Mutated TFEB for treating lysosomal disorders

Abstract

The present invention relates to a mutated transcription factor EB (TFEB) protein that loses the native exon 3. This protein is referred to herein as "TFEB-ex3" or "ex3-TFEB". The invention also relates to polynucleotides encoding such muteins; a vector comprising the polynucleotide; and a biomolecular means for removing TFEB exon 3 in a patient in need thereof. The invention also relates to a pharmaceutical composition comprising the above, for use in the treatment and / or prevention of lysosomal storage diseases and conditions characterized by lysosomal dysfunction.

Description

Technical Field

[0001] This invention relates to a mutated transcription factor EB (TFEB) protein that has lost its native exon 3. This protein is referred to herein as “TFEB-∆ex3” or “∆ex3-TFEB”. The invention also relates to a polynucleotide encoding this mutated protein; a vector comprising said polynucleotide; and a biomolecular tool for removing TFEB exon 3 in patients in need. Particularly, the invention relates to an oligonucleotide molecule capable of mediating exon jumping of TFEB protein exon 3. The invention further relates to said mutated TFEB protein, said polynucleotide, said vector, said molecule, or pharmaceutical composition comprising the above for the treatment and / or prevention of lysosomal storage diseases and diseases characterized by lysosomal dysfunction. Background Technology

[0002] Lysosomes are membrane-bound organelles responsible for breaking down proteins, glycosaminoglycans (GAGs), nucleic acids, lipids, and carbohydrates. Their acidic internal pH is maintained by ATP-dependent proton pumps (called vacuolar ATPases, V-ATPases) and over 60 hydrolases that control waste breakdown and recycling. Initially considered quiescent, terminal, and discarded cells, lysosomes were thought to be passive receptors. However, more recently, lysosomes have emerged as dynamic centers, crucial for gene regulation, secretion, cell remodeling, cell adhesion, development, differentiation, cell migration, apoptosis, and lipid transport. Furthermore, lysosomes are now known to be central to adapting to cellular stress and disease. To this end, lysosomes are capable of transformation, undergoing division and fusion, and interacting with a wide variety of intracellular components.

[0003] In 2009, Sardiello, Ballabio, and their colleagues discovered a network of genes that co-express lysosomal proteins (Sardiello M. et al., Science This hypothesis was confirmed in 2009 (vol.325, pp.473-476). Pattern discovery analysis of lysosomal protein promoter regions revealed a common motif; the researchers named it the Coordinated Lysosomal Expression and Regulation (CLEAR) element. CLEAR is a 10-base palindromic sequence (5'-GTCACGTGAC-3'-SEQ ID NO:139), preferentially located within 200 bp of the transcription start site (TSS). CLEAR is present in gene regulatory regions involved in inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase expression, and lysosomal exocytosis.

[0004] Transcription factor EB (TFEB) is a mammalian transcription factor that binds directly to the shared sequence of CLEAR. By modulating its activity, TFEB coordinates on-demand control of each cellular degradation pathway. In particular, TFEB binding to CLEAR leads to the regulation of lysosomal biogenesis and function, including autophagy activation. Structurally, TFEB contains three functional domains, including a DNA-binding, helix-loop-helix, and leucine zipper (bHLH-Zip) domain, which are also present in other members of the microphthalmia-associated transcription factor (MITF), transcription factor E3 (TFE3), and transcription factor EC (TFEC), and the microphthalmia / transcription factor E (MiT / TFE) family (Da Costa et al., Fundamental & Clinical Pharmacology , 2020, doi:10.1111 / fcp.12634).

[0005] Lysosomal biogenesis is complex and requires the sequential synthesis of approximately 60 hydrolases (glycosidases, proteases, lipases, nucleases, phosphatases, and sulfatases), as well as transmembrane and accessory proteins. To determine the extent of TFEB's control over lysosomal biogenesis, Palmieri et al. performed ChIP-seq analysis of TFEB protein interactors, depicting TFEB-mediated transcriptional regulation, mapping TFEB target sites across the entire genome, and performing co-expression analysis in HeLa cells (Palmieri et al., Hum. Mol. Genet. 2011, 20 3852-66, doi:10.1093 / hmg / ddr306). This study identified 471 TFEB targets. The resulting interactors can be categorized into several classes, ranging from autophagy genes (as expected) to lysosomal enzymes and their transporters, lysosomal membrane proteins, genes responsible for lysosomal acidification and lysosomal localization, and non-lysosomal proteins involved in lysosomal biogenesis.

[0006] TFEB activity is highly dependent on its nuclear localization. Therefore, nuclear signaling pathways strongly regulate TFEB activity. Specifically, TFEB nuclear localization is known to be controlled by specific serine phosphorylation. Similar to starvation, pharmacological or mutation-based inhibition of specific phosphorylation induces autophagy by activating TFEB. Therefore, it has been proposed to substitute or alter serine residues to make them insensitive to phosphorylation, thereby maintaining dephosphorylation (e.g., see US9193755). TFEB nuclear export is also known to be mediated by nuclear localization signals (NLS). Other authors have shown that mutant TFEB, where this localization signal is mutated (corresponding to “∆NLS-TFEB” in the TFEB protein, where the NLS is mutated by replacing two basic arginine residues with alanine), exhibits constitutive cytoplasmic localization (Napolitano et al.). Nature Communication, 2018, 9:3312, doi:10.1038). Other authors have proposed adding NLS to the C-terminus of the TFEB protein to obtain a chimeric molecule that primarily targets the cell nucleus (WO 2010 / 092112). Finally, TFEB is known to contain a nuclear export signal (NES) in its N-terminal region. This NES is a highly evolutionarily conserved hydrophobic sequence shared by CRM1. It has been shown that mutagenesis of three of these conserved residues completely impairs the cytoplasmic relocalization of TFEB (Napolitano et al.). Nature Communication , 2018, 9:3312, doi:10.1038).

[0007] The growing understanding of the role of TFEB and CLEAR in promoting healthy clearance, along with in vitro and in vivo preclinical findings in various animal models of disease, supports the conclusion that pharmacological activation of TFEB can clear toxic proteins to treat rare and common forms of lysosomal diseases and neurodegenerative diseases.

[0008] Currently, there are no curative or approved treatments targeting TFEB. Furthermore, although clinical trials of potential treatments for these diseases are ongoing, there are currently no approved treatments for most lysosomal storage diseases or many conditions characterized by lysosomal dysfunction. Therefore, there remains a need in the art for compositions and methods that effectively treat lysosomal storage diseases and conditions characterized by lysosomal dysfunction based on enhanced lysosomal clearance and removal of cellular aggregates. Summary of the Invention

[0009] Mutant TFEB protein TFEB is a mammalian transcription factor that binds directly to the CLEAR shared sequence (5'-GTCACGTGAC-3'-SEQ ID NO:139) in the regulatory regions of genes involved in inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase expression, and lysosomal exocytosis. By modulating these activities, TFEB coordinates on-demand control of each cellular degradation pathway.

[0010] The polypeptide sequence of human TFEB is shown as SEQ ID NO:1 (corresponding to accession number P19484 in the UniProt database). Variants have been reported, for example, SEQ ID NO:2 (B0QYS6 in the UniProt database) and SEQ ID NO:3 (B0QYS7 in the UniProt database).

[0011] Nuclear signaling pathways regulate cellular energy metabolism through TFEB.

[0012] On one hand, the nuclear output signal is SEQ ID NO:4 (GNSAPNSPMAMLHIGSNP) and is located between amino acids 136 and 153 of SEQ ID NO:1, thus encoding exon 3 of TFEB (Napolitano et al., 2018). This output signal is controlled by CRM1. It is encoded by the third exon.

[0013] The inventors have determined that activating TFEB protein in cellular targets is useful by transfecting the same mutant protein in which the nuclear output signal has been removed by exon 3 skipping or exon 3 deletion, or by removing the signal from an endogenous protein. Such activation of TFEB would enable the clearance of toxic proteins to treat rare and common forms of neurodegenerative diseases (DaCosta et al.). Fundamental & Clinical Pharmacology , 2020, doi:10.1111 / fcp.12634).

[0014] Therefore, in a first aspect, the present invention relates to a mutant TFEB protein that does not contain an amino acid sequence encoded by natural exon 3.

[0015] Exon 3 of TFEB encodes an amino acid sequence containing 85 amino acids. The amino acid sequence encoded by exon 3 has the sequence shown in SEQ ID NO:6 (VQSYLENPTSYHLQQSQHQKVREYLSETYGNKFAAHISPAQGSPKPPPAASPGVRAGHVLSSSAGNSAPNSPMAMLHIGSNPERE). In one embodiment, the amino acid sequence encoded by exon 3 of TFEB is located between amino acid residue 72 (inclusive) and amino acid residue 156 (inclusive) of any one of SEQ ID NO:1, 2, or 3 (in other words, in this embodiment, the amino acid sequence encoded by exon 3 of TFEB consists of the amino acid sequence from amino acid residue 72 (inclusive) to amino acid residue 156 (inclusive) of any one of SEQ ID NO:1, 2, or 3).

[0016] In a preferred embodiment, the TFEB protein is the human protein of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a variant or homolog thereof, the polypeptide sequence of which shows at least 80% identity with SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0017] In a preferred embodiment, the mutant protein of the present invention is a human TFEB protein, wherein the amino acid sequence encoded by exon 3 of SEQ ID NO:6 (or a variant or homolog having at least 80% identity with said sequence) has been removed or deleted.

[0018] The resulting mutant proteins have, for example, SEQ ID NO:7 (corresponding to P19484, wherein the amino acid sequence encoded by exon 3 has been removed), SEQ ID NO:8 (corresponding to B0QYS6, wherein the amino acid sequence encoded by exon 3 has been removed), or SEQ ID NO:9 (corresponding to B0QYS7, wherein the amino acid sequence encoded by exon 3 has been removed).

[0019] The mutant protein may also be a variant or homolog thereof, whose amino acid sequence shows at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, or even more preferably at least 99% percentage identity with SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0020] In other words, the present invention relates to an isolated or recombinant polypeptide whose sequence comprises (or is substantially composed of the following sequences, or is composed of the following sequences) a sequence having at least 80% identity with SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0021] These mutant proteins or their variants preferably still contain the nuclear localization signal SEQ ID NO:5 (NLIERRRRFNIN), which encodes exons 6 and 7 of TFEB (Roczniak-Ferguson et al.). Sci. Signal . 2012; 5(228):ra42. Doi:10.1126).

[0022] Polynucleotides encoding mutant proteins In a second aspect, the present invention relates to any polynucleotide encoding the mutant protein. These polynucleotides are preferably isolated or recombinant nucleic acid molecules.

[0023] Those skilled in the art will understand that, due to the degeneracy of the genetic code, many nucleotide sequences can encode the polypeptides described herein. In particular, the use of codons in a given nucleotide sequence can be adjusted to optimize the expression of the corresponding polypeptide.

[0024] The polynucleotide encoding the human TFEB protein is well known. These are, for example, transcriptomorph 1 (2354 bp), called NM_007162.3; transcriptomorph 2 (2152 bp), called NM_001167827.3; transcriptomorph 3 (2333 bp), called NM_001271944; or transcriptomorph 4 (2163 bp), called NM_001271945. They all contain exon 3 of SEQ ID NO:10 or a variant thereof. SEQ ID NO:10 represents the DNA sequence and RNA sequence (e.g., mRNA) of exon 3 of the TFEB protein.

[0025] This invention relates to a polynucleotide encoding a mutant TFEB protein, the mRNA sequence of which does not contain the mRNA sequence of SEQ ID NO:10 (or a variant or homolog thereof having at least 80% identity with SEQ ID NO:10). Specifically, this invention relates to an mRNA encoding a TFEB protein that lacks the mRNA sequence of SEQ ID NO:10 (or a variant or homolog thereof having at least 80% identity with SEQ ID NO:10).

[0026] These polynucleotides have sequences such as the following: -SEQ ID NO:11, corresponding to NM_007162.3 (variant 1) in which exon 3 has been removed. -SEQ ID NO:12, corresponding to NM_001167827.3 (variant 2) in which exon 3 has been removed. -SEQ ID NO:13, corresponding to NM_001271944 (variant 3) in which exon 3 has been removed. -SEQ ID NO:14, corresponding to NM_001271945 (variant 4) in which exon 3 has been removed.

[0027] The invention also relates to any variants thereof, the sequence of which has 80% or more, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% identity with SEQ ID NO:11-14. The invention also includes all equivalent polynucleotides that, due to codon degeneracy, encode the same protein as the protein encoded by SEQ ID NO:11-14.

[0028] Of course, this invention relates to DNA and RNA sequences, as well as sequences that hybridize with them, and the corresponding double-stranded DNA.

[0029] On the other hand, the present invention relates to vectors comprising the polynucleotides of the present invention as defined above, or vectors encoding the mutant TFEB protein of the present invention as defined above.

[0030] Therefore, in one embodiment, the vector contains a polynucleotide encoding a mutant TFEB protein, wherein the mutant TFEB protein does not contain the amino acid sequence encoded by natural exon 3, and the vector is preferably a plasmid or a viral vector.

[0031] Alternatively / in addition, the vector contains a polynucleotide encoding a mutant TFEB protein, the mRNA sequence of which does not contain the mRNA sequence of SEQ ID NO:10, and the vector is preferably a plasmid or a viral vector.

[0032] Preferably, the amino acid sequence encoded by exon 3 and having the amino acid sequence of SEQ ID NO:6, or a variant thereof, has been removed or deleted.

[0033] In a preferred embodiment, the mutant TFEB protein has a sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and variants or homologs of any one of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0034] In a preferred embodiment, the polynucleotide encoding the mutant TFEB protein has a sequence selected from the group consisting of SEQ ID NO:11-14 or variants thereof.

[0035] These vectors may be cloning and / or expression vectors containing the nucleic acid molecules of the present invention. They may include features that allow the nucleic acid molecules of the present invention to be expressed in and / or secreted from host cells for in vitro production purposes. The vectors of the present invention may be DNA, or RNA, or DNA-RNA hybrids.

[0036] These vectors can also be gene therapy vectors, which can be administered to subjects in need to express the mutant proteins of the present invention in situ.

[0037] The gene therapy vector can be a plasmid or a viral vector, which makes it possible to improve the administration of nucleic acid encoding mutant TFEB to target cells and also increases the stability of the nucleic acid in the cells, thereby enabling a durable effect.

[0038] In the context of this invention, the target cell is any human cell, such as nerve, muscle, or bone cells.

[0039] Pharmaceutical Composition On the other hand, the present invention relates to a pharmaceutical composition comprising a mutant TFEB protein as defined above, or a polynucleotide as defined above, or a carrier as defined above.

[0040] In addition to the proteins, polynucleotides, or carriers of the present invention, the pharmaceutical compositions of the present invention may contain pharmaceutically acceptable excipients.

[0041] As used herein, the term “pharmaceuticalally acceptable excipient” is intended to mean, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or encapsulating agent, such as liposomes, cyclodextrins, encapsulated polymer delivery systems, or polyethylene glycol matrix, which is acceptable for use in subjects, preferably human.

[0042] Pharmaceutically acceptable mediators can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Other examples of pharmaceutically acceptable mediators include, but are not limited to: water for injection (USP); aqueous mediators such as sodium chloride injection, Ringer's solution injection, glucose injection, glucose and sodium chloride injection, and lactated Ringer's solution injection; water-miscible mediators such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous mediators such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0043] Treatment - Gene Therapy In another aspect, the present invention relates to the mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the carrier of the present invention as defined above, the pharmaceutical composition of the present invention as defined above, or any combination thereof, for use as a drug or for preparation of a drug.

[0044] The present invention also relates to the use of the mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the carrier of the present invention as defined above, the pharmaceutical composition of the present invention as defined above, or any combination thereof, as a medicine or for the preparation of a medicine.

[0045] The present invention also relates to a method of treating a disease in a subject in need, comprising administering to the subject a mutant TFEB protein of the present invention as defined above, or a polynucleotide of the present invention as defined above, or a carrier of the present invention as defined above, a pharmaceutical composition of the present invention as defined above, or any combination thereof.

[0046] In particular, the present invention relates to the mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the carrier of the present invention as defined above, the pharmaceutical composition of the present invention as defined above, or any combination thereof, for use as a medicament for treating cell storage disorders involving autophagy defects, particularly lysosomal storage diseases (LSD).

[0047] The present invention also relates to the use of the mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the carrier of the present invention as defined above, the pharmaceutical composition of the present invention as defined above, or any combination thereof, for the treatment of cell storage diseases involving autophagy defects, particularly lysosomal storage diseases (LSD), or for the preparation of a medicament for the treatment of cell storage diseases involving autophagy defects, particularly lysosomal storage diseases (LSD).

[0048] The present invention also relates to a method for treating cellular storage disorders involving autophagy defects, particularly lysosomal storage disorders (LSD), in subjects in need, comprising administering to the subject a mutant TFEB protein of the present invention as defined above, or a polynucleotide of the present invention as defined above, or a carrier of the present invention as defined above, a pharmaceutical composition of the present invention as defined above, or any combination thereof.

[0049] Lysosomal storage disease (LSD) is a condition in which lysosomal function is impaired due to a hereditary condition, resulting in cellular dysfunction. This type of LSD includes, but is not limited to, sphingolipidoses (such as Fabry disease, Faber-Leydig lipogranulomatosis, Gaucher disease (types I / II / III and perinatal lethal forms), GM1 ganglioside storage diseases (types I / II / III), GM2 ganglioside storage diseases (Tessa II disease, Sandhoff disease, GM2 activator deficiency), spheroid cell leukodystrophy (Krabbe disease), metachromatic leukodystrophy, Niemann-Pick disease A / B, prosaposin deficiency, and Saposin B deficiency); and mucopolysaccharidoses (such as MPS I (Hurler / Hurler-Scheie / Scheie syndrome), MPS II (Hunter syndrome), MPS III / A / B / C / D (Sanfilippo syndrome A / B / C / D), MPS... IVA / B (Morquio syndrome A / B), MPS VI (Maroteaux–Lamy syndrome), MPS VII (Sly disease), MPS IX (Natowicz syndrome); glycogen storage diseases (such as GSD0, GSDI (von Gierke's disease), GSD IIa (Pompe's disease), GSD IIb (Danon's disease), GSD III (Cori's disease or Forbes' disease), GSD IV (Andersen's disease), GSD V (McArdle's disease), GSD VI (Hers' disease), GSD VII (Tarui's disease), GSD IX, GSD X, GSD XI, GSD XII, GSD XIII, GSD XV, CDG1T; Glycoproteinose disorders (such as α-mannoside storage (types I / II / III), β-mannoside storage, fucoside storage, aspartate glucosamineuria, Schindler's disease (types I / II / III), sialic acid storage (types I / II), galactosylsialic acid storage); Lipid storage disorders (such as acid lipase deficiency (Wolman's disease, cholesterol ester storage disease)); Post-translational modification defects (such as polysulfatase deficiency, mucolipid storage disease (II α / β, I-cell disease), mucolipid storage disease II (α / β, pseudo-Hurler's dystrophism), mucolipid storage disease III (γ, variant pseudo-Hurler's dystrophism)).Integrated membrane protein disorders (such as cystine storage diseases, myoclonic-renal failure syndrome, sialic acid storage diseases (ISSD, Salla disease), Niemann-Pick disease C1 / C2, mucolipid storage disease IV); neuronal ceroid lipofuscin deposition diseases (CLN) (such as CLN1 (Haltia-Santavuori disease and INCL), CLN2 (Jansky-Bielschowsky disease), CLN3 (Batten-Spielmeyer-Sjogren disease), CLN4 (Parry disease and Kufs disease)). CLN5 (Finnish variant, late infantile form), CLN6 (Lake-Cavanagh or Indian variant), CLN7 (Turkish variant), CLN8 (Northern epilepsy, epileptic intellectual disability), CLN9, CLN10, CLN11, CLN12 (Kufor–Rakeb syndrome), CLN13, CLN14; lysosomal organelle disorders (e.g., Hermansky-Pudlak disease types 1 to 9, Griscelli syndrome 1 (Elejalde syndrome), Griscelli syndrome 2, Chédiak-Higashi disease); polydextrose storage disorders (e.g., Lafora disease, adult-onset PG syndrome, AMP-activated protein kinase deficiency); others (e.g., dense bone development disorder, Papillon–Lefevre syndrome).

[0050] These conditions can also be neurological disorders, leading to impaired lysosomal function and autophagy, resulting in degenerative processes. These diseases include, but are not limited to, Alzheimer's disease; age-related macular degeneration; cerebral β-amyloid angiopathy; prion diseases (such as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, and Kuru); Parkinson's disease; and multiple sclerosis (Gonzalez-Jimenez A. et al.). Int. Mol. Sci. 2022; 23(15):8116. doi:10.3390 / ijms23158116); synucleinopathies (such as multiple system atrophy, Lewy body dementia); tauopathies (such as primary age-related tauopathic dementia, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia (Root J. et al., Neurobiol Dis.2021; 154:105360. doi:10.1016 / j.nbd.2021.105360), chromosome-related Parkinson's syndrome, vacuolating tau proteinosis, Lytico-Bodig disease, gangliocytoma and gangliocytoma, meningoangioma disease, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic acid kinase-related neurodegeneration and lipofuscin deposition disease); frontotemporal degeneration; amyotrophic lateral sclerosis; Huntington's disease; trinucleotide duplication syndrome; familial dementia; hereditary cerebral hemorrhage with amyloidosis; Cadasil syndrome; Alexander's disease; Angelman syndrome; Peyre's disease; Seipin's disease; familial amyloid neuropathy; serine protease inhibitor disease; amyloidosis Diseases such as senile systemic, light chain, heavy chain, secondary, medial aortic, ApoAI, ApoAII, ApoAIV, lysozyme, fibrinogen, dialysis, cardiac atrial, cutaneous lichenification, corneal lactoferrin, apolipoprotein C2, apolipoprotein C3, Lect2, insulin, galactoglobulin-7, keratin or enfviride amyloidosis; type II diabetes; inclusion body myositis / myopathy; cataracts; retinitis pigmentosa with rhodopsin mutations; medullary thyroid carcinoma; pituitary prolactinoma; hereditary phenotyped corneal dystrophy; Mallory bodies; pulmonary alveolar proteinosis; odontogenic (Pindborg) amyloidosis; seminal vesicle amyloidosis; cystic fibrosis; sickle cell disease; plasma cell cachexia; exfoliation syndrome.

[0051] In a preferred embodiment, the disease to be treated is age-related macular degeneration, synucleinosis (preferably multiple system atrophy), or Parkinson's disease; more preferably, the disease is multiple system atrophy.

[0052] In preferred embodiments, the mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the carrier of the present invention as defined above, or the pharmaceutical composition of the present invention, can treat β-mannosin storage disease, cholesterol ester storage disease, CLN1 disease, CLN2 disease, CLN3 disease, CLN7 disease, Fabry disease, GM2-ganglioside storage diseases (Tessa II disease, AB variant, and Sandhoff disease), Krabbe disease, metachromatic leukodystrophy, type I / II / III / IV mucolipid storage diseases, type I mucopolysaccharidosis (Scheie syndrome, Hurler-Scheie syndrome, Hurler syndrome), type II mucopolysaccharidosis (Hunter syndrome), type III mucopolysaccharidosis (Sanfilippo syndrome type A, Sanfilippo syndrome type B), and type IV mucopolysaccharidosis (Morquio syndrome). Syndrome A), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), Niemann-Pick disease type C, Pompe disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, spinocerebellar ataxia, multiple sclerosis, amyotrophic lateral sclerosis, multiple system atrophy, frontotemporal dementia, Lewy body disease, and Friedreich ataxia.

[0053] The mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the vector of the present invention as defined above, or the composition of the present invention, will prevent, reverse, or halt cognitive decline in subjects. Methods for determining cognitive decline in subjects are known in the art. For example, the mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the vector of the present invention as defined above, or the composition of the present invention, can prevent, reverse, or halt visual decline. The mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the vector of the present invention as defined above, or the composition of the present invention, can also prevent, reverse, or halt hearing loss. The compositions of the present invention can also reduce the severity and / or intensity of epileptic seizures. Furthermore, the mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the vector of the present invention as defined above, or the composition of the present invention, can improve or prevent motor dysfunction. The mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the vector of the present invention as defined above, or the composition of the present invention, can also improve or prevent dementia.

[0054] The mutant TFEB protein of the present invention as defined above, or the polynucleotide of the present invention as defined above, or the vector of the present invention as defined above, or the composition of the present invention as defined above, can be safely administered orally, topically, via the oral mucosa, intranasally, intracranially, intraperitoneally, or parenterally (e.g., intraocularly, intravenously, intra-arterially, intrathecally, intramuscularly, intravenously, intracisionally, and subcutaneously). The mutant TFEB protein of the present invention as defined above, the polynucleotide of the present invention as defined above, the vector of the present invention as defined above, or the composition of the present invention as defined above are preferably administered intrathecally or intraocularly (e.g., via intravitreal administration (e.g., intravitreal injection), subretinal administration (e.g., subretinal injection), choroidal administration (e.g., choroidal injection), or any combination thereof), or any combination thereof, wherein intraocular administration is preferably intravitreal administration.

[0055] Exon in situ skipping-gene therapy Exon skipping is a technique used in gene therapy. It involves skipping one or more exons during splicing to allow cells to synthesize truncated but still functional proteins. It has been developed and is now approved for the treatment of several diseases, including Duchenne muscular dystrophy (DMD).

[0056] Exon jumping can be induced in cells using various tools, viral vectors, antisense DNA / RNA, etc. Therefore, this invention relates to any biomolecular tool or device capable of specifically jumping TFEB exon 3 in any human cell, such as nerve, muscle, or bone cells. For example, a biomolecular tool or device capable of specifically jumping TFEB exon 3 of this invention could be morpholino, U7-SNRP, DNA-editing CRISPR, siRNA, antisense oligonucleotides, or small chemical molecules such as TG003 or risdiplam.

[0057] It is well known that antisense oligonucleotides (AON or ASO) mediate highly efficient exon skipping (Rocha C). Methods Mol Biol. 2019; 2036:73-90. doi:10.1007 / 978-1-4939-9670-4_4 and Edinoff et al., Orthop Rev (Pavia). 2021 Jun 19;13(2):24934. doi: 10.52965 / 001c.24934). Therefore, the inventors propose to use ASO-mediated exon skipping to remove exon 3 of TFEB in situ in any human cell, such as nerve, muscle, or bone cells of patients with the aforementioned cell storage disease.

[0058] These ASOs preferably affect exon 3 of TFEB, i.e., SEQ ID NO:10 or a variant thereof, by targeting exon 3 or nearby regions, such as introns 2-3 or 3-4.

[0059] Therefore, this invention relates to oligonucleotide molecules, preferably antisense oligonucleotides (ASOs), capable of mediating exon skipping of exon 3 of the TFEB protein. In a preferred embodiment, the oligonucleotide molecule of the present invention is capable of mediating exon skipping of exon 3 of the TFEB protein, wherein exon 3 encodes the amino acid sequence shown in SEQ ID NO:6 (or a variant / homolog thereof) of the TFEB protein of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 (or variants / homologs thereof). As mentioned above, the variants have, for example, at least 80%, preferably at least 90%, identity with the reference sequence.

[0060] The oligonucleotide molecule is preferably an antisense oligonucleotide (ASO).

[0061] Antisense oligonucleotides (ASOs) are short, single-stranded nucleotide sequences (usually oligodeoxynucleotides), typically 12–40 bases long (typically 15–22 bases), which are most often engineered and synthesized in vitro to be complementary to the target sequence of a transcript (e.g., mRNA or pre-mRNA) to produce a DNA / RNA heteroduplex. Therefore, they inhibit the synthesis of the corresponding protein or mask certain genetic sequences, thereby correcting, regulating, or inhibiting the splicing of the target mRNA, and thus producing or regulating the expression of functional or non-functional proteins. Antisense oligonucleotides can be single-stranded DNA oligonucleotides engineered to be antisense against the target transcript / RNA. Hybridization of ASOs with the target RNA typically triggers cleavage of the RNA by a ribonuclease (e.g., RNase H). Antisense oligonucleotides are preferably chemically modified to resist nuclease activity (e.g., by adding phosphate thioester (PS) to the oligonucleotide for modification / substitution and / or adding modified bases, such as 2' sugar modification, including 2'-O-methyl (2'-OMe)), and to stably hybridize with their target (e.g., by adding modified bases, such as 2'-O-methyl (2'-OMe)), and / or by replacing dC in the CpG motif with 5-methyl dC).

[0062] In one embodiment, the molecules of the present invention are capable of mediating exon skipping in exon 3 of a nucleic acid sequence encoding the TFEB protein.

[0063] In one embodiment, after exon 3 skipping, the resulting TFEB protein has a sequence selected from the group consisting of: variants or homologs of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and any one of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0064] In one embodiment, the molecules of the present invention are capable of mediating exon skipping of exon 3 of SEQ ID NO:6 of the nucleic acid sequence encoding the TFEB protein of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 or a variant or homology thereof.

[0065] In a preferred embodiment, exon 3 encodes the amino acid sequence shown in SEQ ID NO:6. In a preferred embodiment, exon 3 is encoded by the nucleic acid sequence shown in SEQ ID NO:10 (which is the nucleic acid sequence encoding exon 3 of the TFEB protein of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or variants or homologs thereof).

[0066] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence encoding introns 2-3, exon 3, introns 3-4, or any combination thereof (preferably located in a nucleic acid sequence from the nucleic acid sequence encoding introns 2-3 to the nucleic acid sequence encoding introns 3-4, including the nucleic acid sequence of exon 3; in other words, preferably located in a polynucleotide of a nucleic acid sequence encoding introns 2-3 to introns 3-4, including the nucleic acid sequence of exon 3).

[0067] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence encoding introns 2-3, exon 3, introns 3-4, or any combination thereof (preferably located in a nucleic acid sequence from the nucleic acid sequence encoding introns 2-3 to the nucleic acid sequence encoding introns 3-4, including the nucleic acid sequence of exon 3; in other words, preferably located in a polynucleotide of a nucleic acid sequence encoding introns 2-3 to introns 3-4, including the nucleic acid sequence of exon 3).

[0068] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding exon 2 to intron 10 of TFEB, of which nucleic acid residues 236 to 1424 (inclusive) are present (i.e., without exon 1 and introns 1-2 (introns 1-2 correspond to introns located between exon 1 and exon 2 of TFEB, in other words, correspond to introns bridging exon 1 and exon 2)).

[0069] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 236 to 1424 (inclusive) of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB (i.e., without exon 1 and introns 1-2 (introns 1-2 correspond to introns located between exon 1 and exon 2 of TFEB, in other words, correspond to introns bridging exon 1 and exon 2)).

[0070] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding exon 2 to intron 10 of TFEB, comprising nucleic acid residues 269 to 621 (inclusive).

[0071] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence of exon 2 to intron 10 encoding TFEB, comprising nucleic acid residues 269 to 621 (inclusive).

[0072] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding exon 2 to intron 10 of TFEB, comprising nucleic acid residues 339 to 436 (inclusive).

[0073] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding exon 2 to intron 10 of TFEB, at nucleic acid residues 339 to 436.

[0074] The molecule of the present invention preferably targets (or is complementary to, or hybridizes with, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located at nucleic acid residues 339 to 435 of the nucleic acid sequence encoding exons 2 to introns 10 of TFEB; preferably 339 to 434, more preferably 339 to 433, more preferably 339 to 432, more preferably 340 to 431, more preferably 340 to 430 ... 40 to 429, more preferably 340 to 428, more preferably 340 to 427, more preferably 340 to 426, more preferably 340 to 425, more preferably 340 to 424, more preferably 340 to 423, more preferably 340 to 422, more preferably 341 to 421, more preferably 341 to 420, more preferably 341 to 419, more preferably 341 to 418, more preferably 341 to 417, more preferably 341 to 416, more preferably 341 to 415, more preferably 341 to 414, more preferably 341 to 413, more preferably 341 to 412, more preferably 342 to 411, more preferably 342 to 410, more preferably 342 to 409, more preferably 342 to 408, more preferably 342 to 407, more preferably 342 to 406, more preferably 342 to 405, more preferably 342 to 404, more preferably 342 to 403, more preferably 342 to 402, more preferably 342 to 401, more preferably 342 to 400, more preferably 342 to 399, more preferably 342 to 398, even better Among 342 to 397, more preferably 342 to 396, more preferably 342 to 395, more preferably 342 to 394, more preferably 342 to 393, more preferably 342 to 392, more preferably 342 to 391, more preferably 342 to 390, more preferably 342 to 389, more preferably 342 to 388, more preferably 342 to 387, more preferably 342 to 386, more preferably 342 to 385, more preferably 342 to 384, more preferably 342 to 383, and more preferably 342 to 382.

[0075] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located at nucleic acid residues 339 to 435 of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB; preferably 339 to 434, more preferably 339 to 433, more preferably 339 to 432, more preferably 340 to 431, more preferably 340 to 430 ... 340 to 429, more preferably 340 to 428, more preferably 340 to 427, more preferably 340 to 426, more preferably 340 to 425, more preferably 340 to 424, more preferably 340 to 423, more preferably 340 to 422, more preferably 341 to 421, more preferably 341 to 420, more preferably 341 to 419, more preferably 341 to 418, more preferably 341 to 417, more preferably 341 to 416, more preferably 341 to 415, more preferably 341 to 414, even better Select 341 to 413, more preferably 341 to 412, more preferably 342 to 411, more preferably 342 to 410, more preferably 342 to 409, more preferably 342 to 408, more preferably 342 to 407, more preferably 342 to 406, more preferably 342 to 405, more preferably 342 to 404, more preferably 342 to 403, more preferably 342 to 402, more preferably 342 to 401, more preferably 342 to 400, more preferably 342 to 399, more preferably 342 to 398, more preferably... Among the following: 342 to 397, 342 to 396, 342 to 395, 342 to 394, 342 to 393, 342 to 392, 342 to 391, 342 to 390, 342 to 389, 342 to 388, 342 to 387, 342 to 386, 342 to 385, 342 to 384, 342 to 383, and 342 to 382.

[0076] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding exon 2 to intron 10 of TFEB at nucleic acid residues 342 to 369 or nucleic acid residues 358 to 381.

[0077] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding exon 2 to intron 10 of TFEB at nucleic acid residues 342 to 369 or nucleic acid residues 358 to 381.

[0078] Therefore, the nucleic acid sequence encoding exons 2 to intron 10 of TFEB consists of the following: from 5' to 3', exon 2, introns 2-3, exon 3, introns 3-4, exon 4, introns 4-5, exon 5, introns 5-6, exon 6, introns 6-7, exon 7, introns 7-8, exon 8, introns 8-9, exon 9, and intron 10.

[0079] An exemplary nucleic acid sequence (DNA, RNA (e.g., mRNA), or a DNA / RNA hybrid thereof) encoding exon 2 to intron 10 of the TFEB protein is shown in SEQ ID NO: 15. SEQ ID NO: 15 does not contain exon 1, nor introns 1-2 (introns 1-2 correspond to introns located between exon 1 and exon 2 of the TFEB protein; in other words, they correspond to introns connecting exon 1 and exon 2). The exemplary nucleic acid sequence SEQ ID NO: 15 extends from the first nucleic acid residue of exon 2 (including the first residue of exon 2) to the last nucleic acid residue of intron 10 (including the last residue of intron 10) of the exemplary TFEB protein.

[0080] The positions of introns and exons in SEQ ID NO:15 are shown in Table 1 below.

[0081] Table 1: Locations of introns and exons in SEQ ID NO:15 In a preferred embodiment, the nucleic acid sequence encoding exons 2 to introns 10 of TFEB has at least 85% identity with the nucleic acid sequence of SEQ ID NO:15, preferably wherein the nucleic acid sequence encoding exons 2 to introns 10 of TFEB has at least 90% identity with the nucleic acid sequence of SEQ ID NO:15, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and even more preferably, the nucleic acid sequence encoding exons 2 to introns 10 of TFEB has the nucleic acid sequence of SEQ ID NO:15.

[0082] Preferably, the nucleic acid sequence of SEQ ID NO:15 is the nucleic acid sequence of exon 2 to intron 10 of the TFEB protein of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or their variants or homologs.

[0083] More preferably, the molecules of the present invention target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12-40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of 34 to 386 (inclusive) nucleic acid residues encoding introns 2-3, exon 3, and introns 3-4 of TFEB (i.e., a nucleic acid sequence from the nucleic acid sequence encoding introns 2-3 to the nucleic acid sequence encoding introns 3-4, including exon 3; in other words, a polynucleotide encoding a nucleic acid sequence from introns 2-3 to introns 3-4, including exon 3; in other words, a nucleic acid sequence encoding introns 2-3 to introns 3-4 (including exon 3) of TFEB).

[0084] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence of nucleic acid residues 34 to 386 (inclusive) of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB.

[0085] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence of 104 to 201 (inclusive) nucleic acid residues of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB.

[0086] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence of nucleic acid residues 104 to 201 of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB.

[0087] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located at nucleic acid residues 104 to 200 of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB; preferably 104 to 199, more preferably 104 to 198, more preferably 104 to 197, more preferably 105 to 196 of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB. More preferably 105 to 195, more preferably 105 to 194, more preferably 105 to 193, more preferably 105 to 192, more preferably 105 to 191, more preferably 105 to 190, more preferably 105 to 189, more preferably 105 to 188, more preferably 105 to 187, more preferably 106 to 186, more preferably 106 to 185, more preferably 106 to 184, more preferably 106 to 183, more preferably 106 to 182, more preferably 106 to 181, more preferably 106 to 180, more preferably 1 06 to 179, more preferably 106 to 178, more preferably 106 to 177, more preferably 107 to 176, more preferably 107 to 175, more preferably 107 to 174, more preferably 107 to 173, more preferably 107 to 172, more preferably 107 to 171, more preferably 107 to 170, more preferably 107 to 169, more preferably 107 to 168, more preferably 107 to 167, more preferably 107 to 166, more preferably 107 to 165, more preferably 107 to 164, more preferably 107 to 1 63, more preferably 107 to 162, more preferably 107 to 161, more preferably 107 to 160, more preferably 107 to 159, more preferably 107 to 158, more preferably 107 to 157, more preferably 107 to 156, more preferably 107 to 155, more preferably 107 to 154, more preferably 107 to 153, more preferably 107 to 152, more preferably 107 to 151, more preferably 107 to 150, more preferably 107 to 149, more preferably 107 to 148, more preferably 107 to 147.

[0088] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in the nucleic acid residues 104 to 200 of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB; preferably 104 to 199, more preferably 104 to 198, more preferably 104 to 197, more preferably 105 to 108. 96, more preferably 105 to 195, more preferably 105 to 194, more preferably 105 to 193, more preferably 105 to 192, more preferably 105 to 191, more preferably 105 to 190, more preferably 105 to 189, more preferably 105 to 188, more preferably 105 to 187, more preferably 106 to 186, more preferably 106 to 185, more preferably 106 to 184, more preferably 106 to 183, more preferably 106 to 182, more preferably 106 to 181, more preferably 106 to 180, even better Select 106 to 179, more preferably 106 to 178, more preferably 106 to 177, more preferably 107 to 176, more preferably 107 to 175, more preferably 107 to 174, more preferably 107 to 173, more preferably 107 to 172, more preferably 107 to 171, more preferably 107 to 170, more preferably 107 to 169, more preferably 107 to 168, more preferably 107 to 167, more preferably 107 to 166, more preferably 107 to 165, more preferably 107 to 164, more preferably 107 to Among 163, more preferably 107 to 162, more preferably 107 to 161, more preferably 107 to 160, more preferably 107 to 159, more preferably 107 to 158, more preferably 107 to 157, more preferably 107 to 156, more preferably 107 to 155, more preferably 107 to 154, more preferably 107 to 153, more preferably 107 to 152, more preferably 107 to 151, more preferably 107 to 150, more preferably 107 to 149, more preferably 107 to 148, and more preferably 107 to 147.

[0089] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB, at nucleic acid residues 107 to 134 (inclusive) or nucleic acid residues 123 to 146 (inclusive).

[0090] The molecules of the present invention preferably target (or complement, or hybridize, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence encoding TFEB introns 2-3 to 3-4 (including exon 3) of nucleic acid residues 107 to 134 (inclusive) or nucleic acid residues 123 to 146 (inclusive).

[0091] Therefore, the nucleic acid sequence encoding TFEB introns 2-3 to 3-4 (including exon 3) consists of the following from 5' to 3': introns 2-3, exon 3, and introns 3-4.

[0092] An exemplary nucleic acid sequence (DNA, RNA (e.g., mRNA), or a DNA / RNA hybrid thereof) encoding introns 2-3 to 3-4 (including exon 3) of the exemplary TFEB protein is shown in SEQ ID NO: 16. The exemplary nucleic acid sequence SEQ ID NO: 16 extends from the first nucleic acid residue of introns 2-3 (including the first residue of introns 2-3) to the last nucleic acid residue of introns 3-4 (including the last residue of introns 3-4) of the exemplary TFEB protein.

[0093] The positions of introns and exons in SEQ ID NO:16 are shown in Table 2 below.

[0094] Table 2: Locations of introns and exons in SEQ ID NO:16 In a preferred embodiment, the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB has at least 85% identity with the nucleic acid sequence of SEQ ID NO:16. Preferably, the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB has at least 90% identity with the nucleic acid sequence of SEQ ID NO:16. More preferably, it has at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and even more preferably, the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB has the nucleic acid sequence of SEQ ID NO:16.

[0095] Preferably, the nucleic acid sequence of SEQ ID NO:16 is the nucleic acid sequence of introns 2-3 to 3-4 (including exon 3) of the TFEB protein of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or their variants or homologs.

[0096] The molecule of the present invention preferably has (or comprises, or substantially consists of, or consists of) at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78). The nucleic acid sequence shown in 39 (ASO#16.3) has at least 85% identity.

[0097] The molecule of the present invention preferably has (or comprises, or substantially consists of, or consists of) at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78). The nucleic acid sequence shown in 39 (ASO#16.3) has at least 90% identity.

[0098] The molecule of the present invention preferably has (or comprises, or substantially consists of, or consists of) at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78). The nucleic acid sequence shown in 39 (ASO#16.3) has at least 95% identity.

[0099] The molecule of the present invention preferably has (or comprises, or substantially consists of, or consists of) at least 96% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably at least 96% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably at least 96% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably at least 96% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably at least 96% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78). The nucleic acid sequence shown in 39 (ASO#16.3) has at least 96% identity.

[0100] The molecule of the present invention preferably has (or comprises, or substantially consists of, or consists of) at least 97% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably at least 97% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably at least 97% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably at least 97% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably at least 97% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78). The nucleic acid sequence shown in 39 (ASO#16.3) has at least 97% identity.

[0101] The molecule of the present invention preferably has (or comprises, or substantially consists of, or consists of) at least 98% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably at least 98% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably at least 98% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably at least 98% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably at least 98% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78). The nucleic acid sequence shown in 39 (ASO#16.3) has at least 98% identity.

[0102] The molecule of the present invention preferably has (or comprises, or substantially consists of, or consists of) at least 99% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably at least 99% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably at least 99% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably at least 99% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably at least 99% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78). The nucleic acid sequence shown in 39 (ASO#16.3) has at least 99% identity.

[0103] In a particularly preferred embodiment, the molecule of the invention has (or comprises, or is substantially composed of, or is composed of) a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably having a nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), and even more preferably having a nucleic acid sequence shown in SEQ ID NO: 39 (ASO#16.3).

[0104] The sequences of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78) are shown in Table 3 below.

[0105] Table 3: Sequences of preferred molecules of the present invention (preferably ASO) The molecule of the present invention (preferably ASO) preferably contains (or is substantially composed of, or is composed of) 12 to 40 nucleic acid residues (or nucleotides / bases), more preferably 13 to 39 nucleic acid residues, more preferably 14 to 38 nucleic acid residues, more preferably 15 to 37 nucleic acid residues, more preferably 16 to 36 nucleic acid residues, more preferably 17 to 35 nucleic acid residues, more preferably 18 to 34 nucleic acid residues, more preferably 14 to 22 nucleic acid residues, more preferably 15 to 21 nucleic acid residues, more preferably 16 to 20 nucleic acid residues, more preferably 17 to 19 nucleic acid residues, and even more preferably 18 nucleic acid residues.

[0106] The molecules of the present invention (preferably ASO) can carry any chemical modifications known in the art, particularly through skeletal modifications (such as aminophosphates, methylphosphonates, thiophosphates (PS)); or through nucleobase modifications (such as 5-methylcytidine, 5-methylcytosine (m)). 5 C), 5-methyluridine; or by 2'-ribose substitution (e.g., 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-fluoro (2'F), 2'-O-alkyl, 2-O-methylcarbamoylethyl (2-O-MCE)); or by ribose modification (e.g., 5-dehydrated hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threonine nucleic acid (TNA), ethylene glycol nucleic acid (GNA), locked nucleic acid (LNA), FANA (fluoroarabinonucleotide), restricted ethyl (cET), or 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)); or by alternative chemical methods (e.g., phosphoryl diamine morpholino (PMO), N-acetylgalactosamine (tri-antenna GalNAc3), peptide nucleic acid (PNA), tricyclic DNA (tc-DNA), aptamers); or any combination of these chemical modifications (e.g., gapmer, LNA). PS chimeras, 2'-O-alkyl PS chimeras (Roberts TC et al., Nat Rev Drug Discov .2020, 19(10):673-694;Barresi V. et al, Int. J. Mol. Sci 2022, 23, 8875).

[0107] The molecules of the present invention are preferably chemically modified to enhance their anti-nuclease activity (preferably by adding phosphate thioester (PS) to the main chain and / or adding modified bases, such as 2' sugar modification, including 2'-O-methyl (2'-OMe)), and / or to enhance stable hybridization with their targets (e.g. by adding modified bases, such as 2'-O-methyl (2'-OMe)), and / or to replace dC in the CpG motif with 5-methyl dC).

[0108] The oligonucleotide molecules of the present invention, or pharmaceutical compositions containing them, can be used as pharmaceuticals. Therefore, in one aspect, the present invention also relates to the molecules of the present invention as defined above, or pharmaceutical compositions containing them, for use as pharmaceuticals or for the preparation of pharmaceuticals.

[0109] Therefore, the present invention also relates to the use of the molecules of the present invention (as defined above) or pharmaceutical compositions containing them as medicines or for the preparation of medicines.

[0110] The present invention also relates to a method of treating a disease in a subject in need, comprising administering to the subject a molecule of the present invention as defined above or a pharmaceutical composition containing thereas.

[0111] The molecules of the present invention are preferably used as a medicine in subjects who require it.

[0112] The molecules of this invention can be safely administered orally, topically, via the oral mucosa, intranasally, intracranially, intraperitoneally, or parenterally, such as intraocularly, intravenously, intra-arterially, intrathecally, intramuscularly, intravenously, intravenously, intracisionally, and subcutaneously. The molecules of this invention are preferably administered intrathecally or intraocularly (e.g., via intravitreal administration, subretinal administration, choroidal administration, or any combination thereof), or any combination thereof, wherein intraocular administration is preferably intravitreal. It can also be conjugated with delivery systems such as cell-penetrating peptides (CPPs) or liposomes to facilitate access to and entry into target cells (Fabrega C). Pharmaceutics . 2023; 15(2):320).

[0113] Therefore, it can be introduced into pharmaceutical compositions together with pharmaceutically acceptable excipients as defined above. It can be used to manufacture remedies for treating cell storage disorders involving autophagy defects.

[0114] Therefore, the oligonucleotide molecules of the present invention or pharmaceutical compositions containing them can be used as medicines for treating cell storage disorders involving autophagy defects.

[0115] Therefore, the present invention relates to molecules of the present invention as defined above or pharmaceutical compositions containing the thereof, for use as medicaments for treating cell storage disorders involving autophagy defects, particularly lysosomal storage disorders (LSD).

[0116] The present invention also relates to the use of the molecules of the present invention as defined above or pharmaceutical compositions comprising them for the treatment of cell storage disorders involving autophagy defects, particularly lysosomal storage disorders (LSD), or for the preparation of a medicament for the treatment of cell storage disorders involving autophagy defects, particularly lysosomal storage disorders (LSD).

[0117] The present invention also relates to a method of treating cell storage disorders involving autophagy defects, particularly lysosomal storage disorders (LSD), in subjects in need, comprising administering molecules of the present invention as defined above or pharmaceutical compositions containing them.

[0118] These conditions include, for example, lysosomal storage disease (LSD), in which lysosomal function is impaired due to a hereditary condition, leading to cellular dysfunction. This type of LSD includes, but is not limited to, sphingolipid storage disorders (such as Fabry disease, Faber-Leyer lipogranulomatosis, Gaucher disease (types I / II / III and perinatal lethal forms), GM1 ganglioside storage disorders (types I / II / III), GM2 ganglioside storage disorders (Tessa II disease, Sandhoff disease, GM2 activator deficiency), spheroidal leukodystrophy (Krabbe disease), metachromatic leukodystrophy, Niemann-Pick disease A / B, sphingolipid activator proteinogen deficiency, and Saposin B deficiency); mucopolysaccharide storage disorders (such as MPS I (Hurler / Hurler-Scheie / Scheie syndrome), MPS II (Hunter syndrome), MPS III / A / B / C / D (Sanfilippo syndrome A / B / C / D), MPS IVA / B (Morquio syndrome A / B), MPS VI (Maroteaux-Lamy syndrome), MPS VII (Sly disease), MPS IX (Natowicz syndrome); Glycogen storage diseases (such as GSD0, GSDI (von Gierke's disease), GSD IIa (Pompe's disease), GSD IIb (Danon's disease), GSD III (Cori's disease or Forbes' disease), GSD IV (Andersen's disease), GSD V (McArdle's disease), GSDVI (Hers' disease), GSD VII (Tarui's disease), GSD IX, GSD X, GSD XI, GSD XII, GSD XIII, GSD XV, CDG1T; glycoprotein storage disorders (such as α-mannoside storage disorders (types I / II / III), β-mannoside storage disorders, fucoside storage disorders, aspartic acid glucosamineuria, Schindler's disease (types I / II / III), sialic acid storage disorders (types I / II), galactosylsialic acid storage disorders); lipid storage disorders (such as acid lipase deficiency disorders (Wolman's disease, cholesterol ester storage disorders)); post-translational modification defects (such as polysulfatase deficiency, mucolipid storage disorders (type II)). α / β, I-cell disease), mucolipid storage disease II (α / β, pseudo-Hurler's dystrophic disease), mucolipid storage disease III (γ, variant pseudo-Hurler's dystrophic disease)); integrated membrane protein disorders (such as cystine storage disease, myoclonic-renal failure syndrome, sialic acid storage diseases (ISSD, Salla disease), Niemann-Pick disease C1 / C2 type, mucolipid storage disease IV);Neuronal ceroid lipofuscin deposition diseases (CLNs) such as CLN1 (Haltia-Santavuori disease and INCL), CLN2 (Jansky-Bielschowsky disease), CLN3 (Batten-Spielmeyer-Sjogren disease), and CLN4 (Parry disease and Kufs disease). CLN5 (Finnish variant, late infantile form), CLN6 (Lake-Cavanagh or Indian variant), CLN7 (Turkish variant), CLN8 (Northern epilepsy, epileptic intellectual disability), CLN9, CLN10, CLN11, CLN12 (Kufor–Rakeb syndrome), CLN13, CLN14; lysosomal organelle disorders (e.g., Hermansky-Pudlak disease types 1 to 9, Griscelli syndrome 1 (Elejalde syndrome), Griscelli syndrome 2, Chédiak-Higashi disease); polydextrose storage disorders (e.g., Lafora disease, adult-onset PG syndrome, AMP-activated protein kinase deficiency); others (e.g., dense bone development disorder, Papillon–Lefevre syndrome).

[0119] These conditions can also be neurological disorders, which impair lysosomal function and autophagy, leading to degenerative processes. These diseases include, but are not limited to, Alzheimer's disease; age-related macular degeneration; cerebral β-amyloid angiopathy; prion diseases (such as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, Kuru); Parkinson's disease; multiple sclerosis; synucleinopathies (such as multiple system atrophy, Lewy body dementia); tau diseases (such as primary age-related tau dementia, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, chromosome-associated Parkinson's syndrome, vacuolet tau disease, Lytico-Bodig disease, gangliocytomas and ganglioneuromas, meningoangioma, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-associated neurodegeneration, and lipofuscinosis); frontotemporal degeneration; amyotrophic lateral sclerosis; Huntington's disease; trinucleotide repeat disorder; familial dementia; hereditary cerebral hemorrhage with thrombocytopenic purpura. Amyloidosis; Cadilla syndrome; Alexander's disease; Angelman's syndrome; Peyrenia; Seipin's disease; familial amyloid neuropathy; serine protease inhibitor lesions; amyloidosis (such as senile systemic, light chain, heavy chain, secondary, medial aortic, ApoAI, ApoAII, ApoAIV, lysozyme, fibrinogen, dialysis, cardiac atrium, cutaneous lichenification, corneal lactoferrin, apolipoprotein C2, apolipoprotein C3). Lect2, insulin, galactoglucan-7, keratin or enfviride amyloidosis); type II diabetes; inclusion body myositis / myopathy; cataract; retinitis pigmentosa with rhodopsin mutation; medullary thyroid carcinoma; pituitary prolactinoma; hereditary geniomorphic corneal dystrophy; Mallory bodies; pulmonary alveolar proteinosis; odontogenic (Pindborg) amyloid tumors; seminal vesicle amyloid; cystic fibrosis; sickle cell disease; plasma cell cachexia; exfoliation syndrome.

[0120] In a preferred embodiment, the disease to be treated is age-related macular degeneration, synucleinosis (preferably multiple system atrophy), or Parkinson's disease.

[0121] In a preferred embodiment, the antisense oligonucleotides of the present invention or pharmaceutical compositions containing them are capable of treating β-mannosin storage diseases, cholesterol ester storage diseases, CLN1 disease, CLN2 disease, CLN3 disease, CLN7 disease, Fabry disease, GM2-ganglioside storage diseases (Tessa II disease, AB variant, and Sandhoff disease), Krabbe disease, metachromatic leukodystrophy, type I / II / III / IV mucolipid storage diseases, type I mucopolysaccharidosis (Scheie syndrome, Hurler-Scheie syndrome, Hurler syndrome), type II mucopolysaccharidosis (Hunter syndrome), type III mucopolysaccharidosis (Sanfilippo syndrome type A, Sanfilippo syndrome type B), and type IV mucopolysaccharidosis (Morquio syndrome). Syndrome A), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), Niemann-Pick disease type C, Pompe disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, spinocerebellar ataxia, multiple sclerosis, amyotrophic lateral sclerosis, multiple system atrophy, frontotemporal dementia, Lewy body disease, and Friedreich ataxia.

[0122] Treatment The present invention also relates to treatment methods involving any pharmaceutical composition of the present invention in subjects in need, particularly those suffering from cellular storage disorders involving autophagy defects, such as those suffering from any of the aforementioned diseases.

[0123] These pharmaceutical compositions contain a nucleic acid or vector encoding the mutant TFEB of the present invention, or the mutant polypeptide itself, or the molecule of the present invention (especially ASO) or any other tool capable of in situ skipping exon 3 of TFEB.

[0124] The pharmaceutical compositions of the present invention can generally be administered via parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intrathecal, intracranial, intraperitoneal, intranasal, or intramuscular routes. The typical route of administration for the compositions of the present invention is intravenous or intrathecal, although other routes are equally effective.

[0125] Therefore, the treatment method of the present invention preferably includes intrathecal administration of the pharmaceutical composition (which has been proven effective in humans, see Miller et al.). Lancet Neurol. 2013 May;12(5):435-42; and in primates, see Peters S. et al., Pharmaceutics . 2022 Jan 15;14(1):200).

[0126] definition Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the fields of chemistry, biochemistry, cell biology, molecular biology, and medicine.

[0127] As used throughout this document, the terms “a” and “an” mean, in their meaning, “at least one,” “at least first,” “one or more,” or “one or more” of the compounds or steps referenced, unless the context otherwise requires.

[0128] As used herein, the term “and / or” includes the meanings of “and,” “or,” and “all or any other combination of elements connected by the term.”

[0129] As used herein, when used to define products, compositions, cells, uses, and methods, the terms “comprising” (and any form of inclusion, such as “comprising”), “having” (and any form of having, such as “having”), “including” (and any form of including, such as “including”), or “containing” (and any form of containing, such as “containing”) are open-ended and do not exclude additional, unreferenced elements or method steps. Thus, a polypeptide “comprising” the amino acid sequence when the amino acid sequence may be part of the final (and / or complete) amino acid sequence of the polypeptide. Such a polypeptide may have up to several hundred additional amino acid residues (e.g., the linker and antioxidant portion described herein). “Comprising of” means excluding any other components or steps, and “consistently comprising” means excluding any other essential and important components or steps (however, not excluding other minor / unimportant components or steps). In this disclosure, the terms “comprising,” “comprising,” and “consistently comprising” may be used interchangeably if necessary.

[0130] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” or “sequence of nucleic acid,” “polynucleotide,” “oligonucleotide,” “polynucleotide sequence,” and “nucleotide sequence” are used equivalently in this specification to refer to double-stranded DNA, single-stranded DNA, and transcripts of said DNA.

[0131] As used herein, the term "oligonucleotide molecule" refers to a short polymer of nucleic acids (single-stranded or double-stranded DNA or RNA molecules), typically 10-100 nucleotides, bases, or base pairs (preferably 12 to 40 nucleic acid residues, more preferably 13 to 39 nucleic acid residues, more preferably 14 to 38 nucleic acid residues, more preferably 15 to 37 nucleic acid residues, more preferably 16 to 36 nucleic acid residues, more preferably 17 to 35 nucleic acid residues, more preferably 18 to 34 nucleic acid residues, more preferably 14 to 22 nucleic acid residues, more preferably 15 to 21 nucleic acid residues, more preferably 16 to 20 nucleic acid residues, more preferably 17 to 19 nucleic acid residues, and even more preferably 18 nucleic acid residues). It includes, for example, aptamers, interfering RNA, and other well-known polymers.

[0132] In this specification, the term "polypeptide" will be used to refer to "protein" or "peptide" interchangeably.

[0133] It should be understood that this invention does not relate to the genomic nucleotide sequences in their natural chromosomal environment, i.e., their natural state. It includes sequences that have been “isolated” and / or “purified,” i.e., sequences that have been removed directly or indirectly from their natural chromosomal environment, for example, through replication, synthesis, etc.

[0134] The term "variant" here is intended to refer to a polynucleotide or polypeptide whose sequence contains an individual variation (SNP) compared to the reference nucleic acid sequence or amino acid sequence of the present invention.

[0135] In contrast, the term "homologous" refers to polypeptides or polynucleotides whose sequences have been significantly modified relative to a reference nucleic acid or amino acid sequence, such as deletions, truncations, extensions, chimeric fusions, and / or numerous mutations, such that their nucleic acid or amino acid sequences ultimately show at least 80%, preferably 90% or 95% identity with the reference nucleic acid or amino acid sequence.

[0136] For the purposes of this invention, the term "percentage of identity" between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotide or amino acid residues between two sequences to be compared, obtained after optimal alignment, said percentage being purely statistical, and the differences between the two sequences being randomly distributed and spanning their length. Sequence comparisons between two nucleic acid or amino acid sequences are conventionally performed by comparing these sequences after optimal alignment, said comparison being done through fragments or "comparison windows" to identify and compare local regions of sequence similarity. Optimal alignments for the sequences used for comparison can be generated, in addition to manually, by the global homology algorithm of Needleman and Wunsch (1970) [J. Mol. Biol. 48:443]. The percentage of identity between the two sequences is calculated by determining the number of identical positions of identical nucleotide or amino acid residues between the two sequences, dividing said number of identical positions by the total number of positions, and multiplying the result by 100. For example, the needle program, available on the website ebi.ac.uk, can be used with the default parameters (specifically for the parameters “vacancy open”: 10 and “vacancy spread”: 0.5; the chosen matrix is, for example, the “BLOSUM 62” matrix proposed by the program), and the percentage of identity between the two sequences to be compared is calculated directly by the program.

[0137] The present invention also relates to nucleic acid molecules that specifically hybridize with the nucleic acid molecules of the present invention (particularly oligonucleotide molecules of the present invention that specifically hybridize with their nucleic acid molecular targets, which are preferably RNA or transcripts, more preferably messenger RNA (mRNA), and even more preferably precursor mRNA (premRNA; i.e., primary transcripts that become messenger RNA (mRNA) after processing (particularly splicing)). Specific hybridization is preferably observed under highly stringent conditions, i.e., when temperature and ionic strength conditions are selected to allow hybridization between two complementary nucleic acid molecules / fragments. For example, highly stringent conditions can be as follows. DNA-DNA or DNA-RNA hybridization is performed in two steps: (1) in a phosphate buffer (20 mM, pH) containing 5*SSC (1*SSC corresponds to 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10*Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA. (7.5) Pre-hybridize at 42°C for 3 hours; (2) Perform actual hybridization for 20 hours at a temperature depending on the probe size (i.e., 42°C for probes >100 nucleotides), followed by two washes at 20°C in 2*SSC + 2% SDS for 20 minutes each, and one wash at 20°C in 0.1*SSC + 0.1% SDS for 20 minutes each. For probes >100 nucleotides, perform a final wash at 60°C in 0.1*SSC + 0.1% SDS for 30 minutes. Based on the teachings of Sambrook et al., 1989, those skilled in the art will adjust the above-described highly stringent hybridization conditions for determining the size of polynucleotides for larger or smaller oligonucleotides. Examples of the nucleic acid molecules are given below as “diagnostic tools”.

[0138] As used herein, the term "treatment" may be used to describe the prevention, improvement, prevention, or cure of lysosomal storage diseases and conditions characterized by lysosomal dysfunction and / or one or more related symptoms. For example, treatment of existing lysosomal storage diseases and conditions characterized by lysosomal dysfunction may alleviate, improve, or completely eliminate said conditions, or prevent their worsening. Preventive treatment may reduce the risk of disease progression and / or mitigate the severity of disease progression.

[0139] As used herein, the term "vector" refers to a vehicle, preferably a nucleic acid molecule or viral particle, containing elements necessary to allow the delivery, proliferation, and / or expression of any nucleic acid molecule within a host cell or subject. This term includes cloning vectors (vectors for maintenance), expression vectors (vectors that direct the expression of nucleic acid molecules linked to them in various host cells or subjects), extrachromosomal vectors (e.g., multicopy plasmids), integration vectors (e.g., nucleic acid molecules designed to integrate into the host cell genome and generate additional copies during host cell replication), shuttle vectors (e.g., functional in both prokaryotic and / or eukaryotic hosts), and transfer vectors (e.g., for transferring nucleic acid molecules from a viral genome). As part of the description, vectors can be naturally occurring, genetically derived, synthetic, or artificial, or some combination of natural and artificial genetic elements. Each vector contains different components depending on its function (e.g., expression of heteropolynucleotides) and its compatibility with the specific host cell in which it resides.

[0140] Vector components typically include, but are not limited to: origin of replication (OCR), multiple cloning site containing restriction sites, selection marker genes and / or reporter genes, one or more regulatory sequences such as, for example, promoters, ribosome binding sites (RBS), signal sequences, nucleic acid molecule insertion sequences, and transcription termination sequences. Selection marker genes and reporter genes are used to select cells that have already inserted and expressed the desired vector. RBS is used to initiate ribosome recruitment at the initiation stage of nucleic acid molecule translation. Signal sequences allow the translated protein to be recognized and processed by the host cell (i.e., cleaved by signal peptidases).

[0141] Furthermore, the term "vector" must be broadly understood to include both natural and non-natural vectors, such as mRNA, plasmids, viruses, retroviruses, EBV-derived free organisms, granules, bacteriophages, and artificial chromosomes. Typically, such vectors are commercially available (e.g., at Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or obtainable from depositary institutions such as the American Center for Type Culture Collection (ATCC, Rockville, Md.), or are already the subject of numerous publications describing their sequences, organization, and production methods, allowing those skilled in the art to apply them. This invention also includes vectors (e.g., plasmid DNA and mRNA) that are complexed with lipids or polymers to form particulate structures such as liposomes, lipid complexes, or nanoparticles. The selection of a suitable vector will depend primarily on the size of the nucleic acid molecule to be inserted and the specific host cell to which the vector will be transformed.

[0142] Vectors can remain free (i.e., not integrated into the host cell genome) or they can integrate ("stable" or "temporarily") into the host cell genome, either as a result of the initial transformation of the host cell or as a result of subsequent recombination and / or repair events. However, they remain "heterogeneous" compared to the host cell and its natural genome.

[0143] As used herein, the term "subject" refers to any mammal that may benefit from the treatment of the present invention. In particular, the subject is a human. Attached Figure Description

[0144] Figure 1 This study demonstrates the effect of lentiviral infection with TFEB exon 3 deletion (TFEB-∆Ex3) and TFEB with exon 3 (TFEB-WT) on TFEB translocation in HeLa cells. a. HeLa cells were transiently infected with lentiviruses encoding TFEB-WT or TFEB-∆Ex3 and analyzed by microscopy. b. Cells were analyzed to calculate the percentage of TFEB translocation, i.e., the ratio between the nuclear fluorescence intensity and the cellular c-Myc fluorescence intensity. Results are presented as mean ± SEM; ****p<0.0001, two-tailed unpaired test. WT: wild-type.

[0145] Figure 2 Evaluation of the effect of TFEB exon 3 deletion (TFEB-∆Ex3) on TFEB translocation in iPSC-derived neurons. a. iPSC-derived neurons were transiently infected with TFEB-WT or TFEB-∆Ex3, treated with 100 mM trehalose or a carrier, and analyzed by microscopy. b. The neurons described in a. were analyzed to calculate the percentage of TFEB translocation, i.e., the ratio between the nuclear and cellular TFEB fluorescence intensities. c. Mean cell intensity, calculated as the ratio between total TFEB intensity density and total cell area. Results are presented as mean ± SEM; *p < 0.05, one-way ANOVA, Tukey multiple comparison test. ns: not significant, WT: wild-type.

[0146] Figure 3 Evaluation of the effect of stable lentiviral infection with TFEB exon 3 deletion (TFEB-∆Ex3) on translocation in HeLa cells. a. HeLa cells were stably infected with TFEB-WT or TFEB-∆Ex3 and analyzed by microscopy. b. Cells described in a. were analyzed to calculate the percentage of TFEB translocation, i.e., the ratio between the nuclear and cellular TFEB fluorescence intensities. Results are presented as mean ± SEM; ****p<0.0001, two-tailed unpaired test. WT: wild-type.

[0147] Figure 4Evaluation of the effect of stable lentiviral infection with TFEB exon 3 deletion (TFEB-∆Ex3) on CLEAR activation in HeLa cells. a. HeLa cells were stably infected with TFEB-WT or TFEB-∆Ex3, followed by transient infection with CLEAR lentivirus, and analyzed by microscopy. b. Cells described in a. were analyzed to calculate the percentage of GFP fluorescence intensity. Results are presented as mean ± SEM; ****p<0.0001, one-way ANOVA, Tukey multiple comparison test. LV: Lentiviral infection; WT: Wild-type.

[0148] Figure 5 Evaluation of the effect of stable lentiviral infection with TFEB exon 3 deletion (TFEB-∆Ex3) on LAMP1 expression in HeLa cells. a. HeLa cells were stably infected with TFEB-WT or TFEB-∆Ex3 and analyzed by microscopy. b, cb analyses were performed on the cells described in a to calculate the percentage of LAMP1 fluorescence intensity (a) or dot intensity (b). Results are presented as mean ± SEM; p < 0.0001, one-way ANOVA, Tukey multiple comparison test. NT: untransfected; WT: wild-type.

[0149] Figure 6 Evaluation of the effect of stable lentiviral infection with TFEB exon 3 deletion (TFEB-∆Ex3) on beclin-1 expression in HeLa cells. a. HeLa cells were stably infected with TFEB-WT or TFEB-∆Ex3 and analyzed by microscopy. b, cb analyses were performed on the cells described in a to calculate the percentage of beclin-1 fluorescence intensity (a) or dot intensity (b). Results are presented as mean ± SEM; p < 0.0001, one-way ANOVA, Tukey multiple comparison test. NT: untransfected; WT: wild-type.

[0150] Figure 7 Evaluation of the effect of TFEB exon 3 deletion (TFEB-DEx3) overexpression on reducing storage products in a Niemann-Pick disease Hap knockout cell model. Hap1 control or NPC- cells were transiently infected with lentiviruses encoding TFEB-WT (black) or TFEB-DEx3 (white) and analyzed by microscopy. a. GM1 ganglioside spots in each cell were analyzed using cholera toxin labeling. b. Cholesterol spots in each cell were analyzed using filipin labeling. Results are presented as mean ± SEM; **p<0.01, two-way ANOVA, Sidak multiple comparison test. NPC-: Nieman-Pick model; WT: wild type.

[0151] Figure 8: Summary of the power of all tests for exon 3 skipping of TFEB in ASO. Results are expressed as ΔΔCt TFEB-ΔEx3 normalized to invalid scramble. Location of TFEB gene: GRCh38:CM000668.2; chromosome 6: 41,683,978 - 41,736,259 reverse strand; Location of TFEB transcript ENST00000373033: chromosome 6: 41,683,978 - 41,735,608 reverse strand; Location of TFEB exon 3: chromosome 6: 41,690,917 – 41,690,663. We identified a hotspot between ASO#15 and ASO#31, and within this region, two subregions lie between ASO#15.3 to ASO#17.3 and ASO#18.4 to ASO#20. Data ≥ 1 are shown in bold.

[0152] Figure 9 The efficacy of TFEB exon 3 skipping was investigated using the ASO walk method. RT-qPCR analysis of skipping TFEB in HeLa (a) or SH-SY5Y (b) cells after 6 hours and 66 hours of ASO treatment was performed. Curves representing the potential efficacy of 18 ASO skipping TFEB exons 3 are shown. mRNA expression was normalized to GAPDH, and results are expressed as ΔΔCt TFEB-ΔEx3.

[0153] Figure 10 Evaluation of the effects of ASO#16 on PCR, sequencing, and TFEB protein expression in HeLa cells. a. Agarose gels of approximately 300 and 500 bp amplification products obtained by conventional PCR. HeLa cells were treated with ASO null scrambling (ASO#Scr) or ASO#16. b. Sequencing of PCR products obtained under (ASO#16 conditions). Note that skipping was performed without shifting, addition, deletion, or modification of nucleotides in exons 2 and 4. c. Western blots showing protein expression in HeLa cells treated with ASO null scrambling (ASO#Scr) or ASO#16 at eight different doses. Note the expression of truncated TFEB protein, smaller than the expected size of wild-type TFEB protein, corresponding to skipping of exon 3. Scr: null scrambling.

[0154] Figure 11Evaluation of the effect of ASO#16 on CLEAR network activation in HeLa cells. HeLa cells were infected with lentivirus encoding the nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR) and treated with three low doses of either ASO nucleus scrambled (ASO#Scr) or ASO#16. Data showed that ASO#16 activated the CLEAR network even at very low doses compared to ASO#Scr. Results are presented as mean ± SEM; p < 0.0001, one-way ANOVA, Šídák multiple comparison test.

[0155] Figure 12 Evaluation of the effects of ASO#16 and ASO#16.3 on cholesterol deposition in the NPC model. HeLa cells were infected with lentiviral sh-RNA targeting the NPC gene and treated with ASO nullified disorder (ASO#Scr); ASO#16 or ASO#16.3. Cholesterol spots in each cell were analyzed using filipin labeling. Data showed that ASO#16 and ASO#16.3 reduced cholesterol deposition in the NPC model compared with ASO#Scr. Results are presented as mean ± SEM; *p<0.05, one-way ANOVA, Tukey multiple comparison test. NPC: Nieman-Pick model.

[0156] Figure 13 Evaluation of the effect of ASO#16 on ARPE-19 cells. a. Curves representing the potential efficacy of ASO#16 or ASO#Scr skipping TFEB exon 3 in ARPE-19 cells. mRNA expression was normalized to GAPDH, and results are expressed as ΔΔCtTFEB-ΔEx3. b. Percentage of TFEB translocation, i.e., the ratio between the nuclear and cellular TFEB fluorescence intensity in ARPE-19 cells treated with different doses of ASO#16 or ASO#Scr. c. ARPE-19 cells were infected with lentivirus encoding the nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR) and treated with different doses of ASO nucleus scrambled (ASO#Scr) or ASO#16. Results are mean ± SEM; *p<0.05, two-way ANOVA, Šídák multiple comparison test.

[0157] Figure 14Evaluation of the effect of ASO#16 on the ARPE-19 cell model of AMD. ARPE-19 cells were treated with 20 nM ASO#Scr or ASO#16, followed by overloading with ferric ammonium citrate at 48 or 66 h (groups B and D, respectively). Data are expressed as iron spot intensity for each spot. It was noted that ASO#16 significantly reduced iron deposition at both time points compared to ASO#Scr. Results are expressed as mean ± SEM; *p<0.05, two-way ANOVA, Sidak multiple comparison test.

[0158] Figure 15 Evaluation of the effects of ASO#16 and ASO#16.3 on α-synuclein deposition in an α-synuclein precursor filament (PFF) model. Differentiated SH-SY5Y cells were treated with ASO null scrambled (ASO#Scr); ASO#16 or ASO#16.3, along with 5 µg / ml precursor filaments. PFF spots in each cell were analyzed using an anti-phospho-α-synuclein (Ser129) antibody labeling. Data showed that ASO#16 and ASO#16.3 reduced α-synuclein deposition in the PFF model compared to ASO#Scr. Results are presented as mean ± SEM; *p < 0.05, one-way ANOVA, Tukey multiple comparison test. PFF: pre-fabricated precursor filaments. Detailed Implementation

[0159] Example 1. Evaluation of the effect of TFEB exon 3 deletion (TFEB-∆Ex3) on translocation in HeLa cells plan HeLa cells were seeded (5,000 cells / well) in DMEM 96-well plates supplemented with 10% SVF and incubated at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0160] After incubation, cells were infected with lentiviruses encoding TFEB-∆Ex3 or TFEB. Both lentiviruses include a c-Myc tag attached to the TFEB open reading frame, allowing differentiation between exogenously expressed TFEB (wild-type or lacking the third exon) and endogenous TFEB.

[0161] HeLa cells were analyzed 72 hours later.

[0162] Cells were fixed with 4% formaldehyde for 10 min and then washed three times with PBS before immunohistochemistry.

[0163] In short, the primary antibody was diluted in blocking solution (PBS + 2% goat serum + 0.1% triton), incubated overnight at 4°C, then washed three times, and the secondary antibody was incubated in blocking solution at RT for 1 hour for immunostaining.

[0164] To acquire images, at least 30 image domains were obtained in each well of a 96-well plate using a Cellinsight CX7 high content analyzer (Thermo Scientific).

[0165] c-Myc labeling allows for the calculation of the percentage of TFEB translocation in each infected cell (the ratio between the cell nucleus and the cell's c-Myc fluorescence intensity).

[0166] All data are presented as mean ± SEM. Data were analyzed using a two-tailed unpaired Student's t-test to detect significant differences between the two groups. Statistical significance was defined as p < 0.05.

[0167] result like Figure 1 As shown, in HeLa cells, the nuclear translocation of TFEB-∆Ex3 is significantly superior to that of wild-type TFEB.

[0168] 2. Evaluation of the effect of TFEB exon 3 deletion (TFEB-∆Ex3) on translocation in iPSC-derived neurons plan iPSC-derived neurons were seeded at a density of 150 cells / well in N2B27 supplemented with 200 µL of BDNF (20 ng / ml), CDKi (3.3 µM), AMPc (100 µM), and DAPT (10 µM) in 96-well plates coated with poly-L-ornithine and laminin.

[0169] Nine days after thawing, neurons were infected with lentiviruses encoding TFEB-∆Ex3 or TFEB, and analyzed after 72 hours. Both lentiviruses included a c-Myc tag attached to the TFEB open reading frame, allowing differentiation between exogenously expressed TFEB (wild-type or lacking the third exon) and endogenous TFEB.

[0170] After 24 hours, the culture medium was changed, and the neurons were incubated for 4 days before treatment with 100 mM trehalose or the carrier. Two days later, the neurons were fixed with 4% paraformaldehyde for 10 min, and then washed three times with PBS before immunohistochemistry.

[0171] In short, the primary antibody was diluted in blocking solution (PBS + 2% donkey serum + 0.1% triton), incubated overnight at 4°C, then washed three times, and the secondary antibody was incubated in blocking solution at RT for 1 hour for immunostaining.

[0172] To acquire images, at least 40 image domains were obtained in each well of a 96-well plate using a Cellinsight CX7 high content analyzer (Thermo Scientific).

[0173] c-Myc labeling allows for the calculation of the percentage of TFEB translocation (the ratio between the cell nucleus and the cellular TFEB fluorescence intensity) and the mean cellular TFEB intensity (the ratio between the total TFEB intensity density and the total cell area) for each infected cell.

[0174] All data are presented as mean ± SEM. Data were analyzed using one-way ANOVA and Tukey's multiple comparison test to detect significant differences between groups. Statistical significance was defined as p < 0.05.

[0175] result like Figure 2 As shown, the nuclear translocation of TFEB-∆Ex3 is significantly superior to the nuclear translocation of wild-type TFEB in human neurons, and also superior to the nuclear translocation of TFEB induced by the potent translocation agent trehalose.

[0176] Under all conditions, this translocation was observed to have similar TFEB expression levels (Figure c).

[0177] 3. Verification of TFEB translocation in HeLa cells stably expressing TFEB-∆Ex3 plan To generate a stable cell line, HeLa cells were seeded (40,000 cells / well) in DMEM 24-well plates supplemented with 10% SVF and incubated at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0178] After incubation, cells were infected with lentiviruses encoding TFEB-∆Ex3 or TFEB and divided into 6 wells after 48 hours.

[0179] Cell selection was performed by treating cells with geneticin (500 µg / mL; 6 wells) after 24 hours.

[0180] Every 3 days, cells were isolated in T75 flasks and treated with genimycin (500 µg / mL), then frozen for further experiments.

[0181] To evaluate TFEB translocation in these cell lines, HeLa cells were seeded (5,000 cells / well) in DMEM 96-well plates supplemented with 10% SVF and incubated at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0182] 48 hours after incubation, cells were fixed with 4% formaldehyde for 10 min and then washed three times with PBS before immunohistochemistry.

[0183] Cells were permeabilized with 0.1% Triton for 10 minutes at room temperature and incubated in blocking solution (PBS + 2% goat serum + 0.1% Triton) for 30 minutes. The primary antibody was diluted in blocking solution and incubated overnight at 4°C. Cells were then washed three times and incubated with secondary antibody in blocking solution for 1 hour at RT for immunostaining.

[0184] To acquire images, at least 15 image domains were obtained in each well of a 96-well plate using a Cellinsight CX7 high content analyzer (Thermo Scientific).

[0185] TFEB labeling allows for the calculation of the percentage of translocation for each infected cell (the ratio between the TFEB fluorescence intensity in the cell nucleus and the cell).

[0186] All data are presented as mean ± SEM. Data were analyzed using a two-tailed unpaired Student's t-test to detect significant differences between the two groups. Statistical significance was defined as p < 0.05.

[0187] result like Figure 3 As shown, in stably transfected HeLa cells, the nuclear translocation of TFEB-∆Ex3 was significantly superior to that of wild-type TFEB.

[0188] 4. Evaluation of the effect of TFEB exon 3 deletion (TFEB-∆Ex3) overexpression on CLEAR network activation. plan HeLa cells stably expressing TFEB-∆Ex3 or TFEB-WT (as described in Example 3 above) were seeded (5,000 cells / well) in DMEM 96-well plates supplemented with 10% SVF and incubated at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0189] After incubation, the cells are temporarily infected with a lentivirus that encodes the nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR).

[0190] After 48 hours, the cells were fixed with 4% formaldehyde for 10 minutes, and then washed three times with PBS before the GFP intensity was read.

[0191] To acquire images, at least 15 image domains were obtained in each well of a 96-well plate using a Cellinsight CX7 high content analyzer (Thermo Scientific).

[0192] Then the GFP fluorescence intensity in each cell was measured.

[0193] For each cell line (TFEB-∆Ex3 or TFEB-WT), data were calculated as a percentage of controls (i.e., no LV-CLEAR). Results are presented as mean ± SEM. Data were analyzed by one-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was defined as p < 0.05.

[0194] result like Figure 4 As shown, the CLEAR network was significantly more active in cells that stably expressed TFEB-∆Ex3 compared to cells expressing wild-type TFEB.

[0195] 5. Evaluation of the effect of TFEB exon 3 deletion (TFEB-∆Ex3) overexpression on LAMP1 expression plan HeLa cells stably expressing TFEB-∆Ex3 (as described in Example 3 above) or TFEB-WT were seeded (5,000 cells / well) in DMEM 96-well plates supplemented with 10% SVF and incubated at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0196] Four days later, the cells were fixed with 4% formaldehyde for 10 min and then washed three times with PBS before the immunohistochemical protocol.

[0197] Cells were permeabilized with 0.1% Triton for 10 minutes at room temperature and incubated in blocking solution (PBS + 2% goat serum + 0.1% Triton) for 30 minutes. LAMP1 primary antibody was diluted in blocking solution and incubated overnight at 4°C. Cells were then washed three times and incubated with secondary antibody in blocking solution for 1 hour at RT for immunostaining.

[0198] To acquire images, at least 10 image domains were obtained in each well of a 96-well plate using a Cellinsight CX7 high content analyzer (Thermo Scientific).

[0199] For each group, we calculated the total intensity (i.e., LAMP1 intensity) and the intensity of the spot (i.e., LAMP1 spot intensity) in the wells. For each cell line (TFEB-∆Ex3 or TFEB-WT), data were calculated as a percentage of controls (i.e., untransfected, NT). Results are presented as mean ± SEM. Data were analyzed by one-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was set as p < 0.05.

[0200] result like Figure 5 As shown, LAMP1 was significantly more expressed in cells that stably expressed TFEB-∆Ex3 compared to cells expressing wild-type TFEB.

[0201] 6. Evaluation of the effect of TFEB exon 3 deletion (TFEB-∆Ex3) overexpression on Beclin-1 expression plan HeLa cells stably expressing TFEB-∆Ex3 (as described in Example 3 above) or TFEB-WT were seeded (5,000 cells / well) in DMEM 96-well plates supplemented with 10% SVF and incubated at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0202] Four days later, the cells were fixed with 4% formaldehyde for 10 min and then washed three times with PBS before the immunohistochemical protocol.

[0203] Cells were permeabilized with 0.1% Triton for 10 minutes at room temperature and incubated in blocking solution (PBS + 2% goat serum + 0.1% Triton) for 30 minutes. The primary antibody Beclin-1 was diluted in blocking solution and incubated overnight at 4°C. Cells were then washed three times and incubated with secondary antibody in blocking solution for 1 hour at RT for immunostaining.

[0204] To acquire images, at least 10 image domains were obtained in each well of a 96-well plate using a Cellinsight CX7 high content analyzer (Thermo Scientific).

[0205] For each group, total intensity (i.e., Becin-1 intensity) and spot intensity (i.e., Becin-1 spot intensity) in the wells were calculated. For each cell line (TFEB-∆Ex3 or TFEB-WT), data were calculated as a percentage of controls (i.e., untransfected, NT). Results are presented as mean ± SEM. Data were analyzed by one-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was set as p < 0.05.

[0206] result like Figure 6 As shown, compared with cells expressing wild-type TFEB, Beclin-1 was significantly more expressed in cells that stably expressed TFEB-∆Ex3.

[0207] 7. Evaluation of the effect of TFEB exon 3 deletion (TFEB-Ex3) on reducing storage products in a Niemann-Pick disease Hap gene knockout cell model. Target The goal of this study was to evaluate the effects of TFEB or TFEB-∆Ex3 overexpression on storage products in a haploid HAP1 knockout cell line model (edited by CRISPR / Cas, containing edits or deletions encoding target exons). The chosen haploid cell model was the Niemann-Pick disease model, characterized by the deposition of ganglioside GM1 and cholesterol.

[0208] plan Hap1 cells (Horizon Discovery) were seeded in DMEM (5,000 cells / well).

[0209] 24 hours later, cells were infected with lentiviruses encoding TFEB-∆Ex3 or TFEB. Both lentiviruses include a c-myc tag attached to the TFEB open reading frame, allowing differentiation between exogenously expressed TFEB (wild-type or lacking the third exon) and endogenous TFEB. Cells were fixed with 4% formaldehyde 4 days later.

[0210] Cells were permeabilized with 0.1% Triton for 10 min at room temperature. Immunostaining was performed at dilutions of cholera toxin (1 / 1000) or filipin (1 / 50).

[0211] To acquire images, at least 10 image domains were obtained in each well of a 96-well plate using a Cellinsight CX7 high content analyzer (Thermo Scientific).

[0212] For each case, we counted the number of spots per cell (cholera toxin or filipin). Results are presented as mean ± SEM. Data were analyzed by two-way ANOVA and subsequent Sidak multiple comparison test to detect significant differences between groups. Statistical significance was defined as p < 0.05.

[0213] result Figure 7 Overexpression of TFEB-∆Ex3 (rather than TFEB-WT) reduced the accumulation of GM1 gangliosides and cholesterol in the Niemann-PickHap1 cell line.

[0214] 8. Screening of ASOs designed using the ASO-walking method in HeLa cells.

[0215] Target The goal of this study was to evaluate the efficiency of ASOs in skipping TFEB exon 3 in HeLa cells using real-time quantitative PCR. 112 ASOs were designed using the ASO-walking method. Specifically, 18-nucleotide-long ASOs were moved 1 to 5 nucleotides to cover the entire exon 3 of TFEB, as well as 50 nucleotides upstream of exon 3 (in the intron following exon 3) and 48 nucleotides downstream of exon 3 (in the intron preceding exon 3). The sequences of the ASOs (SEQ ID NO: 17 to SEQ ID NO: 128) are shown in Table 3 above.

[0216] plan HeLa cells were seeded (7,500 cells / well) in standard medium (DMEM + 10% SVF).

[0217] Twenty-four hours later, cells were treated with different concentrations of ASO in lipofectamine + DMEM for 6 hours. The cells were then incubated in DMEM + 10% SVF for 66 hours.

[0218] For quantitative real-time PCR, total RNA was extracted using the RNeasy Plus kit (Qiagen) according to the manufacturer's instructions, and reverse transcription (RT) was performed using the high-capacity cDNA reverse transcription kit (Thermofisher).

[0219] The following primers were run using iTAq Sybr green (Biorad): human TFEB (fw: AGCAGCCACCTGAATGTGTA (SEQ ID NO.: 133); rev: GAGCTCTCGCTTCTGGGTC (SEQ ID NO.: 134)); human TFEB skipping (fw: GGGAGGTGTTGAAGTTGGATGA (SEQ ID NO.: 135); rev: TGGGCATCTGCATTTCAGGA (SEQ ID NO.: 136)); GAPDH (fw: ATGACATCAAGAAGGTGGTG (SEQ ID NO.: 137); rev: CATACCAGGAAATGAGCTTG (SEQ ID NO.: 138)). The results were analyzed using CFX Manager software and normalized to the GAPDH mRNA content of each sample.

[0220] result Figure 8 The skipping efficiency of ASO is shown. Results are expressed as ΔΔCt TFEB-ΔEx3 normalized to invalid scrambles. Location of the TFEB gene: GRCh38:CM000668.2; chromosome 6: 41,683,978 - 41,736,259 reverse strand; Location of the TFEB transcript ENST00000373033: chromosome 6: 41,683,978 - 41,735,608 reverse strand; Location of exon 3 of TFEB: chromosome 6: 41,690,917 – 41,690,663. We found a hotspot between ASO#15 and ASO#31, and within this region, two subregions lie between ASO#15.3 to ASO#17.3 and ASO#18.4 to ASO#20. Data ≥ 1 are shown in bold.

[0221] We identified a hotspot between the ASO#15 (start: GRCh38.p14 chromosome 6: 41,690,897 reverse strand) and ASO#31 (end: GRCh38.p14 chromosome 6: 41,690,800 reverse strand) sequences that allowed exon 3 of TFEB to skip. Preferably, we identified two subregions located between ASO #15.3 (start: GRCh38.p14 chromosome 6: 41,690,894 reverse strand) to #17.3 (end: GRCh38.p14 chromosome 6: 41,690,867 reverse strand) and #18.4 (start: GRCh38.p14 chromosome 6: 41,690,878 reverse strand) to #20 (end: GRCh38.p14 chromosome 6: 41,690,855 reverse strand), where 18 ASOs identified in these two regions showed strong potential for exon 3 skipping in TFEB.

[0222] For all humans, the TFEB (ENSG00000112561) gene is located at HGNC:11753; position: GRCh38.p14 chromosome 41,683,978-41,736,259 reverse strand; GRCh38:CM000668.2.

[0223] 9. RT-qPCR confirmed the exon 3 skipping efficiency of TFEB in HeLa and SH-SY5Y cells at low doses. Target The goal of this study was to evaluate the efficiency of our identified 18 ASOs in TFEB exon 3 skipping in HeLa and SH-SY5Y cells using real-time quantitative PCR.

[0224] plan HeLa cells were seeded (7,500 cells / well) in DMEM + 10% SVF.

[0225] SH-SY5Y cells were seeded (16,700 cells / well) in 96-well plates in DMEM + 10% SVF (pre-coated with laminin + poly-L-ornithine).

[0226] After 24 hours, the cells were treated with ASO (#15.3 to #17.3 and #18.4 to #20, at 1.25 and 0.625 nM) in lipofectamine + DMEM for 6 hours. The cells were then incubated in DMEM + 10% SVF for 66 hours.

[0227] For quantitative real-time PCR, total RNA was extracted using the RNeasy Plus kit (Qiagen) according to the manufacturer's instructions and reverse transcribed (RT) using a high-capacity cDNA reverse transcription kit (Thermofisher).

[0228] Run primers for human TFEB, human skipped TFEB, or GAPDH using iTAq Sybr green (Biorad).

[0229] The results were analyzed using CFX Manager software and normalized to the GAPDH mRNA content of each sample.

[0230] result Figure 9 The exon 3 skipping efficiency of the TFEB gene in 18 ASOs tested at low doses is shown. Skipping efficiency varied by ASO, test dose, and cell line. Here, we highlight the significant skipping ability of ASO#16 and ASO#16.3 in low-dose testing and across two cell lines.

[0231] 10. Validation of the effects of ASO#16 on PCR, sequencing, and protein expression in HeLa cells. Target The goal of this study was to evaluate the effect of ASO#16 on skipping exon 3 in HeLa cells using PCR, sequencing of PCR products, and Western blotting.

[0232] plan HeLa cells were seeded (7,500 cells / well) in DMEM + 10%.

[0233] 24 hours later, cells were treated with ASO in 10 nM DMEM + lipofectamine for 6 hours. Then the cells were incubated in DMEM + 10% SVF for 66 hours.

[0234] For PCR, cells were lysed and total RNA extracted using the RNeasy Plus kit (Qiagen) according to the manufacturer's instructions. Reverse transcription (RT) was performed using a high-capacity cDNA reverse transcription kit (Thermofisher). Primers for human TFEB; human skipped TFEB or GAPDH were run using Platinum II hot-start (Green) PCR premix (Thermofisher). Samples were loaded onto agarose gels (1.5%) and purified using the QIAquick PCR purification kit (Qiagen) for Illumina sequencing.

[0235] For Western blotting, cells were seeded (1.7 M cells / T75) in DMEM + 10% SVF. After 24 h, cells were treated with ASO null scrambled (80 nM; SEQ ID NO: 129) or ASO#16 (SEQ ID NO: 36) at 0.625; 1.25; 2.5; 5; 10; 20; 40; 80 nM in DMEM + lipofectamine for 6 h. Cells were then incubated in DMEM + 10% SVF for 66 h.

[0236] Cells were lysed in RIPA buffer. Samples were centrifuged, sonicated, and quantified using a standard procedure following the Pierce BCA protein assay kit. Lysates were incubated in loading buffer at 70°C for 10 min, then loaded onto NuPAGE 10% Bis-Tris microgels and transferred to a membrane (Bio-Rad). A standard IHC protocol was used to expose TFEB.

[0237] result Figure 10 ASO#16 was shown to be able to skip exon 3 of TFEB. Sequencing of the PCR product revealed a TFEB-ΔEx3 sequence lacking the complete exon 3. The sequence did not show any nucleotide shifts, additions, or deletions in exons 2 or 4. Furthermore, ASO#16 allowed dose-dependent expression of the truncated protein of the expected size in HeLa cells.

[0238] 11. The impact of ASO#16 on CLEAR network activation Target The goal of this study was to evaluate the effect of ASO#16 (SEQ ID NO: 36) on the activation of the CLEAR network in HeLa cells.

[0239] plan HeLa cells were seeded in DMEM. After incubation, the cells were temporarily infected with a lentivirus that encodes the nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR).

[0240] Cells were treated for 6 h in DMEM + lipofectamine with ASO null scrambled or ASO#16 at concentrations of 0.625, 1.25, and 2.5 nM. Cells were then incubated in DMEM + 10% SVF for 66 h. Cells were fixed with 4% formaldehyde before GFP intensity readings. Images were acquired using a Cellinsight CX7 high-content analysis instrument (Thermo Scientific). GFP fluorescence intensity in each cell was then measured. For each group (dose), data were calculated as a percentage of the control (i.e., ASO#Scr). Results are presented as mean ± SEM. Data were analyzed by one-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was set as p < 0.05.

[0241] result Compared to invalid, out-of-order ASO, ASO#16 can activate the CLEAR network at a lower dose. Figure 11 ).

[0242] 12. Effects of ASO#16 and ASO#16.3 on cholesterol reduction in the Niemann-Pick cell model Target The goal of this study was to measure the effects of ASO#16 (SEQ ID NO:36) and ASO#16.3 (SEQ ID NO: 39) on deposited products, namely cholesterol, in the Hela Niemann-Pick cell model.

[0243] plan HeLa cells were seeded (40,000 cells / P16) in DMEM + 10% SVF.

[0244] After 24 hours, cells were briefly infected with sh-RNA, which targets the mRNA of one of the genes responsible for Niemann-Pick pathology (NPC).

[0245] After passage and seeding in P96 (5000 cells / well), cells were treated with 10 nM DMEM + lipofectamine containing null scrambled ASO, ASO#16, or ASO#16.3 for 6 h.

[0246] After 72 hours, the cells were fixed with 4% formaldehyde and then incubated with filipin for 2 hours.

[0247] Images were acquired using a Cellinsight CX7 high-content analysis instrument (Thermo Scientific). Data are presented as the number of filipin spots per cell. Results are presented as mean ± SEM. Data were analyzed by one-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was defined as p < 0.05.

[0248] result Both ASO#16 and ASO#16.3 reduced cholesterol deposition in HeLa cells in a Niemann-Pick disease model. Figure 12 ).

[0249] 13. Verify the effect of ASO#16 on ARPE-19 cells. Target The goal of this study is to use RT-qPCR to verify the effects of ASO#16 on TFEB exon 3 skipping, CLEAR network activation, and TFEB translocation in ARPE-19 cells. These ARPE-19 cells will form the basis for our research on age-related macular degeneration (AMD) cell models.

[0250] plan For RT-qPCR, ARPE-19 cells were seeded (10,000 cells / well) in DMEM-F12 + 10% SVF.

[0251] After 24 hours, ARPE-19 cells were treated for 6 hours in lipofectamine + DMEM-F12 with ASO null scrambled or ASO#16 at 0.625, 1.25, 2.5, 5, 10, and 20 nm concentrations. The cells were then incubated in DMEM-F12 + 10% SVF for 66 hours.

[0252] Cells were lysed and total RNA was extracted using the RNeasy Plus kit (Qiagen) according to the manufacturer's instructions, and reverse transcription (RT) was performed using a high-capacity cDNA reverse transcription kit (Thermofisher). Primers for human TFEB and human skipped TFEB were run using iTAq Sybr green (Biorad). Results were analyzed using CFX Manager software and normalized to the GAPDH mRNA content of each sample.

[0253] To evaluate CLEAR network activation and TFEB translocation, ARPE-19 cells were seeded (500,000 cells / T25) in DMEM-F12 + 10% SVF. After incubation, cells were temporarily infected with a lentivirus encoding the nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR). After 48 h, cells were seeded (10,000 cells / well) in DMEM-F12 + 10% SVF. Cells were treated for 6 h in lipofectamine + DMEM-F12 with ASO null scrambled or ASO#16 at 10; 20; 40 and 80 nm or 0.625; 1.25; 2.5; 5; 10; 20; 40 and 80 nm, respectively, for CLEAR and TFEB translocation assays. Cells were then incubated in DMEM-F12 + 10% SVF for 66 h. After incubation, cells were fixed with 4% formaldehyde, and the GFP intensity of each cell was then read. Standard TFEB immunostaining was performed as previously described.

[0254] Images were acquired using a Cellinsight CX7 high content analysis instrument (Thermo Scientific).

[0255] CLEAR data for each dose were calculated as a percentage of the control (i.e., ASO#Scr). TFEB translocation data were calculated as a percentage of TFEB translocation (the ratio between the TFEB fluorescence intensity of the cell nucleus and the cell).

[0256] Results are presented as mean ± SEM. Data were analyzed using two-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was defined as p < 0.05.

[0257] result ASO#16 effectively skips exon 3 of TFEB in a dose-dependent manner. Furthermore, compared to ineffective disordered ASO in ARPE-19 cells, ASO#16 promotes TFEB translocation and activates the CLEAR network. Figure 13 ).

[0258] 14. Evaluation of the effects of ASO#16 on a cell model of age-related macular degeneration (AMD). Target The goal of this study is to evaluate the effects of ASO#16 on the AMD cell model in ARPE-19.

[0259] We chose the iron excess cell model because substantial evidence confirms the role of iron dysregulation in the pathogenesis of AMD. Iron excess is clearly involved in ferroptosis, a newly discovered programmed cell death pathway that has been reported to be associated with the pathogenesis of RPE dysfunction in AMD (Sun et al., 2018; Totsuka et al., 2019; Gupta et al., 2023). Furthermore, iron can be deposited in the macula of AMD patients, particularly in the RPE and Bruch membrane (Hahn et al., 2003).

[0260] plan ARPE-19 cells were seeded (10,000 cells / well) in DMEM-F12 + 10% SVF.

[0261] Twenty-four hours later, ARPE-19 cells were treated with 20 nm ASO ineffective scrambled form or ASO#16 in lipofectamine + DMEM-F12 for 6 hours. After 6 hours, the medium was changed to standard medium (groups A and C) or enriched with 200 µM ferric ammonium citrate (groups B and D). For the latter, the iron medium was removed after 48 hours (group B) or 66 hours (group D). Groups A and C received standard medium replacement at 48 hours and 66 hours, respectively.

[0262] Data were acquired using a Cellinsight CX7 high content analysis instrument (Thermo Scientific). Iron deposition is represented as the intensity of each iron spot.

[0263] Results are presented as mean ± SEM. Data were analyzed using two-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was defined as p < 0.05.

[0264] result Figure 14 The data demonstrated iron deposition in ARPE-19 cells after 48 or 66 hours in the presence of ferric ammonium citrate, and highlighted the efficiency of ASO#16 in reducing this iron deposition in our AMD ARPE-19 model.

[0265] 15. Effects of ASO#16 and ASO#16.3 on α-synuclein aggregation in a SH-SY5Y differentiated cell α-synuclein precursor filament model. Target The goal of this study was to measure the effects of ASO#16 and ASO#16.3 on the deposited product, α-synuclein, in a SH-SY5Y α-synuclein precursor filament (PFF) cell model.

[0266] plan On day 0, SH-SY5Y cells (10,000 cells / well) were seeded in 96-well plates in DMEM + 10% hiSVF (pre-coated with laminin + poly-L-ornithine). ATRA / TPA-differentiated SH-SY5Y cells were treated in DMEM + lipofectamine with 40 nM nullified randomized ASO, ASO#16, or ASO#16.3 for 6 h (day 7). On day 10, cells were treated with precursor fibers (5 µg / ml), fixed with 4% formaldehyde on day 14, and then washed three times with PBS prior to the α-synuclein immunohistochemistry protocol. Images were acquired using a Cellinsight CX7 high-content analysis instrument (Thermo Scientific). Data are expressed as the number of α-synuclein spots per cell. Results are presented as mean ± SEM. Data were analyzed by one-way ANOVA and subsequent Tukey multiple comparison tests to detect significant differences between groups. Statistical significance was set as p < 0.05.

[0267] result Figure 15 Both ASO#16 and ASO#16.3 showed that they reduced α-synuclein aggregation in the α-synuclein precursor filament model of SH-SY5Y differentiated cells.

[0268] Summarize The data presented in Examples 1-15 demonstrate, through proof-of-concept using overexpression of the TFEB protein with exon 3 deletion (TFEB-ΔEx3), that compared to wild-type TFEB protein, TFEB-ΔEx3 protein unexpectedly i) exhibits significant nuclear localization (translocation), ii) increases the expression of BECLIN1 and LAMP-1 proteins, iii) activates the CLEAR network, and iv) reduces cholesterol deposition in the Niemann-Pick C model.

[0269] Furthermore, we demonstrated using the ASO walking method that two hotspots (start: GRCh38.p14 chromosome 6: 41,690,897-41,690,850 and 41,690,842-41,690,8000 reverse strands) were identified in the efficiency of TFEB skipping exon 3. By evaluating the skipping efficiency of TFEB at different doses, 18 ASOs showed strong skipping potential: ASO#15.3 (GRCh38.p14 chromosome 6: 41,690,894 reverse strand) to ASO#17.3 (end: GRCh38.p14 chromosome 6: 41,690,867 reverse strand) and ASO#18.4 (start: GRCh38.p14 chromosome 6: 41,690,878 reverse strand) to ASO#20 (end: GRCh38.p14 chromosome 6: 41,690,855 reverse strand).

[0270] Evaluation of these 18 ASOs at low doses highlighted the potency of two candidates (ASO#16 and ASO#16.3) in skipping TFEB exon 3: ASO#16- sequence: GCTGCAGATGGTAGGATG (SEQ ID NO:36); location: GRCh38.p14 chromosome 6: 41,690,892-41,690,875855 reverse strand.

[0271] ASO#16.3 sequence: ACTGCTGCAGATGGTAGG (SEQ ID NO:39); location: GRCh38.p14 chromosome 6: 41,690,889-41,690,872 reverse strand.

[0272] Based on this, we demonstrate that ASO#16 unexpectedly allows i) perfect exon 3 skipping (verified by sequencing); ii) expression of the protein TFEB-ΔEx3; and iii) activation of the CLEAR network. We also demonstrate that both ASO#16 and ASO#16.3 have the ability to reduce cholesterol deposition in the Niemann-Pick model. Finally, evaluating these data in a cell model of age-related macular degeneration (AMD) surprisingly demonstrates i) strong exon skipping efficiency of TFEB; ii) TFEB translocation; iii) activation of the CLEAR network in ARPE-19 cells; and iv) a significant reduction in iron deposition in the AMD cell model.

Claims

1. A vector containing a polynucleotide encoding a mutant TFEB protein, wherein the mutant TFEB protein does not contain the amino acid sequence encoded by natural exon 3, wherein the vector is preferably a plasmid or a viral vector.

2. A vector containing a polynucleotide encoding a mutant TFEB protein, wherein the mRNA sequence does not contain the mRNA sequence of SEQ ID NO:10, wherein the vector is preferably a plasmid or a viral vector.

3. The vector according to claim 1 or 2, wherein the amino acid sequence encoded by exon 3 and having the amino acid sequence of SEQ ID NO:6 or a variant thereof has been removed or deleted.

4. The vector according to any one of claims 1 to 3, wherein the mutant TFEB protein has a sequence selected from the group consisting of: variants or homologs of any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

5. The vector according to any one of claims 1 to 4, wherein the polynucleotide encoding the mutant TFEB protein has a sequence selected from the group consisting of SEQ ID NO:11-14 or variants thereof.

6. An oligonucleotide molecule capable of mediating exon hopping in exon 3 of the TFEB protein.

7. The molecule according to claim 6, which is capable of mediating exon skipping in exon 3 of the nucleic acid sequence encoding the TFEB protein.

8. The molecule according to any one of claims 6 or 7, wherein it is capable of mediating exon skipping of exon 3 of the nucleic acid sequence encoding the TFEB protein of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or a variant or homology thereof.

9. The molecule according to any one of claims 6 to 8, wherein exon 3 encodes the amino acid sequence shown in SEQ ID NO:

6.

10. The molecule according to any one of claims 6 to 9, wherein it targets (or complements, or hybridizes, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in a nucleic acid sequence encoding introns 2-3, exon 3, introns 3-4, or any combination thereof; preferably located in a nucleic acid sequence from the nucleic acid sequence encoding introns 2-3 to the nucleic acid sequence encoding introns 3-4, including the nucleic acid sequence of exon 3.

11. The molecule according to claims 6 to 10, wherein it targets (or complements, or hybridizes, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, the nucleic acid sequence being located in a nucleic acid sequence encoding introns 2-3, exon 3, introns 3-4, or any combination thereof; preferably located in a nucleic acid sequence from the nucleic acid sequence encoding introns 2-3 to the nucleic acid sequence encoding introns 3-4, including the nucleic acid sequence of exon 3.

12. The molecule according to any one of claims 6 to 11, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 214 to 1402 (inclusive) of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB.

13. The molecule according to any one of claims 6 to 12, wherein it targets a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 236 to 1424 (inclusive) of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB.

14. The molecule according to any one of claims 6 to 13, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 269 to 621 (inclusive) of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB.

15. The molecule according to any one of claims 6 to 14, wherein it targets a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 269 to 621 (inclusive) of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB.

16. The molecule according to any one of claims 6 to 15, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 339 to 436 (inclusive) of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB.

17. The molecule according to any one of claims 6 to 16, wherein it targets a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 339 to 436 (inclusive) of the nucleic acid sequence encoding exon 2 to intron 10 of TFEB.

18. The molecule according to any one of claims 6 to 17, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence encoding exon 2 to intron 10 of TFEB, specifically nucleic acid residues 339 to 435; preferably 339 to 434, more preferably 339 to 433, more preferably 339 to 432, more preferably 340 to 431, more preferably 340 to 430, and even more preferably... 340 to 429, more preferably 340 to 428, more preferably 340 to 427, more preferably 340 to 426, more preferably 340 to 425, more preferably 340 to 424, more preferably 340 to 423, more preferably 340 to 422, more preferably 341 to 421, more preferably 341 to 420, more preferably 341 to 419, more preferably 341 to 418, more preferably 341 to 417, more preferably 341 to 416, more preferably 341 to 415, more preferably 341 to 414, even better. Select 341 to 413, more preferably 341 to 412, more preferably 342 to 411, more preferably 342 to 410, more preferably 342 to 409, more preferably 342 to 408, more preferably 342 to 407, more preferably 342 to 406, more preferably 342 to 405, more preferably 342 to 404, more preferably 342 to 403, more preferably 342 to 402, more preferably 342 to 401, more preferably 342 to 400, more preferably 342 to 399, more preferably 342 to 398, more preferably... Among the following: 342 to 397, 342 to 396, 342 to 395, 342 to 394, 342 to 393, 342 to 392, 342 to 391, 342 to 390, 342 to 389, 342 to 388, 342 to 387, 342 to 386, 342 to 385, 342 to 384, 342 to 383, and 342 to 382.

19. The molecule according to any one of claims 6 to 18, wherein it targets a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in the nucleic acid sequence of nucleic acid residues 339 to 435 encoding exon 2 to intron 10 of TFEB; preferably 339 to 434, more preferably 339 to 433, more preferably 339 to 432, more preferably 340 to 431, more preferably 340 to 430 ... Preferably 340 to 429, more preferably 340 to 428, more preferably 340 to 427, more preferably 340 to 426, more preferably 340 to 425, more preferably 340 to 424, more preferably 340 to 423, more preferably 340 to 422, more preferably 341 to 421, more preferably 341 to 420, more preferably 341 to 419, more preferably 341 to 418, more preferably 341 to 417, more preferably 341 to 416, more preferably 341 to 415, more preferably 341 to 414, and more preferably 340 to 414. Preferred 341 to 413, more preferred 341 to 412, more preferred 342 to 411, more preferred 342 to 410, more preferred 342 to 409, more preferred 342 to 408, more preferred 342 to 407, more preferred 342 to 406, more preferred 342 to 405, more preferred 342 to 404, more preferred 342 to 403, more preferred 342 to 402, more preferred 342 to 401, more preferred 342 to 400, more preferred 342 to 399, more preferred 342 to 398, more... Among the following: 342 to 397, 342 to 396, 342 to 395, 342 to 394, 342 to 393, 342 to 392, 342 to 391, 342 to 390, 342 to 389, 342 to 388, 342 to 387, 342 to 386, 342 to 385, 342 to 384, 342 to 383, and 342 to 382.

20. The molecule according to any one of claims 6 to 19, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence encoding exon 2 to intron 10 of TFEB, or a nucleic acid sequence of nucleic acid residues 342 to 369, or nucleic acid residues 358 to 381.

21. The molecule according to any one of claims 6 to 20, wherein it targets a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in nucleic acid residues 342 to 369, or nucleic acid residues 358 to 381, of a nucleic acid sequence encoding exon 2 to intron 10 of TFEB.

22. The molecule according to any one of claims 6 to 21, wherein the nucleic acid sequence encoding exon 2 to intron 10 of TFEB has at least 85% identity with the nucleic acid sequence of SEQ ID NO:15, preferably wherein the nucleic acid sequence encoding exon 2 to intron 10 of TFEB has at least 90% identity with the nucleic acid sequence of SEQ ID NO:15, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and even more preferably, the nucleic acid sequence encoding exon 2 to intron 10 of TFEB has the nucleic acid sequence of SEQ ID NO:

15.

23. The molecule according to any one of claims 6 to 22, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 34 to 386 (inclusive) of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB.

24. The molecule according to any one of claims 6 to 23, wherein it is directed to a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 34 to 386 (inclusive) of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB.

25. The molecule according to any one of claims 6 to 24, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of 104 to 201 (inclusive) nucleic acid residues of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB.

26. The molecule according to any one of claims 6 to 25, wherein it is directed to a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of nucleic acid residues 104 to 201 (inclusive) of the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB.

27. The molecule according to any one of claims 6 to 26, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB, of nucleic acid residues 104 to 200; preferably 104 to 199, more preferably 104 to 198, more preferably 104 to 197, more preferably 105 to 108, more preferably 105 to 109, more preferably 104 to 198, more preferably 105 to 109, more preferably 105 to 109, of nucleic acid residues encoding introns 2-3 to 3-4 (including exon 3) of TFEB. 96, more preferably 105 to 195, more preferably 105 to 194, more preferably 105 to 193, more preferably 105 to 192, more preferably 105 to 191, more preferably 105 to 190, more preferably 105 to 189, more preferably 105 to 188, more preferably 105 to 187, more preferably 106 to 186, more preferably 106 to 185, more preferably 106 to 184, more preferably 106 to 183, more preferably 106 to 182, more preferably 106 to 181, more preferably 106 to 180, even better Select 106 to 179, more preferably 106 to 178, more preferably 106 to 177, more preferably 107 to 176, more preferably 107 to 175, more preferably 107 to 174, more preferably 107 to 173, more preferably 107 to 172, more preferably 107 to 171, more preferably 107 to 170, more preferably 107 to 169, more preferably 107 to 168, more preferably 107 to 167, more preferably 107 to 166, more preferably 107 to 165, more preferably 107 to 164, more preferably 107 to Among 163, more preferably 107 to 162, more preferably 107 to 161, more preferably 107 to 160, more preferably 107 to 159, more preferably 107 to 158, more preferably 107 to 157, more preferably 107 to 156, more preferably 107 to 155, more preferably 107 to 154, more preferably 107 to 153, more preferably 107 to 152, more preferably 107 to 151, more preferably 107 to 150, more preferably 107 to 149, more preferably 107 to 148, and more preferably 107 to 147.

28. The molecule according to any one of claims 6 to 27, wherein it targets a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence of 104 to 200 nucleic acid residues encoding introns 2-3 to 3-4 (including exon 3) of TFEB; preferably 104 to 199 nucleic acid residues, more preferably 104 to 198, more preferably 104 to 197 ...9, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 109, more preferably 104 to 1 5 to 196, more preferably 105 to 195, more preferably 105 to 194, more preferably 105 to 193, more preferably 105 to 192, more preferably 105 to 191, more preferably 105 to 190, more preferably 105 to 189, more preferably 105 to 188, more preferably 105 to 187, more preferably 106 to 186, more preferably 106 to 185, more preferably 106 to 184, more preferably 106 to 183, more preferably 106 to 182, more preferably 106 to 181, more preferably 106 to 180, More preferably 106 to 179, more preferably 106 to 178, more preferably 106 to 177, more preferably 107 to 176, more preferably 107 to 175, more preferably 107 to 174, more preferably 107 to 173, more preferably 107 to 172, more preferably 107 to 171, more preferably 107 to 170, more preferably 107 to 169, more preferably 107 to 168, more preferably 107 to 167, more preferably 107 to 166, more preferably 107 to 165, more preferably 107 to 164, more preferably 107 Among 163, more preferably 107 to 162, more preferably 107 to 161, more preferably 107 to 160, more preferably 107 to 159, more preferably 107 to 158, more preferably 107 to 157, more preferably 107 to 156, more preferably 107 to 155, more preferably 107 to 154, more preferably 107 to 153, more preferably 107 to 152, more preferably 107 to 151, more preferably 107 to 150, more preferably 107 to 149, more preferably 107 to 148, and more preferably 107 to 147.

29. The molecule according to any one of claims 6 to 28, wherein it targets a nucleic acid sequence of 12 to 40 nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB, of nucleic acid residues 107 to 134 (inclusive), or nucleic acid residues 123 to 146 (inclusive).

30. The molecule according to any one of claims 6 to 29, wherein it is directed to a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues, said nucleic acid sequence being located in a nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB, of nucleic acid residues 107 to 134 (inclusive), or nucleic acid residues 123 to 146 (inclusive).

31. The molecule according to any one of claims 6 to 30, wherein the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB has at least 85% identity with the nucleic acid sequence of SEQ ID NO:16, preferably wherein the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB has at least 90% identity with the nucleic acid sequence of SEQ ID NO:16, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and even more preferably, the nucleic acid sequence encoding introns 2-3 to 3-4 (including exon 3) of TFEB has SEQ ID NO:

16. The nucleic acid sequence of NO:

16.

32. The molecule according to any one of claims 6 to 31, having a nucleic acid sequence having at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 to SEQ ID NO: 128, preferably having at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 to SEQ ID NO: 91, more preferably having at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 to SEQ ID NO: 44 or SEQ ID NO: 50 to SEQ ID NO: 56, more preferably having at least 85% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 or SEQ ID NO: 39, and even more preferably having at least 85% identity with the nucleic acid sequence shown in SEQ ID NO:

39.

33. The molecule according to any one of claims 6 to 32, having at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 to SEQ ID NO: 128, preferably having at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 to SEQ ID NO: 91, more preferably having at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 to SEQ ID NO: 44 or SEQ ID NO: 50 to SEQ ID NO: 56, more preferably having at least 90% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 or SEQ ID NO: 39, and even more preferably having at least 90% identity with the nucleic acid sequence shown in SEQ ID NO:

39.

34. The molecule according to any one of claims 6 to 33, having at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably having at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably having at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably having at least 95% identity with the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#18.4). The nucleic acid sequence shown in SEQ ID NO: 39 (ASO#) has at least 95% identity, more preferably with SEQ ID NO: 39 (ASO#). The nucleic acid sequence shown in 16.3) has at least 95% identity.

35. The molecule according to any one of claims 6 to 34, comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably having SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#18.4). The nucleic acid sequence shown in SEQ ID NO: 39 (ASO#) is preferred. The nucleic acid sequence shown in 16.3).

36. A pharmaceutical composition comprising a mutant TFEB protein as defined in any one of claims 1 to 5, or a polynucleotide as defined in any one of claims 1 to 5, or a carrier as defined in any one of claims 1 to 5, a molecule as defined in any one of claims 6 to 35, or any combination thereof.

37. The mutant TFEB protein as defined in any one of claims 1 to 5, or the polynucleotide as defined in any one of claims 1 to 5, or the vector as defined in any one of claims 1 to 5, or the molecule as defined in any one of claims 6 to 35, used as a drug.

38. The pharmaceutical composition according to claim 36, used as a medicine.

39. The mutant TFEB protein as defined in any one of claims 1 to 5, or the polynucleotide as defined in any one of claims 1 to 5, or the vector as defined in any one of claims 1 to 5, or the molecule as defined in any one of claims 6 to 35, for the treatment of cell storage disorders involving autophagy defects, particularly lysosomal storage disorders (LSD).

40. The pharmaceutical composition of claim 36 for treating cell storage disorders involving autophagy defects, particularly lysosomal storage disorders (LSD).

41. The mutant TFEB protein of claim 39, or the polynucleotide, or the carrier, or the molecule, or the pharmaceutical composition of claim 40, wherein the cell storage disease is preferably selected from the neurodegenerative diseases of: Alzheimer's disease; age-related macular degeneration; cerebral β-amyloid angiopathy; prion diseases (such as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, Kuru); Parkinson's disease; multiple sclerosis; synucleinopathies (such as multiple system atrophy, Lewy body dementia). (Dementia); tau protein diseases (such as primary age-related tau protein dementia, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, chromosome-associated Parkinson's syndrome, vacuolar tau protein disease, Lytico-Bodig disease, gangliocytomas and ganglioneuromas, meningoangioma, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic acid kinase-associated neurodegeneration, and lipofuscin deposition disease); frontotemporal degeneration; amyotrophic lateral sclerosis; Huntington's disease Trinucleotide repeat syndrome; familial dementia; hereditary cerebral hemorrhage with amyloidosis; Cadasil syndrome; Alexander's disease; Angelman's syndrome; Peyre's disease; Seipin's disease; familial amyloid neuropathy; serine protease inhibitor syndrome; amyloidosis (such as senile systemic, light chain, heavy chain, secondary, medial aortic, ApoAI, ApoAII, ApoAIV, lysozyme, fibrinogen, dialysis, cardiac atrium, cutaneous lichenification, corneal lactoferrin, apolipoprotein C2, apolipoprotein C3, Lect 2. Insulin, galactoglobulin-7, keratin or enfviride amyloidosis); type II diabetes; inclusion body myositis / myopathy; cataract; retinitis pigmentosa with rhodopsin mutation; medullary thyroid carcinoma; pituitary prolactinoma; hereditary genotypic corneal dystrophy; Mallory bodies; pulmonary alveolar proteinosis; odontogenic (Pindborg) tumor amyloid; seminal vesicle amyloid; cystic fibrosis; sickle cell disease; plasma cell cachexia; exfoliation syndrome; preferably age-related macular degeneration, synucleinosis (preferably multiple system atrophy) or Parkinson's disease.

42. The mutant TFEB protein according to claim 39 or 41, or the polynucleotide, or the carrier, or the molecule, or the pharmaceutical composition according to claim 40 or 41, wherein the cell storage disease is selected from the group consisting of: β-mannosinolate storage disease, cholesterol ester storage disease, CLN1 disease, CLN2 disease, CLN3 disease, CLN7 disease, Fabry disease, GM2-ganglioside storage diseases (Tessa II disease, AB variant, and Sandhoff disease), Krabbe disease, metachromatic leukodystrophy, type I / II / III / IV mucolipid storage diseases, type I mucopolysaccharidosis (Scheie syndrome, Hurler-Scheie syndrome, Hurler syndrome), type II mucopolysaccharidosis (Hunter syndrome), type III mucopolysaccharidosis (Sanfilippo syndrome type A, Sanfilippo syndrome type B), and type IV mucopolysaccharidosis (Morquio syndrome). Syndrome A), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), Niemann-Pick disease type C, Pompe disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, spinocerebellar ataxia, multiple sclerosis, amyotrophic lateral sclerosis, multiple system atrophy, frontotemporal dementia, Lewy body disease, and Friedreich ataxia.

43. The mutant TFEB protein, or the polynucleotide, or the carrier, or the molecule, or the pharmaceutical composition according to any one of claims 37, 39, 41, or 42, for administration via oral, topical, oral mucosal, intranasal, intracranial, intraperitoneal, or parenteral route; such as intraocular, intravenous, intraarterial, intrathecal, intracerebral, intramuscular, intravenous, intracisional, and subcutaneous administration; preferably for intrathecal or intraocular administration, wherein intraocular administration is preferably selected from the group consisting of: intravitreal administration, subretinal administration, suprachoroidal administration, and any combination thereof, wherein intraocular administration is preferably intravitreal administration.

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