. The sample(s) are preferably derived from newborn blood. Early diagnosis tends to result in efficacious treatment and prevention of irreversible brain damage. The patient(s) are preferably at risk of or predisposed to a Le/Lys disorder. For instance, the method is suitable for screening patient(s) who have one or more family member(s) with a Le/Lys disorder. The patient can be asymptomatic.

Drug screening The present invention also provides methods for screening compounds for their ability to increase or decrease the volume and/or pH of the Le/Lys system. Compounds screened in accordance with the invention are preferably able to increase or decrease the volume and pH of the Le/Lys system. Compounds that increase or decrease the volume and/or pH of the Le/Lys system are suitable for treating Le/Lys disorders, such as Le/Lys lipid disorders. Compounds that decrease the volume and/or pH of the Le/Lys system are suitable for treating age-related disorders. Such disorders are discussed in more detail below. The cellular aging process involves the Le/Lys system and leads to a downturn in its molecule clearing activity, which in turn causes a swelling within the compartment.

The drug screening methods of the invention have several advantages. First, they allow large numbers of compounds to be screened quickly. As mentioned above, the classical assay for diagnosis of NPCl is a skin biopsy followed by elevated staining of cellular cholesterol levels. This method is also sometimes used as a method for screening for novel drugs; a compound that reduces the cholesterol levels has the potential to treat NPC 1. However, staining for cellular cholesterol levels takes much longer than the screening methods of the present invention and cannot screen as many compounds simultaneously. Second, the screening methods of the present invention allow the identification of drugs that will reduce the volume and/or pH of the Le/Lys system irrespective of the mechanism by which the relevant disease increases storage. It is worthy of note that NB-DNJ, a known treatment for NPCl, does not alter the cellular cholesterol levels, but improves the disease course in another manner (by reducing secondary glycolipid storage. As a result, NPCl would not be identified in the classical method for screening for drugs to treat NPC. Example 6 indicates that the screening methods of the present invention do identify NB-DNJ as a potential therapeutic agent for NPC 1.

In one embodiment, the method is for screening two or more compounds for their ability to increase or decrease the volume and/or pH of the Le/Lys system. This method is based on the method of the first embodiment described above. The method involves incubating two or more test samples with one of the two or more compounds. Any compound may be used. Suitable compounds include, but are not limited to, proteins, polynucleotides, small molecules, natural products and lipophilic drugs. The cells in each sample may be incubated with the compound in any volume, for any length of time and at any temperature. Suitable volumes, times and temperatures include, but are not limited to, those discussed above with reference to the marker in the first and second embodiments. Each test sample is incubated with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells and the signal from the marker is concurrently or simultaneously measured in each sample.

An increased signal in a test sample compared with the control value identifies the compound used to treat the test sample as being able to increase the volume and/or pH of the Le/Lys system. An increased signal in a test sample compared with the control value preferably identifies the compound used to treat the test sample as being able to increase the volume and pH of the Le/Lys system.

A decreased signal in a test sample compared with the control value identifies the compound used to treat the test sample as being able to decrease the volume and/or pH of the Le/Lys system. A decreased signal in a test sample compared with the control value preferably identifies the compound used to treat the test sample as being able to decrease the volume and pH of the Le/Lys system. The control volume and/or pH is determined as described above using a sample of cells which has not been incubated with any of the two or more compounds.

In another embodiment, the method is for screening a compound for its ability to increase or decrease the volume and/or pH of the Le/Lys system. This method is based on the method of the second embodiment described above. The method involves incubating one of two samples with the compound. Again, any compound and conditions described above may be used. The two samples are incubated with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells. The signal from the marker is concurrently or simultaneously measured in both samples.

An increased signal in the sample incubated with the compound compared with the sample which has not been incubated with the compound identifies the compound as being able to increase the volume and/or pH of the Le/Lys system. An increased signal in the sample incubated with the compound compared with the sample which has not been incubated with the compound preferably identifies the compound as being able to increase the volume and pH of the Le/Lys system.

A decreased signal in the sample incubated with the compound compared with the sample which has not been incubated with the compound identifies the compound as being able to decrease the volume and/or pH of the Le/Lys system. A decreased signal in the sample incubated with the compound compared with the sample which has not been incubated with the compound preferably identifies the compound as being able to decrease the volume and pH of the Le/Lys system. In both screening methods, any of the methods of the first and second embodiments described above may be used. In both screening methods, the concentration of the marker and/or the number of cells has preferably been calibrated as described above.

If compound(s) are being screened for their ability to increase the volume and/or pH of the Le/Lys system, the cells in the sample(s) preferably have a decreased volume and/or pH of the Le/Lys system compared with wild-type cells. Suitable cells include, but are not limited to, Krabbe and Sialidosis cells. If compound(s) are being screened for their ability to decrease the volume and/or pH of the Le/Lys system, the cells in the sample(s) preferably have an increased volume and/or pH of the Le/Lys system compared with wild-type cells. Suitable cells include, but are not limited to, those isolated from a patient/animal model with a Le/Lys disorder, cells/animals treated with pharmacological agents that induce Le/Lys dysfunction, such as U18666A or enzyme/protein inhibitors, cells/animals treated with siRNA/shRNA to induce Le/Lys dysfunction.

Compounds and their use The present invention also provides compounds capable of increasing or decreasing the volume and/or pH of the Le/Lys system identified using a screening method of the invention. The compounds preferably increase or decrease the volume and pH of the Le/Lys system.

Compounds that are identified in accordance with the invention as being capable of increasing the volume and/or pH of the Le/Lys system may be used to increase the volume and/or pH of the Le/Lys system of a cell. They are preferably used to increase or decrease the volume and pH of the Le/Lys system of a cell.

The present invention provides compounds capable of increasing the volume and/or pH of the Le/Lys system identified using a screening method of the invention for use in increasing or decreasing the volume and/or pH of the Le/Lys system. The present invention also provides an in vitro method of increasing or decreasing the volume and/or pH of the Le/Lys system of a cell, the method comprising administering to the cell a compound identified in accordance with the invention as being capable of increasing or decreasing the volume and/or pH of the Le/Lys system. This method may involve the use of any cell including any of those mentioned above. Suitable conditions, such as temperature and compound concentration, are known to a person skilled in the art. Suitable conditions include, but are not limited to, those mentioned above with reference to the screening methods of the invention.

Compounds that are identified in accordance with the invention as being capable of increasing or decreasing the volume and/or pH of the Le/Lys system may be used to treat or prevent a Le/Lys disorder. Compounds that are identified in accordance with the invention as being capable of decreasing the volume and/or pH of the Le/Lys system may be used to treat or prevent a Le/Lys storage disorder, such as a Le/Lys lipid storage disorder. The present invention provides compounds capable of increasing or decreasing the volume and/or pH of the Le/Lys system identified using a screening method of the invention for use in treating or preventing a Le/Lys disorder. The present invention also provides a method of treating or preventing a Le/Lys disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of a compound identified in accordance with the invention as being capable of increasing or decreasing the volume and/or pH of the Le/Lys system. In a preferred embodiment, the compound is capable of decreasing the volume and/or pH of the Le/Lys system and the Le/Lys disorder is a Le/Lys storage disorder, such as a Le/Lys lipid storage disorder.

The present invention also provides compounds capable of decreasing the volume and/or pH of the Le/Lys system identified using a screening method of the invention for use as anti-aging drugs. The present invention also provides a method of treating or preventing an age-related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of a compound identified in accordance with the invention as being capable of decreasing the volume and/or pH of the Le/Lys system.

The subject is typically a mammalian subject, such as a human, mouse, rat or primate (e.g. a marmoset or monkey). The subject may be a zebrafish, Xenopns or even C. elegans or Drosophila. The subject is preferably human. Treatment can be therapeutic or prophylactic. The Le/Lys disorder is any of those mentioned above.

The compound can be administered to the subject in order to prevent the onset of one or more symptoms of the disorder. In this embodiment, the subject can be asymptomatic. The subject can have a genetic predisposition to the Le/Lys disorder. The subject may have one or more family member(s) with a Le/Lys disorder. A prophylactically effective amount of the compound is administered to such a subject. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the disorder.

A therapeutically effective amount of the compound is an amount effective to ameliorate one or more symptoms of the disorder. Typically, such an amount reduces the volume and/or pH of the Le/Lys system in the subject. This can be confirmed as described above. The compound can be administered to the subject by any suitable means. The compound can be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes. The formulation of any of the compounds will depend upon factors such as the nature of the compound and the disorder to be treated. The compound may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The compound may also be administered as a suppository. A physician will be able to determine the required route of administration for each particular patient.

Typically, the compound is formulated for use with a pharmaceutically acceptable carrier or diluent and this may be carried out using routine methods in the pharmaceutical art. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar- coating, or film coating processes. Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.

Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used.

A therapeutically or prophylactically effective amount of the compound is administered. The dose may be determined according to various parameters, especially according to the compound used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50mg per kg, preferably from about 0.1mg/kg to 10mg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5mg to 2g.

Compounds identified in accordance with the invention may be polynucleotides. Preferably, the polynucleotide, such as RNA or DNA, in particular DNA, is provided in the form of an expression vector, which may be expressed in the cells of the subject to be treated. The polynucleotides maybe naked nucleotide sequences or be in combination with cationic lipids, polymers or targeting systems. The polynucleotides may be delivered by any available technique. For example, the polynucleotide may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the polynucleotide may be delivered directly across the skin using a polynucleotide delivery device such as particle-mediated gene delivery. The polynucleotide may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of polynucleotide constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the polynucleotide is administered in the range of lpg to lmg, preferably to lpg to 10μg nucleic acid for particle mediated gene delivery and lOμg to lmg for other routes.

Monitoring the progressive effect of a compound

The present invention also provides methods for monitoring the progressive effect of a compound that is capable of increasing or decreasing the volume and/or pH of the Le/Lys system. The compound is preferably capable of increasing or decreasing the volume and pH of the Le/Lys system. In combination with other experiments, such as staining for particular lipids, this method allows the mechanism by which the compound affects the Le/Lys system to be elucidated.

This method is based on the method of the first embodiment described above. The method involves incubating two or more test samples with the compound for a different amount of time within a range of times, such as from 0 to 24 hours. A person skilled in the art can determine a suitable amount of time. Each test sample is incubated with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells and the signal from the marker is concurrently or simultaneously measured. The signal obtained from each test sample is plotted against the corresponding time for which the sample was incubated with the compound. This allows the progressive effect of the compound on the volume and/or pH of the Le/Lys system to be monitored. The signal at each time point provides an indication of the volume and/or pH of the volume and/or pH of the Le/Lys system of the cells. This method is exemplified in Example 4.

In this method, any of the methods of the first embodiment described above may be used. The concentration of the marker and/or the number of cells has preferably been calibrated as described above.

Monitoring the progression of a disorder in a patient

The present invention also provides methods for monitoring progression of a Le/Lys disorder or an age-related disorder in a patient. The disorder is preferably a Le/Lys storage disorder, such as a Le/Lys lipid storage disorder.

The patient has typically been diagnosed with the disorder and is typically symptomatic. The patient may be any of those described above. Using these methods, a physician can tell whether the volume and/or pH of the patient's Le/Lys system is increasing (the disorder is getting worse) or decreasing (the patient is getting better) over time. The patient may be undergoing treatment for the disorder.

This method is based on the method of the first embodiment described above. Two or more test samples are taken from the patient at different times. A physician can determine suitable time periods. Each test sample is incubated with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells and the signal from the marker is concurrently or simultaneously measured. The signal obtained from each test sample is plotted against the time at which sample was taken from the patient. This allows the progression of the disorder to be monitored. The signal at each time point provides an indication of the volume and/or pH in the Le/Lys system of the patient. The signal at each time point preferably provides an indication of the volume and pH in the Le/Lys system of the patient. An increase in the volume and/or pH of the Le/Lys system of the patient between time points indicates that the disorder is getting worse. A decrease in the volume and/or pH of the Le/Lys system of the patient between time points indicates that the patient with the disorder is getting better. An improvement in the disorder may be indicative of the effectiveness of any therapy that the patient is undergoing. Similarly, a worsening of the disorder may be indicative of the ineffectiveness of any therapy that the patient is undergoing. No significant change in the volume and/or pH of the Le/Lys system of the patient indicates that the disease is not getting worse. In other words, no significant change in the volume and/or pH of the Le/Lys system of the patient indicates that the patient is stable. In this method, any of the methods of the first embodiment described above may be used. The concentration of the marker and/or the number of cells has preferably been calibrated as described above. The disorder may be any of those discussed above.

Determining the effectiveness of a treatment

The present invention also provides methods for determining the effectiveness of a treatment of a Le/Lys disorder or an age-related disorder in patients. The disorder is preferably a Le/Lys storage disorder, such as a Le/Lys lipid storage disorder.

The patient(s) have typically been diagnosed with the disorder and have been prescribed a treatment. The treatment may involve any of those discussed above. The patient(s) may be any of those described above. Using these methods, a physician can tell whether the treatment is effective at decreasing the volume and/or pH of the patient's Le/Lys system.

In one embodiment, the method is for determining the effectiveness of treatment of a Le/Lys disorder in two or more patients. The method is based on the method of the first embodiment described above. The method involves incubating a test sample taken from each patient after the treatment has begun with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells. The signal from marker is concurrently or simultaneously measured in each sample. The signal measured in each test sample is compared with a control value obtained using a control sample of cells taken from the same patient before the treatment was begun. A decreased signal in a test sample compared with the control value indicates that the treatment is effective in the patient. In other words, a decreased signal in a test sample compared with the control value indicates that the patient is getting better. No significant difference between the signal in a test sample and the control value also indicates that the treatment is effective in the patient. No significant difference between the signal in a test sample and the control value indicates that the patient is stable. An increased signal in a test sample compared with the control value indicates that the treatment is ineffective in the patient. In other words, a increased signal in a test sample compared with the control value indicates that the disease is getting worse.

In another embodiment, the method is for determining the effectiveness of a treatment of a Le/Lys disorder or an age-related disorder in a patient. This method is based on the second embodiment described above. The method involves incubating a control sample taken from the patient before the treatment was begun and a test sample taken from the patient after the treatment has begun with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells. The signals provided by the marker in the test sample and in the control sample are concurrently or simultaneously measured. The signal in the test sample is compared with the signal in the control sample. A decreased signal in the test sample compared with the control sample indicates that the treatment is effective in the patient. No significant difference between the signal in the test sample and the signal in the control sample also indicates that the treatment is effective in the patient. No significant difference between the signal in the test sample and the signal in the control sample indicates that the patient is stable. An increased signal in the test sample compared with the control sample indicates that the treatment is ineffective in the patient. In other words, an increased signal in the test sample compared with the control sample indicates that the patient is getting worse.

In these methods, any of the methods of the first and second embodiments described above may be used. In both methods, the concentration of the marker and/or the number of cells has preferably been calibrated as described above. The disorder and treatment may be any of those discussed above. Detennining the continued effectiveness of a treatment

The present invention also provides methods for determining the continued effectiveness of a treatment of a Le/Lys disorder or an age-related disorder in patients. The disorder is preferably a Le/Lys storage disorder, such as a Le/Lys lipid storage disorder. The patient(s) have typically been diagnosed with the disorder and are undergoing treatment.

The treatment may involve any of those discussed above. The patient(s) may be any of those described above. Using these methods, a physician can tell whether the treatment is continuing to be effective by continuing to maintain or continuing to decrease the volume and/or pH of the patient's Le/Lys system. The ultimate aim of the treatment is of course to return the volume and/or pH of the patient's Le/Lys system towards a normal volume and/or pH. A normal volume and/or pH is the mean value including standard errors in a population of the same organism as the patient in which none of the population have a Le/Lys disorder.

In one embodiment, the method is for determining the continued effectiveness of treatment of a Le/Lys disorder or an age-related disorder in two or more patients. The method is based on the method of the first embodiment described above. The method involves incubating a test sample from each of the patients with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells. The signal from the marker is concurrently or simultaneously measured in each test sample. The signal measured in each test sample is compared with a control value obtained using a control sample of cells taken from the same patient earlier in the treatment. A decreased signal in a test sample compared with the control value indicates that the treatment is continuing to be effective in the patient. No significant difference between the signal in a test sample and the control value also indicates that the treatment is continuing to be effective in the patient. An increased signal in a test sample compared with the control value indicates that the treatment is not continuing to be effective in the patient.

In another embodiment, the method is for determining the continued effectiveness of treatment of a Le/Lys disorder or an age-related disorder in a patient. The method is based on the method of the second embodiment described above. The method involves incubating two or more test samples each taken from the patient at different times with a marker that is capable of measuring the volume and/or pH of the Le/Lys system in the cells. The signal from the marker is concurrently or simultaneously measured in each test sample. The signal obtained from each test sample is plotted against the time at which the sample was taken from the patient. A decreased signal in a test sample compared with the signal in a test sample taken earlier during the treatment indicates that the treatment is continuing to be effective in the patient. No significant difference between the signal in a test sample and the signal in a test sample taken earlier during the treatment also indicates that the treatment is continuing to be effective in the patient. An increased signal in a test sample compared with the signal in a test sample taken earlier during the treatment indicates that the treatment is continuing to be effective in the patient. In these methods, any of the methods of the first and second embodiments described above may be used. In both methods, the concentration of the marker and/or the number of cells has preferably been calibrated as described above. The disorder and treatment may be any of those discussed above.

Kits

The invention also provides a kit for carrying out a method of the invention, which kit comprises a marker that is capable of measuring the volume and/or pH of the Le/Lys system in cells and a fiat plate comprising two or more wells. The flat plate is preferably a standard 96 or 384 well plate. The plate may be any of those described above.

The kit may additionally comprise medium for the cells and/or washing buffers to be used in the method steps.

The kit may also comprise controls, such as those described above, or a link to a website containing ranges for control values or ranges for the volume and/or pH in patients with specific Le/Lys disorders. The kit may also comprise a means to take a sample containing cells from a subject, such as a blood sample. The kit may comprise a means to separate mononuclear cells or T-cells from a blood sample.

The following Examples illustrate the invention.

Example 1 - Confirmation that LysoTracker® loading of cells is dependent upon LysoTracker® concentration and cell number

Initially, a fixed number of Chinese hamster ovary (CHO) cells, 40,000 (enough to completely cover the surface of each well of a 96 well tissue culture plate) were used and the concentration of

LysoTracker® was increased incrementally from 5OnM to 50OnM (Fig. IA). LysoTracker® saturated at 200 to 30OnM and as such was used in all subsequent experiments at 20OnM (to allow for elevations in lysosomal expansion). Higher concentrations (50OnM) can induce alterations in compartment pH and were not used. To analyse the effect of cell number on LysoTracker® loading, the LysoTracker® concentration was fixed at 20OnM and cell number was varied from 1,000 to 100,000 (Fig. IB). The range was linear from 20,000 to 60,000, but non-linear at low cell numbers (1,000 to 5,000) and high cell numbers (100,000 to 150,000). This was a novel discovery about LysoTracker® that was only achievable via a plate assay; a fluorescence-activated cell sorting (FACS) method measures each cell individually. 20OnM LysoTracker® and 40,000 cells per well were therefore used for all subsequent assays. The plate assay allows these parameters to be established easily and quickly during each experiment. This would be incredibly time consuming using FACS.

Example 2 - Confirmation by confocal microscopy double labeling against pulse/chased high molecular weight dextrans that LysoTracker® loads into lysosomes and late endosomes but not early endosomes

A high degree of overlap between lysosomes and LysoTracker® (89%) was observed and a slightly lower degree of overlap between late endosomes and LysoTracker® (78%) was observed (Fig. 2A). Therefore, only changes in the acidic compartments of the endolysosomal system can be measured with LysoTracker®. The acidic compartments of the endolysosomal system are the compartments that are affected in endolysosomal disorders, i.e. late endosomes and lysosomes (Le/Lys). To further confirm this, inhibitors that specifically destroy the acidic Le/Lys compartments were used to see if they depleted LysoTracker® fluorescence. GPN (a substrate of cathepsin C which when cleaved induces osmotic lysis of the Le/Lys), nigericin and monensin (both of which collapse the proton gradient that normally acidifies the Le/Lys) all depleted LysoTracker® fluorescence to the background level (Fig. 2B). This further indicates that LysoTracker® only loads into the acidic Le/Lys system and not into other organelles (ensuring its specificity for this assay as a measure of Le/Lys storage). Again, this kind of assay development was only possible as a result of the high throughput nature of the 96 well plate assay format and would have been incredibly difficult to do by FACS. Furthermore, the microscopy and combined plate reader results indicated that LysoTracker® can in fact record both the lysosomal and late endosomal compartments. This was an unexpected result, which would not have been discovered using a FACS method. This result ensures that all of the endolysosomal storage diseases, including the late endosome-specific ones such as Mucolipidosis type IV(or the cholesterol ester storage disease, Wolman Syndrome), can be assayed.

Example 3 - Mechanism of LysoTracker® action

In order to confirm the mechanism by which LysoTracker® measures Le/Lys storage, CHO cells were incubated with sucrose to induce swelling of the Le/Lys system. LysoTracker® accumulates in acidic compartments within the cells courtesy of protonation of one of the compounds chemical groups (an amine). Provided there is enough free protons, LysoTracker® will continue to load into cells. Under conditions of saturation (lOmin at 37°C with 20OnM LysoTracker®), loading was at a maximal level and as such very little variation in the results was seen when using control CHO cells (Fig. 3). Under conditions where the Le/Lys system is disrupted (for example decreased pH, see Fig. 2B above), there was a change in LysoTracker® fluorescence. An increase in staining would represent not an elevation in protonation, but rather an expansion in the size of the store (increased capacity to uptake LysoTracker®). Sucrose-induced swelling did indeed lead to an elevation in LysoTracker® fluorescence (Fig. 3). It was confirmed that this increase was related to an expansion of the Le/Lys system by using GPN to abolish the signal (Fig. 3). Using the plate assay, it has therefore been shown that changes in LysoTracker® fluorescence is dependent on the size and acidity of the Le/Lys compartment, rather than the lipids stored in this system.

Example 4 - Sensitivity of the assay

The sensitivity of the assay was tested using a drug (Ul 8666A) that induces Niemann-Pick Cl (NPCl) phenotypes in normal cells. The order of events following treatment with Ul 8666A have been well documented (Lloyd-Evans et al, Nature Medicine in press) as initial storage of sphingosine (lOmin to 2h, pmol quantities) then at 4-8h some secondary accumulation of cholesterol and glycosphmgolipids (GSL), which becomes more dramatic at 24h (Fig. 4A). Using LysoTracker® (Fig. 4B), these events could be followed. During the first 0-2h, there was a statistically significant elevation in LysoTracker® staining (indicative of the small pmol elevation in sphingosine). Next, there was a slightly greater elevation at 2-8h (indicative of the beginning of cholesterol and GSL storage), followed by a dramatic elevation at 8-24h (mimicking the accurate biochemical findings exactly). These results indicated that the LysoTracker® plate assay is sensitive enough to detect small nmol changes in lysosomal lipid storage, but also has the capacity to detect further large elevations in storage and faithfully represents the biochemical changes that occur. These results with Ul 8666A also indicated that LysoTracker® can be used as a screening approach to both detect Le/Lys disease progression and also as a pharmaceutical test to detect drugs that might induce dysfunction in the Le/Lysosome compartment (a common finding of many 'amine' drugs known as phospholipidosis).

Example 5 - Screening of diseased cells Next, fibroblasts isolated from many different storage disease patients were screened using the assay. Gaucher disease human patient fibroblasts do not store any lipid (glucosylceramide) and as such would be predicted to appear normal. They did using the assay (an internal control, Fig. 5). All other lysosomal storage disorder fibroblasts did have elevated staining with LysoTracker® as expected. Interestingly, there was a difference in severity of storage; NPCl, NPC2, mucolipidosis type IV and mucopolysaccharidosis fibroblasts had the highest LysoTracker® fluorescence values. This possibly echoes the increased diversity of LE/Lysosome storage material that accumulates in these diseases and the increased dysfunction in both late endosomal and lysosomal compartments. Wild-type cells had very low LysoTracker® fluorescence, which remained in a tight range on all occasions.

Krabbe cells have an alteration in their lysosomal pH and as such there is a decrease in LysoTracker® fluorescence. This indicates that the assay can detect such changes.

Example 6 - Monitoring therapeutic improvement

Having shown that the assay works to distinguish bonaflde lysosomal storage disorder cells from normal cell, the assay was used to monitor therapeutic improvement. Using NPCl fibroblast cells, the effect of NB-DNJ (Fig. 6A) or curcumin (Fig. 6B) on lysosomal dysfunction was tested. In both cases, a decrease in lysosomal fluorescence was observed, over a 3-5 day period with NB-DNJ (as has been reported biochemically; te Vruchte et al. , Journal of Biological Chemistry 2004) and 24h with curcumin (Lloyd-Evans et al. , Nature Medicine, in press). Therefore, the assay was capable of faithfully representing the reduction in lysosomal lipid storage and improved Le/Lys function following drug treatment.

Example 7 - High throughput method for screening drug libraries

Finally, the potential of the assay as a high throughput method for screening drug libraries for lysosomal storage disease therapies was tested. Using a compound library of 24 natural products, one that was capable of reducing LysoTracker® fluorescence in NPCl patient fibroblasts was identified (Fig. 7). This "hit" was confirmed by biochemical analysis of the cells, which indicated a reduction in cholesterol storage and correction in endocytic transport defects. Thus, the assay was confirmed as a novel method for screening new therapies that can reduce lysosomal storage/dysfunction.

Example 8 - Antibody capture method

Normal and NPCl -null human B-lymphoblasts (200,000 cells per well) were loaded (Ih at 4°C) onto 96 well plates pre-coated with streptavidin (Pierce) and pre-incubated with biotinylated anti human-CD19 antibody (Ih at room temperature, eBiosciences). Following capture, cells were incubated with 20OnM LysoTracker® red for 15 minutes at 37 ⁰ C and fluorescence measured on a BMG labtech plate reader. NPCl -null B-lymphoblasts were 3 -times brighter than normal controls (indicating storage). The results are shown in Fig. 8. At low antibody concentrations (O.lμg/ml) fewer cells were captured and the resulting LysoTracker® signals were dimmer indicating the importance of establishing optimal conditions for the experiment.

Example 9 - Lysosomal storage in Danon disease and cystic fibrosis

The assay described above was used to measure lysosomal volume in fibroblast cells from patients with Danon disease (Lampl deficiency) and cystic fibrosis (CFTR deficiency, abnormal lysosomal pH). The results are shown in Figure 10. The results indicate that fibroblasts from both Danon disease (column C) and cystic fibrosis (column B) display lysosomal storage when compared with control cells (column A). These results indicate that the assay of the invention may be used in connection with Danon disease and cystic fibrosis.

Example 10 - Lysosomal storage in chloroquine-induced phospholipidosis The assay described above was used to measure lysosomal volume in cells treated with chloroquine, which induces phospholipidosis. The results are shown in Figure 11. After 10 minutes (column B), chloroquine reduces the LysoTracker® signal because it deacidifϊes the lysosomes. However, after 24 hours (column C), chloroquine induces lysosomal storage which is measured as an increase in LysoTracker® staining compared to untreated control cells (column A). These results indicate that the assay of the invention may be used in connection with drug-induced phospholipidosis.