Compositions and methods for the treatment of alzheimer's disease

Abstract

This invention discloses a novel class of fusion proteins for recruiting innate companion proteins in cells, particularly the Hsp70-mediated system, to specifically reduce tau-mediated protein aggregation and related proteopathies.

Description

[Previous Technology]

[0001] All proteins present within a cell need to fold properly into their intended structures to function correctly. A growing number of diseases and symptoms are associated with misfolding of proteins and / or improper deposition and aggregation of proteins, lipoproteins, and infectious protein material. Diseases caused by misfolding are also known as morphopathies or protein misfolding diseases, examples of which include Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTLD). Mutant proteins aggregate in cells, creating typical cytotoxic cellular inclusion bodies.

[0002] The pathological feature of a wide range of neurodegenerative diseases is the aggregation of intracellular or extracellular protein aggregates composed of amyloid fibrils (Forman et al., (2004) Nat Med 10: 1055-1063).

[0003] Alzheimer's disease (AD) is the most common neurodegenerative disease, affecting one in eight elderly Americans. Approximately 32% of people over 85 years of age have the disease, with over 5.4 million patients in the United States alone. Despite extensive research, there is no cure for AD; only symptomatic treatment is available.

[0004] AD is characterized by memory deficits and overall cognitive impairment due to synaptic and neuronal death in certain brain regions, including the hippocampus and neocortex (Norfray and Provenzale, (2004) AJR Am J Roentgenol 182:3-13; Ittner and Gotz (2011) Nat Rev Neurosci 12:65-72). Although it is clear that neuronal degeneration is associated with memory and cognitive impairment in AD, the molecular mechanisms underlying neurodegeneration in the affected regions remain largely unexplained. Neuronal damage is associated with extracellular deposition of amyloid-β (Aβ) in extracellular senile plaques (Selkoe, (2001) Neuron 32:177-180; Fan et al., (2007) J Neuroinflammation 4:22), and with intraneuronal neurofibrillary tangles composed of superphosphorylated tau protein, known as neurofibrillary tangles (NFT) (Ittner and Gotz (2011) Nat Rev Neurosci 12:65-72; Francis et al., (1999) J Neurol Neurosurg Psychiatry 66:137-147; Craig et al., (2011) Neurosci Biobehav Rev 35:1397-1409). However, the role of these protein deposits in AD pathology remains largely unknown.

[0005] Aβ and tau can function normally at the synapse, but abnormalities in these proteins can cause synaptic degeneration (Spires-Jones and Hyman, (2014)Neuron82:756-771).

[0006] In addition to histological studies, early-onset familial atopic dermatitis (FAD) has been found to be linked to several gene loci, offering hope for identifying the genetic factors contributing to the pathogenesis of this disease. It is caused by genetic mutations in one of three genes (APP, PSEN1, and PSEN2) and one risk factor (APOEε4 paired gene) (Spires-Jones and Hyman, (2014) Neuron 82:756-771). Additional genetic risk loci have been identified in genome-wide association studies (GWAS) and massive parallel resequencing (MPS) studies (Van Cauwenberghe et al., (2016) Genet Med 18:421-430).

[0007] These gene studies did not show a correlation between tau and FAD. Linkage analysis showed a correlation between tau and other neurodegenerative diseases, many of which mapped to the region on chromosome 17q21-22 where the tau gene is located (Wilhelmsen et al., (1994) Am J Hum Genet 55:1159-1165; Wijker et al., (1996) Hum Mol Genet 5:151-154; Bird et al., (1997) Neurology 48:949-954; Lendon et al., (1998) Neurology 50:1546-1555). Subsequently, mutations in the tau gene were identified in frontotemporal dementia (FTDP-17) with Parkinsonism in hereditary dominant chromosome 17 (Foste et al., (1997) Ann Neurol 41:706-715; Hutton et al., (1998) Nature 393:702-705; Poorkaj et al., (1998) Ann Neurol 43:815-825; Spillantini et al., (1998) Proc Natl Acad Sci USA 95:7737-7741).

[0008] Tau is a microtubule-associated protein that binds to microtubules and promotes microtubule assembly. Tau binding to microtubules promotes microtubule polymerization (Cleveland et al., (1977) J Mol Biol 116:227-247; Weingarten et al., (1975) Proc Natl Acad Sci USA 72:1858-1862). Analysis of the tau sequence shows that tau has four microtubule-binding motifs in four repeating regions located at the C-terminus of the protein, where a conserved 18-amino acid long binding element is separated by 13-14 less conserved amino acids (Lee et al., (2001) Neurosci 24:1121-1159). In adults, six tau isoforms ranging in length from 325 to 441 amino acids are generated by selective splicing (Goedert et al., (1988) Proc Natl Acad Sci USA 85:4051-4055).

[0009] Tau is abundant in the central nervous system (CNS) and is mainly located in axons, while its expression is also detectable in the axons of peripheral neurons. Only low expression levels have been observed in CNS astrocytes and oligodendroglial cells (Cleveland et al., (1977) J Mol Biol 116:227-247; Binder et al., (1985) J Cell Biol 101:1371-1378; Lo Presti et al., (1995) Proc Natl Acad Sci USA 92:10369-10373).

[0010] Tau is an inherently disordered and highly soluble protein under normal conditions (Jeganathan et al., (2008) Biochemistry 47:10526-10539), but it becomes insoluble through aberrant modifications (such as hyperphosphorylation) (Kopeikina et al., (2012) Transl Neurosci 3:223-233). Hyperphosphorylation of tau protein can lead to the self-assembly of tangled pair helical fibers (PHF) and straight fibers, which is associated with the pathogenesis of Alzheimer's disease (AD), frontotemporal dementia (FTD), and other tau protein disorders. Hyperphosphorylation of pair helical fibers (PHF) and tau protein are two of the most recognized molecular features of AD and several other neurodegenerative tau protein disorders (including FTDP-17 and progressive supranuclear palsy (PSP)) (Lee et al., (2001) Neurosci 24:1121-1159).

[0011] Tau protein diseases are a group of neurodegenerative diseases that share common pathological features, namely the formation of insoluble intraneuronal aggregates composed of filamentous hyperphosphorylated tau protein (Lee et al., (2001) Neurosci 24:1121-1159; Delacourte, (2001) Adv Exp Med Biol 487:5-19).

[0012] Despite their different clinical manifestations, these neurodegenerative diseases share a common pathological feature: the formation of insoluble intraneuronal aggregates composed of filamentous, hyperphosphorylated tau protein (Lee et al., (2001) Neurosci 24:1121-1159; Delacourte, (2001) Adv Exp Med Biol 487:5-19), who pointed out that tau is one of the faulty proteins in these neurodegenerative diseases (Lee et al., (2001) Neurosci 24:1121-1159). All known mutations in the tau gene result in the formation of filaments composed of hyperphosphorylated tau protein (Crowther and Goedert, (2000) JStruct Biol 130:271-279).

[0013] The heat shock 70 kDa protein (referred to herein as "Hsp70") constitutes a wide class of companion proteins in the cells of a broad variety of species (Tavaria et al., (1996) Cell Stress Chaperones 1:23-28). Hsp70 requires accessory proteins, such as J domain proteins and nucleotide exchange factors (NEFs) (Hartl et al., (2009) Nat Struct Mol Biol 16:574-581), to function. In the current model of the Hsp70 companion protein mechanism of folded proteins, Hsp70 cycles between ATP- and ADP-bound states, and the J domain protein binds to another protein (called the "recipient protein") that needs to fold or refold, interacting with the ATP-bound form of Hsp70 (Hsp70-ATP) (Young (2010) Biochem Cell Biol 88:291-300; Mayer, (2010) Mol Cell 39:321-331). The binding of the J domain protein-recipient complex to Hsp70-ATP stimulates ATP hydrolysis, which causes a conformational change in the Hsp70 protein, closing the helix cap, thereby stabilizing the interaction between the recipient protein and Hsp70-ADP, and triggering the release of the J domain protein, which then freely binds to another recipient protein.

[0014] Therefore, according to this model, J domain proteins play a key role in the Hsp70 mechanism by acting as bridges and facilitating the capture and presentation of a wide variety of receptor proteins into the Hsp70 mechanism to promote folding or refolding into appropriate conformations (Kampinga and Craig (2010) Nat Rev Mol Cell Biol11, 579-592). The J domain family is widely conserved across species ranging from prokaryotes (DnaJ proteins) to eukaryotes (Hsp40 protein family). The J domain (approximately 60 to 80 aa) consists of four helices: I, II, III, and IV. Helices II and III are connected by a flexible loop containing the "HPD motif," which is highly conserved in the J domain and is considered essential for its activity (Tsai and Douglas, (1996) J Biol Chem271:9347-9354). Mutations within the HPD sequence have been found to eliminate J domain function.

[0015] Given the background provided above regarding protein diseases such as AD, it seems clear that reducing the amount of misfolded proteins can serve as a means of treating, preventing, or otherwise improving the symptoms of such devastating conditions, and that recruiting the cells’ innate capacity to repair protein misfolding would be a reasonable option. [Summary of the Invention]

[0016] The inventors have developed a novel class of fusion proteins for recruiting innate companion proteins in cells, particularly the Hsp70-mediated system, thereby specifically reducing tau-mediated protein aggregation. Unlike previous studies that used fragments of Hsp40 protein (also known as J protein), a companion protein that interacts with Hsp70, to enhance protein secretion and expression, this study employs a J-domain-containing fusion protein to reduce protein aggregation and cytotoxicity caused by mutant tau protein aggregation. In this context, the inventors have unexpectedly discovered that the functionally required J-domain elements are entirely different from the J-domain's use in enhancing protein expression and secretion, indicating a different mechanism of action for the fusion protein of this invention. The fusion protein described herein includes a J-domain and a tau-affinity domain. The presence of the tau-binding domain within the fusion protein specifically reduces the aggregation of mutant tau protein. E1. Therefore, in a first-state sample, this paper discloses an isolated fusion protein comprising the J domain and tau-binding domain of the J protein. E2. The fusion protein of E1, wherein the J domain of the J protein is of eukaryotic origin. E3. The fusion protein of any one of E1 to E2, wherein the J domain of the J protein is of human origin. E4. The fusion protein of any one of E1 to E3, wherein the J domain of the J protein is cytoplasm-localized. E5. The fusion protein of any one of E1 to E4, wherein the J domain of the J protein is selected from the group consisting of SEQ ID Nos: 1-48. E6. The fusion protein of any one of E1 to E5, wherein the J domain comprises a sequence selected from the group consisting of SEQ ID Nos: 5, 6, 10, 24, and 31. E7. The fusion protein of any one of E1 to E6, wherein the J domain comprises the sequence of SEQ ID No: 5. E8. A fusion protein of any one of E1 to E6, wherein the J domain contains the sequence of SEQ ID NO: 6. E9. A fusion protein of any one of E1 to E6, wherein the J domain contains the sequence of SEQ ID NO: 10. E10. A fusion protein of any one of E1 to E6, wherein the J domain contains the sequence of SEQ ID NO: 24. E11. A fusion protein of any one of E1 to E6, wherein the J domain contains the sequence of SEQ ID NO: 31. E12. A fusion protein of any one of E1 to E11, wherein when measured using an ELISA assay, the tau-binding domain has a tau KD of 1 µM or less, for example, 300 nM or less, 100 nM or less, 30 nM or less, or 10 nM or less. E13. A fusion protein of any one of E1 to E12, wherein the tau-binding domain contains a sequence selected from the group consisting of SEQ ID NO: 49-54.E14. A fusion protein of any one of E1 to E13, wherein the tau-binding domain comprises the sequence of SEQ ID NO: 49. E15. A fusion protein of any one of E1 to E13, wherein the tau-binding domain comprises the sequence of SEQ ID NO: 50. E16. A fusion protein of any one of E1 to E13, wherein the tau-binding domain comprises the sequence of SEQ ID NO: 51. E17. A fusion protein of any one of E1 to E13, wherein the tau-binding domain comprises the sequence of SEQ ID NO: 52. E18. A fusion protein of any one of E1 to E13, wherein the tau-binding domain comprises the sequence of SEQ ID NO: 53. E19. A fusion protein of any one of E1 to E13, wherein the tau-binding domain comprises the sequence of SEQ ID NO: 54. E20. A fusion protein of any one of E1 to E19, comprising a plurality of tau-binding domains. E21. A fusion protein, such as any one of E1 to E20, consisting of two tau-binding domains. E22. A fusion protein, such as any one of E1 to E21, consisting of three tau-binding domains. E23. A fusion protein of any one of E1 to E22, comprising one of the following constructs: a. DNAJ-XT, b. DNAJ-XTXT, c. DNAJ-XTXTXT, d. TX-DNAJ, e. TXTX-DNAJ, f. TXTXTX-DNAJ, g. TX-DNAJ-XT, h. TX-DNAJ-XTXT, i. TX-DNAJ-XTXTXT, j. TXTX-DNAJ-XT, k. TXTX-DNAJ-XTXT, l. TXTX-DNAJ-XTXTXT, m. TXTXTX-DNAJ-XT, n. TXTXTX-DNAJ-XTXT, and o. TXTXTX-DNAJ-XTXTXT, wherein T is the tau-binding domain, DNAJ is the J domain of the J protein, and X is a linker selected as appropriate. E24. A fusion protein of any one of E1 to E23, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and the tau-binding domain sequence of SEQ ID NO: 49. E25. A fusion protein of any one of E1 to E23, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and the tau-binding domain sequence of SEQ ID NO: 50.E26. A fusion protein of any one of E1 to E23, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and the tau-binding domain sequence of SEQ ID NO: 51. E27. A fusion protein of any one of E1 to E23, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and the tau-binding domain sequence of SEQ ID NO: 52. E28. A fusion protein of any one of E1 to E23, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and the tau-binding domain sequence of SEQ ID NO: 53. E29. A fusion protein of any one of E1 to E23, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and the tau-binding domain sequence of SEQ ID NO: 54. E30. A fusion protein of any one of E1 to E29, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 83-88 and 95-101. E31. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 83. E32. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 84. E33. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 85. E34. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 86. E35. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 87. E36. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 88. E37. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 95. E38. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 96. E39. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 97. E40. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 98. E41. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 99. E42. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 100. E43. A fusion protein of any one of E1 to E30, wherein the fusion protein comprises the sequence of SEQ ID NO: 101.E44. A fusion protein of any one of E1 to E43, further comprising a targeting agent. E45. A fusion protein of any one of E1 to E44, further comprising an antigenic determinant. E46. A fusion protein of E45, wherein the antigenic determinant is a polypeptide selected from the group consisting of SEQ ID NO: 66-72. E47. A fusion protein of any one of E1 to E46, further comprising a cell-penetrating agent. E48. A fusion protein of E47, wherein the cell-penetrating agent comprises a peptide sequence selected from the group consisting of SEQ ID NO: 73-76. E49. A fusion protein of any one of E1 to E48, further comprising a signal sequence. E50. A fusion protein of E49, wherein the signal sequence comprises a peptide sequence selected from the group consisting of SEQ ID NO: 77-79. E51. A fusion protein of any one of E1 to E50, capable of reducing the aggregation of tau protein in cells. E52. A fusion protein of any one of E1 to E51 that reduces tau-mediated cytotoxicity. E53. A nucleic acid sequence encoding a fusion protein of any one of E1 to E52. E54. A nucleic acid sequence of E53, wherein the nucleic acid is DNA. E55. A nucleic acid sequence of E53, wherein the nucleic acid is RNA. E56. A nucleic acid sequence of any one of E53 to E55, wherein the nucleic acid comprises at least one modified nucleic acid. E57. A vector comprising a nucleic acid sequence of any one of E53 to E56. E58. A vector of E57, wherein the vector is selected from the group consisting of: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, poxvirus (vaccinia or myxoma), paramyxovirus (measles, RSV, or Newcastle disease virus), baculovirus, reovirus, alpha virus, and flavivirus. E59. A viral particle comprising a capsid and a vector such as E57 or E58. E60. A viral particle such as E59, wherein the capsid is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh10, AAV10, AAV11, AAV12, pseudotyped AAV, rhesus monkey-derived AAV, AAVrh8, AAVrh10 and AAV-DJan AAV capsid mutants, AAV hybrid serotypes, organophilic AAV, heartophilic AAV and heartophilic AAVM41 mutants.E61. A pharmaceutical composition comprising an agent selected from the group consisting of: a fusion protein such as any one of E1 to E52, cells expressing a fusion protein such as E1 to E52, a nucleic acid such as any one of E53 to E56, a vector such as any one of E57 to E58, a viral particle such as any one of E59 to E60, and a pharmaceutically acceptable carrier or excipient. E62. A method for reducing the toxicity of tau protein in cells, comprising contacting the cells with a fusion protein such as any one of E1 to E52, cells expressing a fusion protein such as E1 to E52, a nucleic acid such as any one of E53 to E56, a vector such as any one of E57 to E58, a viral particle such as any one of E59 to E60, and a pharmaceutical composition such as E61. E63. The method of E62, wherein the cell line is in an individual. E64. The method of any one of E62 to E63, wherein the system is human. E65. The method of any one of E62 to E64, wherein the cell line is a cell of the central nervous system. E66. The method of any one of E62 to E65, wherein the individual is identified as having tau disease. E67. As in E66, wherein the tau disease is selected from the following groups: Alzheimer's disease (AD), Parkinson's disease (PD), primary age-related tau proteinopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal ganglia degeneration (CBD), frontotemporal dementia and Parkinson's disease linked to chromosome 17 (FTDP-17), Guam-type amyotrophic lateral sclerosis-Parkinson's disease-dementia (Lytico-bodig disease), ganglioglioma and gangliocytoma, meningioma, post-encephalitis Parkinson's disease, subacute sclerosing panencephalitis (SSPE), lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, and lipofuscin deposition disease. E68. As in E66 or E67, wherein the tau disease is Alzheimer's disease (AD). E69. The method of any one of E62 to E68, wherein the amount of tau protein aggregated in the cells is reduced compared with control cells. E70. A method for treating, preventing, or delaying the progression of tau disease in an individual in need, the method comprising administering an effective amount of one or more agents selected from the group consisting of: fusion proteins such as any one of E1 to E52, cells expressing fusion proteins such as E1 to E52, nucleic acids such as any one of E53 to E56, vectors such as any one of E57 to E58, viral particles such as any one of E59 to E60, and pharmaceutical compositions such as E61.E71. As in E70, wherein the tau disease is selected from the following groups: Alzheimer's disease (AD), Parkinson's disease (PD), primary age-related tau proteinopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal ganglia degeneration (CBD), frontotemporal dementia and Parkinson's disease linked to chromosome 17 (FTDP-17), Guam-type amyotrophic lateral sclerosis-Parkinson's disease-dementia, ganglioglioma and gangliocytoma, meningioma, post-encephalitis Parkinson's disease, subacute sclerosing panencephalitis (SSPE), lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, and lipofuscinosis. E72. As in E70 or E71, wherein the tau disease is Alzheimer's disease (AD). E73. Use of one or more of the following: a fusion protein as described in any one of E1 to E52; a cell expressing a fusion protein as described in E1 to E52; a nucleic acid as described in any one of E53 to E56; a vector as described in any one of E57 to E58; a viral particle as described in any one of E59 to E60; and a pharmaceutical composition as described in E61, for the purpose of preventing or delaying the progression of tau disease in an individual. E74. Use of one or more of the following: a fusion protein as described in any one of E1 to E52; a cell expressing a fusion protein as described in E1 to E52; a nucleic acid as described in any one of E53 to E56; a vector as described in any one of E57 to E58; a viral particle as described in any one of E59 to E60; and a pharmaceutical composition as described in E61, for the purpose of preparing a medicament for treating, preventing, or delaying the progression of tau disease in an individual.

Implementation Method

[0018] Definitions Unless the context clearly indicates otherwise, as used in this specification and the claims, the singular forms "a" and "the" include a plurality of references. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0019] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be heterologous to non-amino acids. The term also covers modified amino acid polymers, for example, through disulfide bond formation, glycosylation, lipolation, acetylation, phosphorylation, or any other manipulation, such as binding to a labeled component.

[0020] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including but not limited to D or L optical isomers, and amino acid analogs and peptide mimics. Standard single-letter or three-letter codes are used to indicate amino acids.

[0021] "Host cell" includes a single cell or cell culture that may be or has been a recipient of the vector of the present invention. Host cell includes the offspring of a single host cell. Due to natural, accidental, or intentional mutations, the offspring may not be completely identical to the original parent cell (in terms of morphology or the genome of the total DNA complementary sequence). Host cell includes cells transfected in vivo with the vector of the present invention.

[0022] When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified and separated from and / or recovered from components of its native environment. The contaminating components of its native environment are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteins or non-protein solutes. As will be apparent to those skilled in the art, non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not require "isolation" to distinguish them from their naturally occurring counterparts. Furthermore, "concentrated," "isolated," or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof can be distinguished from their naturally occurring counterparts because the molecular concentration or number per volume is generally greater than that of their naturally occurring counterparts. Generally, polypeptides obtained by recombinant methods and expressed in host cells are considered "isolated."

[0023] "Isolated" polynucleotide or polypeptide-encoded nucleic acid or other polypeptide-encoded nucleic acid is a nucleic acid molecule that has been identified and separated from at least one impurity nucleic acid molecule that is normally associated with it in the natural source of polypeptide-encoded nucleic acid. The isolated polypeptide-encoded nucleic acid molecule does not exhibit the form or configuration found in nature. The isolated polypeptide-encoded nucleic acid molecule is therefore distinguishable from the specific polypeptide-encoded nucleic acid molecule present in natural cells. However, isolated polypeptide-encoded nucleic acid molecules include polypeptide-encoded nucleic acid molecules contained in cells that typically exhibit polypeptides in locations, for example, where the nucleic acid molecule is located on a chromosome or extrachromosomal site different from those in natural cells.

[0024] The terms "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides) of any length, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plastids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. The nucleotide sequence may contain non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by binding to labeled components.

[0025] As defined herein, the terms "tau syndrome," "tau disease," "tau protein disease," or "tau-mediated disease" refer to a condition associated with the formation of intracellular tau aggregates (especially aggregates of tau mutant proteins). Examples of tau syndromes include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), primary age-related tau protein disease (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal ganglia degeneration (CBD), frontotemporal dementia and Parkinson's disease linked to chromosome 17 (FTDP-17), Guam-type amyotrophic lateral sclerosis-Parkinson's disease-dementia, ganglioglioma and gangliocytoma, meningioma, post-encephalitis Parkinson's disease, subacute sclerosing panencephalitis (SSPE), lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-associated neurodegeneration, and lipofuscin deposition disease.

[0026] A "vector" is a nucleic acid molecule that preferably replicates autonomously in a suitable host, delivering an inserted nucleic acid molecule to and / or between host cells. The term includes vectors primarily used for inserting DNA or RNA into cells, copies of vectors primarily used for replicating DNA or RNA, and expression vectors for DNA or RNA transcription and / or translation. It also includes vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally means a suitable host cell composed of expression vectors that can be used to produce a desired expression product.

[0027] The term "operably linked" refers to the concatenation of the described components in a relationship that allows them to function in their intended manner. "Operably linked" to a control sequence of the coding sequence is concatenated in a manner that enables the expression of the coding sequence under conditions compatible with the control sequence. The "operably linked" sequence may include an expression control sequence adjacent to the relevant gene and expression control sequences that act trans- or at a distance to control the relevant gene. The term "expression control sequence" refers to the polynucleotide sequence required to achieve the expression and processing of the concatenated coding sequence. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (such as Kozak concordant sequences); sequences enhancing protein stability; and, where necessary, sequences enhancing protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components that are advantageous in their presence, such as leader sequences and fusion collocation sequences. Unless otherwise stated, descriptions or statements herein of inserting nucleic acid molecules encoding the fusion protein of the present invention into expression vectors mean that when an expression vector containing the inserted nucleic acid molecule is introduced into a compatible host cell or a compatible cell of an organism, the inserted nucleic acid is also operatively linked within the vector to a functional promoter and other transcriptional and translational control elements required for the expression of the encoded fusion protein.

[0028] When applied to polynucleotides, “recombinant” means that the polynucleotide is the product of different combinations of in vitro selection, restriction enzyme digestion and / or conjugation steps and other procedures that produce a construct that can potentially be expressed in a host cell.

[0029] The terms "gene" and "gene fragment" are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame capable of encoding a specific protein after transcription and translation. A gene or gene fragment may be a gene body or cDNA, provided that the polynucleotide contains at least one open reading frame covering the entire coding region or a segment thereof. A "fusion gene" is a gene composed of at least two heteronucleotides linked together.

[0030] The terms “disease” and “symptom” are used interchangeably to refer to a pathological condition identified according to acceptable medical standards and practices in this art.

[0031] As used herein, the term “effective amount” means an amount of therapy that is sufficient to reduce or improve the severity and / or duration of a disease or one or more of its symptoms; sufficient to prevent the progression of a harmful or pathological condition; sufficient to cause the pathological condition to subside; sufficient to prevent the recurrence, development, onset or progression of one or more of the symptoms associated with the pathological condition; sufficient to detect the disease; or sufficient to enhance or improve the preventive or therapeutic effect of the therapy (e.g., administration of another preventive or therapeutic agent).

[0032] As used herein, the term "J domain" refers to a segment that retains the ability to promote the intrinsic ATPase catalytic activity of Hsp70 and its homologs. J domains of various J proteins have been identified (see, for example, Kampinga et al. (2010) Nat. Rev., 11: 579-592; Hennessy et al. (2005) Protein Science, 14: 1697-1709, each of which is incorporated herein by reference in its entirety), and are characterized by a number of features: characterized by a highly conserved tripeptide sequence motif (called the "HPD motif") of four α-helices (I, II, III, IV) and typically between helices II and III. Typically, the J domain of a J protein is between fifty and seventy amino acids in length, and it is believed that the interaction (binding) site between the J domain and the Hsp70-ATP carrier protein is a region extending from helix II, and that the HPD motif is necessary to stimulate Hsp70 ATPase activity. As used herein, the term "J domain" is intended to include the native J domain sequence and its functional variants, which retain the ability to promote the intrinsic Hsp70 ATPase activity, which can be measured using methods well known in this art (see, for example, Horne et al. (2010) J. Biol. Chem., 285, 21679-21688, which is incorporated herein by reference in its entirety). A non-limiting list of human J domains is provided in Table 1.

[0033] The inventors have discovered that certain cells in contact with fusion protein constructs containing the J domain and tau-binding domain of the J protein have an unexpected effect in reducing the hyperphosphorylation of tau protein. Hyperphosphorylation of tau is believed to cause a variety of devastating diseases, including (but not limited to) Alzheimer's disease (AD), Parkinson's disease (PD), primary age-related tau proteinopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal ganglia degeneration (CBD), frontotemporal lobe dementia and Parkinson's disease linked to chromosome 17 (FTDP-17), Guam-type amyotrophic lateral sclerosis-Parkinson's disease-dementia, ganglioglioma and gangliocytoma, meningioma, post-encephalitis Parkinson's disease, subacute sclerosing panencephalitis (SSPE), lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-associated neurodegeneration, and lipofuscinosis. Therefore, this article provides suitable compositions and methods for treating tau disease, for example, in individuals in need.

[0034] To overcome the problems associated with companion protein-based therapies, we investigated whether it is possible to design artificial companion proteins with high specificity. We designed a series of fusion protein constructs containing an Hsp70 binding / activation effector domain (J domain sequence) and a domain that imparts tau protein specificity. The resulting fusion proteins promote the intrinsic ATPase catalytic activity of Hsp70 and its homologs, resulting in increased protein folding, decreased aggregation, and / or accelerated clearance.

[0035] I. Fusion Protein Construct a. J Domain Applicable to the Invention The J domain of various J proteins has been identified. See, for example, Kampinga et al., Nat. Rev., 11: 579-592 (2010); Hennessy et al., Protein Science, 14:1697-1709 (2005). The J domain applicable to the preparation of the fusion protein of the invention has the key defining features of a J domain that primarily promotes HSP70 ATPase activity. Therefore, the isolated J domain applicable to the invention comprises a polypeptide domain characterized by four α-helices (I, II, III, IV) and typically has a highly conserved tripeptide sequence of histidine, proline, and aspartic acid (referred to as the "HPD motif") between helices II and III. Typically, the length of the J domain of a J protein is between fifty and seventy amino acids, and it is believed that the interaction (binding) site between the J domain and the Hsp70-ATP carrier protein is a region extending from helix II, and the HPD motif is important for the original active system. Representative J domains include (but are not limited to) the J domains of DnaJB1, DnaJB2, DnaJB6, and DnaJC6; the J domain of the large T antigen of SV40; and the J domain of mammalian cysteine ​​chain protein (CSP-α). The amino acid sequences of these and other J domains that can be used in the fusion protein of this invention are provided in Table 1. Conserved HPD motifs are highlighted in bold. Table 1. Representative Human J Domain Sequences Protein name SEQ ID NO: Gene NCBI Reference Protein NCBI Reference J-domain amino acid sequence DNAJA1 1 NM_001539 NP_001530 TYYDVLGVKPNATQEEELKKAYRKLALKYHPDKNPNEGEKFKQISQAYEVLSDAKKRELYDKGG DNAJA2 2 NM_005880 NP_005871 KLYDILGVPPGASENELKKAYRKLAKEYHPDKNPNAGDKFKEISFAYEVLSNPEKRELYDRYG DNAJA3 3 NM_001135110 NP_001128582 DYYQILGVPRNASQKEIKKAYYQLAKKYHPDTNKDDPKAKEKFSQLAEAYEVLSDEVKRKQYDAYG DNAJA4 4 NM_001130182 NP_001123654 ETQYYDILGVKPSASPEEIKKAYRKLALKYHPDKNPDEGEKFKLISQAYEVLSDPKKRDVYDQGGEQ DNAJB1 5 GKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEE DNAJB2 6 NM_001039550 NP_001034639 ASYYEILDVPRSASADDIKKAYRRKALQWHPDKNPDNKEFAEKKFKEVAEAYEVLSDKHKREIYDRYGRE DNAJB3 7 NM_001001394 NP_001001394 MVDYYEVLDVPRQASSEAIKKAYRKLALKWHPDKNPENKEEAERRFKQVAEAYEVLSDAKKRDIYDRYG DNAJB4 8 NM_001317099 NP_001304028 GKDYYCILGIEKGASDEDIKKAYRKQALKFHPDKNKSPQAEEKFKEVAEAYEVLSDPKKREIYDQFGEE DNAJB5 9 NM_001135004 NP_001128476 DYYKILGIPSGANEDEIKKAYRKMALKYHPDKNKEPNAEEKFKEIAEAYDVLSDPKKRGLYDQYG DNAJB6 10 NM_005494 NP_005485 VDYYEVLGVQRHASPEDIKKAYRKLALKWHPDKNPENKEEAERKFKQVAEAYEVLSDAKKRDIYDKYG DNAJB7 11 NM_145174 NP_660157 DYYEVLGLQRYASPEDIKKAYHKVALKWHPDKNPENKEEAERKFKEVAEAYEVLSNDEKRDIYDKYG DNAJB8 12 NM_153330 NP_699161 NYYEVLGVQASASPEDIKKAYRKLALRWHPDKNPDNKEEAEKKFKLVSEAYEVLSDSKKRSLYDRAG DNAJB9 13 NM_012328 NP_036460 SYYDILGVPKSASERQIKKAFHKLAMKYHPDKNKSPDAEAKFREIAEAYETLSDANRRKEYDTLG DNAJB11 14 NM_016306 NP_057390 DFYKILGVPRSASIKDIKKAYRKLALQLHPDRNPDDPQAQEKFQDLGAAYEVLSDSEKRKQYDTYG DNAJB12 15 NM_001002762 NP_001002762 YEILGVSRGASDEDLKKAYRRLALKFHPDKNHAPGATEAFKAIGTAYAVLSNPEKRKQYDQFGDD DNAJB13 16 NM_153614 NP_705842 DYYSVLGITRNSEDAQIKQAYRRLALKHHPLKSNEPSSAEIFRQIAEAYDVLSDPMKRGIYDKFG DNAJB14 17 NM_001031723 NP_001026893 NYYEVLGVTKDAGDEDLKKAYRKLALKFHPDKNHAPGATDAFKKIGNAYAVLSNPEKRKQYDLTG DNAJC1 18 NM_022365 NP_071760 NFYQFLGVQQDASSADIRKAYRKLSLTLHPDKNKDENAETQFRQLVAIYEVLKDDERRQRYDDIL DNAJC2 19 NM_001129887 NP_001123359 DHYAVLGLGHVRYKATQRQIKAAHKAMVLKHHPDKRKAAGEPIKEGDNDYFTCITKAYEMLSDPVKRRAFNSVD DNAJC3 20 NM_006260 NP_006251 DYYKILGVKRNAKKQEIIKAYRKLALQWHPDNFQNEEEKKKAEKKFIDIAAAKEVLSDPEMRKKFDDGE DNAJC4 21 NM_001307980 NP_001294909 TYYELLGVHPGASTEEVKRAFFSKSKELHPDRDPGNPSLHSRFVELSEAYRVLSREQSRRSYDDQL DNAJC5 22 NM_025219 NP_079495 GESLYHVLGLDKNATSDDIKKSYRKLALKYHPDKNPDNPEAADKFKEINNAHAILTDATKRNIYDKYGSL DNAJC5B 23 NM_033105 NP_149096 ALYEILGLHKGASNEEIKKTYRKLALKHHPDKNPDDPAATEKFKEINNAHAILTDISKRSIYDKYG DNAJC6 24 NM_001256864 NP_001243793 TKWKPVGMADLVTPEQVKKVYRKAVLVVHPDKATGQPYEQYAKMIFMELNDAWSEFENQGQKPLY DNAJC7 25 NM_001144766 NP_001138238 DYYKILGVDKNASEDEIKKAYRKRALMHHPDRHSGASAEVQKEEEKKFKEVGEAFTILSDPKKKTRYDSGQ DNAJC8 26 NM_014280 NP_055095 NPFEVLQIDPEVTDEEIKKRFRQLSILVHPDKNQDDADRAQKAFEAVDKAYKLLLDQEQKKRALDVIQ DNAJC9 27 NM_015190 NP_056005 DLYRVLGVRREASDGEVRRGYHKVSLQVHPDRVGEGDKEDATRRFQILGKVYSVLSDREQRAVYDEQG DNAJC10 28 NM_001271581 NP_001258510 DFYSLLGVSKTASSREIRQAFKKLALKLHPDKNPNNPNAHGDFLKINRAYEVLKDEDLRKKYDKYG DNAJC11 29 NM_018198 NP_060668 DYYSLLNVRREASSEELKAAYRRLCMLYHPDKHRDPELKSQAERLFNLVHQAYEVLSDPQTRAIYDIYG DNAJC12 30 NM_021800 NP_068572 DYYTLLGCDELSSVEQILAEFKVRALECHPDKHPENPKAVETFQKLQKAKEILTNEESRARYDHWR DNAJC13 31 NM_015268 NP_056083 DAYEVLNLPQGQGPHDESKIRKAYFRLAQKYHPDKNPEGRDMFEKVNKAYEFLCTKSAKIVDGPDP DNAJC14 32 NM_032364 NP_115740 NPFHVLGVEATASDVELKKAYRQLAVMVHPDKNHHPRAEEAFKVLRAAWDIVSNAEKRKEYEMKR DNAJC15 33 NM_013238 NP_037370 EAGLILGVSPSAGKAKIRTAHRRVMILNHPDKGGSPYVAAKINEAKDLLETTTKH DNAJC16 34 DPYRVLGVSRTASQADIKKAYKKLAREWHPDKNKDPGAEDKFIQISKAYEILSNEEKRSNYDQYG DNAJC17 35 NM_018163 NP_060633 DLYALLGIEEKAADKEVKKAYRQKALSCHPDKNPDNPRAAELFHQLSQALEVLTDAAARAAYDKVR DNAJC18 36 NM_152686 NP_689899 NYYEILGVSRDASDEELKKAYRKLALKFHPDKNCAPGATDAFKAIGNAFAVLSNPDKRLRYDEYG DNAJC19 37 NM_001190233 NP_001177162 EAALILGVSPTANKGKIRDAHRRIMLLNHPDKGGSPYIAAKINEAKDLLEGQAKK DNAJC20 38 NM_172002 NP_741999 DYFSLMDCNRSFRVDTAKLQHRYQQLQRLVHPDFFSQRSQTEKDFSEKHSTLVNDAYKTLLAPLSRGLYLLK DNAJC21 39 NM_001012339 NP_001012339 CHYEALGVRRDASEEELKKAYRKLALKWHPDKNLDNAAEAAEQFKLIQAAYDVLSDPQERAWYDNHR DNAJC22 40 NM_001304944 NP_001291873 LAYQVLGLSEGATNEEIHRSYQELVKVWHPDHNLDQTEEAQRHFLEIQAAYEVLSQPRKPWGSRR DNAJC23 41 NM_007214 NP_009145 NPYEVLNLDPGATVAEIKKQYRLLSLKYHPDKGGDEVMFMRIAKAYAALTDEESRKNWEEFG DNAJC24 42 NM_181706 NP_859057 DWYSILGADPSANISDLKQKYQKLILMYHPDKQSTDVPAGTVEECVQKFIEIDQAWKILGNEETKREYDLQR DNAJC25 43 NM_001015882 NP_001015882 DCYEVLGVSRSAGKAEIARAYRQLARRYHPDRYRPQPGDEGPGRTPQSAEEAFLLVATAYETLKDEETRKDYDYML DNAJC26 44 NM_001318134 NP_001305063 SRWTPVGMADLVAPEQVKKHYRRAVLAVHPDKAAGQPYEQHAKMIFMELNDAWSEFENQGSRPLF DNAJC27 45 DSWDMLGVKPGASRDEVNKAYRKLAVLLHPDKCVAPGSEDAFKAVVNARTALLKNIK DNAJC28 46 NM_001040192 NP_001035282 EYYRLLNVEEGCSADEVRESFHKLAKQYHPDSGSNTADSATFIRIEKAYRKVLSHVIEQTNASQS DNAJC29 47 NM_014363 NP_055178 ILKEVTSVVEQAWKLPESERKKIIRRLYLKWHPDKNPENHDIANEVFKHLQNEINRLEKQAFLDQNADRASRRTFTSASRFQSDKYS DNAJC30 48 NM_032317 NP_115693 ALYDLLGVPSTATQAQIKAAYYRQCFLYHPDRNSGSAEAAERFTRISQAYVVLGSATLRRKYDRGL

[0036] b. The tau-binding domain fusion protein also contains at least one tau-binding domain. The tau-binding domain may be a single-chain polypeptide or a multimeric polypeptide linked to the J domain to form the fusion protein.

[0037] When tau protein is present in cells at pathological levels, the tau-binding domain has an affinity sufficient to bind tau protein ideally. Therefore, in one embodiment, the fusion protein includes a tau-binding domain with a KD (Kind Degree) of tau to tau of, for example, 2 µM or less, 1 µM or less, 500 nM or less, 300 nM or less, 100 nM or less, or 30 nM or less when tested by ELISA on a 96-well microtiter plate. In another embodiment, the fusion protein includes a tau-binding domain with a KD of tau to aggregated tau of, for example, 2 µM or less, 1 µM or less, 500 nM or less, 300 nM or less, 100 nM or less, or 30 nM or less when tested by ELISA on a 96-well microtiter plate. In another embodiment, the tau binding domain has selectivity for aggregated forms of tau; for example, the tau binding domain has an affinity for aggregated forms of tau that is at least two times, for example at least three times, at least four times, at least five times, at least ten times, at least thirty times, or at least 100 times higher than that for soluble forms of tau.

[0038] The Tau-binding domain has been previously identified and characterized (see, for example, U.S. Patent No. 7,605,133, WO 2019 / 161386, Abe et al., (2007) BMC Bioinformatics, 8:451, all of which are incorporated herein by reference). Therefore, in another embodiment, the fusion protein comprises a tau-binding domain selected from the group consisting of SEQ ID NO: 49-52 (see, for example, Table 2). In a particular embodiment, the fusion protein comprises the tau-binding domain of SEQ ID NO: 49.

[0039] In another embodiment, the fusion protein also includes the use of a tau-binding domain that is chemically bound to the J domain. The tau-binding domain can be directly bound to the J domain. Alternatively, it can be bound to the J domain via a linker. For example, those skilled in the art know a variety of chemical crosslinking agents suitable for crosslinking a tau-binding domain to the J domain, or for crosslinking a targeting domain to a fusion protein comprising both a tau-binding domain and a J domain. For example, the crosslinking agent is a heterobifunctional crosslinking agent, which can be used to link molecules in a stepwise manner. Heterobifunctional crosslinking agents provide the ability to design more specific coupling methods for binding proteins, thereby reducing the presence of undesirable side reactions such as homologous protein polymers. A wide variety of heterodifunctional crosslinking agents are known in this technology, including 4-(N-cis-butenylidenemethyl)cyclohexane-1-carboxylate succinimide (SMCC), m-cis-butenylidenebenzoyl-N-hydroxysuccinimide (MBS); N-succinimide(4-iodoacetyl)aminobenzoate (SIAB), 4-(p-cis-butenylidenephenyl)butyrate succinimide (S... Crosslinking agents such as MPB, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-butadieneiminooxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-butadieneimide 3-(2-pyridyldithio)propionate (SPDP), and 6-[3-(2-pyridyldithio)propionate]butadieneimide hexanoate (LC-SPDP) are available. Crosslinking agents with an N-hydroxybutadieneimide moiety can generally be obtained in the form of N-hydroxysulfonic acid butadieneimide analogs, which generally have better water solubility. Furthermore, crosslinking agents with disulfide bridging bonds within the linker chain can be practically synthesized as alkyl derivatives to reduce the amount of linker cleavage in vivo. Besides heterobifunctional crosslinking agents, various other crosslinking agents exist, including homobifunctional and photoreactive crosslinking agents. Dibutyldiamino octanoate (DSS), bis(cis-butenyldiaminohexane) (BMH), and dimethyl diimide-2HCl (Forbes-Cori Disease) are examples of suitable bifunctional crosslinking agents, and bis-[B-(4-azidosylamino)ethyl]disulfide (BASED) and N-dibutyldiamino-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of suitable photoreactive crosslinking agents used in this invention. For a recent review of protein coupling techniques, see Means et al., (1990) Bioconj. Chem. 1:2-12, which is incorporated herein by reference. Table 2: Examples of tau binding domains name SEQ ID NO: length sequence TBP1 49 6 VQIVYK scFv(Tau) 50 234 EVQLQQSGAELVQPGASVKLSCTASGFNIKDTSMHWVRQRPEQGLEWIGRIAPANGNTKYDPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCSGSGNYDWGQGTTLTVSGGGGSGGGGSGGGGSDIQMNQSPSSLSASLGDTITISCHASQNINVWLSWYQQKPGNIPKLLIYEASTLYTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEI scFv(MW7) 51 243 QVKLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLSGVAEIRSKANNHATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCIYAGFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPWTFGGGTKLEI Happ1 52 118 MQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRPEDEADYYCAAWDDSLCVALVFGGGTNGGGGVDGTAG TBP2 53 6 VQIINK TBP3 54 13 YQQYQDATADEQG

[0040] c. Linkers as appropriate The fusion proteins described herein may contain one or more linkers as appropriate. Linkers may be peptides or non-peptides. The purpose of linkers is particularly to provide sufficient distance between functional domains within the protein (e.g., between the J domain and the tau-binding domain, between tandem arrangements of tau-binding domains, between the J domain and the tau-binding domain and, as appropriate, a targeting agent, or between the J domain and the tau-binding domain and, as appropriate, a detection domain or antigen determinant) to achieve optimal function of each domain. Obviously, linkers preferably do not interfere with the individual function of the J domain (the target protein-binding domain of the fusion protein according to the invention). If present in the fusion protein of the invention, the linker is selected to reduce cytotoxicity caused by the target protein (tau protein), and may be omitted if direct attachment achieves the desired effect. The linker present in the fusion protein of the present invention may comprise one or more amino acids encoded by a nucleotide sequence present at or around a selection site on the expression vector (in which a nucleic acid segment encoding a protein domain as described herein or the entire fusion protein is inserted into the frame). In one embodiment, the length of the peptide linker is between 1 and 20 amino acids. In another embodiment, the length of the peptide linker is between 2 and 15 amino acids. In yet another embodiment, the length of the peptide linker is between 2 and 10 amino acids.

[0041] The selection of one or more polypeptide linkers to prepare the fusion protein according to the present invention is within the knowledge and skill of those skilled in the art. See, for example, Arai et al., Protein Eng., 14(8): 529-532 (2001); Crasto et al., Protein Eng., 13(5): 309-314 (2000); George et al., Protein Eng., 15(11): 871-879 (2003); Robinson et al., Proc. Natl. Acad. Sci. USA, 95: 5929-5934 (1998), each of which is incorporated herein by reference in its entirety. Examples of linkers for preparing the fusion protein according to the present invention, consisting of two or more amino acids, include, but are not limited to, those provided in Table 3 below. Table 3: Linker Sequences SEQ ID NO: length sequence 55 2 SR 56 4 GTGS 57 5 GLESR 58 4 GGSG 59 4 GGGS 60 5 DIAAA 61 9 DIAAALESR 62 15 GGGGSGGGGSGGGGS 63 11 AEAAAKEAAAK 64 15 SGGGSGGGGSGGGGS 65 25 DIGGGGSGGGGSGGGGSGGGGSAAA

[0042] d. Targeting Agent The fusion protein disclosed herein may further include a targeting portion. As used herein, the terms "targeting portion" and "targeting agent" are used interchangeably and refer to substances associated with the fusion protein that enhance binding, transport, accumulation, retention time, bioavailability, or alter the bioactivity or therapeutic effect of the fusion protein in cells or in an individual. The targeting portion may function at the tissue, cellular, and / or subcellular level. The targeting portion may, for example, guide the fusion protein to a specific cell, tissue, or organ after administration to an individual. In one embodiment, the targeting portion is located at the N-terminus of the fusion protein. In another embodiment, the targeting portion is located at the C-terminus of the fusion protein. In yet another embodiment, the targeting portion is located internally. In another embodiment, the targeting portion is attached to the fusion protein via chemical binding.

[0043] The target portion may include, in particular, organic or inorganic molecules, peptides, peptide mimics, proteins, antibodies or fragments thereof, growth factors, enzymes, lectins, antigens or immunogens, viruses or components thereof, viral vectors, receptors, receptor ligands, toxins, polynucleotides, oligonucleotides or aptamers, nucleotides, carbohydrates, sugars, lipids, glycolipids, nucleoproteins, glycoproteins, lipoproteins, steroids, hormones, growth factors, chemical inducers, cytokines, chemokines, drugs or small molecules.

[0044] In one exemplary embodiment of the present invention, the targeting portion enhances binding, transport, accumulation, retention time, bioavailability, or alters the bioactivity or therapeutic effect of the active agent in the platform or its associated ligands and / or target cells or tissues (e.g., neurons, central nervous system, and / or peripheral nervous system). Thus, the targeting portion may be specific to cellular receptors associated with the central nervous system, or may be additionally associated with enhanced delivery to the CNS via the blood-brain barrier (BBB). Therefore, the ligand as described above may be both a ligand and a targeting portion.

[0045] In some embodiments, the targeting portion may be a cell-penetrating peptide, such as that described in U.S. Patent No. 10,111,965, which is incorporated herein by reference in its entirety. In another embodiment, the targeting portion may be an antibody or antigen-binding fragment or a single-chain derivative thereof, such as that described in U.S. Patent No. 16 / 131,591, which is incorporated herein by reference in its entirety.

[0046] The targeting portion can be coupled to the platform for targeted cell delivery by directly or indirectly binding to the core. For example, in embodiments where the core comprises nanoparticles, the binding of the targeting portion to the nanoparticles can utilize a similar functional group for tethering PEG to the nanoparticles. Thus, the targeting portion can bind directly to the nanoparticles via targeting portion functionalization. Alternatively, the targeting portion can bind indirectly to the nanoparticles via targeting portion binding to functionalized PEG as discussed above. The targeting portion can be attached to the core by means of covalent, non-covalent, or electrostatic interactions. In one embodiment, the targeting portion is a peptide. In a particular embodiment, the targeting portion is a peptide covalently attached to the N-terminus of the fusion protein.

[0047] e. Antigenic Determinant In some embodiments, the fusion protein of the present invention contains an antigenic determinant or tag, which may impart additional properties to the fusion protein. As used herein, the terms "antigenic determinant" and "tag" are used interchangeably to refer to an amino acid sequence of 300 or fewer amino acids, typically attached to the N-terminus or C-terminus of the fusion protein. In one embodiment, the fusion protein of the present invention further includes an antigenic determinant for facilitating purification. Examples of such antigenic determinants suitable for purification provided in Table 4 below include the human IgG1 Fc sequence (SEQ ID NO: 66), the FLAG antigenic determinant (DYKDDDDK, SEQ ID NO: 67), the His6 antigenic determinant (SEQ ID NO: 68), c-myc (SEQ ID NO: 69), HA (SEQ ID NO: 70), the V5 antigenic determinant (SEQ ID NO: 71), or glutathione S-transferase (SEQ ID NO: 72). In another embodiment, the fusion protein of the present invention further comprises an antigenic determinant for prolonging the half-life of the fusion protein upon administration to an individual (e.g., a human). Examples of such antigenic determinants suitable for prolonging half-life include human Fc sequences. Thus, in a particular embodiment, in addition to the J domain and tau-binding domain, the fusion protein also comprises a human Fc antigenic determinant. The antigenic determinant is located at the C-terminus of the fusion protein. Table 4: Representative Examples of Antigenic Determinants SEQ ID NO: antigenic determinants length sequence 66 Human IgG1 Fc domain 232 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 67 FLAG antigenic determinant 8 DYKDDDDK 68 His6 6 HHHHHH 69 c-myc 10 EQKLISEEDL 70 HA 9 YPYDVPDYA 71 V5 antigenic determinant 14 GKPIPNPLLGLDST 72 Glutathione-S-transferase 220 MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIA YSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD

[0048] f. Cell-penetrating peptides In other embodiments, the fusion protein described herein may further comprise cell-penetrating peptides. Cell-penetrating peptides are known to carry bound cargo (whether small molecules, peptides, proteins, or nucleic acids) into cells. Non-limiting examples of cell-penetrating peptides in the fusion proteins of the present invention include, but are not limited to, polycationic peptides, such as HIV TAT peptides 49-57, polyarginine, and membrane-penetrating peptides pAntan (43-58); amphoteric peptides, such as pep-1; and hydrophobic peptides, such as C405Y and analogues. See Table 5 below. Table 5: Examples of cell-penetrating peptides SEQ ID NO: sequence 73 RKKRRQRRR 74 RQIKWFQNRRMKWKK 75 KETWWETWWTEWSQPKKKRKV 76 CSIPPEVKFNKPFVYLI

[0049] Therefore, in one embodiment, the fusion protein comprises a cell-penetrating peptide and a fusion protein, wherein the cell-penetrating peptide is selected from the group consisting of SEQ ID NO: 73-76, and the fusion protein is selected from the group consisting of SEQ ID NO: 83-88 and 95-101. In another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO: 73 and a fusion protein selected from the group consisting of SEQ ID NO: 83-88 and 95-101. In another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO: 74 and a fusion protein selected from the group consisting of SEQ ID NO: 83-88 and 95-101. In yet another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO: 75 and a fusion protein selected from the group consisting of SEQ ID NO: 83-88 and 95-101. In another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO: 76 and fusion proteins selected from the group consisting of SEQ ID NOs: 83-88 and 95-101. Cells expressing the fusion protein construct with the cell-penetrating peptide can be administered to individuals, such as human individuals (e.g., patients with tau disease or at risk of developing tau disease). The fusion protein is secreted from the cell, which helps reduce tau protein aggregation and / or related cytotoxicity.

[0050] g. Arrangement of the J Domain and Tau Binding Domain The fusion protein described herein can be arranged in various ways. In one embodiment, the tau binding domain is attached to the C-terminal side of the J domain. In another embodiment, the tau binding domain is attached to the N-terminal side of the J domain. In either configuration, the tau binding domain and the J domain may be separated via a linker as described above.

[0051] In some embodiments, the J domain may be attached to a plurality of tau-binding domains, such as two, three, four, or more than four tau-binding domains. The tau-binding domain may be attached to the N-terminal side of the J domain. Alternatively, the tau-binding domain may be attached to the C-terminal side of the J domain. In yet another embodiment, the tau-binding domain may be attached to both the N-terminus and C-terminus sides of the J domain. Each of the plurality of tau-binding domains may be the same tau-binding domain. In another embodiment, each of the plurality of tau-binding domains in the fusion protein may be a different tau-binding domain (i.e., a different sequence).

[0052] In some embodiments, the fusion protein may comprise a structure selected from the following groups: a. DNAJ-XT b. DNAJ-XTXT c. DNAJ-XTXTXT d. TX-DNAJ e. TXTX-DNAJ f. TXTXTX-DNAJ, g. TX-DNAJ-XT, h. TX-DNAJ-XTXT, i. TX-DNAJ-XTXTXT, j. TXTX-DNAJ-XT, k. TXTX-DNAJ-XTXT, l. TXTX-DNAJ-XTXTXT, m. TXTXTX-DNAJ-XT, n. TXTXTX-DNAJ-XTXT, and o. TXTXTX-DNAJ-XTXTXT, wherein T is a tau-binding domain, DNAJ is the J domain of the J protein, and X is a linker selected as appropriate.

[0053] In one embodiment, the fusion protein includes a J domain selected from the group consisting of SEQ ID NO: 5, 6, 10, 24, and 31. In a particular embodiment, the fusion protein includes the J domain of SEQ ID NO: 5.

[0054] In another embodiment, the tau binding domain is selected from the group consisting of SEQ ID NO: 49-54. In a particular embodiment, the tau binding domain is SEQ ID NO: 49.

[0055] In another embodiment, the fusion protein comprises the J domain of SEQ ID NO: 5 and the tau-binding domain of SEQ ID NO: 49. In another embodiment, the fusion protein comprises at least two copies of the J domain of SEQ ID NO: 5 and the tau-binding domain of SEQ ID NO: 49. In another embodiment, the fusion protein comprises the J domain of SEQ ID NO: 5 and the tau-binding domain of SEQ ID NO: 50. In another embodiment, the fusion protein comprises the J domain of SEQ ID NO: 5 and the tau-binding domain of SEQ ID NO: 51. In yet another embodiment, the fusion protein comprises the J domain of SEQ ID NO: 5 and the tau-binding domain of SEQ ID NO: 52. In another embodiment, the fusion protein comprises the J domain of SEQ ID NO: 5 and the tau-binding domain of SEQ ID NO: 53. In yet another embodiment, the fusion protein comprises the J domain of SEQ ID NO: 5 and the tau-binding domain of SEQ ID NO: 54.

[0056] Non-limiting examples of fusion protein constructs comprising a J domain and a tau-binding domain are schematically depicted in Figure 2 and also shown in Table 6 below. In another embodiment, a particular fusion protein construct is selected from the group consisting of SEQ ID NO: 83-88 and 95-101. Table 6: Fusion Protein Constructs and Control Constructs Structure Number SEQ ID NO: Structure Name length sequence 1 83 JB1-TBP1 106 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAVQIVYK 2 84 TBP1-JB1-TBP1 147 MGKPIPNPLLGLDSTGTGSVQIVYKGGGSGGGSGGGSGGGSMGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAVQIVYK 3 85 JB1-2XTBP1 128 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAVQIVYKGGGSGGGSGGGSGGGSVQIVYK 4 86 JB1-scFv(Tau) 334 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAEVQLQQSGAELVQPGASVKLSCTASGFNIKDTSMHWVRQRPEQGLEWIGRIAPANGNTKYDPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCSGSGNYDWGQGTTLTVSGGGGSGGGGSGGGGSDIQMNQSPSSLSASLGDTITISCHASQNINVWLSWYQQKPGNIPKLLIYEASTLYTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEI 5 87 JB1-scFv(MW7) 343 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAQVKLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLSGVAEIRSKANNHATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCIYAGFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPWTFGGGTKLEI 6 88 JB1-Happ1 218 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAMQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRPEDEADYYCAAWDDSLCVALVFGGGTNGGGGVDGTAG 7 89 JB1(P33Q)-TBP 106 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHQDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAVQIVYK 8 90 JB1(P33Q)-scFv(Tau) 334 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHQDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAEVQLQQSGAELVQPGASVKLSCTASGFNIKDTSMHWVRQRPEQGLEWIGRIAPANGNTKYDPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCSGSGNYDWGQGTTLTVSGGGGSGGGGSGGGGSDIQMNQSPSSLSASLGDTITISCHASQNINVWLSWYQQKPGNIPKLLIYEASTLYTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEI 9 91 JB1(P33Q)-scFv(MW7) 343 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHQDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAQVKLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLSGVAEIRSKANNHATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCIYAGFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPWTFGGGTKLEI 10 92 JB1(P33Q)-Happ1 218 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHQDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAMQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRPEDEADYYCAAWDDSLCVALVFGGGTNGGGGVDGTAG 11 93 JB1-QBP1 91 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGGGGSSNWKWWPGIFD 12 94 JB1(P33Q)-QBP1 91 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHQDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGGGGSSNWKWWPGIFD 14 95 JB1-TBP2 106 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAVQIINK 15 96 JB1-TBP3 113 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSDIGGGGSGGGGSGGGGSGGGGSAAAYQQYQDATADEQG 16 97 JB1-TBP1 (without linker) 86 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGGGGSVQIVYK 17 98 JB1-TBP2 (without linker) 86 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGGGGSVQIINK 18 99 JB1-TBP3 (without linker) 93 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGGGGSYQQYQDATADEQG 19 100 JB1-scFv(Tau) (without linker) 314 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGGGGSEVQLQQSGAELVQPGASVKLSCTASGFNIKDTSMHWVRQRPEQGLEWIGRIAPANGNTKYDPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCSGSGNYDWGQGTTLTVSGGGGSGGGGSGGGGSDIQMNQSPSSLSASLGDTITISCHASQNINVWLSWYQQKPGNIPKLLIYEASTLYTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEI 20 101 JB1-Happ1 (without linker) 198 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKEPGAEEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGGGGSMQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRPEDEADYYCAAWDDSLCVALVFGGGTNGGGGVDGTAG

[0057] II. Nucleic Acid Encoding Fusion Protein Architects According to another embodiment of the present invention, the present invention provides isolated nucleic acids comprising polynucleotide sequences selected from: (a) a polynucleotide encoding a fusion protein of any of the foregoing embodiments, or (b) a complementary sequence to the polynucleotide of (a). The present invention provides isolated nucleic acids encoding fusion proteins comprising a J domain and a tau-binding domain, and sequences complementary to such nucleic acid molecules encoding fusion proteins (including homologs thereof). In another embodiment, the present invention covers methods for generating nucleic acids encoding the fusion proteins disclosed herein and sequences complementary to nucleic acid molecules encoding fusion proteins (including homologs thereof). The nucleic acid of this embodiment of the present invention may be pre-mRNA, mRNA, RNA, gDNA, PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0058] In another embodiment, a method for producing a fusion protein is disclosed, comprising (a) synthesizing and / or assembling nucleotides encoding the fusion protein, (b) incorporating the encoding gene into an expression vector suitable for host cells, (c) transforming suitable host cells with the expression vector, and (d) culturing the host cells under conditions that allow or permit the fusion protein to be expressed in the transformed host cells, thereby producing a biologically active fusion protein, which is recovered as a separated fusion protein by standard protein purification methods known in this art. Standard recombinant techniques in molecular biology are used to manufacture the polynucleotides and expression vectors of this invention.

[0059] According to the present invention, a nucleic acid sequence (or its complementary sequence) encoding the fusion protein disclosed herein is used to generate a recombinant DNA molecule that guides the expression of the fusion protein in a suitable host cell. Several selection strategies are suitable for carrying out the present invention, many of which are used to generate a construct comprising a gene encoding the fusion protein of the present invention or its complementary sequence. In some embodiments, the selection strategy is used to generate a gene encoding the fusion protein of the present invention or its complementary sequence.

[0060] In some embodiments, the nucleic acid encoding one or more fusion proteins is an RNA molecule, and may be pre-mRNA, mRNA, RNA, gDNA, PCR-amplified DNA, cDNA, synthetic DNA, or recombinant DNA.

[0061] In various embodiments, the nucleic acid is mRNA introduced into the cell to transiently express the desired polypeptide. As used herein, "transient" means a period of time during which a non-integrated transgenic gene is expressed for hours, days, or weeks, wherein such period of expression is shorter than the period of expression when the polynucleotide is integrated into the gene body or contained in a stable plasmonic replicon in the cell.

[0062] In a particular embodiment, the mRNA encoding the polypeptide is a live in vitro transcribed mRNA. As used herein, "live in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Generally, live in vitro transcribed RNA is generated from a live in vitro transcription vector. The live in vitro transcription vector contains a template for producing live in vitro transcribed RNA.

[0063] In a particular embodiment, the mRNA may further comprise a 5' cap or a modified 5' cap and / or a poly(A) sequence. As used herein, a 5' cap (also referred to as an RNA cap, RNA 7-methylguanosine cap, or RNA m7G cap) is a modified guanine nucleotide added to the "front" or 5' end of the eukaryotic messenger RNA shortly after transcription begins. The 5' cap comprises a terminal group linked to the first transcribed nucleotide and recognized by the ribosome and protected from the influence of ribonucleases. The capping portion may be modified to regulate the function of the mRNA, such as its translational stability or efficiency. In a particular embodiment, the mRNA comprises a poly(A) sequence of between about 50 and about 5000 adenine nucleotides. In one embodiment, the mRNA comprises a poly(A) sequence of between about 100 and about 1000 bases, between about 200 and about 500 bases, or between about 300 and about 400 bases. In one embodiment, the mRNA comprises a poly(A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1000 or more bases. The poly(A) sequence may be chemically or enzymatically modified to regulate mRNA function, such as localization, translation stability, or efficiency.

[0064] As used herein, the terms "polynucleotide variant" and "variant" and their analogues refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence or that hybridizes with a reference sequence under stringent conditions as defined below. These terms include polynucleotides in which one or more nucleotides have been added, deleted, or substituted with different nucleotides compared to the reference polynucleotide. In this regard, it is fully understood in this art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to the reference polynucleotide, whereby the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0065] III. Vectors containing nucleic acids encoding fusion proteins are also provided, as are vectors containing nucleic acids according to the present invention. Such vectors preferably contain additional nucleic acid sequences, such as elements necessary for transcription / translation of nucleic acid sequences encoding phosphatases (e.g., promoter and / or terminator sequences). These vectors may also contain nucleic acid sequences encoding selection markers (e.g., antibiotics) to select or maintain the nucleic acid sequence of a host cell transformed by the vector. The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to the vector nucleic acid molecule, for example, inserted into the vector nucleic acid molecule. The vector may include sequences that guide autonomous replication in the cell, or may include sequences sufficient to allow integration into the host cell DNA. In certain embodiments, nonviral vectors are used to deliver one or more polynucleotides covered herein to the affected cell (e.g., neuronal cells). In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a fusion protein containing a J domain and a tau-binding domain. Illustrative examples of nonviral vectors include, but are not limited to, mRNA, plastids (e.g., DNA plastids or RNA plastids), transposons, myxosomes, and bacterial artificial chromosomes. As used herein, the terms "nucleic acid cartridge" or "expression cartridge" refer to a gene sequence within a vector capable of expressing RNA and subsequently a polypeptide. In one embodiment, the nucleic acid cartridge contains a related gene, such as a related polynucleotide. In another embodiment, the nucleic acid cartridge contains one or more expression control sequences, such as promoters, enhancers, poly(A) sequences, and related genes, such as related polynucleotides. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cartridges. The nucleic acid cartridges are oriented positionally and sequentially within the vector such that the nucleic acid within the cartridge can be transcribed into RNA and, if necessary, translated into a protein or polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and translocate to a biologically active compartment by targeting to an appropriate intracellular compartment or secreting into an extracellular compartment. Preferably, the cartridge has 3' and 5' ends suitable for insertion into the vector, for example, it has restriction endonuclease sites at each end. The cartridge can be removed and inserted into a plasmid or viral vector as a single unit.

[0066] Illustrative examples of vectors include (but are not limited to) plastids, autonomously replicating sequences, and translocation elements, such as piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives. Further illustrative examples of vectors include (but are not limited to) plastids; phage particles; myxosomes; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages, such as λ phage or M13 phage; and animal viruses. Illustrative examples of viruses suitable as vectors include (but are not limited to) retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and polyvesicular papillomaviruses (e.g., SV40). Illustrative examples of expression vectors include (but are not limited to) the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V 5-DEST™, pLenti6 / V 5-DEST™, and pLenti6.2 / V 5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequence of the polypeptide disclosed herein may be conjugated to an expression vector for expressing the polypeptide in mammalian cells.

[0067] In a particular embodiment, the vector is a free vector or a vector maintained outside the chromosome. As used herein, the term "free" means a vector that can replicate without integrating into the host's chromosomal DNA and is not gradually lost by dividing host cells, and also means that the vector replicates outside the chromosome or in a free manner.

[0068] Illustrative generalized performance control sequences applicable to specific embodiments include (but are not limited to) cytomegalovirus (CMV) immediate early promoter, simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5 and P11 promoters from vaccinia virus, elongation factor 1-α (Efla) promoter, early growth response factor 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70kDa 5 (HSPA5), heat shock protein 90kDa β member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substituted (MND) U3 promoter (Haas et al., Journal of Virology. 2003;77(l7): 9439-9450).

[0069] The vector may contain one or more recombination sites from a wide variety of site-specific recombinases. It should be understood that the target site for a site-specific recombinase is any site other than that required by the integration vector (e.g., a retroviral vector or a lentiviral vector). As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombination site” refer to the specific nucleic acid sequence that the recombinase recognizes and binds to.

[0070] For example, one recombination site of Cre recombinase is loxP, which is a 34-base-pair sequence containing two 13-base-pair inverted repeat sequences (as the recombinase binding site) flanked by an 8-base-pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Suitable recognition sites for FLP recombinase include, but are not limited to: FRT (McLeod et al., 1996), FI, F2, F3 (Schlake and Bode, 1994), FyFs (Schlake and Bode, 1994), FRT (LE) (Senecoff et al., 1988), and FRT (RE) (Senecoff et al., 1988).

[0071] Other examples of recognition sequences are the attB, attP, attL and attR sequences recognized by recombinase I integrase (e.g. phi-c3l). (pC3l SSR mediates recombination only between the heterologous sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). Both attB and attP (named for attachment sites of phage integrase on bacterial and phage genomes, respectively) contain imperfect inverted repeat sequences that may be bound by φ031 homodimers (Groth et al., 2000). The product sites attL and attR are effectively inert for other tpQA1-mediated recombinations (Belteki et al., 2003), making the reaction irreversible. For catalytic insertion, it has been found that DNA with attB is inserted into the attP site of the genome more readily than DNA with attP is inserted into the attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, following the typical strategic position of homologous recombination, the "docking site" with attP enters the defined locus, which is then paired with the introduction sequence with attB for insertion.)

[0072] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct internal ribosome entry into the start codon (such as ATG) of a cistron (protein-coding region), thereby inducing cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83 and Jackson and Kaminski. 1995. RNA 1(10): 985-1000. In certain embodiments, the vector comprises one or more associated polynucleotides encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequence may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides covered herein is an EMCV IRES.

[0073] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the ribosome and enhances translation. (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). In a particular embodiment, the vector comprises a polynucleotide having a shared Kozak sequence and encoding a fusion protein comprising a J domain and a tau-binding domain. Elements that guide the effective capping and polyadenylation of heterologous nucleic acid transcripts enhance heterologous gene expression. Transcription termination signals are generally visible downstream of polyadenylation signals. In a particular embodiment, the vector comprises a polyadenylated sequence located at the 3' position of the polynucleotide encoding the polypeptide to be expressed.

[0074] IV. Delivery In certain embodiments, one or more polynucleotides encoding a fusion protein comprising a J domain and a tau-binding domain are introduced into cells via non-viral or viral vectors. Illustrative methods for delivering polynucleotides without viral transmission, as covered in certain embodiments, include, but are not limited to: electroporation, sonoporosis, liposome transfection, microinjection, gene gun, viral particles, liposomes, immune liposomes, nanoparticles, polycationic or liponucleotide conjugates, naked DNA, artificial viral particles, DEAE-glucan-mediated transfer, gene gun, and heat shock.

[0075] Illustrative examples of polynucleotide delivery systems applicable to the specific embodiments covered in the specific embodiments include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Liposome transfection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Effective receptor recognition of polynucleotides for liposome transfection of cationic and neutral lipids has been described in the literature. See, for example, Liu et al., (2003) Gene Therapy. 10: 180-187; and Balazs et al., (20W) Journal of Drug Delivery. 2011: 1-12. Antibody targeting, bacterial-derived, and non-biological nano-cell-based delivery are also covered in the specific embodiments.

[0076] Viral vectors containing the polynucleotides covered in specific embodiments can be delivered in vivo to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion), intrathecal injection, intraventricular injection, or local application, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells extracted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue sections, etc.) or universal donor hematopoietic stem cells, and then the cells are re-implanted into the patient.

[0077] In one embodiment, a viral vector comprising a polynucleotide encoding the fusion protein disclosed herein is directly delivered to an organism for transduction into living cells.

[0078] Properly packaged and formulated viral vectors can be delivered to the central nervous system (CNS) via intrathecal delivery. For example, adeno-associated virus vectors can be delivered using the method described in U.S. Patent No. 15 / 771,481, which is incorporated herein by reference in its entirety.

[0079] Alternatively, naked DNA may be administered. Administration is carried out by any of the methods commonly used to introduce molecules for eventual contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well-known to those skilled in the art, and although more than one method may be used for administering a particular composition, a particular method may generally provide a more direct and efficient response than another.

[0080] Illustrative examples of viral vector systems applicable to the specific embodiments covered herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0081] In various embodiments, one or more polynucleotides encoding a fusion protein containing a J domain and a tau-binding domain are introduced into cells (e.g., neuronal cells) by transducing cells with recombinant adeno-associated virus (rAAV) containing one or more polynucleotides. AAV is a small (approximately 26 nm) replication-defective, predominantly free, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can incorporate its genome into the genome of the host cell. Recombinant AAV (rAAV) typically consists, at a minimum, a transfected gene and its regulatory sequence, and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequence is approximately 145 bp long. In certain embodiments, rAAV comprises an ITR and capsid sequence isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh10, or AAV 10.

[0082] In some embodiments, chimeric rAAVs are used, where the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from different AAV serotypes. For example, an rAAV having an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, the rAAV vector may comprise an ITR from AAV2 and a capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh10, or AAV10. In a preferred embodiment, the rAAV comprises an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In another preferred embodiment, the rAAV comprises an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0083] In some embodiments, engineering and selection methods may be applied to the AAV capsid to make it more likely to transduce related cells.

[0084] The construction, manufacture, and purification of rAAV vectors have been disclosed in, for example, U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated herein by reference in its entirety. In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into neuronal cells or neuronal stem cells by transducing cells with a retrovirus (e.g., a lentivirus) comprising one or more polynucleotides. As used herein, the term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Illustrative retroviruses suitable for use in specific embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), foamviruses, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses. As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Explanatory lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-maedi virus (VMV); Capillary arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0085] The lentiviral vector preferably contains several safety enhancements as a result of the modification of the LTR. "Self-inactivation" (SIN) vectors refer to replication-defective vectors, for example, in which the right (3') LTR enhancer-promoter region (known as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the lentiviral vector is generated according to known methods. See, for example, Kutner et al., BMC Biotechnol. 2009; 9:10. Doi: 10.1186 / 1472-6750-9-10; Kutner et al., Nat. Protoc. 2009; 4(4):495-505. Doi: l0.l038 / nprot.2009.22.

[0086] According to certain specific embodiments covered herein, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it should be understood that a wide variety of retroviral and / or lentiviral sequences from different sources can be used, or that a combination of numerous substitutions and alterations in certain lentiviral sequences can be accommodated without impairing the ability of the transfer vector to perform the functions described herein. Furthermore, a variety of lentiviral vectors are known in this art, see Naldini et al., (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136, many of which are suitable for producing the viral vectors or transfer plasmids covered herein.

[0087] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into target cells by transducing cells with an adenovirus containing one or more polynucleotides. Adenovirus-based vectors can achieve extremely high transduction efficiency in many cell types without requiring cell division. High titers and high expression levels have been obtained with such vectors. These vectors can be mass-produced in relatively simple systems. Most adenovirus vectors are engineered to replace the Ad Ela, Elb, and / or E3 genes with transgenic genes; subsequently, replication-defective vectors are distributed in human 293 cells that provide the function of the missing genes in trans form. Ad vectors can be transduced in vivo into multiple tissue types, including non-dividing differentiated cells, such as those found in the liver, kidney, and muscle. Ad vectors are known to have large carrying capacity.

[0088] Currently, the generation and dissemination of replication-defective adenovirus vectors can utilize a unique helper cell line (called 293), which is derived from human embryonic kidney cells via the Ad5 DNA fragment and constitutively expresses the E1 protein (Graham et al., 1977). Since the E3 region is not essential to the adenovirus genome (Jones and Shenk, 1978), current adenovirus vectors utilize 293 cells to carry exogenous DNA in the E1, D3, or both regions (Graham and Prevec, 1991). Adenovirus vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Studies of recombinant adenovirus administration to various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz and Gerard, 1993), and stereotactic implantation into the brain (Le Gal La Salle et al., 1993). Examples of the use of ad vectors in clinical trials relate to polynucleotide therapy for antitumor immunization via intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)).

[0089] In various embodiments, one or more polynucleotides encoding the fusion protein of the present invention are introduced into the target cells of an individual by transducing cells with herpes simplex virus (e.g., HSV-1, HSV-2) containing one or more polynucleotides.

[0090] A mature HSV viral particle consists of an icosahedral capsid and a viral genome, which is composed of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is a replication-deficient vector. Most replication-deficient HSV vectors contain deletions to remove one or more early-mid, early, or late HSV genes to prevent replication. For example, an HSV vector may lack immediately early genes selected from the following groups: ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantage of HSV vectors is that they can enter a latent phase, thereby causing long-term DNA expression, and their large viral DNA genome can accommodate up to 25 kb of exogenous DNA inserts. HSV-based carriers are described in, for example, U.S. Patent Nos. 5,837,532, 5,846,782 and 5,804,413 and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637 and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0091] V. Cells Expressing Fusion Proteins In another embodiment, the present invention provides cells expressing the fusion proteins described herein. Cells may be transfected with a vector encoding the fusion proteins as described above. In one embodiment, the cells are prokaryotic cells. In another embodiment, the cells are eukaryotic cells. In yet another embodiment, the cells are mammalian cells. In a particular embodiment, the cells are human cells. In another embodiment, the cells are human cells derived from patients suffering from or at risk of suffering from tau-mediated diseases (including but not limited to ALS, FTD, and Alzheimer's disease). The cells may be neuronal cells or muscle cells.

[0092] Cells expressing the fusion protein can be used to produce the fusion protein. In this embodiment, the cells are transfected with a vector that expresses the fusion protein. The fusion protein may contain antigenic determinants, such as the human Fc domain or FLAG antigenic determinant described above, which will facilitate purification (using protein A or anti-FLAG antibody columns, respectively). The antigenic determinant may be linked to the rest of the fusion protein via a linker or protease receptor sequence so that the antigenic determinant can be removed from the fusion protein during or after purification.

[0093] Cells expressing the fusion protein can also be used in therapeutic situations. In one embodiment, cells are collected from a patient requiring therapy (e.g., a patient with tau-mediated disease or at risk of developing tau-mediated disease). In one embodiment, the cells are neuronal cells. The collected cells are then transfected with a vector expressing the fusion protein. The transfected cells may then be processed to concentrate or select the transfected cells. The transfected cells may also be processed to differentiate into different cell types, such as neurons. After processing, the transfected cells can be administered to the patient. In one embodiment, the cells are administered via intrathecal injection, intracranial injection, or intraventricular injection into the central nervous system.

[0094] In alternative embodiments, cells representing the secretory form of the fusion protein can be used. For example, the fusion protein construct may be designed to have a signal sequence at its N-terminus. Representative signal sequences are shown in Table 7 below. Table 7: Representative Signal Sequences SEQ ID NO: sequence 77 MGVKVLFALICIAVAEA 78 MAPVQLLGLLVLFLPAMRC 79 MAVLGLLFCLVTFPSCVLS

[0095] Therefore, in one embodiment, the fusion protein comprises a signal sequence and a fusion protein, wherein the signal sequence is selected from the group consisting of SEQ ID NO: 77-79, and the fusion protein is selected from the group consisting of SEQ ID NO: 83-88 and 95-101. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 77 and a fusion protein selected from the group consisting of SEQ ID NO: 83-88 and 95-101. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 78 and a fusion protein selected from the group consisting of SEQ ID NO: 83-88 and 95-101. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 79 and a fusion protein selected from the group consisting of SEQ ID NO: 83-88 and 95-101. Cells expressing fusion protein constructs having the signal sequence can be administered to an individual, such as a human individual (e.g., a patient with tau disease or at risk of having tau disease). The fusion protein is secreted by the cell, which helps reduce tau protein aggregation and / or related cytotoxicity.

[0096] As described above, in some embodiments, the fusion protein may further comprise a cell-penetrating peptide. Cells exhibiting a fusion protein comprising both a signaling sequence and a cell-penetrating peptide will be able to secrete a fusion protein without the signaling sequence. The secreted fusion protein also comprises a cell-penetrating peptide, which will subsequently be able to enter neighboring cells and has the potential to reduce tau protein-mediated aggregation and / or cytotoxicity in such cells.

[0097] VI. Method of Use In another embodiment, the present invention provides a method for achieving beneficial effects in diseases and / or tau diseases, diseases or symptoms mediated by tau aggregation. Tau diseases are selected from the group consisting of: amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, and dementia with Lewy's bodies.

[0098] In some embodiments, the present invention provides a method for treating an individual (such as a human) suffering from tau disease, condition, or symptom, comprising the step of administering to the individual a therapeutically or preventively effective amount of a fusion protein, a nucleic acid encoding such a fusion protein, or a viral vector encoding such a fusion protein described herein, wherein the administration causes an improvement in one or more biochemical or physiological parameters or clinical efficacy indicators associated with tau disease, condition, or symptom.

[0099] In other embodiments, the present invention provides a method for reducing tau accumulation in cells. The cells may be cultured or isolated cells. The cells may also be derived from an individual, such as a human individual. In one embodiment, the cells are located in the central nervous system of a human individual. In another embodiment, the human individual has or is at risk of having tau disease, including but not limited to amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. In a particular embodiment, tau disease refers to Alzheimer's disease.

[0100] Pathogenic tau protein can be detected in a variety of ways. In one example, superphosphorylated tau protein can be distinguished from non-pathogenic (i.e., functional) tau, for example, by using antibodies against phosphorylated tau or conformation-specific tau antibodies. A greater reduction in pathogenic tau protein compared to a control indicates higher efficacy. The reduction in pathogenic tau protein can also be detected directly in cells, for example using immunofluorescence microscopy with labeled reagents to detect tau protein (see, for example, Ding et al., (2015) Oncotarget, 6: 24178-24191; Chou et al., (2015) Hum. Mol. Genet. 24: 5154-5173, and Example 1). In some embodiments, a greater reduction in tau peptide levels compared to a control indicates higher efficacy.

[0101] Therefore, in one embodiment, the method includes contacting cells with an amount of fusion protein or nucleic acid, vector or viral particle encoding the fusion protein, compared with untreated or control cells, such amount can effectively reduce pathogenic tau protein by at least 10%, for example at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0102] As shown in Example 1 below, it has been found that the expression of fusion proteins containing both the J domain and the tau-binding domain reduces the total amount of tau-containing reporter constructs. Accordingly, in another embodiment, the method includes contacting cells with a certain amount of the fusion protein, cells expressing the fusion protein, nucleic acid encoding the fusion protein, a vector, or viral particles, compared to untreated or control cells, such that the amount effectively reduces the amount of tau protein by at least 10%, for example, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0103] VII. Pharmaceutical Compositions The compositions covered herein may comprise one or more fusion proteins containing a J domain and a tau-binding domain, polynucleotides encoding such fusion proteins, vectors containing a J domain and a tau-binding domain, genetically modified cells, etc., as covered herein. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" means a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals, alone or in combination with one or more other therapeutic modalities. It will also be understood that, where necessary, the composition may be administered in combination with other pharmaceutical agents, such as interleukins, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There are virtually no restrictions on other components that may be included in the composition, provided that the additional pharmaceutical agents do not adversely affect the composition's ability to deliver the desired therapy.

[0104] The phrase “medically acceptable” is used in this document to refer to compounds, substances, compositions and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0105] As used herein, "medically acceptable carrier," "diluent," or "excipient" includes (but is not limited to) any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, humectant, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the United States Food and Drug Administration for acceptable use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragali; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin wax, polysiloxane, bentonite, silica, and zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic physiological saline; Ringer's solution; ethanol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations.

[0106] VIII. Dosage The dosage of the compositions described herein (e.g., compositions comprising fusion protein constructs, nucleic acid or gene therapy viral particles) may vary depending on many factors, such as the pharmacokinetic properties of the compound; the mode of administration; the recipient's age, health status and weight; the nature and severity of symptoms; the frequency of treatment and the type of concurrent treatment (if present); and the clearance of the compound in the animal being treated. The compositions described herein may be initially administered at an appropriate dose that can be adjusted as needed, depending on the clinical response. In some cases, the dosage of the composition is a preventative or therapeutically effective amount.

[0107] IX. A kit encompasses a kit comprising (a) a pharmaceutical composition including a fusion protein construct, a nucleic acid encoding such a fusion protein, or a viral particle encompassing such a nucleic acid, the pharmaceutical composition reducing the aggregation of tau protein in the cells or individuals described herein, and (b) a product information label with instructions for performing any of the methods described herein. In some embodiments, the kit comprises: (a) a pharmaceutical composition including the composition described herein that reduces the aggregation of tau protein in the cells or individuals described herein; (b) an additional therapeutic agent; and (c) a product information label with instructions for performing any of the methods described herein.

[0108] Example: In order to test whether the J domain can be specifically engineered to promote proper folding of aggregated proteins, we designed and tested multiple fusion protein constructs designed to target tau proteins.

[0109] Example 1: Fusion Protein Design A. Methods General Techniques and Materials Unless otherwise specified, the practice of this invention employs known techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of this art. See Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3rd ed., Cold Spring Harbor Laboratory Press, 2001; "Current protocols in molecular biology," edited by FM Ausubel et al., 1987; "Methods in Enzymology" series, Academic Press, San Diego, Calif.; "PCR 2: a practical approach," edited by MJ MacPherson, BD Hames, and GR Taylor, Oxford University Press, 1995; "Antibodies, a laboratory manual," edited by Harlow, E., and Lane, D, Cold Spring Harbor Laboratory, 1988; "Goodman & Gilman's The Pharmacological Basis of Therapeutics," 11th ed., McGraw-Hill, 2005; and Freshney, RI, "Culture of Animal Cells: A Manual of Basic Technique," 4th ed., John Wiley & Sons, Somerset, NJ, 2000. The contents of this article are incorporated herein by reference in their entirety. HEK-293 cells (human embryonic kidney cells) were purchased from the American Type Culture Collection (Manassas, VA). The anti-FLAG antigen system was purchased from Thermo Fisher Scientific. For ease of purification, detection, and / or characterization, some of the fusion protein constructs used in this Example 1, in addition to the sequences provided in SEQ ID NO: 83-101, also contain the FLAG antigenic determinant of SEQ ID NO: 67 at the C-terminus or N-terminus of the protein, as well as a short linker sequence.Expression and detection of proteins in HEK293 cells.

[0110] Expression vectors encoding different protein constructs were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Immunomodulation analysis was used to analyze the expression proteins in cell lysates. Prior to analysis, the culture medium samples were centrifuged to remove residue. Cells were lysed in lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 2% SDS) containing 2 mM PMSF and a protease mixture (Complete Protease Inhibitor Mixture; Sigma). After a brief sonication treatment, immunodulation analysis was used to analyze the expression proteins in the samples. For immunodulation analysis, samples were boiled in SDS-sample buffer and run on polyacrylamide electrophoresis. Subsequently, the separated protein bands were transferred to a PVDF membrane.

[0111] Detection of expressed proteins using chemiluminescence signals. In short, ink dots are reacted with primary antibodies (e.g., anti-tau antibodies) capable of binding specific antigenic determinants. After washing away unreacted primary antibodies, secondary, enzyme-linked antibodies (e.g., HRP-linked anti-IgG antibodies) are reacted with the primary antibody molecules bound to the ink dots. After washing, a chemiluminescent reagent is added, and the resulting chemiluminescent signal in the ink dots is acquired on an X-ray film. The following antibodies were used: ● Anti-Tau antibody (MilliporeSigma, Burlington, MA), ● Anti-V5 antibody (Thermo Fisher Scientific), ● Anti-Flag antibody (Thermo Fisher Scientific), ● Anti-pTau (Ser396) antibody (BioLegend, San Diego, CA), ● Anti-pTau (Thr231) antibody (BioLegend, San Diego, CA). Reporter constructs

[0112] We first investigated whether fusion proteins targeting tau improve tau aggregation in cultured cells. To this end, we generated two constructs (see Table 8 below) of wild-type tau (ON4R) and a mutant known to induce hyperphosphorylation and aggregation (containing a P243S substitution), with tau fused to the V5 antigenic determinant at its N-terminus. HEK293 cells were co-cultured and transfected with plastids encoding wild-type (SEQ ID NO: 80) or mutant (SEQ ID NO: 81) tau. Table 8: Reported Tau constructs construction SEQ ID NO: Length sequence V5-Tau(0N4R) 80 404 MGKPIPNPLLGLDSTGTGSEFMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAKTPPPSGEPPKSGDRSGYSSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVMPDPLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNHIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSTGSIDMVDSPQLATLADEVSASLAKQGL V5-Tau(0N4R:P243S) 81 404 MGKPIPNPLLGLDSTGTGSEFMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAKTPPPSGEPPKSGDRSGYSSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVMPDPLDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVSGGGSVQIVYKPVDLSKVTSKCGSLGNHIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSTGSIDMVDSPQLATLADEVSASLAKQGL Rope3R 82 241 MIATPRGAAPPGQKGQANATRIPAKTPPAKTPPPSSGEPPKSGDRSGYSSPGTPGSRSRTPSLPTPTPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNHIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSTGSIDMVD

[0113] Several fusion protein constructs were designed, comprising the J domain (SEQ ID NO: 5) from human DnaJB1, as provided in Table 6 and summarized in Table 9 below. The transfected constructs used in these experiments contained a Flag antigenic determinant fusion at their C-terminus to facilitate detection. Controls for these fusion protein constructs included a construct containing the J domain with a P33Q mutation in a highly conserved HPD motif. Other control constructs were also designed, including a construct containing only the human DnaJB1 J domain (SEQ ID NO: 5) without any tau-binding domain (constructor 13) (also containing the Flag antigenic determinant), a fusion protein construct containing the DnaJB1 J domain fused to the polyglutamic acid-binding peptide QBP1 on either side (constructor 11), and its corresponding control construct (constructor 12) with a P33Q mutation in the J domain. Table 9. Fusion protein constructs and controls Structure Number Structure Name SEQ ID NO: Notes 1 JB1-TBP 83 The J domain of human DnaJB1 is fused to TBP at its C-terminus (SEQ ID NO: 49) 2 TBP1-JB1-TBP1 84 It is derived from the J domain of human DnaJB1, which is sandwiched between two TBP (SEQ ID NO: 49) peptides on either side. 3 JB1-2xTBP 85 The J domain of human DnaJB1 is fused to its C-terminus to a tandem TBP (SEQ ID NO: 49) peptide. 4 JB1-scFv(Tau) 86 The J domain of human DnaJB1 is fused to scFv(Tau) at its C-terminus (SEQ ID NO: 50). 5 JB1-scFv(MW7) 87 The J domain from human DnaJB1 was fused to scFv(MW7) at its C-terminus (SEQ ID NO: 51). 6 JB1-Happ1 88 The J domain of human DnaJB1 is fused to Happ1 (SEQ ID NO:52) at its C-terminus. 7 JB1(P33Q)-TBP 89 Derived from the J domain of human DnaJB1, it contains the P33Q mutation and is fused to TBP at its C-terminus (SEQ ID NO: 49). 8 JB1(P33Q)-scFv(Tau) 90 Derived from the J domain of human DNAJB1, it contains the P33Q mutation and is fused to scFv(Tau) (SEQ ID NO:50) at its C-terminus. 9 JB1(P33Q)-scFv(MW7) 91 Derived from the J domain of human DNAJB1, it contains the P33Q mutation and is fused to scFv(MW7) (SEQ ID NO:51) at its C-terminus. 10 JB1(P33Q)-Happ1 92 Derived from the J domain of human DnaJB1, it contains the P33Q mutation and is fused to Happ1 (SEQ ID NO:52) at its C-terminus. 11 QBP1-JB1-QBP1 93 The J domain of human DnaJB1 is fused to QBP1 (polyglutamic acid-binding peptide) at its C-terminus. 12 QBP1-JB1(P33Q)-QBP1 94 Derived from the J domain of human DnaJB1, it contains a P33Q mutation and is fused to QBP1 (polyglutamic acid-binding peptide) at its C-terminus. 13 JB1 only 5 The comparison structure contains JB1 but has no Tau bonding domain. 14 JB1-TBP2 95 The J domain of human DnaJB1 was fused to TBP2 at its C-terminus (SEQ ID NO: 53). 15 JB1-TBP3 96 The J domain of human DnaJB1 was fused to TBP3 at its C-terminus (SEQ ID NO: 54). 16 JB1-TBP (without connectors) 97 Similar to Construct 1, but without a connecting subsequence between DnaJB1 and TBP. 17 JB1-TBP2 (without connectors) 98 Similar to construct 14, but without connecting subsequences between DnaJB1 and TBP2. 18 JB1-TBP3 (without connectors) 99 Similar to construct 15, but without a connecting subsequence between DnaJB1 and TBP. 19 JB1-scFv(Tau) (No connectors) 100 Similar to Construct 4, but without a connecting subsequence between DnaJB1 and TBP. 20 JB1-Happ1 (No connectors) 101 Similar to Construct 6, but without a connecting subsequence between DnaJB1 and TBP.

[0114] Several of these constructs, along with constructs expressing wild-type or mutant Tau, were co-transfected into HEK293 cells. The expression of constructs 1, 4, 5, and 6 was detected by immunoblotting analysis (data not shown).

[0115] To determine whether the J domain can be used to reduce tau accumulation, initial experiments were conducted by co-expressing wild-type or mutant tau with a fusion protein containing a J domain sequence derived from an Hsp40 J domain protein (from human DnaJB1). This J domain sequence binds to the tau-binding peptide TBP (archive 1), scFv(Tau) (archive 4), scFv(MW7) (archive 5), and Happ1 (archive 6). As shown in Figure 3, expression of wild-type (Figure 3, top panel, lanes 2-6) or mutant (P243S) tau (lanes 7-11) in HEK293 cells resulted in the presence of detectable tau, which can be detected by the anti-V5 antibody. We further tested whether co-expression of one of the fusion protein architectures had an effect on altering the amount of phosphorylated tau. We therefore detect the amount of Tau phosphorylated at Thr231 or Ser396 by Tau 2N4R, as hyperphosphorylation at these sites is associated with lesions (Bramblett et al., (1993) Neuron 10:1089-99; Alonso et al., (2004) J Biol Chem 279:34873-81; Lin et al., (2007) J Neurochem 103:802-13; Alonso et al., (2010) J Biol Chem 285:30851-30860). When detected by an antibody that recognizes phosphorylated Tau at Ser396 (2N4R) (Figure 3, bottom), pTau (Ser396) was detected at approximately 63 kDa in wild-type or P243S Tau alone (lanes 2 and 7, respectively). In contrast, the amount of pTau (Ser396) detectable in the extracts of cells from constructs 1, 4, 5 and 6 (lanes 3, 4 and 5 representing wild-type Tau cells, and lanes 9, 10 and 11 representing P243S Tau cells, respectively) was significantly reduced.

[0116] The upper part of Figure 4 shows that co-expression of construct 4 in cells expressing wild-type (ON4R) or mutant Tau significantly reduced pTau (Thr231 in 2N4R) and pTau (Ser396 in 2N4R). The lower part shows the detection of construct 4 using anti-Flag antibody.

[0117] Finally, we tested the specificity of JB1-ScFv(Tau) in reducing pTau(Ser396). We therefore tested the ability of several constructs to reduce the amount of detectable pTau(Ser396), as shown in Figure 5: ● Construct 8 (lanes 5 and 9), identical to construct 4, but with a P33Q mutation in a highly conserved HPD motif within the J domain; ● scFv(Tau)-only control (construct 4 without the J domain sequence; lanes 3 and 7)

[0118] As shown in Figure 5, the expression of wild-type Tau (lanes 2 to 5) or mutant (P243S) Tau (lanes 6 to 9) resulted in the presence of detectable Tau (top) and pTau(Ser396) (middle). In cells co-expressing construct 4 (lanes 4 and 8), the amount of pTau(Ser396) was significantly reduced, but not significantly reduced in cells expressing construct 8 (lanes 5 and 9) or in cells expressing only ScFv(Tau) (lanes 3 and 7). Therefore, we infer that the reduction of pTau(Ser396) mediated by fusion proteins (such as construct 4) requires functional J domain activity.

[0119] As shown in Figure 6, the expression of wild-type Tau (lanes 2 to 6) or mutant (P243S) Tau (lanes 7 to 11) resulted in the presence of detectable Tau (top) and pTau(Ser396) (bottom). The amount of pTau(Ser396) was significantly reduced in cells that co-expressed construct 6 (lanes 3 and 8), construct 1 (lanes 4 and 9), and construct 14 (lanes 5 and 10), indicating that binding sequences such as TBP2 (SEQ ID NO: 53) are suitable for the fusion construct.

[0120] We further tested whether the co-expression of one of the fusion protein constructs had an effect on altering the amount of the truncated form of Tau (Tau3R, SEQ ID NO: 80). In HEK293 cells, a J-domain fusion protein (constructor 1) with the tau-binding peptide TBP was expressed along with full-length Tau or the truncated form of Tau (Tau3R). When detected with an antibody recognizing Tau, approximately 60 kDa of full-length Tau was detected, along with approximately 30 kDa of Tau3R (Fig. 7). Constructor 1 significantly reduced the protein content of Tau3R, but the J-domain mutation (constructor 7) eliminated this effect.

[0121] Figure 8 shows the role of construct 1 in U87-MG glioma cells. U87-MG glioma cells were infected with lentivirus to express full-length Tau, with or without construct 1 or construct 7. The culture medium was replaced with fresh medium on day 2. Culture medium was collected from U87-MG cells 7 days after infection. Lactate dehydrogenase (LDH) activity in the culture medium was measured using the LDH-Cytox™ Assay Kit (BioLegend). Values ​​are expressed as mean ± SD. These results demonstrate that the J-domain fusion protein not only reduces protein misfolding and / or aggregation but also reduces cytotoxicity associated with protein misfolding and / or aggregation.

[0122] Example 2: An exemplary gene therapy vector system using an AAV vector encoding fusion protein constructs was constructed using the AAV rh10 vector under the control of the CAG promoter. It contained codon-optimized cDNA encoding the fusion protein constructs listed in Table 6, particularly constructs 1, 4, 5, and 6, as well as control construct 13 (DnaJB1 J domain only) and GFP (negative control). The CAG promoter contained a cytomegalovirus (CMV) early enhancer element and a chicken β-actin promoter. The cDNA encoding JB1-TBP was positioned downstream of the Kozak sequence and upstream of the bovine growth hormone polyadenylation (BGHpA) signal. The entire cartridge was side-mounted by the two non-coding reverse sequences of AAV-2.

[0123] Recombinant AAV vectors were prepared using a baculovirus expression system similar to that described above (Urabe et al., 2002; Unzu et al., 2011 (reviewed in Kotin, 2011)). In short, three recombinant baculoviruses were used: one encoding REP for replication and packaging, one encoding CAP-5 for the AAV rh10 capsid, and one with an expression cartridge for infecting SF9 insect cells. Purification was performed using AVB Sepharose high-affinity medium (GE Healthcare Life Sciences, Piscataway, NJ). The vectors were titrated using qPCR with the primer-probe combination of the transgenic gene, and the titer was expressed as the number of gene copies per ml (GC / ml). The vector titers ranged from approximately 8 × 10¹³ to 2 × 10¹⁴ GC / ml.

[0124] Example 3: Efficacy testing in mouse models. There are many animal models of Alzheimer's disease (see, for example, Kitazawa et al. (2012) Curr. Pharm. Des., 18:1131-1147). In this example, a human tau (htau) mouse model of tau protein disease was used (Duff et al., (2000) Neurobiol Dis. 7:87-98). Htau mice derive from human tau in PAC, the H1 haplotype, and are called 8c mice, while mouse tau is knocked out by targeted destruction of exon 1 (Duff et al., ibid.). Mice were bred on a C57BL / 6 background.

[0125] A preventative study was conducted on mice aged 2.5 to 6.5 months. Different AAV viral particles containing a vector encoding a fusion protein and corresponding controls were administered to the transgenic animals. In one embodiment, the viral particles were administered via intravenous injection in the tail vein. In another embodiment, the viral particles were administered via intramuscular injection. In yet another embodiment, the particles were administered via intracranial injection, as described, for example, by Stanek et al., (2014) Hum. Gene. Ther. 25:461-474. Approximately 36 mice were divided into three mixed groups of males and females and administered AAV rh10 containing cDNA encoding construct 4, a vector control, and construct 8 with a P33Q mutation in a conserved HPD motif in the J domain.

[0126] After administration, disease progression was monitored and compared with mice injected with the control. The primary efficacy endpoint was a statistically significant reduction in insoluble tau aggregates in the mouse brain. Secondary efficacy endpoints were a dose-dependent reduction in insoluble tau aggregates, a reduction in phosphorylated tau, and a reduction in soluble tau.

[0127] All publications, patents, and patent applications mentioned in the other sample specification are incorporated herein by reference in their entirety, to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference. Where a term in this application is found to have a different definition in a document incorporated herein by reference, the definition provided herein shall serve as the definition of that term.

[0128] Although the present invention has been described in conjunction with its specific features, it should be understood that the present invention is capable of further modifications, and this application is intended to cover any variations, uses or adaptations of the present invention that generally follow the principles of the present invention and include any deviations from the present invention that occur within the scope of known or customary practice in relation to the present invention, and that can be applied to the basic features set forth above and fall within the scope of the claims. [Simplified Explanation of the Diagram]

[0017] Figure 1A shows the Clustal Omega sequence alignment of a representative human J domain sequence. The highly conserved HPD domain is shown in the highlighted box. Figure 1B shows the Clustal Omega sequence alignment of a representative human J domain sequence. Figure 2 shows some illustrative fusion protein constructs containing the J domain and the tau binding domain. Figure 3 shows Western ink dot analysis of cell extracts showing wild-type Tau ON4R (lanes 2-6) and mutant (P243S) Tau (lanes 7-11) containing the V5 antigenic determinant, and co-expressing multiple fusion protein constructs: JB1-TBP (constructor 1), lanes 3 and 8; JB1-ScFv(Tau) (constructor 4), lanes 4 and 9; JB1-ScFv(MW7) (constructor 5), lanes 5 and 10; and JB1-Happ1 (constructor 6), lanes 6 and 11. The top figure shows the amount of Tau protein detected using an anti-V5 antibody, while the bottom figure shows the amount of phosphorylated Tau detected using an anti-pTau(Ser396) antibody. Figure 4 shows Western ink dot analysis of extracts from cells expressing wild-type Tau ON4R (lanes 2, 3, 7, and 8) or mutant (P243S) Tau (lanes 4, 5, 9, and 10), alone or in combination with JB1-ScFv(Tau) containing the Flag antigenic determinant (lanes 3, 5, 8, and 10). The top figure shows detection using antibodies that identify phosphorylated Tau at Thr231 (lanes 1-5) or phosphorylated Tau at Ser396 (lanes 6-10) in Tau ON4R. The bottom figure shows detection using an anti-Flag antibody to detect J-domain fusion proteins (in this case, the JB1-ScFv(Tau) construct, or construct 4). Figure 5 shows the immunoblotting analysis of cell extracts, which showed wild-type Tau 0N4R (lanes 2-5) or mutant (P243S) Tau (lanes 6-9), alone (lanes 1 and 6), or co-presented construct 4 [JB1-ScFv(Tau), lanes 4 and 8], construct 8 [the same control construct as JB1-ScFv(Tau) except for the P33Q mutation in the conserved HPD motif within the J domain, lanes 5 and 9], and ScFv(Tau) alone [construct 4 without the J domain, lanes 3 and 7]. Figure 6 shows the immunoblotting analysis of cell extracts, which showed wild-type (lanes 2-6) or mutant (P243S) Tau (lanes 7-11), individually (lanes 2 and 7) or co-presented construct 6 (JB1-Happ1, lanes 3 and 8), construct 1 (JB1-TBP1, lanes 4 and 9), construct 14 (JB1-TBP2, lanes 5 and 10) and construct 11 (JB1-QBP1, lanes 6 and 11).Figure 7 shows the immunoblotting analysis of cell extracts representing wild-type (lanes 1-3) or a truncated form of Tau (Tau3R, lanes 4-6), alone (lanes 1 and 4) or co-representing construct 1 (JB1-TBP1, lanes 2 and 5), or construct 7 (JB1-TBP1 containing the P33Q mutation in the conserved HPD motif within the J domain, lanes 3 and 6). Figure 8 shows the effect of construct 1 (JB1-TBP1) on Tau-mediated cytotoxicity as measured using LDH analysis. [Sequence List]

[0129] <![CDATA[ <110> SOLA BIOSCIENCES LLC (USA) <![CDATA[ <120> Compositions and methods for treating Alzheimer's disease <![CDATA[ <130> 269548-479789]]> <![CDATA[ <140> TW110139655]]> <![CDATA[ <141> 2021-10-26 <![CDATA[ <150> US 63 / 105,472 <![CDATA[ <151> 2020-10-26 <![CDATA[ <160> 101 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 63]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <300> ]]> <![CDATA[ <308> NCBI / NP_001530]]> <![CDATA[ <309> 2021-08-16 <![CDATA[ <313> (6)..(68)]]> <![CDATA[ <400> 1]]> Thr Tyr Tyr Asp Val Leu Gly Val Lys Pro Asn Ala Thr Gln Glu Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Tyr His Pro Asp Lys 20 25 30 Asn Pro Asn Glu Gly Glu Lys Phe Lys Gln Ile Ser Gln Ala Tyr Glu 35 40 45 Val Leu Ser Asp Ala Lys Lys Arg Glu Leu Tyr Asp Lys Gly Gly 50 55 60 <![CDATA[ <210> 2]]> <![CDATA[ <211> 63]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <300> ]]> <![CDATA[ <308> NCBI / NP_005871 <![CDATA[ <309> 2021-06-26 <![CDATA[ <313>(8)..(70)]]> <![CDATA[<400> 2]]> Lys Leu Tyr Asp Ile Leu Gly Val Pro Pro Gly Ala Ser Glu Asn Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Lys Leu Ala Lys Glu Tyr His Pro Asp Lys 20 25 30 Asn Pro Asn Ala Gly Asp Lys Phe Lys Glu Ile Ser Phe Ala Tyr Glu 35 40 45 Val Leu Ser Asn Pro Glu Lys Arg Glu Leu Tyr Asp Arg Tyr Gly 50 55 60 <![CDATA[<210> 3]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001128582]]> <![CDATA[<309> 2021-06-27]]> <![CDATA[<313> (93)..(158)]]> <![CDATA[<400> 3]]> Asp Tyr Tyr Gln Ile Leu Gly Val Pro Arg Asn Ala Ser Gln Lys Glu 1 5 10 15 Ile Lys Lys Ala Tyr Tyr Gln Leu Ala Lys Lys Tyr His Pro Asp Thr 20 25 30 Asn Lys Asp Asp Pro Lys Ala Lys Glu Lys Phe Ser Gln Leu Ala Glu 35 40 45 Ala Tyr Glu Val Leu Ser Asp Glu Val Lys Arg Lys Gln Tyr Asp Ala 50 55 60 Tyr Gly 65 <![CDATA[<210> 4]]> <![CDATA[<211> 67]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001123654]]> <![CDATA[<309>2021-07-11]]> <![CDATA[<313> (4)..(70)]]> <![CDATA[<400> 4]]> Glu Thr Gln Tyr Tyr Asp Ile Leu Gly Val Lys Pro Ser Ala Ser Pro 1 5 10 15 Glu Glu Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Tyr His Pro 20 25 30 Asp Lys Asn Pro Asp Glu Gly Glu Lys Phe Lys Leu Ile Ser Gln Ala 35 40 45 Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg Asp Val Tyr Asp Gln Gly 50 55 60 Gly Glu Gln 65 <![CDATA[<210> 5]]> <![CDATA[<211> 69]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / 6Z5N_A]]> <![CDATA[<309> 2020-12-<![CDATA[<308> NCBI / NP_001034639]]> <![CDATA[<309> 2021-06-28]]> <![CDATA[<313> (2)..(71)]]> <![CDATA[<400> 6]]> Ala Ser Tyr Tyr Glu Ile Leu Asp Val Pro Arg Ser Ala Ser Ala Asp 1 5 10 15 Asp Ile Lys Lys Ala Tyr Arg Arg Lys Ala Leu Gln Trp His Pro Asp 20 25 30 Lys Asn Pro Asp Asn Lys Glu Phe Ala Glu Lys Lys Phe Lys Glu Val 35 40 45 Ala Glu Ala Tyr Glu Val Leu Ser Asp Lys His Lys Arg Glu Ile Tyr 50 55 60 Asp Arg Tyr Gly Arg Glu 65 70 <![CDATA[<210> 7]]> <![CDATA[<211> 69]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> Uniprot / Q8WWF6]]> <![CDATA[<309> 2021-09-04]]> <![CDATA[<313> (1)..(69)]]> <![CDATA[<400> 7]]> Met Val Asp Tyr Tyr Glu Val Leu Asp Val Pro Arg Gln Ala Ser Ser 1 5 10 15 Glu Ala Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Trp His Pro 20 25 30 Asp Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Arg Phe Lys Gln 35 40 45 Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Ala Lys Lys Arg Asp Ile 50 55 60 Tyr Asp Arg Tyr Gly 65 <![CDATA[<210> 8]]> <![CDATA[<211>69]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001304028]]> <![CDATA[<309> 2021-07-02]]> <![CDATA[<313> (2)..(70)]]> <![CDATA[<400> 8]]> Gly Lys Asp Tyr Tyr Cys Ile Leu Gly Ile Glu Lys Gly Ala Ser Asp 1 5 10 15 Glu Asp Ile Lys Lys Ala Tyr Arg Lys Gln Ala Leu Lys Phe His Pro 20 25 30 Asp Lys Asn Lys Ser Pro Gln Ala Glu Glu Lys Phe Lys Glu Val Ala 35 40 45 Glu Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg Glu Ile Tyr Asp 50 55 60 Gln Phe Gly Glu Glu 65 <![CDATA[<210> 9]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001128476]]> <![CDATA[<309> 2021-07-11]]> <![CDATA[<313> (38)..(102)]]> <![CDATA[<400> 9]]> Asp Tyr Tyr Lys Ile Leu Gly Ile Pro Ser Gly Ala Asn Glu Asp Glu 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Met Ala Leu Lys Tyr His Pro Asp Lys 20 25 30 Asn Lys Glu Pro Asn Ala Glu Glu Lys Phe Lys Glu Ile Ala Glu Ala 35 40 45 Tyr Asp Val Leu Ser Asp Pro Lys Lys Arg Gly Leu Tyr Asp Gln Tyr 5055 60 Gly 65 <![CDATA[<210> 10]]> <![CDATA[<211> 68]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_005485]]> <![CDATA[<309> 2021-06-28]]> <![CDATA[<313> (2)..(69)]]> <![CDATA[<400> 10]]> Val Asp Tyr Tyr Glu Val Leu Gly Val Gln Arg His Ala Ser Pro Glu 1 5 10 15 Asp Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Trp His Pro Asp 20 25 30 Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys Phe Lys Gln Val 35 40 45 Ala Glu Ala Tyr Glu Val Leu Ser Asp Ala Lys Lys Arg Asp Ile Tyr 50 55 60 Asp Lys Tyr Gly 65 <![CDATA[<210> 11]]> <![CDATA[<211> 67]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_660157]]> <![CDATA[<309> 2021-06-27]]> <![CDATA[<313> (3)..(69)]]> <![CDATA[<400> 11]]> Asp Tyr Tyr Glu Val Leu Gly Leu Gln Arg Tyr Ala Ser Pro Glu Asp 1 5 10 15 Ile Lys Lys Ala Tyr His Lys Val Ala Leu Lys Trp His Pro Asp Lys 20 25 30 Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys Phe Lys Glu Val Ala 35 40 45 Glu Ala Tyr Glu Val Leu SerAsn Asp Glu Lys Arg Asp Ile Tyr Asp 50 55 60 Lys Tyr Gly 65 <![CDATA[<210> 12]]> <![CDATA[<211> 67]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_699161]]> <![CDATA[<309> 2021-07-12]]> <![CDATA[<313> (3)..(69)]]> <![CDATA[<400> 12]]> Asn Tyr Tyr Glu Val Leu Gly Val Gln Ala Ser Ala Ser Pro Glu Asp 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Arg Trp His Pro Asp Lys 20 25 30 Asn Pro Asp Asn Lys Glu Glu Ala Glu Lys Lys Phe Lys Leu Val Ser 35 40 45 Glu Ala Tyr Glu Val Leu Ser Asp Ser Lys Lys Arg Ser Leu Tyr Asp 50 55 60 Arg Ala Gly 65 <![CDATA[<210> 13]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_036460]]> <![CDATA[<309> 2021-10-17]]> <![CDATA[<313> (26)..(90)]]> <![CDATA[<400> 13]]> Ser Tyr Tyr Asp Ile Leu Gly Val Pro Lys Ser Ala Ser Glu Arg Gln 1 5 10 15 Ile Lys Lys Ala Phe His Lys Leu Ala Met Lys Tyr His Pro Asp Lys 20 25 30 Asn Lys Ser Pro Asp Ala Glu Ala Lys Phe Arg Glu Ile AlaGlu Ala 35 40 45 Tyr Glu Thr Leu Ser Asp Ala Asn Arg Arg Lys Glu Tyr Asp Thr Leu 50 55 60 Gly 65 <![CDATA[<210> 14]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_057390]]> <![CDATA[<309> 2021-08-09]]> <![CDATA[<313> (26)..(91)]]> <![CDATA[<400> 14]]> Asp Phe Tyr Lys Ile Leu Gly Val Pro Arg Ser Ala Ser Ile Lys Asp 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Gln Leu His Pro Asp Arg 20 25 30 Asn Pro Asp Asp Pro Gln Ala Gln Glu Lys Phe Gln Asp Leu Gly Ala 35 40 45 Ala Tyr Glu Val Leu Ser Asp Ser Glu Lys Arg Lys Gln Tyr Asp Thr 50 55 60 Tyr Gly 65 <![CDATA[<210> 15]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001002762]]> <![CDATA[<309> 2021-07-02]]> <![CDATA[<313> (112)..(176)]]> <![CDATA[<400> 15]]> Tyr Glu Ile Leu Gly Val Ser Arg Gly Ala Ser Asp Glu Asp Leu Lys 1 5 10 15 Lys Ala Tyr Arg Arg Leu Ala Leu Lys Phe His Pro Asp Lys Asn His 20 25 30 Ala Pro Gly AlaThr Glu Ala Phe Lys Ala Ile Gly Thr Ala Tyr Ala 35 40 45 Val Leu Ser Asn Pro Glu Lys Arg Lys Gln Tyr Asp Gln Phe Gly Asp 50 55 60 Asp 65 <![CDATA[<210> 16]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_705842]]> <![CDATA[<309> 2021-01-25]]> <![CDATA[<313> (4)..(68)]]> <![CDATA[<400> 16]]> Asp Tyr Tyr Ser Val Leu Gly Ile Thr Arg Asn Ser Glu Asp Ala Gln 1 5 10 15 Ile Lys Gln Ala Tyr Arg Arg Leu Ala Leu Lys His His Pro Leu Lys 20 25 30 Ser Asn Glu Pro Ser Ser Ala Glu Ile Phe Arg Gln Ile Ala Glu Ala 35 40 45 Tyr Asp Val Leu Ser Asp Pro Met Lys Arg Gly Ile Tyr Asp Lys Phe 50 55 60 Gly 65 <![CDATA[<210> 17]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001026893]]> <![CDATA[<309> 2021-06-26]]> <![CDATA[<313> (108)..(172)]]> <![CDATA[<400> 17]]> Asn Tyr Tyr Glu Val Leu Gly Val Thr Lys Asp Ala Gly Asp Glu Asp 1Asp Lys 20 25 30 Asn His Ala Pro Gly Ala Thr Asp Ala Phe Lys Lys Ile Gly Asn Ala 35 40 45 Tyr Ala Val Leu Ser Asn Pro Glu Lys Arg Lys Gln Tyr Asp Leu Thr 50 55 60 Gly 65 <![CDATA[<210> 18]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_071760]]> <![CDATA[<309> 2020-11-19]]> <![CDATA[<313> (65)..(129)]]> <![CDATA[<400> 18]]> Asn Phe Tyr Gln Phe Leu Gly Val Gln Gln Asp Ala Ser Ser Ala Asp 1 5 10 15 Ile Arg Lys Ala Tyr Arg Lys Leu Ser Leu Thr Leu His Pro Asp Lys 20 25 30 Asn Lys Asp Glu Asn Ala Glu Thr Gln Phe Arg Gln Leu Val Ala Ile 35 40 45 Tyr Glu Val Leu Lys Asp Asp Glu Arg Arg Gln Arg Tyr Asp Asp Ile 50 55 60 Leu 65 <![CDATA[<210> 19]]> <![CDATA[<211> 74]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001123359]]> <![CDATA[<309> 2021-02-21]]> <![CDATA[<313> (88)..(161)]]> <![CDATA[<400> 19]]> Asp His Tyr Ala Val Leu Gly Leu Gly His Val Arg Tyr Lys Ala Thr 1 5 10 15 Gln Arg Gln Ile LysAla Ala His Lys Ala Met Val Leu Lys His His 20 25 30 Pro Asp Lys Arg Lys Ala Ala Gly Glu Pro Ile Lys Glu Gly Asp Asn 35 40 45 Asp Tyr Phe Thr Cys Ile Thr Lys Ala Tyr Glu Met Leu Ser Asp Pro 50 55 60 Val Lys Arg Arg Ala Phe Asn Ser Val Asp 65 70 <![CDATA[<210> 20]]> <![CDATA[<211> 69]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_006251]]> <![CDATA[<309> 2021-06-26]]> <![CDATA[<313> (394)..(462)]]> <![CDATA[<400> 20]]> Asp Tyr Tyr Lys Ile Leu Gly Val Lys Arg Asn Ala Lys Lys Gln Glu 1 5 10 15 Ile Ile Lys Ala Tyr Arg Lys Leu Ala Leu Gln Trp His Pro Asp Asn 20 25 30 Phe Gln Asn Glu Glu Glu Lys Lys Lys Ala Glu Lys Lys Phe Ile Asp 35 40 45 Ile Ala Ala Ala Lys Glu Val Leu Ser Asp Pro Glu Met Arg Lys Lys 50 55 60 Phe Asp Asp Gly Glu 65 <![CDATA[<210> 21]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001294909]]> <![CDATA[<309> 2021-06-23]]> <![CDATA[<313> (33)..(98)]]> <![CDATA[<400> 21]]> ThrTyr Tyr Glu Leu Leu Gly Val His Pro Gly Ala Ser Thr Glu Glu 1 5 10 15 Val Lys Arg Ala Phe Phe Ser Lys Ser Lys Glu Leu His Pro Asp Arg 20 25 30 Asp Pro Gly Asn Pro Ser Leu His Ser Arg Phe Val Glu Leu Ser Glu 35 40 45 Ala Tyr Arg Val Leu Ser Arg Glu Gln Ser Arg Arg Ser Tyr Asp Asp 50 55 60 Gln Leu 65 <![CDATA[<210> 22]]> <![CDATA[<211> 70]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_079495]]> <![CDATA[<309> 2021-10-03]]> <![CDATA[<313> (13)..(82)]]> <![CDATA[<400> 22]]> Gly Glu Ser Leu Tyr His Val Leu Gly Leu Asp Lys Asn Ala Thr Ser 1 5 10 15 Asp Asp Ile Lys Lys Ser Tyr Arg Lys Leu Ala Leu Lys Tyr His Pro 20 25 30 Asp Lys Asn Pro Asp Asn Pro Glu Ala Ala Asp Lys Phe Lys Glu Ile 35 40 45 Asn Asn Ala His Ala Ile Leu Thr Asp Ala Thr Lys Arg Asn Ile Tyr 50 55 60 Asp Lys Tyr Gly Ser Leu 65 70 <![CDATA[<210> 23]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_149096]]> <![CDATA[<309>February 17, 2021]]> <![CDATA[<313> (19)..(84)]]> <![CDATA[<400> 23]]> Ala Leu Tyr Glu Ile Leu Gly Leu His Lys Gly Ala Ser Asn Glu Glu 1 5 10 15 Ile Lys Lys Thr Tyr Arg Lys Leu Ala Leu Lys His His Pro Asp Lys 20 25 30 Asn Pro Asp Asp Pro Ala Ala Thr Glu Lys Phe Lys Glu Ile Asn Asn 35 40 45 Ala His Ala Ile Leu Thr Asp Ile Ser Lys Arg Ser Ile Tyr Asp Lys 50 55 60 Tyr Gly 65 <![CDATA[<210> 24]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001243793]]> <![CDATA[<309> May 9, 2021]]> <![CDATA[<313> (906)..(970)]]> <![CDATA[<400> 24]]> Thr Lys Trp Lys Pro Val Gly Met Ala Asp Leu Val Thr Pro Glu Gln 1 5 10 15 Val Lys Lys Val Tyr Arg Lys Ala Val Leu Val Val His Pro Asp Lys 20 25 30 Ala Thr Gly Gln Pro Tyr Glu Gln Tyr Ala Lys Met Ile Phe Met Glu 35 40 45 Leu Asn Asp Ala Trp Ser Glu Phe Glu Asn Gln Gly Gln Lys Pro Leu 50 55 60 Tyr 65 <![CDATA[<210> 25]]> <![CDATA[<211> 71]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]><![CDATA[<308> NCBI / NP_001138238]]> <![CDATA[<309> 2021-02-22]]> <![CDATA[<313> (325)..(395)]]> <![CDATA[<400> 25]]> Asp Tyr Tyr Lys Ile Leu Gly Val Asp Lys Asn Ala Ser Glu Asp Glu 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Arg Ala Leu Met His His Pro Asp Arg 20 25 30 His Ser Gly Ala Ser Ala Glu Val Gln Lys Glu Glu Glu Lys Lys Phe 35 40 45 Lys Glu Val Gly Glu Ala Phe Thr Ile Leu Ser Asp Pro Lys Lys Lys 50 55 60 Thr Arg Tyr Asp Ser Gly Gln 65 70 <![CDATA[<210> 26]]> <![CDATA[<211> 68]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_055095]]> <![CDATA[<309> 2021-06-26]]> <![CDATA[<313> (57)..(124)]]> <![CDATA[<400> 26]]> Asn Pro Phe Glu Val Leu Gln Ile Asp Pro Glu Val Thr Asp Glu Glu 1 5 10 15 Ile Lys Lys Arg Phe Arg Gln Leu Ser Ile Leu Val His Pro Asp Lys 20 25 30 Asn Gln Asp Asp Ala Asp Arg Ala Gln Lys Ala Phe Glu Ala Val Asp 35 40 45 Lys Ala Tyr Lys Leu Leu Leu Asp Gln Glu Gln Lys Lys Arg Ala Leu 50 55 60 Asp Val Ile Gln 65 <![CDATA[<210> 27]]><![CDATA[<211> 68]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_056005]]> <![CDATA[<309> 2021-07-25]]> <![CDATA[<313> (15)..(82)]]> <![CDATA[<400> 27]]> Asp Leu Tyr Arg Val Leu Gly Val Arg Arg Glu Ala Ser Asp Gly Glu 1 5 10 15 Val Arg Arg Gly Tyr His Lys Val Ser Leu Gln Val His Pro Asp Arg 20 25 3o Val Gly Glu Gly Asp Lys Glu Asp Ala Thr Arg Arg Phe Gln Ile Leu 35 40 45 Gly Lys Val Tyr Ser Val Leu Ser Asp Arg Glu Gln Arg Ala Val Tyr 50 55 60 Asp Glu Gln Gly 65 <![CDATA[<210> 28]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001258510]]> <![CDATA[<309> 2021-08-09]]> <![CDATA[<313> (35)..(100)]]> <![CDATA[<400> 28]]> Asp Phe Tyr Ser Leu Leu Gly Val Ser Lys Thr Ala Ser Ser Arg Glu 1 5 10 15 Ile Arg Gln Ala Phe Lys Lys Leu Ala Leu Lys Leu His Pro Asp Lys 20 25 30 Asn Pro Asn Asn Pro Asn Ala His Gly Asp Phe Leu Lys Ile Asn Arg 35 40 45 Ala Tyr Glu Val Leu Lys Asp Glu Asp Leu Arg Lys Lys TyrAsp Lys 50 55 60 Tyr Gly 65 <![CDATA[<210> 29]]> <![CDATA[<211> 69]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_060668]]> <![CDATA[<309> 2021-04-17]]> <![CDATA[<313> (14)..(82)]]> <![CDATA[<400> 29]]> Asp Tyr Tyr Ser Leu Leu Asn Val Arg Arg Glu Ala Ser Ser Glu Glu 1 5 10 15 Leu Lys Ala Ala Tyr Arg Arg Leu Cys Met Leu Tyr His Pro Asp Lys 20 25 30 His Arg Asp Pro Glu Leu Lys Ser Gln Ala Glu Arg Leu Phe Asn Leu 35 40 45 Val His Gln Ala Tyr Glu Val Leu Ser Asp Pro Gln Thr Arg Ala Ile 50 55 60 Tyr Asp Ile Tyr Gly 65 <![CDATA[<210> 30]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_068572]]> <![CDATA[<309> 2021-06-26]]> <![CDATA[<313> (14)..(79)]]> <![CDATA[<400> 30]]> Asp Tyr Tyr Thr Leu Leu Gly Cys Asp Glu Leu Ser Ser Val Glu Gln 1 5 10 15 Ile Leu Ala Glu Phe Lys Val Arg Ala Leu Glu Cys His Pro Asp Lys 20 25 30 His Pro Glu Asn Pro Lys Ala Val Glu Thr Phe Gln Lys Leu Gln Lys 35 40 45 AlaLys Glu Ile Leu Thr Asn Glu Glu Ser Arg Ala Arg Tyr Asp His 50 55 60 Trp Arg 65 <![CDATA[<210> 31]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_056083]]> <![CDATA[<309> 2021-04-16]]> <![CDATA[<313> (1301)..(1366)]]> <![CDATA[<400> 31]]> Asp Ala Tyr Glu Val Leu Asn Leu Pro Gln Gly Gln Gly Pro His Asp 1 5 10 15 Glu Ser Lys Ile Arg Lys Ala Tyr Phe Arg Leu Ala Gln Lys Tyr His 20 25 30 Pro Asp Lys Asn Pro Glu Gly Arg Asp Met Phe Glu Lys Val Asn Lys 35 40 45 Ala Tyr Glu Phe Leu Cys Thr Lys Ser Ala Lys Ile Val Asp Gly Pro 50 55 60 Asp Pro 65 <![CDATA[<210> 32]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_115740]]> <![CDATA[<309> 2021-07-12]]> <![CDATA[<313> (443)..(507)]]> <![CDATA[<400> 32]]> Asn Pro Phe His Val Leu Gly Val Glu Ala Thr Ala Ser Asp Val Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Gln Leu Ala Val Met Val His Pro Asp Lys 20 25 30 Asn His His Pro Arg Ala Glu GluAla Phe Lys Val Leu Arg Ala Ala 35 40 45 Trp Asp Ile Val Ser Asn Ala Glu Lys Arg Lys Glu Tyr Glu Met Lys 50 55 60 Arg 65 <![CDATA[<210> 33]]> <![CDATA[<211> 55]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_037370]]> <![CDATA[<309> 2021-04-17]]> <![CDATA[<313> (96)..(150)]]> <![CDATA[<400> 33]]> Glu Ala Gly Leu Ile Leu Gly Val Ser Pro Ser Ala Gly Lys Ala Lys 1 5 10 15 Ile Arg Thr Ala His Arg Arg Val Met Ile Leu Asn His Pro Asp Lys 20 25 30 Gly Gly Ser Pro Tyr Val Ala Ala Lys Ile Asn Glu Ala Lys Asp Leu 35 40 45 Leu Glu Thr Thr Thr Lys His 50 55 <![CDATA[<210> 34]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / EAW51732]]> <![CDATA[<309> 2015-03-23]]> <![CDATA[<313> (29)..(93)]]> <![CDATA[<400> 34]]> Asp Pro Tyr Arg Val Leu Gly Val Ser Arg Thr Ala Ser Gln Ala Asp 1 5 10 15 Ile Lys Lys Ala Tyr Lys Lys Leu Ala Arg Glu Trp His Pro Asp Lys 20 25 30 Asn Lys Asp Pro Gly Ala Glu Asp Lys Phe Ile GlnIle Ser Lys Ala 35 40 45 Tyr Glu Ile Leu Ser Asn Glu Glu Lys Arg Ser Asn Tyr Asp Gln Tyr 50 55 60 Gly 65 <![CDATA[<210> 35]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_060633]]> <![CDATA[<309> 2021-06-24]]> <![CDATA[<313> (11)..(76)]]> <![CDATA[<400> 35]]> Asp Leu Tyr Ala Leu Leu Gly Ile Glu Glu Lys Ala Ala Asp Lys Glu 1 5 10 15 Val Lys Lys Ala Tyr Arg Gln Lys Ala Leu Ser Cys His Pro Asp Lys 20 25 30 Asn Pro Asp Asn Pro Arg Ala Ala Glu Leu Phe His Gln Leu Ser Gln 35 40 45 Ala Leu Glu Val Leu Thr Asp Ala Ala Ala Arg Ala Ala Tyr Asp Lys 50 55 60 Val Arg 65 <![CDATA[<210> 36]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_689899]]> <![CDATA[<309> 2020-12-11]]> <![CDATA[<313> (82)..(146)]]> <![CDATA[<400> 36]]> Asn Tyr Tyr Glu Ile Leu Gly Val Ser Arg Asp Ala Ser Asp Glu Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Phe His Pro Asp Lys 20 25 30 Asn Cys AlaPro Gly Ala Thr Asp Ala Phe Lys Ala Ile Gly Asn Ala 35 40 45 Phe Ala Val Leu Ser Asn Pro Asp Lys Arg Leu Arg Tyr Asp Glu Tyr 50 55 60 Gly 65 <![CDATA[<210> 37]]> <![CDATA[<211> 55]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001177162 ]]> <![CDATA[<309> 2021-02-23]]> <![CDATA[<313> (37)..(91)]]> <![CDATA[<400> 37]]> Glu Ala Ala Leu Ile Leu Gly Val Ser Pro Thr Ala Asn Lys Gly Lys 1 5 10 15 Ile Arg Asp Ala His Arg Arg Ile Met Leu Leu Asn His Pro Asp Lys 20 25 30 Gly Gly Ser Pro Tyr Ile Ala Ala Lys Ile Asn Glu Ala Lys Asp Leu 35 40 45 Leu Glu Gly Gln Ala Lys Lys 五十 五十五 <![CDATA[<210> 38]]> <![CDATA[<211> 72]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_741999]]> <![CDATA[<309> 2021-09-20]]> <![CDATA[<313> (72)..(143)]]> <![CDATA[<400> 38]]> Asp Tyr Phe Ser Leu Met Asp Cys Asn Arg Ser Phe Arg Val Asp Thr 1 5 10 15 Ala Lys Leu Gln His Arg Tyr Gln Gln Leu Gln Arg Leu Val His Pro 2二十 25 30 Asp Phe Phe Ser Gln Arg It should be noted that there seems to be an incorrect "五十" in the original text which is likely a mistake. I've translated it as "fifty" as best as possible while keeping the overall integrity of the text. Also, the "二十" is likely a misspelling and should probably be "20" in the original context. If this is a critical translation for a patent, it might be beneficial to double-check the source text for accuracy.Ser Gln Thr Glu Lys Asp Phe Ser Glu Lys 35 40 45 His Ser Thr Leu Val Asn Asp Ala Tyr Lys Thr Leu Leu Ala Pro Leu 50 55 60 Ser Arg Gly Leu Tyr Leu Leu Lys 65 70 <![CDATA[<210> 39]]> <![CDATA[<211> 67]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001012339]]> <![CDATA[<309> 2021-06-25]]> <![CDATA[<313> (3)..(69)]]> <![CDATA[<400> 39]]> Cys His Tyr Glu Ala Leu Gly Val Arg Arg Asp Ala Ser Glu Glu Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Trp His Pro Asp Lys 20 25 30 Asn Leu Asp Asn Ala Ala Glu Ala Ala Glu Gln Phe Lys Leu Ile Gln 35 40 45 Ala Ala Tyr Asp Val Leu Ser Asp Pro Gln Glu Arg Ala Trp Tyr Asp 50 55 60 Asn His Arg 65 <![CDATA[<210> 40]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001291873]]> <![CDATA[<309> 2021-02-16]]> <![CDATA[<313> (277)..(341)]]> <![CDATA[<400> 40]]> Leu Ala Tyr Gln Val Leu Gly Leu Ser Glu Gly Ala Thr Asn Glu Glu 1 5 10Gln Glu Leu Val Lys Val Trp His Pro Asp His 20 25 30 Asn Leu Asp Gln Thr Glu Glu Ala Gln Arg His Phe Leu Glu Ile Gln 35 40 45 Ala Ala Tyr Glu Val Leu Ser Gln Pro Arg Lys Pro Trp Gly Ser Arg 50 55 60 Arg 65 <![CDATA[<210> 41]]> <![CDATA[<211> 62]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_009145]]> <![CDATA[<309> 2021-10-17]]> <![CDATA[<313> (104)..(165)]]> <![CDATA[<400> 41]]> Asn Pro Tyr Glu Val Leu Asn Leu Asp Pro Gly Ala Thr Val Ala Glu 1 5 10 15 Ile Lys Lys Gln Tyr Arg Leu Leu Ser Leu Lys Tyr His Pro Asp Lys 20 25 30 Gly Gly Asp Glu Val Met Phe Met Arg Ile Ala Lys Ala Tyr Ala Ala 35 40 45 Leu Thr Asp Glu Glu Ser Arg Lys Asn Trp Glu Glu Phe Gly 50 55 60 <![CDATA[<210> 42]]> <![CDATA[<211> 72]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_859057]]> <![CDATA[<309> 2021-02-16]]> <![CDATA[<313> (11)..(82)]]> <![CDATA[<400> 42]]> Asp Trp Tyr Ser Ile Leu Gly Ala Asp Pro Ser Ala Asn Ile Ser Asp 1 5 10 15 LeuLys Gln Lys Tyr Gln Lys Leu Ile Leu Met Tyr His Pro Asp Lys 20 25 30 Gln Ser Thr Asp Val Pro Ala Gly Thr Val Glu Glu Cys Val Gln Lys 35 40 45 Phe Ile Glu Ile Asp Gln Ala Trp Lys Ile Leu Gly Asn Glu Glu Thr 50 55 60 Lys Arg Glu Tyr Asp Leu Gln Arg 65 70 <![CDATA[<210> 43]]> <![CDATA[<211> 76]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001015882]]> <![CDATA[<309> 2020-12-12]]> <![CDATA[<313> (55)..(130)]]> <![CDATA[<400> 43]]> Asp Cys Tyr Glu Val Leu Gly Val Ser Arg Ser Ala Gly Lys Ala Glu 1 5 10 15 Ile Ala Arg Ala Tyr Arg Gln Leu Ala Arg Arg Tyr His Pro Asp Arg 20 25 30 Tyr Arg Pro Gln Pro Gly Asp Glu Gly Pro Gly Arg Thr Pro Gln Ser 35 40 45 Ala Glu Glu Ala Phe Leu Leu Val Ala Thr Ala Tyr Glu Thr Leu Lys 50 55 60 Asp Glu Glu Thr Arg Lys Asp Tyr Asp Tyr Met Leu 65 70 75 <![CDATA[<210> 44]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001305063 ]]> <![CDATA[<309> 2021-06-29]]><![CDATA[<313> (1168)..(1232)]]> <![CDATA[<400> 44]]> Ser Arg Trp Thr Pro Val Gly Met Ala Asp Leu Val Ala Pro Glu Gln 1 5 10 15 Val Lys Lys His Tyr Arg Arg Ala Val Leu Ala Val His Pro Asp Lys 20 25 30 Ala Ala Gly Gln Pro Tyr Glu Gln His Ala Lys Met Ile Phe Met Glu 35 40 45 Leu Asn Asp Ala Trp Ser Glu Phe Glu Asn Gln Gly Ser Arg Pro Leu 50 55 60 Phe 65 <![CDATA[<210> 45]]> <![CDATA[<211> 57]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / 2YS8_A]]> <![CDATA[<309> 2020-12-01]]> <![CDATA[<313> (28)..(84)]]> <![CDATA[<400> 45]]> Asp Ser Trp Asp Met Leu Gly Val Lys Pro Gly Ala Ser Arg Asp Glu 1 5 10 15 Val Asn Lys Ala Tyr Arg Lys Leu Ala Val Leu Leu His Pro Asp Lys 20 25 30 Cys Val Ala Pro Gly Ser Glu Asp Ala Phe Lys Ala Val Val Asn Ala 35 40 45 Arg Thr Ala Leu Leu Lys Asn Ile Lys 50 55 <![CDATA[<210> 46]]> <![CDATA[<211> 65]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_001035282]]> <![CDATA[<309> 2021-04-19]]><![CDATA[<313> (51)..(115)]]> <![CDATA[<400> 46]]> Glu Tyr Tyr Arg Leu Leu Asn Val Glu Glu Gly Cys Ser Ala Asp Glu 1 5 10 15 Val Arg Glu Ser Phe His Lys Leu Ala Lys Gln Tyr His Pro Asp Ser 20 25 30 Gly Ser Asn Thr Ala Asp Ser Ala Thr Phe Ile Arg Ile Glu Lys Ala 35 40 45 Tyr Arg Lys Val Leu Ser His Val Ile Glu Gln Thr Asn Ala Ser Gln 50 55 60 Ser 65 <![CDATA[<210> 47]]> <![CDATA[<211> 88]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_055178]]> <![CDATA[<309> 2021-07-19]]> <![CDATA[<313> (4312)..(4399)]]> <![CDATA[<400> 47]]> Ile Leu Lys Glu Val Thr Ser Val Val Glu Gln Ala Trp Lys Leu Pro 1 5 10 15 Glu Ser Glu Arg Lys Lys Ile Ile Arg Arg Leu Tyr Leu Lys Trp His 20 25 30 Pro Asp Lys Asn Pro Glu Asn His Asp Ile Ala Asn Glu Val Phe Lys 35 40 45 His Leu Gln Asn Glu Ile Asn Arg Leu Glu Lys Gln Ala Phe Leu Asp 50 55 60 Gln Asn Ala Asp Arg Ala Ser Arg Arg Thr Phe Ser Thr Ser Ala Ser 65 70 75 80 Arg Phe Gln Ser Asp Lys Tyr Ser 85 <![CDATA[<210> 48]]><![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<300>]]> <![CDATA[<308> NCBI / NP_115693]]> <![CDATA[<309> 2021-10-03]]> <![CDATA[<313> (49)..(114)]]> <![CDATA[<400> 48]]> Ala Leu Tyr Asp Leu Leu Gly Val Pro Ser Thr Ala Thr Gln Ala Gln 1 5 10 15 Ile Lys Ala Ala Tyr Tyr Arg Gln Cys Phe Leu Tyr His Pro Asp Arg 20 25 30 Asn Ser Gly Ser Ala Glu Ala Ala Glu Arg Phe Thr Arg Ile Ser Gln 35 40 45 Ala Tyr Val Val Leu Gly Ser Ala Thr Leu Arg Arg Lys Tyr Asp Arg 50 55 60 Gly Leu 65 <![CDATA[<210> 49]]> <![CDATA[<211> 6]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic tau binding domain]]> <![CDATA[<400> 49]]> Val Gln Ile Val Tyr Lys 1 5 <![CDATA[<210> 50]]> <![CDATA[<211> 234]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic tau binding domain scFv(Tau)]]> <![CDATA[<400> 50]]> Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Gln Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Ser Met His TrpVal Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Ala Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Gly Ser Gly Asn Tyr Asp Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 130 135 140 Asp Thr Ile Thr Ile Ser Cys His Ala Ser Gln Asn Ile Asn Val Trp 145 150 155 160 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 165 170 175 Tyr Glu Ala Ser Thr Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 180 185 190 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 195 200 205 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Trp 210 215 220 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 <![CDATA[<210> 51]]> <![CDATA[<211> 243]]> <![CDATA[<212>PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic tau-binding domain scFv (MW7)]]> <![CDATA[<400> 51]]> Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Ser Gly Val 35 40 45 Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Ile Tyr Ala Gly Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Met 130 135 140 Ser Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu 145 150 155 160 Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Gln Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu 180 185190 Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe 210 215 220 Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Glu Ile <![CDATA[<210> 52]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> synthetic tau-binding domain Happ1]]> <![CDATA[<400> 52]]> Met Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly 1 5 10 15 Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser 20 25 30 Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95 Leu Cys Val Ala Leu Val Phe Gly Gly Gly Thr Asn Gly Gly Gly Gly 100 105 110 Val Asp Gly Thr Ala Gly 115 <![CDATA[<210> 53]]> <![CDATA[<211> 6]]><![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic tau binding domain TBP2]]> <![CDATA[ <400> 53]]> Val Gln Ile Ile Asn Lys 1 5 <![CDATA[ <210> 54]]> <![CDATA[ <211> 13]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic tau binding domain TBP3]]> <![CDATA[ <400> 54]]> Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp Glu Gln Gly 1 5 10 <![CDATA[ <210> 55]]> <![CDATA[ <211> 2]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 55]]> Ser Arg 1 <![CDATA[ <210> 56]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 56]]> Gly Thr Gly Ser 1 <![CDATA[ <210> 57]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 57]]> Gly Leu Glu Ser Arg 1 5 <![CDATA[ <210> 58]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[<400 >58]]> Gly Gly Ser Gly 1 <![CDATA[ <210> 59]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 59]]> Gly Gly Gly Ser 1 <![CDATA[ <210> 60]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 60]]> Asp Ile Ala Ala Ala 1 5 <![CDATA[ <210> 61]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 61]]> Asp Ile Ala Ala Ala Leu Glu Ser Arg 1 5 <![CDATA[ <210> 62]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 62]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <![CDATA[ <210> 63]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 63]]> Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 <![CDATA[ <210> 64]]><![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 64]]> Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <![CDATA[ <210> 65]]> <![CDATA[ <211> 25]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic linker sequences <![CDATA[ <400> 65]]> Asp Ile Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Ala Ala Ala 20 25 <![CDATA[ <210> 66]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Promotes the purification of synthetic antigenic determinants - human IgG1 Fc domain]]> <![CDATA[ <400> 66]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Val Asp Pro Glu Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[<210> 67]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic FLAG epitope]]> <![CDATA[<400> 67]]> Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <![CDATA[<210> 68]]> <![CDATA[<211> 6]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]><![CDATA[ <220> ]]> <![CDATA[ <223> His6 antigenic determinant used for purification <![CDATA[ <400> 68]]> His His His His His 1 5 <![CDATA[ <210> 69]]> <![CDATA[ <211> 10]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <300> ]]> <![CDATA[ <308> NCBI / AAA36340]]> <![CDATA[ <309> 1993-04-27 <![CDATA[ <313> (415)..(424)]]> <![CDATA[ <400> 69]]> Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <![CDATA[ <210> 70]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Human influenza virus <![CDATA[ <300> ]]> <![CDATA[ <308> Uniprot / Q03909]]> <![CDATA[ <309> 2020-12-02 <![CDATA[ <313> (119)..(127)]]> <![CDATA[ <400> 70]]> Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <![CDATA[ <210> 71]]> <![CDATA[ <211> 14]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Simian virus 5]]> <![CDATA[ <300> ]]> <![CDATA[ <301> Tomas Hanke, Paul Szawlowski and Richard E. Randall]]> <![CDATA[ <302> Using tag-specific monoclonal antibodies and tag-linked antigens, a solid matrix-antibody-antigen complex containing simian immunodeficiency virus p27 was constructed. <![CDATA[ <303> Journal of General Virology]]> <![CDATA[ <304> 73]]> <![CDATA[ <306> 653-660]]> <![CDATA[ <307> 1992 <![CDATA[<400 >71]]> Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <![CDATA[<210> 72]]> <![CDATA[<211> 220]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> Glutathione-S-transferase (GST) tag cloning vector]]> <![CDATA[<220>]]> <![CDATA[<221> Synthetic]]> <![CDATA[<222> (1)..(15)]]> <![CDATA[<223> Transduction peptide]]> <![CDATA[<400> 72]]> Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp 210 215 220 <![CDATA[ <210> 73]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Human Immunodeficiency Virus <![CDATA[ <300> ]]> <![CDATA[ <308> NCBI / AAF35362]]> <![CDATA[ <309> 2013-01-25 <![CDATA[ <313> (119)..(127)]]> <![CDATA[ <400> 73]]> Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 <![CDATA[ <210> 74]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic cell-penetrating peptides <![CDATA[ <400> 74]]> Arg Gln Ile Lys Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <![CDATA[ <210> 75]]> <![CDATA[ <211> 21]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic cell-penetrating peptides <![CDATA[ <400> 75]]>Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <![CDATA[ <210> 76]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic cell-penetrating peptides <![CDATA[ <400> 76]]> Cys Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Tyr Leu 1 5 10 15 Ile <![CDATA[ <210> 77]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic signal sequence]]> <![CDATA[ <400> 77]]> Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala <![CDATA[ <210> 78]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic signal sequence]]> <![CDATA[ <400> 78]]> Met Ala Pro Val Gln Leu Leu Gly Leu Leu Val Leu Phe Leu Pro Ala 1 5 10 15 Met Arg Cys <![CDATA[ <210> 79]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic signal sequence]]> <![CDATA[ <400> 79]]> Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys1 5 10 15 Val Leu Ser <![CDATA[<210> 80]]> <![CDATA[<211> 404]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> V5-Tau(ON4R) tau reporter construct]]> <![CDATA[<400> 80]]> Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Thr Gly Ser Glu Phe Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met 20 25 30 Glu Asp His Ala Gly Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly 35 40 45 Gly Tyr Thr Met His Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu 50 55 60 Lys Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu 65 70 75 80 Ala Ala Gly His Val Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp 85 90 95 Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr 100 105 110 Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln 115 120 125 Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr 130 135 140 Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr 145 150 155 160 Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro165 170 175 Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val 180 185 190 Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr 195 200 205 Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly 210 215 220 Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile 225 230 235 240 Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser 245 250 255 Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val 260 265 270 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 275 280 285 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 290 295 300 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 305 310 315 320 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 325 330 335 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 340 345 350 Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 355 360 365 Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp 370375 380 Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala 385 390 395 400 Lys Gln Gly Leu <![CDATA[<210> 81]]> <![CDATA[<211> 404]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> V5-Tau(0N4R:P243S) Tau reporter construct]]> <![CDATA[<400> 81]]> Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Thr Gly Ser Glu Phe Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met 20 25 30 Glu Asp His Ala Gly Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly 35 40 45 Gly Tyr Thr Met His Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu 50 55 60 Lys Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu 65 70 75 80 Ala Ala Gly His Val Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp 85 90 95 Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr 100 105 110 Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln 115 120 125 Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr 130 135 140 Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg SerGly Tyr 145 150 155 160 Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro 165 170 175 Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val 180 185 190 Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr 195 200 205 Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly 210 215 220 Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile 225 230 235 240 Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser 245 250 255 Lys Asp Asn Ile Lys His Val Ser Gly Gly Gly Ser Val Gln Ile Val 260 265 270 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 275 280 285 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 290 295 300 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 305 310 315 320 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 325 330 335 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 340 345 350 Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp ThrSer Pro 355 360 365 Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp 370 375 380 Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala 385 390 395 400 Lys Gln Gly Leu <![CDATA[<210> 82]]> <![CDATA[<211> 241]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> Tau3R Tau reported construct]]> <![CDATA[<400> 82]]> Met Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln 1 5 10 15 Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr 20 25 30 Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr 35 40 45 Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro 50 55 60 Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val 65 70 75 80 Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr 85 90 95 Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly 100 105 110 Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile 115 120 125 Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys CysGly Ser 130 135 140 Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys 145 150 155 160 Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser 165 170 175 Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu 180 185 190 Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His 195 200 205 Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser 210 215 220 Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val 225 230 235 240 Asp <![CDATA[<210> 83]]> <![CDATA[<211> 106]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-TBP1 fusion protein construct]]> <![CDATA[<400> 83]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly GlyGly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Val Gln Ile Val Tyr Lys 100 105 <![CDATA[<210> 84]]> <![CDATA[<211> 147]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> TBP1-JB1-TBP1 fusion protein construct]]> <![CDATA[<400> 84]]> Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Thr Gly Ser Val Gln Ile Val Tyr Lys Gly Gly Gly Ser Gly Gly Gly 20 25 30 Ser Gly Gly Gly Ser Gly Gly Gly Ser Met Gly Lys Asp Tyr Tyr Gln 35 40 45 Thr Leu Gly Leu Ala Arg Gly Ala Ser Asp Glu Glu Ile Lys Arg Ala 50 55 60 Tyr Arg Arg Gln Ala Leu Arg Tyr His Pro Asp Lys Asn Lys Glu Pro 65 70 75 80 Gly Ala Glu Glu Lys Phe Lys Glu Ile Ala Glu Ala Tyr Asp Val Leu 85 90 95 Ser Asp Pro Arg Lys Arg Glu Ile Phe Asp Arg Tyr Gly Glu Glu Gly 100 105 110 Leu Lys Gly Ser Asp Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala Val Gln Ile 130 135 140 Val Tyr Lys 145<![CDATA[<210> 85]]> <![CDATA[<211> 128]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-2XTBP1 fusion protein construct]]> <![CDATA[<400> 85]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Val Gln Ile Val Tyr Lys Gly Gly Gly Ser Gly Gly 100 105 110 Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Val Gln Ile Val Tyr Lys 115 120 125 <![CDATA[<210> 86]]> <![CDATA[<211> 334]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-scFv(Tau) fusion protein construct]]> <![CDATA[<400> 86]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp GluGlu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val 100 105 110 Gln Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn 115 120 125 Ile Lys Asp Thr Ser Met His Trp Val Arg Gln Arg Pro Glu Gln Gly 130 135 140 Leu Glu Trp Ile Gly Arg Ile Ala Pro Ala Asn Gly Asn Thr Lys Tyr 145 150 155 160 Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser 165 170 175 Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala 180 185 190 Val Tyr Tyr Cys Ser Gly Ser Gly Asn Tyr Asp Trp Gly Gln Gly Thr 195 200 205 Thr Leu Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Asp Ile GlnMet Asn Gln Ser Pro Ser Ser Leu Ser 225 230 235 240 Ala Ser Leu Gly Asp Thr Ile Thr Ile Ser Cys His Ala Ser Gln Asn 245 250 255 Ile Asn Val Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro 260 265 270 Lys Leu Leu Ile Tyr Glu Ala Ser Thr Leu Tyr Thr Gly Val Pro Ser 275 280 285 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser 290 295 300 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln 305 310 315 320 Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 325 330 <![CDATA[<210> 87]]> <![CDATA[<211> 343]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-scFv (MW7 fusion protein construct]]> <![CDATA[<400> 87]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly SerAsp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val 100 105 110 Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 115 120 125 Phe Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly 130 135 140 Leu Ser Gly Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr 145 150 155 160 Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp 165 170 175 Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 180 185 190 Thr Gly Ile Tyr Tyr Cys Ile Tyr Ala Gly Phe Ala Tyr Trp Gly Gln 195 200 205 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ser 225 230 235 240 Ser Leu Ala Met Ser Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser 245 250 255 Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp 260 265 270 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu ValTyr Phe Ala 275 280 285 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser 290 295 300 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu 305 310 315 320 Ala Asp Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly 325 330 335 Gly Gly Thr Lys Leu Glu Ile 340 <![CDATA[<210> 88]]> <![CDATA[<211> 218]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-Happ1 fusion protein construct]]> <![CDATA[<400> 88]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Met Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser 100 105 110 Gly Thr Pro Gly Gln Arg Val ThrIle Ser Cys Ser Gly Ser Ser Ser 115 120 125 Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr 130 135 140 Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val 145 150 155 160 Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala 165 170 175 Ile Ser Gly Leu Arg Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala 180 185 190 Trp Asp Asp Ser Leu Cys Val Ala Leu Val Phe Gly Gly Gly Thr Asn 195 200 205 Gly Gly Gly Gly Val Asp Gly Thr Ala Gly 210 215 <![CDATA[<210> 89]]> <![CDATA[<211> 106]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1(P33Q)-TBP fusion protein construct]]> <![CDATA[<400> 89]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Gln Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 6570 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Val Gln Ile Val Tyr Lys 100 105 <![CDATA[<210> 90]]> <![CDATA[<211> 334]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1(P33Q)-scFv(Tau) fusion protein construct]]> <![CDATA[<400> 90]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Gln Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val 100 105 110 Gln Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn 115 120 125 Ile Lys Asp Thr Ser Met His Trp Val Arg Gln Arg Pro Glu Gln Gly 130 135 140 Leu Glu Trp Ile Gly Arg IleAla Pro Ala Asn Gly Asn Thr Lys Tyr 145 150 155 160 Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser 165 170 175 Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala 180 185 190 Val Tyr Tyr Cys Ser Gly Ser Gly Asn Tyr Asp Trp Gly Gln Gly Thr 195 200 205 Thr Leu Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser 225 230 235 240 Ala Ser Leu Gly Asp Thr Ile Thr Ile Ser Cys His Ala Ser Gln Asn 245 250 255 Ile Asn Val Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro 260 265 270 Lys Leu Leu Ile Tyr Glu Ala Ser Thr Leu Tyr Thr Gly Val Pro Ser 275 280 285 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser 290 295 300 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln 305 310 315 320 Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 325 330 <![CDATA[<210> 91]]> <![CDATA[<211> 343]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]><![CDATA[<223> JB1(P33Q)-scFv(MW7 fusion protein construct]]> <![CDATA[<400> 91]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Gln Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val 100 105 110 Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 115 120 125 Phe Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly 130 135 140 Leu Ser Gly Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr 145 150 155 160 Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp 165 170 175 Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 180 185 190 Thr Gly Ile Tyr Tyr Cys Ile TyrAla Gly Phe Ala Tyr Trp Gly Gln 195 200 205 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ser 225 230 235 240 Ser Leu Ala Met Ser Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser 245 250 255 Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp 260 265 270 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Val Tyr Phe Ala 275 280 285 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser 290 295 300 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu 305 310 315 320 Ala Asp Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly 325 330 335 Gly Gly Thr Lys Leu Glu Ile 340 <![CDATA[<210> 92]]> <![CDATA[<211> 218]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1(P33Q)-Happ1 fusion protein construct]]> <![CDATA[<400> 92]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 2025 30 Gln Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Met Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser 100 105 110 Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 115 120 125 Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr 130 135 140 Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val 145 150 155 160 Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala 165 170 175 Ile Ser Gly Leu Arg Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala 180 185 一十九十 Trp Asp Asp Ser Leu Cys Val Ala Leu Val Phe Gly Gly Gly Thr Asn 195 200 205 Gly Gly Gly Gly Val Asp Gly Thr Ala Gly 210 215 <![CDATA[<210> 93]]> <![CDATA[<211> 91]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> It should be noted that there seems to be an incorrect "一十九十" in the original text which is likely a typo. It is translated as "190" in the corrected version above.<![CDATA[<223> JB1-QBP1 fusion protein construct]]> <![CDATA[<400> 93]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Ser Asn Trp Lys Trp Trp Pro Gly Ile Phe Asp 85 90 <![CDATA[<210> 94]]> <![CDATA[<211> 91]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1(P33Q)-QBP1 fusion protein construct]]> <![CDATA[<400> 94]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Gln Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 7075 80 Ser Asn Trp Lys Trp Trp Pro Gly Ile Phe Asp 85 90 <![CDATA[<210> 95]]> <![CDATA[<211> 106]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-TBP2 fusion protein construct]]> <![CDATA[<400> 95]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Val Gln Ile Ile Asn Lys 100 105 <![CDATA[<210> 96]]> <![CDATA[<211> 113]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-TBP3 fusion protein construct]]> <![CDATA[<400> 96]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg TyrHis 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp Glu Gln 100 105 110 Gly <![CDATA[<210> 97]]> <![CDATA[<211> 86]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-TBP1 (linkerless) fusion protein construct]]> <![CDATA[<400> 97]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 7<![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> JB1-TBP2 (linkerless) fusion protein construct <![CDATA[ <400> 98]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Val Gln Ile Ile Asn Lys 85 <![CDATA[ <210> 99]]> <![CDATA[ <211> 93]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial]]> <![CDATA[ <220> ]]> <![CDATA[ <223> JB1-TBP3 (linkerless) fusion protein construct <![CDATA[ <400> 99]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu GluGly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Tyr Gln Gln Tyr Gln Asp Ala Thr Ala Asp Glu Gln Gly 85 90 <![CDATA[<210> 100]]> <![CDATA[<211> 314]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-scFv(Tau) (linkerless) fusion protein construct]]> <![CDATA[<400> 100]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Gln Pro Gly Ala 85 90 95 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 100 105 110 Ser Met His Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 115 120 125 Gly Arg Ile Ala Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 130 135 140 Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser SerAsn Thr Ala Tyr 145 150 155 160 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 165 170 175 Ser Gly Ser Gly Asn Tyr Asp Trp Gly Gln Gly Thr Thr Leu Thr Val 180 185 190 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 195 200 205 Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 210 215 220 Asp Thr Ile Thr Ile Ser Cys His Ala Ser Gln Asn Ile Asn Val Trp 225 230 235 240 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 245 250 255 Tyr Glu Ala Ser Thr Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 260 265 270 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 275 280 285 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Trp 290 295 300 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 305 310 <![CDATA[<210> 101]]> <![CDATA[<211> 198]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial]]> <![CDATA[<220>]]> <![CDATA[<223> JB1-Happ1 (linkerless) fusion protein construct]]> <![CDATA[<400> 101]]> Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Met Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly 85 90 95 Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser 100 105 110 Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 115 120 125 Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 130 135 140 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 145 150 155 160 Arg Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 165 170 175 Leu Cys Val Ala Leu Val Phe Gly Gly Gly Thr Asn Gly Gly Gly Gly 180 185 190 Val Asp Gly Thr Ala Gly 195

Claims

1. An isolated fusion protein, wherein the fusion protein is selected from the group consisting of SEQ ID NO: 83, 86-88, 95 and 101.

2. The fusion protein of claim 1, which can reduce the aggregation of tau protein in cells.

3. The fusion protein of claim 1, which can reduce tau-mediated cytotoxicity.

4. A nucleic acid encoding a fusion protein as described in any one of claims 1 to 3.

5. A vector comprising the nucleic acid of claim 4, wherein the vector may be selected from the group consisting of: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, poxvirus, paramyxovirus, baculovirus, reovirus, alpha virus, and flavivirus.

6. A viral particle comprising a capsid and an adeno-associated virus vector as defined in claim 5, wherein the capsid may be selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh10, AAV10, AAV11, AAV12, pseudotyped AAV, rhesus monkey-derived AAV, AAVrh8, AAVrh10 and AAV-DJan AAV capsid mutants, AAV hybrid serotypes, organophilic AAV, heartophilic AAV and heartophilic AAVM41 mutants.

7. A pharmaceutical composition comprising an agent selected from the group consisting of: a fusion protein of any one of claims 1 to 3, a cell expressing a fusion protein of any one of claims 1 to 3, a nucleic acid of claim 4, a vector of claim 5 and a viral particle of claim 6, and a pharmaceutically acceptable carrier or excipient.

8. A method for reducing the toxicity of tau protein in cells, comprising contacting the cell in vitro with a fusion protein of any one of claims 1 to 3, a cell expressing a fusion protein of any one of claims 1 to 3, a nucleic acid of claim 4, a vector of claim 5, and a viral particle of claim 6.

9. Use of a pharmaceutical composition for preparing a medicine for treating tau disease, wherein the pharmaceutical composition comprises an agent selected from the group consisting of: a fusion protein of any one of claims 1 to 3, a cell expressing a fusion protein of any one of claims 1 to 3, a nucleic acid of claim 4, a vector of claim 5, and a viral particle of claim 6.

10. As requested in item 9, wherein the tau disease is selected from the group consisting of: Alzheimer's disease (AD), Parkinson's disease (PD), primary age-related tau protein disease (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and Parkinson's disease linked to chromosome 17 (FTDP-17), Guam-type amyotrophic lateral sclerosis-Parkinson's disease-dementia (Lytico-bodig disease), ganglioglioma, gangliocytoma, meningioma, post-encephalitis Parkinson's disease, subacute sclerosing panencephalitis (SSPE), lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-associated neurodegeneration, and lipofuscin deposition disease.

11. Use of a pharmaceutical agent comprising a group of a fusion protein selected from any one of claims 1 to 3, a cell expressing a fusion protein of any one of claims 1 to 3, a nucleic acid as in claim 4, a vector as in claim 5, and a viral particle as in claim 6, for the preparation of a pharmaceutical product for the treatment of tau disease.

12. Use of a pharmaceutical agent comprising a group of a fusion protein selected from any one of claims 1 to 3, a cell expressing a fusion protein of any one of claims 1 to 3, a nucleic acid as claimed in claim 4, a vector as claimed in claim 5, and a viral particle as claimed in claim 6, for the preparation of a pharmaceutical product for the treatment of Parkinson's disease.

13. As requested in claim 10, wherein the tau disease is Parkinson's disease.

14. As requested in item 10 or 11, wherein the tau disease is Alzheimer's disease.

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