Polypeptides, nucleic acid constructs and vectors and methods of treating, preventing or alleviating disease using same

Abstract

The present invention relates to a nucleic acid construct having a polynucleotide encoding a secreted protein, the non-secreted portion of which has a signal sequence domain and a transmembrane peptide (CPP) sequence domain, and to the use of the nucleic acid construct in the treatment of disease.

Description

[Technical field]

[0001] The present invention relates generally to polypeptides, nucleic acid constructs and vectors for use in the treatment, amelioration or prevention of disease. [Background technology]

[0002] Currently, there are no approved neuromodulatory therapies for most neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Nearly all neurodegenerative diseases involve the deposition of protein aggregates, which are thought to be due to an inability to degrade these aggregates. For example, AD is characterized by the accumulation of pathological forms of β-amyloid (Abeta) and tau, along with a lack of the autophagy / lysosomal system for clearance [1-3]. Similarly, PD is characterized by the deposition of α-synuclein (aSyn) in the brain, along with defects in lysosomal clearance, including a large deficiency in lysosomes [4]. Others have suggested that lysosomal defects are common to other neurodegenerative diseases, including frontotemporal dementia [5].

[0003] Transcription factor EB (TFEB) is a master regulator of autophagosome / lysosomal function that shuts out hundreds of genes into the Coordinated Lysosomal Expression and Regulation (CLEAR) network [6-8]. TFEB normally remains inactive in the cytoplasm bound to cytoplasmic proteins. Upon activation, TFEB is released and translocates to the nucleus where TFEB binds to DNA and directs gene expression.

[0004] Patients and AD animal models show a failure to shut out lysosomes (lysosomal clearance) with a decrease in active TFEB [1-3, 9]. Conversely, increased TFEB expression has been shown to reduce Abeta and tau deposition and improve cognitive function in mouse models [9-12]. Similarly, TFEB overexpression drives the shutout of aSyn aggregates from cell lines

[13] , from cultured dopaminergic / midbrain neurons overexpressing aSyn [14, 15], or from cells treated with the dopaminergic toxin 6-hydroxydopamine

[14] . TFEB overexpression can also protect neurons from neurodegenerative disease in rat models or in PD

[16] , namely the PD-causing toxins rotenone [17, 18] and MPTP / MPP

[19] . + [4, 19] Therefore, enhancing TFEB function is a promising strategy for treating neurodegenerative diseases.

[0005] Despite decades of research, gene transfer into the CNS remains a significant challenge. The main challenge in their use is the reliance on either cell penetrating peptides (CPPs) or viral transduction. CPPs, such as the “TAT” peptide derived from the HIV virus, allow macromolecules to cross cell membranes and the blood-brain barrier, but this approach requires the delivery of large amounts of protein and has had little clinical success to date

[20] . Our own work has shown that TAT-tagged peptides rapidly transduce into the brain, but these same TAT peptides cause rapid clearance of material from the brain

[21] . Viral transduction, on the other hand, uses a virus to deliver genetic DNA into individual cells and is highly effective in cellular and isolated mouse models. Many viral vectors are under investigation, including adenoviruses, lentiviruses, and adeno-associated viruses (AAVs). AAVs are considered by many to be the preferred vector for therapy in the CNS. The reasons are reduced oncogenicity (not integrated into the genome), longer expression time, safety in handling, relative freedom from immune reactions, and neurophilicity (especially AAV9 variants)

[22] . However, the core problem remains unsolved, namely that viruses injected into solid tissues only expand a few millimeters. Similarly, systemically administered viruses that are diluted and spread throughout the body only introduce a fraction of cells into the target tissue. We still do not have an answer for diseases that require the introduction of every single cell in the tissue [20, 22, 23]. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 6,184,344 [Patent Document 2] U.S. Patent No. 6,174,530 [Non-patent literature]

[0007]

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[0008] Disclosed herein is a system for improving the spread of virally delivered polypeptides. In some embodiments of the invention, non-secreted proteins (proteins normally found only inside cells) are modified to be secreted from the virally transduced cell and taken up by surrounding cells. Unlike conventional configurations, the secreted polypeptides of the invention can be deployed from cell to cell. In one embodiment, the non-secreted protein is a transcription factor that is modified to a secreted transcription factor (sTF). In one embodiment, the sTF is secreted TFEB (sTFEB), which is deployed from cell to cell, increasing the size and number of lysosomes and reducing β-amyloid, tau and α-synuclein aggregates. sTFEB delivery using the system of the invention in the brain is significantly more robust than conventional viral delivery, increasing lysosomal protein expression as well as reducing aggregated α-synuclein, aggregated tau and β-amyloid. Nucleic acid constructs encoding the secreted polypeptide(s) of the invention can be incorporated into vector systems that can be used to treat, alleviate or prevent various diseases / conditions affecting the body. In some embodiments, the disease / condition is associated with dysfunctional lysosomal clearance, for example affecting skeletal, mucosal skin, cardiac, muscular tissue, peripheral nervous system, and central nervous system. Diseases / conditions that affect dysfunctional lysosomal clearance include one or more neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and muscle diseases, such as muscular dystrophies. The constructs of the invention can also be used to treat, alleviate, or prevent infectious diseases caused by pathogens. Furthermore, the constructs of the invention have broad applicability to other neurodegenerative diseases and gene therapy paradigms.

[0009] In one embodiment, the invention relates to a nucleic acid construct comprising a polynucleotide encoding a secreted protein variant having a signal sequence domain and a non-secreted protein having a cell-penetrating peptide (CPP) sequence domain.

[0010] In one embodiment of the nucleic acid construct of the invention, the non-secreted protein is a nuclear protein, a cytoplasmic protein or an organellar protein.

[0011] In another embodiment of the nucleic acid construct of the invention, the non-secreted protein is a transcription factor.

[0012] In another embodiment of the nucleic acid construct of the invention, the secreted protein variant is a secreted TFEB (sTFEB) having wild-type TFEB with a signal sequence and a self-transmitting sequence.

[0013] In another embodiment of the nucleic acid construct of the invention, the non-secreted protein is an HSP or Cox8.

[0014] In another embodiment of the nucleic acid construct of the invention, the sequence encoding the cytoskeletal attachment site is deleted from the polynucleotide.

[0015] In another embodiment of the nucleic acid construct of the invention, the sequence encoding the DNA binding domain is deleted from the polynucleotide.

[0016] In another embodiment of the nucleic acid construct of the invention, the self-threading sequence comprises a TAT sequence, Pep-1, PVEC, polyarginine (R9, R8), penetratin, PVEC, MPG, R6 / W3, SAP, CyloP-1, gH625, GALA, TP10, CADY, L17E, MPPs, Ac-1, Entac-1, Ac-2, Peptide 3, RR5-App, RR4-App, RR3-aPP, TATp-D, R4-R4, R5, R5, [WR]4, Cyclic Tat, cFΦR4, Danamide D, Pro-(Xaa)4-Tyr, Cyclic sC18, RRRRΦF, BIM SAHB9 SAH-SOS1, 4-R, 4-W, Sp-CC-PEG2000, K10(QW)6, YTA4, v2 or W3.

[0017] In another embodiment of the nucleic acid construct of the present invention, the signal sequence domain is derived from the human amyloid precursor protein.

[0018] In another embodiment of the nucleic acid construct of the invention, the construct comprises an inducible or constitutive promoter operatively linked to a polynucleotide encoding a non-secreted protein.

[0019] In another embodiment of the nucleic acid construct of the invention, the promoter drives expression of the polynucleotide in the central nervous system.

[0020] In another embodiment of the nucleic acid construct of the present invention, the construct comprises a GFAP (Glial Fibrillary Acidic Protein) promoter, a CAG promoter, an SV40 promoter, a neuronal promoter, or an EFla promoter.

[0021] In another embodiment of the nucleic acid construct of the invention, the polynucleotide comprises, consists essentially of, or consists of SEQ ID NO:5.

[0022] In another embodiment of the nucleic acid construct of the invention, the polynucleotide comprises, consists essentially of, or consists of SEQ ID NO:6.

[0023] In another embodiment of the nucleic acid construct of the invention, the polynucleotide comprises, consists essentially of, or consists of SEQ ID NO:7.

[0024] Another embodiment of the present invention provides a recombinant vector comprising the nucleic acid construct of the present invention.

[0025] In one embodiment, the recombinant vector is a viral vector.

[0026] In another embodiment, the viral vector is a retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes simplex virus.

[0027] In another embodiment, the viral vector is an adeno-associated virus (AAV).

[0028] Another embodiment of the present invention relates to recombinant secreted protein variants having a signal sequence domain and a non-secreted protein having a cellular cleavage peptide (CPP) domain.

[0029] In one embodiment of the recombinant secreted protein variant of the present invention, the non-secreted protein is a nuclear protein, a cytoplasmic protein or an organellar protein.

[0030] In another embodiment of the nucleic acid construct of the invention, the non-secreted protein is a transcription factor.

[0031] In another embodiment of the recombinant secreted protein variant of the invention, the recombinant secreted protein is recombinant secreted transcription factor EB (sTFEB) having wild-type TFEB with a signal sequence and a self-transforming peptide domain.

[0032] In another embodiment of the recombinant secreted protein variant of the present invention, the recombinant secreted protein is a recombinant secreted HSP or a recombinant secreted Cox8.

[0033] In another embodiment of the recombinant secreted protein variant of the invention, the CPP domain is selected from the group consisting of TAT sequence, Pep-1, PVEC, polyarginine (R9, R8), penetratin, PVEC, MPG, R6 / W3, SAP, CyloP-1, gH625, GALA, TP10, CADY, L17E, MPPs, Ac-1, Entac-1, Ac-2, Peptide3, RR5-App, RR4-App, RR3-aPP, TATp-D, R4-R4, R5, R5, [WR]4, Cyclic Tat, cFФR4, DanamideD, Pro-(Xaa)4-Tyr, Cyclic sC18, RRRRФF, BIM SAHB9. With SAH-SOS1, 4-R, 4-W, Sp-CC-PEG2000, K10(QW)6, YTA4, v2 or W3.

[0034] In another embodiment of the recombinant secreted protein variant of the present invention, the signal sequence domain comprises the signal peptide of human amyloid precursor protein.

[0035] In another embodiment of the recombinant secreted protein variant of the present invention, the recombinant secreted protein is deleted for the cytoskeleton binding site (sTFEB del30).

[0036] In another embodiment of the recombinant secreted protein variant of the present invention, the recombinant secreted protein is deleted for the DNA binding site (sTFEB-DD).

[0037] In another embodiment of the recombinant secreted protein variant of the present invention, the recombinant secreted protein comprises, consists essentially of, or consists of SEQ ID NO:2.

[0038] In another embodiment of the recombinant secreted protein variant of the present invention, the recombinant secreted protein comprises, consists essentially of, or consists of SEQ ID NO:3.

[0039] In another embodiment of the recombinant secreted protein variant of the present invention, the recombinant secreted protein comprises, consists essentially of, or consists of SEQ ID NO:4.

[0040] In another embodiment, the present invention relates to a method for treating, alleviating or preventing a disease in a subject, comprising administering to the subject a vector having a polynucleotide construct encoding a secreted protein variant having a signal sequence domain and a non-secreted protein having a cell-penetrating peptide (CPP) domain.

[0041] In one embodiment of this method, the vector is a viral vector.

[0042] In another embodiment of the method, the viral vector is a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, or a herpes simplex virus.

[0043] In another embodiment of the method, the viral vector is AAV.

[0044] In another embodiment of the method, the non-secreted protein is a nuclear protein, an organellar protein, or a cytoplasmic protein.

[0045] In another embodiment of the method, the non-secreted protein is a transcription factor.

[0046] In another embodiment of the method, the transcription factor is transcription factor EB (TFEB) and the recombinant secreted protein variant has wild-type TFEB with a signal sequence domain and a cellular cleavage peptide domain.

[0047] In another embodiment of the method, the non-secreted protein is an HSP or Cox8.

[0048] In another embodiment of the method, the CPP domain has a TAT sequence, Pep-1, PVEC, polyarginine (R9, R8), penetratin, PVEC, MPG, R6 / W3, SAP, CyloP-1, gH625, GALA, TP10, CADY, L17E, MPPs, Ac-1, Entac-1, Ac-2, Peptide 3, RR5-App, RR4-App, RR3-aPP, TATp-D, R4-R4, R5, R5, [WR]4, Cyclic Tat, cFΦR4, Danamide D, Pro-(Xaa)4-Tyr, Cyclic sC18, RRRRΦF, BIM SAHB9 SAH-SOS1, 4-R, 4-W, Sp-CC-PEG2000, K10(QW)6, YTA4, v2, or W3.

[0049] In another embodiment of the method, the signal sequence domain is the signal peptide of the human amyloid precursor protein.

[0050] In another embodiment of the method, the recombinant secreted protein variant is defective in the cytoskeletal binding site (sTFEB del30).

[0051] In another embodiment of the method, the recombinant secreted protein comprises, consists essentially of, or consists of SEQ ID NO:2.

[0052] In another embodiment of the method, the recombinant secreted protein has, consists essentially of, or consists of SEQ ID NO:3.

[0053] In another embodiment of the method, the recombinant secreted protein has, consists essentially of, or consists of SEQ ID NO:4.

[0054] In another embodiment of the method, the disease is associated with dysfunctional lysosomal clearance.

[0055] In another embodiment of the method, the disease affects the skeleton, skin, mucous membranes, heart, liver, hematopoietic system, muscle, peripheral nervous system, and central nervous system.

[0056] In another embodiment of the method, the disease is a neurodegenerative disease.

[0057] In another embodiment of the method, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0058] In another embodiment of the method, the disease is caused by a pathogen.

[0059] In another embodiment of the method, the pathogen is a virus, a bacterium, or a fungus.

[0060] In another embodiment of the method, the disease is muscular dystrophy or MS.

[0061] Another embodiment of the present invention relates to the use of the vectors disclosed herein in the treatment of disease.

[0062] In one embodiment of the use, the disease is associated with dysfunctional lysosomal clearance.

[0063] In another embodiment of the use, the disease affects the skeleton, skin, mucous membranes, heart, liver, hematopoietic system, muscle, peripheral nervous system and central nervous system.

[0064] In another embodiment of the use, the disease is a neurodegenerative disease.

[0065] In another embodiment of the use, said neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0066] In another embodiment of the use, the disease is caused by a pathogen.

[0067] In another embodiment of the use, the pathogen is a virus, a bacterium or a fungus. [Brief description of the drawings]

[0068] The following drawings illustrate various aspects of the present invention and alternative preferred embodiments. [Figure 1]FIG. 1 is a schematic diagram of a construct according to the invention. The vector is designed to express sTFEB (secreted TFEB). The vector in these embodiments is designed to express sTFEB fused to green fluorescent protein (GFP). This system uses GFP, which allows easy tracking of the spread of sTFEB using a fluorescent microscope. In some embodiments, a non-secreted red fluorescent protein (RFP) is used to distinguish transduced cells from non-transduced cells that have taken up sTFEB (if tracking the spread or distinguishing between transduced and non-transduced is not required, this fluorescent protein can be removed to make room for the vector, improving expression and removing a potential target for the immune system). In some embodiments, the construct has a secretion signal sequence and a cell penetrating peptide (CPP; in this case the TAT peptide from HIV). In some embodiments, sTFEB lacks a cytoskeleton binding site (CC; sTFEB del30). In some embodiments, the vectors contain luciferase for live animal imaging instead of GFP. In some embodiments, a signal sequence (here the signal peptide of the human amyloid precursor protein) 'SS' guides TFEB to the secretion system. When secretion occurs, the secreted protein is taken up from the extracellular space by untransduced cells due to the presence of the 'CPP' domain. The 'CC' domain of TFEB binds cytoskeletal structures. In the absence of this binding, TFEB constitutively translocates to the nucleus and drives gene expression (so TFEB is always active). The HLH domain is required for DNA binding and loss of this domain inactivates TFEB-mediated gene activation and can be used as a control for proof-of-principle studies. [Diagram 2]Figure 2 shows cell-to-cell spread of sTFEB-GFP in neuroblastoma. Micrographs of N2A cells transfected with sTFEB-GFP (top), sTFEB-del130-GFP (middle) and sTFEB-DD-GFP (bottom). Cells were immunostained with LAMP1 (magenta). The rightmost panel shows the colocalization analysis channel comparing the overlap of sTFEB-GFP and lysosomes using Imaris software (Bitplane). Cells transfected with sTFEB-GFP also express RFP and are marked by an arrowhead. All other cells, which are green, take up GFP from the medium. Cells transfected with sTFEB-GFP show punctate anti-cytoplasmic / nuclear staining depending on how much sTFEB-GFP they have taken up. Some sTFEB-GFP in the cytoplasm colocalizes with lysosomes. Deletion of the cytoskeleton-binding domain (sTFEB-del30) increased the number of cells with nuclear GFP signal and reduced the amount of sTFEB in lysosomes, whereas deletion of both the DNA-binding and cytoskeleton-binding domains (sTFEB-DD) resulted in cytoplasmic GFP expression but reduced lysosomal labeling. [Diagram 3] Figure 3 shows that sTFEB moves from cell to cell with high efficiency. These micrographs show that it is sTFEB (green) but not adenovirus (red) that spreads from cell to cell. The cells in the left panel are transfected with sTFEB-GFP and an adenovirus encoding a non-secreted red fluorescent protein. The medium conditioned by these cells is then added to a second cell dish (right panel - neuroblastoma cell line and cultured human iPSC-derived human neurons). After this, the second cell dish turns green (instead of red), indicating that they have taken up sTFEB-GFP and not the viral particles themselves. [Figure 4A-4C]Figures 4A-C show that sTFEB induces expression of cathepsin B, lysosome size, and lysosome number. Figure 4A shows micrographs of N2a cells (Figure 4A-upper panel) or iPSC-derived neurons (Figure 4A-lower panel) that incorporated sTFEB Ddel130 or TFEB / inactive sTFEB-DD-GFP from conditioned media over a 48-h period. Magic Red (Immunochemistry Technologies) fluorescent substrate for cathepsin B was added to the media and cells were imaged live. Lysosome size and number were quantified using Imaris software. N2A cells that incorporated constitutively active sTFEB-del30 had significantly more lysosomes (compared to control cells), significantly larger lysosome size, and significantly stronger CatB enzymatic activity (generated by integration of intensity*area) (see Figure 4B). Imaris software was used to quantitate lysosome size and number in human iPSC-derived neurons. Neurons that had incorporated constitutively active sTFEB-del30 had significantly higher lysosome numbers (compared to control cells), significantly larger size, and significantly stronger CatB enzymatic activity (generated by intensity*area integration) (see Figure 4C ). [Figure 5A-5B] Figures 5A and 5B show that sTFEB increases the transcription levels of lysosomal genes (Figure 5A) and lysosomal proteins (Figure 5B). As shown in Figure 5A, N2A cells were treated with constitutively active sTFEB-del30 or inactive sTFEB-DD and gene expression was analyzed by rtPCR and normalized to cells exposed to normal medium. sTFEB increases lysosomal gene expression. As shown in Figure 5B, N2A cells were treated with sTFEB, constitutively active TFEB-del30 or inactive sTFEB-DD and cells were analyzed by Western blotting. sTFEB increases the expression of LAMP1 and CatD4 proteins, but not tubulin (control). [Figure 6A-6B]Figures 6A and 6B show that constitutively active sTFEB-del30 reduces intracellular β-amyloid. N2A neuroblastoma cells were transfected with inactive TFEB-DD-GFP (Figure 6A) or constitutively active sTFEB-del30 (Figure 6B). After 48 hours, they were loaded overnight with 500 nM fluorescently labeled β-amyloid-42 (shown in red). After another 48 hours post-seeding, cells were fixed and imaged. Micrographs showed that cells containing inactive TFEB-DD-GFP had a much more intense red signal from β-amyloid than cells containing constitutively active TFEB-GFP. [Figure 7A-7B] Figures 7A and 7B show that constitutively active sTFEB-del30 reduces neurofibrillary tangles. Human iPSC-derived neurons were transfected with mutant tau (tauP301L)-blue fluorescent protein (shown in the micrograph in cyan) and treated with preformed tau fibrils to elicit neurofibrillary tangles. Cells were then exposed to medium containing inactive TFEB-DD-GFP (Figure 7A) or constitutively active sTFEB-del30 (Figure 7B). Micrographs show that cells exposed to inactive TFEB are loaded with tau filaments (Figure 7A), whereas cells exposed to active TFEB-del30 have few visible filaments (Figure 7B). [Figure 8A-8D] Figures 8A-8D show that constitutively active sTFEB reduces α-synuclein deposition. SHSY5Y neuroblastoma cells (8A and 8B) and human iPSC-derived neurons (8C and 8D) were transfected with aSyn A53T-BFP (green) and either TFEB-DD-GFP (inactive, 8A and 8C) or sTFEB-del30-GFP (constitutively active, 8B and 8D). After 5 days, cells were fixed and imaged by confocal microscopy. Micrographs show that cells that had incorporated inactive sTFEB-DD-GFP had cytoplasmic synuclein inclusions (arrowheads 8A and 8C), whereas cells that had incorporated sTFEB-del30-GFP did not (8B and 8D). [Figure 9]Figure 9 shows that sTFEB-del30-GFP significantly enhances TFEB expression, enhances lysosomal staining, and reduces α-synuclein in the brain compared to wild-type TFEB-GFP. On the left, 14-month-old homozygous M83 mice were intracerebroventricularly injected with 10∧7 infectious units of adenovirus overexpressing constitutively active sTFEB-del30-GFP, and on the right, normal wt-TFEB-GFP was injected. Seven days later, mouse brains were perfused, fixed, cryosectioned, immunostained with antibodies against GFP (green) and LAMP1 (red), aggregated phosphorylated synuclein, and imaged on a Leica TSC SP8 confocal microscope. The top row of Figure 9 shows micrographs showing that TFEB-GFP expression is significantly higher on the sTFEB-treated side. The middle row of Figure 9 shows that the constitutively active TFEB side generally exhibits stronger LAMP1 staining (more lysosomes) and, at higher magnification, more lysosomes. The bottom row of Figure 9 shows tissue stained with an antibody against phosphorylated α-synuclein, which is specific to pathological synuclein deposits and is reduced in the sTFEB-treated area. [Figure 10A-10B] FIG. 10A is a photomicrograph of a mouse brain showing β-amyloid clearing by sTFEB-del30. On the left, 12-month-old 3XTG Alzheimer's mouse hippocampi were injected with adenovirus expressing TFEB-del30-GFP, and on the right, with adenovirus expressing wt-GFP alone. After 1 week, the mice were sacrificed and targeted stained for GFP (green) and β-amyloid (yellow). The left side treated with sTFEB-del30-GFP shows greater expression and diffusion compared to non-diffuse GFP expression (see FIG. 10A). The number of amyloid plaques in each hippocampus and in the outer layer on the hippocampus were counted, and the results are shown in the graph in FIG. 10B. Figures 10A and 10B show that few plaques were observed in the hippocampus and tegument on the sTFEB-del30 side (left side of Figure 10A, sTFEB) compared to the control side (right side of Figure 10A, GFP). [Figures 11A-11D]Figures 11A-11D show that sTFEB spreads beyond the injection site, crowding out β-amyloid. Figure 11A shows GFP staining at the level of injection, and Figure 11B shows GFP staining 3 mm posterior to the injection site. Figure 11C shows amyloid-42 immunostaining at the level of injection, and Figure 11D shows a magnification of the small rectangle in Figure 11C. [Figure 12] FIG. 12 shows the alignment of SEQ ID NO:2 and SEQ ID NO:5. Detailed Description of the Invention

[0069] Abbreviation Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Also, unless otherwise indicated, except in the claims, "or" also means "and" and vice versa. Non-limiting terms are not limiting unless otherwise specified or the context indicates otherwise (e.g., "including, having, and comprising" generally mean "without limitation"). In the claims, the singular terms (a, an, and the) also refer to the plural unless otherwise specified. "Consisting essentially of" means that the recited elements are necessarily included, excluding elements that materially affect the basic and novel characteristics of the listed elements, and that other elements may be used as appropriate. Also, "consisting of" means that all elements other than the listed elements are excluded. All embodiments defined by each of these terms are within the scope of the present invention.

[0070] All numerical expressions herein, such as ranges, levels, amounts, and concentrations, are approximations and include (+) or (-) increments of 0.1, 1.0, or 10.0, as the case may be. Numerical values ​​recited should be construed as "about."

[0071] The term "administration" includes any method of delivery of a construct, vector or composition of the invention, including delivery of a pharmaceutical composition, vaccine or therapeutic agent to a system of a subject or to a particular area of ​​a subject. As used herein, the terms "systemic administration", "administered systemically", "local administration" and "administered locally" refer to administration other than direct administration to the central nervous system, such as subcutaneous administration, so that a compound, drug or other substance can enter the patient's body and be metabolized or otherwise processed. The terms "parenteral administration" and "parenterally administered" refer to forms of administration other than enteral or local administration, such as injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, cardiac, intradermal, peritoneal, transorgan aspiration, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrapleural administration.

[0072] The term "amino acid" is well known. In general, the abbreviations used herein to refer to amino acids and protecting groups are based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For the amino acids relevant to the present invention, M refers to methionine, R refers to arginine, G refers to glycine, E refers to glutamic acid, L refers to lecithin, and F refers to phenylalanine. In some embodiments, the amino acids used in the present invention are the naturally occurring amino acids present in proteins, or the naturally occurring anabolic or catabolic products of such amino acids that contain amino and carboxyl groups. Particularly preferred amino acid side chains have side chains selected from the group of amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.

[0073] The term "fragment" as used herein refers to a peptide or polypeptide having an amino acid sequence having at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, and at least 200 contiguous amino acid residues, and at least 250 contiguous amino acid residues of a full-length peptide, polypeptide, or protein. In one embodiment, a fragment of a full-length protein retains an activity of the full-length protein, e.g., an immunogenic activity. In some embodiments, a fragment of a full-length protein does not have an activity of the full-length protein, e.g., a non-immunogenic activity.

[0074] As used herein, the terms "subject" and "patient" are interchangeable, and the term "subject(s)" refers to either a human or non-human animal.

[0075] As used herein, the phrase "pharmacologically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, for transporting a compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Illustrative examples of materials that can be used as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols, such as polyethylene glycol. (11) glycols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic counterparts used in pharmaceutical formulations.

[0076] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotides of any length in polymeric form, either deoxyribonucleotides or ribonucleotides, or their analogs. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) obtained by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides or nucleotide analogs. The nucleotide structure, if present, may be modified prior to or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide moieties. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling moiety. The term "recombinant" polynucleotide refers to a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, which is not naturally occurring and is linked to another polynucleotide in a non-natural configuration. "Oligonucleotide" refers to a single-stranded polynucleotide of less than about 100 nucleotides, e.g., 75, 50, 25, or 10 nucleotides.

[0077] The terms "polypeptide", "peptide" and "protein" (when single chain) are used interchangeably herein to refer to a polymer of amino acids, which may be linear, branched or interrupted by non-amino acids. The terms also refer to modified amino acid polymers, such as those that have been subjected to any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or conjugation with a labeling moiety.

[0078] In some embodiments, the polypeptides of the present invention can be chemically synthesized or ribosomal synthesized in a cell-free system or in a cell. Chemical synthesis of the polypeptides of the present invention can be performed using a variety of conventional methods, such as stepwise solid-phase synthesis, semi-synthesis by conformation-assisted religation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation utilizes the chemoselective reaction of two unprotected peptide segments to form a transient thioester bond intermediate. This transient thioester bond intermediate spontaneously rearranges to produce a full-length ligation product with a native peptide bond at the ligation site. The full-length ligation product is chemically identical to the protein synthesized by cell-free synthesis.The full-length ligation products can be optionally refolded and / or oxidized to generate native disulfide-containing protein molecules (see US Pat. Nos. 6,184,344 and 6,174,530, and TW Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; LH Huang, et al., Biochemistry (1991): vol. 30, p 7402; M. Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p 90; RE Offord, “Chemical Approaches to Protein Engineering”, in Protein Design and the Development of New therapeutics and Vaccines, JB Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; CJA Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; TK Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91: 12544-12548; M. Schnlzer, etal., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).

[0079] "Recombinant expression vector" refers to a genetically engineered polynucleotide construct that expresses an mRNA, protein, polypeptide, peptide, or fragment thereof, upon contact of the vector with a host cell under conditions suitable for expression of the mRNA, protein, polypeptide, peptide, or fragment thereof in the host cell. The disclosed vectors are not naturally occurring as a whole.

[0080] Overview The present invention provides novel secreted / diffusible protein variants or versions that are designed to expand from the site of delivery and be taken up by surrounding cells for efficient tissue distribution. To this end, nucleotide constructs have been designed to express the novel protein variants of the present invention.

[0081] In one embodiment, the invention provides secreted variants of non-secreted proteins that improve tissue distribution upon delivery by a vector, such as a viral vector.

[0082] In one embodiment, the secreted variant protein has a signal sequence and a cell membrane penetrating peptide (CPP) at its N-terminus, such as the TAT peptide derived from the transcription factor of HIV, which can import the produced variant secreted protein through the cell membrane. The signal peptide at the amino-terminal region of the variant secreted protein targets the protein to the ER and Golgi network (secretory pathway) for synthesis and secretion of the variant protein. As shown in Figure 1, the signal sequence and the CPP sequence can be attached to the N-terminus of the secreted diffusible protein.

[0083] The signal sequence may be any suitable signal sequence that directs the protein to the ER and the Golgi network, such as the signal peptide of human amyloid precursor protein.

[0084] Non-limiting examples of CPPs suitable for the constructs of the invention are Pep-1, PVEC, polyarginine (R9, R8), penetratin, PVEC, MPG, R6 / W3, SAP, CyloP-1, gH625, GALA, TP10, CADY, L17E, MPPs, Ac-1, Ent ac-1, Ac-2, peptide 3, RR5-App, RR4-App, RR3-aPP, TATp-D, R4-R4, R5, R5, [WR]4, Cyclic Tat, cFΦR4, Danamide D, Pro-(Xaa)4-Tyr, Cyclic sC18, RRRRΦF, BIM SAHB9 SAH-SOS1, 4-R, 4-W, Sp-CC-PEG2000, K10(QW)6, YTA4, v2 or W3 (Daniela Kalafatovic and Ernest Giralt, Molecules. 2017 Nov;22(11):1929).

[0085] Exemplary non-secreted proteins that can be secreted variants of the present invention include, but are not limited to, non-secreted nuclear proteins, cytoplasmic proteins and organelle proteins such as TFEB, stress response proteins (such as heat shock proteins HSPs) or mitochondrial proteins (such as Cox8).

[0086] In one embodiment, the non-secreted protein to be modified into a secreted variant is a transcription factor, and in some embodiments, the secreted variant is a secreted transcription factor (sTF). In one embodiment, the sTF is a secreted / diffusible TFEB (sTFEB). In one embodiment of the novel sTFEB variants of the invention, wild-type TFEB (SEQ ID NO:1) has a signal sequence (resulting in secretion of the resulting protein) and a cell membrane penetrating peptide (CPP; in this case, the TAT peptide from HIV) at its N-terminus, allowing the resulting variant protein to be imported across the cell membrane (see TFEB, SEQ ID NO:2 in Figure 1). The signal peptide in the amino-terminal region of the variant secreted protein targets the protein to the ER and Golgi network (secretory pathway) for synthesis and secretion of the variant protein. As shown in Figure 1, a signal sequence and a CPP sequence can be attached to the N-terminus of TFEB.

[0087] In one embodiment, the secreted protein variant of the invention is constitutively active by eliminating its cytoskeletal binding site (if present). Thus, using sTFEB as an example, removing the cytoskeletal binding site will cause the protein variant to translocate to the nucleus due to its absence.

[25] In another embodiment, the secreted protein variant is further modified by eliminating the DNA binding site (e.g., in the case where the secreted protein variant is a transcription factor) so that the secreted protein variant of this embodiment does not activate gene expression.

[0088] In one embodiment, the novel sTFEB is constitutively active (sTFEB-Delta30; SEQ ID NO:3) by erasing the cytoskeletal binding site of wild-type TFEB, such that the absence of this site results in the protein being translocated to the nucleus.

[25] By erasing both the cytoskeletal and DNA binding sites, an inactive form of sTFEB (sTFEB-DD; SEQ ID NO:4) is generated, such that the TFEB protein cannot bind DNA and activate gene expression.

[0089] In some embodiments, the polynucleotide constructs of the invention encoding novel secreted / diffusible transcription factors can be inserted into any suitable vector, such as an expression plasmid, or a virus, such as an adenovirus, an adeno-associated virus 9 (AAV9), a retrovirus, a lentivirus, or a herpes simplex virus. In some embodiments, the invention also relates to vectors carrying the nucleotide constructs of the invention. The viral construct used in this example is a minimal EFla promoter that drives expression in the central nervous system, although other cell type specific promoters can be used. Non-limiting examples of promoters that can be used include the Glial Fibrillary Acidic Promoter (GFAP), the CAG promoter, the SV40 promoter, the neuronal promoter, or the EFla promoter.

[0090] In some embodiments, a set of constructs have been engineered with sTFEB in an adeno-associated virus 5 (AAV5) vector that is driven by the glial fibrillary acidic promoter (GFAP) and preferentially expressed in glia / astrocytes.

[27] These GFAP constructs reduce the metabolic burden of sTFEB production on neurons. Adenovirus has been used in many preliminary experiments, but AAV requires several weeks for expression, cell culture experiments are difficult, and AAV is too small to incorporate RFP to confirm transduction of cells.

[0091] In one embodiment, sTFEB of the invention has, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2.

[0092] In another embodiment, the sTFEB of the invention has, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:3.

[0093] In another embodiment, the sTFEB of the invention has, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:4.

[0094] In one embodiment, a construct of the invention comprises, consists essentially of, or consists of a nucleotide sequence encoding sTFEB (SEQ ID NO:5).

[0095] In another embodiment, a construct of the invention comprises, consists essentially of, or consists of a nucleotide sequence encoding sTFEBdel30 (SEQ ID NO:6).

[0096] In another embodiment, a construct of the invention comprises, consists essentially of, or consists of a nucleotide sequence encoding sTFEB-DD (SEQ ID NO:7).

[0097] In another embodiment, the construct of the invention comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 linked to green fluorescent protein (GFP), or a small peptide tag useful for detection (e.g., an HA tag), or any other protein useful for detection.

[0098] The secreted protein variants of the invention can be isolated or purified from cells present individually in the medium or from recombinant sources. For example, a DNA fragment encoding a polypeptide construct of the invention can be subcloned into an appropriate vector using known molecular genetic techniques. The fragment is transcribed and the polypeptide is then translated in vitro. Commercially available kits can also be used. PCR can be optionally used for manipulating nucleic acids.

[0099] In some embodiments, the invention provides a vector comprising a nucleic acid construct of the invention. The nucleic acid construct of the invention can be inserted into a suitable vector. Methods for the selection of vectors and the construction of vectors are known and described in the general technical literature.

[0100] Suitable vectors may be designed for propagation and / or expansion and include, for example, plasmids, plasmid-liposome complexes, CELid vectors, and viral vectors, such as parvovirus-based vectors (i.e., AAV vectors), retroviral vectors, herpes simplex virus (HSV)-based vectors, adenovirus-based vectors, lentiviruses, and poxviruses. All of these expression constructs may be prepared using standard recombinant DNA techniques.

[0101] In one embodiment, the vector is an AAV vector, including any AAV of any suitable serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and variants thereof.

[0102] The AAV vector can be packaged within a capsid protein, a fragment thereof, or any of the AAV serotypes described herein.

[0103] In one embodiment of the invention, the vector is a recombinant expression vector.

[0104] In some embodiments, vectors of the invention contain regulatory sequences that allow for one or more of the transcription, translation, and expression of the nucleic acid contained in the vector in a cell transfected with the vector or infected with a virus containing the vector. As used herein, "operably linked" sequences include both regulatory sequences that flank the nucleotide sequence encoding sTFEB of the invention and regulatory sequences that act in trans or at a distance to control the nucleotide sequence encoding sTFEB.

[0105] The regulatory sequences include suitable transcription initiation and termination promoter and enhancer sequences, RNA processing signals such as splicing and polyadenylation (polyA) signal sequences, sequences which stabilize cytoplasmic mRNA, sequences which enhance translation efficiency (e.g., Kozak consensus sequences), sequences which enhance protein stability, and, if desired, sequences which enhance secretion of the encoded product. The polyA signal sequences may be synthetic or derived from a number of suitable species, such as human SV-40 and bovine SV-40.

[0106] Where the vector is intended for administration to a host (such as a human), the vector (such as an AAV) is preferably dead (i.e., replication incompetent) or may be attenuated or have low replication efficiency in target cells (e.g., less than 1 progeny per cell, or less than 0.1 progeny per cell), which can be easily determined by experimental determination of viral titer following injection of target cells.

[0107] Other therapeutic proteins that can be used as non-secreted proteins in the nucleic acid constructs of the invention include heat shock proteins such as HSP70, which aid in cellular stress, and mitochondrial cytochrome c oxidase subunit 8 (Cox8), which targets mitochondria within the cell.

[0108] Without being bound to the mechanism of action, FIG. 2 shows how the construct of the invention encoding sTFEB works in cultured neuroblastoma cells (N2A cells). In this experiment, when DNA is introduced into the plasmid, only a small proportion of cells take up the DNA. Cells that take up from the plasmid DNA are green from the TFEB construct with green fluorescent protein (GFP) and red due to the non-secreted RFP signal as shown by the arrowhead. All other green cells (no red staining) have taken up sTFEB-GFP from the medium. In the case of full-length TFEB-GFP, the cells have a punctate green signal in the cytoplasm and variable nuclear staining. Here, an antibody against LAMP1 was used to stain the lysosomes. Using Imaris software, it is possible to select the brightest GFP and LAMP1 pixels and generate a white colocalization channel indicating that sTFEB has been partially colocalized in LAMP1-labeled lysosomes. Deletion of the cytoplasmic retention domain (del30-deficient) renders the construct constitutively active, increasing the number of cells with TFEB-del30-GFP in the nucleus and increasing the amount of sTFEB-del30-GFP colocalized with lysosomes. sTFEB DD is primarily cytoplasmic when both the cytoskeleton and DNA-binding domains are deleted, resulting in reduced colocalization with lysosomes.

[0109] The vectors of the present invention reach nearly 100% transduction efficiency in cell lines and human iPSC-derived neurons (Figure 3). By removing the medium from a dish of sTFEB-transduced cells, sTFEB can be transferred to nearly 100% of recipient cells with human neurons generated from inducible programmable stem cells (iPSC-derived neurons). TFEB-del30 is present in the nuclei of most recipient cells. Uptake of sTFEB from the medium (especially constitutively active sTFEB-del30) increases the size and number of lysosomes (see Figure 4) and increases the transcription or translation of eisosome genes (see Figure 5). Uptake of sTFEB-del30 also results in the clearance of β-amyloid (blockage, Figure 6), clearance of neurofibrillary tangles (Figure 7), and clearance of α-synuclein inclusions. Stereotactic injection of the vector of the invention into third generation Alzheimer's (APP / PS1 / Tau) and Parkinson's (M83) mice demonstrates dramatic spread of the vector into the brain (compared to wild-type non-secreted TFEB) (see FIG. 9 and FIG. 11A-C), increased expression of the lysosomal protein LAMP1, and reduced α-synuclein and β-amyloid plaques (see FIG. 9, FIG. 10A-B, FIG. 11C-D). The mice in FIG. 10A-B were treated for only one week, yet showed reduced plaques.

[0110] The constructs of the present invention provide a novel gene delivery method that solves one of the major problems of gene therapy. Currently, there are no widely accepted disease-modifying treatments for muscle and neurodegenerative diseases. The present invention provides novel constructs expressing secreted protein variants, such as secreted transcription factors, that have been shown to enhance lysosomal clearance in human neurons and mouse brain. The system of the present invention allows the transfer of engineered forms of TFEB from virally transduced cells to non-transduced cells, enhancing the tissue distribution of this gene and increasing lysosomal gene expression and lysosomal size and number. This results in greater clearance of proteins such as β-amyloid, tau, and α-synuclein, which are involved in Alzheimer's and Parkinson's diseases. Surprisingly, these changes are evident as soon as one week after administration.

[0111] In another embodiment, the viral system of the invention serves as the basis for the treatment or prevention of a disease suffered by a subject (animal, including human). In one embodiment, the invention relates to a method for treating, alleviating or preventing a disease or condition in a subject, comprising administering to the subject a vector carrying a construct encoding a secreted protein variant of the invention. Where the disease is a neurodegenerative disease, the vector of the invention can be administered by any suitable mode of administration, such as by stereotactic injection into the brain, or by delivery into the patient's CSF, or by intravenous delivery.

[0112] In one embodiment, the disease / disease that can be treated, ameliorated or prevented using the method of the present invention is associated with lysosomal dysfunction. The disease / disease affects the skeleton, skin, mucous membranes, heart, liver, hematopoietic system, muscle, peripheral nervous system and central nervous system. In one embodiment, the disease / disease is a neurodegenerative disease such as Alzheimer's disease or Parkinson's disease. The construct of the present invention can also be used for other diseases or conditions. For example, if the construct has HSP70 that helps cell stress or Cox8, it has mitochondrial cytochrome oxidase subunit 8 (Cox8) to target mitochondria-associated conditions.

[0113] In another embodiment, the disease / condition that can be treated, alleviated, or prevented using the methods of the present invention is a viral infection, bacterial infection, or fungal infection caused by a pathogen.

[0114] The advantages of the present system over conventional systems in neurodegenerative diseases are: 1) Amyloid deposition is necessary to diagnose AD, but amyloid is not the actual cause of AD. Amyloid is considered to be a side effect of some metabolic problems, such as defective protein clearance. The present invention targets the lack of underlying global biochemical knowledge in neurodegenerative diseases (defective lysosomal clearance) and does not depend on the specific deposited protein involved. For example, the present invention may be effective in cases where β-amyloid deposition is not the primary problem in Alzheimer's disease. 2) AAV expresses genes for a long period of time, so patients only need a single dose. The present invention is also applicable to many other muscle and neurodegenerative diseases.

[0115] To aid in the understanding of the present invention, the following illustrative, non-limiting examples are provided.

[0116] [Example] Materials and Methods DNA construct: To generate a TFEB protein that can spread from transduced to non-transduced cells, the construct contains a signal sequence for protein secretion, such as that derived from the human amyloid precursor protein [MLPGLALLLLAAWTARAL] (SEQ ID NO:8), and a cell membrane penetrating peptide (the TAT peptide from HIV TVTVVRSVVSVVV (SEQ ID NO:9)). In addition, the TFEB protein lacks a cytoskeleton binding site that inhibits nuclear transport, a DNA-binding helix-loop-helix domain, and a C-terminal green fluorescent protein that can be removed to allow space in the vector, enhance expression, and eliminate potential targets for the immune system. Initial experiments will use constructs expressing sTFEB alone or in combination with a non-secreted red fluorescent protein (tdTomatoFP), which can be transcribed from its own on-promoter or in tandem with sTFEB on the same transcript, with an E2a cleavage site between the two proteins, allowing cleavage of the tdTomatoFP and sTFEB proteins during translation. Currently, these constructs exist within adenoviruses and within AAV9.

[0117] In Vivo Experiments To demonstrate functionally active propagation of TFEB in a mouse model of AD, we used 3×Tg mice (Jackson laboratory; B6; 129-Tg(APPSwe, tauP301L)1LfaPsen1 tmMpm We will conduct experiments using tau.3×Tg mice (Charles River Canada; C57B / 6) with amyloid plaques and phosphorylated tau.3×Tg mice exhibit Aβ deposition as early as 6 months, with widespread cortical deposits by 12 months. Phosphorylated tau is present in hippocampus for 12–15 months. We will use WT mice (Charles River Canada; C57B / 6) as control animals.

[0118] Transgenic mice are injected with the adenoviral constructs of the present invention to drive expression of propagating TFEB (WT or mutant) or GFP as a negative control. WT control mice are also injected with the adenoviral constructs to assess the effect of TFEB delivery on endogenous Aβ and tau. Animals are stereotaxically injected bilaterally into the hippocampus or lateral ventricle. Injection volumes are 10 7 ~10 12 Viral particle / side. Since the distribution of viral particles is the main concern of the in vivo study, two injection sites are selected to visualize and determine the most efficient intracerebral distribution of the propagated TFEB construct of the present invention. Briefly, surgery is performed under 2% isoflurane anesthesia, and 5 μL of virus is pumped into the lateral ventricle (AP-0.6 mm, ML ± 1.2 mm, DV-2 mm) or hippocampus (AP-2.4 mm, ML ± 2 mm, DV-1.5 mm) at a flow rate of 0.5 μL / min for 10 min via a 26 G Hamilton syringe attached to an infusion pump.

[0119] To determine whether the role of TFEB in lysosomal clearance exhibits neuroprotective vs. neurorescue effects, animals of three different ages will be used: 3 months (pre-phenotypic abnormalities), 5-6 months (abnormal development), and 12 months (post-phenotypic abnormalities). Furthermore, to determine the timeline of TFEB effects and efficiency, animals of these ages will be sacrificed at three end points: 1 week, 1 month, and 3 months after surgery. Thus, to test each injected dose of TFEB virus, 324 mice will be used (6 replicates x 3 mouse strains x 2 injection sites x 3 starting points x 3 end points). All care of animal specimens and surgeries will be performed in accordance with the guidelines of the Animal Care Committee at Western University (AUP 2016-069).

[0120] To visualize the distribution of the propagation-enhanced TFEB, the brains are fixed and the parietal section is cut for tissue analysis. In the case of the protein of the present invention (TFEB), since it is fluorescently tagged, no extra TFEB detection is necessary, but immunostaining directed against GFP and RFP may be used to amplify the original TFEB signal. In addition, commercially available antibodies are used to detect LAMP1, Aβ 40 , Aβ 42 Immunostaining will be performed for phosphorylated tau.

[0121] To assess the ability of TFEB to propagate and modulate lysosomal activity, confocal microscopy is performed to measure the expression, number and size of lysosomes in each TFEB condition (WT vs. mutant). In addition, amyloid plaques or tau neurofibrillary tangles are modified using NIH ImageJ and Imaris software to investigate the distribution of TFEB activity in lowering tau and Aβ levels. Stereological analysis is performed on sections stained with cresyl violet solution and neurons are also counted. Briefly, unbiased stereological analysis is performed using the optical dissector method and data analysis is performed using Stereo Investigator software (MBF Bioscience).

[0122] result Micrograph showing sTFEB-GFP spreading from cell to cell in neuroblastoma cells Figure 2 shows microphotographs of N2A cells transfected with sTFEB-GFP (top), sTFEB-del30-GFP (middle) and sTFEB-DD-GFP (bottom). Cells were immunostained with LAMP1 (magenta). The rightmost panel shows the colocalization analysis channels comparing the overlap of sTFEB-GFP and lysosomes (LAMP1) using Imaris software (Bitplane). Cells transfected with sTFEB-GFP also express RFP and are marked by an arrowhead. All other cells, which are green, take up GFP from the medium. Cells transfected with sTFEB-GFP show punctate anti-cytoplasmic / nuclear staining depending on how much sTFEB-GFP they have taken up. Some sTFEB-GFP in the cytoplasm colocalizes with lysosomes. Deletion of the cytoskeleton-binding domain (sTFEB-del30) increases the number of cells with nuclear GFP signal and reduces the amount of sTFEB in lysosomes. Deletion of both the DNA-binding and cytoskeleton-binding domains (sTFEB-DD) results in cytoplasmic GFP expression but reduced lysosomal labeling.

[0123] sTFEB is delivered from cell to cell with high efficiency Figure 3 shows micrographs demonstrating that sTFEB (green), but not adenovirus (red), spreads from cell to cell. The cells in the left panel of Figure 3 are transfected with adenovirus encoding sTFEB-GFP and a non-secreted red fluorescent protein. The medium conditioned by these cells is then added to a second cell dish (right panel - neuroblastoma cell line and cultured human iPSC-derived human neurons). After this, the second cell dish turns green (instead of red), indicating that it has taken up sTFEB-GFP and not the viral particles themselves.

[0124] sTFEB enhances the expression of cathepsin-B, lysosomal size and lysosomal number Figure 4A shows micrographs of N2a cells (Figure 4A-upper panel) or iPSC-derived neurons (Figure 4A-lower panel) that had taken up sTFEB Ddel130 or TFEB / inactive sTFEB-DD-GFP from conditioned medium over a period of 48 hours. Magic Red (Immunochemistry Technologies) fluorescent substrate for cathepsin B was added to the medium and cells were imaged live.

[0125] Lysosome size and number were quantified using Imaris software. N2A cells that had incorporated constitutively active sTFEB-del30 had significantly more lysosomes, which were significantly larger in size and intensity (compared to control cells). * CatB enzyme activity) generated by area integration was also quite strong (see Fig. 4B ).

[0126] Lysosome size and number were quantified in human iPSC-derived neurons using Imaris software. Neurons incorporating constitutively active sTFEB-del30 had significantly more lysosomes, larger size (compared to control cells), and higher lysosome counts (intensity * CatB enzyme activity (generated by area integration) was also quite strong (see Fig. 4C ).

[0127] sTFEB increases levels of lysosomal genes and proteins As shown in Figure 5A, N2A cells were treated with constitutively active sTFEB-del30 or inactive sTFEB-DD and gene expression was analyzed by rtPCR and normalized to cells exposed to normal medium. sTFEB enhances lysosomal gene expression. As shown in Figure 5B, N2A cells were treated with sTFEB, constitutively active TFEB-del30, or inactive sTFEB-DD, and cells were analyzed by Western blotting. sTFEB increases the expression of LAMP1 and CatD proteins, but not tubulin (control).

[0128] Constitutively active sTFEB-del30 reduces intracellular β-amyloid N2A neuroblastoma cells were transfected with inactive TFEB-DD-GFP (Figure 6A) or constitutively active sTFEB-del30 (Figure 6B). After 48 h, they were loaded overnight with 500 nM fluorescently labeled β-amyloid-42 (shown in red). Cells were fixed and imaged 48 h after further seeding. Micrographs showed that cells containing inactive TFEB-DD-GFP had a much more intense red signal from β-amyloid than cells containing constitutively active TFEB-GFP.

[0129] Constitutively active sTFEB-del30 reduces neurofibrillary tangles Human iPSC-derived neurons were transfected with mutant tau (tau P301L)-blue fluorescent protein (shown in the micrograph in cyan) and treated with preformed tau fibrils to elicit neurofibrillary tangles. Cells were then exposed to medium containing inactive TFEB-DD-GFP (Figure 7A) or constitutively active sTFEB-del30 (Figure 7B). Micrographs show that cells exposed to inactive TFEB are loaded with tau filaments (Figure 7A), whereas cells exposed to active TFEB-del30 have few visible filaments (Figure 7B).

[0130] Constitutively active sTFEB reduces α-synuclein deposition SHSY5Y neuroblastoma cells (8A and 8B) and human iPSC-derived neurons (8C and 8D) were transfected with aSyn A53T-BFP (green) and either TFEB-DD-GFP (inactive, 8A and 8C) or sTFEB-del30-GFP (constitutively active, 8B and 8D). After 5 days, cells were fixed and imaged by confocal microscopy. Micrographs show that cells that incorporated inactive sTFEB-DD-GFP had cytoplasmic synuclein inclusions (arrowheads 8A and 8C), whereas cells that incorporated sTFEB-del30-GFP did not (8B and 8D).

[0131] sTFEB-del30-GFP significantly increases TFEB expression in the brain, enhances lysosomal staining, and reduces α-synuclein compared to wild-type TFEB-GFP On the left, 10 μg of adenovirus overexpressing constitutively active sTFEB-del30-GFP was administered into the ventricles of 14-month-old homozygous M83 mice. ∧ sTFEB-GFP was administered locally on the right side of the mouse brain, followed by local administration of 7 infectious units of TFEB on the right side. Seven days later, mouse brains were perfused, fixed, cryosectioned, immunostained with antibodies against GFP (green), LAMP1 (red), aggregated phosphorylated synuclein, and imaged on a Leica TSC SP8 confocal microscope. The top row of Fig. 9 shows photomicrographs demonstrating that TFEB expression is significantly higher on the sTFEB-treated side. The middle row of Fig. 9 shows that the constitutively active TFEB side generally exhibits stronger LAMP1 staining (increased lysosome numbers) and, at higher magnification, increased lysosome numbers. The bottom row of Fig. 9 shows tissue stained with an antibody against phosphorylated α-synuclein, which is specific for pathological synuclein deposits, and is decreased on the sTFEB-treated side.

[0132] Clearing β-amyloid by sTFEB-del30 12 month old 3XTG Alzheimer's mouse hippocampi were injected with adenovirus expressing TFEB-del30-GFP on the left side and with adenovirus expressing wt-GFP alone on the right side. After 1 week, mice were sacrificed and stained for GFP (green) and β-amyloid (yellow). The left side treated with sTFEB-del30-GFP shows greater expression and diffusion compared to non-diffuse GFP expression (see Figure 10A). The number of amyloid plaques in each hippocampus and in the outer layer on the hippocampus was counted, and the results are shown in the graph of FIG. 10B. Figures 10A and 10B show that few plaques were observed in the hippocampus and tegument on the sTFEB-del30 side (left side of Figure 10A, sTFEB) compared to the control side (Figure 10A, GFP).

[0133] sTFEB spreads beyond its administration site, leading to β-amyloid crowding out Five-month-old 3xTG (APP / PS1 / tau) hippocampi were injected with adenovirus expressing sTFEB-GFT on the left side and with an equal amount of adenovirus expressing GFP on the right side. Mice were sacrificed 2 weeks later, perfused, fixed, cryosectioned, and immunostained with antibodies against GFP and β-amyloid-42 (Aβ42). Figure 11A shows GFP staining at the level of injection, demonstrating that the sTFEB-GFP signal covers the entire hemisphere, covering the hemisphere more than the standard GFP-expressing adenovirus. Figure 11B shows that 3 mm posterior to the injection site, sTFEB-GFP still covers most of the hemisphere, whereas the regular adenovirus-GFP is only found in deeper brain structures. When compared to the GFP-adenovirus, sTFEB-GFP injection dramatically reduces the amyloid-42 immunostained Aβ signal at the level of injection (see Figure 11C). A small portion of Figure 11C is shown enlarged in Figure 11D, which shows that July AD animals have significant intracellular amyloid as well as early plaques (right side), both of which are dramatically reduced by sTFEB-GFP (left side).

[0134] Through the illustrated and described embodiments, the best currently contemplated modes for making and using the present disclosure are described. Without further elaboration, it is believed that a person skilled in the art can make the most of the present disclosure based on the description provided herein. All publications cited herein are incorporated by reference.

[0135] While the above description contains many specificities, these should not be construed as limiting the scope of the disclosure, but merely as illustrative of some of the current embodiments of the disclosure.

[0136] Sequence Listing SEQ ID NO: 1 TFEB (wild type protein) Met Ala Ser Arg Ile Gly Leu Arg Met Gln Leu Met Arg Glu Gln Ala Gln Gln Glu Glu Gln Arg Glu Arg Met Gln Gln Gln Ala Val Met His Tyr Met Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Gly Gly Pro Pro Thr Pro Ala Ile Asn Thr Pro Val His Phe Gln Ser Pro Pro Pro Val Pro Gly Glu Val Leu Lys Val Gln Ser Tyr Leu Glu Asn Pro Thr Ser Tyr His Leu Gln Gln Ser Gln His Gln Lys Val Arg Glu Tyr Leu Ser Glu Thr Tyr Gly Asn Lys Phe Ala Ala His Ile Ser Pro Ala Gln Gly Ser Pro Lys Pro Pro Pro Ala Ala Ser Pro Gly Val Arg Ala Gly His Val Leu Ser Ser Ser Ala Gly Asn Ser Ala Pro Asn Ser Pro Met Ala Met Leu His Ile Gly Ser Asn Pro Glu Arg Glu Leu Asp Asp Val Ile Asp Asn Ile Met Arg Leu Asp Asp Val Leu Gly Tyr Ile Asn Pro Glu Met Gln Met Pro Asn Thr Leu Pro Leu Ser Ser Ser His Leu Asn Val Tyr Ser Ser Asp Pro Gln Val Thr Ala Ser Leu Val Gly Val Thr Ser Ser Ser Cys Pro Ala Asp Leu Thr Gln Lys Arg Glu Leu Thr Asp Ala Glu Ser Arg Ala Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Arg Phe AsnIle Asn Asp Arg Ile Lys Glu Leu Gly Met Leu Ile Pro Lys Ala Asn Asp Leu Asp Val Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Arg Met Gln Lys Asp Leu Gln Lys Ser Arg Glu Leu Glu Asn His Ser Arg Arg Leu Glu Met Thr Asn Lys Gln Leu Trp Leu Arg Ile Gln Glu Leu Glu Met Gln Ala Arg Val His Gly Leu Pro Thr Thr Ser Pro Ser Gly Met Asn Met Ala Glu Leu Ala Gln Gln Val Val Lys Gln Glu Leu Pro Ser Glu Glu Gly Pro Gly Glu Ala Leu Met Leu Gly Ala Glu Val Pro Asp Pro Glu Pro Leu Pro Ala Leu Pro Pro Gln Ala Pro Leu Pro Thr Gln Pro Pro Ser Pro Phe His His Leu Asp Phe Ser His Ser Leu Ser Phe Gly Gly Arg Glu Asp Glu Gly Pro Pro Gly Tyr Pro Glu Pro Leu Ala Pro Gly His Gly Ser Pro Phe Pro Ser Leu Ser Lys Lys Asp Leu Asp Leu Met Leu Leu Asp Ser Leu Leu Leu Pro Ala Ser Asp Pro Leu Leu Ser Thr Met Ser Pro Glu Ala Ser Lys Ala Ser Arg Arg Ser Ser Phe Ser Met Glu Glu Gly Asp Val Leu Lys Val Pro Arg Ala Arg Asp Pro Pro Val Ala Thr

[0137] <h2 style=";text-align:left;direction:ltr">SEQ ID NO: 2 (protein) sTFEB SS-TAT-TFEB<h2 style=";text-align:left;direction:ltr"> Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Asn Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Met Ala Ser Arg Ile Gly Leu Arg Met Gln Leu Met Arg Glu Gln Ala Gln Gln Glu Glu Gln Arg Glu Arg Met Gln Gln Gln Ala Val Met His Tyr Met Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Gly Gly Pro Pro Thr Pro Ala Ile Asn Thr Pro Val His Phe Gln Ser Pro Pro Pro Val Pro Gly Glu Val Leu Lys Val Gln Ser Tyr Leu Glu Asn Pro Thr Ser Tyr His Leu Gln Gln Ser Gln His Gln Lys Val Arg Glu Tyr Leu Ser Glu Thr Tyr Gly Asn Lys Phe Ala Ala His Ile Ser Pro Ala Gln Gly Ser Pro Lys Pro Pro Pro Ala Ala Ser Pro Gly Val Arg Ala Gly His Val Leu Ser Ser Ser Ala Gly Asn Ser Ala Pro Asn Ser Pro Met Ala Met Leu His Ile Gly Ser Asn Pro Glu Arg Glu Leu Asp Asp Val Ile Asp Asn Ile Met Arg Leu Asp Asp Val Leu Gly Tyr Ile Asn Pro Glu Met Gln Met Pro Asn Thr Leu Pro Leu Ser Ser Ser His Leu Asn Val Tyr Ser Ser Asp Pro Gln Val Thr Ala Ser Leu Val Gly Val Thr Ser Ser Ser Cys Pro Ala Asp Leu Thr GlnLys Arg Glu Leu Thr Asp Ala Glu Ser Arg Ala Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Arg Arg Phe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly Met Leu Ile Pro Lys Ala Asn Asp Leu Asp Val Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Arg Met Gln Lys Asp Leu Gln Lys Ser Arg Glu Leu Glu Asn His Ser Arg Arg Leu Glu Met Thr Asn Lys Gln Leu Trp Leu Arg Ile Gln Glu Leu Glu Met Gln Ala Arg Val His Gly Leu Pro Thr Thr Ser Pro Ser Gly Met Asn Met Ala Glu Leu Ala Gln Gln Val Val Lys Gln Glu Leu Pro Ser Glu Glu Gly Pro Gly Ala Leu Met Leu Gly Ala Glu Val Pro Asp Pro Glu Pro Leu Pro Ala Leu Pro Pro Gln Ala Pro Leu Pro Leu Pro Thr Gln Pro Pro Ser Pro Phe His Leu Asp Phe Ser His Ser Leu Ser Phe Gly Gly Arg Glu Asp Glu Gly Pro Pro Gly Tyr Pro Glu Pro Leu Ala Pro Gly His Gly Ser Pro Phe Pro Ser Leu Ser Lys Lys Asp Leu Asp Leu Met Leu Leu Asp Ser Leu Leu Pro Leu Ala Ser Asp Pro Leu Leu Ser Thr Met Ser Pro Glu Ala Ser Lys Ala Ser Ser Arg Arg Ser Ser PheDear Met Glu Glu Gly Asp Val Leu Lys Val Pro Arg Ala Arg Asp Pro Pro Val Ala Thr

[0138] SEQ ID NO: 3 (protein) sTFEB-del30 (SS-TAT-TFEB-del 30) Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Asn Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Met His Tyr Met Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Gly Gly Pro Pro Thr Pro Ala Ile Asn Thr Pro Val His Phe Gln Ser Pro Pro Pro Val Pro Gly Glu Val Leu Lys Val Gln Ser Tyr Leu Glu Asn Pro Thr Ser Tyr His Leu Gln Gln Ser Gln His Gln Lys Val Arg Glu Tyr Leu Ser Glu Thr Tyr Gly Asn Lys Phe Ala Ala His Ile Ser Pro Ala Gln Gly Ser Pro Lys Pro Pro Pro Ala Ala Ser Pro Gly Val Arg Ala Gly His Val Leu Ser Ser Ser Ala Gly Asn Ser Ala Pro Asn Ser Pro Met Ala Met Leu His Ile Gly Ser Asn Pro Glu Arg Glu Leu Asp Asp Val Ile Asp Asn Ile Met Arg Leu Asp Asp Val Leu Gly Tyr Ile Asn Pro Glu Met Gln Met Pro Asn Thr Leu Pro Leu Ser Ser Ser His Leu Asn Val Tyr Ser Ser Asp Pro Gln Val Thr Ala Ser Leu Val Gly Val Thr Ser Ser Ser Cys Pro Ala Asp Leu Thr Gln Lys Arg Glu Leu Thr Asp Ala Glu Ser Arg Ala Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg ArgPhe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly Met Leu Ile Pro Lys Ala Asn Asp Leu Asp Val Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Arg Met Gln Lys Asp Leu Gln Lys Ser Arg Glu Leu Glu Asn His Ser Arg Arg Leu Glu Met Thr Asn Lys Gln Leu Trp Leu Arg Ile Gln Glu Leu Glu Met Gln Ala Arg Val His Gly Leu Pro Thr Thr Ser Pro Ser Gly Met Asn Met Ala Glu Leu Ala Gln Gln Val Val Lys Gln Glu Leu Pro Ser Glu Glu Gly Pro Gly Glu Ala Leu Met Leu Gly Ala Glu Val Pro Asp Pro Glu Pro Leu Pro Ala Leu Pro Pro Gln Ala Pro Leu Pro Leu Pro Thr Gln Pro Pro Ser Pro Phe His His Leu Asp Phe Ser His Ser Leu Ser Phe Gly Arg Glu Asp Glu Gly Pro Pro Gly Tyr Pro Glu Pro Leu Ala Pro Gly His Gly Ser Pro Phe Pro Ser Leu Ser Lys Lys Asp Leu Asp Leu Met Leu Leu Asp Ser Leu Leu Pro Leu Ala Ser Asp Pro Leu Leu Ser Thr Met Ser Pro Glu Ala Ser Lys Ala Ser Ser Arg Arg Ser Ser Phe Ser Met Glu Glu Gly Asp Val Leu Lys Val Pro Arg Ala Arg Asp Pro Pro Val Ala Thr

[0139] <h2 style=";text-align:left;direction:ltr">SEQ ID NO: 4 (Protein) sTFEB-DD (SS-TAT-TFEB-DD)<h2 style=";text-align:left;direction:ltr"> Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Asn Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Met His Tyr Met Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Gly Gly Pro Pro Thr Pro Ala Ile Asn Thr Pro Val His Phe Gln Ser Pro Pro Pro Val Pro Gly Glu Val Leu Lys Val Gln Ser Tyr Leu Glu Asn Pro Thr Ser Tyr His Leu Gln Gln Ser Gln His Gln Lys Val Arg Glu Tyr Leu Ser Glu Thr Tyr Gly Asn Lys Phe Ala Ala His Ile Ser Pro Ala Gln Gly Ser Pro Lys Pro Pro Pro Ala Ala Ser Pro Gly Val Arg Ala Gly His Val Leu Ser Ser Ser Ala Gly Asn Ser Ala Pro Asn Ser Pro Met Ala Met Leu His Ile Gly Ser Asn Pro Glu Arg Glu Leu Asp Asp Val Ile Asp Asn Ile Met Arg Leu Asp Asp Val Leu Gly Tyr Ile Asn Pro Glu Met Gln Met Pro Asn Thr Leu Pro Leu Ser Ser Ser His Leu Asn Val Tyr Ser Ser Asp Pro Gln Val Thr Ala Ser Leu Val Gly Val Thr Ser Ser Ser Cys Pro Ala Asp Leu Thr Gln Lys Arg Glu Leu Thr Asp Ala Glu Ser Arg Ala Asp Arg Ile Lys Glu Leu Gly Met Leu Ile Pro Lys Ala Asn Asp Leu Asp Val ArgTrp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Arg Met Gln Lys Asp Leu Gln Lys Ser Arg Glu Leu Glu Asn His Ser Arg Arg Leu Glu Met Thr Asn Lys Gln Leu Trp Leu Arg Ile Gln Glu Leu Glu Met Gln Ala Arg Val His Gly Leu Pro Thr Thr Ser Pro Ser Gly Met Asn Met Ala Glu Leu Ala Gln Gln Val Val Lys Gln Glu Leu Pro Ser Glu Glu Gly Pro Gly Glu Ala Leu Met Leu Gly Ala Glu Val Pro Asp Pro Glu Pro Leu Pro Ala Leu Pro Pro Gln Ala Pro Leu Pro Thr Gln Pro Pro Ser Pro Phe His His Leu Asp Phe Ser His Ser Leu Ser Phe Gly Gly Arg Glu Asp Glu Gly Pro Gly Tyr Pro Glu Pro Leu Ala Pro Gly His Gly Ser Pro Phe Pro Ser Leu Ser Lys Lys Asp Leu Asp Leu Met Leu Leu Asp Asp Ser Leu Leu Leu Pro Leu Ala Ser Asp Pro Leu Leu Ser Thr Met Ser Pro Glu Ala Ser Lys Ala Ser Arg Arg Ser Phe Ser Met Glu Glu Gly Asp Val Leu Lys Val Arg Ala Arg Asp Pro Pro Val Ala Thr

[0140] SEQ ID NO: 5 DNA sTFEB (SS TAT_TFEB) <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0141] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 6 DNA sTFEB-del30 (SS-TAT-TFEB-del30)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0142] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 7 DNA sTFEB-DD (SS-TAT-TFEB-DD)<h2 style=";text-align:left;direction:ltr">

Claims

1. A nucleic acid construct having a polynucleotide encoding a secreted protein, the secreted protein comprising a non-secreted protein having a signal sequence domain and a cell-penetrating peptide (CPP) sequence domain.

2. The nucleic acid construct of claim 1, wherein the non-secreted protein is a nuclear protein, a cytoplasmic protein, or an organelle protein.

3. The nucleic acid construct of claim 1, wherein the non-secreted protein is a transcription factor.

4. 2. The nucleic acid construct of claim 1, wherein the secreted protein variant is a secreted TFEB (sTFEB) having a wild-type TFEB with a signal sequence and a self-threading sequence.

5. The nucleic acid construct of claim 1, wherein the non-secreted protein is HSP or Cox8.

6. The nucleic acid structure according to any one of claims 1 to 5, wherein the polynucleotide lacks a sequence encoding a cytoskeleton-binding site.

7. The nucleic acid structure of any one of claims 1 to 6, wherein the polynucleotide is deleted of a sequence encoding a DNA binding domain.

8. The self-threading sequence is a TAT sequence, Pep-1, PVEC, polyarginine (R ９ , R ８ ), Penetratin, PVEC, MPG, R ６ / W ３ , SAP, CyloP-1, gH625, GALA, TP10, CADY, L17E, MPPs, Ac-1, Entac-1, Ac-2, Peptide 3, RR5-App, RR4-App, RR3-aPP, TATp-D, R4-R4, R5, R5, [WR]4, Cyclic Tat, cFФR4, Danamide D, Pro-(Xaa)4-Tyr, Cyclic sC18, RRRRФF, BIM SAHB9 SAH-SOS1, 4-R, 4-W, Sp-CC-PEG ２０００ , K １０ (QW) ６ , YTA4, v2 or W ３ The nucleic acid structure according to any one of claims 1 to 7, comprising:

9. The nucleic acid structure according to any one of claims 1 to 8, wherein the signal sequence domain is derived from human amyloid precursor protein.

10. The construct of any one of claims 1 to 9, wherein the construct comprises an inducible or constitutive promoter that acts to link a polynucleotide encoding a non-secreted protein.

11. The construct of claim 10 , wherein the promoter drives expression of the polynucleotide in the central nervous system.

12. The nucleic acid construct according to any one of claims 1 to 9, wherein the construct has a GFAP promoter, a CAG promoter, an SV40 promoter, a neurana promoter or an EFla promoter.

13. The nucleic acid construct of claim 1, wherein the polynucleotide has SEQ ID NO:

5.

14. The nucleic acid construct of claim 1, wherein the polynucleotide has SEQ ID NO:

6.

15. The nucleic acid construct of claim 1, wherein the polynucleotide has SEQ ID NO:

7.

16. A recombinant vector comprising the nucleic acid construct according to any one of claims 1 to 15.

17. The recombinant vector according to claim 16, wherein the recombinant vector is a viral vector.

18. 18. The recombinant vector of claim 17, wherein the viral vector is a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, or a herpes simplex virus.

19. The recombinant vector of claim 17, wherein the viral vector is an adeno-associated virus (AAV).

20. A recombinant secreted protein variant having a signal sequence domain and a non-secreted protein having a cell penetrating peptide (CPP) domain.

21. The recombinant secreted protein variant of claim 20, wherein the non-secreted protein is a nuclear protein, a cytoplasmic protein or an organelle protein.

22. The recombinant secreted protein variant of claim 20, wherein the non-secreted protein is a transcription factor.

23. 21. The recombinant secreted protein of claim 20, wherein the recombinant secreted protein is a recombinant secreted transcription factor EB (sTFEB) having a wild-type TFEB with a signal sequence and a self-transducing peptide domain.

24. The recombinant secreted protein of claim 20, wherein the recombinant secreted protein is a recombinant secreted HSP or a recombinant secreted Cox8.

25. The CPP domain may be a TAT sequence, Pep-1, PVEC, polyarginine (R ９ , R ８ ), Penetratin, PVEC, MPG, R ６ / W ３ , SAP, CyloP-1, gH625, GALA, TP10, CADY, L17E, MPPs, Ac-1, Entac-1, Ac-2, P eptide3, RR5-App, RR4-App, RR3-aPP, TATp-D, R4-R4, R5, R5, [WR]4, Cyclic Tat, cFФR4, DanamideD, Pro-(Xaa)4-Tyr, Cyclic sC18, RRRRФF, BIM SAHB9 SAH-SOS1, 4-R, 4-W, Sp-CC-PEG ２０００ , K １０ (QW) ６ , YTA4, v2 or W ３ The recombinant secreted protein according to any one of claims 20 to 24, comprising:

26. The recombinant secreted protein according to any one of claims 20 to 25, wherein the signal sequence domain comprises a signal peptide of human amyloid precursor protein.

27. The recombinant secretory protein according to any one of claims 20 to 26, wherein the recombinant secretory protein lacks a cytoskeleton binding site (sTFEB del30).

28. The recombinant secreted protein according to any one of claims 20 to 27, wherein the recombinant secreted protein lacks the DNA binding domain (sTFEB-DD).

29. 21. The recombinant secreted protein of claim 20, wherein the recombinant secreted protein has SEQ ID NO:

2.

30. The sTFEB of claim 20, wherein the recombinant secreted protein has SEQ ID NO:

3.

31. The sTFEB of claim 20, wherein the recombinant secreted protein has SEQ ID NO:

4.

32. A method for treating, alleviating or preventing a disease by administering a vector to a subject, the vector comprising a polynucleotide encoding a recombinant secreted protein variant having a signal sequence domain and a non-secreted protein having a cell-penetrating peptide (CPP) domain.

33. The method of claim 32, wherein the vector is a viral vector.

34. 34. The method of claim 33, wherein the viral vector is a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, or a herpes simplex virus.

35. The method of claim 33, wherein the viral vector is AAV.

36. The method according to any one of claims 32 to 35, wherein the non-secreted protein is a nuclear protein, a cytoplasmic protein or an organelle protein.

37. The method according to any one of claims 32 to 35, wherein the non-secreted protein is a transcription factor.

38. 38. The method of claim 37, wherein the transcription factor is transcription factor EB (TFEB) and the recombinant secreted protein variant has a wild-type TFEB having a signal sequence domain and a cell penetrating peptide domain.

39. The method according to any one of claims 32 to 35, wherein the non-secreted protein is HSP or Cox8.

40. The CPP domain is selected from the group consisting of TAT sequence, Pep-1, PVEC, polyarginine (R9, R8), penetratin, PVEC, MPG, R6 / W3, SAP, CyloP-1, gH625, GALA, TP10, CADY, L17E, MPPs, Ac-1, Entac-1, Ac-2, Peptide 3, RR5-App, RR4-App, RR3-aPP, TATp-D, R4-R4, R5, R5, [WR]4, Cyclic Tat, cFΦR4, Danamide D, Pro-(Xaa)4-Tyr, Cyclic sC18, RRRRΦF, BIM SAHB9 40. The method of any one of claims 32 to 39, having SAH-SOS1, 4-R, 4-W, Sp-CC-PEG2000, K10(QW)6, YTA4, v2 or W3.

41. The method according to any one of claims 32 to 40, wherein the signal sequence domain is a signal peptide of human amyloid precursor protein.

42. The method according to any one of claims 32 to 41, wherein the recombinant secreted protein variant is deleted for the cytoskeleton binding site (sTFEB del30).

43. 33. The method of claim 32, wherein the recombinant secreted protein has SEQ ID NO:

2.

44. 33. The method of claim 32, wherein the recombinant secreted protein has SEQ ID NO:

3.

45. 33. The method of claim 32, wherein the recombinant secreted protein has SEQ ID NO:

4.

46. The method of any one of claims 32 to 45, wherein the disease is associated with dysfunctional lysosomal clearance.

47. 46. ​​The method of any one of claims 32 to 45, wherein the disease affects the skeleton, skin, mucous membranes, heart, liver, kidneys, hematopoietic system, muscles, peripheral nervous system and central nervous system.

48. 48. The method of claim 47, wherein the disease is a neurodegenerative disease.

49. 49. The method of claim 48, wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

50. The method of any one of claims 32 to 49, wherein the disease is caused by a pathogen.

51. 51. The method of claim 50, wherein the pathogen is a virus, a bacterium or a fungus.

52. 33. The method of claim 32, wherein the disease is muscular dystrophy or MS.

53. Use of a vector according to any one of claims 33 to 48 in the treatment of a disease.

54. 54. The use according to claim 53, wherein the disease is associated with dysfunctional lysosomal clearance.

55. 54. The use according to claim 53, wherein the disease affects the skeleton, skin, mucous membranes, heart, liver, kidneys, hematopoietic system, muscles, peripheral nervous system and central nervous system.

56. 54. The use according to claim 53, wherein the disease is a neurodegenerative disease.

57. 54. The use according to claim 53, wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

58. 54. The use according to claim 53, wherein the disease is caused by a pathogen.

59. 59. The use according to claim 58, wherein the pathogen is a virus, a bacterium or a fungus.

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