Treatment for batten disease

Abstract

The disclosure concerns use of a compound for treating a lysosomal storage disorder, increasing lysosomal clearance, reducing a neurological pathology or reducing seizures. The disclosure further concerns a method for treating a lysosomal storage disorder, increasing lysosomal clearance, reducing a neurological pathology or reducing seizures, which comprises administering a compound to a subject. The disclosure also concerns use of a compound in the manufacture of a medicament for use in treating one or more symptoms of a lysosomal storage disorder, increasing lysosomal clearance, reducing a neurological pathology or reducing seizures. In any case, the compound is steroid, a GABA receptor ligand, a Sigma-1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

Description

[0001] TREATMENT FOR BATTEN DISEASE

[0002] FIELD OF THE DISCLOSURE

[0003] The disclosure concerns use of a compound for treating a lysosomal storage disorder, increasing lysosomal clearance, reducing a neurological pathology or reducing seizures. The disclosure further concerns a method for treating a lysosomal storage disorder, increasing lysosomal clearance, reducing a neurological pathology or reducing seizures, which comprises administering a compound to a subject. The disclosure also concerns use of a compound in the manufacture of a medicament for use in treating one or more symptoms of a lysosomal storage disorder, increasing lysosomal clearance, reducing a neurological pathology or reducing seizures. In any case, the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0004] BACKGROUND

[0005] The Neuronal Ceroid Lipofuscinoses (NCL) are a group of lysosomal storage disorders (LSDs), also known as Batten disease, which collectively constitute the most common cause of inherited paediatric neurodegenerative disorders. There are currently 13 known forms of NCL, each caused by mutation of individual genes. The majority of affected proteins in NCLs localise to the lysosomal lumen or membrane. However, others are localised to other cellular compartments, including the endoplasmic reticulum (ER), Golgi, cytosol and plasma membrane. Despite diversity in the roles of the affected proteins, the 13 currently classified NCLs share a hallmark accumulation of auto- fluorescent lysosomal storage material and common characteristics including progressive neurodegeneration resulting in motor and cognitive decline, retinopathy leading to blindness, myoclonic epilepsy and severely premature death. The lysosomal storage material in the NCLs predominantly consists of ceroid-lipopigments, as well as subunit c of mitochondrial ATP synthase (SCMAS) or sphingolipid activator proteins A and D.

[0006] CLN2 disease, otherwise known as the classic Late Infantile form of NCL (cLINCL), is caused by autosomal recessive inherited mutation in the CLN2 gene, leading to dysfunction of the encoded lysosomal serine protease enzyme Tripeptidyl Peptidase 1 (TPP1). The disease causes cell death in the central nervous system (CNS) and neural retina. Children with CLN2 disease will typically present at 2-4 years old. Early symptoms can include seizures, language delay, motor dysfunction, behavioural problems and dementia. Progression of the disease leads to rapid motor and cognitive deterioration, progressive visual impairment, the development of refractory seizures and death normally within early adolescence. Current treatments for NCLs are limited and focus on symptom management. CLN2 disease is currently the only NCL in which there is a clinically approved treatment that has been shown to effectively attenuate disease progression, in the form of enzyme replacement therapy for the brain. However, peripheral disease is likely to emerge in patients receiving enzyme replacement therapy, which has already been noted in the retina. Improved treatments are therefore needed.

[0007] SUMMARY OF THE DISCLOSURE

[0008] The present inventors have used a model of a lysosomal storage disorder, namely CLN2 disease, to screen for treatments for Batten disease. By doing so, the inventors have identified that steroids (such as pregnenolone) and related compounds have anti-seizure and neuroprotective effects, and can rescue lysosomal impairment to enhance clearance. The inventors have further identified potential mechanisms for these beneficial effects. One such mechanism is the modulation of GABA receptors by downstream metabolites of the steroid. Another is the agonistic effect of the steroid or a metabolite thereof on the sigma- 1 receptor. A further mechanism is the modulation of microtubules, which may have downstream effects on lysosome motility. Another is function as a TRP channel ligand.

[0009] Accordingly, the invention provides:

[0010] - a compound for use in a method of treating a lysosomal storage disorder, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0011] - a compound for use in increasing lysosomal clearance, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0012] - a compound for use in reducing a neurological pathology, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator; - a compound for use in reducing seizures, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0013] - a method of treating a lysosomal storage disorder comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0014] - a method of increasing lysosomal clearance comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0015] - a method of reducing a neurological pathology comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma-1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0016] - a method of reducing seizures comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0017] - use of a compound in the manufacture of a medicament for use in treating one or more symptoms of a lysosomal storage disorder, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0018] - use of a compound in the manufacture of a medicament for use in increasing lysosomal clearance, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator;

[0019] - use of a compound in the manufacture of a medicament for use in reducing a neurological pathology, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator; and - use of a compound in the manufacture of a medicament for use in reducing seizures, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0020] DESCRIPTION OF THE FIGURES

[0021] Figure 1: Locomotion assay drug screen in tppl~~ zebrafish identifies pregnenolone as hit compound. (A) Drug screen design for FDA approved drug library compounds. (B) Representative locomotion assay plate. 96-well plate containing one fish per well in 200pL aquarium water with treatment. Locomotion tracks are shown. White tracks are predicted movements that were not measured. (C) Locomotion assay plots from blind testing of re-sourced pregnenolone in tppl~ ~ zebrafish compared to WT siblings. Pregnenolone (preg) treatment for 24 h from 48 hpf significantly improved seizure-like locomotion phenotype in tppl~ ~ zebrafish, demonstrated by reduced movement duration (s), distance (mm) and mean velocity (mm / s) [LogLn] compared to DMSO treated tppl~ ~ zebrafish. Results from two replicate experiments. (D) Single representative EEG recordings of tppl~ ~ zebrafish at 3 dpf treated with preg or DMSO control for 24 hr. (E) Plot showing significant reduction in mean EEG amplitude in preg treated tppl~ ~ zebrafish compared to DMSO control. Results are combined from three days of experiments. (C and E) Data are shown by box and whisker plot (minimum to maximum) with individual points each representing an individual animal. (C) Linear mixed model (fixed: genotype, treatment / random: date of experiment). (E) Lfripaired t-test. *P < 0.033, **P < 0.002, ***p < 0.001. (F) Representative TUNEL stained sagittal sections from WT and tppl~ ~ zebrafish treated with preg or DMSO control from 52 hpf for 3 hours, with the mesencephalon indicated as the region within the dotted white line. (G) Quantification of mean fluorescent intensity of TUNEL staining in the mesencephalon. Results from two replicate experiments. Data are shown by box and whisker plot (minimum to maximum) with individual points each representing an individual fish. Two-way ANOVA. *p < 0.033, **p < 0.002, *** / ? < 0.001.

[0022] Figure 2: Pregnenolone ameliorates lysosomal abnormalities and dysfunction in tppl ~ zebrafish. (A-B) ZsGreen-Lampl WT and tppl'1' zebrafish at 3 dpf treated with DMSO or pregnenolone (preg) for 24 hr. (A) Representative 40x max-projection confocal images, enlarged area indicated by white box. (B) Quantification of lysosomal number (left) and size (right) from ZsGreen-Lampl confocal images. Results from three replicate experiments. Data are shown by box and whisker plot (minimum to maximum) with individual points each representing an individual fish. (C) Representative image of Western blot analysis from whole lysates of de-yolked non-transgenic WT and tppl’1' zebrafish at 4 dpf treated with DMSO or preg for 48 hr, stained with antibodies against P62 normalized to HSC70 and SCMAS normalized to Tom20. Data are shown by box and whisker plot (minimum to maximum) with individual points each representing an individual sample. (D, E) Seizure-like locomotion assay from 3 dpf WT and tppl’1' mutant zebrafish treated with pregnenolone (Preg) + 3-MA for 24 hr, with DMSO, pregnenolone, or 3-MA alone as controls. (D) Representative locomotion assay plate. 96-well plate containing one fish per well in 200 pL aquarium water with treatment. Locomotion tracks are shown. White tracks are predicted movements that were not measured. (E) Plot showing duration of movement(s). No significant difference between pregnenolone with and without 3-MA, in tppl' ' zebrafish. Results from three replicate experiments. Data are shown by box and whisker plot (min to max) with individual points each representing an individual fish. (B, C) Linear mixed model (fixed: genotype, treatment / random: date of experiment / plate). (E) Linear mixed model on log transformed data (fixed: genotype, treatment / random: date of experiment). P* <0.05, **<0.01, P*** <0.001.

[0023] Figure 3: Lysosomal abnormalities and dysfunction in tppl'1' zebrafish are ameliorated by pregnenolone. (A) Representative image of Western blot analysis from whole lysates of de-yolked non-transgenic WT and tppP1' zebrafish at 3 dpf treated with DMSO or pregnenolone (preg) for 24 hr, stained with antibodies against S6 and phosphorylated(p)-S6 normalized to HSC70. Data are shown as mean ± SD with individual points each representing an individual sample. (B, C) ZsGreen- TFEB;mCherry-nls WT and tppl'1' zebrafish at 3 dpf treated with DMSO or preg for 24 hr. (B) Representative cropped 40x single-plane images showing distribution of ZsGreen- TFEB, alongside mCherry-nls nuclear marker. ZsGreen-TFEB / mCherry-nls merge shown with corresponding ZsGreen-TFEB-only image below. Examples of nucleus indicated by yellow arrow. (C) Quantification of ZsGreen-TFEB intensity within nucleus over intensity of total area of muscle measured. (A, C) Linear mixed model (fixed: genotype, treatment / random: date of experiment / plate). P* <0.05, **<0.01, P*** <0.001. (D, E) RNA Sequencing data of a custom lysosomal gene set analysed in head (left) and tail (right) tissue of 3 dpf (D) tppl'1' vs WT zebrafish (24 hr DMSO treatment) and (E) tppl'1' zebrafish with preg vs DMSO treatment (treated for 24 hr). Results from 6 replicate samples, n=10 fish per sample. Barcode plots from CAMERA gene set enrichment analysis. Each bar represents a single gene; red and blue sections are representations of the portion of genes that most contribute to up- and down-regulated enrichment respectively. The curves show the enrichment score of the bars in the barcode plot; parts above and below the dashed line signify up- and down-regulated enrichment respectively. CAMERA p value result and highest enriched direction indicated on each plot.

[0024] Figure 4: Steroid hormone biosynthesis is dysregulated in tppl'1' zebrafish and boosted by pregnenolone treatment. Steroid hormone measurements from 3 dpf WT and tppl'1' zebrafish treated with pregnenolone (preg) or DMSO for 24 hr. Results from 6 replicate samples, n=50 fish per sample. (A) Heatmap representing hormone measurements. Each block represents a single hormone from an individual sample. (B) Individual hormone measurement plots show picograms (pg) of hormones per gram of fish tissue. Data are shown by box and whisker plot (minimum to maximum) with individual points each representing an individual sample. Linear mixed model (fixed: genotype, treatment / random: date of experiment). / ?* <0.05, / ?**<0.01, / ?*** <0.001.

[0025] Figure 5: Overview of changes in steroid hormone levels in tppl'1' zebrafish compared with WT siblings. An overview of the steroid hormone synthesis pathway with blue (ringed) and red (unringed) circles representing hormones that were significantly increased or decreased respectively in tppl'1' zebrafish compared with WT siblings (treated with DMSO for 24 hr). Grey circles represent hormones that were unchanged. Hormones that are not circled were not measured. Hormones were measured by mass spectrometry in 3 dpf zebrafish (adapted from76).

[0026] Figure 6: Cholesterol and hormone related genes are enriched in tppl'1' zebrafish and further boosted by pregnenolone treatment. RNA Sequencing data from head (left) and tail (right) tissue of 3 dpf zebrafish treated with DMSO or pregnenolone (Preg) for 24 hr. Results from 6 replicate samples, n=10 fish per sample. (A-C) Barcode plots from CAMERA gene set enrichment analysis. Each bar represents a single gene; red and blue sections are representations of the portion of genes that most contribute to up- and down-regulated enrichment respectively. The curves show the enrichment score of the bars in the barcode plot; parts above and below the dashed line signify up- and down- regulated enrichment respectively. CAMERA p value result and highest enriched direction indicated on each plot. (A) tppl'1' vs WT zebrafish (B) pregnenolone vs DMSO treated WT zebrafish (C) pregnenolone vs DMSO tppl'1' zebrafish.

[0027] Figure 7: Overview of major pathways of cholesterol metabolism in the cell.

[0028] Cholesterol availability in the cell depends predominantly on uptake of circulating LDL by the LDL-Receptor (LDLR) and de novo biosynthesis from acetyl-coA, mediated by the rate-limiting enzymes 3 -hydroxy-3 -m ethyl glutaryl coenzyme A reductase (HMGCR) and squalene mono-oxygenase (SM). Cholesterol biosynthesis is regulated by the transcription factor SREBF2. Excess cholesterol is exported to the blood through ABCA1 or ABCG1. Imported or stored cholesterol esters are unesterified in the lysosome and free cholesterol can be distributed to other organelles in the cell via membrane contact sites, mediated by NPC1 and NPC2, and StARD3. Free cholesterol can be delivered to the inner mitochondrial membrane, mediated by StARDl, for use in hormone biosynthesis. Excess cholesterol can be esterified by ACAT or LCAT for storage in lipid droplets or excretion as HDL.

[0029] Figure 8: p62 and SCMAS loci mRNA analysis. RNA Sequencing data from head (top panel) and tail (bottom panel) tissue of 3 dpf WT and tppl- / - mutant zebrafish treated with DMSO or pregnenolone (Preg) for 24 hr. Results from 6 replicate samples, n=10 fish per sample. Plots show Log2 -transformed counts per million (CPM) for (A) p62; (B) SCMAS loci: atp5mcl, atp5mc3a, atp5mc3b. Data are shown by box and whisker plot (min to max) with individual points each representing an individual sample. Linear mixed model (fixed: genotype, treatment / random: plate). P* <0.05, P**<0.01, P*** <0.001.

[0030] Figure 9: Preliminary ZsGreen-LC3 flux assay suggests pregnenolone increases autophagic flux and tppl- / -mutant zebrafish. ZsGreen-LC3WT and tpp 1 - / - mutant zebrafish at 3 dpf treated with DMSO or pregnenolone (Preg) for 24 hr, with NH4C1 or H2O control for the last 4 hr. (A) Representative 40xmax -projection images. (B) Quantification of ZsGreen-LC3 confocal images: increase in ZsGreen-LC3 area in both WT and tppl- / - mutant zebrafish treated with pregnenolone +NH4C1, compared to pregnenolone+control demonstrates increase in autophagic flux with pregnenolone treatment. Results from one experiment. Data are shown by box and whisker plot

[0031] (minimum to maximum) with individual points each representing an individual fish. Linear mixed model (fixed: genotype, treatment / random: date of experiment), p* <0.05, p**<0.01, p*** <0.001.

[0032] Figure 10: Hormone measurements in WT and tppl- / - mutant zebrafish. Hormone measurements from 3 dpf WT and tppl- / - mutant zebrafish treated with pregnenolone (Preg) or DMSO for 24 hr. Results from 6 replicate samples, n=50 fish per sample. Individual hormone measurement plots show picograms (pg) of hormones per gram of fish tissue. Hormones are grouped by those that are increased (A), decreased (B) or unchanged (C) in tppl- / - mutant zebrafish compared with WT (treated with DMSO). Data are shown by box and whisker plot (minimum to maximum) with individual points each representing an individual sample. Linear mixed model (fixed: genotype, treatment / random: date of experiment). P* <0.05, P**<0.01, P*** <0.001.

[0033] Figure 11: Dysregulation of key genes in the cholesterol and hormone pathway in tppl- / - mutant zebrafish and the effect of pregnenolone (Preg) treatment. (A-G) Individual gene analysis from RNA Sequencing data in head and tail tissue. Plots show Log2-transformed counts per million (CPM) for genes encoding key proteins involved in (A) cholesterol biosynthesis (srebf2, hmgcra, hmgcrb), (B) esterification (Icat, acatl, acat2), (C) import (Idlra) and (D) export (abcgl, abcala, abcalb), (E) regulation of lysosomal cholesterol (npcl, npc2) and (F) hormone synthesis (cyp3cl, hsdl lb2, stard3, srd5a2a, cypl7al, akrlc), as indicated by left panel. Data are shown by box and whisker plot (minimum to maximum) with individual points each representing an individual sample. Linear mixed model (fixed: genotype, treatment / random: plate), p* <0.05, p**<0.01, p*** <0.001.

[0034] Figure 12: Metabolomics of tppl- / - zebrafish. (A) Principal component analysis (PCA) of metabolites shows that pregnenolone (RVC1) causes a shift in normal and tppl~ ~ zebrafish. (B) OPLS-DA modelling and univariate analysis indicate 6 / 61 metabolites that approach normal when tppl- / - zebrafish are treated with pregnenolone.

[0035] DETAILED DESCRIPTION

[0036] It is to be understood that different applications of the disclosed methods and products 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 disclosure only, and is not intended to be limiting. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0037] General definitions

[0038] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs.

[0039] 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 steroid” includes “steroids”, reference to “an agonist” includes two or more such agonists, and the like.

[0040] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “a method comprising administering a compound to the individual” should be interpreted to mean that the method contains a step of administering such a compound, but that the method may contain additional steps such as, for example, administering a further therapeutic agent.

[0041] In some aspects of the disclosure, the word “comprising” is replaced with the phrase “consisting of’. The term “consisting of’ is intended to be limiting. For example, the phrase “a method consisting of administering a compound to the individual” should be interpreted to mean that the method contains a step administering such a compound, and no additional steps.

[0042] The term “alkyl”, as used herein, refers to a linear or branched chain saturated hydrocarbon radical. A “Cn-m alkyl” refers to an alkyl having from n to m carbon atoms. Examples of C1.3 alkyl groups are methyl, ethyl, i-propyl, and n-propyl. For the avoidance of doubt, where two alkyl moieties are present in a group, the alkyl moieties may be the same or different.

[0043] The term “carbocyclyl”, as used herein, refers to a saturated or partially unsaturated cyclic hydrocarbon group. A “6-membered carbocyclyl” refers to a carbocyclyl having 6 carbon atoms. Examples of a 6-membered carbocyclyl group include cyclohexyl, cyclohexenyl, and cyclohex-1, 3-dienyl.

[0044] The term “aryl”, as used herein, refers to a monocyclic, bicyclic or polycyclic aromatic group which contains up to 14 carbon atoms, typically from 6 to 10 carbon atoms, in the cyclic portion. A 6-memebered aryl is typically a monocyclic ring which contains 6 carbon atoms. Examples of aryl groups include phenyl, naphthyl, indenyl and indanyl groups. Phenyl is preferred.

[0045] As used herein the term oxo represents a group of formula: =0, the term nitrile represents a group of formula: -CN, and the term sulfate represents a group of formula:

[0046] A Ci-3 alkoxy group is a C1.3 alkyl group attached to an oxygen atom.

[0047] Unless otherwise specified, included in the substituent definitions are ionic, salt, solvate, and protected forms of these substituents. For example, a reference to a hydroxyl group also includes the anionic form (-O'), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to a sulfate group also includes the anionic form (- OSOs'), a salt (e.g. a sodium salt) or solvate thereof, as well as conventional protected forms.

[0048] The compounds for use according to the invention can exist in various tautomeric forms and it is to be understood that the invention encompasses all such tautomeric forms.

[0049] In certain of the compounds for use according to the invention, dependant on the nature of the substituents present, there may be chiral carbon atoms and therefore the compounds may exist as stereoisomers. The invention extends to all such stereoisomeric forms of the compounds for use according to the invention, including enantiomers, diastereomers and mixtures thereof, such as racemates. The different stereoisomeric forms may be separated or resolved one from the other by conventional methods or any given isomer may be obtained by conventional stereoselective or stereospecific syntheses.

[0050] It is also to be understood that reference to an isotope thereof means that any atom present in a compound for use according to the invention may be present in any available isotopic form. For instance, a carbon atom may be12C or13C. A hydrogen atom may be (deuterium).

[0051] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising a formulation, and / or the patient being treated therewith. As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or / ?-toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines.

[0052] Treatment of a lysosomal storage disorder

[0053] The disclosure provides a compound for use in a method of treating a lysosomal storage disorder, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The method may comprise administering the compound (for example an effective amount of the compound) to the subject. The disclosure also provides a method of treating a lysosomal storage disorder comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The disclosure further provides use of a compound in the manufacture of a medicament for use in treating one or more symptoms of a lysosomal storage disorder, wherein the compound is a steroid, a GABA receptor ligand, a Sigma-1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. Use of the medicament may comprise (for example an effective amount of the compound) administering the compound to the subject.

[0054] Lysosomal storage disorders (LSDs) are a group of inherited metabolic disorders that result from defects in lysosomal function. Lysosomes are sacs of enzymes within cells. Normally, the enzymes digest large molecules and pass the fragments on to other parts of the cell for recycling. However, if a critical enzyme is defective due to a mutation, the digestion process may be adversely affected. This may lead to the accumulation of undigested or partially digested molecules, leading to impaired cell function and ultimately cell death. The neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders which collectively constitute the most common cause of inherited paediatric neurodegenerative disorders. NCLs are also known as Batten disease. There are currently 13 known forms of NCL or Batten disease, each caused by a mutation of an individual gene. As a result of the mutation, toxic metabolites may accumulate within the cell; lipid trafficking may be impaired; signaling may be interrupted; calcium homeostasis may be disturbed; and / or the unfolded protein response may be activated.

[0055] Accordingly, the lysosomal storage disorder may be a neuronal ceroid lipofuscinosis. In other words, the lysosomal storage disorder may be Batten disease. The lysosomal storage disorder may be any known form of Batten disease. For instance, the lysosomal storage disorder may be CLN1 disease, CLN2 disease, CLN3 disease, CLN4 disease, CLN5 disease, CLN6 disease, CLN7 disease, CLN8 disease, CLN10 disease, CLN11 disease, CLN12 disease, CLN13 disease or CLN14 disease.

[0056] The lysosomal storage disorder may, for example, be a form of Batten disease in which a gene encoding a soluble lysosomal enzyme is mutated. For instance, the lysosomal storage disorder may be CLN1 disease, CLN2 disease, CLN10 disease or CLN13 disease. CLN1 disease is caused by a mutation in the gene encoding PPT1. CLN2 is caused by a mutation in the gene encoding TPP1. CLN10 disease is caused by a mutation in the gene encoding CTSD. CLN11 disease is caused by a mutation in the gene encoding CTSF.

[0057] The lysosomal storage disorder may, for example, be a form of Batten disease in which a gene encoding a soluble, secretable lysosomal protein is mutated. For instance, the lysosomal storage disorder may be CLN5 disease. CLN5 disease is caused by a mutation in the CLN5 gene, which encodes such a protein.

[0058] The lysosomal storage disorder may, for example, be a form of Batten disease in which a gene encoding a protein in the secretory pathway is mutated. For instance, the lysosomal storage disorder may be CLN11 disease. CLN11 disease is caused by a mutation in the gene encoding GRN.

[0059] The lysosomal storage disorder may, for example, be a form of Batten disease in which a gene encoding a cytoplasmic protein is mutated. For instance, the lysosomal storage disorder may be CLN4 disease or CLN14 disease. CLN4 disease is caused by a mutation in the gene encoding DNAJC5. CLN14 disease is caused by a mutation in the gene encoding KCTD7.

[0060] The lysosomal storage disorder may, for example, be a form of Batten disease in which a gene encoding a transmembrane protein in the endoplasmic reticulum (ER) is mutated. For instance, the lysosomal storage disorder may be CLN6 disease. CLN6 disease is caused by a mutation in the CLN6 gene, which encodes such a protein.

[0061] The lysosomal storage disorder may, for example, be a form of Batten disease in which a gene encoding a transmembrane protein in the ER / c / .s Golgi is mutated. For instance, the lysosomal storage disorder may be CLN8 disease. CLN8 disease is caused by a mutation in the CLN8 gene, which encodes such a protein.

[0062] The lysosomal storage disorder may, for example, be a form of Batten disease in which a gene encoding a transmembrane protein in lysosomes is mutated. For instance, the lysosomal storage disorder may be CLN3 disease, CLN7 disease or CLN12 disease. CLN3 disease, CLN7 disease and CLN12 disease are caused by a mutation in the CLN3, CLN7 or CLN12 gene respectively, which encode such proteins.

[0063] In CLN3 disease and CLN7 disease, globotriaosylceramide (Gb3) is known to accumulate in lysosomes. Globotriaosylceramide is also thought to accumulate in CLN5 disease. In one aspect of the disclosure, the lysosomal storage disorder is not a disorder associated with increased levels of globotriaosylceramide. In other words, the lysosomal storage disorder may be a lysosomal storage disorder in which globotriaosylceramide does not accumulate in lysosomes. The lysosomal storage disorder may, for example, be a disease other than CLN3 disease. That is, in one aspect of the disclosure, the lysosomal storage disorder is not CLN3 disease. The lysosomal storage disorder may, for example, be a lysosomal storage disorder other than CLN3 disease, CLN7 disease, or CLN5 disease. That is, in one aspect of the disclosure, the lysosomal storage disorder is not CLN3 disease. CLN7 disease or CLN5 disease.

[0064] In a preferred aspect of the disclosure, the lysosomal storage disorder is a form of Batten disease in which a gene encoding a soluble lysosomal enzyme is mutated. The lysosomal storage disorder may, for example, be CLN1 disease, CLN2 disease, CLN10 disease, or CLN13 disease. For instance, the lysosomal storage disorder may be CLN2 disease. While different lysosomal storage disorders have different underlying mutations, there are certain similarities in the presentation of each disease. For example, neuropathological changes are a common feature, and the diseases often lead to seizures. It follows that a treatment that is demonstrated to reduce neurological pathology and / or seizures for one lysosomal storage disorder may also be used to treat another lysosomal storage disorder. A treatment that is demonstrated to reduce neurological pathology and / or seizures for one NCL may also be used to treat another NCL.

[0065] In the context of the present disclosure, treating a lysosomal storage disorder may comprise reducing or eliminating one or more clinical signs or symptoms of the lysosomal storage disorder. For example, treating a lysosomal storage disorder may comprise reducing or eliminating neurological pathology associated with the disorder. For example, treating a lysosomal storage disorder may comprise reducing seizures caused by the lysosomal storage disorder. A reduction in seizures may comprise a reduction in the number, frequency, duration and / or severity of seizures, for instance.

[0066] Treating a lysosomal storage disorder may comprise counteracting the pathogenesis of the lysosomal storage disorder. For example, treating a lysosomal storage disorder may comprise improving lysosomal clearance. A reduction in or elimination of one or more clinical signs or symptoms of the lysosomal storage disorder (e.g. seizures) may result from such lysosomal clearance. Improvement of lysosomal clearance is discussed in more detail below, in the section “Increasing lysosomal clearance”.

[0067] One or more other mechanisms may contribute, or further contribute, to treatment of the lysosomal storage disorder. For instance, the lysosomal storage disorder may be treated by stimulating steroid hormone biosynthesis. That is, the compound may stimulate steroid hormone biosynthesis and thereby treat the lysosomal storage disorder. Steroid hormones, such as neurosteroids, are known to modulate neuronal excitability which has important implications in seizure susceptibility and control. Provision of an upstream member of the steroid hormone synthesis pathway may, for example, beneficially result in increased production of a downstream steroid having anti-convulsant effects.

[0068] In another example, the lysosomal storage disorder may be treated by modulation of GABA receptors. GABA receptors may, for example, be modulated by the compound itself (e.g. a GABA receptor ligand) or a metabolite or derivative of the compound (e.g. a metabolite or derivative of a steroid). In a further example, the lysosomal storage disorder may be treated by an agonistic effect of the compound or a metabolite or derivative thereof on the sigma- 1 receptor. In another example, the lysosomal storage disorder may be treated by modulation of microtubules, which may have downstream effects on lysosome motility. The lysosomal storage disorder may also be treated by function of the compound or a metabolite or derivative thereof as a TRP channel ligand. TRP channels are implicated in hyperexcitability and seizures.

[0069] In any case, treatment may be effected by administering the compound to a subject having the lysosomal storage disorder. Administration, compounds and subjects are discussed in more detail below.

[0070] Increasing lysosomal clearance

[0071] The disclosure provides a compound for use in increasing lysosomal clearance, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The method may comprise administering the compound to the subject. The disclosure also provides a method of increasing lysosomal clearance comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The disclosure further provides use of a compound in the manufacture of a medicament for use in increasing lysosomal clearance, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. Use of the medicament may comprise administering the compound (for example an effective amount of the compound) to the subject. Lysosomal clearance may, for example, be increased relative to the level of lysosomal clearance prior to use or administration of the compound.

[0072] The function of lysosomes is well-known in the art. Briefly, lysosomes contain a variety of enzymes that enable the cell to break down various biomolecules it engulfs, such as peptides, nucleic acids, carbohydrates, and lipids. Lysosomes are also capable of fusing with other organelles and digesting large structures or cellular debris. Through cooperation with phagosomes, they are able to conduct autophagy, clearing out damaged structures. In the context of the present disclosure, increasing lysosomal clearance may refer to improving lysosomal function. For example, increased lysosomal clearance may refer to an improved ability of the lysosome to break down polymers, such as peptides, nucleic acids, carbohydrates, and / or lipids. Increased lysosomal clearance may refer to an improved ability of the lysosome to digest large structures or cellular debris. Increased lysosomal clearance may refer to improved autophagic activity of the lysosome.

[0073] In any case, methods for determining lysosomal clearance are known in the art and described herein. For instance, assays that measure lysosomal enzyme activity are disclosed in Strovel et al. (Measurement of lysosomal enzyme activities: A technical standard of the American College of Medical Genetics and Genomics (ACMGf Genetics in Medicine, Volume 24, Issue 4, 2022, Pages 769-783). In addition, commercially- available lysosome activity assay kits use a lysosome-specific self-quenched substrate that acts as endocytic cargo and is degraded in lysosomes. The fluorescence signal from the substrate is proportional to lysosomal activity, indicative of clearance.

[0074] Furthermore, clearance of autophagic material in the lysosome may be used as to indicate lysosomal clearance, as in the present Examples. In particular, one or more autophagy-associated proteins, such as P62, can be measured using a quantitative technique such as Western blotting or RNA-seq. Higher levels of the protein may indicate accumulation, associated with reduced lysosomal clearance. Lower levels of the protein may indicate that there is no accumulation, associated with effective lysosomal clearance. Thus, increasing lysosomal clearance may refer to reducing the amount of one or more autophagy-associated proteins. The amount of the one or more proteins may, for example, be reduced in a sample obtained from a subject to whom the compound is administered. The reduction may, for example, be relative to the amount of the one or more proteins present in a sample obtained from the subject prior to administration of the compound. The sample may, for example, comprise one or more cells, such as one or more neurological cells.

[0075] The number and / or size of lysosomes in a cell may also be used to indicate the level of lysosomal clearance. The number and / or size of lysosomes may, for example, be determined using a fluorescent imaging technical such as confocal microscopy, as in the present Examples. A reduced number and / or increased size of lysosomes may indicate defective lysosomal clearance. Thus, increasing lysosomal clearance may refer to increasing the number of lysosomes and / or reducing the size of lysosomes. The number of lysosomes may, for example, be increased in a sample obtained from a subject to whom the compound is administered. The increase may, for example, be relative to the number of lysosomes present in a sample obtained from the subject prior to administration of the compound. The size of lysosomes may, for example, be reduced in a sample obtained from a subject to whom the compound is administered. The reduction may, for example, be relative to the size of lysosomes present in a sample obtained from the subject prior to administration of the compound. The sample may, for example, comprise one or more cells, such as one or more neurological cells.

[0076] Lysosomal clearance may be increased by any mechanism that is elicited by the compound. In any case, increasing lysosomal clearance in accordance with the disclosure may have beneficial effects in a subject administered with the compound. In particular, increasing lysosomal clearance may treat a lysosomal storage disorder. As set out above, treatment of a lysosomal storage disorder may refer to reducing or eliminating one or more clinical signs or symptoms of the disorder, such as seizures. Treatment of a lysosomal storage disorder may refer to reducing or eliminating neurological pathology associated with the disorder.

[0077] Reducing a neurological pathology

[0078] The disclosure provides a compound for use in reducing a neurological pathology, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The method may comprise administering the compound to the subject. The disclosure also provides a method of reducing a neurological pathology comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The disclosure further provides use of a compound in the manufacture of a medicament for use in reducing a neurological pathology, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. Use of the medicament may comprise administering the compound to the subject. In the context of the present disclosure, a neurological pathology may refer to any defect of the nervous system. The defect may be a defect affecting the central nervous system, peripheral nervous system or both. The defect may be caused by structural and / or functional abnormalities in the nervous system. Exemplary neurological pathologies include seizures, impaired vision, learning difficulties, weakness and poor co-ordination, for example.

[0079] In one aspect of the disclosure, the neurological pathology is seizures. Preferably, the seizures are epileptic seizures. An epileptic seizure may be defined as a seizure that starts in the brain. Other types of seizure include hypoglycaemic seizures, febrile convulsions, and seizures caused by heart problems.

[0080] Other neurological pathologies include, for example, reduced lifespan and neurological cell death.

[0081] Reducing a neurological pathology may refer to improving or resolving the neurological pathology. For instance, one or more anatomical, metabolic or physiological defects associated with the neurological pathology may be improved or resolved. One or more clinical signs or symptoms of the neurological pathology may be improved or resolved. The improvement or resolution in the neurological pathology may, for example, be relative to the neurological pathology prior to use or administration of the compound. The neurological pathology may be treated by use or administration of the compound.

[0082] When the neurological pathology is seizures, such as epileptic seizures, reduction or treatment of the neurological pathology may, for example, refer to a reduction in the number of seizures experienced by a subject having the neurological pathology. The number of seizures may, for example, be expressed as the number of seizures within a given time frame, such as a day, week or month. Reduction or treatment of the neurological pathology may, for example, refer to a reduction in the frequency of seizures experienced by a subject having the neurological pathology. That is, seizures may occur less often following use or administration of the compound. Reduction or treatment of the neurological pathology may, for example, refer to a reduction in the duration of seizures experienced by a subject having the neurological pathology. That is, each seizure may occur for a shorter period of time following use or administration of the compound. Reduction or treatment of the neurological pathology may, for example, refer to a reduction in the severity of seizures experienced by a subject having the neurological pathology. For instance, seizures may become partial rather than generalised following use or administration of the compound.

[0083] The neurological pathology may be reduced by any mechanism that is elicited by the compound. For example, increasing lysosomal clearance may reduce the neurological pathology. Stimulating steroid hormone biosynthesis may reduce the neurological pathology. Modulating GABA receptors may reduce the neurological pathology. Agonistic effects on the sigma- 1 receptor may reduce the neurological pathology. Function of the compound or a metabolite or derivative thereof as a TRP channel ligand may reduce the neurological pathology.

[0084] Reducing a neurological pathology in accordance with the disclosure may have beneficial effects in a subject administered with the compound. In particular, reducing a neurological pathology may treat a lysosomal storage disorder. As set out above, treatment of a lysosomal storage disorder may refer to reducing or eliminating one or more clinical signs or symptoms of the disorder, such as seizures.

[0085] Reducing seizures

[0086] The disclosure provides a compound for use in reducing seizures, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The method may comprise administering the compound (for example an effective amount of the compound) to the subject. The disclosure also provides a method of reducing seizures comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. The disclosure further provides use of a compound in the manufacture of a medicament for use in reducing seizures, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. Use of the medicament may comprise administering the compound to the subject.

[0087] The seizures may be any type of seizure, but are preferably epileptic seizures. As set out above, an epileptic seizure may be defined as a seizure that starts in the brain. Other types of seizure include hypoglycaemic seizures, febrile convulsions, and seizures caused by heart problems. Reducing seizures may refer to improving or resolving the seizures. In this context, improving seizures may refer to reducing the number, frequency, duration or severity of seizures. Resolving seizures may refer to the prevention of future seizures. The improvement or resolution in seizures may, for example, be relative to the seizures prior to use or administration of the compound. The seizures may be treated by use or administration of the compound.

[0088] Reducing or treating seizures may, for example, comprise reducing the number of seizures experienced by a subject. The number of seizures may, for example, be expressed as the number of seizures within a given time frame, such as a day, week or month. Reducing or treating seizures may, for example, comprise reducing the frequency of seizures experienced by a subject. That is, seizures may occur less often following use or administration of the compound. Reducing or treating seizures may, for example, comprise reducing the duration of seizures experienced by a subject. That is, each seizure may occur for a shorter period of time following use or administration of the compound. Reducing or treating seizures may, for example, comprise reducing the severity of seizures experienced by a subject. For instance, seizures may become partial rather than generalised following use or administration of the compound.

[0089] The seizures may be reduced by any mechanism that is elicited by the compound. For example, increasing lysosomal clearance may reduce the seizures. Stimulating steroid hormone biosynthesis may reduce the seizures. Modulating GABA receptors may reduce the seizures. Agonistic effects on the sigma- 1 receptor may reduce seizures. Function of the compound or a metabolite or derivative thereof as a TRP channel ligand may reduce the seizures.

[0090] Reducing seizures in accordance with the disclosure may have beneficial effects in a subject administered with the compound. In particular, treatment of a lysosomal storage disorder may refer to reducing or eliminating one or more clinical signs or symptoms of the disorder, as set out above. Thus, reducing seizures may treat a lysosomal storage disorder.

[0091] Compound

[0092] As set out above, the compound is a steroid, a GABA receptor ligand, a Sigma-1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator. Accordingly, the compound may act in various ways to treat a lysosomal storage disorder, increase lysosomal clearance, reduce a neurological pathology and / or reduce seizures.

[0093] The compound may, for example, be able to stimulate steroid hormone synthesis. The steroid hormone synthesis pathway is known in the art, and shown in Figure 5. Provision of an upstream member of the steroid hormone synthesis pathway may, for example, result in increased production of a downstream steroid. The downstream steroid may, for example treat a lysosomal storage disorder, increase lysosomal clearance, reduce a neurological pathology and / or reduce seizures. Thus, the compound may be a member of the steroid hormone synthesis pathway, such as an upstream member. The compound may be a precursor to a member of the steroid synthesis pathway.

[0094] The compound may, for example, be able to activate a GABA receptor or to enhance the function of a GABA receptor. For instance, the compound may be a GABA receptor ligand, i.e. a molecule that interacts with (e.g. binds to) a GABA receptor to stimulate or enhance its function. GABA receptors are a class of receptors that respond to the neurotransmitter gamma-aminobutyric acid (GABA), which is the chief inhibitory compound in the mature vertebrate central nervous system. The GABA receptor may, for example, be a GABAA receptor. GABAA receptors are ligand-gated ion channels. Binding of ligand triggers opening of a chloride ion-selective pore. The increased chloride conductance drives the membrane potential towards the reversal potential of the Cl ion, inhibiting the firing of new action potentials. As set out in the Examples, steroids such as allopregnanolone, progesterone, androsterone and 11 -deoxycorticosterone are all known to enhance GABAA receptor function, eliciting anti-convulsant actions. The GABA receptor may, for example, be a GAB AB receptor. GAB AB receptors are G protein-coupled receptors, in which the ligand-binding part is linked by a G-protein to a potassium channel. Ligand binding stimulates the opening of the potassium channel, which brings the neuron closer to the equilibrium potential of potassium and thereby reduces the frequency of action potentials. Preferably, the GABA receptor is a GABAA receptor.

[0095] The compound may, for example, be able to activate a Sigma- 1 receptor. For instance, the compound may be a Sigma-1 receptor ligand, i.e. a molecule that interacts with (e.g. binds to) a Sigma- 1 receptor to stimulate its function. In other words, the compound may be a Sigma-1 receptor agonist. The sigma-1 receptor is a chaperone protein at the endoplasmic reticulum. This transmembrane protein is particularly concentrated in certain regions of the central nervous system, and has been implicated in several neurological disorders such as schizophrenia, clinical depression, and bipolar disorder. It modulates calcium signalling through the IP3 receptor and inhibits voltage gated potassium channels. The Sigma-1 receptor may be involved in preventing endoplasmic reticulum stress in neurodegenerative diseases, and has been implicated in autophagosome formation and maturation. Sigma-1 receptor is an anti-epileptic target. Pregnenolone (a steroid) and its downstream metabolite DHEA, as well as their sulfated forms, act as sigma-1 receptor agonists.

[0096] The compound may, for example, be able to modulate transient receptor potential channels (TRP channels). For instance, the compound may be a TRP channel ligand. A TRP channel ligand may be defined as a molecule that can interact (e.g. bind to) one or more TRP channels to exert a modulatory effect. TRP channels are a group of ion channels located mostly on the plasma membrane of animal cells. TRP channels modulate ion entry driving forces and Ca2+and Mg2+transport machinery in the plasma membrane. Many TRP channels have important roles in mediating sensations, such as pain, temperature, vision, pressure and taste. TRP channels have been implicated in hyperexcitability and seizures, and mutations in TRPs have been linked to neurodegenerative disorders.

[0097] The compound may, for example, be able to modulate microtubules. For instance, the compound may be a microtubule modulator. Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules are important for lysosome motility. In particular, lysosomes move along microtubules in a highly-orchestrated, retrograde and anterograde fashion to enable them to exert their functions. For instance, completion of the autophagic process requires the fusion of the autophagosome with the lysosome, which is facilitated through physical proximity of the organelles, slowing of vesicular trafficking and positioning of these degradative compartments in the vicinity of the nucleus in a microtubule-dependent manner. A microtubule modulator may be defined as a molecule that influences the assembly or function of microtubules. The function may, for instance, be microtubuledependent lysosome motility. For instance, a microtubule modulator may improve microtubule-dependent lysosome motility. A microtubule modulator may reduce microtubule-dependent lysosome motility. A microtubule modulator may speed up microtubule-dependent lysosome motility. A microtubule modulator may slow down microtubule-dependent lysosome motility. The microtubule modulator may exert its function directly on microtubules. Alternatively or additionally, the microtubule modulator may exert its function on a microtubule-associated molecule, such as a microtubule based motor protein or a factors (e.g. protein) that contributes to microtubule assembly.

[0098] The compound may, for example, be able to modulate a cannabinoid receptor, such as CBi or CB2. For instance, the compound may stimulate a cannabinoid receptor, such as CBi or CB2. The compound may, for example, be a cannabinoid receptor ligand, such as a cannabinoid. Well-known cannabinoids include tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN).

[0099] Further mechanisms are also possible. The compound may, for example, be able to reduce cell death, such as neurological cell death. An improvement in cell death is demonstrated in the Examples. The compound may, for example, regulate sub-cellular cholesterol levels, or modulate the cholesterol pathway. The cholesterol pathway is well- known in the art. The compound may, for example, modulate cellular respiration. For instance, the compound increase anaerobic respiration, for instance in a neurological cell. The compound may, for example, restore reduced levels of lactic acid to normal levels, for instance in a neurological cell.

[0100] Preferably, the compound does not achieve its effects on lysosomal storage disorders, lysosomal clearance, neurological pathology and / or seizures by actions on Transcription factor EB (TFEB). That is, the compound preferably has a TFEB- independent action. The compound is preferably not a TFEB activator. The compound preferably does not stimulate TFEB activity. TFEB is a master gene for lysosomal biogenesis. TFEB responds to nutrient cues at the surface of the lysosome to regulate lysosomal gene expression and autophagy, as explained in the Examples. Under aberrant lysosomal storage conditions, TFEB translocates from the cytoplasm to the nucleus, resulting in the activation of its target genes. TFEB-independence of a compound may be investigated by measuring nuclear translocation of TFEB, for instance using fluorescently- tagged TFEB in confocal microscopy as set out in the Examples. A TFEB -dependent compound will have an effect on nuclear translocation, but a TFEB-independent compound will not.

[0101] In a preferred aspect of the disclosure the compound is a steroid. The steroid may, for example, have the following formula (I), or be an isotope and / or pharmaceutically acceptable salt of a molecule having the formula: Formula (I) wherein ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6-membered aryl, wherein ring A is optionally substituted by one or more groups selected from halogen, hydroxyl, oxo, nitrile, sulfate, C1.3 alkyl and C1.3 alkoxyl, wherein said C1.3 alkyl and C1.3 alkoxyl are each optionally substituted with 1, 2 or 3 groups independently selected from OH and halogen, and / or wherein two adjacent carbons of ring A are joined by an -O- such that they form an epoxy group;

[0102] X is CH(R2) or C(O);

[0103] Y is C(C(O)R3)(R4), CH(R5) or C(O);

[0104] R1is unsubstituted C1.3 alkyl, unsubstituted C1.3 alcohol, OR6or C(O)R7;

[0105] R2is H, OR8or unsubstituted C1.3 alcohol;

[0106] R3is unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol;

[0107] R4is H, OR9or unsubstituted C1.3 alcohol;

[0108] R5is H, OR10or unsubstituted C1.3 alcohol; wherein each of R6, R7, R8, R9and R10is independently selected from H or unsubstituted C1.3 alkyl; wherein indicates a bond which may be a double bond or a single bond; and wherein each of ring B, C and D may be optionally substituted with one or more group selected from halogen, hydroxyl, nitrile, C1.3 alkyl and C1.3 alkoxyl, wherein said C1.3 alkyl and C1.3 alkoxyl are each optionally substituted with 1, 2 or 3 groups independently selected from OH and halogen; and / or wherein optionally two adjacent carbons of each of ring B, C and D are joined by an -O- such that they form an epoxy group.

[0109] Ring A is preferably a 6-membered saturated or unsaturated carbocyclic ring or a 6- membered aryl, wherein ring A is optionally substituted by one or more groups optionally selected from hydroxyl, oxo, and unsubstituted C1.3 alkyl.

[0110] Typically Ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6- membered aryl, wherein ring A is substituted with oxo or hydroxyl and is optionally substituted by unsubstituted C1.3 alkyl. For example, often Ring A is selected from a 6- membered saturated carbocyclic ring substituted by unsubstituted C1.3 alkyl and hydroxyl; a 6-membered mono-unsaturated carbocyclic ring substituted by unsubstituted C1.3 alkyl and oxo or hydroxyl; a 6-membered di-unsaturated carbocyclic ring substituted by C1.3 alkyl and oxo; or a 6-membered aryl ring substituted with hydroxyl.

[0111] Preferably ring A is selected from one of the following formulae:

[0112] Formula (Aa);

[0113] Formula (Ab); and

[0114] Formula (Ac); wherein R11is OR12or unsubstituted C1.3 alcohol, wherein R12is H or unsubstituted C1.3 alkyl.

[0115] More preferably, ring A is selected from Formula (Aa); and Formula (Ab); wherein R11is OR12and R12is H.

[0116] Most preferably Ring A is of formula: Formula (Aa); wherein R11is OR12, and wherein R12is H.

[0117] X is CH(R2) or C(O). Preferably X is CH(R2).

[0118] R2is H, OR8or unsubstituted C1.3 alcohol. Preferably R2is H or OR8.

[0119] R8is H or unsubstituted C1.3 alkyl. Preferably R8is H.

[0120] Thus typically R2is H or OH.

[0121] Preferably R2is H.

[0122] Preferably, therefore, X is CH(R2) or C(O), wherein R2is H or OR8, wherein R8is H. More preferably X is CH2 or CH(OH). X is most preferably CH2.

[0123] Y is C(C(O)R3)(R4), CH(R5) or C(O). Preferably Y is C(C(O)R3)(R4).

[0124] R3is unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol. Preferably R3is CH3 or

[0125] CH2OH.

[0126] R4is H, OR9or unsubstituted C1.3 alcohol. Preferably R4is H or OR9.

[0127] R9is H or unsubstituted C1.3 alkyl. Preferably R9is H.

[0128] Thus typically R4is H or OH.

[0129] Preferably, therefore, Y is C(C(O)R3)(R4), wherein R3is CH3 or CH2OH, and R4is H or OH. In some preferred embodiments Y is CH(C(O)Me). R5is H, OR10or unsubstituted C1.3 alcohol, wherein R10is H or unsubstituted C1.3 alkyl. Preferably R5is H or OR10.

[0130] R10is preferably H. Therefore R5is preferably H or OH.

[0131] R1is unsubstituted C1.3 alkyl, unsubstituted C1.3 alcohol, OR6or C(O)R7.

[0132] Preferably R1is CH3, CH2OH, or C(O)R7.

[0133] R6is H or unsubstituted C1.3 alkyl. Preferably R6is H.

[0134] R7is H or unsubstituted C1.3 alkyl. Preferably R7is H.

[0135] Thus, in some preferred embodiments R1is CH3, CH2OH, or C(O)H.

[0136] Most preferably R1is CH3.

[0137] Each of ring B, C and D may be optionally substituted with one or more group selected from halogen, hydroxyl, nitrile, C1.3 alkyl and C1.3 alkoxyl, wherein said C1.3 alkyl and C1.3 alkoxyl are each optionally substituted with 1, 2 or 3 groups independently selected from OH and halogen; and / or or wherein two adjacent carbons of each of ring B, C and D are joined by an -O- such that they form an epoxy group. Preferably Ring B, C and D are not further substituted.

[0138] In some embodiments, the steroid is compound of formula (I) or pharmaceutically acceptable salt thereof: Formula (I) wherein ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6-membered aryl, wherein ring A is optionally substituted by one or more groups selected from halogen, hydroxyl, oxo, C1.3 alkyl and C1.3 alkoxyl, wherein said C1.3 alkyl and C1.3 alkoxyl are each optionally substituted with 1, 2 or 3 groups independently selected from OH and halogen;

[0139] X is CH(R2) or C(O);

[0140] Y is C(C(O)R3)(R4), CH(R5) or C(O);

[0141] R1is unsubstituted C1.3 alkyl, unsubstituted C1.3 alcohol, OR6or C(O)R7; R2is H, OR8or unsubstituted C1.3 alcohol;

[0142] R3is unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol;

[0143] R4is H, OR9or unsubstituted C1.3 alcohol;

[0144] R5is H, OR10or unsubstituted C1.3 alcohol; wherein each of R6, R7, R8, R9and R10is independently selected from H or unsubstituted C1.3 alkyl; wherein indicates a bond which may be a double bond or a single bond; and wherein each of ring B, C and D may be optionally substituted with one or more group selected from halogen, hydroxyl, C1.3 alkyl optionally substituted with 1 to 3 halogen groups, and C1.3 alkoxyl optionally substituted with 1 to 3 halogen groups.

[0145] In some embodiments, the steroid is compound of formula (I) or a pharmaceutically acceptable salt thereof: Formula (I) wherein: ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6-membered aryl, wherein ring A is substituted with oxo or OR11and is optionally substituted by unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol;

[0146] X is CH(R2) or C(O);

[0147] Y is C(C(O)R3)(R4), CH(R5) or C(O);

[0148] R1is unsubstituted C1.3 alkyl, unsubstituted C1.3 alcohol, OR6or C(O)R7;

[0149] R2is H, OR8or unsubstituted C1.3 alcohol;

[0150] R3is unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol;

[0151] R4is H, OR9or unsubstituted C1.3 alcohol;

[0152] R5is H, OR10or unsubstituted C1.3 alcohol; each of R6, R7, R8, R9, R10, and R11is independently selected from H or unsubstituted C1.3 alkyl; indicates a bond which may be a double bond or a single bond; and each of rings B, C and D may be independently optionally further substituted with one or more group independently selected from halogen, hydroxyl, C1.3 alkyl and C1.3 alkoxyl, wherein said C1.3 alkyl and C1.3 alkoxyl are each optionally substituted with 1, 2 or 3 groups independently selected from OH and halogen. Preferably each of Rings B, C and D are not further substituted.

[0153] In some preferred embodiments:

[0154] Ring A is selected from a 6-membered saturated carbocyclic ring substituted by C1.3 alkyl and hydroxyl; a 6-membered mono-unsaturated carbocyclic ring substituted by C1.3 alkyl and oxo or hydroxyl; or a 6-membered di-unsaturated carbocyclic ring substituted by C1.3 alkyl and oxo;

[0155] X is CH(R2);

[0156] Y is C(C(O)R3)(R4);

[0157] R1is CH3, CH2OH, or C(O)R7;

[0158] R2is H or OR8;

[0159] R3is CH3or CH2OH;

[0160] R4is H or OR9;

[0161] R7is H;

[0162] R8is H;

[0163] R9is H; and

[0164] Rings B, C and D are not further substituted.

[0165] In some preferred embodiments: ring A is selected from one of the following formulae: Formula (Aa); Formula (Ab); g A is of formula: Formula (Aa);

[0166] X is CH2 or CH(OH), preferably wherein X is CH2; Y is CH(C(O)Me);

[0167] R1is CH3;

[0168] R11is OR12, and wherein R12is H.

[0169] Formula (I) may, for example, be further defined as formula (la): Formula (la) wherein ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6-membered aryl selected from one of the following formulae: Formula (Aa); Formula (Ab); and Formula (Ac);

[0170] X is CH(R2) or C(O);

[0171] Y is C(C(O)R3)(R4), CH(R5) or C(O);

[0172] R1is unsubstituted C1.3 alkyl, unsubstituted C1.3 alcohol, or C(O)R7;

[0173] R2is H or OR8;

[0174] R3is unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol;

[0175] R4is H or OR9;

[0176] R5is H or OR10;

[0177] R11is OR12or unsubstituted C1.3 alcohol; wherein each of R6, R7, R8, R9, R10and R12is independently selected from H or unsubstituted C1.3 alkyl; and wherein indicates a bond which may be a double bond or a single bond.

[0178] In Formula (la), the following may apply:

[0179] X is CH2, CH(OH) or C(O);

[0180] Y is C(O), or a group represented by

[0181] R1is CH3, CH2OH, or C(O)H;

[0182] R3is CH3or CH2OH;

[0183] R4is H or OH; R11is OH.

[0184] In some embodiments of formula (la) ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6-membered aryl selected from one of the following formulae:

[0185] Formula (Aa); and Formula (Ab);

[0186] X is CH2, CH(OH) or C(O);

[0187] Y is C(O), or a group represented by

[0188] R1is CH3, CH2OH, or C(O)H;

[0189] R3is CH3or CH2OH;

[0190] R4is H or OH;

[0191] R11is OH; and wherein indicates a bond which may be a double bond or a single bond.

[0192] In any case, the steroid may be a steroid of the steroid biosynthesis pathway. For instance, the steroid may be a steroid shown in Figure 5. The steroid may, for example, be selected from the group consisting of pregnenolone, progesterone, allopregnanolone, 11- deoxy-corticosterone, corticosterone, 18-OH-corticosterone, 17a-OH-pregnenolone, 17a- OH-progesterone, 21 -deoxycortisol, cortisol, 20a-DH-cortisol, cortisone, TH-aldosterone, l ip,21-DH-5P-pregnane-3, 20-dione, TH-corticosterone, aldosterone, 11 -deoxycorti sol, TH-l l-deoxycortisol, urocortisone, 5a-DH progesterone, 21-OH-pregnenolone, and 18- OH-cortisol. For instance, the steroid may be selected from (i) the group consisting of pregnenolone, progesterone, allopregnanolone, 11 -deoxy-corticosterone, corticosterone, 18-OH-corticosterone, 17a-OH-progesterone, TH aldosterone, cortisol, 20a-DH-cortisol, cortisone, and urocortisone; (ii) the group consisting of 18-OH-corticosterone, cortisol, cortisone, and 20a-DH-cortisol or (iii) the group consisting of pregnenolone, progesterone, and allopregnanolone. The steroid may, for instance be pregnenolone or allopregnanolone, or an analogue of pregnenolone or allopregnanolone. Allopregnanolone is a naturally occurring neurosteroid which is made in the body from the hormone progesterone. Furthermore, allopregnanolone, like pregnenolone, is a positive allosteric modulator of the action of GABA at GAB AA receptor. Beneficial effects demonstrated for pregnenolone may therefore be considered to extend to allopregnanolone.

[0193] The compound may, for example, be selected from the following group: Pregnenolone, Pregnenolone 16a-carbonitrile, Pregnenolone-d4-l, Pregnenolone monosulfate, Pregnenolone-d4, Pregnenolone monosulfate sodium, Pregnenolone monosulfate-d4 sodium, Pregnenolone-13C2,d2, Pregnenolone monosulfate sodium- 13C2,d2, MAP4343, Dehydroepiandrosterone sulfate sodium salt, MAP4343-d4, Trilostane, Progesterone, Progesterone (Standard), Progesterone-d9, Progesterone- 13 C3, Progesterone- 13 C5, Progesterone- 13 C2, Pregnanediol, PF-02413873, Dexamethasone oxetanone, Fadrozole hydrochloride hemihydrate, Pregnanediol 3 -glucuronide, ZK 216348, Norgestimate, (-)-ZK 216348, Glucocorticoid receptor agonist, Melengestrol acetate, Liquiritin, Asoprisnil, Estradiol dipropionate, Medroxyprogesterone, Medroxyprogesterone-d3, Nomegestrol, Pregnanediol-d5, Ulipristal, AKR1C1-IN-1, Isocupressic acid, 5a-Pregnane-3, 20-dione, Dy droge sterone, Medroxyprogesterone acetate, Promegestone, Dydrogesterone-D6, Fluorogestone acetate, Medroxyprogesterone acetate- d3, Mifepristone, Cridanimod, Tanaproget, Medroxyprogesterone acetate (Standard), Norgestrel, Nestoron, Norgestimate D6, Fadrozole hydrochloride, Mifepristone-d3, 5a- Pregnane-3p,6a-diol-20-one, Ulipristal acetate, Mifepristone (Standard), OP-3633, 5a- Pregnane-3,20-dione-d6, Mifepristone-13C,d3, PF-3882845, Ovalbumins, Megestrol acetate, AL-438, Ulipristal acetate-d6, Eplerenone-d3, (Rac)-Finerenone, Nomegestrol acetate, PF-998425, Dienogest, Megestrol acetate-d3, Megestrol acetate-d3-l, ARD-61, Trilostane, Melengestrol acetate-d6, Melengestrol acetate-d3, Finerenone, Melengestrol acetate-d2, (rac)-AG-205, Trimegestone, Cyclofenil, Deoxycorticosterone acetate, Deoxycorticosterone acetate (Standard), Pyrrhocoricin, Corticosterone, Corticosterone-d8, Corticosterone-d4, Acetyl-ACTH (7-24) (human, bovine, rat), RU28362, 21- Hydroxypregnenolone, 18-Hydroxycorticosterone, 18-Hydroxycorticosterone-d4, d[Cha4]- AVP TFA, d[Cha4]-AVP, Prosaikogenin F, Effusol, 1 ip-HSDl-IN-6, Darglitazone, Lutein, Osilodrostat phosphate, Osilodrostat, SR-31747, SR-31747 free base, Darglitazone Sodium, 21 -Deoxycortisol, 21-Deoxycortisol-d8, Pyrrhocoricin, Hydrocortisone, Cortisol sulfate sodium, Cortisol sulfate, Cortisol-21-glucuronide-d4 sodium, Cortisol-21-sulfate-d4 sodium salt, Hydrocortisone (Standard), Hydrocorti sone-d7, Hydrocorti sone-d3, Hydrocorti sone-d4, Hydrocorti sone-d2, Hydrocortisone phosphate, Hydrocortisone 17- butyrate, Hydrocortisone acetate, Hydrocortisone phosphate sodium, 6P-Hydroxy Cortisol- d4, Adrenocorticotropic hormone, Adrenocorticotropic hormone TFA, CYP11B1-IN-2, 18-Hydroxycortisol, BC11-38, 5P-Dihydrocortisol, ACTH (11-24) (acetate), ACTH (11- 24), Allotetrahydrocortisol, Allotetrahydrocortisol-d5, Cortodoxone, ACTH (1-14), ACTH (1-14) (TFA), Prednisolone hemi succinate, Cortodoxone-d2, Tetrahydrocortisol, Cortisone acetate, Tetrahydrocortisol-d5, BI-135585, 18-Oxocortisol, Cortisone, (Des-His6)-ACTH (1-24) (human, bovine, rat), (Des-Glu5)-ACTH (1-24) (human, bovine, rat), Cortisone-d7, Cortisone-d8, Cortisone-13C3, Cortisone-d2, 1 ip-HSDl-IN-6, 5P-Dihydrocortisol-d6, PF- 915275, ABT-384, Cortisone, Cortisone acetate, Cortisone-d8, Cortisone-d7, Cortisone- 13C3, Cortisone-d2, 5P-Dihydrocortisone, Tetrahydrocortisone, Tetrahydrocorti sone-d5, Tetrahydrocorti sone-d6, BI-135585, 1 lp-HSDl-IN-6, PF-915275, 11-Beta- hydroxyandrostenedione, 1 l-Beta-hydroxyandrostenedione-d7, ent-1 ip-Hydroxyatis-16- ene-3, 14-dione, 1 l-Beta-hydroxyandrostenedione-d4, 5a-Pregnan-l ip,17a,21-triol-3,20- dione-l,2,4,5-d4, 1 Ip-Hydroxyandrosterone, 1 lp-HSDl-IN-1, 1 lp-HSDl-IN-9, l lp- HSD1-IN-6, 1 Ip-Prostaglandin E2, 1 lp-HSDl-IN-8, 1 lp-HSDl-IN-7, 1 lp-HSDl-IN-11, l lp-HSDl-IN-12, 5a-Pregnane-3,20-dione-d6, 1 lp-HSDl-IN-10, l lbeta- Hydroxyprogesterone, 5a-Pregnane-3, 20-dione, 1 ip-Hydroxycedrelone, 1 lp-13,14- Dihydro-15-keto Prostaglandin F2a-d4, 9a,l ip-Prostaglandin F2a-d9, 2,3-Dinor-l ip- prostaglandin F2a-d9, Lup-20(29)-ene-3p,l lp-diol, 9a,l ip-Prostaglandin F2a, AZD8329, (4a,l lp,15p)-l l,15-Dihydroxykaur-16-en-19-oic acid, BVT-14225, AZD 4017, BMS- 823778 hydrochloride, Tetrahydrocortisol, BMS-770767, HSD-016, INCB 13739, Tetrahydrocortisol-d5, BMS-816336, SKI2852, Clofutriben, (R)-BMS-816336, AMG-221, BI-135585, ABT-384, (Rac)-BMS-816336, BVT 2733, AL-8810, Emodin, Necroptosis- IN-3, Emodin-d4, 18P-Glycyrrhetyl-3-O-sulfate, PF-915275, Carbenoxolone-d4, Carbenoxolone disodium, 11 -Ketodihydrotestosterone, Metyrapone, Thalidomide-NH- (CH2)3-NH-Boc, Metyrapone Tartrate, 1 l-Ketodihydrotestosterone-d3, Cycloheptane-1,3- dione, Cyclohexane- 1, 3-dione, 2-Methylcyclopentane-l, 3-dione, 5-(tert- Butyl)isobenzofuran-l,3-dione, (S)-3-Aminopiperidine-2,6-dione-dl hydrochloride, (R)-3- Aminopiperidine-2,6-dione-dl hydrochloride, (25R)-Spirost-4-ene-3, 12-dione, 6,7- Diaminoquinoxaline-2, 3-dione dihydrochloride, Androsta-1, 4, 6-triene-3, 17-dione, 1- Phenylpropane- 1,2-dione, Cyclo-(Pro-Gly), 5-Ethyl-5-phenylimidazolidine-2,4-dione-d5, 5,5-Dimethylcyclohexane-l,3-dione, (5a)-Stigmastane-3, 6-dione, 1 -Benzylpyrrol e-2, 5- dione, 5P-Cholanic acid 3,7-dione methyl ester, 2-(ethylamino)cyclohexa-2,5-diene-l,4- di one-d5 , 1 ,4-Dioxacycl oheptadecane-5 , 17 -di one, 5 a-Pregnane-3 p, 6a-diol -20-one, Periplocoside N, 5-(4-Hydroxyphenyl)-5-phenylimidazolidine-2,4-dione-d5,13C3, 2,2- Dimethyl-l,3-dioxane-4, 6-dione, Isatin, Trimethadione, Senecionine, Glaucoside A, SPA70, Pteridine-2,4(lH,3H)-dione, TCID, 1, 2, 4-Triazine-3, 5-dione 2-p-D- xylopyranoside, 21-Acetoxypregna-l,4,9(l l),16-tetraene-3, 20-dione, Qingyangshengenin, 1 ,2,6,7, 8,9-Hexahydro- 1 ,6,6-trimethyl-3 , 11 -dioxanaphth[2, 1 -e]azulene- 10,12-dione, 4, 10- Dioxatri cyclo[5.2. 1.02.6]dec-8-ene-3, 5-dione, rel-3a,4,7,7a-Tetrahydro-lH-isoindole- l,3(2H)-dione-d6, Forskolin, TTA-A2, 1,3 -Cyclopentanedi one, Deacylmetaplexigenin, 3- Hydroxyphthalic anhydride, 4-Fluorophthalic anhydride, Benzoylacetone, Diglycolic anhydride, 17a-Hydroxy pregnenolone, Aldosterone, Aldosterone-d7, Lorundrostat, Aldosterone-d4, Aldosterone-d8, Acetyl-ACTH (7-24) (human, bovine, rat), Dexfadrostat, Deoxycorticosterone acetate, Eplerenone, Metyrapone, Metyrapone Tartrate, Dicirenone, Osilodrostat phosphate, Osilodrostat, Baxdrostat, Canrenone, (S)-Baxdrostat, Deoxycorticosterone, Canrenone-d4, Canrenone-d6, Canrenoate potassium, Deoxycorticosterone-d8, BI 689648, Torsemide, Spironolactone, 18-Oxocortisol, Spironolactone-d3, Spironolactone-d7, Torsemide-d7, Spironolactone-d3-l, Deoxycorticosterone acetate (Standard), Deserpidine, Eplerenone-d3, Medetomidine hydrochloride, Medetomidine, CYP11B2-IN-1, Glucose-6-phosphate dehydrogenase, Cenderitide, Allisartan isoproxil, ML365, Angiotensin II human-13C6,15N TFA, Angiotensin II human, Angiotensin II human TFA, Angiotensin II human acetate, Angiotensin II human, 5-Valine-angiotensin I, Acetyl-ACTH (7-24) (human, bovine, rat), [D-Pro4,D-Trp7,9] Substance P (4-11), Cenderitide, Angiotensin II human TFA, Angiotensin II human acetate, Cortodoxone, Tetrahydro-11 -deoxycortisol, Cortodoxone- d5, Cortodoxone-d2, Pyrrhocoricin, Progesterone (Standard), Progesterone-d9, Progesterone, Progesterone- 13 C3, Progesterone-13C5, Progesterone- 13 C2, Pregnanediol, PF-02413873, Dexamethasone oxetanone, Fadrozole hydrochloride hemihydrate, Pregnanediol 3 -glucuronide, ZK 216348, Norgestimate, (-)-ZK 216348, Glucocorticoid receptor agonist, Melengestrol acetate, Liquiritin, Asoprisnil, Estradiol dipropionate, Medroxyprogesterone, Medroxyprogesterone-d3, Nomegestrol, Pregnanediol-d5, Ulipristal, AKR1C1-IN-1, Isocupressic acid, 5a-Pregnane-3, 20-dione, Dydrogesterone, Medroxyprogesterone acetate, Promegestone, Dydrogesterone-D6, Fluorogestone acetate, Medroxyprogesterone acetate-d3, Mifepristone, Cridanimod, Tanaproget, Medroxyprogesterone acetate (Standard), Norgestrel, Nestoron, Norgestimate D6, Fadrozole hydrochloride, Mifepristone-d3, 5a-Pregnane-3p,6a-diol-20-one, Ulipristal acetate, Mifepristone (Standard), OP-3633, 5a-Pregnane-3,20-dione-d6, Mifepristone- 13C,d3, PF-3882845, Ovalbumins, Megestrol acetate, AL-438, Ulipristal acetate-d6, Eplerenone-d3, (Rac)-Finerenone, Nomegestrol acetate, PF-998425, Dienogest, Megestrol acetate-d3, Megestrol acetate-d3-l, ARD-61, Trilostane, Melengestrol acetate-d6, Melengestrol acetate-d3, Finerenone, Melengestrol acetate-d2, (rac)-AG-205, Trimegestone, and Cyclofenil.

[0194] Subject

[0195] The compound may be administered to a subject to treat a lysosomal storage disorder, increase lysosomal clearance, reduce a neurological pathology and / or reduce seizures. Preferably, the subject is an animal. The animal may, for example, be a human or a non-human mammal. Exemplary non-human mammals include companion animals, such as cats, dogs, horses, rabbits and guinea pigs, and farm animals such as cattle, sheep and pigs.

[0196] The subject may be a juvenile. For instance, the subject may be a human juvenile or child. A child may, for example, be defined as a human of 0 to 18 years of age. Thus, the subject may be a human between 0 and 18 years old. For certain conditions, such as lysosomal storage disorders, it may be beneficial to implement treatment when the subject is as young as possible, to minimise the progression of disease and / or development of pathologies. Therefore, the subject may advantageously be between 0 and 10 years old, such as between 0 and 5 years old or between 0 and 2 years old.

[0197] Alternatively, the subject may be an adult. For humans, an adult may be defined as an individual of over 18 years of age

[0198] Administration

[0199] The compound may be administered by any suitable route. For instance, the route may be oral, intravenous, intramuscular, subcutaneous, intradermal, transdermal, intracerebral ventricular, or intraperitoneal.

[0200] The compound may, for example, be administered as part of a composition which further comprises a pharmaceutically acceptable excipient. The exact nature of the composition will depend upon several factors including the compound to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19thEdition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0201] Aspects of the disclosure

[0202] The disclosure provides:

[0203] 1. A compound for use in a method of treating a lysosomal storage disorder, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0204] 2. A compound for use in increasing lysosomal clearance, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0205] 3. A compound for use in reducing a neurological pathology, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0206] 4. A compound for use in reducing seizures, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0207] 5. A method of treating a lysosomal storage disorder comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0208] 6. A method of increasing lysosomal clearance comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0209] 7. A method of reducing a neurological pathology comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0210] 8. A method of reducing seizures comprising administering a compound to a subject, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0211] 9. Use of a compound in the manufacture of a medicament for use in treating one or more symptoms of a lysosomal storage disorder, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0212] 10. Use of a compound in the manufacture of a medicament for use in increasing lysosomal clearance, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0213] 11. Use of a compound in the manufacture of a medicament for use in reducing a neurological pathology, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0214] 12. Use of a compound in the manufacture of a medicament for use in reducing seizures, wherein the compound is a steroid, a GABA receptor ligand, a Sigma- 1 receptor ligand, a cannabinoid receptor ligand, a TRP channel ligand and / or a microtubule modulator.

[0215] 13. The compound for use, method, or use of any one of the preceding aspects, wherein the steroid is compound of formula (I) or an isotope and / or pharmaceutically acceptable salt thereof:

[0216] Formula (I) wherein ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6-membered aryl, wherein ring A is optionally substituted by one or more groups selected from halogen, hydroxyl, nitrile, oxo, sulfate, C1.3 alkyl and C1.3 alkoxyl, wherein said C1.3 alkyl and C1.3 alkoxyl are each optionally substituted with 1, 2 or 3 groups independently selected from OH and halogen, and / or wherein two adjacent carbons of ring A are joined by an -O- such that they form an epoxy group;

[0217] X is CH(R2) or C(O);

[0218] Y is C(C(O)R3)(R4), CH(R5) or C(O);

[0219] R1is unsubstituted C1.3 alkyl, unsubstituted C1.3 alcohol, OR6or C(O)R7;

[0220] R2is H, OR8or unsubstituted C1.3 alcohol;

[0221] R3is unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol;

[0222] R4is H, OR9or unsubstituted C1.3 alcohol;

[0223] R5is H, OR10or unsubstituted C1.3 alcohol; wherein each of R6, R7, R8, R9and R10is independently selected from H or unsubstituted C1.3 alkyl; wherein indicates a bond which may be a double bond or a single bond; and wherein each of ring B, C and D may be optionally substituted with one or more group selected from halogen, hydroxyl, nitrile, C1.3 alkyl optionally substituted with 1 to 3 halogen groups, and C1.3 alkoxyl optionally substituted with 1 to 3 halogen groups; and / or wherein optionally two adjacent carbons of each of ring B, C and D are joined by an -O- such that they form an epoxy group.

[0224] 14. The compound for use of aspect 13, wherein formula (I) is further defined as formula (la): wherein ring A is a 6-membered saturated or unsaturated carbocyclic ring or a 6-membered aryl selected from one of the following formulae:

[0225] Formula (Aa);

[0226] Formula (Ab); and

[0227] Formula (Ac);

[0228] X is CH(R2) or C(O);

[0229] Y is C(C(O)R3)(R4), CH(R5) or C(O);

[0230] R1is unsubstituted C1.3 alkyl, unsubstituted C1.3 alcohol, or C(O)R7; R2is H or OR8;

[0231] R3is unsubstituted C1.3 alkyl or unsubstituted C1.3 alcohol;

[0232] R4is H or OR9;

[0233] R5is H or OR10;

[0234] R11is OR12or unsubstituted C1.3 alcohol; wherein each of R6, R7, R8, R9, R10and R12is independently selected from H or unsubstituted C1.3 alkyl; and wherein indicates a bond which may be a double bond or a single bond.

[0235] 15. The compound for use of aspect 14, wherein

[0236] X is CH2, CH(OH) or C(O);

[0237] Y is C(O), or a group represented by

[0238] R1is CH3, CH2OH, or C(O)H;

[0239] R3is CH3or CH2OH;

[0240] R4is H or OH;

[0241] R11is OH.

[0242] 16. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is a steroid of the steroid biosynthesis pathway.

[0243] 16a. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is a steroid.

[0244] 17. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is a steroid selected from the group consisting of pregnenolone, progesterone, allopregnanolone, 11 -deoxy-corticosterone, corticosterone, 18-OH-corticosterone, 17a- OH-pregnenolone, 17a-OH-progesterone, 21 -deoxycortisol, cortisol, 20a-DH-cortisol, cortisone, TH-aldosterone, l ip,21-DH-5P-pregnane-3, 20-dione, TH-corticosterone, aldosterone, 11 -deoxycortisol, TH- 11 -deoxycortisol, urocortisone, 5 a-DH progesterone, 21-OH-pregnenolone, and 18-OH-cortisol.

[0245] 17a. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is a steroid selected from the group consisting of pregnenolone, progesterone, 11 -deoxy-corticosterone, corticosterone, 18-OH-corticosterone, 17a-0H- pregnenolone, 17a-OH-progesterone, 21 -deoxycortisol, cortisol, 20a-DH-cortisol, cortisone, TH-aldosterone, l ip,21-DH-5P-pregnane-3, 20-dione, TH-corticosterone, aldosterone, 11 -deoxycortisol, TH- 11 -deoxycortisol, urocortisone, 5 a-DH progesterone, 21-OH-pregnenolone, and 18-OH-cortisol.

[0246] 18. The compound for use, method, or use of aspect 17, wherein the compound is a steroid selected from the group consisting of pregnenolone, progesterone, allopregnanolone, 11- deoxy-corticosterone, corticosterone, 18-OH-corticosterone, 17a-OH-progesterone, TH aldosterone, cortisol, 20a-DH-cortisol, cortisone, and urocortisone.

[0247] 18a. The compound for use, method, or use of aspect 17, wherein the compound is a steroid selected from the group consisting of pregnenolone, progesterone, 11 -deoxycorticosterone, corticosterone, 18-OH-corticosterone, 17a-OH-progesterone, TH aldosterone, cortisol, 20a-DH-cortisol, cortisone, and urocortisone.

[0248] 18b. The compound for use, method, or use of aspect 18 or 18a, wherein the compound is a steroid selected from the group consisting of 18-OH-corticosterone, cortisol, cortisone, and 20a-DH-cortisol.

[0249] 19. The compound for use, method, or use of aspect 18, wherein the compound is a steroid selected from the group consisting of pregnenolone, progesterone, and allopregnanolone. 19a. The compound for use, method, or use of aspect 19, wherein the compound is pregnenolone or progesterone.

[0250] 20. The compound for use, method, or use of aspect 19, wherein the compound is pregnenolone or allopregnanolone.

[0251] 21. The compound for use, method, or use of aspect 20, wherein the compound is pregnenolone. 22. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is able to stimulate steroid hormone synthesis.

[0252] 23. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is a GABA receptor modulator.

[0253] 24. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is an agonist of the sigma- 1 receptor.

[0254] 25. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is a steroid.

[0255] 26. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is not a TFEB activator.

[0256] 27. The compound for use, method, or use of any one of aspects 1, 5, 9, or 13 to 26, wherein the lysosomal storage disorder is a neuronal ceroid lipofuscinoses disorder and / or Batten disease.

[0257] 28. The compound for use, method, or use of any one of aspects 1, 5, 9, or 13 to 27, wherein the lysosomal storage disorder is not CLN3 disease.

[0258] 29. The compound for use, method, or use of any one of aspects 1, 5, 9, or 13 to 28, wherein the lysosomal storage disorder is not CLN3 disease, CLN7 disease, or CLN5 disease.

[0259] 30. The compound for use, method, or use of any one of aspects 1, 5, 9, or 13 to 29, wherein the lysosomal storage disorder is CLN1 disease, CLN2 disease, CLN5 disease, CLN10 disease, or CLN13 disease.

[0260] 31. The compound for use, method, or use any one any one of aspects 1, 5, 9, or 13 to 30, wherein the lysosomal storage disorder is CLN1 disease, CLN2 disease, CLN10 disease, or CLN13 disease.

[0261] 32. The compound for use, method or use of any one of aspects 1, 5, 9, or 13 to 31, wherein the lysosomal storage disorder is CLN2 disease.

[0262] 33. The compound for use, method, or use of any one of aspects 1, 5, 9, or 13 to 32, wherein the lysosomal storage disorder is not a disorder associated with increased levels of gl ob otri ao sy 1 c erami de .

[0263] 34. The compound for use, method, or use of any one of aspects 1, 5, 9, or 13 to 33, wherein treating a lysosomal storage disorder comprises reducing seizures caused by the lysosomal storage disorder. 35. The compound for use, method, or use of any one of aspects 1, 5, 9, or 13 to 34, wherein treating a lysosomal storage disorder comprises improving lysosomal clearance.

[0264] 36. The compound for use, method, or use of any one of aspects 3, 7, 11, or 13 to 35, wherein the neurological pathology is seizures.

[0265] 37. The compound for use, method, or use of any one of aspects 4, 8, or 12 to 36, wherein the seizures are epileptic seizures.

[0266] 38. The compound for use, method, or use of any one of the preceding aspects, wherein the lysosomal storage disorder, the neurological pathology and / or the seizures are treated or reduced by increasing lysosomal clearance.

[0267] 39. The compound for use, method, or use of any one of the preceding aspects, wherein the lysosomal storage disorder, the neurological pathology and / or the seizures are treated or reduced by stimulating steroid hormone biosynthesis.

[0268] 40. The compound for use, method, or use of any one of the preceding aspects, wherein the compound is administered as part of a composition which further comprises a pharmaceutically acceptable excipient.

[0269] 41. The compound for use, or use of any one of aspects 1 to 4, or 8 to 40, wherein treating a lysosomal storage disorder, reducing a neurological pathology, or reducing seizures comprises administering the steroid to a subject.

[0270] 42. The compound for use, method, or use of any one of aspects 5 to 9, or 13 to 41, wherein the subject is an animal.

[0271] 43. The compound for use, method, or use of aspect 42, wherein the subject is a human.

[0272] 44. The compound for use, method, or use of aspect 42, wherein the subject is a human adult or a human child.

[0273] 45. The compound for use, method, or use of aspect 42, wherein the subject is a human between 0 and 18 years old.

[0274] EXAMPLES

[0275] Example 1

[0276] Overview A zebrafish model of CLN2 disease was used to perform a drug screen of 640 FDA-approved compounds. The zebrafish model harbours a premature stop codon mutation in exon 3 of the tppl gene, owing to a single T > A point mutation in the tpp Monape|etppl800011-1-’ herein referred to as tppl^'). The mutation results in deficiency of the Tppl enzyme, confirmed by Western blot and an enzyme activity assay. Various aspects of the human CLN2 disease are replicated in the tppl' ' zebrafish, including storage accumulation and enlarged lysosomes, motor defects and retinal and neuronal degeneration. These disease phenotypes can be observed from 2 days postfertilisation (dpf) and the zebrafish do not survive past 7 dpf . Another notable phenotype of tppl' ' zebrafish is a period of increased locomotion, indicative of seizures.

[0277] By using a combination of qualitative and quantitative phenotypes, a drug screen was performed. As set out in detail below, the drug screen identified the neurosteroid pregnenolone as a potential therapeutic for CLN2 disease. In particular, pregnenolone reduces duration of movement, distance and velocity of seizure-like locomotion activity in tppl'1' zebrafish, demonstrating a rescue of the phenotype. The anti-epileptic effect of pregnenolone was confirmed by electroencephalogram (EEG) measurement of epileptiform activity in the tppl'1' zebrafish. Live imaging of a novel transgenic zebrafish line expressing a Lampl lysosomal reporter showed that pregnenolone alleviates the lysosomal phenotype in tppl'1' zebrafish. Furthermore, pregnenolone treatment was shown to lead to a reduction in cell death in the CNS and enhance clearance of autophagic material.

[0278] Given that pregnenolone is a direct metabolite of cholesterol and the precursor of all steroids, the cholesterol-hormone pathway in tppl'1' zebrafish was investigated. Measurement of steroid hormone levels and whole genome RNA sequencing revealed dysregulation of hormone biosynthesis and related genes in tppl'1' zebrafish compared with their healthy (phenotypically wild-type) ‘WT’ siblings. This observation indicates perturbation in the cholesterol pathway in CLN2 disease pathogenesis, providing a target for therapeutic intervention.

[0279] Materials and methods

[0280] Generation and maintenance of zebrafish Adult zebrafish were maintained in a multi-rack aquarium system and kept on a constant 14 / 10 hr light / dark cycle at 27-29 °C and pH 6.0-7.5. Adult zebrafish were fed 2-3 times per day a diet of Hikari, krill and live brineshrimp. Where required, embryos were mechanically dechori onated with forceps. For Schedule 1 culling, adult and embryonic / larval zebrafish were exposed to 0.15 % 2-phenoxyethanol for 10 minutes or 24 hr respectively. Adult zebrafish were then decapitated and embryos / larvae were crushed.

[0281] Generation of tpplsa0011homozygotes and normal healthy siblings (WT)

[0282] Zebrafish carrying a T / A point mutation in the tpplsa00nallele were generated by N-ethyl-N-nitrosourea mutagenesis in the Tubingen strain and originally obtained from the Sanger Institute (Cambridge, UK: www.sanger.ac.uk / Projects / D_rerio / zmp / ) as an outcross to WT Tupfel long fin fish and subsequently maintained for 12 years by backcrossing to WT Tupfel long fin fish. Adult heterozygous carriers of the tppfa'mmutation (herein referred to as tppl+ / ') were identified by KASP genotyping. Briefly, this method involves allelic discrimination determined by qPCR reaction ratio of fluorescent cassettes, FAM and HEX, which correspond to T / A allele-specific primers developed by LGC Biosearch Technologies (https: / / biosearch-cdn.azureedge.net / assetsv6 / KASP-genotyping-chemistry- User-guide.pdf). Embryos for experiments were generated by in-cross of tppl+ / ' zebrafish and raised at 28 °C under standard husbandry conditions. Homozygous embryonic genotypes were assigned owing to the presence of a retinal phenotype on or after 2 dpf. Normal (WT) siblings were used as controls. Prior to any experiment, zebrafish embryos / larvae were viewed under a dissecting microscope to remove unfertilised embryos and to ensure they were healthy, or in the case of tppl'1', that they did not have any abnormal phenotypes beyond the expected ones (i.e. reduced retina size, small head, small and curved body, heart oedema, seizure-like locomotion). If any other phenotypes or injuries were observed in either WT or tppl'1' zebrafish, they were excluded from the study.

[0283] Generation o f transgenic zebra fish and confocal imaging

[0284] Generation of lines Transgenic zebrafish Tg(actclb:lampl-ZsGreen)ne,°8, Tg(actclb:lc3-ZsGreen)ne, 8,rg(actclb:tfeb-ZsGreen)riei<)Sand Tg(actclb:nls-mCherry)nei09were independently generated using I-SCEI meganuclease-mediated transgenic insertion into 1-cell stage embryos with tppl+!' background as previously described by Thermes et al. (2002). Individual founders were selected as ZsGreen-positive or mCherry-positive for propagation of each transgenic line. For ZsGreen-LC3 and ZsGreen-Lampl experiments, subsequent generations were crossed with non-transgenic tppl+!' and the progeny of the second generation were selected for fluorescence and used for imaging experiments. For ZsGreen-TFEB experiments, subsequent generations of Hg(actclb:tfeb- ZsGreeri)net08and Tg(actclb:nls-mCherry)ne'09were crossed to each other and the progeny were selected for both ZsGreen and mCherry fluorescence and used for imaging experiments.

[0285] Image acquisition and analysis

[0286] Dechori onated embryos, anaesthetised with 0.016 % tricaine (made in aquarium water, buffered with 4 % 1 M Tris pH 9 and adjusted to pH 7 with NaOH), were mounted laterally on 1.5mm glass coverslips with 1 % low-melting point agarose gel in aquarium water. Live imaging was conducted using a 40x water objective on either a Leica™ SP5 or SP8 confocal microscope.

[0287] ZsGreen -LC3 and ZsGreen-Lampl

[0288] Confocal images were analysed as maximum projections of 8-10 z-stack images using FIJI software. Binary images were generated by applying a threshold to highlight fluorescent signal and eliminate background; the threshold value was the mean fluorescent intensity of all fish in one experiment multiplied by 1.5 for ZsGreen-LC3 and 2 for ZsGreen-Lampl. Region of interest was defined manually using the freehand drawing tool or by applying a threshold to outline the total area of fish muscle. Measurements of ZsGreen-Lampl were derived using the ‘Analyse Particles’ feature on FIJI. ZsGreen-LC3 was measured as a total area of that identified by threshold. Area and puncta count measurements were normalised to the total area of fish muscle per image.

[0289] ZsGreen-TFEB and mCherry-nls Confocal images consisting of 6 z-stack images were acquired and analysed individually. Nuclei were identified by a binary image generated by applying a threshold to highlight fluorescent signal of mCherry-nls and eliminate background; the threshold value was kept consistent per experiment. Region of interest was identified by applying a threshold using the ZsGreen-TFEB channel to find the total area of fish muscle. ZsGreen- TFEB was measured as total raw fluorescent units over raw fluorescent units within the identified nuclei. Area and puncta count measurements were normalised to the total area of fish muscle per image.

[0290] Drug screen

[0291] At 48 hpf, dechorionated tppl~ ~ and WT siblings were placed one embryo per well of a flat bottomed 96 round well plate in 49.5 pl aquarium water as shown (Fig 1). Each plate contained one set of WT + DMSO, one set of tppl~ ~ + DMSO and the rest of the plates were sets of tppl~ ~ + each compound. 0.5 pl of DMSO or compound (Enzo Life Sciences FDA-approved library of known bioactives; 400 pg / ml in DMSO) was added to each well to a final compound concentration of 4 pg / ml. In Screen 1, two tppl~ ~ zebrafish were used per compound and plates were incubated until the fish were 120 hpf and then placed in the DanioVision chamber (Noldus) for a 20 minute recording. Outputs from Etho Vision tracking software (Noldus) for each fish were distanced moved, mean velocity, max velocity, time spent moving and number of movement bouts (start velocity 0.4 mm / s, stop velocity 0.2 mm / s). Fish were then qualitatively scored for morphology, and quantitatively survival and touch response before culling. In Screen 2, the number of tppl~ ’ zebrafish per compound was increased to three to improve reliability, and the assays were expanded to gain richer data and to include seizure-like locomotion which is obscured at later stages by loss of locomotion. Welfare was improved by reducing the age of zebrafish at the last assay time point. Plates were incubated at 28 °C until scored qualitatively for retinal size and quantitatively for survival at 54 hpf, 73 hpf and 79 hpf and then incubated at 22 °C until scoring for retinal size and survival at 97 hpf, and then culled. They were also assessed quantitatively for seizure-like locomotion using DanioVision for 1 hour (with temperature controlled to 25 °C and the light on) at 72 hpf (total distance moved, time spent moving and number of movement bouts; start velocity 4.5 mm / s, stop velocity 2 mm / s) and for loss of locomotion for 20 minutes at 28 °C at 96 hpf (total distance moved, time spent moving and number of movement bouts; start velocity 0.4 mm / s, stop velocity 0.2 mm / s). Compounds that appeared to improve at least one phenotype were re-tested on 10 animals per group (tppE ' + DMSO, tppl~ ~ + compound, WT + DMSO) using the same screen protocol but compound purchased from a new source. The compound that still had beneficial effects after re-screen (pregnenolone) was retested blind in two replicate experiments following the screen protocol except using approx. Ten zebrafish per group (tppl~ ~ + DMSO, tppl~ ~ + pregnenolone, WT + DMSO, WT + pregnenolone).

[0292] Chemical treatments

[0293] Embryos were dechorionated mechanically with forceps prior to any treatment. Pregnenolone (Preg, Sigma, P9129) treatment was performed at 2 dpf for 24-48 hr, as indicated. 0.4 mg / mL pregnenolone dissolved in DMSO was added to aquarium water for a final concentration of 4 pg / mL in 6 well plates with 4.5 mL total volume. 1 % DMSO was used as a control. D echori onated embryos were incubated in treatment for 24 hr at 28 °C. For 48 hr treatment, treatments were refreshed at 24 hr.

[0294] 3 -Methyladenine (3 -MA, Sigma, M9281) treatment was performed at 2 dpf for 24 hr, alongside or in combination with pregnenolone as above. 3-MA was dissolved in ddFFO with gentle heating for a stock concentration of 0.33 M, then added to aquarium water for a final concentration of 10 mM in 6 well plates with 4.5 mL total volume. 1 % DMSO + ddFFO was used as a control.

[0295] For LC3-ZsGreen flux assay, 500 pL 1 M NH4CI was added directly into 6 well plate containing 4.5 mL of 4 pg / mL pregnenolone (or DMSO control), for a final concentration of 100 mM. NH4CI was added in the last 4 hr of pregnenolone treatment prior to imaging. 10 % ddFFO was used as a control.

[0296] Electroencephalogram (EEG) measurements

[0297] The method used was adapted from Zdebik et al. (2013). Following pregnenolone treatment at 2 dpf, 3 dpf larvae were placed in 5mM of D-tubocurarine (Fluka) for 10 minutes, rinsed in aquarium water, and then stabilised in 1.8 % low-melting point agarose gel in aquarium water before placing an electrode onto the optic tectum of the midbrain. Electroencephalogram (EEG) measurements were then recorded for an average of 45 minutes using Axioscope software. Recordings were generated using Clampfit 10 and analysed using Origin 7 software to obtain the mean amplitude.

[0298] Immunoblotting

[0299] Dechori onated 4 dpf larvae maintained on ice, were deyolked by repeat pipetting in Phosphate Buffered Saline (PBS) with 0.1 mM EDTA through a p200 pipette. Samples were then homogenized in RIPA buffer (Sigma) containing lx protease and phosphatase inhibitors (ThermoSci entific), then centrifuged at 5,000 g for 15 minutes at 4 °C to collect the soluble fraction. Protein concentrations were quantified by BCA protein assay (ThermoSci entific) and equal concentrations of lysates were mixed with NuPAGE LDS Sample Buffer (4x) (Invitrogen) and NuPAGE Sample Reducing Agent (lOx) (Invitrogen), then heated at 70 °C for 10 minutes. Samples were resolved on 4-12 % NuPAGE BiS- Tris gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen), which were blocked with 5 % milk powder in TBS-Tween. The membrane was probed with primary antibodies (anti-HSC70 [SantaCruz, sc-7298]; anti-Tom20 [SantaCruz, sc-11415; anti-Subunit C of ATP Synthase (SCMAS) [Abeam, abl 10273], anti-P62 [Cell Signalling, 5114S]) overnight at 4 °C and secondary antibodies (anti-mouse / rabbit IgG, HRP-labelled [Perkin Elmer, NEF822001EA / NEF812001EA]) for 1 hr at room temperature, with washing in TBS-Tween in between. Following washing, blots were visualised by ECL kit (ThermoSci entific) as directed by manufacturer’s instructions.

[0300] TUNEL assay

[0301] Apoptosis was detected by the DeadEnd™ Fluorometric TUNEL system (Promega), performed based on principles described by Gavrieli et al. (1992) using a protocol developed from Promega (2009). Larvae were maintained in 0.003 % 1 -phenyl 2- thiourea (PTU) after 24 hpf. Following pregnenolone treatment larvae at 48 hpf, 72 hpf zebrafish were fixed in 4 % paraformaldehyde in PBS (PF A), and stored overnight at 4 °C, before storage in methanol at -20 °C. Larvae were rehydrated in PBT (50:50 % methanol / PBS solution containing 0.1 % Triton™ X-100 (Sigma-Aldrich)) prior to permeabilization with 10 pg / mL Proteinase K (Promega) in PBT for 60 minutes. Larvae were then washed in PBT, fixed for 20 minutes in 4 % PF A, then chilled for 10 minutes at -20 °C in a 2: 1 solution of ethanol and acetone. The larvae were then washed again in PBT before incubation in 100 pl Equilibration Buffer (Promega) on a rocker for 30 minutes, before incubation in TUNEL stain buffer (35 pL Equilibration Buffer, 10 pL Nucleotide Mix, 2 pL Terminal Deoxynucleotidyl Transferase, Recombinant enzyme (Promega) at 37 °C for 60 minutes inside a humified chamber, protected from light. The reaction was terminated by the addition of 500 pl of 2 x SSC (Promega) for 15 minutes, followed by washes in PBT, after which the larvae were cleared in 70 % glycerol / PBS solution. Larvae were mounted laterally on 1.5mm glass coverslips with 2 % low-melting point agarose gel in aquarium water. TUNEL staining was imaged using a Leica SP5 confocal microscope at lOx magnification. Fluorescent intensity was quantified using Volocity software version 6.3 (PerkinElmer). Mean fluorescent intensity was measured within a cuboid region of interest, located within the mesencephalon, identified as the region within the head of the larvae between the visually identifiable structures of the eye and otic capsule.

[0302] Seizure-like locomotion assay

[0303] Following dechorionation and 24 hr treatment with pregnenolone and / or 3MA, 3 dpf larvae were transferred to individual wells of a 96 square well plate in 200 pL. The plate was mounted in a DanioVision (Noldus) tracking system at 25 °C with the chamber light on and movement of the larvae was tracked for 20 minutes. Movement parameters of individual larvae were quantified using Etho Vision XT (Noldus) software. Zebrafish were assayed for seizure-like activity as in the drug screen.

[0304] RNA Sequencing

[0305] Following dechorionation and 24 hr pregnenolone treatment from 2 dpf, 3 dpf larvae were anaesthetised with 0.016 % tricaine (made in aquarium water, buffered with 4 % IM Tris pH 9 and adjusted to pH 7 with NaOH) and transferred to a 50 mL petri dish in a drop of water for dissection to separate heads and tails, and remove the yolk. The larvae were dissected with a 0.13 - 0.17 mm thick square glass coverslip. Two transverse cuts were made: 1, caudal to the hindbrain and rostral to the heart; 2, caudal to the yolk sac. Heads and tails were separated and all water was removed before flash freezing in liquid nitrogen. Six replicates of each condition were treated and processed in batches on the same day within a two-hour period.

[0306] Samples were disrupted and lysed using the FastPrep-24 speed 6 2 x 60” in 500 pL of kit lysis buffer. RNA was extracted from 400 pL of lysate and eluted in 50 pL. The RNA was then quantified using Quant-it RiboGreen assay (Life Technologies) and underwent quality control on a Fragment Analyzer.

[0307] Libraries were prepared with 150 ng of RNA input using the QuantSeq 3’ mRNA- Seq Library Prep Kit (FWD) HT for Illumina (Lexogen) and then quantified with Quant-it Picogreen (Life Technologies). Library sizes were controlled with the High Sensitivity NHS Fragment Analysis kit on a Fragment Analyzer (Agilent). RNA sequencing was performed on HiSeq 2500 with Rapid V2 chemistry SR 65 cycles (Illumina) loaded with 3 % Phix, using two Flow cells.

[0308] Raw count table was produced by HTseq-count and differential expression analysis was performed using Bioconductor R package edgeR (version 3.36). Gene set enrichment analysis was performed using CAMERA (limma Bioconductor R package, version 3.50.3) with custom gene sets (Tables 1 and 2).

[0309] Table 1: Lysosome gene set

[0310] Table 2: Cholesterol gene set

[0311] Hormone analysis in zebrafish larvae

[0312] Dechorionated 48 hpf zebrafish were treated in groups of 25 with DMSO or pregnenolone for 24 hr. A t 3 dpf, following treatment, two groups of samples were pooled so that 50 larvae were transferred to 1.5 mL cryotubes and all water was removed before flash freezing in liquid nitrogen. Samples were shipped on dry ice and further sample processing and hormone analysis using a Sciex QTRAP 6500+ mass spectrometry system was performed by The Metabolomics Innovation Centre, Canada. Hormone measurements were normalised to an average weight of 3 dpf WT or tppl~ ~ mutant larvae.

[0313] Statistical analysis

[0314] Data are expressed as box-and-whisker plot (minimum to maximum) or mean ± SD, as indicated. Statistical analysis for all data were performed using unpaired t-test or linear mixed model in GraphPad Prism 9 and SPSS respectively. Differences between groups were considered statistically significant when P < 0.05. Statistical significance is indicated in figures.

[0315] Results

[0316] As locomotion of zebrafish is easily and rapidly monitored, locomotion in tppl~ ~ zebrafish was used as the primary quantitative measure to screen 640 FDA-approved compounds. Locomotion assays were supplemented with other quantitative and qualitative assays (Fig 1 A, Table 3). Among the 640 screened compounds, 34 of these were identified as potential hits due to improvement in at least one assay, and re-tested using a new batch of compound. Among these, pregnenolone (from screen 2) was identified as the only hit due to apparent improvement in retinal size, seizure-like locomotion at 72 hpf, locomotion at 96 hpf and survival in 96 hpf. No other compounds showed a reduction in any phenotypes when re-tested. Blind testing validated the effect of pregnenolone on seizurelike locomotion, as demonstrated by a significant reduction in movement duration, distance and velocity in tppl'1' zebrafish (Fig 1 B,C).

[0317] The anti-seizure effect of pregnenolone on tppl'1' zebrafish was verified by electroencephalogram (EEG) analysis at 3 dpf after a 24hr treatment with 4 pg / ml pregnenolone (Fig 1 D, E). Typical seizure-like brain activity in tppl'1' zebrafish can be seen in Fig ID, showing single representative EEG recordings from tppl'1' zebrafish treated with DMSO or pregnenolone, alongside healthy WT siblings. Mean EEG amplitude from all measured tppl' ' zebrafish (n=6 DMSO treated, n=8 pregnenolone treated) show a significant reduction in EEG amplitude in those treated with pregnenolone compared with the DMSO control. These results support the locomotion assay results described above, showing an anti-seizure effect of pregnenolone in tppl'1' zebrafish.

[0318] To investigate whether cell death in tppl' ' zebrafish brains was altered by pregnenolone treatment, a TUNEL cell death assay was performed. This revealed a significant reduction of cell death in the mesencephalon of tppl'1' zebrafish treated with pregnenolone for 3 hours from 52 hpf, compared with DMSO (Fig 1 F, G). This finding suggests pregnenolone results in an improvement in pathology as well as seizures.

[0319] Table 3

[0320] Two or three tppl- / - zebrafish were treated with each drug from the FDA-approved drug library in one of two screens (Screen 1 and 2 respectively). Improvements in phenotypes were noted in several compounds from Screen 1 (A) and Screen 2 (B). Compounds in Tables A and B were re-sourced and re-tested using 10 tppl- / - zebrafish but otherwise the same protocol as before. Only one compound from Screen 2 (pregnenolone) still resulted in phenotypic improvements (C). Performing the assays blinded in replicate using 10 tppl- / - zebrafish per replicate revealed that pregnenolone only significantly improved seizure like locomotion at 76 hpf (D and Figure 1).

[0321] A. Summary of results from Screen 1

[0322] B. Summary of results from Screen 2 results.

[0323] C. Summary of results from re-testing compounds that cause phenotypic improvement in

[0324] Screens 1 and 2. D. Summary of results from blind testing of pregnenolone

[0325] Pregnenolone ameliorates lysosomal structure and function in tppl~ ~ zebrafish Pregnenolone is an endogenous neurosteroid, a direct metabolite of cholesterol and the precursor of all steroid hormones. Pregnenolone is also an agonist of the sigma-1 receptor, which binds and regulates cholesterol levels within subcellular compartments, especially in lipid rafts. Pregnenolone was recently identified in another screen for compounds improving lysosomal storage in CLN3 disease, also known as juvenile Neuronal Ceroid Lipofuscinosis: in ARPE-19 CLN3 knockout cells pregnenolone was shown to reduce storage accumulation in the lysosomes. To investigate whether pregnenolone has a therapeutic effect on the cell pathology of tppP1' zebrafish, transgenic zebrafish with ZsGreen-tagged lysosomal membrane protein Lampl under the muscle-specific actclb promoter in the background of tppP1' zebrafish were generated. The muscle reporter provides a large area of homogenous tissue allowing for accurate high-throughput measurement of the number and average size of lysosomes in live zebrafish, which would be more complicated in brain tissue owing to its smaller size and vast differences in lysosomal function between different neuronal cell populations. Quantification of confocal images show a strong lysosomal phenotype in the muscle of tppl'1' zebrafish; the average size of lysosomes is significantly increased and the number of lysosomes is significantly decreased compared to healthy WT siblings (Fig 2 A, B). Both these parameters were significantly improved in tppl'1' zebrafish treated with pregnenolone for 24 hr, compared to the DMSO control. Interestingly, pregnenolone had the same effect on the lysosomes of WT zebrafish, namely a significant decrease in average lysosome size and significant increase in lysosome number (Fig 2 A, B).

[0326] Autophagy is a key function of the lysosome and a process that has been shown to be impaired in various LSDs, including CLN2 disease. The neuroprotective effect of autophagy induction and the inverse detriment caused by defects in autophagy have been widely demonstrated in the literature among a multitude of neurodegenerative disease. Given the observed decrease in lysosomal size with pregnenolone treatment, P62 (an autophagic adaptor protein) was measured to test whether pregnenolone treatment enhances clearance of autophagic material in the lysosome, leading to the observed reduction in lysosomal size. Western blot analysis of de-yolked zebrafish tissue revealed a significant accumulation of P62 in tppl'1' zebrafish compared to WT siblings at 4 dpf (treated with DMSO), which was significantly reduced by 48 hr pregnenolone treatment (Fig 2C). Western blot analysis showed pregnenolone had no effect on the accumulation of SCMAS, the major component of storage material in CLN2 disease (Fig 5 A). RNA sequencing analysis of p62 shows that pregnenolone does not alter mRNA quantity at 3 dpf following 24 hr treatment, suggesting the reduction in P62 protein accumulation is indicative of enhanced clearance in tppl'1' zebrafish (Fig 8A). Similarly, mRNA quantity of three different SCMAS loci, atp5mcl. atp5mc3a and atp5mc3b, is also unaffected by pregnenolone treatment (Fig 8B), indicating that the lack of change in SCMAS protein represents no change in clearance.

[0327] Reduction in the accumulation of P62 protein in tppl'1' zebrafish treated with pregnenolone compared with DMSO suggests enhanced autophagic clearance. To test this hypothesis further, transgenic zebrafish with ZsGreen-tagged autophagosome marker LC3, again under the muscle-specific actclb promoter in the background of tppl'1' zebrafish, were generated. Autophagic flux was measured in zebrafish by live imaging of ZsGreen- LC3 with and without a 4 hr treatment of NH4CI to block lysosomal degradation. At 3 dpf, tppl' ' zebrafish did not yet show altered ZsGreen-LC3 flux compared with WT. However, preliminary results from one experiment showed that both WT and tppl'1' zebrafish revealed a significant accumulation of ZsGreen-LC3 following 24 hr pregnenolone treatment in the presence of NH4CI, compared to the DMSO + NH4CI control (Fig 9 A, B). These results, in combination with the reduction of P62 protein accumulation, suggest pregnenolone treatment induces autophagy in both WT and tppl' ' zebrafish.

[0328] To test whether the induction of autophagy by pregnenolone is responsible for the alleviation of seizures in tppl'1' zebrafish, a locomotion assay was performed on the zebrafish with pregnenolone treatment in combination with autophagy induction inhibitor, 3-MA. Results from 3 dpf zebrafish following 24 hr concurrent treatment of pregnenolone and 3-MA show duration of movement is comparable to pregnenolone treatment only (Fig 2 D, E). This finding therefore suggests that induction of autophagy is not the mechanism of action of pregnenolone’s anti-seizure effect.

[0329] Pregnenolone acts via an mTORC 1 / TFEB-independent mechanism

[0330] Lysosomal biogenesis and function are largely regulated by mammalian target of rapamycin 1 (mTORCl) and transcription factor EB (TFEB), which respond to nutrient cues at the surface of the lysosome to regulate lysosomal gene expression and autophagy. Given the effect on lysosomes in both WT and tppl'1' zebrafish, experiments were undertaken to determine whether pregnenolone treatment acts via an mTORC 1 / TFEB- dependent mechanism. Western blot analysis of mTORCl metabolite S6, revealed significantly increased phosphorylation of S6 in tppl'1' zebrafish compared with WT. However, pregnenolone had no effect on this result (Fig 3 A). Furthermore, measurement of nuclear translocation of ZsGreen-TFEB using 3 dpf ZsGreen-TFEB ;mCherry-nls transgenic zebrafish, as has been published previously, suggested suppression of TFEB nuclear translocation (Fig 3 B, C). 24 hr treatment with pregnenolone showed no effect in either WT or tppl'1' zebrafish. These results highlight mTORCl / TFEB signaling abnormalities in tppl'1' zebrafish, but indicate that pregnenolone functions independently of this pathway. Enrichment analysis of a lysosomal gene set (Table 1) supported these results, showing upregulation of lysosomal genes in tppl'1' zebrafish compared with WT, which was not further altered by pregnenolone treatment (Fig 3 D, E). Perturbance in cholesterol and hormone metabolism in tppP ' zebrafish

[0331] Pregnenolone is a direct metabolite of cholesterol; the conversion of cholesterol to pregnenolone precedes the production of all steroid hormones and is a rate-limiting step in steroidogenesis. In this regard, the endogenous concentrations of pregnenolone and a number of downstream steroid hormones were analysed in WT and tppP1' zebrafish, and the effect of pregnenolone treatment on these assessed. Hormone analysis by mass spectrometry was conducted in 3 dpf zebrafish following 24 hr treatment with pregnenolone or DMSO control. Results showed dysregulation of steroid hormones in tppP1' zebrafish compared with WT siblings in control conditions (Fig 4A and Fig 10), including significantly higher levels of pregnenolone (Fig 4B). Four out of the twenty-two measured hormones - 18-OH-corticosterone, cortisol, cortisone and 20a-DH-cortisol - were decreased in tppP1' zebrafish compared with WT siblings under DMSO conditions, and were rescued by pregnenolone treatment (Fig 4B and Fig 11). Seven steroids were significantly increased in tppP1' zebrafish compared with WT siblings: and eleven were unchanged (Supplementary Fig 10). The changes in steroid hormone levels are summarised in Fig 5.

[0332] To further investigate the cholesterol and steroid pathway, enrichment analysis of a custom set of genes related to this pathway was performed (Table 2). The RNA Seq data from the zebrafish revealed significant enrichment of the cholesterol and steroid pathway in tppP1' zebrafish compared with WT siblings in both head and tail tissue (Fig 6A). Pregnenolone treatment stimulated this pathway in WT zebrafish (Fig 6B) and lead to further enrichment of the pathway in tppP1' zebrafish (Fig 6C).

[0333] Looking at individual genes involved in the various steps of cholesterol metabolism and hormone biosynthesis, tppP1' zebrafish show significant changes in several major genes with diverse roles (Fig 11, Tables 4 and 5). These changes are summarised in Fig 7, which highlights the diverse functions within the cholesterol metabolism pathway of the proteins encoded by the mRNAs with altered expression in tppP1' zebrafish and tppP1' zebrafish treated with pregnenolone. Three genes are of particular interest because their mRNA levels are increased in tppP1' but brought nearer to normal levels by treatment with pregnenolone. These are leal. stard3 and npc2. encoding Lecithin-cholesterol acyltransferase (LCAT), StAR-Related Lipid Transfer Domain Containing 3 (StARD3) and Niemann-Pick C2 (NPC2). StARD3 resides on the late endosome / lysosome membrane and NPC2 is an internal late endosome / lysosome protein, but both are both important for the transfer of cholesterol from late endosomes / lysosomes to other subcellular membrane compartments such as the plasma membrane, ER and mitochondria. LCAT, however, is found in the ER where it converts free cholesterol to cholesterol esters for storage in lipid droplets.

[0334] Table 4: Top 25 differentially expressed genes (DEG) in the cholesterol gene set in the head tissue of tppl~!' mutant zebrafish compared with WT siblings. Upregulated genes are underlined and downregulated genes are not underlined. Genes are ranked by their F value, showing most DEG in decreasing order from top to bottom. *Also in top 25 DEG in tail tissue of tppl’1' mutant zebrafish compared with WT siblings (Table 5).

[0335] Table 5: Top 25 differentially expressed genes (DEG) in the cholesterol gene set in the tail tissue of tppl ' mutant zebrafish compared with WT siblings. Upregulated genes are underlined and downregulated genes are not underlined. Genes are ranked by their F value, showing most DEG in decreasing order from top to bottom. *Also in top 25 DEG in head tissue of tppl’1' mutant zebrafish compared with WT siblings (Table 4).

[0336] Discussion

[0337] Seizures are a major symptom of CLN2 disease that worsen with disease progression and present a challenge for management of the condition as they often become resistant to anti-epileptic drugs. The present Example demonstrates the use of the seizurelike locomotion assay in the tppl'1' zebrafish model of CLN2 disease to identify potential candidate compounds to alleviate seizures. The screen described herein identified pregnenolone as a hit compound, shown here to elicit an anti-seizure effect in tppl'1' zebrafish. Furthermore, reduced cell death in the CNS of tppl'1' zebrafish treated with pregnenolone reveal a neuroprotective effect. The results here show a partial rescue of lysosomal impairment in tppl'1' zebrafish treated with pregnenolone. This is evident from improved morphology of the lysosome in muscle cells of the zebrafish and a decrease in P62 accumulation following treatment of pregnenolone. These results present pregnenolone as a powerful candidate for further testing as a novel therapeutic for CLN2 disease. Pregnenolone is an FDA-approved compound and has been shown to be safely administered in adults for other conditions, and so could be safely administered to children to treat Batten disease.

[0338] In line with the results presented herein, pregnenolone was recently identified in another screen, in ARPE-19 CLN3 knockout cells, where it was shown to reduce storage accumulation in the lysosomes. The preliminary LC3 flux results provided herein suggest storage reduction is associated with induction of autophagy by pregnenolone. Although reduction in SCMAS protein was not observed with pregnenolone treatment in the tppl'1' zebrafish, the decrease in lysosomal size and reduction of P62 protein accumulation suggest an overall reduction in storage accumulation. SCMAS is believed to be a direct substrate of TPPl / Tppl and Tppl is almost entirely absent in tppl'1' zebrafish; thus, the persistent accumulation of SCMAS despite improvements in lysosomal function is unsurprising.

[0339] Autophagy induction has been shown to have significant therapeutic effects in other models of LSDs caused by specific enzyme deficiency. In a. Drosophila model of Gaucher disease lacking the glucocerebrosidase enzyme, for example, induction of autophagy by means of mTORCl inhibition by rapamycin treatment was shown to significantly increase lifespan and improve locomotor and oxidative stress phenotypes. Extended lifespan and phenotypic improvement by autophagy induction in the Gaucher disease model indicates a sustained therapeutic effect that can lead to significant improvements in disease outcome. Despite these benefits associated with autophagy induction, inhibiting autophagy with 3-MA in tppl'1' zebrafish here did not affect pregnenolone’s anti-seizure effect, indicating that autophagy is unlikely to be the mechanism of action responsible for this particular therapeutic effect.

[0340] Pregnenolone might act via other mechanisms to induce the anti-seizure effect. Potential mechanisms include modulation of GABAA receptors by downstream metabolites of pregnenolone and / or agonistic effects on the sigma-1 receptor, which has been shown to elicit neuroprotective effects. Neurosteroids are known for their role in modulation of neuronal excitability which has important implications in seizure susceptibility and control. Allopregnanolone, progesterone, androsterone and 11 -deoxycorticosterone are all known to enhance GABAA receptor function, eliciting anti-convulsant actions. Several studies have demonstrated the anti-seizure effect of allopregnanolone, and Ganaxolone, an analogue of allopregnanolone, has shown promising results in clinical trials for a number of epilepsies. These findings present an alternative mechanism for the seizure rescue observed in tppl'1' zebrafish treated with pregnenolone, as raised pregnenolone levels might lead to increases in anti-convulsant hormones. Consistent with this hypothesis, an increase in the concentration of a number of hormones was seen following pregnenolone treatment, including anti-convulsant steroids, progesterone and 11 -deoxycorticosterone. GABAA activation has also been shown to have neuroprotective effects, thus, action through GABAA signalling could also be responsible for the attenuation of cell death in tppl'1' zebrafish treated with pregnenolone. Pregnenolone and its downstream metabolite DHEA, as well as their sulfated forms, act as sigma-1 receptor agonists. The sigma-1 receptor is an anti-epileptic target. Positive modulators of the receptor, fenfluramine and E1R, have been shown to elicit anticonvulsant effects. Fenfluramine has demonstrated substantial efficacy in reducing seizures in a phase 3 clinical trial for patients with Dravet syndrome, a refractory paediatric epilepsy. This finding suggests that actions through the sigma-1 receptor could therefore underlie the anti-seizure effect in tppl'1' zebrafish treated with pregnenolone.

[0341] The sigma-1 receptor is widely expressed in different tissues, including brain and muscle. It binds cholesterol and regulates a variety of protein binding partners in a variety of membranes. Sigma-1 receptor localisation to mitochondrial -associated membrane (MAM) microdomains of the ER is most studied. Here it regulates calcium flux into the mitochondria via the inositol trisphosphate receptor 3 (InsP3R3) calcium channel. Calcium regulation is involved in various cell death pathways and sigma-1 agonists have been shown to attenuate neuronal death in a number of models of neurodegenerative diseases. In a cell model of Huntington’s disease for example, the sigma-1 receptor agonist PRE-084 (2-(4-morpholinoethyl)- 1 -phenyl cyclohexane 1 -carboxylate hydrochloride) increased antioxidants and reduced reactive oxygen species (ROS). In line with a role in regulating oxidative stress, knockdown of the sigma- 1 receptor has been shown to increase ROS production, likely through dysregulation of calcium homeostasis. As increased levels of ROS have been documented in CLN2 disease patient cells, and oxidative stress can lead to cell death, pregnenolone might ameliorate cell death in tppl'1' zebrafish by alleviating oxidative stress through its action on the sigma-1 receptor. At the plasma membrane, the sigma- 1 receptor decreases voltage-gated sodium and calcium channel activity whilst increasing activity of some voltage-gated potassium channels, thereby limiting exci totoxi city. This relationship provides an additional possible mechanism of action of pregnenolone in limiting both seizures and cell death in tppl' ' zebrafish.

[0342] Identification of pregnenolone as a hit compound led to investigation of the cholesterol and steroid hormone pathway in the zebrafish model described herein because 1) pregnenolone is a steroid that is synthesised from cholesterol in the mitochondria, and 2) cholesterol is bound by the sigma-1 receptor, for which pregnenolone is an agonist. This investigation led to findings of dysregulation throughout the cholesterol and steroid hormone pathways that have not been previously noted in CLN2 disease. Increased endogenous pregnenolone in tppl~ ~ zebrafish was found. As pregnenolone reduces seizures in those zebrafish, pregnenolone is the precursor of many steroids, and pregnenolone and those steroids can have anti-epileptic effects, this increase in endogenous pregnenolone could be a compensatory mechanism. On the other hand, given that pregnenolone is a metabolite of cholesterol, elevated levels of endogenous pregnenolone in tppl~ ~ zebrafish could result in depleted cholesterol stores, and pregnenolone treatment may reduce cholesterol depletion in addition to negating the need for the proposed compensatory increase in pregnenolone.

[0343] Upregulation of npc2. stard3 and Icat mRNAs and their restoration to normal mRNA levels by pregnenolone treatment was also observed in tppl~ ~ zebrafish. This indicates that cholesterol handling is affected. Cholesterol is crucial for membrane organisation, neuronal differentiation and myelination. Furthermore, cholesterol is important for the integrity of the lysosomal membrane and modulates ion permeability. Disruption to the cholesterol pathway could contribute to CLN2 disease pathology via disturbance of its function in any one of these roles, highlighting cholesterol abnormality as an important area for further research in tppl~ ' zebrafish and other models of CLN2 disease.

[0344] Pregnenolone is further known to be involved in a number of other pathways. For instance, pregnenolone synthesis may be stimulated by a cannabinoid or a cannabinoid receptor ligand. Cannabinoids and cannabinoid receptor ligands are known in the art to be good anti-epileptics for drug-resistant seizures. In addition, pregnenolone may act as a TRP channel ligand. TRP channels are implicated in hyperexcitability and seizures. Pregnenolone is also known to regulate microtubule dynamics, which are important for lysosome motility. Thus, cannabinoid receptor ligands, TRP channel ligand and microtubule modulators are expected to have similar benefits to those demonstrated herein for pregnenolone.

[0345] In summary, the present Example employed a zebrafish model of CLN2 disease to perform a drug screen which identified pregnenolone as a potential therapeutic for the disease. Pregnenolone was shown to reduce seizures in the model and improve cell pathology, including improvement in lysosome morphology and function and reduced cell death. The safety profile of pregnenolone make it an appealing candidate for further testing into the use of it as a therapeutic for CLN2 disease and other NCLs. The novel Lampl-ZsGreen transgenic zebrafish presented here revealed a striking phenotype in tppl' ’ zebrafish compared to healthy WT siblings. This transgenic line provides a powerful tool to investigate potential therapeutics that can improve lysosomal biology in vivo, as demonstrated by the effect of pregnenolone. The use of the actlb muscle promoter provides a substantial area of tissue that can be automatically detected and imaged using a high-content confocal imaging system. These lines can therefore be used as the basis of a screen similar to the one presented in the current study, through automated imaging in a 96 well format.

[0346] Example 2

[0347] Principal component analysis (PCA) of metabolites was used for metabolomic analysis of tppl' ' zebrafish and normal controls. Administration of pregnenolone (RVC1) was demonstrated to cause a shift in normal and tppl' ' zebrafish (Fig. 12A). OPLS-DA modelling and univariate analysis indicated 6 / 61 metabolites that approach normal when tppl- / - zebrafish are treated with pregnenolone (Fig 12. B). Notably, tppl' ' zebrafish have reduced glucose-6-phosphate and fructose-6-phosphate suggesting reduced glycolysis and reduced generation of pyruvate. tppl' ' zebrafish also have reduced lactic acid, which is formed from anaerobic respiration of pyruvate. Administration of pregnenolone increase lactic acid as well as threonine in tppl' ' zebrafish, such that they approach normal levels. Accordingly, pregnenolone appears to increase anaerobic respiration in tppl' ' zebrafish. This may contribute to pregnenolone’s anti-seizure effect, especially given that glucose transporter type 1 (GLUT1) is needed to get glucose required for aerobic respiration into the brain, and deficiency causes seizures.

Claims

CLAIMS1. A compound for use in a method of treating a lysosomal storage disorder, wherein the compound is pregnenolone.

2. A compound for use in increasing lysosomal clearance, wherein the compound is pregnenolone.

3. A compound for use in reducing a neurological pathology, wherein the compound is pregnenolone.

4. A compound for use in reducing seizures, wherein the compound is pregnenolone.

5. The compound for use of claim 1, wherein the lysosomal storage disorder is a neuronal ceroid lipofuscinoses disorder and / or Batten disease.

6. The compound for use of claim 1 or claim 5, wherein:(i) the lysosomal storage disorder is not CLN3 disease;(ii) the lysosomal storage disorder is not CLN3 disease, CLN7 disease, or CLN5 disease;(iii) the lysosomal storage disorder is CLN1 disease, CLN2 disease, CLN5 disease, CLN10 disease, or CLN13 disease;(iv) the lysosomal storage disorder is CLN1 disease, CLN2 disease, CLN10 disease, or CLN13 disease; or(v) the lysosomal storage disorder is CLN2 disease.

7. The compound for use of any one of claims 1 or 5 to 6, wherein the lysosomal storage disorder is not a disorder associated with increased levels of globotriaosylceramide.

8. The compound for use of any one of claims 1 or 5 to 7, wherein treating a lysosomal storage disorder comprises reducing seizures caused by the lysosomal storage disorder.

9. The compound for use of any one of claims 1 or 5 to 8, wherein treating a lysosomal storage disorder comprises improving lysosomal clearance.

10. The compound for use of claim 3, wherein the neurological pathology is seizures.

11. The compound for use of claim 4, wherein the seizures are epileptic seizures.

12. The compound for use of any one of the preceding claims, wherein the lysosomal storage disorder, the neurological pathology and / or the seizures are treated or reduced by increasing lysosomal clearance.

13. The compound for use of any one of the preceding claims, wherein the lysosomal storage disorder, the neurological pathology and / or the seizures are treated or reduced by stimulating steroid hormone biosynthesis.

14. The compound for use of any one of the preceding claims, wherein the compound is administered as part of a composition which further comprises a pharmaceutically acceptable excipient.

15. The compound for use of any one of the preceding claims, wherein treating a lysosomal storage disorder, increasing lysosomal clearance, reducing a neurological pathology, or reducing seizures comprises administering the compound to a subject.

16. The compound for use of claim 15, wherein the subject is an animal, a human, a human adult or a human child; optionally wherein the subject is a human between 0 and 18 years old.

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