WO2025026988A1

WO2025026988A1 - Mutated tfeb for treating lysosomal disorders

Info

Publication number: WO2025026988A1
Application number: PCT/EP2024/071492
Authority: WO
Inventors: Marine DROGUERRE; Mathieu Charveriat; Julien VEYS
Original assignee: Theranexus
Priority date: 2023-07-28
Filing date: 2024-07-29
Publication date: 2025-02-06

Abstract

The present invention relates to a mutated transcription factor EB (TFEB) protein, that is deprived of its natural exon 3. This protein is herein referred to as "TFEB-∆ex3" or "∆ex3-TFEB". The present invention also relates to polynucleotides encoding this mutated protein; to vectors comprising said polynucleotides; and to biomolecular tools to remove the exon 3 of TFEB in patients in need thereof. The present invention also relates to pharmaceutical compositions comprising same, for use in the treatment and/or prevention of lysosomal storage disorders and disorders characterized by lysosomal dysfunction.

Description

MUTATED TFEB FOR TREATING LYSOSOMAL DISORDERS

SUMMARY OF THE INVENTION

The present invention relates to a mutated transcription factor EB (TFEB) protein, that is deprived of its natural exon 3. This protein is herein referred to as “TFEB-Aex3” or “Aex3-TFEB”. The present invention also relates to polynucleotides encoding this mutated protein; to vectors comprising said polynucleotides; and to biomolecular tools to remove the exon 3 of TFEB in patients in need thereof. The present invention relates in particular to oligonucleotide molecules able to mediate exon skipping of exon 3 of the TFEB protein. The present invention also relates to said mutated TFEB protein, said polynucleotide, said vector, said molecule, or a pharmaceutical composition comprising same, for use in the treatment and/or prevention of lysosomal storage disorders and disorders characterized by lysosomal dysfunction.

BACKGROUND OF THE INVENTION

Lysosomes are membrane-bound organelles responsible for the breakdown of proteins, glycosaminoglycans (GAGs), nucleic acids, lipids, and carbohydrates. An acidic internal pH is maintained by an ATP-dependent proton pump, known as the vacuolar-type ATPase (V-ATPase) and over 60 hydrolytic enzymes which gov-ern the breakdown and recycling of waste material. Initially considered to be static, terminal, waste cell, lysosomes were thought of passive recipients. However, lysosomes have recently emerged as dynamic centers, critical for gene regulation, secretion, cellular remodeling, cell adhesion, development, differentiation, cell migration, apoptosis, and lipid transport. Moreover, they are now known to be central for adaptation to cellular stress and disease. To this end, lysosomes are capable of transforming, undergoing fission, fusion, as well as interacting with various intra-cellular components.

In 2009, Sardiello, Ballabio, and their colleagues confirmed this hypothesis by uncovering a coordinately expressed gene network of lysosomal proteins (Sardiello M. et al., Science 2009, vol.325, p.473-476). Pattern discovery analysis of lysosomal protein promoter regions revealed a common motif; the investigators named the Coordinated Lysosomal Expression and Regulation (CLEAR) element. CLEAR is a palindromic 10-base sequence (5’ -GTCACGTGAC-3’ - SEQ ID NO: 139) preferentially located within 200 bp from the transcription start site (TSS). It is present in the regulatory regions of genes inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase enzyme expression, and lysosomal exocytosis.

The Transcription Factor EB (TFEB) is a mammalian transcription factor that binds directly to this CLEAR consensus sequence. By modulating its activities, TFEB coordinates on-demand control over each cell’s degradation pathway. In particular, TFEB binding to CLEAR results in modulation of lysosome biogenesis and function, including autophagy activation. Structurally, TFEB contains three functional domains, including the DNA binding, helix-loop-helix, and leucine zipper (bHLH-Zip) domains, which are also found in microphthalmia-associated transcription factor (MITF), transcription factor E3 (TFE3), and transcription factor EC (TFEC), the other members of the microphthalmia/transcription factor E (MiT/TFE) family (Da Costa et al., Fundamental & Clinical Pharmacology, 2020, doi:10.1 111/fcp.12634).

The biogenesis of lysosomes is complex and requires continuous synthesis of hydrolases (glycosidases, proteases, lipases, nucleases, phosphatases, and sulfatases), of which there are approximately 60, as well as transmembrane and accessory proteins. To determine the breadth of TFEB control over lysosomal biogenesis, Palmieri and colleagues performed ChlP-seq analysis of TFEB protein interactors profiling TFEB-mediated transcriptional regulation, and genome-wide mapping of TFEB target sites and co-expression analyses in HeLa cells (Palmieri et al., Hum. Mol. Genet. 2011 , 20 3852-66, doi:10.1093/hmg/ddr306). This study revealed 471 TFEB targets. The resultant interactors can be grouped into several categories from autophagy genes, as expected, to lysosomal enzymes and their transporters, lysosomal membrane proteins, genes responsible for lysosomal acidification, lysosomal positioning, and non-lysosomal proteins involved in lysosomal biogenesis.

TFEB activity is highly dependent on its nuclear localization. Thus, a nuclear signaling pathway strongly regulates TFEB activity. In particular, it is known that nuclear localization of TFEB is controlled by specific serine phosphorylation. Similar to starvation, pharmacological or gene mutation- based inhibition of specific phosphorylation induces autophagy by activating TFEB. It was therefore proposed to substitute or alter the serine residues to render them insensitive to phosphorylation, so that they remain dephosphorylated (see e.g. US9193755). It is also known that TFEB nuclear export is mediated through a nuclear localization signal (NLS). Other authors showed that mutated TFEB in which this localization signal is mutated (“ANLS-TFEB” corresponding to the TFEB protein, in which the NLS was mutated by replacing two basic arginine residues by alanine) displays constitutive cytosolic localization (Napolitano et al. Nature Communication, 2018, 9:3312, doi: 10.1038). Other authors proposed to add a NLS to the C-terminal end of the TFEB protein, to obtain a chimeric molecule that mainly targets the nucleus (WO 2010/0921 12). Finally, it is known that TFEB contains a nuclear export signal (NES) in its N-terminal part. This NES is a CRM1 consensus hydrophobic sequence that is highly evolutionary conserved. It has been shown that the mutagenesis of three of these conserved residues completely impairs cytosolic relocalization of TFEB (Napolitano et al. Nature Communication, 2018, 9:3312, doi:10.1038).

The growing understanding of the role of TFEB and CLEAR in the promotion of healthy clearance together with in vitro and in vivo preclinical findings in various animal models of disease supports the conclusion that the pharmacological activation of TFEB could clear toxic proteins to treat both rare and common forms of lysosomal and neurodegenerative diseases.

At the moment, no cures or approved treatments targeting TFEB currently exist. Additionally, while clinical trials are in progress on possible treatments for some of these diseases, there is currently no approved treatment for the majority of lysosomal storage disorders, or many disorders characterized by lysosomal dysfunction. Therefore, there is still a need in the art for compositions and methods of treating efficiently lysosomal storage disorders and disorders characterized by lysosomal dysfunction based on an enhancement of lysosomal clearance and the removal of cellular aggregates.

DESCRIPTION OF THE INVENTION

Mutated TFEB protein

TFEB is a mammalian transcription factor that binds directly to the CLEAR consensus sequence (5’ -GTCACGTGAC-3’ - SEQ ID NO:139) present in the regulatory regions of genes inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase enzyme expression, and lysosomal exocytosis. By modulating these activities, TFEB coordinates on-demand control over each cell’s degradation pathway.

The polypeptide sequence of human TFEB is shown on SEQ ID NO:1 (corresponding to accession number P19484 in the UniProt Database). Variants thereof have been reported, see e.g. SEQ ID NO:2 (B0QYS6 in the UniProt Database) and SEQ ID NO:3 (B0QYS7 in the UniProt Database).

A nuclear signaling pathway regulates cellular energy metabolism through TFEB.

On a one hand, the Nuclear Export Signal is of SEQ ID NO:4 (GNSAPNSPMAMLHIGSNP) and is located between amino acids 136 and 153 of SEQ ID NO:1 , therefore encoded in exon 3 of TFEB (Napolitano et al, 2018). This export signal is controlled by CRM1 . It is encoded in the third exon.

The present inventors determined that it would be useful to activate the TFEB protein in cellular targets by transfecting in same mutated proteins in which the Nuclear Export Signal has been removed by exon 3 skipping or exon 3 deletion, or by removing said signal in the endogenous protein. This activation of TFEB will enable to clear toxic proteins to treat both rare and common forms of neurodegenerative disease (Da Costa et al. Fundamental & Clinical Pharmacology, 2020, doi:10.1111/fcp.12634).

In a first aspect, the present invention therefore concerns a mutated TFEB protein that does not contain the amino acid sequence encoded by natural exon 3.

Exon 3 of TFEB encodes an amino acid sequence which contains 85 amino acids. The amino acid sequence encoded by exon 3 has the sequence displayed in SEQ ID NO:6 (VQSYLENPTSYHLQQSQHQKVREYLSETYGNKFAAHISPAQGSPKPPPAASPGVRAGHVLSSSA GNSAPNSPMAMLHIGSNPERE). In one embodiment, the amino acid sequence encoded by exon 3 of TFEB is located between amino acid residue 72 (included) and amino acid residue 156 (included) 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 ranging from amino acid residue 72 (included) to amino acid residue 156 (included) of any one of SEQ ID NO:1 , 2 or 3).

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 variants or homologous thereof whose polypeptide sequences display at least 80% identity with SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3.

In a preferred embodiment, the mutated protein of the invention is the human TFEB protein in which the amino acid sequence encoded by exon 3 of SEQ ID NO:6 (or a variant or homologous thereof sharing at least 80% identity with said sequence) has been removed or is absent.

The resulting mutated protein has for example the SEQ ID NOT (corresponding to P19484 in which the amino acid sequence encoded by exon 3 has been removed), SEQ ID NO:8 (corresponding to B0QYS6 in which the amino acid sequence encoded by exon 3 has been removed) or SEQ ID NO:9 (corresponding to B0QYS7 in which the amino acid sequence encoded by exon 3 has been removed).

The mutated protein can also be a variant or a homologous thereof, whose amino acid sequence displays a percentage identity of 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%, and even more preferably of at least 99% with SEQ ID NOT, SEQ ID NO:8 or SEQ ID NO:9.

In other terms, the present invention relates to an isolated or recombinant polypeptide whose sequence comprises (or essentially consists of, or consists of) a sequence sharing at least 80% identity with SEQ ID NOT, SEQ ID NO:8 or SEQ ID NO:9. These mutated proteins or variants thereof preferably still contain the Nuclear Localisation Signal is of SEQ ID NO:5 (NLIERRRRFNIN) which is encoded in exons 6 and 7 of TFEB (Roczniak-Ferguson et al, Sc/. Signal. 2012; 5(228):ra42. Doi:10.1126).

Polynucleotides encoding the mutated protein

In a second aspect, the present invention relates to any polynucleotides encoding the mutated protein described above. These polynucleotides are preferably isolated or recombinant nucleic acid molecules.

The skilled person will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that may encode a polypeptide as described herein. In particular, codon usage within a given nucleotide sequence may be adapted for optimized expression of the corresponding polypeptide.

Polynucleotides encoding the human TFEB protein are well-known. They are for example the transcript variant 1 of 2354 bp referenced as NM_007162.3, the transcript variant 2 of 2152 bp referenced as NM_001167827.3, the transcript variant 3 of 2333 bp referenced as NM_001271944, or the transcript variant 4 of 2163 bp referenced as NM_001271945. All of them contain the exon 3 of SEQ ID NO:10 or variants thereof. SEQ ID NQ:10 represents both the DNA sequence and the RNA sequence (e.g., mRNA) of the exon 3 of TFEB protein.

The present invention concerns a polynucleotide encoding a mutated TFEB protein, whose mRNA sequence does not contain the mRNA sequence of SEQ ID NQ:10 (or a variant or homologous thereof having at least 80% identity with SEQ ID NQ:10). In particular, the invention relates to a mRNA encoding the TFEB protein, deprived of the mRNA sequence of SEQ ID NQ:10 (or a variant or homologous thereof having at least 80% identity with SEQ ID NQ:10).

These polynucleotides have for example the following sequences:

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. The present invention also concerns any variants thereof, whose sequence share more than 80% identity, preferably at least 85%, more preferably at least 90%, and even more preferably of at least 95% identity with SEQ ID NO:11-14. The invention also encompasses all the equivalent polynucleotides that, due to codon degeneracy, encode the same proteins as SEQ ID NO:11-14 do.

The present invention relates, of course, to both the DNA and RNA sequences, and also to the sequences which hybridize with them, as well as the corresponding double-stranded DNAs.

In another aspect, the present invention concerns vectors comprising the polynucleotides of the invention, as defined above, or vectors encoding the mutated TFEB protein of the invention, as defined above.

Accordingly, in one embodiment, the vector contains a polynucleotide encoding a mutated TFEB protein, wherein the mutated TFEB protein does not contain the amino acid sequence encoded by natural exon 3, said vector being preferably a plasmid or a viral vector.

Alternatively or in combination, the vector contains a polynucleotide encoding a mutated TFEB protein, whose mRNA sequence does not contain the mRNA sequence of SEQ ID NQ:10, said vector being preferably a plasmid or a viral vector.

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 is absent.

In a preferred embodiment, the mutated TFEB protein has a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and a variant or a homologous of any one of SEQ ID NOT, SEQ ID NO:8, or SEQ ID NO:9.

In a preferred embodiment, the polynucleotide encoding the mutated TFEB protein has a sequence selected in the group consisting of SEQ ID NO:11-14, or variants thereof.

These vectors can be cloning and/or expression vectors containing the nucleic acid molecule of the invention. They can include features which allow the expression and/or the secretion of the nucleic acid molecule of the invention in I from a host cell, for in vitro production purpose. The vector of the invention may be DNA, or RNA, or a DNA-RNA hybrid.

These vectors can also be gene therapy vectors that can be administered to subjects in need thereof, in order to express the mutated protein of the invention in situ. Said gene therapy vector can be a plasmid or a viral vector, that makes it possible to improve the administration of the nucleic acid encoding the mutated TFEB in the target cells, and also to increase the stability of said nucleic acid into said cells, thereby enabling a long-lasting effect to be obtained.

Said target cells are, in the context of the invention, any human cells, for example neural, muscular or skeletal cells.

Pharmaceutical composition

In another aspect, the present invention relates to a pharmaceutical composition comprising the mutated TFEB protein as defined above, or a polynucleotide as defined above, or a vector as defined above.

Apart from the protein, polynucleotide or vector of the invention, the pharmaceutical composition of the invention can contain a pharmaceutically acceptable excipient.

As used herein, the term "pharmaceutically acceptable excipient" is intended to mean, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or encapsulating agent, such as a liposome, cyclodextrins, encapsulating polymeric delivery systems or polyethyleneglycol matrix, which is acceptable for use in subjects, preferably humans.

The pharmaceutically acceptable vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Additional examples of pharmaceutically acceptable vehicles include, but are not limited to: water for Injection USP; aqueous vehicles such as, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Therapy - Gene therapy

In another aspect, the present invention concerns the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, orthe vector ofthe invention, as defined above, the pharmaceutical composition of the invention, as defined above, or any combination thereof, for its use as a medicament, or for the manufacture of a medicament.

The present invention also concerns a use of the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, the pharmaceutical composition of the invention, as defined above, or any combination thereof, as a medicament, or for the manufacture of a medicament.

The present invention also concerns a method of treating a disease in a subject in need thereof, comprising administering the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, the pharmaceutical composition of the invention, as defined above, or any combination thereof, in said subject.

In particular, the present invention concerns the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, the pharmaceutical composition of the invention, as defined above, or any combination thereof, for its use as a medicament for treating cell storage disorders involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs).

The present invention also concerns a use of the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, the pharmaceutical composition of the invention, as defined above, or any combination thereof, for treating cell storage disorders involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs), or for the manufacture of a medicament for a treating cell storage disorder involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs).

The present invention also concerns a method of treating a cell storage disorder involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs) in a subject in need thereof, comprising administering the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, the pharmaceutical composition of the invention, as defined above, or any combination thereof, in said subject.

Said lysosomal storage disorders (LSDs) are disorders wherein the lysosomal function is impaired due to an inherited condition, thereby conducting to cellular dysfunction. Such LSDs include but are not limited to Sphingolipidoses (such as Fabry disease, Farber lipogranulomatosis, Gaucher disease (type l/ll/lll and perinatal lethal form), GM1 gangliosidosis (type l/ll/lll), GM2 gangliosidosis (Tay- Sachs disease, Sandhoff disease, GM2 activator deficiency), Globoid cell leukodystrophy (Krabbe disease), Metachromatic leukodystrophy, Niemann-Pick disease types A/B, Prosaposin Deficiency and Saposin B Deficiency); Mucopolysaccharidoses (such as MPS I (Hurler/Hurler-Scheie/Scheie syndrome), MPS II (Hunter syndrome), MPS lll/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 disease (such as GSDO, GSDI (von Gierke's disease), GSD Ila (Pompe disease), GSD lib (Danon 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; Glycoproteinoses (such as a-Mannosidosis (type l/ll/lll), p-Mannosidosis, Fucosidosis, Aspartylglucosaminuria, Schindler disease (type l/ll/lll), Sialidosis (type l/ll), Galactosialidosis); Lipid storage diseases (such as Acid lipase deficiency (Wolman disease, cholesterol ester storage disease)); Post-translational modification defects (such as Multiple sulfatase deficiency, Mucolipidosis (II a/p, l-cell disease), Mucolipodosis II (a/p, pseudo-Hurler polydystrophy), Mucolipidosis III (y, variant pseudo-Hurler polydystrophy)); Integral membrane protein disorders (such as Cystinosis, Action myoclonus-renal failure syndrome, Sialic acid storage disease (ISSD, Salla disease), Niemann-Pick disease types C1/C2, Mucolipidosis IV); Neuronal ceroid lipofuscinoses (CLNs) (such as CLN1 (Haltia-Santavuori disease and INCL), CLN2 (Jansky-Bielschowsky disease), CLN3 (Batten-Spielmeyer- Sjogren disease), CLN4 (Parry disease and Kufs type A/B), CLN5 (Finnish variant late infantile), CLN6 (Lake- Cavanagh or Indian variant), CLN7 (Turkish variant), CLN8 (northern epilepsy, epilepsy mental retardation), CLN9, CLN10, CLN11 , CLN12 (Kufor-Rakeb syndrome), CLN13, CLN14); Lysosome- related organelles disorders (such as Hermansky-Pudlak disease type 1 to type 9, Griscelli syndrome 1 (Elejalde syndrome), Griscelli syndrome 2, Chediak-Higashi disease); Polyglucosan storage diseases (such as Lafora disease, adult PG body disease, AMP-activated protein kinase deficiency); Others (such as Pycnodysostosis, Papillon-Lefevre syndrome).

These disorders can also be diseases of the nervous system whereby lysosomal function and autophagy are impaired, hence contributing to a degenerative process. Such diseases include but are not limited to Alzheimer's disease; Age-related macular degeneration; Cerebral p-amyloid angiopathy; Prion diseases (such as Creutzfeldt- Jakob disease, Gerstmann-Straussler-Scheinker Syndrome, Kuru); Parkinson's disease; Multiple sclerosis (Gonzalez-Jimenez A. et al. Int. Mol. Sci. 2022; 23(15):8116. doi:10.3390/ijms231581 16); Synucleinopathies (such as multiple system atrophy, dementia with Lewy bodies); Tauopathies (such as Primary age-related tauopathy 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), parkinsonism linked to chromosome, Vacuolar tauopathy, Lytico- bodig disease, Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis); Frontotemporal lobar degeneration; Amyotrophic lateral sclerosis; Huntington's disease; trinucleotide repeat disorders; Familial dementia; Hereditary cerebral hemorrhage with amyloidosis; CADASIL syndrome; Alexander disease; Angelman syndrome; Pelizaeus-Merzbacher disease; Seipinopathies; Familial amyloidotic neuropathy; Serpinopathies; Amyloidosis (such as Senile systemic, light chain, heavy chain, secondary, Aortic medial, ApoAl, ApoAII, ApoAIV, Lysozyme, Fibrinogen, Dialysis, Cardiac atrial, Cutaneous lichen, Corneal lactoferrin, Apolipoprotein C2, Apolipoprotein C3, Lect2, Insulin, Galectin-7, Corneodesmosin or Enfuvirtide amyloidosis); Type II diabetes; Inclusion body myositis/myopathy; Cataracts; Retinitis pigmentosa with rhodopsin mutations; Medullary thyroid carcinoma; Pituitary prolactinoma; Hereditary lattice corneal dystrophy; Mallory bodies; Pulmonary alveolar proteinosis; Odontogenic (Pindborg) tumor amyloid; Seminal vesicle amyloid; Cystic fibrosis; Sickle cell disease; Plasma cell dyscrasias; Exfoliation syndrome.

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

In a preferred embodiment, the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, or the pharmaceutical composition of the invention, enables to treat Beta-mannosidosis, Cholesteryl ester storage disease, CLN1 disease, CLN2 disease, CLN3 disease, CLN7 disease, Fabry disease, GM2-gangliosidosis (Tay-Sachs disease, AB variant, and Sandhoff disease), Krabbe disease, Metachromatic leukodystrophy, Mucolipidosis Type l/ll/lll/IV, Mucopolysaccharidosis type I (Scheie syndrome, Hurler-Scheie syndrome, Hurler syndrome), Mucopolysaccharidosis type II (Hunter syndrome), Mucopolysaccharidosis type III (Sanfilippo syndrome type A, Sanfilippo syndrome type B), Mucopolysaccharidosis type IV (Morquio A syndrome), Mucopolysaccharidosis type VI (Ma rote aux- La my 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.

The mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, or the composition of the invention will prevent, reverse, or arrest cognitive decline in a subject. Methods of determining cognitive decline in a subject are known in the art. For instance, the mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, or the composition of the invention may prevent, reverse, or arrest vision failure. The mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector of the invention, as defined above, or the composition of the invention may also prevent, reverse, or arrest hearing loss. The composition of the invention may also reduce the severity and/or intensity of seizures. Additionally, the mutated TFEB protein of the invention, as defined above, orthe polynucleotide of the invention, as defined above, or the vector ofthe invention, as defined above, or the composition of the invention may improve or prevent motor dysfunction. The mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, or the vector ofthe invention, as defined above, or the composition ofthe invention may also improve or prevent dementia.

The mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, orthe vector ofthe invention, as defined above, orthe composition ofthe invention, as defined above, can be safely administered by oral, topic, oromucosal, intranasal, intracranial, intraperitoneal, or parenteral route, such as intraocularly, intravenously, intraarterially, intrathecally, intracerebrally, intramuscularly, intraventricularly, intracisternally and subcutaneously. The mutated TFEB protein of the invention, as defined above, or the polynucleotide of the invention, as defined above, orthe vector ofthe invention, as defined above, orthe composition ofthe invention, as defined above, is preferably administered intrathecally or intraocularly (for example by intravitreal administration (e.g. intravitreal injection), subretinal administration (e.g. subretinal injection), suprachoroidal administration (e.g. suprachoroidal injection), or any combination thereof), or any combination thereof, wherein the intraocular administration is preferably intravitreal administration.

Exon skipping in situ - Gene therapy

Exon skipping is a technique used in gene therapy. It consists in skipping one or more exons during splicing, in order to allow a cell to synthesize a truncated, but usually still functional protein. It has been developed - and is now approved - for treating several diseases, among which the Duchenne Muscular Dystrophy (DMD).

Exon skipping can be induced in the cell through different tools, by viral vectors, by antisense DNA/RNA, etc. Thus, the present invention concerns any biomolecular tool or device enabling to skip specifically the exon 3 of TFEB, in any human cells, for example neural, muscular or skeletal cells. For example, the biomolecular tool or device enabling to skip specifically the exon 3 of TFEB of the invention can be a morpholino, a U7-SNRP, a DNA editing type CRISPR, a siRNA, an antisense oligonucleotide, or a small chemical molecule like TG003 or risdiplam.

Antisense oligonucleotides (AON or ASO) are well known to mediate very 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: 10.52965/001 c.24934). The present inventors thus propose to use ASO-mediated exon skipping in order to remove exon 3 of TFEB in situ in any human cells, for example neural, muscular or skeletal cells of patients suffering from the cell storage disorders mentioned above.

These ASOs preferably affect the exon 3 of TFEB, that is, SEQ ID NO:10, or a variant thereof, by targeting exon 3 or nearby regions, for example intron 2-3 or intron 3-4 regions.

Thus, the present invention concerns an oligonucleotide molecule, preferably an antisense oligonucleotide (ASO), that is able to mediate exon skipping of exon 3 of the TFEB protein. In a preferred embodiment, the oligonucleotide molecule of the invention is able to mediate exon skipping of exon 3 of the TFEB protein, wherein the exon 3 encodes the amino acid sequence shown in SEQ ID NO:6 (or a variant I homologous thereof) of the TFEB protein of SEQ ID NO:7, SEQ ID NO:8 or of SEQ ID NO:9 (or of a variant I homologous thereof). Said variants have e.g. at least 80%, preferably at least 90% identity with the said reference sequences, as disclosed above.

The oligonucleotide molecule is preferably an antisense oligonucleotide (ASO).

Antisense oligonucleotides are short-length single-stranded sequences of nucleotides (typically oligodeoxynucleotides), generally of 12-40 bases (typically 15-22 bases long), that are designed and synthesized in vitro to be complementary to, most often, a target sequence of a transcript (e.g., a mRNA or a pre-mRNA) to generate a DNA/RNA heteroduplex. They thus inhibit synthesis of the corresponding protein or mask certain genetic sequences so as to correct, modulate or inhibit splicing of the target mRNA, and thus generate or regulate the expression of a functional or non-functional protein. Antisense oligonucleotides may be single stranded DNA oligonucleotides, designed in antisense orientation to the transcript/RNA of interest. Hybridization of the ASO to the target RNA generally triggers cleavage of the RNA by a ribonuclease (e.g., RNase H). Antisense oligonucleotides are preferably modified chemically in order to resist nuclease activity (for example by adding phosphorothioate (PS) modifications/substitutions to the oligonucleotide and/or adding modified bases, such as 2' sugar modifications, including 2'-O-methyl (2'-OMe)) and to stably hybridize with its targets (for example by adding modified bases, such as 2'-O-methyl (2'-OMe)), and/or substituting 5-methyl dC for dC in CpG motifs).

In one embodiment, the molecule of the invention is able to mediate exon skipping of exon 3 in a nucleic acid sequence encoding TFEB protein.

In one embodiment, the resulting TFEB protein has a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and a variant or a homologous of any one of SEQ ID NOT, SEQ ID NO:8, or SEQ ID NO:9, after exon 3 skipping. In one embodiment, the molecule of the invention is able to mediate exon skipping of the exon 3 of SEQ ID NO:6 of a nucleic acid sequence encoding the TFEB protein of SEQ ID NO:7, of SEQ ID NO:8, or of SEQ ID NO:9, or of a variant or homologous thereof.

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 NQ:10 (which is the nucleic acid sequence encoding the exon 3 of TFEB protein of SEQ ID NO:7, of SEQ ID NO:8, of SEQ ID NO:9, or of a variant or homologous thereof).

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence encoding intron 2-3, exon 3, intron 3-4, or any combination thereof (preferably located in the nucleic acid sequence ranging from the nucleic acid sequence encoding intron 2-3 to the nucleic acid sequence encoding intron 3-4, including exon 3; in other words, preferably located in the polynucleotide encoding the nucleic acid sequences ranging from intron 2-3 to intron 3-4, including exon 3).

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence encoding intron 2-3, exon 3, intron 3-4, or any combination thereof (preferably located in the nucleic acid sequence ranging from the nucleic acid sequence encoding intron 2-3 to the nucleic acid sequence encoding intron 3-4, including exon 3; in other words, preferably located in the polynucleotide encoding the nucleic acid sequences ranging from intron 2-3 to intron 3-4, including exon 3).

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 236 to 1424 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB (i.e., without exon 1 and intron 1-2 (intron 1-2 corresponds to the intron located between exon 1 and exon 2 of TFEB, in other words to the intron bridging exon 1 to exon 2)).

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 236 to 1424 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB (i.e., without exon 1 and intron 1-2 (intron 1-2 corresponds to the intron located between exon 1 and exon 2 of TFEB, in other words to the intron bridging exon 1 to exon 2)). The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 269 to 621 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 269 to 621 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 339 to 436 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 339 to 436 of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 339 to 435 of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB; preferably from 339 to 434, more preferably from 339 to 433, more preferably from 339 to 432, more preferably from 340 to 431 , more preferably from 340 to 430, more preferably from 340 to 429, more preferably from 340 to 428, more preferably from 340 to 427, more preferably from 340 to 426, more preferably from 340 to 425, more preferably from 340 to 424, more preferably from 340 to 423, more preferably from 340 to 422, more preferably from 341 to 421 , more preferably from 341 to 420, more preferably from 341 to 419, more preferably from 341 to 418, more preferably from 341 to 417, more preferably from 341 to 416, more preferably from 341 to 415, more preferably from 341 to 414, more preferably from 341 to 413, more preferably from 341 to 412, more preferably from 342 to 411 , more preferably from 342 to 410, more preferably from 342 to 409, more preferably from 342 to 408, more preferably from 342 to 407, more preferably from 342 to 406, more preferably from 342 to 405, more preferably from 342 to 404, more preferably from 342 to 403, more preferably from 342 to 402, more preferably from 342 to 401 , more preferably from 342 to 400, more preferably from 342 to 399, more preferably from 342 to 398, more preferably from 342 to 397, more preferably from 342 to 396, more preferably from 342 to 395, more preferably from 342 to 394, more preferably from 342 to 393, more preferably from 342 to 392, more preferably from 342 to 391 , more preferably from 342 to 390, more preferably from 342 to 389, more preferably from 342 to 388, more preferably from 342 to 387, more preferably from 342 to 386, more preferably from 342 to 385, more preferably from 342 to 384, more preferably from 342 to 383, more preferably from 342 to 382, of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 339 to 435 of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB; preferably from 339 to 434, more preferably from 339 to 433, more preferably from 339 to 432, more preferably from 340 to 431 , more preferably from 340 to 430, more preferably from 340 to 429, more preferably from 340 to 428, more preferably from 340 to 427, more preferably from 340 to 426, more preferably from 340 to 425, more preferably from 340 to 424, more preferably from 340 to 423, more preferably from 340 to 422, more preferably from 341 to 421 , more preferably from 341 to 420, more preferably from 341 to 419, more preferably from 341 to 418, more preferably from 341 to 417, more preferably from 341 to 416, more preferably from 341 to 415, more preferably from 341 to 414, more preferably from 341 to 413, more preferably from 341 to 412, more preferably from 342 to 411 , more preferably from 342 to 410, more preferably from 342 to 409, more preferably from 342 to 408, more preferably from 342 to 407, more preferably from 342 to 406, more preferably from 342 to 405, more preferably from 342 to 404, more preferably from 342 to 403, more preferably from 342 to 402, more preferably from 342 to 401 , more preferably from 342 to 400, more preferably from 342 to 399, more preferably from 342 to 398, more preferably from 342 to 397, more preferably from 342 to 396, more preferably from 342 to 395, more preferably from 342 to 394, more preferably from 342 to 393, more preferably from 342 to 392, more preferably from 342 to 391 , more preferably from 342 to 390, more preferably from 342 to 389, more preferably from 342 to 388, more preferably from 342 to 387, more preferably from 342 to 386, more preferably from 342 to 385, more preferably from 342 to 384, more preferably from 342 to 383, more preferably from 342 to 382, of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 342 to 369, or from nucleic acid residues 358 to 381 , of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 342 to 369, or from nucleic acid residues 358 to 381 , of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB. The nucleic acid sequence encoding from exon 2 to intron 10 of TFEB thus consists of, from 5’ to 3’, exon 2, intron 2-3, exon 3, intron 3-4, exon 4, intron 4-5, exon 5, intron 5-6, exon 6, intron 6-7, exon 7, intron 7-8, exon 8, intron 8-9, exon 9, and intron 10.

An exemplary nucleic acid sequence (DNA, RNA (e.g. mRNA) or DNA/RNA hybrid thereof) encoding TFEB protein, from exon 2 to intron 10, is shown in SEQ ID NO: 15. SEQ ID NO: 15 does not contain exon 1 , nor intron 1-2 (intron 1-2 corresponds to the intron located between exon 1 and exon 2 of TFEB protein, in other words to the intron bridging exon 1 to exon 2). Exemplary nucleic acid sequence SEQ ID NO:15 ranges from the first nucleic acid residue of the exon 2 (first residue of exon 2 included) of exemplary TFEB protein to the last nucleic acid residue of the intron 10 (last residue of intron 10 included).

Positions of introns and exons in SEQ ID NO:15 are shown in Table 1 below.

Table 1 : Positions of introns and exons in SEQ ID NO:15

In a preferred embodiment, the nucleic acid sequence encoding from 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 from 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 % identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleic acid sequence of SEQ ID NO:15, even more preferably the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB of TFEB has the nucleic acid sequence of SEQ ID NO:15.

Preferably, the nucleic acid sequence of SEQ ID NO:15 is the nucleic acid sequence from exon 2 to intron 10 of TFEB protein of SEQ ID NO:7, of SEQ ID NO:8, of SEQ ID NO:9, or of a variant or homologous thereof).

The molecule of the invention is more preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 34 to 386 (included) of the nucleic acid sequence encoding intron 2-3, exon 3, and intron 3-4 of TFEB (i.e., the nucleic acid sequence ranging from the nucleic acid sequence encoding intron 2-3 to the nucleic acid sequence encoding intron 3-4, including exon 3; in other words, the polynucleotide encoding the nucleic acid sequences ranging from intron 2-3 to intron 3-4, including exon 3; in other words, the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB).

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 34 to 386 (included) of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 104 to 201 (included) of the nucleic acid sequence encoding the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 104 to 201 of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 104 to 200 of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB; preferably from 104 to 199, more preferably from 104 to 198, more preferably from 104 to 197, more preferably from 105 to 196, more preferably from 105 to 195, more preferably from 105 to 194, more preferably from 105 to 193, more preferably from 105 to 192, more preferably from 105 to 191 , more preferably from 105 to 190, more preferably from 105 to 189, more preferably from 105 to 188, more preferably from 105 to 187, more preferably from 106 to 186, more preferably from 106 to 185, more preferably from 106 to 184, more preferably from 106 to 183, more preferably from 106 to 182, more preferably from 106 to 181 , more preferably from 106 to 180, more preferably from 106 to 179, more preferably from 106 to 178, more preferably from 106 to 177, more preferably from 107 to 176, more preferably from 107 to 175, more preferably from 107 to 174, more preferably from 107 to 173, more preferably from 107 to 172, more preferably from 107 to 171 , more preferably from 107 to 170, more preferably from 107 to 169, more preferably from 107 to 168, more preferably from 107 to 167, more preferably from 107 to 166, more preferably from 107 to 165, more preferably from 107 to 164, more preferably from 107 to 163, more preferably from 107 to 162, more preferably from 107 to 161 , more preferably from 107 to 160, more preferably from 107 to 159, more preferably from 107 to 158, more preferably from 107 to 157, more preferably from 107 to 156, more preferably from 107 to 155, more preferably from 107 to 154, more preferably from 107 to 153, more preferably from 107 to 152, more preferably from 107 to 151 , more preferably from 107 to 150, more preferably from 107 to 149, more preferably from 107 to 148, more preferably from 107 to 147, of the nucleic acid sequence from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 104 to 200 of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB; preferably from 104 to 199, more preferably from 104 to 198, more preferably from 104 to 197, more preferably from 105 to 196, more preferably from 105 to 195, more preferably from 105 to 194, more preferably from 105 to 193, more preferably from 105 to 192, more preferably from 105 to 191 , more preferably from 105 to 190, more preferably from 105 to 189, more preferably from 105 to 188, more preferably from 105 to 187, more preferably from 106 to 186, more preferably from 106 to 185, more preferably from 106 to 184, more preferably from 106 to 183, more preferably from 106 to 182, more preferably from 106 to 181 , more preferably from 106 to 180, more preferably from 106 to 179, more preferably from 106 to 178, more preferably from 106 to 177, more preferably from 107 to 176, more preferably from 107 to 175, more preferably from 107 to 174, more preferably from 107 to 173, more preferably from 107 to 172, more preferably from 107 to 171 , more preferably from 107 to 170, more preferably from 107 to 169, more preferably from 107 to 168, more preferably from 107 to 167, more preferably from 107 to 166, more preferably from 107 to 165, more preferably from 107 to 164, more preferably from 107 to 163, more preferably from 107 to 162, more preferably from 107 to 161 , more preferably from 107 to 160, more preferably from 107 to 159, more preferably from 107 to 158, more preferably from 107 to 157, more preferably from 107 to 156, more preferably from 107 to 155, more preferably from 107 to 154, more preferably from 107 to 153, more preferably from 107 to 152, more preferably from 107 to 151 , more preferably from 107 to 150, more preferably from 107 to 149, more preferably from 107 to 148, more preferably from 107 to 147, of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 107 to 134 (included), or from nucleic acid residues 123 to 146 (included), of the nucleic acid sequence encoding from intron

2-3 to intron 3-4 (including exon 3) of TFEB.

The molecule of the invention is preferably directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 107 to 134 (included), or from nucleic acid residues 123 to 146 (included), of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

The nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB thus consists of, from 5’ to 3’, intron 2-3, exon 3, and intron 3-4.

An exemplary nucleic acid sequence (DNA, RNA (e.g. mRNA) or DNA/RNA hybrid thereof) encoding TFEB protein, from intron 2-3 to intron 3-4 (including exon 3), is shown in SEQ ID NO: 16. Exemplary nucleic acid sequence SEQ ID NO:16 ranges from the first nucleic acid residue of the intron 2-3 (first residue of intron 2-3 included) of exemplary TFEB protein to the last nucleic acid residue of the intron

3-4 (last residue of intron 3-4 included).

Positions of introns and exons in SEQ ID NO:16 are shown in Table 2 below.

Table 2 : Positions of introns and exons in SEQ ID NO:16

In a preferred embodiment, the nucleic acid sequence encoding from intron 2-3 to intron 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 from intron 2-3 to intron 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 % identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, with the nucleic acid sequence of SEQ ID NO:16, even more preferably the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB has the nucleic acid sequence of SEQ ID NO:16.

Preferably, the nucleic acid sequence of SEQ ID NO:16 is the nucleic acid sequence from intron 2- 3 to intron 3-4 (including exon 3) of TFEB protein of SEQ ID NO:7, of SEQ ID NO:8, of SEQ ID NO:9, or of a variant or homologous thereof).

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

The molecule of the invention preferably has (or comprises, or consists essentially of, or consists of) at least 90% identity with the nucleic acid sequences shown in any one of SEQ ID NO: 17 (ASO#59) to SEQ ID NO: 128 (ASO#78), preferably having at least 90% identity with the nucleic acid sequences shown in any one of SEQ ID NO: 31 (ASO#15) to SEQ ID NO: 91 (ASO#31), more preferably having at least 90% identity with the nucleic acid sequences shown in any one of SEQ ID NO: 34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or of SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably having at least 90% identity with the nucleic acid sequences shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), more preferably having at least 90% identity with the nucleic acid sequence shown in SEQ ID NO: 39 (ASO#16.3). The molecule of the invention preferably has (or comprises, or consists essentially of, or consists of) at least 95% identity with the nucleic acid sequences 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 sequences 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 sequences shown in any one of SEQ ID NO34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or of 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 sequences shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), more preferably having at least 95% identity with the nucleic acid sequence shown in SEQ ID NO: 39 (ASO#16.3).

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

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

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

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

In a particularly preferred embodiment, the molecule of the invention has (or comprises, or consists essentially of, of consists 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 the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), more preferably having the nucleic acid sequence shown in SEQ ID NO: 39 (ASO#16.3).

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

Table 3 : Sequences of preferred molecules of the invention (which are preferably ASOs)

The molecule (preferably the ASO) of the invention preferably comprises (or consists essentially of, or consists 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, even more preferably 18 nucleic acid residues. The molecule (preferably the ASO) of the invention may carry any chemical modification known in the art, in particular it can be through backbone modifications (such as phosphoramidate, methylphosphonate, phosphorothioate (PS)); or through nucleobase modifications (such as 5- methylcytidine, 5-methylcytosine (m⁵C), 5-methyluridine); or through 2’-ribose substitutions (such as 2’-O-methyl (2’-OMe), 2’-0-methoxyethyl (2’-MOE), 2’-Fluoro (2'F), 2’-O-alkyl, 2-0- methylcarbamoylethyl (2-O-MCE)); orth rough ribose modifications (such as 5-anhydrohexitol nucleic acid (HNA), Cyclohexene nucleic acid (CeNA), Threose nucleic acid (TNA), Glycol nucleic acid (GNA), Locked nucleic acid (LNA), FANA (Fluoro Arabino nucleic acid) , constrained ethyl (cET) or a 2'-O,4'-C-Ethylene-bridged nucleic acid (ENA)); or through alternative chemistries (such as phosphorodiamidate morpholino (PMO), N-acetylgalactosamine (triantennary GalNAc3), peptide nucleic acid (PNA), Tricyclo-DNA (tc-DNA), aptamer); or any combination of these chemical modifications (such as gapmer, LNA PS chimera, 2’-O-alkyl PS chimera) (Roberts T.C. et al, Nat Rev Drug Discov. 2020, 19(10):673-694; Barresi V. et al, Int. J. Mol. Sci. 2022, 23, 8875).

The molecule of the invention is preferably modified chemically in order to enhance its resist to nuclease activity (preferably by adding phosphorothioate (PS) to the backbone, and/or adding modified bases, such as 2' sugar modifications, including 2'-O-methyl (2'-OMe)) and/or to enhance stable hybridization with its target (for example by adding modified bases, such as 2'-O-methyl (2'- OMe)), and/or substituting 5-methyl dC for dC in CpG motifs).

The oligonucleotide molecule of the invention or a pharmaceutical composition containing same can be used as a medicament. Thus, in one aspect, the present invention also relates to the molecule of the invention, as defined above, or the pharmaceutical composition containing same, for use as a medicament, or for the manufacture of a medicament.

Accordingly, the present invention also concerns a use of the molecule of the invention (as defined above), or the pharmaceutical composition containing same, as a medicament, or for the manufacture of a medicament.

The present invention also concerns a method of treating a disease in a subject in need thereof, comprising administering the molecule of the invention, as defined above, or the pharmaceutical composition containing same, in said subject.

The molecule of the invention is preferably for use as a medicament, in subjects in need thereof.

The molecule of the invention can be safely administered by oral, topic, oromucosal, intranasal, intracranial, intraperitoneal, or parenteral route, such as intraocularly, intravenously, intraarterially, intrathecally, intracerebrally, intramuscularly, intraventricularly, intracisternally and subcutaneously. The molecule of the invention is preferably administered intrathecally, or intraocularly (for example by intravitreal administration (e.g. intravitreal injection), subretinal administration (e.g. subretinal injection), suprachoroidal administration (e.g. suprachoroidal injection), or any combination thereof), or any combination thereof, wherein the intraocular administration is preferably intravitreal administration. It can also be associated with a delivery system, such as a Cellular-Penetrating Peptide (CPP) or a liposome structure, to reach and enter the target cells more easily (Fabrega C. Pharmaceutics. 2023; 15(2):320).

As such, it can be introduced in a pharmaceutical composition, together with a pharmaceutically acceptable excipient as defined above. It can be used to manufacture a medicament that is intended for treating cell storage disorders involving an autophagy deficit.

Thus, the oligonucleotide molecule of the invention or the pharmaceutical composition containing same can be used as a medicament for treating cell storage disorders involving an autophagy deficit.

Accordingly, the present invention concerns the molecule of the invention or the pharmaceutical composition containing same, as defined above, for its use as a medicament for treating cell storage disorders involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs).

The present invention also concerns a use of the molecule of the invention or the pharmaceutical composition containing same, as defined above, for treating cell storage disorders involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs), or for the manufacture of a medicament for a treating cell storage disorder involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs).

The present invention also concerns a method of treating a cell storage disorder involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs) in a subject in need thereof, comprising administering the molecule of the invention or the pharmaceutical composition containing same, as defined above.

These disorders are for example lysosomal storage disorders (LSD) wherein the lysosomal function is impaired due to an inherited condition, thereby conducting to cellular dysfunction. Such LSDs include but are not limited to Sphingolipidoses (such as Fabry disease, Farber lipogranulomatosis, Gaucher disease (type l/ll/lll and perinatal lethal form), GM1 gangliosidosis (type l/ll/lll), GM2 gangliosidosis (Tay-Sachs disease, Sandhoff disease, GM2 activator deficiency), Globoid cell leukodystrophy (Krabbe disease), Metachromatic leukodystrophy, Niemann-Pick disease types A/B, Prosaposin Deficiency and Saposin B Deficiency); Mucopolysaccharidoses (such as MPS I (Hurler/Hurler-Scheie/Scheie syndrome), MPS II (Hunter syndrome), MPS lll/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 disease (such as GSDO, GSDI (von Gierke's disease), GSD Ila (Pompe disease), GSD lib (Danon 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; Glvcoproteinoses (such as a-Mannosidosis (type l/ll/lll), p-Mannosidosis, Fucosidosis, Aspartylglucosaminuria, Schindler disease (type l/ll/lll), Sialidosis (type l/ll), Galactosialidosis); Lipid storage diseases (such as Acid lipase deficiency (Wolman disease, cholesterol ester storage disease)); Post-translational modification defects (such as Multiple sulfatase deficiency, Mucolipidosis (II a/p, l-cell disease), Mucolipodosis II (a/p, pseudo-Hurler polydystrophy), Mucolipidosis III (y, variant pseudo-Hurler polydystrophy)); Integral membrane protein disorders (such as Cystinosis, Action myoclonus-renal failure syndrome, Sialic acid storage disease (ISSD, Salla disease), Niemann-Pick disease types C1/C2, Mucolipidosis IV); Neuronal ceroid lipofuscinoses (CLNs) (such as CLN1 (Haltia-Santavuori disease and INCL), CLN2 (Jansky- Bielschowsky disease), CLN3 (Batten-Spielmeyer- Sjogren disease), CLN4 (Parry disease and Kufs type A/B), CLN5 (Finnish variant late infantile), CLN6 (Lake-Cavanagh or Indian variant), CLN7 (Turkish variant), CLN8 (northern epilepsy, epilepsy mental retardation), CLN9, CLN10, CLN11 , CLN12 (Kufor-Rakeb syndrome), CLN13, CLN14); Lysosome-related organelles disorders (such as Hermansky-Pudlak disease type 1 to type 9, Griscelli syndrome 1 (Elejalde syndrome), Griscelli syndrome 2, Chediak-Higashi disease); Polyglucosan storage diseases (such as Lafora disease, adult PG body disease, AMP-activated protein kinase deficiency); Others (such as Pycnodysostosis, Papillon-Lefevre syndrome).

They can also be diseases of the nervous system whereby lysosomal function and autophagy are impaired, hence contributing to a degenerative process. Such diseases include but are not limited to Alzheimer's disease; Age-related macular degeneration; Cerebral p-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, dementia with Lewy bodies); Tauopathies (such as Primary age-related tauopathy dementia, Chronic traumatic encephalopathy, Progressive supranuclear palsy, Corticobasal degeneration, Frontotemporal dementia, parkinsonism linked to chromosome, Vacuolar tauopathy, Lytico-bodig disease, Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis); Frontotemporal lobar degeneration; Amyotrophic lateral sclerosis; Huntington's disease; trinucleotide repeat disorders; Familial dementia; Hereditary cerebral hemorrhage with amyloidosis; CADASIL syndrome; Alexander disease; Angelman syndrome; Pelizaeus-Merzbacher disease; Seipinopathies; Familial amyloidotic neuropathy; Serpinopathies; Amyloidosis (such as Senile systemic, light chain, heavy chain, secondary, Aortic medial, ApoAl, ApoAII, ApoAIV, Lysozyme, Fibrinogen, Dialysis, Cardiac atrial, Cutaneous lichen, Corneal lactoferrin, Apolipoprotein C2, Apolipoprotein C3, Lect2, Insulin, Galectin- 7, Corneodesmosin or Enfuvirtide amyloidosis); Type II diabetes; Inclusion body myositis/myopathy; Cataracts; Retinitis pigmentosa with rhodopsin mutations; Medullary thyroid carcinoma; Pituitary prolactinoma; Hereditary lattice corneal dystrophy; Mallory bodies; Pulmonary alveolar proteinosis; Odontogenic (Pindborg) tumor amyloid; Seminal vesicle amyloid; Cystic fibrosis; Sickle cell disease; Plasma cell dyscrasias; Exfoliation syndrome.

In a preferred embodiment, the disease to be treated is age-related macular degeneration, a synucleinopathy (preferably multiple system atrophy), or Parkinson’s disease.

In a preferred embodiment, the antisense oligonucleotide of the invention or the pharmaceutical composition containing same enables to treat Beta-mannosidosis, Cholesteryl ester storage disease, CLN1 disease, CLN2 disease, CLN3 disease, CLN7 disease, Fabry disease, GM2-gangliosidosis (Tay-Sachs disease, AB variant, and Sandhoff disease), Krabbe disease, Metachromatic leukodystrophy, Mucolipidosis Type l/ll/lll/IV, Mucopolysaccharidosis type I (Scheie syndrome, Hurler-Scheie syndrome, Hurler syndrome), Mucopolysaccharidosis type II (Hunter syndrome), Mucopolysaccharidosis type III (Sanfilippo syndrome type A, Sanfilippo syndrome type B), Mucopolysaccharidosis type IV (Morquio A syndrome), 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.

Treatment methods

The present invention also relates to treatment methods involving any of the pharmaceutical compositions of the invention in subjects in need thereof, in particular, in human suffering from cell storage disorders involving an autophagy deficit, e.g., from any of the above-mentioned diseases.

These pharmaceutical compositions contain either the nucleic acids or vectors encoding the mutated TFEB of the invention, or the mutated polypeptides themselves, or the molecule (in particular ASOs) of the invention or any other tool enabling to skip exon 3 of TFEB in situ.

The pharmaceutical compositions of the invention can usually be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intrathecal, intracranial, intraperitoneal, intranasal or intramuscular means. A typical route of administration of the composition of the invention is intravenous or intrathecal, although other routes can be equally effective.

The treatment methods of the invention therefore preferably involve the intrathecal administration of said pharmaceutical compositions (which has been proved efficient in human, 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).

Definitions

Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in chemistry, biochemistry, cellular biology, molecular biology, and medical sciences.

As used herein throughout the entire text, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "one or a plurality" of the referenced compounds or steps, unless the context dictates otherwise.

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

As used herein, when used to define products, compositions, cells, uses and methods, the term "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") is open- ended and do not exclude additional, unrecited elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence might be part of the final (and/or whole) amino acid sequence of the polypeptide. Such a polypeptide can have up to several hundred additional amino acid residues (e.g., linker and antioxidant moiety as described herein). "Consisting of" means excluding any other components or steps "consisting essentially of mean excluding other components or steps of any essential significance (however, other minor/insignificant components or steps are not excluded). In the present disclosure, the terms “comprising”, “consisting of and “consisting essentially of may be replaced with each other, if required.

As used herein, the terms "nucleic acid", "nucleic acid sequence" or "sequence of nucleic acid", "polynucleotide", "oligonucleotide", "polynucleotide sequence", and "nucleotide sequence", which will be used equally in the present description, will be intended to refer to double-stranded DNA, singlestranded DNA and products of transcription of said DNAs.

As used herein, the term “oligonucleotide molecule” designates a short polymer of nucleic acid (single - or double-stranded - DNA or RNA molecule), typically of 10-100 nucleotides, bases or base pairs (preferably 12 to 40 nucleic acid residues, more preferably 13 to 39 nucleic acid residues, 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, even more preferably 18 nucleic acid residues). It encompasses for example aptamers, interfering RNAs, and other well known polymers.

In the present description, the term "polypeptide" will be used to refer equally to a “protein” or a “peptide”.

It should be understood that the present invention does not relate to the genomic nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. It involves sequences which have been “isolated” and/or “purified”, i.e., they have been removed, directly or indirectly, from their natural chromosomal environment, for example by copying, synthetizing, etc.

The term "variant" is hereby intended to refer to a polynucleotide or a polypeptide whose sequence contains individual variations (SNPs) as compared with the reference nucleic acid sequence or amino acid sequence of the invention.

By contrast, the term "homologous" is intended to refer to a polypeptide or polynucleotide whose sequence has, with respect to the reference nucleic acid or amino acid sequence, bigger modifications, such as in particular a deletion, a truncation, an extension, a chimeric fusion and/or a number of mutations, so that their nucleic acid or amino acid sequence eventually shows at least 80%, preferably 90% or 95%, identity with the reference nucleic acid sequence or amino acid sequence.

For the purpose of the present invention, the term "percentage of identity" between two nucleic acid or amino acid sequences is intended to refer to a percentage of nucleotides or of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and throughout their length. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "window of comparison" in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison can be produced, besides manually, by means of the global homology algorithm of Needleman and Wunsch (1970) [J. Mol. Biol. 48:443], The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions and multiplying the result obtained by 100 so as to obtain the percentage of identity between these two sequences. For example, the needle program available on the site ebi.ac.uk, may be used, the parameters used being those given by default (in particular for the parameters "Gap open":10, and "gap extend":0.5; the matrix chosen being, for example, the "BLOSUM 62" matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.

The present invention also relates to nucleic acid molecules which hybridize specifically with the nucleic acid molecules of the invention (in particular to an oligonucleotide molecule of the invention which hybridizes specifically with its nucleic acid molecule target, said nucleic acid molecule target being preferably a RNA or a transcript, more preferably a messenger RNA (mRNA), more preferably a precursor mRNA (pre-mRNA; i.e., a primary transcript that becomes a messenger RNA (mRNA) after processing (in particular splicing)). Specific hybridization is preferably observed under high stringency conditions, i.e., when the temperature and ionic strength conditions are chosen so as to allow the hybridization between two complementary nucleic acid molecules/fragments. By way of illustration, high stringency conditions can be as follows. The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42°C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5*SSC (1*SSC corresponds to a 0.15 M NaCI+0.015 M sodium citrate solution), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10*Denhardt's, 5% of dextran sulfate and 1 % of salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature dependent on the size of the probe (i.e. 42°C. for a probe of size>100 nucleotides), followed by two 20-minute washes at 20°C. in 2*SSC+2% SDS and one 20-minute wash at 20°C. in 0.1*SSC+0.1 % SDS. The final wash is carried out in 0.1 *SSC+0.1 % SDS for 30 minutes at 60°C for a probe of size>100 nucleotides. The high stringency hybridization conditions described above for a polynucleotide of defined size will be adjusted by those skilled in the art for oligonucleotides of greater or smaller size, according to the teaching of Sambrook et al., 1989. Examples of said nucleic acid molecules are given below as “diagnostic tools”.

As used herein, the term “treat” may be used to describe prophylaxis, amelioration, prevention or cure of a lysosomal storage disorder and disorders characterized by lysosomal dysfunction and/or one or more of its associated symptoms. For instance, treatment of an existing lysosomal storage disorder and disorders characterized by lysosomal dysfunction may reduce, ameliorate or altogether eliminate the disorder, or prevent it from worsening. Prophylactic treatment may reduce the risk of developing a disorder and/or lessen its severity if the disorder later develops.

The term “vector” as used herein refers to a vehicle, preferably a nucleic acid molecule or a viral particle, that contains the elements necessary to allow delivery, propagation and/or expression of any of nucleic acid molecule(s) within a host cell or subject. This term encompasses cloning vectors (vectors for maintenance), expression vectors (vectors directing the expression of nucleic acid molecules to which they are linked in various host cells or subjects), extrachromosomal vectors (e.g. multicopy plasmids), integration vectors (e.g. designed to integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates), shuttle vectors (e.g. functioning in both prokaryotic and/or eukaryotic hosts) and transfer vectors (e.g. for transferring nucleic acid molecule(s) in a viral genome). As part of the description, the vectors may be of naturally occurring genetic sources, synthetic or artificial, or some combination of natural and artificial genetic elements. Each vector contains various components, depending on its function (e.g., expression of heterologous polynucleotide) and its compatibility with the particular host cell in which it resides.

The vector components generally include, but are not limited to: an origin of replication (at which replication is initiated), a multicloning site containing restriction sites, a selection marker gene and/or a reporter gene, one or more regulatory sequence(s) such as for example a promoter, a ribosome binding site (RBS), a signal sequence, the nucleic acid molecule insert and a transcription termination sequence. The selection marker gene and the reporter gene are used for selection of cells for which the desired vector has been inserted and is expressed. RBS is used to initiate the recruitment of a ribosome during the initiation of translation of the nucleic acid molecule. The signal sequence allows the protein expressed by the translation to be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.

Furthermore, the term “vector” has to be understood broadly as including non-natural or natural vectors such as mRNA, plasmids, viruses, retrovirus, EBV-derived episomes, cosmids, bacteriophage, and artificial chromosomes. Typically, such vectors are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them. The present invention also encompasses vectors (e.g. plasmid DNA and mRNA) complexed to lipids or polymers to form particulate structures such as liposomes, lipoplexes or nanoparticles. Selection of an appropriate vector will depend mainly on the size of the nucleic acid molecules to be inserted into the vector and the particular host cell to be transformed with the vector.

Vectors may remain free (i.e., non-integrated into a host cell genome), or may become integrated ("stably” or “temporary” incorporated) into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events. Still, they remain “heterologous” as compared to the host cell and its natural genome.

As used herein, the term “subject” designates any mammal that may benefit from the treatment of the invention. In particular, said subject is a human being. FIGURE LEGENDS

Figure 1 shows the effect of lentiviral infection of TFEB exon 3 deletion (TFEB-AEx3) and TFEB with Exon 3 (TFEB-WT) on TFEB translocation in Hela cells, a. Hela cells were transitory infected with lentivirus coding either TFEB-WT or TFEB-AEx3 and analyzed by microscopy, b. Cells were analyzed to calculate the percentage of TFEB translocation i.e. the ratio between nuclear and cellular c-Myc fluorescence intensity. Results are mean ± SEM; ****p<0.0001 , Two-tailed Unpaired test. WT: wild-type.

Figure 2: Evaluation of the effect of TFEB exon 3 deletion (TFEB-AEx3) on TFEB translocation iPSC-derived neurons, a. iPSC-derived neurons were transitory infected with either TFEB-WT or TFEB-AEx3, treated with 100mM of trehalose or vehicle and analyzed by microscopy, b. Neurons described in a. were analyzed to calculate the percentage of TFEB translocation i.e. the ratio between nuclear and cellular TFEB fluorescence intensity c. Mean cellular intensity calculated as the ratio between the total TFEB intensity density and the total area of the cell. Results are mean ± SEM; *p<0.05, One-Way ANOVA, Tukey's multiple comparisons test, ns: not significant, WT: wild-type.

Figure 3: Evaluation of the effect of stable lentiviral infection of TFEB exon 3 deletion (TFEB- AEx3) on its translocation in Hela cells, a. Hela cells were stably infected with either TFEB-WT or TFEB-AEx3 and analyzed by microscopy, b. Cells described in a. were analyzed to calculate the percentage of TFEB translocation i.e. the ratio between nuclear and cellular TFEB fluorescence intensity. Results are mean ± SEM; ****p<0.0001 , Two-tailed Unpaired test. WT: wild-type.

Figure 4: Evaluation of the effect of stable lentiviral infection of TFEB exon 3 deletion (TFEB- AEx3) on CLEAR activation in Hela cells, a. Hela cells were stably infected with either TFEB-WT or TFEB-AEx3 then transitory infected with a CLEAR lentivirus and analyzed by microscopy, b. Cells described in a. were analyzed to calculate the percentage of GFP fluorescence intensity. Results are mean ± SEM; ****p<0.0001 , One-way ANOVA, Tukey’s multiple comparison test. LV: Lentiviral infection; WT: wild-type.

Figure 5: Evaluation of the effect of stable lentiviral infection of TFEB exon 3 deletion (TFEB- AEx3) on LAMP1 expression in Hela cells, a. Hela cells were stably infected with either TFEB-WT or TFEB-AEx3 and analyzed by microscopy. b,c. Cells described in a. were analyzed to calculate the percentage of LAMP1 fluorescence intensity (a) or spot intensity (b). Results are mean ± SEM; ****p<0.0001 , One-way ANOVA, Tukey’s multiple comparison test. NT: Not transfected; WT: wildtype. Figure 6: Evaluation of the effect of stable lentiviral infection of TFEB exon 3 deletion (TFEB- AEx3) on beclin-1 expression in Hela cells, a. Hela cells were stably infected with either TFEB- WT or TFEB-AEx3 and analyzed by microscopy. b,c. Cells described in a were analyzed to calculate the percentage of beclin-1 fluorescence intensity (a) or spot intensity (b). Results are mean ± SEM; ****p<0.0001 , One-way ANOVA, Tukey’s multiple comparison test. NT: Not transfected; WT: wildtype.

Figure 7: Evaluation of the effect of TFEB exon 3 deletion (TFEB-DEx3) overexpression in a Hap knockout cellular model of Niemann-Pick disease on reducing storage products. Hap1 control or NPC- cells were transitory infected with lentivirus coding either TFEB-WT (black) or TFEB- DEx3 (white) and analyzed by microscopy, a. GM1 ganglioside spots per cell were analyzed using cholera toxin labelling, b. Cholesterol spots per cell were analyzed using filipin labelling. Results are mean ± SEM; **p<0.01 , Two-way ANOVA, Sidak's multiple comparisons test. NPC-: Nieman-Pick model; WT: wild-type.

Figure 8: Summary of efficacy of exon 3 skipping of TFEB across all tested ASOs. Results are expressed in AACt TFEB-AEx3 normalized to 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 exon 3 of TFEB: Chromosome 6: 41 ,690,917 - 41 ,690,663. We identified a hotspot located between ASO#15 and ASO#31 and within this zone, the two subzones located between ASO#15.3 to ASO#17.3 and the ASO#18.4 to ASO#20. Data > 1 are in bold.

Figure 9: Efficacy of exon 3 skipping of TFEB using the ASO walk approach. RT-qPCR analysis of skipped TFEB in Hela (a) or SH-SY5Y (b) cells 66h after a 6-hour treatment with ASOs. Curves representing the potential efficacy of the 18 ASOs to skip exon 3 of TFEB. mRNA expression was normalized to GAPDH and results are expressed in AACt TFEB-AEx3.

Figure 10: Evaluation of the effect of ASO#16 on PCR, sequencing and TFEB protein expression in Hela cells, a. Agarose gel of ~300 and ~500 bp amplification products obtained by conventional PCR. Hela cells were treated with either ASO scramble (ASO#Scr) or ASO#16. b. Sequencing of the PCR products obtained in a (ASO#16 condition). Note that the skipping is performed without shifting, adding, deleting, or modifying the nucleotides of exon 2 and 4. c. Western blot showing protein expression in Hela cells treated with either ASO scramble (ASO#Scr) or ASO#16 at eight different doses. Note the expression of a truncated TFEB protein of an expected size, smallerthan the TFEB wild-type protein, corresponding to the skipping of exon 3. Scr: Scramble.

Figure 11 : Evaluation of the effect of ASO#16 in CLEAR network activation in HeLa cells. Hela cells were infected with a lentivirus coding for a nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR) and treated with either ASO scramble (ASO#Scr) or ASO#16 at three low doses. The data demonstrates that the ASO#16 activates CLEAR network, even at very low doses, in comparison to ASO#Scr. Results are mean ± SEM; ****p<0.0001 , One-way ANOVA, Sidak's multiple comparisons test.

Figure 12: Evaluation of the effect of ASO#16 and ASO#16.3 in cholesterol accumulation on NPC model. Hela cells were infected with a lentivirus sh-RNA targeting the NPC gene and treated with either ASO scramble (ASO#Scr); ASO#16 or ASO#16.3. Cholesterol spots per cell were analyzed using filipin labelling. The data demonstrates that the ASO#16 and ASO#16.3 reduces cholesterol accumulation in the NPC model in comparison to ASO#Scr. Results are mean ± SEM; *p<0.05, One-way ANOVA, Tukey's multiple comparisons test. NPC-: Nieman-Pick model.

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 to skip exon 3 of TFEB on ARPE-19 cells. mRNA expression was normalized to GAPDH and results are expressed in AACt TFEB-AEx3. b. Percentage of TFEB translocation i.e. the ratio between nuclear and cellular TFEB fluorescence intensity in ARPE-19 cells treated with either ASO#16 or ASO#Scr at different dose. c. ARPE-19 cells were infected with a lentivirus coding for a nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR) and treated with either ASO scramble (ASO#Scr) or ASO#16 at different dose. Results are mean ± SEM; *p<0.05, Two-way ANOVA, Sidak's multiple comparisons test.

Figure 14: Evaluation of the effect of ASO#16 on ARPE-19 cell model of AMD. ARPE-19 cells were treated with either ASO#Scr or ASO#16 at 20nM and then overload with ferric ammonium citrate during 48 or 66h (group B and D, respectively). Data are expressed as intensity of iron spots per spot. Note that ASO#16 is able to significantly reduce the iron accumulation at both time points in comparison to ASO#Scr. Results are expressed as mean ± SEM; *p<0.05, Two-way ANOVA, Sidak’s multiple comparisons test.

Figure 15: Evaluation of the effect of ASO#16 and ASO#16.3 on alpha-synuclein accumulation in alpha-Syn pre-formed fibril (PFF) model. Differentiated SH-SY5Y cells were treated with either ASO scramble (ASO#Scr); ASO#16 or ASO#16.3 and with 5pg/ml of pre-formed fibrils. PFF spots per cell were analyzed using anti-phospho-a-Synuclein (Ser129) antibody labelling. The data demonstrates that the ASO#16 and ASO#16.3 reduces alpha-synuclein accumulation in the PFF model in comparison to ASO#Scr. Results are mean ± SEM; *p<0.05, One-way ANOVA, Tukey's multiple comparisons test. PFF: pre-formed fibrils. EXAMPLES

1. Evaluation of the effect of TFEB exon 3 deletion (TFEB-AEx3) on its translocation in Hela cells

Protocol

Hela cells were seeded (5.000 cells / well) in DMEM supplemented with 10% of SVF in 96-well plates and incubated for 24h at 37°C, 5% CO2 and 95% humidity.

After incubation, cells where infected with lentivirus coding forTFEB-AEx3 or TFEB. Both lentiviruses include a c-Myc tag attached to the TFEB open reading frame allowing to differentiate the exogenously expressed TFEB (either WT or with the deletion of the third Exon) from the endogenous TFEB.

After 72h, Hela cells were analyzed.

Cells were fixed for 10 min with 4% formaldehyde and then rinsed 3 times with PBS before immunohistochemistry protocol.

Briefly, immunostainings were performed upon dilution of primary antibodies in blocking solution (PBS + 2% goat serum + 0.1 % triton) overnight at 4 °C, followed by three washes and secondary antibody incubation in blocking solution for 1 h at RT.

For the acquisition of the images, at least 30 image fields were acquired per well of the 96-well plate by using Cellinsight CX7 High Content Analysis device (Thermo Scientific).

The c-Myc labeling allowed to calculate for each infected cell, the percentage of TFEB translocation (the ratio between nuclear and cellular c-Myc fluorescence intensity).

All data are shown as mean ± SEM. Data were analyzed by two-tailed unpaired Student’s t test for detecting significant differences between two groups. Statistical significance was set at p < 0.05.

Results

As disclosed on Figure 1 , the nuclear translocation of TFEB-AEx3 is significantly superior to the nuclear translocation of the wild-type form of TFEB in Hela cells.

2. Evaluation of the effect of TFEB exon 3 deletion (TFEB-AEx3) on its translocation in iPSC-derived neurons

Protocol iPSCs-derived neurons were plated into Poly-L-ornithine and Laminine coated 96-well plates at a density of 150K cells I well in 200pL of N2B27 supplemented with BDNF (20ng/ml), CDKi (3,3pM), AMPc (100pM) and DAPT (10pM). 9 days post thawing, neurons where infected with lentivirus coding for TFEB-AEx3 or TFEB and analyzed after 72h. Both lentiviruses include a c-Myc tag attached to the TFEB open reading frame allowing to differentiate the exogenously expressed TFEB (either WT or with the deletion of the third Exon) from the endogenous TFEB.

After 24 hours, the medium was changed and the neurons were incubated for 4 days before being treated with 100mM of trehalose or vehicle. After 2 days, neurons were fixed for 10 min with 4% paraformaldehyde and then rinsed 3 times with PBS before immunohistochemistry protocol.

Briefly, immunostainings were performed upon dilution of primary antibodies in blocking solution (PBS + 2% donkey serum + 0.1% triton) overnight at 4 °C, followed by three washes and secondary antibody incubation in blocking solution for 1 h at RT.

For the acquisition ofthe images, at least 40 image fields were acquired per well of the 96-well plate by using Cellinsight CX7 High Content Analysis device (Thermo Scientific).

The c-Myc labeling allowed to calculate for each infected cell, the percentage of TFEB translocation (the ratio between nuclear and cellular TFEB fluorescence intensity) and the mean cellular TFEB intensity (the ratio between the total TFEB intensity density and the total area of the cell).

All data are shown as mean ± SEM. Data were analyzed by One-Way ANOVA and Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

As disclosed on Figure 2, nuclear translocation of TFEB-AEx3 is significantly superior to the nuclear translocation of the wild-type form of TFEB in human neurons, and also superior to the nuclear translocation of TFEB induced by a potent translocator, trehalose.

This translocation is observed at similar levels of TFEB expression in all the conditions (panel c).

3. Validation of TFEB translocation on Hela cells stably expressing TFEB-AEx3

Protocol

To generate a stable cell line, Hela cells were seeded (40.000 cells I well) in DMEM supplemented with 10% of SVF in 24-well plates and incubated for 24h at 37°C, 5% CC^ and 95% humidity.

After incubation, cells where infected with lentivirus coding for TFEB-AEx3 or TFEB, and splitted in 6-well after 48h. The selection of cells was carried out by geneticin treatment (500|jg/mL; 6-well) after 24h.

Every 3 days, cells were split in T75 flasks and treated with geneticin (500pg/mL) before being frozen for further experiments.

To evaluate TFEB translocation in these cell line, Hela cells were seeded (5.000 cells /well) in DMEM supplemented with 10% of SVF in 96-well plates and incubated for 24h at 37°C, 5% CO2 and 95% humidity.

48h after incubation, cells where fixed for 10 min with 4% formaldehyde and then rinsed 3 times with PBS before immunohistochemistry protocol.

Cells were permeabilized for 10min with 0.1 % triton at room temperature and incubated for 30 min in a blocking solution (PBS + 2% goat serum + 0.1 % triton). Immunostaining was performed upon dilution of primary antibody in blocking solution overnight at 4 °C, followed by three washes and secondary antibody incubation in blocking solution for 1 h at RT.

For the acquisition of the images, at least 15 image fields were acquired per well of the 96-well plate by using Cellinsight CX7 High Content Analysis device (Thermo Scientific).

The TFEB labeling allowed to calculate for each infected cell, the percentage of translocation (the ratio between nuclear and cellular TFEB fluorescence intensity).

Results

As disclosed on Figure 3, nuclear translocation of TFEB-AEx3 is significantly superior to the nuclear translocation of the wild-type form of TFEB in stably-transfected Hela cells.

4. Evaluation of the effect of TFEB exon 3 deletion (TFEB-AEx3) overexpression on CLEAR network activation

Protocol

Hela cells stably expressing either TFEB-AEx3 or TFEB-WT (as in example 3 above) were seeded (5.000 cells / well) in DMEM supplemented with 10% of SVF in 96-well plates and incubated for 24h at 37°C, 5% CO2 and 95% humidity.

After incubation, cells where transitory infected with a lentivirus coding for a nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR). After 48h, cells were fixed for 10 min with 4% formaldehyde and then rinsed 3 times with PBS before reading the GFP intensity.

The GFP fluorescence intensity was then measured in each cell.

For each cell line (TFEB-AEx3 or TFEB-WT), data are calculated as percentage of control (ie. NO LV-CLEAR). Results are mean ±SEM. Data were analyzed by One-Way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

As disclosed on Figure 4, CLEAR network is significantly more activated in cells stably expressing TFEB-AEx3 in comparison to cells expressing the wild-type form of TFEB.

5. Evaluation of the effect of TFEB exon 3 deletion (TFEB-AEx3) overexpression on LAMP1 expression

Protocol

Hela cells stably expressing either TFEB-AEx3 (as in example 3 above) or TFEB-WT were seeded (5.000 cells / well) in DMEM supplemented with 10% of SVF in 96-well plates and incubated for 24h at 37°C, 5% CO2 and 95% humidity.

After 4 days, cells where fixed for 10 min with 4% formaldehyde and then rinsed 3 times with PBS before immunohistochemistry protocol.

Cells were permeabilized for 10min with 0.1 % triton at room temperature and incubated for 30 min in a blocking solution (PBS + 2% goat serum + 0.1 % triton). Immunostaining was performed upon dilution of LAMP1 primary antibody in blocking solution overnight at 4 °C, followed by three washes and secondary antibody incubation in blocking solution for 1 h at RT.

For the acquisition of the images, at least 10 image fields were acquired per well of the 96-well plate by using Cellinsight CX7 High Content Analysis device (Thermo Scientific).

For each group, we calculated the total intensity in the well (i.e LAMP1 intensity) and the intensity of the spot (i.e LAMP1 spot intensity). For each cell line (TFEB-AEx3 or TFEB-WT), data are calculated as percentage of control (ie. Not transfected, NT). Results are mean ± SEM. Data were analyzed by One-Way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

As disclosed on Figure 5, LAMP1 is significantly more expressed in cells stably expressing TFEB- AEx3 in comparison to cells expressing the wild-type form of TFEB.

6. Evaluation of the effect of TFEB exon 3 deletion (TFEB-AEx3) overexpression on Beclin-1 expression

Protocol

Cells were permeabilized for 10min with 0.1 % triton at room temperature and incubated for 30 min in a blocking solution (PBS + 2% goat serum + 0.1 % triton). Immunostaining was performed upon dilution of Beclin-1 primary antibody in blocking solution overnight at 4 °C, followed by three washes and secondary antibody incubation in blocking solution for 1 h at RT.

For each group, the total intensity in the well (i.e Becin-1 intensity) and the intensity of the spot (i.e Beclin-1 spot intensity) were calculated. For each cell line (TFEB-AEx3 or TFEB-WT), data are calculated as percentage of control (ie. Not transfected, NT). Results are mean ± SEM. Data were analyzed by One-Way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

As shown on Figure 6, Beclin-1 is significantly more expressed in cells stably expressing TFEB-AEx3 in comparison to cells expressing the wild-type form of TFEB. 7. Evaluation of the effect of TFEB exon 3 deletion (TFEB-AEx3) in a Hap knockout cellular model of Niemann-Pick disease on reducing storage products

Aim

The aim of this study is to evaluate the effect of TFEB orTFEB-AEx3 overexpression in haploid HAP1 knockout cell line model (edited by CRISPR/Cas to contain edition or deletion in a coding exon of interest) on storage products. The chosen haploid cellular model is that of Niemann-Pick disease, characterized by an accumulation of ganglioside GM1 and cholesterol.

Protocol

Hap1 cells (Horizon Discovery) were seeded (5.000 cells / well) in DMEM.

After 24h, cells were infected with lentivirus coding forTFEB-AEx3 or TFEB. Both lentiviruses include a c-myc tag attached to the TFEB open reading frame allowing to differentiate the exogenously expressed TFEB (either WT or with the deletion of the third Exon) from the endogenous TFEB. After 4 days, cells were fixed with 4% formaldehyde.

Cells were permeabilized for 10 min with 0.1 % triton at room temperature. Immunostaining was performed upon dilution of cholera toxin (1/1000) or filipin (1/50).

For each condition, we calculated the number of spots (cholera toxin or filipin) per cell. Results are mean ± SEM. Data were analyzed by two-way ANOVA followed by Sidak's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

Figure 7 shows that overexpression of TFEB-AEx3 but not TFEB-WT reduced the storage products GM1 ganglioside and cholesterol, accumulated in Niemann-Pick Hap1 cell line.

8. Screening of ASOs designed by the ASO-walk approach for exon 3 skipping of TFEB in HeLa cells.

Aim

The aim of this study was to assess the efficiency of ASOs in exon 3 skipping of TFEB in HeLa cells using the quantitative real time PCR technique. 112 ASOs were designed using the ASO-walk approach. Specifically, the 18-nucleotide-long ASOs were shifted by 1 to 5 nucleotides to cover the entire exon 3 as well as 50 nucleotides upstream (in the intron after exon 3) and 48 nucleotides downstream (in the intron before exon 3) of exon 3 of TFEB. The sequences of the ASOs (SEQ ID NO: 17 to SEQ ID NO: 128) are shown in Table 3 above.

Protocol

Hela cells were seeded (7.500 cells / well) in standard culture medium (DMEM + 10%SVF).

After 24 hours, the cells were treated with ASOs at various concentrations in lipofectamine + DMEM for 6 hours. The cells were then incubated for 66 hours in DMEM + 10%SVF.

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 with High- capacity cDNA reverse transcription kit (Thermofisher).

Primers for human TFEB (fw: AGCAGCCACCTGAATGTGTA (SEQ ID NO.: 133) ; rev: GAGCTCTCGCTTCTGGGTC (SEQ ID NO.: 134)); human TFEB skipped (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)) were run with iTAq Sybr green (Biorad). Results were analyzed with the CFX Manager software and normalized to GAPDH mRNA content for each sample.

Results

Figure 8 shows skipping efficiency of ASOs. Results are expressed in AACt TFEB-AEx3 normalized to 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 exon 3 of TFEB: Chromosome 6: 41 ,690,917 - 41 ,690,663. We identified a hotspot located between ASO#15 and ASO#31 and within this zone, the two subzones located between ASO#15.3 to ASO#17.3 and the ASO#18.4 to ASQ#20. Data > 1 are in bold.

We identified a hotspot located between sequences of 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) enabling the skipping of exon 3 of TFEB. Preferentially, we identified the two subzones 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 the 18 ASOs identified in these two areas, show a strong potential for exon 3 skipping of TFEB. For all, human TFEB (ENSG00000112561) gene HGNC:11753; location: GRCh38.p14 Chromosome 6: 41 ,683,978-41 ,736,259 reverse strand; GRCh38:CM000668.2.

9. Confirmation of the exon 3 skipping efficiency of TFEB by RT-qPCR on HeLa and SH-SY5Y cells at low dose

Aim

The aim of this study was to assess the efficiency of our 18 identified ASOs in exon 3 skipping of TFEB in HeLa and SH-SY5Y cells using the quantitative real time PCR technique.

Protocol

Hela cells were seeded (7.500 cells / well) in DMEM + 10%SVF.

SH-SY5Y cells were seeded (16 700 cells / well) in DMEM + 10%SVF in 96-well plates (previously coated with laminin + Poly-L-Ornithine).

After 24 hours, Hela or SH-SY5Y cells were treated with ASOs (#15.3 to #17.3 and #18.4 to #20 at 1 .25; and 0,625nM) in lipofectamine + DMEM for 6 hours. The cells were then incubated for 66 hours in DMEM + 10%SVF.

Primers for human TFEB; human TFEB skipped or GAPDH were run with iTAq Sybr green (Biorad).

Results were analyzed with the CFX Manager software and normalized to GAPDH mRNA content for each sample.

Results

Figure 9 demonstrates exon 3 skipping efficiency of gene TFEB across the 18 ASOs tested at low doses. The skipping efficiency varies depending on the ASOs, doses tested, and cell line. Here, we highlight significant skipping capability for ASO#16 and ASO#16.3 at both low doses tested and across the two cell lines.

10. Validation of the effect of ASO#16 on PCR, sequencing and protein expression on Hela cells

Aim The aim of this study is to evaluate the effect of ASO#16 in Hela cells on skipping Exon 3 using PCR, sequencing of the PCR products and western blot.

Protocol

Hela cells were seeded (7.500 cells I well) in DMEM + 10%.

After 24h, cells were treated with ASOs at 10nM in DMEM + lipofectamine for 6h. The cells were then incubated for 66 hours in DMEM + 10%SVF.

For PCR, cells were lysed and total RNA was extracted using the RNeasy Plus Kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription (RT) was performed with High- capacity cDNA reverse transcription kit (Thermofisher). Primers for human TFEB; human TFEB skipped or GAPDH were run with Platinum II hot start (Green) PCR master Mix (Thermofisher). The samples were loaded onto an agarose gel (1 ,5%) and purified using the QIAquick PCR Purification Kit (Qiagen) for illumina sequencing.

For western blot, cells were seeded (1 ,7 M cells I T75) in DMEM + 10%SVF. After 24h, cells were treated with either ASO scramble (80nM; SEQ ID NO: 129) or ASO#16 (SEQ ID NO: 36) at 0.625;

I .25; 2.5; 5; 10; 20; 40; 80 nM in DMEM + lipofectamine for 6h. The cells were then incubated for 66 hours in DMEM + 10%SVF.

Cells were lysed in RIPA buffer. Samples underwent the standard steps of centrifugation, sonication, and quantification using Pierce BCA Protein Assay Kit. Lysates were incubated for 10 minutes at 70°C in sample loading buffer and then loaded onto NuPAGE 10% Bis-Tris Mini Gel, then transferred to membranes (Bio-Rad). Standard IHC protocol was used to reveal TFEB.

Results

Figure 10 shows that the ASO#16 is capable of skipping exon 3 of TFEB. Sequencing of the PCR products shows an TFEB-AEx3 sequence lacking the entire exon 3. This sequence does not exhibit any shifting, addition, or deletion of nucleotides from exon 2 or exon 4. Moreover, the ASO#16 allows the dose-dependent expression of a truncated protein of the expected size in Hela cells.

II. Effect of the ASO#16 on CLEAR network activation

Aim

The aim of this study is to evaluate the effect of ASO#16 (SEQ ID NO: 36) on CLEAR network activation in HeLa cells.

Protocol Hela cells were seeded in DMEM. After incubation, cells were transitory infected with a lentivirus coding for a nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR).

Cells were treated with either ASO scramble or ASO#16 at 0.625; 1.25 and 2.5 nM in DMEM + lipofectamine for 6h. The cells were then incubated for 66 hours in DMEM + 10%SVF. Cells were fixed with 4% formaldehyde before reading the GFP intensity. Images were acquired using Cellinsight CX7 High Content Analysis device (Thermo Scientific). The GFP fluorescence intensity was then measured in each cell. For each group (dose), data are calculated as percentage of control (i.e. ASO#Scr). Results are mean ± SEM. Data were analyzed by One-Way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

ASO#16 is able to activate the CLEAR network at low dose in comparison to the scramble ASO (Figure 11).

12. Effect ASO#16 and ASO#16.3 on cholesterol reduction in a Niemann-Pick cellular model

Aim

The aim of this study is to measure the effect of the ASO#16 (SEQ ID NO: 36) and ASO#16.3 (SEQ ID NO: 39) on the accumulation product, i.e. cholesterol, in a Niemann-Pick cellular model of Hela.

Protocol

Hela cells were seeded (40.000 cells I P16) in DMEM + 10%SVF.

After 24h, cells were transitory infected with a sh-RNA targeting the mRNA of one of the genes responsible for the Niemann-Pick pathology (NPC).

After passage and seeding in P96 (5000 cells I well), cells were treated with either scramble ASO, ASO#16 or ASO#16.3 at 10nM in DMEM + lipofectamine during 6h.

After 72h, cells were fixed with 4% formaldehyde then incubated with filipin 2h.

Images were acquired using Cellinsight CX7 High Content Analysis device (Thermo Scientific). Data are expressed as number of filipin spots per cell. Results are mean ± SEM. Data were analyzed by One-way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results Both ASO#16 and AS0#16.3 reduces cholesterol accumulation in a Niemann-Pick disease model of Hela cells (Figure 12).

13. Validation of the effect of the AS0#16 on ARPE-19 cells

Aim

The aim of this study is to validate the effects of the ASO#16 on the skipping of exon 3 of TFEB using the RT-qPCR approach, the activation of the CLEAR network, and the translocation of TFEB in ARPE-19 cells. These ARPE-19 cells will form the basis of our studies on the Age-related macular degeneration (AMD) cellular model.

Protocol

For RT-qPCR, ARPE-19 cells were seeded (10.000 cells I well) in DMEM-F12 + 10%SVF.

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

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 with High-capacity cDNA reverse transcription kit (Thermofisher). Primers for human TFEB; human TFEB skipped or were run with iTAq Sybr green (Biorad). Results were analyzed with the CFX Manager software and normalized to GAPDH mRNA content for each sample.

For evaluation of the CLEAR network activation and TFEB translocation, ARPE-19 cells were seeded (500.000 cells I T25) in DMEM-F12 + 10%SVF. After incubation, cells were transitory infected with a lentivirus coding for a nuclear form of GFP under the CLEAR promoter 4X sequence (LV-CLEAR). After 48h cells were seeded (10.000 cell I well) in DMEM-F12 + 10%SVF. Cells were treated with either ASO Scramble or ASO#16 at 10; 20; 40 and 80nm or 0.625; 1 .25; 2.5; 5; 10; 20; 40 and 80nm respectively for CLEAR and TFEB translocation experiment in lipofectamine + DMEM-F12 for 6 hours. The cells were then incubated for 66 hours in DMEM-F12 + 10%SVF. After incubation, cells were fixed with 4% formaldehyde before reading the GFP intensity in each cell. Standard Immunostaining of TFEB was performed as previously described.

Images were acquired using Cellinsight CX7 High Content Analysis device (Thermo Scientific).

CLEAR data were calculated for each group (dose) as percentage of control (i.e. ASO#Scr). TFEB translocation data were calculated as the percentage of TFEB translocation (the ratio between nuclear and cellular TFEB fluorescence intensity). Results are mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

The ASO#16 is efficient in skipping exon 3 of TFEB in a dose-dependent manner. In addition, ASO#16 promotes TFEB translocation and activate the CLEAR network in comparison to the scramble ASO in ARPE-19 cells (Figure 13).

14. Evaluation of the effect of the ASO#16 on age-related macular degeneration (AMD) cellular model

Aim

The aim of this study is to evaluate the effect of the ASO#16 on a cellular model of AMD in ARPE- 19.

We have chosen the iron overload cellular model due to numerous pieces of evidence confirming the involvement of iron dysregulation in the pathogenic mechanisms of AMD. Iron overload is notably involved in ferroptosis, a newly identified programmed cell death pathway, reported to be associated with the pathogenesis of RPE dysfunction in AMD (Sun et al., 2018; Totsuka et al., 2019; Gupta et al., 2023). In addition, Iron could accumulate in the macula of AMD patients, particularly in RPE and Bruch’s membrane (Hahn et al., 2003).

Protocol

ARPE-19 cells were seeded (10.000 cells I well) in DMEM-F12 + 10%SVF.

After 24 hours, ARPE-19 cells were treated with either ASO Scramble or ASO#16 at 20nm in lipofectamine + DMEM-F12 for 6 hours. After 6 hours, the medium was changed and replaced with a standard medium (groups A and C) or enriched with 200pM of ferric ammonium citrate (group 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 a standard medium change at 48 and 66 hours, respectively.

Data were acquired using Cellinsight CX7 High Content Analysis device (Thermo Scientific). Iron accumulation is represented as the intensity of iron spots per spot.

Results are mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05. Results

The data in Figure 14 demonstrate iron accumulation in ARPE-19 cells in the presence of ferric ammonium citrate after 48 or 66 hours and highlight the efficiency of ASO#16 in reducing this iron accumulation in our ARPE-19 model of AMD.

15. Effect of ASO#16 and ASO#16.3 on alpha-synuclein aggregation in alpha-Syn pre-formed fibril model of SH-SY5Y differentiated cells

Aim

The aim of this study is to measure the effect of the ASO#16 and ASO#16.3 on the accumulation product, i.e. alpha-synuclein, in an alpha-synuclein pre-formed fibril (PFF) cellular model of SH- SY5Y.

Protocol

At DO, SH-SY5Y cells were seeded (10 000 cells I well) in DMEM + 10%hiSVF in 96-well plates (previously coated with laminin + Poly-L-Ornithine). ATRA/TPA-differentiated SH-SY5Y cells were treated (D7) with either scramble ASO, ASO#16 or ASO#16.3 at 40nM in DMEM + lipofectamine during 6h. At D10, cells were treated with pre-formed fibrils (5pg/ml), fixed with 4% formaldehyde at D14 and then rinsed 3 times with PBS before alpha-synuclein immunohistochemistry protocol. Images were acquired using Cellinsight CX7 High Content Analysis device (Thermo Scientific). Data are expressed as number of alpha-synuclein spots per cell. Results are mean ± SEM. Data were analyzed by One-way ANOVA followed by Tukey's multiple comparisons test for detecting significant differences between groups. Statistical significance was set at p < 0.05.

Results

Figure 15 shows that both ASO#16 and ASO#16.3 reduces alpha-synuclein aggregates in alpha- Syn pre-formed fibril model of SH-SY5Y differentiated cells.

General observations

The data presented in examples 1 - 15 demonstrate through a proof of concept using the overexpression of the protein TFEB lacking exon 3 (TFEB-AEx3) that the TFEB-AEx3 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 accumulation in a Niemann-Pick type C model, in comparison to the WT TFEB protein. Furthermore, we demonstrated through an ASO walk approach that two hot spots (Start: GRCh38.p14 Chromosome 6: 41 ,690,897-41 ,690,850 and 41 ,690,842-41 ,690,800 reverse strand) were identified in the efficiency of skipping exon 3 of TFEB. Through an evaluation of the skipping efficiency of TFEB at different doses, 18 ASOs showed a strong potential for skipping: 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).

The evaluation of these 18 ASOs at low doses highlighted the efficacy of two candidates (ASO#16 and ASO#16.3) in skipping exon 3 of TFEB:

ASO#16 - sequence: GCTGCAGATGGTAGGATG (SEQ ID NO:36); location: GRCh38.p14 Chromosome 6: 41 ,690,892- 41 ,690,875855 reverse strand.

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

Based on this, we demonstrated that the ASO#16 unexpectedly allowed i) a perfect skipping of exon 3 (verified by sequencing); ii) the expression of a protein TFEB-AEx3 and iii) activates the CLEAR network. We also demonstrated that both ASO#16 and ASO#16.3 possess the capability to reduce cholesterol accumulation in Niemann-Pick model. Finally, these data were evaluated in a cellular model of age-related macular degeneration (AMD), surprisingly demonstrating i) a strong exon skipping efficiency of TFEB ii) a translocation of TFEB iii) an activation of the CLEAR network in ARPE-19 cells and iv) a significant reduction in iron accumulation in AMD cellular model.

Claims

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

2. A vector containing a polynucleotide encoding a mutated TFEB protein, whose mRNA sequence does not contain the mRNA sequence of SEQ ID NO:10, said vector being preferably a plasmid or a viral vector.

3. The vector of 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 is absent.

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

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

6. An oligonucleotide molecule that is able to mediate exon skipping of exon 3 of the TFEB protein.

7. The molecule of claim 6, which is able to mediate exon skipping of exon 3 in a nucleic acid sequence encoding TFEB protein.

8. The molecule of any one of claim 6 or 7, wherein it is able to mediate exon skipping of the exon 3 of a nucleic acid sequence encoding the TFEB protein of SEQ ID NO:7, of SEQ ID NO:8, of SEQ ID NO:9, or of a variant or homologous thereof.

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

10. The molecule any one of claims 6 to 9, wherein it is directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence encoding intron 2-3, exon 3, intron 3-4, or any combination thereof; preferably located in the nucleic acid sequence ranging from the nucleic acid sequence encoding intron 2-3 to the nucleic acid sequence encoding intron 3-4, including exon 3.

11 . The molecule of claims 6 to 10, wherein it is directed to (or targeting, or complementary to, or hybridizing to, or any combination thereof) a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence encoding intron 2-3, exon 3, intron 3-4, or any combination thereof; preferably located in the nucleic acid sequence ranging from the nucleic acid sequence encoding intron 2-3 to the nucleic acid sequence encoding intron 3-4, including exon 3.

12. The molecule of any one of claims 6 to 11 , wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 214 to 1402 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

13. The molecule of any one of claims 6 to 12, wherein it is directed to a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 236 to 1424 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

14. The molecule of any one of claims 6 to 13, wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 269 to 621 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

15. The molecule of any one of claims 6 to 14, wherein it is directed to a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 269 to 621 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

16. The molecule of any one of claims 6 to 15, wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 339 to 436 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

17. The molecule of any one of claims 6 to 16, wherein it is directed to a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 339 to 436 (included) of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

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

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

20. The molecule of any one of claims 6 to 19, wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 342 to 369, or from nucleic acid residues 358 to 381 , of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

21 . The molecule of any one of claims 6 to 20, wherein it is directed to a nucleic acid sequence of 12 to 40 consecutive nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 342 to 369, or from nucleic acid residues 358 to 381 , of the nucleic acid sequence encoding from exon 2 to intron 10 of TFEB.

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

23. The molecule of any one of claims 6 to 22, wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 34 to 386 (included) of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

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

25. The molecule of any one of claims 6 to 24, wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 104 to 201 (included) of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

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

27. The molecule of any one of claims 6 to 26, wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 104 to 200 of the nucleic acid sequence encoding from intron 2-3 to intron 3- 4 (including exon 3) of TFEB; preferably from 104 to 199, more preferably from 104 to 198, more preferably from 104 to 197, more preferably from 105 to 196, more preferably from 105 to 195, more preferably from 105 to 194, more preferably from 105 to 193, more preferably from 105 to 192, more preferably from 105 to 191 , more preferably from 105 to 190, more preferably from 105 to 189, more preferably from 105 to 188, more preferably from 105 to 187, more preferably from 106 to 186, more preferably from 106 to 185, more preferably from 106 to 184, more preferably from 106 to 183, more preferably from 106 to 182, more preferably from 106 to 181 , more preferably from 106 to 180, more preferably from 106 to 179, more preferably from 106 to 178, more preferably from 106 to 177, more preferably from 107 to 176, more preferably from 107 to 175, more preferably from 107 to 174, more preferably from 107 to 173, more preferably from 107 to 172, more preferably from 107 to 171 , more preferably from 107 to 170, more preferably from 107 to 169, more preferably from 107 to 168, more preferably from 107 to 167, more preferably from 107 to 166, more preferably from 107 to 165, more preferably from 107 to 164, more preferably from 107 to 163, more preferably from 107 to 162, more preferably from 107 to 161 , more preferably from 107 to 160, more preferably from 107 to 159, more preferably from 107 to 158, more preferably from 107 to 157, more preferably from 107 to 156, more preferably from 107 to 155, more preferably from 107 to 154, more preferably from 107 to 153, more preferably from 107 to 152, more preferably from 107 to 151 , more preferably from 107 to 150, more preferably from 107 to 149, more preferably from 107 to 148, more preferably from 107 to 147, of the nucleic acid sequence from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

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

29. The molecule of any one of claims 6 to 28, wherein it is directed to a nucleic acid sequence of 12 to 40 nucleic acid residues located in the nucleic acid sequence ranging from nucleic acid residues 107 to 134 (included), or from nucleic acid residues 123 to 146 (included), of the nucleic acid sequence encoding from intron 2-3 to intron 3-4 (including exon 3) of TFEB.

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

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

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

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

34. The molecule of any one of claims 6 to 33, having at least 95% identity with the nucleic acid sequences 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 sequences 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 sequences shown in any one of SEQ ID NO34 (ASO#15.3) to SEQ ID NO: 44 (ASO#17.3) or of 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 sequences shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), more preferably having at least 95% identity with the nucleic acid sequence shown in SEQ ID NO: 39 (ASO#16.3).

35. The molecule of any one of claims 6 to 34, having 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 of SEQ ID NO: 50 (ASO#18.4) to SEQ ID NO: 56 (ASO#20), more preferably having the nucleic acid sequence shown in any one of SEQ ID NO: 36 (ASO#16) or SEQ ID NO: 39 (ASO#16.3), more preferably having the nucleic acid sequence shown in SEQ ID NO: 39 (ASO#16.3).

36. A pharmaceutical composition comprising the mutated 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 vector of any one of claims 1 to 5, the molecule of any one of claims 6 to 35, or any combination thereof.

37. Mutated TFEB protein as defined in any one of claims 1 to 5, or polynucleotide as defined in any one of claims 1 to 5, or vector of any one of claims 1 -5, or molecule of any one of claims 6 to 35, for its use as a medicament.

38. The pharmaceutical composition of claim 36, for its use as a medicament.

39. Mutated TFEB protein as defined in any one of claims 1 to 5, or polynucleotide as defined in any one of claims 1 to 5, or vector of any one of claims 1 -5, or molecule of any one of claims 6 to 35, for use for treating cell storage disorders involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs).

40. The pharmaceutical composition of claim 36, for use for treating cell storage disorders involving an autophagy deficit, in particular Lysosomal Storage Disorders (LSDs).

41 . The mutated TFEB protein for use, or the polynucleotide for use, or the vector for use, or the molecule for use, according to claim 39, or the pharmaceutical composition for use according to claim 40, wherein said cell storage disorders are neurodegenerative disorders, preferably chosen among: Alzheimer's disease; Age-related macular degeneration; Cerebral p-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, dementia with Lewy bodies); Tauopathies (such as Primary age-related tauopathy dementia, Chronic traumatic encephalopathy, Progressive supranuclear palsy, Corticobasal degeneration, Frontotemporal dementia, parkinsonism linked to chromosome, Vacuolar tauopathy, Lytico-bodig disease, Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis); Frontotemporal lobar degeneration; Amyotrophic lateral sclerosis; Huntington's disease; trinucleotide repeat disorders; Familial dementia; Hereditary cerebral hemorrhage with amyloidosis; CADASIL syndrome; Alexander disease; Angelman syndrome; Pelizaeus-Merzbacher disease; Seipinopathies; Familial amyloidotic neuropathy; Serpinopathies; Amyloidosis (such as Senile systemic, light chain, heavy chain, secondary, Aortic medial, ApoAl, ApoAII, ApoAIV, Lysozyme, Fibrinogen, Dialysis, Cardiac atrial, Cutaneous lichen, Corneal lactoferrin, Apolipoprotein C2, Apolipoprotein C3, Lect2, Insulin, Galectin-7, Corneodesmosin or Enfuvirtide amyloidosis); Type II diabetes; Inclusion body myositis/myopathy; Cataracts; Retinitis pigmentosa with rhodopsin mutations; Medullary thyroid carcinoma; Pituitary prolactinoma; Hereditary lattice corneal dystrophy; Mallory bodies; Pulmonary alveolar proteinosis; Odontogenic (Pindborg) tumor amyloid; Seminal vesicle amyloid; Cystic fibrosis; Sickle cell disease; Plasma cell dyscrasias; Exfoliation syndrome; preferably age-related macular degeneration, a synucleinopathy (preferably multiple system atrophy), or Parkinson’s disease.

42. The mutated TFEB protein for use, or the polynucleotide for use, or the vector for use, or the molecule for use, according to claim 39 or 41 , or the pharmaceutical composition for use according to claim 40 or 41 , wherein said cell storage disorder is chosen in the group consisting of: Beta-mannosidosis, Cholesteryl ester storage disease, CLN1 disease, CLN2 disease, CLN3 disease, CLN7 disease, Fabry disease, GM2-gangliosidosis (Tay-Sachs disease, AB variant, and Sandhoff disease), Krabbe disease, Metachromatic leukodystrophy, Mucolipidosis Type 1/II/III/IV, Mucopolysaccharidosis type I (Scheie syndrome, Hurler-Scheie syndrome, Hurler syndrome), Mucopolysaccharidosis type II (Hunter syndrome), Mucopolysaccharidosis type III (Sanfilippo syndrome type A, Sanfilippo syndrome type B), Mucopolysaccharidosis type IV (Morquio A syndrome), 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 mutated TFEB protein for use, or the polynucleotide for use, or the vector for use, or the molecule for use, according to any one claims 37, 39, 41 or 42, or the pharmaceutical composition for use according to any one of claims 38 or 40 to 42, for an administration via oral, topic, oromucosal, intranasal, intracranial, intraperitoneal, or parenteral route; such as intraocularly, intravenously, intraarterially, intrathecally, intracerebrally, intramuscularly, intraventricularly, intracisternally and subcutaneously; preferably for intrathecal administration or for 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.