Compositions and methods for the treatment of TDP-43 proteinopathy

Abstract

A novel class of fusion proteins is disclosed that recruits the cell's innate chaperone machinery, specifically the Hsp70-mediated system, to specifically reduce TDP-43-mediated protein aggregation and associated proteopathies.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 63 / 016,707, filed on April 28, 2020, and also to U.S. Provisional Patent Application No. 63 / 035,437, filed on June 5, 2020. The entire contents of the foregoing applications are hereby incorporated by reference in their entirety.

Background Art

[0002] All proteins expressed within cells must fold correctly into their intended structures in order to function properly. An increasing number of diseases and disorders have been shown to be associated with inappropriate protein folding and / or inappropriate deposition and aggregation of proteins and lipoproteins, as well as infectious proteinaceous substances. Examples of diseases caused by misfolding, also known as conformational diseases or proteopathies, include Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal lobe dementia (FTLD). Mutant proteins aggregate in cells, resulting in typical cytotoxic cell inclusions.

[0003] Various neurodegenerative diseases are pathologically characterized by the accumulation of intracellular or extracellular protein aggregates composed of amyloid fibrils (Forman et al., (2004) Nat Med. 10:1055~1063). For example, the pathogenesis of Alzheimer's disease (AD) is defined by senile plaques and neurofibrillary condensates composed of β-amyloid and microtubule-associated protein tau, respectively, while Lewy bodies, composed of α-synuclein, are the disease-defining lesions of Parkinson's disease. Until recently, the neuropathology of both frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) (Kumar-Singh & Van (2007) Brain Pathol., 17:104~114), the most common phenotype associated with FTLD syndrome, and amyotrophic lateral sclerosis (ALS) (Xiao et al., (2006) Biochim Biophys Acta, 1762:1001~1012) was defined by non-amyloidogenic ubiquitinated inclusions (UBIs).

[0004] FTLD, the second most common type of presenile dementia, refers to a heterogeneous group of neurodegenerative disorders that commonly involve behavioral and / or language impairments (Kumar-Singh & Van, ibid.). Some affected individuals exhibit motor impairments such as parkinsonism or motor neuron disease (MND). While the name FTLD reflects marked degeneration of the frontal and temporal lobes, numerous neuropathological abnormalities have been identified in these patients (Cairns et al., (2007) Acta Neuropathol., 1145:5~22). Two broad pathological subdivisions of FTLD are recognized: brains with tau-positive inclusions (i.e., tauopathy), and brains with UBI undetectable by antibodies against tau, α-synuclein, and β-amyloid (i.e., FTLD-U). Up to 40% of FTLD cases exhibit a familial inheritance pattern with three distinct genetic abnormalities associated with the FTLD-U pathology, including mutations in progranulin (PGRN) and balocin-containing protein (VCP), as well as linkage to a novel locus on chromosome 9p.

[0005] Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig's disease, is a neurodegenerative disease characterized by progressive degeneration of both upper and lower motor neurons in the brainstem and spinal cord, leading to progressive muscle wasting and weakness (Al-Chalabi et al., (2016) Amyotrophic lateral sclerosis., 15(11):1182~1194; Robberecht & Philips, (2013) Nat Rev Neurosci., 14(4):248~264; Talbot et al., (2018) Nucleic Acids Res., 37(8):e64). ALS has a median incidence of approximately 5.4 cases per 100,000 people with a median age of onset between 54 and 67 years, and the risk is slightly higher in men than in women (Chio et al., (2009) Amyotroph Lateral Scler., 10(5-6):310-323; Chio et al., (2013) Neuroepidemiology, 41(2):118-130; McCombe & Henderson, (2010) Gend Med., 7(6):557-570). ALS is a catastrophic neurodegenerative disease for which there is no effective treatment, and patients usually die within 2 to 4 years of the onset of the disease, mainly from respiratory failure and swallowing problems (Chio et al., (2009) Amyotroph Lateral Scler., 10(5-6):310~323; del Aguila et al., (2003) Neurology, 60(5):813~819; Tabata et al., (2009) Nucleic Acids Res., 7(8):e64).

[0006] The majority of ALS cases are sporadic (sALS) of unknown cause, and only about 10% of cases involve a Mendelian inheritance pattern of familial gene mutations known as familial ALS (fALS) (Renton et al., (2014) Nat Neurosci., 17(1):17~23; Taylor et al., (2016) Nature, 539(7628):197~206; Turner et al., (2017) J Neurol Neurosurg Psychiatry, 88(12):1042~1044). To date, up to 30 genes have been described as monogenic causes of ALS, with the most frequent being C9orf72, SOD1, FUS, and TARDBP / TDP43 (Chia et al., (2018) Lancet Neurol., 17(1):94~102; Nicolas et al., (2018) Neuron, 97(6):1268~1283; Volk et al., (2018) Med Genet., 30(2):252~258).

[0007] The variability in penetrants in ALS, coupled with the identification of gene mutations in numerous genes, suggests that multiple factors, including multiple gene components and environmental factors, may underlie disease susceptibility. In fact, factors contributing to oxidative stress include protein misfolding / aggregation (Ross & Poirier, (2004) Nat Med., 10 Suppl:S10~17), glutamate-mediated excitotoxicity (Blasco et al., (2014) Curr Med Chem., 21(31):3551~3575), mitochondrial dynamics abnormalities (Cappello & Francolini, (2017) Int J Mol Sci., 18(10); Delic et al., (2018) J Neurosci Res., 96(8):1353~1366; Onesto et al., (2016) Acta Neuropathol Commun., 4(1):47), and oxidative stress (Anand et al., (2013) Oxid Med Cell Longev., 2013:635831; Sharma et al., (2016) Neurochem It has been suggested that combinations of cellular pathways, such as those described in Res., 41(5):965~984), may lead to neurotoxic consequences in ALS.

[0008] Transactive response (TAR)-DNA binding protein (TDP-43), with a molecular weight of 43 kDa, was identified as a major disease protein in FTLD-U and UBI of ALS (Neumann et al., (2006) Science 314:130~133). The identification of TDP-43 pathology in both of these disorders provided a mechanistic link to the following: 1) the majority of ALS patients exhibit a range of behavioral and cognitive changes that fall within the scope of FTLD (Murphy et al., (2007) Arch. Neurol., 64:330-334); 2) MND is commonly observed in FTLD-U patients (McKhann et al., (2001) Arch. Neurol., 58:1803-1809); 3) there is significant overlap in ubiquitin pathology observed in ALS and FTLD-U (MacKenzie & Feldman (2005) J. Neuropathol. Exp. Neurol. 64:730-739); and 4) identification of loci and specific gene mutations in families with simultaneous segregation of both ALS and FTLD (Talbot & Ansorge (2006) Hum. Mol. Genet., 15:R183~R187). TDP-43 has also been shown to be a histopathological marker for several other neurodegenerative diseases, including Alzheimer's disease (Amador-Ortiz et al., (2007), Ann Neurol., 61:435~45), Parkinson's disease (Lin and Dickson, (2008), Acta Neuropathol., 116:205~13), Huntington's disease (Schwab et al., (2008), J Neuropathol Exp Neurol., 67:1159~65), hippocampal sclerosis (Amador-Ortiz et al., (2007), ibid.), and Lewy body dementia (Lin and Dickson, (2008), ibid.); (Lagier-Tourenne et al., (2010), Human Molecular Genetics, 19:R46~R64). Therefore, there is a need to develop novel therapeutic modalities optimized to target specific antigens, proteins, glycoproteins, or lipoproteins, particularly for diseases with pathological conditions based on protein misfolding and aggregation, as well as diseases involving heterogeneous aggregates.

[0009] Heat shock 70kDa proteins (referred to herein as "Hsp70s") constitute a ubiquitous class of chaperone proteins in cells of various species (Tavaria et al., (1996) Cell Stress Chaperones 1, 23-28). Hsp70s require assistant proteins called co-chaperone proteins, such as J-domain proteins and nucleotide exchange factors (NEFs), to function (Hartl et al., (2009) Nat Struct Mol Biol 16, 574-581). In the latest model of the Hsp70 chaperone mechanism for protein folding, Hsp70 cycles between ATP-bound and ADP-bound states, and the J-domain protein interacts with the ATP-bound form of Hsp70 (Hsp70-ATP) by binding to another protein (called a "client protein") that requires folding or refolding (Young (2010) Biochem Cell Biol 88, 291-300; Mayer, (2010) Mol Cell 39, 321-331). The binding of the J-domain protein-client protein complex to Hsp70-ATP stimulates ATP hydrolysis, which causes a conformational change in the Hsp70 protein, closing the helix lid, thereby stabilizing the interaction between the client protein and Hsp70-ADP, and also inducing the release of the J-domain protein, which can then freely bind to another client protein.

[0010] Therefore, this model suggests that J-domain proteins play a crucial role in the Hsp70 mechanism by acting as crosslinks and facilitating the capture of various client proteins and their submission to the Hsp70 mechanism, thereby promoting folding or refolding into the appropriate three-dimensional structure (Kampinga & Craig (2010) Nat Rev Mol Cell Biol 11, 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 60-80aa) consists of four helices: I, II, III, and IV. Helices II and III are connected via a mobile loop containing an "HPD motif," which is highly conserved across the J-domain and is thought to be important for activity (Tsai & Douglas, (1996) J Biol Chem 271, 9347-9354). Mutations within the HPD sequence have been found to cause loss of J-domain function.

[0011] Given the background information provided above regarding proteopathies such as ALS, it seems clear that reducing levels of misfolded proteins could be a useful means of treating, preventing, or otherwise improving the symptoms of these devastating disorders, and that mobilizing the cells' innate ability to repair protein misfolding would be a natural choice to pursue. [Overview of the project]

[0012] The inventors have developed a novel class of fusion proteins that mobilize the cell's innate chaperone mechanism, specifically the Hsp70-mediated system, to specifically reduce TDP-43-mediated protein aggregation. Unlike previous studies by the inventors that used fusion proteins containing a fragment of the Hsp40 protein (also known as the J protein), a co-chaperone that interacts with Hsp70, to enhance protein secretion and expression, this study uses a J-domain-containing fusion protein with the aim of reducing protein aggregation and cytotoxicity caused by the aggregation of mutant TDP-43 protein. In this regard, the inventors have made the surprising discovery that the J-domain elements required for function are entirely different from the use of the J-domain in enhancing protein expression and secretion, demonstrating a distinct mechanism for the mode of action of this fusion protein. The fusion proteins described herein contain a J-domain and a domain having affinity for TDP-43. The presence of a TDP-43-binding protein within the fusion protein results in a specific reduction in the aggregation of mutant TDP-43 protein.

[0013] E1. Accordingly, in a first embodiment, an isolated fusion protein comprising the J domain and the TDP-43 binding domain of the J protein is disclosed herein. E2. A fusion protein described in E1, in which the J domain of the J protein is of eukaryotic origin. E3. A fusion protein described in any one of E1-E2, in which the J domain of the J protein is of human origin. E4. A fusion protein described in any one of E1-E3, in which the J domain of the J protein is localized in the cytoplasm. E5. A fusion protein described in any one of E1 to E4, wherein the J domain of the J protein is selected from the group consisting of Sequence IDs 1 to 50. E6. A fusion protein described in any one of E1 to E5, wherein the J domain contains a sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49. E7. A fusion protein described in any one of E1-E6, wherein the J domain contains the sequence of Sequence ID No. 5. E8. A fusion protein described in any one of E1-E6, wherein the J domain contains the sequence of SEQ ID NO: 10. E9. A fusion protein described in any one of E1 to E6, wherein the J domain contains the sequence of Sequence ID No. 16. E10. A fusion protein described in any one of E1 to E6, wherein the J domain contains the sequence of SEQ ID NO: 25. E11. A fusion protein described in any one of E1 to E6, wherein the J domain contains the sequence of Sequence ID No. 31. E12. When the TDP-43 binding domain is measured, for example, using an ELISA assay, the K content for TDP-43 (for example, using a reporter construct containing the 207 amino acids at the C-terminus of TDP-43) is 1 μM or less, for example, 300 nM or less, 100 nM or less, 30 nM or less, or 10 nM or less. D A fusion protein having any one of the following characteristics: E1 to E11. E13. A fusion protein described in any one of E1 to E12, wherein the TDP-43 binding domain contains a sequence selected from the group consisting of SEQ ID NOs. 51 to 55. E14. A fusion protein described in any one of E1 to E13, wherein the TDP-43 binding domain contains the sequences of SEQ ID NOs. 51-53. E15. A fusion protein described in any one of E1 to E13, wherein the TDP-43 binding domain contains the sequence of Sequence ID No. 51. E16. A fusion protein described in any one of E1 to E13, wherein the TDP-43 binding domain contains the sequence of Sequence ID No. 53. E17. A fusion protein described in any one of E1 to E16, containing multiple TDP-43 binding domains. E18. A fusion protein described in any one of E1 to E17, consisting of two TDP-43 binding domains. E19. A fusion protein described in any one of E1 to E18, consisting of three TDP-43 binding domains. E20. The following structures: a. DNAJ-X-T, b. DNAJ-X-T-X-T, c. DNAJ-X-T-X-T-X-T, d. T-X-DNAJ, e. T-X-T-X-DNAJ, f. T-X-T-X-T-X-DNAJ, g. T-X-DNAJ-X-T, h. T-X-DNAJ-X-T-X-T, i. TDNAJ-X-TTTTTDNAJ-X-T, j. T-X-T-X-DNAJ-X-TT, k. TTDNAJ-X-T-X-TTTTTDNAJ-X-T, l. T-X-T-X-DNAJ-X-T-X-T-X-T, m. T-X-T-X-T-X-DNAJ-X-T, n. T-X-T-X-T-X-DNAJ-X-T-X-T, o. T-X-T-X-T-X-DNAJ-X-T-X-T-X-T, p. DnaJ-X-DnaJ-X-T-X-T, q. T-X-DnaJ-X-DnaJ, r. T-X-T-X-DnaJ-X-DnaJ, and s. T-X-TDnaJ-X-TDnaJ-X-TTTT comprises one of the following: wherein T is a TDP-43 binding domain, DNAJ is the J domain of the J protein, X is an optional linker, and the fusion protein described in any one of E1 to E19. E21. The fusion protein according to any one of E1 to E20, comprising the J domain sequence of SEQ ID NO: 5 and the TDP-43 binding domain sequence of SEQ ID NO: 51. E22. The fusion protein according to any one of E1 to E21, comprising the J domain sequence of SEQ ID NO: 5 and two copies of the TDP-43 binding domain sequence of SEQ ID NO: 53. The fusion protein according to any one of E1 to E22, comprising a sequence selected from the group consisting of SEQ ID NOs: 80 to 85, and 89 to 97. The fusion protein according to any one of E1 to E23, comprising a sequence selected from the group consisting of SEQ ID NOs: 80, 82 to 85, 89 to 90, and 92 to 97. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 80. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 90. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 92. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 94. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 95. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 96. The fusion protein according to any one of E1 to E30, further comprising a targeting reagent. The fusion protein according to any one of E1 to E31, further comprising an epitope. The fusion protein according to E32, wherein the epitope is a polypeptide selected from the group consisting of SEQ ID NOs: 67 to 73. The fusion protein according to any one of E1 to E33, further comprising a cell-penetrating agent. The fusion protein according to E34, wherein the cell-penetrating agent is selected from the group consisting of SEQ ID NOs: 74 to 77. The fusion protein according to any one of E1 to E35, further comprising a signal sequence. The fusion protein according to E36, wherein the signal sequence comprises a peptide sequence selected from the group consisting of SEQ ID NOs: 98 to 100. E38. A fusion protein described in any one of E1-E37 that has the ability to reduce TDP-43 protein aggregation in cells. E39. A fusion protein described in any one of E1-E38 that has the ability to reduce TDP-43-mediated cytotoxicity. E40. A nucleic acid sequence encoding a fusion protein described in any one of E1 to E39. E41. The nucleic acid sequence described in E40, wherein the nucleic acid is DNA. E42. The nucleic acid sequence described in E40, wherein the nucleic acid is RNA. E43. A nucleic acid sequence according to any one of E40 to E42, wherein the nucleic acid comprises at least one modified nucleic acid. E44. A nucleic acid sequence described in any one of E40-E43, further comprising a promoter region, a 5'UTR, and a 3'UTR such as a poly(A) signal. E45. The nucleic acid sequence described in E44, wherein the promoter region comprises a sequence selected from the group consisting of a CMV enhancer sequence, a CMV promoter, a CBA promoter, a UBC promoter, a GUSB promoter, a NSE promoter, a synapsin promoter, a MeCP2 promoter, and a GFAP promoter. E46. A vector containing one of the nucleic acid sequences listed in E40 to E45. E47. A vector as described in E46, selected from the group consisting of adeno-associated viruses (AAV), adenoviruses, lentiviruses, retroviruses, herpesviruses, poxviruses (vaccinia or myxoma), paramyxoviruses (measles, RSV, or Newcastle disease virus), baculoviruses, reoviruses, alphaviruses, and flaviviruses. E48. AAV, as described in E46 or E47. E49. A viral particle containing a capsid and one of the vectors described in E46-E48. E50. A viral particle as described in E49, wherein the capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, pseudotyped AAV, rhesus monkey-derived AAV, AAVrh8, AAVrh10, and AAV-DJan AAV capsid variants, AAV hybrid serotypes, organotropic AAV, cardiacotropic AAV, and cardiacotropic AAVM41 variants. E51. A viral particle as described in E49 or E50, wherein the capsid is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, and AAVrh10. E52. A viral particle listed in any one of E49-E51, whose capsid is AAV2. E53. A viral particle listed in any one of E49-E51, whose capsid is AAV5. E54. A viral particle listed in any one of E49-E51, whose capsid is AAV8. E55. A viral particle listed in any one of E49-E51, whose capsid is AAV9. E56. A viral particle listed in any one of E49-E51, whose capsid is AAVrh10. E57. A pharmaceutical composition comprising a fusion protein described in any one of E1 to E39, cells expressing the fusion protein described in E1 to E39, a nucleic acid described in any one of E40 to E45, a vector described in any one of E46 to E48, a viral particle described in any one of E49 to E56, and a pharmaceutically acceptable carrier or excipient. E58. A method for reducing the toxicity of TDP-43 protein in cells, comprising the step of contacting the cells with an effective amount of one or more active substances selected from the group consisting of a fusion protein according to any one of E1 to E39, cells expressing the fusion protein according to E1 to E39, a nucleic acid according to any one of E40 to E45, a vector according to any one of E46 to E48, a viral particle according to any one of E49 to E56, and a pharmaceutical composition according to E57. E59. The method described in E58, wherein the cells are within the target area. E60. A method described in any one of E58-E59, wherein the subject is a human. E61. The method according to any one of E58-E60, wherein the cells are cells of the central nervous system and / or peripheral nervous system. E62. The method described in any one of E58-E61, wherein the subject has been identified as having TDP-43 disease. E63. The method according to E62, wherein the TDP-43 disease is selected from the group consisting of ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and limbic-dominant age-related TDP-43 encephalopathy. E64. The method described in E62 or E63, wherein the TDP-43 disease is ALS. E65. A method according to any one of E58-E64, which results in a decrease in the amount of aggregated TDP-43 protein in cells compared to control cells. E66. A method for treating, preventing, or delaying the progression of TDP-43 disease in a subject requiring treatment, prevention, or delay of its progression, comprising the step of administering an effective amount of one or more active substances selected from the group consisting of a fusion protein described in any one of E1 to E39, cells expressing a fusion protein described in E1 to E39, a nucleic acid described in any one of E40 to E45, a vector described in any one of E46 to E48, a viral particle described in any one of E49 to E56, and a pharmaceutical composition described in E57. E67. The method according to E66, wherein the TDP-43 disease is selected from the group consisting of ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and limbic-dominant age-related TDP-43 encephalopathy. E68. The method described in E67, where TDP-43 disease is ALS. E69. Use of one or more of the fusion protein described in any one of E1 to E39, cells expressing the fusion protein described in E1 to E39, nucleic acid described in any one of E40 to E45, vector described in any one of E46 to E48, viral particle described in any one of E49 to E56, and pharmaceutical composition described in E57 in the preparation of a pharmaceutical useful for preventing or delaying the progression of TDP-43 disease in a subject. E70. Use as described in E69, wherein TDP-43 disease is selected from the group consisting of ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and limbic-dominant age-related TDP-43 encephalopathy. E71. Use of TDP-43 as described in E69 or E70, when the disease is ALS. [Brief explanation of the drawing]

[0014] [Figure 1A-1] This figure shows the Clustal Omega sequence alignment of representative human J-domain sequences. Highly conserved HPD domains are indicated within the highlighted rectangles. [Figure 1A-2] This figure shows the Clustal Omega sequence alignment of representative human J-domain sequences. Highly conserved HPD domains are indicated within the highlighted rectangles. [Figure 1B-1] This figure shows the culcal omega sequence alignment of representative human J-domain sequences. [Figure 1B-2] This figure shows the culcal omega sequence alignment of representative human J-domain sequences. [Figure 2-1] This figure shows several exemplary fusion protein constructs containing the J domain and the TDP-43 binding domain. [Figure 2-2] This figure shows several exemplary fusion protein constructs containing the J domain and the TDP-43 binding domain. [Figure 3]This figure shows the visualization of TDP43-GFP fusion construct aggregation in cells as measured by fluorescence microscopy, and the effect of J-domain fusion proteins in reducing said aggregation. Fluorescence microscopy observations of cells transfected with a GFP reporter construct containing either full-length TDP-43 (GFP-TDP43FL; panels 1-4) or the C-terminal fragment of TDP-43 (GFP-TDPCTF; panels 5-8), and further containing scFv control (panels 2 and 6), DnaJB1-scFv(3B12A) fusion protein (panels 3 and 7), and DnaJB1-scFv(3B12A) fusion protein containing a P33Q mutation in the conserved HPD domain (panels 4 and 8), were compared to cells transfected with the reporter construct alone (panels 1 and 5). [Figure 4] This figure shows the quantification of aggregation in different constructs from Figure 3, standardized against control cells expressing only the GFP-TDP43CTF construct. [Figure 5] This figure shows immunoblot analysis of cell extracts expressing the GFP reporter construct with and without the fusion protein construct. The upper panel is a Western blot analysis using an anti-GFP antibody to detect the larger GFP-TDP43FL (lanes 1-4) and the smaller GFP-TDP43CTF (lanes 5-8). The lower panel is a Western blot analysis using an anti-FLAG epitope antibody to detect scFv(3B12A) control (lanes 2 and 6), the fusion protein construct (DnaJB1-scFv(3B12A), lanes 3 and 7), and the DnaJB1-scFv(3B12A) fusion protein containing the P33Q mutation within the conserved HPD domain (panels 4 and 8). [Figure 6] This figure shows immunoblot detection of GFP reporter constructs from soluble or insoluble fractions derived from cell extracts expressing either the GFP-TDP43FL or GFP-TDP43CTF reporter construct and nothing else (negative control), or construct 2 or 3. [Figure 7]This figure shows the visualization of TDP43-GFP fusion construct aggregation in cells as measured by fluorescence microscopy, and the effect of the J-domain fusion protein in reducing said aggregation. Fluorescence microscopy observations of cells transfected with a GFP reporter construct containing either full-length TDP-43 (GFP-TDP43FL) or the C-terminal fragment of TDP-43 (GFP-TDPCTF), and further containing constructs 2, 3, 5, 6, and 7, were compared to a control (none) expressing only the reporter. [Figure 8] This figure shows the visualization of TDP43-GFP fusion construct aggregation in cells as measured by fluorescence microscopy, and the effect of the J-domain fusion protein in reducing said aggregation. Fluorescence microscopy observations of cells transfected with a GFP reporter construct containing either full-length TDP-43 (GFP-TDP43FL) or the C-terminal fragment of TDP-43 (GFP-TDPCTF), and further containing constructs 1, 2, 3, 4, 9, 10, 11, or 14, were compared to a control (none) expressing only the reporter. [Figure 9A] This figure shows the quantification and detection of the TDP-43 reporter construct. Figure 9A shows the quantitative difference in aggregation in cells from the experiment shown in Figure 8. Figure 9B shows immunoblotting analysis of cell extracts using an anti-GFP antibody to quantify reporter construct levels in cell extracts. [Figure 9B] This figure shows the quantification and detection of the TDP-43 reporter construct. Figure 9A shows the quantitative difference in aggregation in cells from the experiment shown in Figure 8. Figure 9B shows immunoblotting analysis of cell extracts using an anti-GFP antibody to quantify reporter construct levels in cell extracts. [Figure 10-1]This figure shows the effect of bafilomycin A1 (BFA) (a potent inhibitor of the delayed phase of autophagy) or MG132 (a proteasome inhibitor) on reducing GFP-TDP43CTF in cells co-expressing construct 3. Cells were transfected with either the reporter construct GFP-TDP43FL (lane 2) or GFP-TDP43CTF (lanes 3-8), and co-transfected with the nucleic acid encoding construct 3 (lanes 4-8). Treatment of cells with either BFA (0.01 μM in lane 5, and 0.1 μM in lane 6) resulted in higher levels of the GFP-TDP43CTF reporter construct, as detected by immunoblotting. In contrast, treatment with 0.1 μM or 1.0 μM of MG132 (lanes 7 and 8, respectively) had little to no effect. [Figure 10-2] This figure shows the effect of bafilomycin A1 (BFA) (a potent inhibitor of the delayed phase of autophagy) or MG132 (a proteasome inhibitor) on reducing GFP-TDP43CTF in cells co-expressing construct 3. Cells were transfected with either the reporter construct GFP-TDP43FL (lane 2) or GFP-TDP43CTF (lanes 3-8), and co-transfected with the nucleic acid encoding construct 3 (lanes 4-8). Treatment of cells with either BFA (0.01 μM in lane 5, and 0.1 μM in lane 6) resulted in higher levels of the GFP-TDP43CTF reporter construct, as detected by immunoblotting. In contrast, treatment with 0.1 μM or 1.0 μM of MG132 (lanes 7 and 8, respectively) had little to no effect. [Figure 11A] This figure shows the effect of co-expressing construct 3 (lanes 4 and 8) on the levels of the GFP-TDP43CTF reporter in the soluble (non-aggregated) and insoluble (aggregated) fractions of cell extracts when explored with anti-TDP43 antibody (Figure 11A) and anti-phospho-TDP43 antibody (Figure 11B). [Figure 11B]This figure shows the effect of co-expressing construct 3 (lanes 4 and 8) on the levels of the GFP-TDP43CTF reporter in the soluble (non-aggregated) and insoluble (aggregated) fractions of cell extracts when explored with anti-TDP43 antibody (Figure 11A) and anti-phospho-TDP43 antibody (Figure 11B). [Figure 12] This figure shows the effects of the simultaneous expression of constructs 3 (lane 3), 7 (lane 4), 2 (lane 5), 15 (lane 6), and 16 (lane 7) on reducing the level of phosphorylated GFP-TDP43CTF reporter in cell extracts when detected with an anti-phospho TDP43 antibody. [Figure 13] This figure shows the effects of co-expressing construct 3 (lanes 3, 5, 8, and 10) in cells expressing either TDP43FL (lanes 2, 3, 7, and 8) or the TDP43ΔNLS construct (lanes 4, 5, 9, and 10) on the levels of TDP-43 (top panel when searched with anti-TDP43 antibody), phosphorylated TDP-43 (second panel when searched with anti-phospho TDP-43 antibody), Flag epitope (third panel when searched with anti-FLAG antibody), and tubulin (bottom panel, anti-tubulin antibody). [Figure 14] This figure shows additional constructs tested for their ability to reduce phosphorylated TDP-43. Cells expressing either GFP-TDP43FL (lanes 1 and 7) or GFP-TDP43CTF (lanes 2-6, 8-12) were co-transfected with construct 3 (lanes 3 and 9), construct 17 (lanes 4 and 10), construct 18 (lanes 5 and 11), and construct 19 (lanes 6 and 12). The soluble fraction (lanes 1-6) and insoluble fraction (lanes 7-12) were explored with an anti-phosphoTDP43 antibody. [Figure 15A]This figure summarizes the results of C57 mice injected with AAV rh10 containing either a control or a vector encoding construct 3, administered by injection either intraarachnoid (IT) or intraventricular (ICV). Figure 15A summarizes the study schedule. Figure 15B shows immunoblots of cerebral extracts from mice 3 weeks after IT administration of AAV rh10 containing either a control (lanes 1 and 2) or a vector containing construct 3 (lanes 3 and 4). Figure 15C shows immunoblots of cerebral extracts from mice after ICV injection. Lanes 1-3 show immunoblots of cerebral extracts from mice 3 weeks after ICV administration of AAV rh10 containing either a control (lanes 1 and 2) or a vector containing construct 3 (lane 3). Lanes 4-8 show immunoblots of mice at 8 weeks from control (lanes 4-6) and construct 3 (lanes 7 and 8) mice. [Figure 15B] This figure summarizes the results of C57 mice injected with AAV rh10 containing either a control or a vector encoding construct 3, administered by injection either intraarachnoid (IT) or intraventricular (ICV). Figure 15A summarizes the study schedule. Figure 15B shows immunoblots of cerebral extracts from mice 3 weeks after IT administration of AAV rh10 containing either a control (lanes 1 and 2) or a vector containing construct 3 (lanes 3 and 4). Figure 15C shows immunoblots of cerebral extracts from mice after ICV injection. Lanes 1-3 show immunoblots of cerebral extracts from mice 3 weeks after ICV administration of AAV rh10 containing either a control (lanes 1 and 2) or a vector containing construct 3 (lane 3). Lanes 4-8 show immunoblots of mice at 8 weeks from control (lanes 4-6) and construct 3 (lanes 7 and 8) mice. [Figure 15C]This figure summarizes the results of C57 mice injected with AAV rh10 containing either a control or a vector encoding construct 3, administered by injection either intraarachnoid (IT) or intraventricular (ICV). Figure 15A summarizes the study schedule. Figure 15B shows immunoblots of cerebral extracts from mice 3 weeks after IT administration of AAV rh10 containing either a control (lanes 1 and 2) or a vector containing construct 3 (lanes 3 and 4). Figure 15C shows immunoblots of cerebral extracts from mice after ICV injection. Lanes 1-3 show immunoblots of cerebral extracts from mice 3 weeks after ICV administration of AAV rh10 containing either a control (lanes 1 and 2) or a vector containing construct 3 (lane 3). Lanes 4-8 show immunoblots of mice at 8 weeks from control (lanes 4-6) and construct 3 (lanes 7 and 8) mice. [Figure 16A] This figure summarizes the results using ΔNLS8 mice injected with AAV rh10 containing either a control or a vector encoding construct 3, administered via ICV injection. Figure 16A shows the study schedule. Figure 16B shows the mean body weight of males from each group. Figure 16C shows the survival rates of mice from different groups. [Figure 16B] This figure summarizes the results using ΔNLS8 mice injected with AAV rh10 containing either a control or a vector encoding construct 3, administered via ICV injection. Figure 16A shows the study schedule. Figure 16B shows the mean body weight of males from each group. Figure 16C shows the survival rates of mice from different groups. [Figure 16C] This figure summarizes the results using ΔNLS8 mice injected with AAV rh10 containing either a control or a vector encoding construct 3, administered via ICV injection. Figure 16A shows the study schedule. Figure 16B shows the mean body weight of males from each group. Figure 16C shows the survival rates of mice from different groups. [Modes for carrying out the invention]

[0015] definition As used herein and in the claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, the term “a cell” includes multiple cells, including a mixture thereof.

[0016] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, and may contain modified amino acids, which may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified by any other operation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component.

[0017] As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including, but not limited to, both D- and L-type optical isomers, as well as amino acid analogs and peptide mimes. Standard one-letter or three-letter codes are used to name amino acids.

[0018] "Host cells" include individual cells or cell cultures that may or may have been recipients of the vector of interest. Host cells include offspring of a single host cell. These offspring are not necessarily identical (in morphology or in the genome of the whole DNA complement) to the original parent cell due to natural, accidental, or planned mutations. Host cells include cells transfected in vivo with the vector of this invention.

[0019] Where used to describe the various polypeptides disclosed herein, “isolated” means a polypeptide that has been identified and separated from components of its natural environment, and / or recovered. These contaminating components of its natural environment are typically materials that interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not exist naturally do not require “isolation” to distinguish them from their naturally occurring counterparts. In addition, “concentrated,” “isolated,” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are distinguishable from their naturally occurring counterparts by generally having a higher concentration or number of molecules per unit volume than their naturally occurring counterparts. Generally, polypeptides produced by recombinant means and expressed in host cells are considered “isolated.”

[0020] An "isolated" polynucleotide, or nucleic acid encoding a polypeptide, or any other nucleic acid encoding a polypeptide, is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule that is normally associated with the natural source of the nucleic acid encoding that polypeptide. An isolated polypeptide-encoding nucleic acid molecule takes on a form or configuration other than that which is found in nature. Thus, an isolated polypeptide-encoding nucleic acid molecule is distinguished from the nucleic acid molecule encoding that particular polypeptide as it would be if it were present in a natural cell. However, an isolated polypeptide-encoding nucleic acid molecule is one that is contained in a cell that normally expresses that polypeptide, where, for example, the nucleic acid molecule is located in a chromosomal or extrachromosomal location different from its location in a natural cell.

[0021] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “nucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. 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 from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, 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 analogues. Modifications to the nucleotide structure may be given before or after the assembly of the polymer, if present. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components.

[0022] As defined herein, the terms “TDP-43 disorder” or “TDP-43-mediated disease” refer to disorders associated with the formation of intracellular TDP-43 aggregates, particularly aggregates of TDP-43 mutant proteins. Examples of TDP-43 disorders include, but are not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, hippocampal sclerosis, Lewy body dementia, and limbic-dominant age-related TDP-43 encephalopathy.

[0023] A “vector” is a nucleic acid molecule that preferably self-replicates in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for the insertion of DNA or RNA into cells, replication vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. 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 polypeptides. An “expression system” usually implies a suitable host cell composed of an expression vector capable of functioning to produce a desired expression product.

[0024] The term “operatably ligated” refers to the juxtaposition of the described components, where they are related in a way that allows them to function in their intended manner. A coding sequence and an “operatably ligated” regulatory sequence are ligated so that the expression of the coding sequence is achieved under conditions compatible with the regulatory sequence. “Operatably ligated” sequences may include both expression regulatory sequences that are close to the gene of interest and expression regulatory sequences that act trans or at a distance to control the gene of interest. The term “expression regulatory sequence” refers to a polynucleotide sequence that is necessary for the expression and processing of the coding sequence to which it is ligated. Expression regulatory sequences include appropriate transcription start, terminate, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, where necessary, sequences that enhance protein secretion. The properties of such regulatory sequences vary depending on the host organism; in prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such regulatory sequences generally include promoters and transcription termination sequences. The term “regulatory sequence” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Unless otherwise specified, any description or reference herein to inserting a nucleic acid molecule encoding the fusion protein of the present invention into an expression vector means that the inserted nucleic acid is also operably linked within the vector to a functional promoter and other transcription and translation regulatory elements required for the expression of the encoded fusion protein when the expression vector containing the inserted nucleic acid molecule is introduced into a compatible host cell or a compatible cell of an organism.

[0025] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of in vitro cloning, restriction and / or ligation steps, and various combinations of other procedures that produce a construct that can potentially be expressed in host cells.

[0026] The terms “gene” and “gene fragment” are used interchangeably herein. They refer to polynucleotides containing at least one open reading frame that, after transcription and translation, has the ability to code for a particular protein. A gene or gene fragment can be genomic DNA or cDNA, insofar as its polynucleotides contain at least one open reading frame, and it may encompass an entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterogeneous polynucleotides linked together.

[0027] The terms “disease” and “disorder” are used interchangeably to refer to pathological conditions identified by an acceptable level of medical care and practice in the art.

[0028] As used herein, the term “effective dose” means an amount of treatment that is sufficient to reduce or improve the severity and / or duration of a disease or one or more symptoms; prevent the progression of an adverse or pathological condition; cause regression of a pathological condition; prevent the recurrence, development, onset or progression of one or more symptoms associated with a pathological condition; detect a disorder; or enhance or improve the prophylactic or therapeutic effect of a treatment (e.g., administration of another prophylactic or therapeutic agent).

[0029] As used herein, the term “J domain” refers to the fragment that retains the ability to accelerate the catalytic activity of Hsp70 and its congeners' endogenous ATPases. The J domains of various J proteins have been determined (see, e.g., Kampinga et al. (2010) Nat. Rev., 11:579-592; Hennessy et al. (2005) Protein Science, 14:1697-1709 (each incorporated as a whole by reference)), and are characterized by several hallmarks: four α-helices (I, II, III, IV), and a highly conserved tripeptide sequence motif of histidine, proline, and aspartic acid (called the “HPD motif”), usually between helices II and III. Typically, the J-domain of a J-protein is between 50 and 70 amino acids long, and the site of interaction (binding) of the J-domain with the Hsp70-ATP chaperone protein is thought to be a region extending from within helix II, where the HPD motif is required for stimulation of Hsp70 ATPase activity. As used herein, the term “J-domain” is intended to include native J-domain sequences and their functional variants that retain the ability to accelerate the endogenous ATPase activity of Hsp70, the ability of which can be measured using methods well known in the art (e.g., see Horne et al. (2010) J. Biol. Chem., 285, 21679–21688, incorporated herein by reference in whole). An unrestricted list of human J-domains is provided in Table 1.

[0030] Detailed explanation The inventors have found that contacting cells to some extent with a fusion protein construct containing the J domain and TDP-43 binding domain of the J protein produces an unexpected effect of reducing the aggregation of mutant TDP-43 protein. Aggregation of mutant TDP-43 is thought to cause several devastating diseases, including, but not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and Alzheimer's disease. Therefore, useful compositions and methods for treating TDP-43 disorders, for example in subjects where such treatment is needed, are provided herein.

[0031] To overcome the problems associated with chaperone-based therapy, the inventors investigated whether it was possible to design highly specific artificial chaperone proteins. The inventors designed a series of fusion protein constructs comprising an effector domain (J domain sequence) for Hsp70 binding / activation and a domain conferring specificity to the TDP-43 protein. The resulting fusion proteins act to accelerate the catalytic activity of Hsp70 and its related endogenous ATPases, resulting in increased protein folding, reduced aggregation, and / or accelerated clearance.

[0032] I. Fusion protein constructs a. Useful J domains in the present invention The J domains of various J proteins have been determined. See, for example, Kampinga et al., Nat. Rev., 11:579~592 (2010); Hennessy et al., Protein Science, 14:1697~1709 (2005). The J domains useful in preparing the fusion proteins of the present invention are key to defining the features of the J domain that primarily accelerate Hsp70 ATPase activity. Therefore, the isolated J domains useful in the present invention comprise a polypeptide domain characterized by four α-helices (I, II, III, IV) and a tripeptide sequence of histidine, proline, and aspartic acid (called the "HPD motif"), which is usually highly conserved between helices II and III. Typically, the J domain of a J protein is between 50 and 70 amino acids long, and the site of interaction (binding) of the J domain with the Hsp70-ATP chaperone protein is thought to be a region extending from within helix II, with the HPD motif forming the basis of primitive activity. Representative J domains include, but are not limited to, those of DnaJB1, DnaJB2, DnaJB6, DnaJC6, the J domain of the SV40 large T antigen, and the J domain of mammalian cysteine ​​string protein (CSP-α). Amino acid sequences for these and other J domains that may be used in the fusion proteins of the present invention are provided in Table 1. The conserved HPD motif is highlighted in bold. In one embodiment, the fusion protein disclosed herein includes a J domain selected from the group consisting of SEQ ID NOs: 1 to 50. As shown in the Examples section below, the inventors have found that the use of a J domain lacking the conserved "HPD" motif is not able to reduce protein aggregation. Therefore, in another embodiment, the fusion protein disclosed herein comprises a J domain containing a consensus HPD motif. In one particular embodiment, a selection is made from the group consisting of SEQ ID NOs: 1-15 and 17-50.

[0033] In certain embodiments, the fusion protein includes a J domain selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49.

[0034] [Table 1-1]

[0035] [Table 1-2]

[0036] [Table 1-3]

[0037] b. TDP-43 binding domain The fusion protein also contains at least one TDP-43 binding domain. The TDP-43 binding domain may be a single-chain polypeptide or a multimeric polypeptide linked to the J domain to form the fusion protein.

[0038] Ideally, the TDP-43 binding domain should have sufficient affinity to bind to the TDP-43 protein when it is present at pathological levels within the cell. Therefore, in one embodiment, when the fusion protein is tested by ELISA on a 96-well microtiter plate, it should have K2+, ≤2 μM, ≤1 μM, ≤500 nM, ≤300 nM, ≤100 nM, and ≤30 nM of a TDP-43 reporter construct (e.g., full-length TDP-43 (Novus Biologicals, NBP2-22850, Centennial, CO)). D It contains a TDP-43 binding domain.

[0039] The TDP-43 binding domain has been previously identified and characterized (see, for example, U.S. Patent No. 10,259,866, WO2016 / 53610, WO2018 / 218252, WO2019 / 134981, and WO2019 / 177138, respectively, incorporated herein by reference). Therefore, in another embodiment, the fusion protein includes a TDP-43 binding domain selected from the group consisting of SEQ ID NOs. 51 to 55 (see, for example, Table 2). In one particular embodiment, the fusion protein includes the TDP-43 binding domain of SEQ ID NO. 51. In another embodiment, the fusion protein includes the TDP-43 binding domain of SEQ ID NO. 53. In yet another embodiment, the fusion protein includes the TDP-43 binding domain of SEQ ID NO. 52.

[0040] In another embodiment, the fusion protein also intends to use a TDP-43 binding domain that is chemically conjugated with the J domain. The TDP-43 binding domain may be conjugated directly with the J domain, or it may be conjugated with the J domain by a linker. For example, there are numerous chemical crosslinkers known to those skilled in the art that are useful for crosslinking the TDP-43 binding domain with the J domain, or a targeting domain, with a fusion protein containing the TDP-43 binding domain and the J domain. For example, crosslinkers are heterobifunctional crosslinkers that can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers offer the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of undesirable side reactions such as homoprotein polymers. Various heterobifunctional crosslinking agents are known in the art, including succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Crosslinking agents having an N-hydroxysuccinimide moiety can generally be obtained as N-hydroxysulfosuccinimide analogs with higher water solubility. In addition, crosslinking agents having disulfide crosslinks in the linking chain can be synthesized instead as alkyl derivatives to reduce the amount of linker cleavage in vivo. Several other crosslinking agents exist, including heterobifunctional crosslinking agents, homobifunctional crosslinking agents, and photoreactive crosslinking agents.Disuccinimidyl sberate (DSS), bismaleimide hexane (BMH), and dimethylpimerimidate·2HCl are examples of useful homobifunctional crosslinking agents used in this disclosure, while bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinking agents. For a recent overview of protein coupling techniques, see Means et al., (1990) Bioconj. Chem. 1:2-12, incorporated herein by reference.

[0041] [Table 2]

[0042] c. Optional linker The fusion proteins described herein may optionally contain one or more linkers. The linkers may be peptidogenic or non-peptidogenic. The purpose of the linkers is, among other things, to provide appropriate distances between functional domains within the protein (e.g., between the J domain and the TDP-43 binding domain, between tandem arrangements of TDP-43 binding domains, between the J domain and the TDP-43 binding domain and any of the optional targeting reagents, or between the J domain and the TDP-43 binding domain and any of the optional detection domains or epitopes) for the optimal function of each domain. Clearly, the linkers preferably do not interfere with the function of the J domain, the target protein binding domain, or any of the fusion proteins of the present invention. If the linkers are present in the fusion proteins of the present invention, they are selected to attenuate cytotoxicity caused by the target protein (TDP-43 protein), and they 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 on the nucleic acid segment per cloning site of an expression vector into which a nucleic acid segment encoding a protein domain or the entire fusion protein, as described herein, is inserted in frame. In one embodiment, the peptide linker is between 1 and 20 amino acids long. In another embodiment, the peptide linker is between 2 and 15 amino acids long. In yet another embodiment, the peptide linker is between 2 and 10 amino acids long.

[0043] Selecting one or more polypeptide linkers to construct the fusion proteins according to the present invention is within the scope of the knowledge and skills 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 incorporated herein by reference as a whole). Examples of linkers of two or more amino acids that may be used in preparing the fusion proteins according to the present invention are, but are not limited to, those provided in Table 3 below.

[0044] [Table 3]

[0045] d. Targeting reagents The fusion proteins disclosed herein may further include a targeting moiety. As used herein, the terms “targeting moiety” and “targeting reagent” refer to a substance associated with a fusion protein that is interchangeable and enhances the binding, transport, accumulation, residence time, bioavailability, or modifies its biological activity or therapeutic effect in cells or in the body of a subject. The targeting moiety may be functional at the tissue, cell, and / or intracellular levels. The targeting moiety can, for example, direct the localization or intracellular distribution of the fusion protein to specific cells, tissues, or organs upon administration of the fusion protein to a subject. In one embodiment, the targeting moiety is located at the N-terminus of the fusion protein. In another embodiment, the targeting moiety is located at the C-terminus of the fusion protein. In yet another embodiment, the targeting moiety is located internally. In yet another embodiment, the targeting moiety attaches to the fusion protein via chemical conjugation.

[0046] The targeting moieties may include, but are not limited to, organic or inorganic molecules, peptides, peptide mimes, 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, chemoattractants, cytokines, chemokines, drugs, or small molecules.

[0047] In exemplary embodiments of the present invention, the targeting moiety enhances the binding, transport, accumulation, residence time, and bioavailability of the platform, or its associated ligand and / or active substance, or modifies its biological activity or therapeutic effect, in target cells or tissues, such as nerve cells, the central nervous system, and / or the peripheral nervous system. Therefore, the targeting moiety may have specificity for cellular receptors associated with the central nervous system, or otherwise be related to enhanced delivery to the CNS via the blood-brain barrier (BBB). Consequently, such ligands may be both ligands and targeting moieties.

[0048] In some embodiments, the targeting moiety may be a cell-permeable peptide, such as that described in U.S. Patent No. 10,111,965, which is incorporated herein by reference in whole. In another embodiment, the targeting moiety may be an antibody or its antigen-binding fragment or single-chain derivative, such as that described in U.S. Patent Application No. 16 / 131,591, which is incorporated herein by reference in whole. In a further embodiment, the targeting moiety may be an amino acid sequence relating to a nuclear localization signal or an export-nuclear localization signal.

[0049] The targeting moiety can be linked to a platform for targeted cell delivery by directly or indirectly binding to its core. For example, in embodiments where the core includes nanoparticles, the conjugation of the targeting moiety with the nanoparticles can utilize similar functional groups used to tether PEG to the nanoparticles. Thus, the targeting moiety can be directly bound to the nanoparticles through functionalization of the targeting moiety. Alternatively, the targeting moiety can be indirectly bound to the nanoparticles through conjugation with functionalized PEG of the targeting moiety, as discussed above. The targeting moiety can adhere to the core by covalent, non-covalent, or electrostatic interactions. In one embodiment, the targeting moiety is a peptide. In certain embodiments, the targeting moiety is a peptide that covalently attaches to the N-terminus of a fusion protein.

[0050] e. Epitope In certain embodiments, the fusion protein of the present invention contains an optional epitope or tag that can impart additional properties to the fusion protein. As used herein, the terms “epitope” and “tag” are used interchangeably to refer to an amino acid sequence, typically 300 amino acids or less in length, that typically attaches to the N-terminus or C-terminus of the fusion protein. In one embodiment, the fusion protein of the present invention further includes an epitope used to facilitate purification. Examples of such epitopes useful for purification, provided in Table 4 below, include the human IgG1 Fc sequence (SEQ ID NO: 67), the FLAG epitope (DYKDDDDK, SEQ ID NO: 68), the His6 epitope (SEQ ID NO: 69), the c-myc (SEQ ID NO: 70), the HA (SEQ ID NO: 71), the V5 epitope (SEQ ID NO: 72), or glutathione-s-transferase (SEQ ID NO: 73). In another embodiment, the fusion protein of the present invention further includes an epitope used to increase the half-life of the fusion protein when administered to a subject, e.g., a human. An example of such an epitope useful for increasing half-life is the human Fc sequence. Therefore, in one particular embodiment, the fusion protein includes a human Fc epitope in addition to the J domain and the TDP-43 binding domain. The epitope is located at the C-terminus of the fusion protein.

[0051] [Table 4]

[0052] f. Cell-permeable peptides In other embodiments, the fusion proteins described herein may further include cell-permeable peptides. Cell-permeable peptides are known to deliver conjugated cargo, whether it be small molecules, peptides, proteins, or nucleic acids, into cells. Non-limiting examples of cell-permeable peptides in the fusion proteins of the present invention include polycationic peptides, e.g., HIV TAT peptides 49-57, polyarginine, and penetratin pAntan (43-58); amphiphilic peptides, e.g., pep-1; hydrophobic peptides, e.g., C405Y; and others of the same kind. See Table 5 below.

[0053] [Table 5]

[0054] Therefore, in one embodiment, the fusion protein comprises a cell-permeable peptide and a fusion protein, wherein the cell-permeable peptide is selected from the group consisting of SEQ ID NOs: 74-77, and the fusion protein comprises a J domain and a TDP-43 binding domain. In another embodiment, the fusion protein is selected from the group consisting of SEQ ID NOs: 80-85 and 89-96. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 74, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the cell-permeable peptide of SEQ ID NO: 75, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the cell-permeable peptide of SEQ ID NO: 76, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the cell-permeable peptide of SEQ ID NO: 77, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. Cells expressing this fusion protein construct, including the cell-permeable peptide, may be administered to a subject, such as a human subject (e.g., a patient with or at risk of developing TDP-43 impairment). The fusion protein is secreted from the cells and helps reduce TDP-43-containing protein aggregation and / or associated cytotoxicity.

[0055] g. Arrangement of the J domain and TDP-43 binding domain The fusion proteins described herein can be configured in numerous ways. In one embodiment, the TDP-43 binding domain is attached to the C-terminal side of the J domain. In another embodiment, the TDP-43 binding domain is attached to the N-terminal side of the J domain. The TDP-43 binding domain and the J domain can optionally be separated via a linker as described above in any of the configurations.

[0056] In some embodiments, the J domain may be attached to multiple TDP-43 binding domains, for example, two TDP-43 binding domains, three TDP-43 binding domains, four TDP-43 binding domains, or more. The TDP-43 binding domains may be attached to the N-terminal side of the J domain, or to the C-terminal side of the J domain. In another embodiment, the TDP-43 binding domains may be attached to both the N-terminal and C-terminal sides of the J domain. Each of the multiple TDP-43 binding domains may be the same TDP-43 binding domain. In another embodiment, each of the multiple TDP-43 binding domains in the fusion protein may be a different TDP-43 binding domain (i.e., a different sequence).

[0057] In some embodiments, the fusion protein belongs to the following group: 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. TDNAJ-X-TTTTTDNAJ-XT, j. TXTX-DNAJ-X-TT, k. TTDNAJ-XTX-TTTTTDNAJ-XT, l. TXTX-DNAJ-XTXTXT, m. TXTXTX-DNAJ-XT, n. TXTXTX-DNAJ-XTXT, o. TXTXTX-DNAJ-XTXTXT, p. DnaJ-X-DnaJ-XTXT, q. TX-DnaJ-X-DnaJ, r. TXTX-DnaJ-X-DnaJ, and s. TX-TDnaJ-X-TDnaJ-X-TTTT It may include a structure selected from, Here, T is a TDP-43 binding domain, DNAJ is the J domain of the J protein. X is an optional linker.

[0058] In one embodiment, the fusion protein includes a J domain selected from the group consisting of SEQ ID NOs: 5, 6, 10, 24, and 31. In one particular embodiment, the fusion protein includes the J domain of SEQ ID NO: 5.

[0059] In another embodiment, the TDP-43 binding domain is selected from the group consisting of SEQ ID NOs. 51 to 55. In one particular embodiment, the TDP-43 binding domain is selected from the group consisting of SEQ ID NOs. 51 to 53.

[0060] In another embodiment, the fusion protein includes the J domain of SEQ ID NO: 5 and the TDP-43 binding domain of SEQ ID NO: 51. In yet another embodiment, the fusion protein includes the J domain of SEQ ID NO: 5 and at least two copies of the TDP-43 binding domain of SEQ ID NO: 53.

[0061] Non-limiting examples of fusion protein constructs containing the J domain and the TDP-43 binding domain are schematically illustrated in Figure 2 and also shown in Table 6 below. In another embodiment, a specific fusion protein construct is selected from the group consisting of SEQ ID NOs: 80-85 and 89-96.

[0062] [Table 6-1]

[0063] [Table 6-2]

[0064] [Table 6-3]

[0065] II. Nucleic acids encoding fusion protein constructs In another aspect of the present invention, an isolated nucleic acid is provided comprising (a) a polynucleotide encoding a fusion protein as described in any of the embodiments described above, or (b) a polynucleotide selected from complements of the polynucleotide of (a). The present invention provides an isolated nucleic acid encoding a fusion protein comprising a J domain and a TDP-43 binding domain, as well as sequences complementary to such nucleic acid molecules encoding the fusion protein, in addition to homologous variants thereof. In another aspect, the present invention encompasses methods for producing nucleic acids encoding the fusion proteins disclosed herein, sequences complementary to the nucleic acids encoding the fusion proteins, in addition to homologous variants thereof. Nucleic acids according to this aspect of the present invention may be pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0066] In yet another embodiment, a method for producing a fusion protein is disclosed, comprising the steps of (a) synthesizing and / or assembling nucleotides encoding a 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 cause or enable the expression of the fusion protein in the transformed host cells, thereby producing a bioactive fusion protein, the produced bioactive fusion protein being recovered as an isolated fusion protein by standard protein purification methods known in the art. Standard recombination techniques in molecular biology are used to construct the polynucleotides and expression vectors of the present invention.

[0067] According to the present invention, the nucleic acid sequence encoding the fusion protein disclosed herein (or its complement) is used to construct a recombinant DNA molecule that directs the expression of the fusion protein in a suitable host cell. Several cloning strategies are suitable for carrying out the present invention, many of which are used to construct constructs containing genes encoding the fusion protein or its complement. In some embodiments, cloning strategies are used to produce genes encoding the fusion protein or its complement.

[0068] In a particular embodiment, the nucleic acid encoding one or more fusion proteins is an RNA molecule and may be pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0069] In various embodiments, the nucleic acid is mRNA introduced into a cell to transiently express a desired polypeptide. As used herein, “transient” refers to the expression of an unintegrated transgene for a period of time of several hours, several days, or several weeks, the duration of which is shorter than that of polynucleotide expression when integrated into the genome or contained within a stable plasmid replicon in a cell.

[0070] In certain embodiments, the mRNA encoding the polypeptide is mRNA transcribed in vitro. As used herein, “RNA transcribed in vitro” means RNA, preferably mRNA, that is synthesized in vitro. Generally, RNA transcribed in vitro is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used to produce RNA transcribed in vitro. In certain embodiments, mRNA may further include a 5' cap or a modified 5' cap and / or a poly(A) sequence. As used herein, a 5' cap (also called an RNA cap, RNA 7-methylguanosine cap, or RNA m7G cap) is a modified guanine nucleotide attached to the “front” or 5' end of eukaryotic messenger RNA immediately after the start of transcription. The 5' cap includes a terminal group that is ligated to the first transcribed nucleotide, is recognized by ribosomes, and is protected from Rnases. The capping portion may be modified to modulate the functionality of mRNA, such as its stability or translational efficiency. In certain embodiments, mRNA may include a poly(A) sequence of between about 50 and about 5000 adenines. In one embodiment, mRNA may include a poly(A) sequence between about 100 and about 1000 bases, between about 200 and about 500 bases, or between about 300 and about 400 bases. In one aspect, mRNA contains poly(A) sequences of approximately 65, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more bases. The poly(A) sequences may be chemically or enzymatically modified to regulate the functionality of mRNA, such as localization, stability, or translation efficiency.

[0071] As used herein, the terms “polynucleotide variant” and “variant” and other similar terms refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes with a reference sequence under stringent conditions as defined below. These terms include polynucleotides in which one or more nucleotides are added, deleted, or replaced with different nucleotides compared to the reference polynucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, and that the thereby modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0072] In a particular embodiment, the nucleic acid sequence contains a nucleotide sequence encoding the gene of interest (e.g., a fusion protein comprising a J domain and a polyglutamine-binding domain) within the nucleic acid cassette. As used herein, the terms “nucleic acid cassette” or “expression cassette” refer to the genetic sequence within a vector capable of expressing RNA and subsequently polypeptides. In one embodiment, the nucleic acid cassette contains the gene of interest, e.g., the polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and the gene of interest, e.g., the polynucleotide of interest. The vector may contain one, two, three, four, five, six, seven, eight, nine, or ten or more nucleic acid cassettes. The nucleic acid cassette is locologically and ordinally oriented within the vector so that the nucleic acids within the cassette can be transcribed to RNA, translated into proteins or polypeptides as needed, undergo appropriate post-translational modifications required for activity in transformed cells, and translocated to appropriate compartments for biological activity by targeting to appropriate intracellular compartments or secretion to extracellular compartments. Preferably, the cassette has 3' and 5' ends adapted for immediate insertion into the vector, for example, it has restriction endonuclease sites at each end. The cassette can be removed and inserted into plasmids or viral vectors as a single unit.

[0073] Exemplary ubiquitous expression regulatory sequences suitable for use in specific embodiments include the cytomegalovirus (CMV) early promoter, the simian virus 40 (SV40) (e.g., early or late), the Moloney's mouse leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, and vaccinia virus-derived H5, P7.5, and Pl. I promoter, elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (b-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 (Okabe et al. (1997) FEBS) Examples include, but are not limited to, the (MND)U3 promoter in which the β-actin promoter, myeloproliferative sarcoma virus enhancer, and negative regulatory region are deleted and replaced with the dl587rev primer binding site (Haas et al., Journal of Virology. 2003; 77(l7): 9439-9450).

[0074] In one embodiment, at least one element for enhancing the specificity and expression of the transgene target, such as a promoter (see, for example, Powell et al. (2015) Discovery Medicine 19(102):49-57 (the contents of which are incorporated herein by reference in their entirety)), may be used in conjunction with the polynucleotides described herein. Promoters that promote expression in most tissues include, but are not limited to, human elongation factor la-subunit (EFla), early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements are used to restrict expression to certain cell types, and include, but are not limited to, nervous system promoters that may be used to restrict expression to neurons, astrocytes, or oligodendrocytes. Non-limiting examples of tissue-specific expression elements for neurons include, but are not limited to, the promoters of neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl CpG-binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH, ηβ2, PPE, Enk, and EAAT2. Non-limiting examples of tissue-specific expression elements for astrocytes include the promoters of glial fibrillary acidic protein (GFAP) and EAAT2. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include the myelin basic protein (MBP) promoter. Yu et al. (incorporated collectively by reference in (2011) Molecular Pain, 7:63) evaluated eGFP expression under CAG, EFIa, PGK, and UBC promoters in rat DRG cells and primary DRG cells using lentiviral vectors and found that UBC showed weaker expression than the other three promoters, and that glial expression was only 10-12% for all promoters.Soderblom et al. (incorporated collectively by reference, E. Neuro 2015) evaluated eGFP expression in AAV8 with CMV and UBC promoters and AAV2 with CMV promoter after intracortical injection. Intranasal administration of plasmids containing UBC or EFIa promoters showed higher sustained airway expression than expression with CMV promoter (see, e.g., Gill et al., (2001) Gene Therapy, vol. 8, 1539-1546, incorporated collectively by reference). Husain et al. (incorporated collectively by reference, (2009) Gene Therapy) evaluated HβH constructs with hGUSB promoter, HSV-1LAT promoter, and NSE promoter and found that HβH constructs showed weaker expression than NSE in mouse brains. Passini and Wolfe (incorporated as a whole by reference, J. Virol. 2001, 12382-12392) evaluated the long-term effects of ΗβΗ vectors after intracerebroventricular injection in neonatal mice and found sustained expression for at least one year. Xu et al. (incorporated as a whole by reference, (2001) Gene Therapy, 8, 1323-1332) found low expression in all brain regions when NF-L and NF-H promoters were used, compared to CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE+wpre, NSE(0.3kb), NSE(1.8kb), and NSE(1.8kb+wpre). Xu et al. found that the promoter activity, in descending order, was NSE (1.8kb), EF, NSE (0.3kb), GFAP, CMV, hENK, PPE, NFL, and NFH. NFL is a 650-nucleotide promoter, and NFH is a 920-nucleotide promoter; neither is present in the liver, but NFH is abundant in proprioceptive neurons, the brain, and the spinal cord, and NFH is present in the heart. Scn8a is a 470-nucleotide promoter, expressed in the DRG, spinal cord, and brain, with particularly high expression in hippocampal neurons and cerebellar Purkinje cells, the cortex, the thalamus, and the hypothalamus.(For example, see Drews et al. 2007 and Raymond et al. 2004, which are incorporated by reference as a whole.)

[0075] III. Vectors containing nucleic acids encoding fusion proteins Vectors containing nucleic acids according to the present invention are also provided. Such vectors preferably include additional nucleic acid sequences such as elements necessary for the transcription / translation of the nucleic acid sequence encoding a phosphatase (e.g., promoter and / or terminator sequences). The vector may also include a nucleic acid sequence encoding a selection marker (e.g., an antibiotic) for selecting or maintaining host cells transformed with the vector. The term “vector” refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally ligated to the vector nucleic acid molecule, for example, by insertion therein. The vector may include sequences that direct self-replication in a cell, or may include sequences sufficient to enable integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides intended herein to infected cells (e.g., nerve cells). In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a fusion protein comprising a J domain and a TDP-43 binding domain. Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0076] Examples of vectors include, but are not limited to, plasmids, self-replicating sequences, and transposition factors such as piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives. Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or Pl-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of viruses useful as vectors include, but are not limited to, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus (VIMS)), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). 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 lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of polypeptides disclosed herein may be ligated into such expression vectors for polypeptide expression in mammalian cells.

[0077] In certain embodiments, the vector is an episomal vector, or a vector maintained outside the chromosome. As used herein, the term “episomal” means a vector that can replicate without integration into the host's chromosomal DNA and without being gradually lost from a dividing host cell; that is, the vector replicates outside the chromosome or episomalally.

[0078] A vector may contain one or more recombination sites for any of a variety of site-specific recombinases. It should be understood that the target sites for site-specific recombinases are in addition to any sites required for the incorporation of the vector, e.g., a retroviral vector or a lentiviral vector. As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombinant site” refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0079] For example, one recombination site for Cre recombinase is loxP, a 34-base pair sequence containing two 13-base pair reverse repeats (which act as recombinase binding sites) adjacent to an 8-base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521~527 (1994)). Appropriate 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).

[0080] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme pC3l. (pC3l SSR mediates recombination only between heterotype sites, attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). AttB and attP, named in relation to the attachment site for phage integrase in bacterial and phage genomes, respectively, both contain incomplete reverse repeats, possibly linked by a φ031 homodimer (Groth et al., 2000). The generation sites, attL and attR, are effectively further tpQA It is inactive to 1-mediated recombination (Belteki et al., 2003), making the reaction irreversible. Regarding the catalysis of insertion, it has been found that attB-containing DNA is more easily inserted into genomic attP sites than attP sites are inserted into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to position the attP-containing "docking site" at a limited gene locus via homologous recombination, and then that attP-containing "docking site" is partnered with the sequence into which the attB-containing insertion will enter.

[0081] As used herein, “intrasequence ribosome entry site” or “IRES” refers to an element that facilitates direct intrasequence ribosome entry into a start codon, such as ATG, of a cistron (protein-coding region), thereby resulting in 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 polynucleotides of interest encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences 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 contemplated herein is an EMCV IRES.

[0082] As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome and increases translation (Kozak, 1986, Cell 44(2):283-92, and Kozak, 1987, Nucleic Acids Res. 15(20):8125-48). In certain embodiments, the vector comprises a polynucleotide having a consensus Kozak sequence, as well as a polynucleotide encoding a fusion protein containing a J domain and a TDP-43 binding domain. Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector comprises a polyadenylation sequence on the 3' side of the polynucleotide encoding the polypeptide to be expressed.

[0083] Examples of viral vector systems suitable for use in the specific embodiments envisioned herein include, but are not limited to, vectors of adeno-associated viruses (AAV), retroviruses, herpes simplex viruses, adenoviruses, and vaccinia viruses.

[0084] In various embodiments, one or more polynucleotides encoding a fusion protein containing a J domain and a polyglutamine-binding domain are introduced into cells, such as nerve cells, by transduction of recombinant adeno-associated virus (rAAV) containing one or more polynucleotides into the cells. AAV is a small (about 26 nm), replication-deficient, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the host cell's genome. Recombinant AAV (rAAV) typically consists minimally of a transgene, as well as its regulatory sequence, and 5' and 3' AAV terminal inversion sequences (ITRs). The ITR sequence is about 145 bp long. In certain embodiments, rAAV comprises an ITR and capsid sequence isolated from AAV1, AAV2 (e.g., US6962815B2, incorporated herein by reference as a whole), AAV3, AAV4, AAV5 (e.g., US7479554B2, incorporated herein by reference as a whole), AAV6, AAV7, AAV8 (e.g., US7282199B2, incorporated herein by reference as a whole), AAV9 (e.g., US9737618B2, incorporated herein by reference as a whole), AAV rh10 (e.g., US9790472B2, incorporated herein by reference as a whole), or AAV10. In one embodiment, the vector of the present invention is encapsulated within a capsid selected from the group consisting of AAV2, AAV5, AAV8, AAV9, and AAV rh10. In one embodiment, the vector is encapsulated within AAV2. In one embodiment, the vector is encapsulated within AAV5. In one embodiment, the vector is encapsulated within AAV8. In one embodiment, the vector is encapsulated within AAV9. In one embodiment, the vector is encapsulated within AAV rh10.

[0085] In some embodiments, chimeric rAAVs are used in which the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from a different AAV serotype. For example, an rAAV having an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6 is called AAV2 / AAV6. In certain embodiments, the rAAV vector may contain an ITR derived from AAV2 and a capsid protein derived from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In preferred embodiments, the rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In preferred embodiments, the rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0086] In some embodiments, the manipulation and selection methods can be applied to the AAV capsid to increase the likelihood of transducing the cells of interest.

[0087] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Patents 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 as a whole.

[0088] IV. Delivery In certain embodiments, one or more polynucleotides encoding a fusion protein containing a J domain and a TDP-43 binding domain are introduced into cells by a non-viral or viral vector. Exemplary methods for non-viral delivery of polynucleotides as intended in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, particle guns, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0089] Examples of polynucleotide delivery systems suitable for use in specific embodiments as envisioned in particular embodiments include, but are not limited to, those offered by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in the literature, e.g., Liu et al., (2003) Gene Therapy 10:180-187; and Balazs et al., (20W) Journal of Drug Delivery 2011:1-12. Delivery based on antibody-targeted, bacterial, non-viable nanocells is also envisioned in particular embodiments.

[0090] Viral vectors containing polynucleotides as intended in a particular embodiment may be delivered in vivo by administration to individual patients, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection), subarachnoid injection, intracerebroventricular injection, or topical application, as described below. Alternatively, the vector may be delivered ex vivo to cells explanted from individual patients (e.g., mobilized peripheral blood, lymphocytes, bone marrow fluid, tissue biopsy, etc.) or to hematopoietic stem cells from a pluripotent donor, and then re-implanted into the patient.

[0091] In one embodiment, a viral vector comprising a polynucleotide encoding the fusion protein disclosed herein is administered directly to a living organism for in vivo cell transduction.

[0092] Appropriately packaged and formulated viral vectors can be delivered to the central nervous system (CNS) via subarachnoid delivery. For example, an adeno-associated virus vector can be delivered using the method described in U.S. Patent Application No. 15 / 771,481, which is incorporated herein by reference as a whole.

[0093] Alternatively, naked DNA may be administered. Administration is by one of the routes commonly used to introduce the molecule to final contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Appropriate methods for administering such nucleic acids are available and well known to those skilled in the art, and more than one route may be used to administer a particular composition, although a particular route may provide a more rapid and effective response than another.

[0094] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into cells, such as nerve cells or neural stem cells, by transduction of a retrovirus, such as a lentivirus, containing one or more polynucleotides into the cells. As used herein, the term “retrovirus” refers to an RNA virus that transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in particular embodiments include, but are not limited to, Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses. As used herein, the term “lentivirus” refers to a group (or genus) of complex lentiviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and HIV 2); Visna-Maedivirus (VMV); Caprine arthritis encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Monkey immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., an HIV cis-acting sequence element) is preferred.

[0095] Lentiviral vectors preferably include several safety enhancements as a result of modifying the LTR. “Self-inactivating” (SIN) vectors refer to vectors in which the replication-deficient vector, for example, the right-side (3') LTR enhancer-promoter region known as the U3 region, is 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 that drives transcription of the viral genome during viral particle production. Examples of heterologous promoters that may be used include, for example, promoters for simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., very early), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (vims) (HSV) (thymidine kinase). In certain embodiments, lentiviral vectors are prepared according to known methods. For example, see Kutner et al., BMC Biotechnol.2009;9:10 Doi:10.1186 / 1472-6750-9-10; and Kutner et al., Nat.Protoc.2009;4(4):495~505 Doi:l0.l038 / nprot.2009.22.

[0096] According to certain embodiments contemplated herein, the majority or all of the viral vector backbone sequence is derived from a lentivirus, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences may be used, or that numerous combined substitutions and modifications in certain parts of the lentiviral sequence may be acceptable without impairing the transfer vector's ability to perform the functions described herein. Furthermore, various lentiviral vectors are known in the art; see Naldini et al. (1996a, 1996b, and 1998); Zufferey et al. (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and No. 5,994,136 (many of which can be adapted to construct the viral vectors or transfer plasmids contemplated herein).

[0097] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into target cells by transduction of an adenovirus containing one or more polynucleotides into the cells. Adenovirus-based vectors have a very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. These vectors can be produced in large quantities in relatively simple systems. Most adenovirus vectors are engineered so that the transgene replaces the Ad Ela, Elb, and / or E3 genes, and then the replication-deficient vector is grown in human 293 cells that transduce the deleted gene function. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing differentiated cells such as those found in the liver, kidney, and muscle. Typical Ad vectors have high transport capacity.

[0098] The current production and transmission of replication-deficient adenovirus vectors can utilize a unique helper cell line named 293, which is transformed from human embryonic kidney cells with an Ad5 DNA fragment and constitutively expresses the El protein (Graham et al., 1977). Since the E3 region does not need to be from the adenovirus genome (Jones & Shenk, 1978), current adenovirus vectors with the help of 293 cells have foreign DNA in either the El, E3, or both regions (Graham & Prevec, 1991). Adenovirus vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies involving the administration of recombinant adenovirus to different tissues include tracheal infusion (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Examples of Ad vector use in clinical trials include polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083~9 (1998)).

[0099] In various embodiments, one or more polynucleotides encoding the fusion protein of the present invention are introduced into target cells by transduction of a herpes simplex virus containing one or more polynucleotides, such as HSV-1 or HSV-2, into the cells.

[0100] A mature HSV virion consists of an enveloped icosahedral capsid containing a viral genome consisting of a linear double-stranded DNA molecule of 152 kb. In one embodiment, an HSV-based viral vector is deficient in one or more essential or non-essential HSV genes. In one embodiment, an HSV-based viral vector is replication-deficient. Most replication-deficient HSV vectors contain deletions that remove one or more very early, early, or late HSV genes to prevent replication. For example, an HSV vector may be deficient in very early genes selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter a latent period, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA insertion fragments up to 25 kb. Vectors based on HSV are described, for example, in U.S. Patents 5,837,532, 5,846,782, and 5,804,413, and in International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637, and WO99 / 06583 (each of which is incorporated herein by reference as a whole).

[0101] V. Cells expressing fusion proteins In yet another embodiment, the present invention provides cells expressing the fusion proteins described herein. The cells can be transfected with a vector encoding the fusion protein 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 yet another embodiment, the cells are human cells derived from a patient who has or is at risk of developing a TDP-43-mediated disorder, which includes, but is not limited to, ALS, FTD, and Alzheimer's disease. The cells may be nerve cells or muscle cells.

[0102] Cells expressing the fusion protein may be useful in producing the fusion protein. In this embodiment, cells are transfected with a vector that overexpresses the fusion protein. The fusion protein may optionally contain an epitope, for example, a human Fc domain or a FLAG epitope as described herein, which facilitates purification (using a protein A column or an anti-FLAG antibody column, respectively). The epitope may be linked to the rest of the fusion protein via a linker or a protease substrate sequence so that the epitope can be removed from the fusion protein during or after purification.

[0103] Cells expressing fusion proteins may also be useful in therapeutic applications. In one embodiment, cells are collected from patients in need of treatment (e.g., patients with or at risk of developing TDP-43-mediated disorders). In one embodiment, the cells are nerve cells. The collected cells are then transfected with a vector expressing the fusion protein. The transfected cells may then be processed to enrich or select for transfected cells. The transfected cells may also be treated to differentiate into different cell types, such as nerve cells. After processing, the transfected cells may be administered to the patient. In one embodiment, the cells are administered by directed injection into the central nervous system via subarachnoid injection, intracranial injection, or intracerebroventricular injection.

[0104] In an alternative embodiment, cells expressing the secreted form of the fusion protein may be used. For example, the fusion protein construct may be designed to have a signal sequence at its N-terminus. Typical signal sequences are shown in Table 7 below.

[0105] [Table 7]

[0106] 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 NOs: 98-100, and the fusion protein comprises a J domain and a TDP-43 binding domain. In another embodiment, the signal sequence is selected from the group consisting of SEQ ID NOs: 98-100, and the fusion protein is selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 98, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 99, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 100, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-85, 89-90, and 92-96. Cells expressing a fusion protein construct containing a signal sequence can be administered to a target, such as a human target (e.g., a patient with or at risk of developing TDP-43 impairment). This fusion protein is secreted from the cells and helps reduce TDP-43 protein aggregation and / or associated cytotoxicity.

[0107] As described above, in certain embodiments, the fusion protein may further contain a cell-permeable peptide. Cells expressing a fusion protein containing a signal sequence and a cell-permeable peptide have the ability to secrete a fusion protein lacking the signal sequence. The secreted fusion protein, also containing the cell-permeable peptide, then has the ability to enter nearby cells and, in those cells, has the potential to reduce TDP-43 protein-mediated aggregation and / or cytotoxicity.

[0108] VI. How to Use In another embodiment, the present invention provides a method for achieving beneficial effects in a disorder and / or a TDP-43 disorder, disorder, or condition mediated by TDP-43 aggregation. The TDP-43 disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and limbic-dominant age-related TDP-43 encephalopathy.

[0109] In some embodiments, the present invention provides a method for treating a subject, such as a human, having a TDP-43 disease, disorder, or condition, comprising the step of administering to the subject a therapeutically or prophylactically effective amount of a fusion protein, a nucleic acid encoding such a fusion protein, or a viral vector encoding such a fusion protein as described herein, wherein the administration results in an improvement of one or more biochemical or physiological parameters or clinical endpoints related to the TDP-43 disease, disorder, or condition.

[0110] In other embodiments, the present invention provides a method for reducing TDP-43 aggregation in cells. The cells may be cultured cells or isolated cells. The cells may also be derived from a subject, e.g., a human subject. In one embodiment, the cells are located within the central nervous system of a human subject. In another embodiment, the human subject has or is at risk of developing a TDP-43 disorder, which may include, but is not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. In one particular embodiment, the TDP-43 disorder is amyotrophic lateral sclerosis.

[0111] Aggregation of TDP-43 protein can be detected by several methods. For example, aggregated TDP-43 protein can be distinguished from free (i.e., soluble) TDP-43-containing protein by trapping insoluble aggregates based on solubility, for example, by passing them through a selective filter of cell solubilizers. Non-aggregated proteins pass through these filters, while aggregates remain on the filters and can be detected using various reagents, including antibodies directed against TDP-43 protein. The amount of trapped aggregated protein in a solubilizer of a cell sample treated with a fusion protein, cells expressing a fusion protein, or nucleic acid, vector, or viral particle encoding a fusion protein, as described herein, can be compared to a solubilizer from untreated or control-treated cells. In this case, a decrease in the amount of aggregated TDP-43 protein in the treated sample compared to the control sample indicates the effectiveness of the fusion protein or the nucleic acid, vector, or viral particle encoding the fusion protein (see, for example, Kim et al., (2014) Mol. Cell. Biol., 34:643-652, and Example 1). A greater decrease in aggregated TDP-43 protein compared to the control indicates higher efficacy. The reduction in TDP-43 protein aggregation can also be directly detected in cells, for example, using immunofluorescence microscopy with a labeling reagent for detecting TDP-43 protein (see, e.g., Ding et al., (2015) Oncotarget, 6:24178~24191; Chou et al., (2015) Hum.Mol.Genet. 24:5154~5173, and Example 1). In certain embodiments, a greater reduction in TDP-43 polypeptide levels compared to a control indicates higher efficacy.

[0112] Therefore, in one embodiment, the method includes the step of contacting cells with an amount of a fusion protein or nucleic acid, vector, or viral particles encoding the fusion protein that is effective in reducing TDP-43 protein aggregation 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%, compared to untreated or control cells.

[0113] As shown in Example 1 below, it has been found that the expression of a fusion protein containing a J domain and a TDP-43 binding domain reduces the overall level of the TDP-43-containing reporter construct. Therefore, in another embodiment, the method includes the step of contacting cells with an amount of a fusion protein, cells expressing the fusion protein, or nucleic acid, vector, or viral particles encoding the fusion protein that is effective in reducing the level of TDP-43 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%, compared to untreated or control cells.

[0114] VII. Pharmaceutical Compositions The compositions envisioned herein may include one or more fusion proteins containing a J domain and a TDP-43 binding domain, polynucleotides encoding such fusion proteins, vectors containing them, genetically modified cells, etc. The compositions include, but are not limited to, pharmaceutical compositions. “Pharmaceutical composition” means a composition formulated as a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals, either 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 active agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active substances. There are virtually no limitations on other components that may also be included in the composition, provided that the additional active agents do not adversely affect the composition’s ability to deliver the intended treatment.

[0115] The phrase “pharmaceutically acceptable” is used herein to mean, within the bounds of sound medical judgment, a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0116] As used herein, “pharmaceutically acceptable carrier,” “diluent,” or “excipient” includes, but is not limited to, any adjuvant, carrier, excipient, flow enhancer, sweetener, diluent, preservative, colorant, flavoring, surfactant, humectant, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that is approved by the U.S. Food and Drug Administration as acceptable for use in human or animal husbandry. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn (com) starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, wax, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn (com) oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, 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 saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and any other suitable substances used in pharmaceutical formulations.

[0117] VIII. Dosage The dosage of the compositions described herein (e.g., compositions comprising fusion protein constructs, nucleic acids, or gene therapy virus particles) may vary depending on many factors, including the pharmacodynamic properties of the compound; the mode of administration; the recipient's age, health, and weight; the nature and severity of symptoms; the frequency of treatment, and, if any, the type of concurrent treatment; and the clearance rate of the compound in the animal to be treated. The compositions described herein may be administered initially at an appropriate dosage, which may be adjusted as needed depending on the clinical response. In some embodiments, the dosage of the composition is a prophylactic or therapeutically effective dose.

[0118] IX. Kit A kit is intended to include (a) a pharmaceutical composition comprising a fusion protein construct, a nucleic acid encoding such a fusion protein, or a viral particle containing such nucleic acid, which reduces the aggregation of TDP-43 protein in cells or subjects as described herein, and (b) a package insert containing instructions for use to carry out any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising the composition described herein that reduces the aggregation of TDP-43 protein in cells or subjects as described herein, (b) an additional therapeutic agent, and (c) a package insert containing instructions for use to carry out any of the methods described herein. [Examples]

[0119] To test whether the J domain can be specifically manipulated to promote the proper folding of aggregated proteins, we designed and tested several fusion protein constructs designed to target the TDP-43 protein.

[0120] Example 1: Fusion Protein Design A. Method General technologies and materials The invention will be carried out using the usual techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, unless otherwise indicated, which are within the capabilities of those skilled in the art. Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3rd edition, Cold Spring Harbor Laboratory Press, 2001; "Current protocols in molecular biology," FM Ausubel et al. (eds.), 1987; "Methods in Enzymology" series, Academic Press, San Diego, Calif.; "PCR 2: a practical approach," MJ MacPherson, B.D. Hames, and GR. Taylor (eds.), Oxford University Press, 1995; "Antibodies, a laboratory manual," Harlow, E. and Lane, D. (eds.), Cold Spring Harbor Laboratory, 1988; "Goodman & Gilman's The Pharmacological Basis of Therapeutics," 11th edition, McGraw-Hill, 2005; and Freshney, RI, "Culture of Animal Cells: A Manual of Basic Technique," 4th edition, John Wiley & See Sons, Somerset, NJ, 2000 (their contents 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). Anti-FLAG antibody was purchased from Thermo Fisher Scientific. Rabbit anti-GFP antibody was purchased from GenScripts (Piscataway, NJ).To facilitate purification and characterization, some of the fusion protein constructs used in this Example 1 contain the FLAG epitope of SEQ ID NO: 68, in addition to the sequences provided in SEQ ID NOs: 80-85 and 89-96, either at the C-terminus or N-terminus of the protein, in addition to a short linker sequence.

[0121] Protein expression and detection in HEK293 cells Expression vector plasmids encoding various protein constructs were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Cell solubles were analyzed for expressed proteins using an immunoblot assay. Before analysis, the culture medium samples were centrifuged to remove debris. Cells were lysed in a lysis buffer containing 2 mM PMSF and a protease cocktail (complete protease inhibitor cocktail; Sigma) (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 2% SDS). After short sonication, the samples were analyzed for expressed proteins using an immunoblot assay. For immunoblot analysis, the samples were boiled in SDS sample buffer and subjected to polyacrylamide electrophoresis. The separated protein bands were then transferred to a PVDF membrane.

[0122] The expressed proteins were detected using chemiluminescent signals. Briefly, the blot was reacted with a primary antibody capable of binding to a specific epitope (e.g., GFP). After washing away the unreacted primary antibody, an enzyme-conjugated secondary antibody (e.g., HRP-conjugated anti-IgG antibody) was reacted with the primary antibody molecules bound to the blot. After rinsing, a chemiluminescent reagent was added, and the resulting chemiluminescent signal in the blot was captured on X-ray film.

[0123] Fluorescence microscopy In some cases, aggregation of the TDP-43 (full-length C-terminal fragment) GFP reporter construct (see below) was detected in vivo using fluorescence microscopy. Cultured cells expressing the reporter construct, as well as a fusion protein containing the J domain and TDP-43 binding domain, were washed with PBS and fixed with 4% paraformaldehyde in PBS for 5 minutes. After three 5-minute washes in PBS, nuclear DNA was stained with DAPI. The percentage of cells containing TDP-43 (GFP lesions) in the transfected cells was counted.

[0124] Fractionation assay Transfected HEK293 cells are homogenized in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail, 2 mM PMSF, 10 mM NaF, and 2 mM Na3VO4. After brief sonication, protein concentration is measured using a BCA assay kit (Pierce). Equal amounts of protein are centrifuged at 16,000xg, 4°C, for 30 minutes to separate into a soluble fraction (supernatant) and an insoluble fraction (pellet). The insoluble fraction is further solubilized in SDS lysis buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 2% SDS). Both the soluble and insoluble fractions were applied to SDS-PAGE under reducing conditions, and then subjected to an immunoblotting assay with an anti-GFP antibody.

[0125] B. Reporter structure The inventors first investigated whether the fusion molecule of the present invention, which targets TDP-43, improves its aggregation in cultured cells. To achieve this objective, the inventors constructed GFP-based reporter constructs GFP-TDP43 and GFP-TDP43 in which GFP is fused to the C-terminus of either the full-length human TDP-43 protein or the C-terminal fragment TDP-43 (known to form intracellular aggregation and cytotoxicity) (see Table 8 below). HEK293 cells were cultured and transfected with plasmids encoding either the full-length TDP43 as a GFP fusion [GFP-TDP43FL (SEQ ID NO: 101) or GFP-TDP43CTF (SEQ ID NO: 102)], or the C-terminal fragment of TDP43 containing the C-terminal 207 amino acids of TDP-43 (amino acids 208-414 of human TDP-43). As previously reported (Zhang et al., (2009) Proc Natl Acad Sci USA., 106(18):7607~12), the inventors found that the majority of expressed GFP-TDP43FL localized to the cell nucleus (Figure 3, Panel 1), while GFP-TDP43CTF generated intracellular inclusions (Figure 3, Panel 5).

[0126] [Table 8]

[0127] C. Fusion protein constructs To determine whether the fusion protein of the present invention can be used to reduce TDP-43 aggregation, the initial experiment involved co-expression of a fusion protein containing a J-domain sequence derived from human Hsp40 J-domain protein, conjugated with a GFP-recognizing single-stranded variable fragment (scFv) (data not presented). Surprisingly, when GFP-TDP43CTF was expressed with this construct, most of the aggregation disappeared, but when GFP scFv (without the J-domain sequence) was expressed with GFP-TDP43CTF, no significant effect was observed. This suggests that TDP-43 aggregation can be resolved using the Hsp70-mediated pathway (data not presented).

[0128] Subsequently, the inventors designed a series of fusion protein constructs as shown in Table 9.

[0129] [Table 9]

[0130] The initial experiment was conducted to test the ability of the fusion protein constructs to reduce TDP-43 aggregation. Constructor 3 (JB1-scFv(3B12A)), as well as companion controls Constructor 2 (TDP-43 binding domain only) and Constructor 4 (identical to Constructor 3 except containing mutant P33Q within the conserved HPD motif of the J domain), were transfected into cells expressing either the GFP-TDP43FL reporter construct or the GFP-TDP43CTF reporter construct. Figure 3 shows that aggregation of the GFP construct in cells expressing the GFP-TDP43CTF reporter construct was greater in cells expressing GFP-TDP43FL. Cells expressing Constructor 3 showed a substantial reduction in aggregation, while expression of controls (Constructors 2 and 4) did not reduce aggregation (Figure 4; see also Table 10 below). The lack of activity in construct 4 containing P33Q strongly suggests that the ability to reduce aggregation is driven by the J domain acting through the Hsp70 pathway.

[0131] [Table 10]

[0132] Cell extracts from these cells were analyzed using immunoblotting with either an anti-GFP antibody or an anti-FLAG antibody to determine the levels of either a GFP-containing reporter construct or a FLAG epitope-containing fusion protein construct (Figure 5). Cell extracts expressing GFP-TDP43FL showed a prominent approximately 70 kDa band when searched with an anti-GFP antibody, while cells expressing GFP-TDP43CTF showed a band of approximately 50 kDa. Interestingly, cells possessing the GFP-TDP43CTF reporter and also expressing construct 3 (JB1-scFv(3B12A)) showed a significant decrease in reporter protein levels compared to negative controls, scFv controls (construct 2), or P33Q mutants (construct 4). No difference was observed in the levels of the full-length reporter construct (GFP-TDP43FL).

[0133] To investigate whether the decrease in TDP-43 levels is dependent on protein aggregation, extracts from cells expressing either GFP-TDP43FL or GFP-TDP43CTF, and also expressing construct 2 or 3, were fractionated into soluble (non-aggregated) and insoluble (aggregated) fractions, and the presence of the reporter was detected by searching with an anti-GFP antibody. As shown in Figure 6, in cells expressing the full-length reporter (GFP-TDP43FL), there was no significant change in reporter levels in either the soluble or insoluble fractions. In contrast, in cells expressing the GFP-TDP43CTF reporter, a moderate decrease in reporter was observed in the soluble fraction of cells expressing construct 3 (JB1-scFv(3B12A)), but not in cells expressing construct 2 (scFv(3B12A)). In contrast, in the insoluble fraction (presumably the aggregated form), there is a significant decrease in reporter levels, which is an almost complete disappearance of the reporter.

[0134] In summary, these data strongly suggest that the fusion protein can reduce TDP-43 levels in cells and act to preferentially accelerate the clearance of aggregated TDP43 proteins.

[0135] Based on the above results, several additional constructs (constructs 5-7) containing different stereochemistrys of the TDP-43 binding domain to the J domain were constructed. These constructs were tested for their ability to reduce aggregation. These new constructs were compared to construct 3 (JB1-scFv(3B12A)), as well as cells expressing no expression (negative control) or scFv alone (construct 2). Figure 7 shows the results of these experiments (also summarized in Table 11 below).

[0136] [Table 11]

[0137] As seen in Figure 7, constructs 3 (JB1-scFv(3B12A)), 5 (scFv(3B12A)-JB1), 6 (scFv(3B12A)-JB1-scFv(3B12A)), and 7 (JB6-scFv(3B12A)) show a moderate reduction in aggregation levels in cells expressing the GFP-TDP43FL reporter constructs compared to negative controls and cells expressing construct 2 (scFv alone). In contrast, cells expressing the GFP-TDP43CTF construct show higher overall levels of protein aggregation in negative controls and cells expressing construct 2, consistent with previous observations. Furthermore, cells expressing constructs 3, 5, 6, and 7 also exhibited dramatically reduced levels of protein aggregation, demonstrating the effectiveness of the following conformations of the fusion protein: DNAJ-T, T-DNAJ, and T-DNAJ-T (where DNAJ is the J domain and T is the TDP-43 binding domain). In addition, multiple J domains (e.g., derived from DnaJB1 and DnaJB6) were found to be active in the fusion protein. Subsequently, additional constructs were prepared and tested, as shown in Figures 8 and 9 (see also Table 12 below). Interestingly, expression of full-length DnaJB1 without the TDP-43 binding domain was able to reduce TDP-43 aggregation by approximately 43%, compared to the >90% reduction by construct 3 (JB1-scFv(3B12A)). In addition, expression of construct 14 containing two tandem copies of the QBP1 peptide also showed a substantial reduction in aggregation (approximately 71%). QBP1 has previously been shown to interact with TDP-43 (see, e.g., Mompean et al., (2019) Arch. Biochem. Biophys. 675:108113). Therefore, multiple TDP-43 binding domains (scFv(3B12A) and QBP1) were found to be active when positioned in the fusion protein.Furthermore, when cell extracts were analyzed by immunoblotting using an anti-GFP antibody to quantify the levels of reporter constructs (Figure 9B), cells expressing construct 3 (JB1-scFv(3B12A)), construct 9 (full-length DnaJB1), and construct 14 (JB1-2XQBP1) were found to have lower levels of the reporter constructs.

[0138] [Table 12]

[0139] Additional structures were tested, as shown in Tables 13 and 14 below.

[0140] [Table 13]

[0141] [Table 14]

[0142] As shown above, numerous constructs, including those using alternative J domains (see, e.g., JB6 and JC7 domains), were effective in reducing GFP-TDP43CTF aggregation. Other fusion protein constructs, such as J domains derived from DNAJC6 and SV40, or bacterial J domain protein (DnaJ), were also found to be effective in reducing aggregation of other reporter constructs (data not presented). However, construct 16 (see Table 1, SEQ ID NO: 16), which contained a J domain without a consensus HPD sequence, was unable to reduce aggregation of the GFP-TDP43CTF reporter construct.

[0143] As shown in Table 15 below, additional constructs were tested. Constructor 13 (JB1-scFv(3F10), SEQ ID NO: 90) uses scFv 3F10 fused to the J domain of DNAJB1 and binding to TDP-43. Constructor 20 (JB1-scFv(3B12A)-DD), SEQ ID NO: 97, contains a dimerizing domain derived from human DnaJA1. As shown below, constructor 13 showed a moderate ability to reduce GFP-TDP43CTF aggregation. Expression of constructor 20, which is constructor 3 containing a dimerizing domain, showed a strong reduction in GFP-TDP43CTF aggregation. Further enhancement of the effect by the dimerizing domain is consistent with the domain configuration found in several native J-domain proteins and is likely due to enhanced interaction between dimerizing constructor 3 and GFP-TDP43CTF (Sha (2000) Structure 8(8), 799-807).

[0144] [Table 15]

[0145] The inventors then investigated the mechanism by which the fusion protein reduces aggregation. HEK293 cells were transfected with either the GFP-TDP43FL or GFP-TDP43CTF reporter construct alone or in combination with construct 3 (JB1-scFv(3B12A)), and either BFA (bafilomycin A1, an inhibitor of the delayed phase of autophagy) or MG132 (a proteasome inhibitor). As shown in Figure 10, treatment of cells with 10 nM or 100 nM BFA resulted in the reappearance of pathogenic TDP43 in a dose-dependent manner. In contrast, treatment with 0.1 μM or 1.0 μM MG132 had little to no effect on the accumulation of pathogenic TDP43 forms. Taken together, these results suggest that the fusion protein construct exerts its effects via chaperone-mediated autophagy.

[0146] Example 2 AAV vector encoding a fusion protein construct Exemplary gene therapy vectors are constructed using an AAV9 vector containing codon-optimized cDNA encoding the fusion protein constructs shown in Table 6, specifically constructs 2, 4, 6, 7, 17, and 20-31, in addition to control construct 1 (DnaJB1 J domain only) and GFP (negative control), under the control of a CAG promoter containing a cytomegalovirus (CMV) initial enhancer element and a chicken beta-actin promoter. The cDNA encoding the constructs is located downstream of the Kozak sequence and is polyadenylated by the bovine growth hormone polyadenylation (BGHpA) signal. The entire cassette is flanked by two non-coding terminal inversion sequences of AAV-2.

[0147] Recombinant AAV vectors are prepared using a baculovirus expression system similar to that described above (Urabe et al., 2002; Unzu et al., 2011 (reviewed in Kotin, 2011)). Briefly, three recombinant baculoviruses are used to infect SF9 insect cells: one encoding REP for replication and packaging, one encoding CAP-5 for the AAV9 capsid, and one having an expression cassette. Purification is performed using AVB Sepharose rapid affinity medium (GE Healthcare Life Sciences, Piscataway, NJ). The vectors are titrated using QPCR with primer-probe combinations for the transgene, and the titer is expressed as genome copy number per ml (GC / ml). The titer of the vector is approximately 8 × 10⁶. 13 ~2×10 14 It is between GC / ml.

[0148] Example 3 Testing of expression and efficacy in a mouse model of ALS First, experiments were conducted in wild-type C57BL / 6J mice to confirm the expression of construct 3 and to determine whether its expression would cause adverse effects in animals. 6 × 10⁶ mice containing either the control described above or construct 3 were used. 10The vg AAVrh10 capsid was administered by either intraarachnoid injection or intraventricular injection, as shown in Table 16 below.

[0149] [Table 16]

[0150] Mice were observed for ataxia, hindlimb weakness, or limping. Body weight and clinical observations were performed weekly one week after AAV injection. At three weeks, mice (n=3) were humanely euthanized with CO2 to confirm AAV expression. No difference in body weight was found during the three weeks after ICV or IT injection (data not presented). As shown in Figure 15, mice injected via subarachnoid space (Figure 15B) or ICV (Figure 15C) expressed construct 3 when detected using immunoblotting of cerebral extract, but this was not detected in control mice. Furthermore, expression was found to be significantly higher at eight weeks after ICV injection than at three weeks.

[0151] The vectors prepared above were tested for TDP-43-related pathology in novel AAV NEFH-tTA × hTDP-43ΔNLS 2 recombinant mice (rNLS mice). This model has a doxycycline (DOX)-suppressing construct that expresses pathogenic TDP-43 (human TDP-43ΔNLS). Removal of DOX causes TDP-43ΔNLS expression to lead to rapid and progressive deterioration of the animals, resulting in severe weight loss and death, generally within 6–8 weeks. The efficacy of construct 3 (JB1-scFv(3B12A), SEQ ID NO: 80) in slowing the progression of TDP-43ΔNLS-mediated pathology was tested. Controls or AAV rh10 containing construct 3 were administered unilaterally via ICV in P1 / P2 at a volume of 2 ul (maximum 4 ul) in different groups, as shown in Table 17 below. Except for control group 1, DOX elimination occurred at 5 weeks.

[0152] [Table 17]

[0153] Weight was measured twice a week after weaning. After completion of the experiment, samples were collected from all surviving mice. The whole brain was collected and divided into two hemispheres. One hemisphere was weighed and frozen on dry ice. The second hemisphere was fixed in 4% PFA for histological evaluation.

[0154] As shown in Figure 16B, control mice in Group 2 began to lose a significant amount of body weight over the following weeks after DOX removal, resulting in a statistically significant weight difference compared to Group 1. Surprisingly, Group 3, which expressed construct 3, also showed a statistically significant weight gain compared to Group 2 (results for Groups 2 and 3 show only the weight of males, as Group 1 contains only males).

[0155] When survival rates were examined, the inventors found that animals in control group 2 (DOX off) rapidly deteriorated, resulting in a survival rate of only 37.5% of mice (0% for males) at 10 weeks. However, mice expressing construct 3 showed a 100% survival rate at 10 weeks and were indistinguishable from the control group 1 (DOX on).

[0156] Other embodiments All publications, patents, and patent applications referenced in this specification are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually referenced and incorporated as a whole. If any term in this application is found to be defined differently from a term in any document incorporated herein by reference, the definition provided herein shall be the definition of that term.

[0157] While the present invention is described in relation to its particular aspects, it is intended and understood that this application is intended to cover any variations, uses, or adaptations of the present invention, including departures from this disclosure, that can be further modified and generally applied to the essential features set forth above, falling within the scope of known or customary practices in the art and generally following the principles of the present invention.

Claims

1. An isolated fusion protein comprising the J domain and TDP-43 binding domain of the J protein, The fusion protein having the ability to reduce the aggregation of TDP-43 protein in cells.

2. The fusion protein according to claim 1, wherein the J domain of the J protein is of human origin.

3. The fusion protein according to claim 1, wherein the J domain of the J protein is selected from the group consisting of SEQ ID NOs: 1 to 50.

4. The fusion protein according to claim 3, wherein the J domain contains a sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49.

5. The fusion protein according to claim 1, wherein the TDP-43 binding domain includes a sequence selected from the group consisting of SEQ ID NOs. 51 to 55.

6. The following structures: a. DNAJ-X-T, b. DNAJ-X-T-X-T, c. DNAJ-X-T-X-T-X-T, d. T-X-DNAJ, e. T-X-T-X-DNAJ, f. T-X-T-X-T-X-DNAJ, g. T-X-DNAJ-X-T, h. T-X-DNAJ-X-T-X-T, i. TDNAJ-X-TTTTTTDNAJ-X-T, j. T-X-T-X-DNAJ-X-TT, k. TTDNAJ-X-TX-TTTTDNAJ-X-T, l. T-X-T-X-DNAJ-X-T-X-T-X-T, m. T-X-T-X-T-X-DNAJ-X-T, n. T-X-T-X-T-X-DNAJ-X-T-X-T, o. T-X-T-X-T-X-DNAJ-X-T-X-T-X-T, p. DnaJ-X-DnaJ-X-T-XT, q. T-X-DnaJ-X-DnaJ, r. T-X-T-X-DnaJ-X-DnaJ, and s. T-X-TDnaJ-X-TDnaJ-X-TTTT Including one of the following, Here, T is a TDP-43 binding domain, DNAJ is the J domain of the J protein. The fusion protein according to claim 1, wherein X is an optional linker.

7. The fusion protein according to claim 6, comprising the J domain sequence of SEQ ID NO: 5 and the TDP-43 binding domain of SEQ ID NO:

51.

8. The fusion protein according to claim 1, comprising a sequence selected from the group consisting of SEQ ID NOs: 80, 82-85, 89-92, and 94-97.

9. A fusion protein according to any one of claims 1 to 8, which has the ability to reduce TDP-43-mediated cytotoxicity.

10. A nucleic acid encoding a fusion protein according to any one of claims 1 to 9.

11. The nucleic acid according to claim 10, wherein the nucleic acid is DNA.

12. The nucleic acid according to claim 10, further comprising a promoter region, a 5'UTR, and a 3'UTR such as a poly(A) signal.

13. The nucleic acid according to claim 12, wherein the promoter region comprises a sequence selected from the group consisting of a CMV enhancer sequence, a CMV promoter, a CBA promoter, a UBC promoter, a GUSB promoter, a NSE promoter, a synapsin promoter, a MeCP2 promoter, and a GFAP promoter.

14. A vector comprising the nucleic acid according to any one of claims 10 to 13.

15. The vector according to claim 14, selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, poxvirus, paramyxovirus, baculovirus, reovirus, alphavirus, and flavivirus.

16. The vector according to claim 15, which is an AAV.

17. A viral particle comprising a capsid and a vector according to any one of claims 14 to 16.

18. The viral particle according to claim 17, wherein the capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, pseudotype AAV, rhesus monkey-derived AAV, AAVrh8, AAVrh10, and AAV-DJan AAV capsid variants, AAV hybrid serotypes, organotropic AAV, cardiacotropic AAV, and cardiacotropic AAVM41 variants.

19. The virus particle according to claim 18, wherein the capsid is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, and AAVrh10.

20. The virus particle according to claim 19, wherein the capsid is AAV9.

21. The virus particle according to claim 19, wherein the capsid is AAV rh10.

22. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 9, a cell expressing the fusion protein according to any one of claims 1 to 9, a nucleic acid according to any one of claims 10 to 13, a vector according to any one of claims 14 to 16, an active substance selected from the group consisting of virus particles according to any one of claims 17 to 21, and a pharmaceutically acceptable carrier or excipient.

23. An in vitro method for reducing the toxicity of TDP-43 protein in cells, comprising the step of contacting the cells with an effective amount of one or more active substances selected from the group consisting of a fusion protein according to any one of claims 1 to 9, a cell expressing the fusion protein according to any one of claims 1 to 9, a nucleic acid according to any one of claims 10 to 13, a vector according to any one of claims 14 to 16, a viral particle according to any one of claims 17 to 21, and a pharmaceutical composition according to claim 22.

24. The use of a fusion protein according to any one of claims 1 to 9, a cell expressing a fusion protein according to any one of claims 1 to 9, a nucleic acid according to any one of claims 10 to 13, a vector according to any one of claims 14 to 16, a viral particle according to any one of claims 17 to 21, or a pharmaceutical composition according to claim 22 in the manufacture of a pharmaceutical for treating, preventing, or delaying the progression of TDP-43 disease in subjects requiring treatment, prevention, or delay of the progression thereof.

25. The use according to claim 24, wherein the TDP-43 disease is selected from the group consisting of ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, and Lewy body dementia.

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