Compositions and methods for treating TDP-43 proteinopathy

Abstract

A novel class of fusion proteins is disclosed to recruit cellular innate chaperones, particularly the Hsp70-mediated system, to specifically reduce TDP-43-mediated protein aggregation and associated protein conformation disorders.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 016,707, filed April 28, 2020, and U.S. Provisional Patent Application No. 63 / 035,437, filed June 5, 2020, pursuant to 35 USC § 119(e). The entire contents of the above applications are incorporated herein by reference. Background Technology

[0003] All proteins expressed within a cell need to fold correctly into their intended structures to function properly. A growing number of diseases and conditions are linked to inappropriate protein folding and / or the inappropriate deposition and aggregation of proteins and lipoproteins, as well as infectious protein material. Also known as conformational disorders or protein conformational diseases, examples of diseases caused by misfolding include Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTLD). Mutant proteins aggregate in cells, leading to the characteristic cytotoxic cellular inclusion bodies.

[0004] The pathology of a wide variety of neurodegenerative diseases is 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 pathology of Alzheimer's disease (AD) is defined by senile plaques and neurofibrillary tangles composed of β-amyloid and microtubule-associated protein tau, respectively, while Lewy bodies composed of α-synuclein are the defining lesion of Parkinson's disease. Until recently, the neuropathology of both frontotemporal degeneration with ubiquitin inclusions (FTLD-U) (Kumar-Singh & Van (2007) BrainPathol., 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-amyloid ubiquitinated inclusions (UBIs).

[0005] FTLD, the second most common form of Alzheimer's disease, refers to a heterogeneous group of neurodegenerative disorders with common behavioral and / or language impairments (Kumar-Singh & Van, ibid.). Some affected individuals exhibit motor impairments, such as Parkinson's disease or motor neuron disease (MND). While designated FTLD reflects prominent frontal and temporal lobe degeneration, multiple neuropathological abnormalities have been identified in these patients (Cairns et al., (2007) Acta Neuropathol., 1145:5–22). The two major pathological subdivisions of FTLD are recognized: brain with tau-positive inclusion bodies (i.e., tau proteinopathy), and brain with UBI that is not detected by antibodies against tau, α-synuclein, and β-amyloid (i.e., FTLD-U). Up to 40% of FTLD cases showed a familial inheritance pattern with three distinct genetic abnormalities associated with FTLD-U pathology, including mutations in pregranulin (PGRN) and valine-containing protein (VCP), as well as linkage to a novel locus on chromosome 9p.

[0006] Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig's disease, is a neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons in the brainstem and spinal cord, leading to progressive muscle atrophy 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 age of onset of 54–67 years and a median prevalence of approximately 5.4 cases per 100,000 people, with men at a slightly higher risk compared to 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 devastating neurodegenerative disease with no effective treatment, and patients usually die within 2-4 years of onset, primarily due to respiratory failure and swallowing problems (Chio et al., (2009)). Amyotroph Lateral Scler. 10 (5-6):310-323; del Aguila et al., (2003) Neurology60 (5):813-819; Tabata et al., (2009) Nucleic Acids Res. 7 (8):e64).

[0007] Most ALS cases are sporadic (sALS) of unknown cause, while only ~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 single-gene causes of ALS, 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).

[0008] The variability in the prevalence of ALS and the identification of gene mutations in a large number of genes suggest that multiple factors, such as multigenomic factors and environmental factors, may be the basis for disease susceptibility. Indeed, combinations of cellular pathways, such as protein misfolding / aggregation, have been proposed to lead to neurotoxic outcomes in ALS (Ross & Poirier, (2004)). Nat Med. 10Suppl: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 contributors to oxidative stress (Anand et al., (2013)). Oxid Med Cell Longev., 2013:635831; Sharma et al., (2016) Neurochem Res., 41 (5):965-984).

[0009] A 43 kDa interactive response (TAR)-DNA binding protein (TDP-43) 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 these two conditions has provided insights into the following mechanistic links: 1) a large proportion of ALS patients exhibit a range of behavioral and cognitive changes belonging to the FTLD range (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) the identification of specific gene loci and mutations in families with co-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), and 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.); in (Lagier-Tourenne et al., (2010), Human Molecular Genetics A review was conducted in 19:R46–R64.

[0010] Therefore, there is a need to develop new therapeutic modalities that are optimized for target-specific antigens, proteins, glycoproteins, or lipoproteins, particularly for diseases with pathologies based on protein misfolding and aggregation, and in the case of heterogeneous aggregates.

[0011] Heat shock 70 kDa proteins (referred to as “Hsp70s” in this paper) constitute a class of chaperone proteins that are ubiquitous in the cells of a wide variety of species (Tavaria et al., (1996)). Cell Stress Chaperones 1, 23-28). Hsp70 requires accessory proteins called co-chaperone proteins, such as J domain proteins and nucleotide exchange factors (NEF) (Hartl et al., (2009)). Nat Struct Mol Biol 16, 574-581), in order to function. In the current model of the Hsp70 chaperone mechanism for folding proteins, Hsp70 cycles between ATP- and ADP-bound states, and the J domain protein binds to another protein (called the “client protein”) that needs to be folded or refolded, interacting with the ATP-bound form of Hsp70 (Hsp70-ATP) (Young (2010)). Biochem Cell Biol 88, 291-300; Mayer, (2010) Mol Cell 39, 321-331). The binding of the J domain protein-client protein complex to Hsp70-ATP stimulates ATP hydrolysis, which leads to a conformational change in the Hsp70 protein, closing the helical cap and thereby stabilizing the interaction between the client protein and Hsp70-ADP, and triggering the release of the J domain protein, which then freely binds to another client protein.

[0012] Therefore, according to this model, J-domain proteins play a key role within the Hsp70 mechanism by acting as bridges and facilitating the capture and submission of a wide variety of client proteins into the Hsp70 mechanism to promote folding or refolding into the correct conformation (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 60-80 aa) consists of four helices: I, II, III, and IV. Helices II and III are linked via a flexible loop containing an "HPD motif," which is highly conserved among the J domains and is considered essential for activity (Tsai & Douglas, (1996)). J Biol Chem 271, 9347-9354). Mutations within the HPD sequence have been found to eliminate the function of the J domain.

[0013] Given the background provided above on protein conformation disorders such as ALS, it seems clear that reducing the levels of misfolded proteins can serve as a means of treating, preventing, or otherwise improving the symptoms of these devastating conditions, and that recruiting the cells’ innate ability to repair protein misfolding would be a logical choice. Summary of the Invention

[0014] The inventors have developed a novel class of fusion proteins to recruit cellular innate chaperone mechanisms, particularly Hsp70-mediated systems, to specifically reduce TDP-43-mediated protein aggregation. Unlike previous studies using fusion proteins containing fragments 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 employs fusion proteins containing a J domain to reduce protein aggregation and cytotoxicity induced by the aggregation of mutant TDP-43 protein. In this context, the inventors unexpectedly discovered that the functionally required J domain element serves a distinctly different purpose in enhancing protein expression and secretion, confirming a different mechanism regarding the mode of action of this fusion protein. The fusion protein described herein contains a J domain as well as a domain with affinity for TDP-43. The presence of the TDP-43-binding domain within the fusion protein results in a specific reduction in the aggregation of mutant TDP-43 protein.

[0015] E1. Therefore, in the first aspect, this article discloses an isolated fusion protein containing the J domain of the J protein and the TDP-43 binding domain.

[0016] The fusion protein of E2 and E1, wherein the J domain of the J protein is of eukaryotic origin.

[0017] E3. A fusion protein of any one of E1-E2, wherein the J domain of the J protein is of human origin.

[0018] E4. A fusion protein of any one of E1-E3, wherein the J domain of the J protein is cytoplasm-localized.

[0019] E5. A fusion protein of any one of E1-E4, wherein the J domain of the J protein is selected from SEQ ID No: 1-50.

[0020] E6. A fusion protein of any one of E1-E5, wherein the J domain comprises a sequence selected from SEQ ID NO: 1, 5, 6, 10, 16, 24, 25, 31 and 49.

[0021] E7. A fusion protein of any one of E1-E6, wherein the J domain comprises the sequence of SEQ ID NO: 5.

[0022] E8. A fusion protein of any one of E1-E6, wherein the J domain comprises the sequence of SEQ ID NO: 10.

[0023] E9. A fusion protein of any one of E1-E6, wherein the J domain comprises the sequence of SEQ ID NO: 16.

[0024] E10. A fusion protein of any one of E1-E6, wherein the J domain comprises the sequence of SEQ ID NO: 25.

[0025] E11. A fusion protein of any one of E1-E6, wherein the J domain comprises the sequence of SEQ ID NO: 31.

[0026] E12. A fusion protein of any one of E1-E11, wherein, for example, when measured using an ELISA assay, the TDP-43 binding domain has a Kc of 1 μM or less for TDP-43 (e.g., using a reporter construct containing the C-terminal 207 amino acids of TDP-43), such as 300 nM or less, 100 nM or less, 30 nM or less, or 10 nM or less. D .

[0027] E13. A fusion protein of any one of E1-E12, wherein the TDP-43 binding domain comprises a sequence selected from SEQ ID NO: 51-55.

[0028] E14. A fusion protein of any one of E1-E13, wherein the TDP-43 binding domain comprises the sequence of SEQ ID NO:51-53.

[0029] E15. A fusion protein of any one of E1-E13, wherein the TDP-43 binding domain comprises the sequence of SEQ ID NO:51.

[0030] E16. A fusion protein of any one of E1-E13, wherein the TDP-43 binding domain comprises the sequence of SEQ ID NO:53.

[0031] E17. A fusion protein of any one of E1-E16, which contains multiple TDP-43 binding domains.

[0032] E18. A fusion protein of any one of E1-E17, which consists of two TDP-43 binding domains.

[0033] E19. A fusion protein of any one of E1-E18, which consists of three TDP-43 binding domains.

[0034] E20. A fusion protein of any one of E1-E19, comprising one of the following constructs:

[0035]

[0036] r. TXTX-DnaJ-X-DnaJ, and

[0037] s. TX-TDnaJ-X-TDnaJ-X-TTTT

[0038] in,

[0039] T is the TDP-43 associative domain.

[0040] DNAJ is the J domain of the J protein, and

[0041] X is an optional connector.

[0042] E21. A fusion protein of any one of E1-E20, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and the TDP-43 binding domain sequence of SEQ ID NO: 51.

[0043] E22. A fusion protein of any one of E1-E21, wherein the fusion protein comprises the J domain sequence of SEQ ID NO: 5 and two copies of the TDP-43 binding domain sequence of SEQ ID NO: 53.

[0044] E23. A fusion protein of any one of E1-E22, wherein the fusion protein comprises a sequence selected from SEQ ID NO: 80-85 and 89-97.

[0045] E24. A fusion protein of any one of E1-E23, wherein the fusion protein comprises a sequence selected from SEQ ID NO: 80, 82-85, 89-90 and 92-97.

[0046] E25. A fusion protein of any one of E1-E23, wherein the fusion protein comprises the sequence of SEQ ID NO: 80.

[0047] E26. A fusion protein of any one of E1-E23, wherein the fusion protein comprises the sequence of SEQ ID NO: 90.

[0048] E27. A fusion protein of any one of E1-E23, wherein the fusion protein comprises the sequence of SEQ ID NO: 92.

[0049] E28. A fusion protein of any one of E1-E23, wherein the fusion protein comprises the sequence of SEQ ID NO: 94.

[0050] E29. A fusion protein of any one of E1-E23, wherein the fusion protein comprises the sequence of SEQ ID NO: 95.

[0051] E30. A fusion protein of any one of E1-E23, wherein the fusion protein comprises the sequence of SEQ ID NO: 96.

[0052] E31. A fusion protein of any one of E1-E30, which further includes a targeting agent.

[0053] E32. A fusion protein of any one of E1-E31, which further includes an epitope.

[0054] The fusion protein of E33 and E32, wherein the epitope is a polypeptide selected from SEQ ID NO:67-73.

[0055] A fusion protein of any one of E34, E1, or E33, further comprising a cell penetrant.

[0056] The fusion protein of E35 and E34, wherein the cell penetrant is selected from SEQ ID NO: 74-77.

[0057] E36. A fusion protein of any one of E1-E35, which further includes a signal sequence.

[0058] The fusion protein of E37 and E36, wherein the signal sequence comprises a peptide sequence selected from SEQ ID NO: 98-100.

[0059] E38. A fusion protein of any one of E1-E37, which can reduce the aggregation of TDP-43 protein in cells.

[0060] E39. A fusion protein of any one of E1-E38 that can reduce TDP-43-mediated cytotoxicity.

[0061] E40. A nucleic acid sequence that encodes a fusion protein of any one of E1-E39.

[0062] The nucleic acid sequences of E41 and E40, wherein the nucleic acid is DNA.

[0063] E42. A nucleic acid sequence of any one of E40, wherein the nucleic acid is RNA.

[0064] E43. A nucleic acid sequence of any one of E40-E42, wherein the nucleic acid comprises at least one modified nucleic acid.

[0065] E44. Nucleic acid sequence of any one of E40-E43, which further includes a promoter region, 5' UTR, 3' UTR such as poly(A) signal.

[0066] Nucleic acid sequences of E45 and E44, wherein the promoter region contains sequences selected from CMV enhancer sequences, CMV promoters, CBA promoters, UBC promoters, GUSB promoters, NSE promoters, synapsin promoters, MeCP2 promoters, and GFAP promoters.

[0067] E46. A vector containing a nucleic acid sequence of any one of E40-E45.

[0068] E47. Vectors of E46, wherein the vectors are selected from adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, poxvirus (vaccinia or myxoma), paramyxovirus (measles virus, RSV or Newcastle disease virus), baculovirus, reovirus, alphavirus and flavivirus.

[0069] A carrier of E48, E46 or E47, wherein the carrier is AAV.

[0070] E49. A viral particle comprising a capsid and a vector of any one of E46-E48.

[0071] Viral particles of E50 and E49, wherein the capsid is selected from 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 mutants, AAV heterozygous serotypes, organophilic AAV, cardophilic AAV and cardophilic AAVM41 mutants.

[0072] Viral particles of E51, E49, or E50, wherein the capsid is selected from AAV2, AAV5, AAV8, AAV9, and AAVrh10.

[0073] Viral particles of any one of E52, E49 - E51, wherein the capsid is AAV2.

[0074] Viral particles of any one of E53, E49 - E51, wherein the capsid is AAV5.

[0075] Viral particles of any one of E54, E49 - E51, wherein the capsid is AAV8.

[0076] Viral particles of any one of E55, E49 - E51, wherein the capsid is AAV9.

[0077] Viral particles of any one of E56, E49 - E51, wherein the capsid is AAV rh10.

[0078] E57. A pharmaceutical composition comprising a pharmaceutical agent selected from the following pharmaceutical agents and pharmaceutically acceptable carriers or excipients: a fusion protein of any one of E1-E39, a cell expressing a fusion protein of E1-E39, a nucleic acid of any one of E40-E45, a carrier of any one of E46-E48, and a viral particle of any one of E49-E56.

[0079] E58. A method for reducing the toxicity of TDP-43 protein in cells, comprising contacting the cells with an effective amount of one or more agents selected from: a fusion protein of any one of E1-E39, cells expressing a fusion protein of E1-E39, a nucleic acid of any one of E40-E45, a vector of any one of E46-E48, a viral particle of any one of E49-E56, and a pharmaceutical composition of E57.

[0080] The methods of E59 and E58, wherein the cells are in the subject.

[0081] The method of any one of E60, E58, or E59, wherein the subject is a human being.

[0082] The method of any one of E61, E58 - E60, wherein the cells are cells of the central nervous system and the peripheral nervous system.

[0083] The method of any one of E62, E58 - E61, wherein the subject is identified as having TDP-43 disease.

[0084] E63. The method of E62, wherein the TDP-43 disease is selected from ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia and limbic predominant age-related TDP-43 encephalopathy.

[0085] Methods E64, E62, or E63, wherein the TDP-43 disease is ALS.

[0086] The method of any one of E65, E58 - E64, wherein, when compared with control cells, there is a reduction in the amount of aggregated TDP-43 protein in said cells.

[0087] E66. A method for treating, preventing, or delaying the progression of TDP-43 disease in a subject with this need, said method comprising administering an effective amount of one or more agents selected from: a fusion protein of any one of E1-E39, cells expressing a fusion protein of E1-E39, a nucleic acid of any one of E40-E45, a vector of any one of E46-E48, a viral particle of any one of E49-E56, and a pharmaceutical composition of E57.

[0088] E67. E66 methods, wherein the TDP-43 disease is selected from ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia and age-related TDP-43 encephalopathy with limbic lobe involvement.

[0089] The methods of E68 and E67, wherein the TDP-43 disease is ALS.

[0090] E69. Use of one or more of the following to prepare a medicament for preventing or delaying the progression of TDP-43 disease in a subject: a fusion protein of any one of E1-E39, a cell expressing a fusion protein of E1-E39, a nucleic acid of any one of E40-E45, a vector of any one of E46-E48, a viral particle of any one of E49-E56, and a pharmaceutical composition of E57.

[0091] Uses of E70 and E69, wherein the TDP-43 disease is selected from ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia and age-related TDP-43 encephalopathy with limbic lobe involvement.

[0092] The uses of E71, E69, or E70, wherein the TDP-43 disease is ALS. Attached Figure Description

[0093] Figure 1A The Clustal Omega sequence alignment of representative human J domain sequences is shown. Highly conserved HPD domains are displayed in the highlighted boxes.

[0094] Figure 1B The Clustal Omega sequence alignment of a representative human J domain sequence is shown.

[0095] Figure 2 Some illustrative fusion protein constructs containing the J domain and the TDP-43 binding domain are shown.

[0096] Figure 3Visualizations of TDP43-GFP fusion construct aggregation in cells, as measured by fluorescence microscopy, are shown, along with the effect of the J-domain fusion protein in reducing aggregation. Fluorescence microscopy of cells transfected with the GFP reporter construct (GFP-TDP43FL; insets 1-4) or the C-terminal fragment of TDP-43 (GFP-TDPCTF; insets 1-5) are compared. Figure 5-8 ), further containing scFv control (small Figure 2 and 6 ), DnaJB1-scFv (3B12A) fusion protein (small Figure 3 and 7 ), and the DnaJB1-scFv (3B12A) fusion protein (small) containing the P33Q mutation in the conserved HPD domain. Figure 4 and 8 ).

[0097] Figure 4 This shows the results of normalizing control cells expressing only the GFP-TDP43CTF construct from [a specific source]. Figure 3 Quantification of aggregation in different constructs.

[0098] Figure 5 Immunoblot analysis of cell extracts expressing GFP reporter constructs, with or without fusion protein constructs, is shown. The top panel shows Western blot analysis using an anti-GFP antibody, detecting larger GFP-TDP43FL (lanes 1-4) and smaller GFP-TDP43CTF (lanes 5-8). The bottom panel shows Western blot analysis using an anti-FLAG epitope antibody, detecting scFv (3B12A) control (lanes 2 and 6), fusion protein construct (DnaJB1-scFv (3B12A), lanes 3 and 7), and the DnaJB1-scFv (3B12A) fusion protein containing a P33Q mutation in the conserved HPD domain (smaller). Figure 4 and 8 ).

[0099] Figure 6 Immunoblot detection of GFP reporter constructs from soluble or insoluble fractions of cell extracts, wherein the cells express GFP-TDP43FL or GFP-TDP43CTF reporter constructs and do not express (negative control) or express construct 2 or 3.

[0100] Figure 7Visualization of TDP43-GFP fusion construct aggregation in cells, as measured by fluorescence microscopy, and the effect of the J-domain fusion protein in reducing aggregation are shown. Fluorescence microscopy of cells transfected with GFP reporter constructs, and further containing constructs 2, 3, 5, 6, and 7, is compared with a control (without) expressing a single reporter construct containing full-length TDP-43 (GFP-TDP43FL) or the C-terminal fragment of TDP-43 (GFP-TDPCTF).

[0101] Figure 8 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 aggregation are shown. Fluorescence microscopy of cells transfected with GFP reporter constructs, and further containing constructs 1, 2, 3, 4, 9, 10, 11, or 14, is compared with a control (without) expressing a single reporter construct containing full-length TDP-43 (GFP-TDP43FL) or the C-terminal fragment of TDP-43 (GFP-TDPCTF).

[0102] Figure 9 shows the quantification and detection of the TDP-43 reporter sub-construct: Figure 9A Showing from Figure 8 The quantitative differences in cell aggregation in the experiment shown. Figure 9B The immunoblotting analysis of cell extracts using anti-GFP antibody is shown to quantify the level of reporter constructs in the cell extracts.

[0103] Figure 10 This study demonstrated the effects of bafloxacin A1 (BFA), a potent inhibitor of late autophagy, and the proteasome inhibitor MG132, on reducing GFP-TDP43CTF in cells co-expressing construct 3. Cells were transfected with either construct GFP-TDP43FL (lane 2) or GFP-TDP43CTF (lanes 3-8) using a reporter transfection method, and co-transfected with nucleic acids encoding construct 3 (lanes 4-8). Treatment with either BFA (at 0.01 µM, lane 5, and at 0.1 µM, lane 6) resulted in higher levels of the GFP-TDP43CTF reporter protein, as detected by Western blotting. In contrast, treatment with 0.1 or 1.0 µM MG132 (lanes 7 and 8, respectively) had little to no effect.

[0104] Figure 11 shows the effect of co-expression construct 3 (lanes 4 and 8) on the levels of GFP-TDP43CTF reporter in the soluble (non-aggregated) and insoluble (aggregated) fractions of cell extracts, as shown by anti-TDP43 antibody ( Figure 11A) and antiphospho-TDP43 antibody ( Figure 11B ) detected.

[0105] Figure 12 The effects of co-expression of construct 3 (lane 3), construct 7 (lane 4), construct 2 (lane 5), construct 15 (lane 6), and construct 16 (lane 7) on reducing the level of phosphorylated GFP-TDP43CTF reporter in cell extracts, as detected by anti-phosphorylated TDP43 antibody, are shown.

[0106] Figure 13 The diagram shows the effects of co-expression construct 3 (lanes 3, 5, 8, and 10) on the levels of TDP-43 (as detected with anti-TDP43 antibody, top panel), phosphorylated TDP-43 (as detected with anti-phosphorylated TDP-43 antibody, second inset), Flag epitope (as detected with anti-FLAG antibody, third inset), and tubulin (anti-tubulin antibody, bottom panel) in cells expressing TDP43FL (lanes 2, 3, 7, and 8) and TDP43ΔNLS constructs (lanes 4, 5, 9, and 10).

[0107] Figure 14 Additional constructs were shown to test the ability to reduce phosphorylated TDP-43. Cells expressing GFP-TDP43FL (lanes 1 and 7) or GFP-TDP43CTF (lanes 2-6, 8-12) were co-transfected with constructs 3 (lanes 3 and 9), 17 (lanes 4 and 10), 18 (lanes 5 and 11), and 19 (lanes 6 and 12). Soluble fractions (lanes 1-6) and insoluble fractions (lanes 7-12) were detected with an anti-phosphorylated TDP43 antibody.

[0108] Figure 15 shows a summary of the results in C57 mice injected with AAV rh10 containing either the control or encoding construct 3, administered via intrathecal (IT) or intravascular (ICV) injection. Figure 15A The research timeline is summarized. Figure 15B Immunoblots of brain extracts from mice were shown 3 weeks after administration of AAV rh10 IT containing either the control (lanes 1 and 2) or the vector containing construct 3 (lanes 3 and 4). Figure 15C Immunoblotting of brain extracts from mice following ICV injection is shown. Lanes 1-3 show immunoblotting of brain extracts from mice 3 weeks after administration of AAVrh10 ICV containing either the control (lanes 1 and 2) or the construct 3 (lane 3). Lanes 4-8 show immunoblotting of 8-week-old mice from control (lanes 4-6) and construct 3 (lanes 7 and 8) mice.

[0109] Figure 16 shows a summary of the results using ΔNLS8 mice, which were injected with AAV rh10 containing either the control or the vector encoding construct 3 via ICV injection. Figure 16A The research timeline is shown. Figure 16B The average weight of the males from each group is shown. Figure 16C The survival of mice from different groups is shown.

[0110] definition

[0111] As used in the specification and claims, the singular forms “an,” “a,” and “the” include plural indicators unless the context clearly indicates otherwise. For example, the term “cell” includes a plurality of cells, including mixtures thereof.

[0112] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeled component.

[0113] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptides. Standard single-letter or three-letter codes are used to designate amino acids.

[0114] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of the subject vector. Host cells include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be completely identical to the original parent cell (in terms of morphology or the genome of total DNA complementarity). Host cells include cells transfected in vivo with the vector of the present invention.

[0115] When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified and separated from and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein or non-protein solutes. As will be apparent to those skilled in the art, non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be "isolated" to distinguish them from their naturally occurring counterparts. Furthermore, "concentrated," "separated," or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are distinguishable from their naturally occurring counterparts because the molecular concentration or number per volume is generally greater than that of their naturally occurring counterparts. Generally, polypeptides prepared by recombinant means and expressed in host cells are considered "isolated."

[0116] "Isolated" polynucleotide or polypeptide-encoded nucleic acid (BNI) or other polypeptide-encoded nucleic acid (UNI) are nucleic acid molecules that are identified and separate from at least one contaminant nucleic acid molecule, which typically binds to the contaminant nucleic acid molecule in its natural source. The isolated polypeptide-encoded nucleic acid molecule differs from its form or background found in nature. Therefore, the isolated polypeptide-encoded nucleic acid molecule is different from a specific polypeptide-encoded nucleic acid molecule present in natural cells. However, isolated polypeptide-encoded nucleic acid molecules include polypeptide-encoded nucleic acid molecules contained in cells that normally express polypeptides, in which, for example, the nucleic acid molecule is located on chromosomes or extrachromosomally in a location different from that in natural cells.

[0117] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length, namely, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis (locus), 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 analogs. Modifications to the nucleotide structure can be conferred before or after polymer assembly, if present. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components.

[0118] As defined herein, the term “TDP-43 syndrome” or “TDP-43-mediated disease” refers to a syndrome associated with the formation of intracellular TDP-43 aggregates, particularly aggregates of mutant TDP-43 proteins. Examples of TDP-43 syndromes include, but are not limited to, preferably, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and age-related TDP-43 encephalopathy with predominantly limbic involvement.

[0119] A “vector” is a nucleic acid molecule that preferably replicates autonomously in a suitable host, transferring an inserted nucleic acid molecule into and / or between host cells. This term includes vectors that primarily function to insert DNA or RNA into cells, replication vectors that primarily function to replicate DNA or RNA, and expression vectors that function to transcribe and / or translate 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 a polypeptide. An “expression system” generally means a suitable host cell composed of expression vectors that can function to produce the desired expression product.

[0120] The term "operably linked" refers to the juxtaposition of the components, wherein the components are in a relationship that allows them to function in their intended manner. Control sequences "operably linked" to a coding sequence are linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences can include expression control sequences adjacent to the target gene, or they can include trans- or distance-based expression control sequences that control the expression of the target gene. The term "expression control sequence" refers to the polynucleotide sequence necessary for the expression and processing of the coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (e.g., Kozak concordant sequences); sequences enhancing protein stability; and sequences enhancing protein secretion when needed. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Unless otherwise stated, descriptions or statements herein of nucleic acid molecules encoding the fusion protein of the present invention inserted into expression vectors mean that the inserted nucleic acid has also been operatively linked within the vector to a promoter and other transcriptional and translational control 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 compatible cell of an organism.

[0121] When applied to polynucleotides, “recombination” means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and / or ligation steps and other procedures that result in a construct that may potentially be expressed in a host cell.

[0122] The terms "gene" and "gene segment" are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame (ORF) that, after transcription and translation, encodes a specific protein. A gene or gene segment can be genomic DNA or cDNA, provided that the polynucleotide contains at least one ORF and can cover an entire coding region or a segment thereof. A "fusion gene" is a gene composed of at least two heterologous polynucleotides linked together.

[0123] The terms “disease” and “symptom” are used interchangeably to refer to a pathological condition identified according to acceptable medical standards and practices in the field.

[0124] As used herein, the term “effective amount” refers to an amount of therapy sufficient to reduce or improve the severity and / or duration of a disease or one or more of its symptoms; prevent the progression of a harmful or pathological condition; induce the resolution of a pathological condition; prevent the recurrence, development, onset, or progression of one or more symptoms associated with a pathological condition; detect symptoms; or enhance or improve the preventive or therapeutic effect of a therapy (e.g., by administering another preventive or therapeutic agent).

[0125] As used herein, the term “J domain” refers to a segment that retains the ability to accelerate the intrinsic ATPase catalytic activity of Hsp70 and its homologs. J domains of various J proteins have been identified (see, for example, Kampinga et al. (2010) Nat. Rev., 11: 579-592; Hennessy et al. (2005) Protein Science, 14: 1697-1709, each incorporated herein by reference in its entirety), and are characterized by a number of features: characterized by four α-helices (I, II, III, IV), and typically possessing a highly conserved tripeptide sequence motif (called the “HPD motif”) between helices II and III, consisting of histidine, proline, and aspartic acid. Typically, the J domain of a J protein is 50 to 70 amino acids in length, and the interaction (binding) site of the J domain with the Hsp70-ATP chaperone protein is considered to be a region extending from helix II, and the HPD motif is essential for stimulating Hsp70 ATPase activity. As used herein, the term "J domain" is intended to include native J domain sequences and functional variants thereof that retain the ability to accelerate the intrinsic ATPase activity of Hsp70, an ability that can be measured using methods well known in the art (see, for example, Horne et al. (2010)). J. Biol. Chem. (285, 21679-21688, which are incorporated herein by reference in their entirety). Table 1 provides a non-restrictive list of the J-structure domains. Detailed Implementation

[0126] The inventors have discovered that contacting cells with a fusion protein construct containing the J domain of the J protein and the TDP-43 binding domain has a surprisingly effective effect in reducing the aggregation of mutant TDP43 protein. Aggregation of mutant TDP-43 is thought to contribute to many devastating diseases, including but not limited to amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and Alzheimer's disease. Accordingly, this document provides useful compositions and methods for treating TDP-43 symptoms, for example, in subjects with such needs.

[0127] To overcome the challenges associated with chaperone-based therapies, we investigated the possibility of designing highly specific artificial chaperone proteins. We designed a series of fusion protein constructs comprising an effector domain (J domain sequence) for Hsp70 binding / activation and a domain conferring specificity for TDP-43 protein. The resulting fusion proteins act to accelerate the intrinsic ATPase catalytic activity of Hsp70 and its homologs, leading to increased protein folding, reduced aggregation, and / or accelerated clearance.

[0128] I. Fusion protein construct

[0129] a. The J-structure field can be used in this invention.

[0130] The J domains of various J proteins have been identified. See, for example, Kampinga et al., Nat. Rev., 11: 579-592 (2010); Hennessy et al., Protein Science, 14:1697-1709 (2005). The J domains that can be used to prepare the fusion proteins of the present invention have key defining features of the J domain that primarily accelerates HSP70 ATPase activity. Accordingly, the isolated J domains that can be used in the present invention comprise a polypeptide domain characterized by four α-helices (I, II, III, IV) and typically have a highly conserved tripeptide sequence (referred to as the “HPD motif”) between helices II and III, containing histidine, proline, and aspartic acid. Typically, the J domain of a J protein is 50 to 70 amino acids in length, and the interaction (binding) site of the J domain with the Hsp70-ATP chaperone protein is considered to be a region extending from helix II, and the HPD motif is the basis of the original activity. Representative J domains include, but are not limited to, the J domains of DnaJB1, DnaJB2, DnaJB6, and DnaJC6, the J domain of the large T antigen of SV40, and the J domain of mammalian cysteine ​​string protein (CSP-α). Table 1 provides the amino acid sequences of these and other J domains that can be used in the fusion proteins of this invention. Conserved HPD motifs are highlighted in bold. In one embodiment, the fusion protein disclosed herein comprises a J domain selected from SEQ ID NO: 1-50. As shown in the following example section, the inventors found that using a J domain lacking the conserved "HPD" motif does not reduce protein aggregation. Similarly, in another embodiment, the fusion protein disclosed herein comprises a J domain containing a shared HPD motif. In a particular embodiment, a J domain selected from SEQ ID NO: 1-15, 17-50.

[0131] In one particular embodiment, the fusion protein comprises a J domain selected from SEQ ID NO: 1, 5, 6, 10, 16, 24, 25, 31 and 49.

[0132] Table 1. Representative human J-domain sequences

[0133]

[0134] b. TDP-43 combined structural domain

[0135] 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.

[0136] Ideally, the TDP-43 binding domain has sufficient affinity to bind to the TDP-43 protein when it is present in the cell at pathological levels. Therefore, in one embodiment, the fusion protein comprises a TDP-43 binding domain that, when tested by ELISA on a 96-well microtiter plate, has a Kc for the TDP-43 reporter construct (e.g., full-length TDP-43 (Novus Biologicals, NBP2-22850, Centennial, CO) with, for example, 2 µM or less, 1 µM or less, 500 nM or less, 300 nM or less, 100 nM or less, or 30 nM or less. D .

[0137] The TDP-43 binding domain has been previously identified and characterized (see, for example, U.S. Patent Nos. 10,259,866, WO 2016 / 53610, WO 2018 / 218252, WO 2019 / 134981, and WO 2019 / 177138, each incorporated herein by reference). Therefore, in another embodiment, the fusion protein comprises a TDP-43 binding domain selected from SEQ ID NOs: 51-55 (see, for example, Table 2). In one particular embodiment, the fusion protein comprises the TDP-43 binding domain of SEQ ID NO: 51. In another embodiment, the fusion protein comprises the TDP-43 binding domain of SEQ ID NO: 53. In yet another embodiment, the fusion protein comprises the TDP-43 binding domain of SEQ ID NO: 52.

[0138] In another embodiment, the fusion protein also considers the use of a TDP-43 binding domain chemically conjugated to the J domain. The TDP-43 binding domain can be directly conjugated to the J domain. Alternatively, it can be conjugated to the J domain via a linker. For example, a wide range of chemical cross-linking agents are available, known to those skilled in the art, and can be used to cross-link the TDP-43 binding domain to the J domain, or to cross-link the targeting domain to a fusion protein comprising both the TDP-43 binding domain and the J domain. For example, the cross-linking agent is a heterobifunctional cross-linking agent, which can be used to link molecules in a stepwise manner. Heterobifunctional cross-linking agents provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing unwanted side reactions such as homopolymerization. A variety of heterobifunctional crosslinkers are known in the art, including succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (SMCC), m-maleimidebenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimide-(4-iodoacetyl)aminobenzoate (SIAB), succinimide-4-(p-maleimidephenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimide-oxycarbonyl-a-methyl-a-(2-pyridinedithio)-toluene (SMPT), N-succinimide-3-(2-pyridinedithio)propionate (SPDP), and succinimide-6-[3-(2-pyridinedithio)propionate]hexanoate (LC-SPDP). Crosslinkers having an N-hydroxysuccinimide moiety can be obtained as N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. Conversely, crosslinkers having disulfide bridges within the linker chain can be synthesized as alkyl derivatives to reduce the amount of linker cleavage in the bulk. Besides heterobifunctional crosslinkers, many other crosslinkers exist, including homobifunctional crosslinkers and photoreactive crosslinkers. Disuccinimide octanoate (DSS), bismaleimide hexane (BMH), and dimethyl heptamethinyl ester 2HCl (Forbes-Cori) are examples of useful homobifunctional crosslinkers, and bis-[B-(4-azidosalicylic acid)ethyl] disulfide (BASED) and N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinkers used in this disclosure. For the latest review on protein coupling techniques, see Means et al. (1990), which are incorporated herein by reference. Bioconj. Chem 1:2-12.

[0139] Table 2: Examples of TDP-43 combined with structural domains

[0140]

[0141] c. Optional connector

[0142] The fusion proteins described herein may optionally contain one or more linkers. Linkers may be peptide- or non-peptide-based. The purpose of linkers is, in particular, to provide sufficient distance between functional domains within the protein (e.g., between the J domain and the TDP-43 binding domain, between tandem arrangements of the TDP-43 binding domains, between the J domain and the TDP-43 binding domain and an optional targeting agent, or between the J domain and the TDP-43 binding domain and an optional detection domain or epitope) for optimal function of each domain. Clearly, the linkers preferably do not interfere with the separate function of the J domain, which is the target protein binding domain of the fusion protein according to the invention. If present in the fusion protein of the invention, the linker is selected to attenuate cytotoxicity caused by the target protein (TDP-43 protein), and may be omitted if direct attachment achieves the desired effect. Linkers present in the fusion protein of the invention may contain one or more amino acids encoded by a nucleotide sequence present in or around a cloning site on an expression vector, within which a nucleotide segment encoding a protein domain or the entire fusion protein as described herein is inserted within a frame. In one embodiment, the peptide linker is from 1 amino acid to 20 amino acids in length. In another embodiment, the peptide linker is from 2 amino acids to 15 amino acids in length. In yet another embodiment, the peptide linker is from 2 amino acids to 10 amino acids in length.

[0143] The selection of one or more peptide linkers to generate the fusion protein according to the invention is within the knowledge and skill of those skilled in the art. See, for example, Arai et al. Protein Eng. , 14 (8): 529-532 (2001); Crasto et al., Protein Eng. , 13 (5): 309-314 (2000); George et al., Protein Eng. , 15 (11): 871-879 (2003); Robinson et al., Proc. Natl. Acad. Sci. USA References cited herein are incorporated herein by reference in their entirety. Examples of linkers having two or more amino acids that can be used to prepare fusion proteins according to the invention include, but are not limited to, those provided in Table 3 below.

[0144] Table 3: Connector Sequence

[0145] SEQ ID NO: length sequence 56 2 SR 57 4 GTGS 58 5 GLESR 59 4 GGSG 60 4 GGGS 61 5 DIAAA 62 9 DIAAALESR 63 15 GGGGSGGGGSGGGGS 64 11 AEAAAKEAAAK 65 15 SGGGSGGGGSGGGGS 66 25 DIGGGGSGGGGSGGGGSGGGGSAAA

[0146] d. Targeted reagents

[0147] The fusion proteins disclosed herein may further include a targeting moiety. As used herein, the terms "targeting moiety" and "targeting agent" are used interchangeably and refer to substances associated with the fusion protein that enhance its binding, transport, accumulation, residence time, bioavailability, or modify its biological activity or therapeutic efficacy in cells or a subject. The targeting moiety may be functional at the tissue, cellular, and / or subcellular levels. For example, after administration of the fusion protein to a subject, the targeting moiety may direct the fusion protein to specific cells, tissues, or organs, or to intracellular distribution. 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 internally located. In yet another embodiment, the targeting moiety is attached to the fusion protein via chemical conjugation.

[0148] The target may include, but is not limited to, organic or inorganic molecules, peptides, peptide mimics, proteins, antibodies or fragments thereof, growth factors, enzymes, lectins, antigens or immunogens, viruses or components thereof, viral vectors, receptors, receptor ligands, toxins, polynucleotides, oligonucleotides or aptamers, nucleotides, carbohydrates, sugars, lipids, glycolipids, nucleoproteins, glycoproteins, lipoproteins, steroids, hormones, growth factors, chemical inducers, cytokines, chemokines, drugs or small molecules.

[0149] In one exemplary embodiment of the invention, the targeting portion enhances the binding, transport, accumulation, residence time, and bioavailability of the platform or its associated ligands and / or active agents, or modifies the bioactivity or therapeutic efficacy of the platform or its associated ligands and / or active agents in target cells or tissues, such as neurons, the central nervous system, and / or the peripheral nervous system. Therefore, the targeting portion may be specific to cellular receptors associated with the central nervous system, or otherwise associated with enhanced delivery to the CNS across the blood-brain barrier (BBB). Thus, as described above, the ligand can be both a ligand and a targeting portion.

[0150] In some embodiments, the targeting portion may be a cell-penetrating peptide, such as that described in, for example, U.S. Patent No. 10,111,965, which is incorporated herein by reference in its entirety. In another embodiment, the targeting portion may be an antibody or an antigen-binding fragment or single-chain derivative thereof, such as that described in, for example, U.S. Serial No. 16 / 131,591, which is incorporated herein by reference in its entirety. In a further embodiment, the targeting portion may be an amino acid sequence for nuclear localization or nuclear output signals.

[0151] The targeting portion can be coupled to a platform for targeted cell delivery by binding directly or indirectly to the core. For example, in an embodiment where the core comprises nanoparticles, the conjugation of the targeting portion to the nanoparticles can utilize similar functional groups used for tethering PEG to the nanoparticles. Thus, the targeting portion can bind directly to the nanoparticles through functionalization of the targeting portion. Alternatively, as discussed above, the targeting portion can bind indirectly to the nanoparticles by conjugating the targeting portion to functionalized PEG. The targeting portion can attach to the core via covalent, non-covalent, or electrostatic interactions. In one embodiment, the targeting portion is a peptide. In a particular embodiment, the targeting portion is a peptide covalently attached to the N-terminus of a fusion protein.

[0152] e. Epitope

[0153] In some embodiments, the fusion protein of the present invention contains optional epitopes or tags that can confer 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 is typically attached to the N-terminus or C-terminus of the fusion protein. In one embodiment, the fusion protein of the present invention further comprises epitopes for facilitating purification. Examples of such epitopes that can be used for purification, provided in Table 4 below, include human IgG1 Fc sequence (SEQ ID NO: 67), FLAG epitope (DYKDDDDK, SEQ ID NO: 68), His6 epitope (SEQ ID NO: 69), c-myc (SEQ ID NO: 70), HA (SEQ ID NO: 71), 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 comprises epitopes that, when administered to a subject, such as a human, increase the half-life of the fusion protein. Examples of such epitopes that can be used to increase half-life include human Fc sequences. 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.

[0154] Table 4: Representative Examples of Epitopes

[0155]

[0156]

[0157] f. Cell-penetrating peptides

[0158] In other embodiments, the fusion protein described herein may further comprise a cell-penetrating peptide. Cell-penetrating peptides are known to carry conjugated cargo into cells, whether the conjugated cargo is a small molecule, peptide, protein, or nucleic acid. Non-limiting examples of cell-penetrating peptides in the fusion protein of the present invention include, but are not limited to, polycationic peptides such as HIVTAT peptides 49-57, polyarginine and penetrantin pAntan (43-58), amphiphilic peptides such as pep-1, hydrophobic peptides such as C405Y, etc. See Table 5 below.

[0159] Table 5: Examples of cell-penetrating peptides

[0160] SEQ ID NO: length sequence 74 9 RKKRRQRRR 75 15 RQIKWFQNRRMKWKK 76 21 KETWWETWWTEWSQPKKKRKV 77 17 CSIPPEVKFNKPFVYLI

[0161] Therefore, in one embodiment, the fusion protein comprises a cell-penetrating peptide and a fusion protein, wherein the cell-penetrating peptide is selected from SEQ ID NO: 74-77, and the fusion protein comprises a J domain and a TDP-43 binding domain. In one embodiment, the fusion protein is selected from SEQ ID NO: 80-85 and 89-96. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 74, and the fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. In another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO: 75, and the fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO: 76, and the fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO: 77, and said fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. Cells expressing the fusion protein construct having the cell-penetrating peptide can be administered to subjects, such as human subjects (e.g., patients with or at risk of having TDP-43 syndrome). The fusion protein is secreted from the cells, which helps reduce the aggregation of TDP-43-containing proteins and / or associated cytotoxicity.

[0162] g. Arrangement of J domain and TDP-43 combined domain

[0163] The fusion protein described herein can be arranged in several 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. In either configuration, the TDP-43 binding domain and the J domain can optionally be separated via a linker as described above.

[0164] In some embodiments, the J domain may attach to multiple TDP-43 binding domains, such as two, three, four, or more TDP-43 binding domains. The TDP-43 binding domains may attach to the N-terminal side of the J domain. Alternatively, the TDP-43 binding domain may attach to the C-terminal side of the J domain. In another embodiment, the TDP-43 binding domain may attach to both the N-terminus and C-terminus sides of the J domain. Each of the multiple TDP-43 binding domains may be the same TDP-43 binding domain. In yet 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).

[0165] In some implementations, the fusion protein may comprise structures selected from the group consisting of:

[0166]

[0167]

[0168] r. TXTX-DnaJ-X-DnaJ, and

[0169] s. TX-TDnaJ-X-TDnaJ-X-TTTT

[0170] in,

[0171] T is the TDP-43 associative domain.

[0172] DNAJ is the J domain of the J protein, and

[0173] X is an optional connector.

[0174] In one embodiment, the fusion protein includes a J domain selected from SEQ ID NO: 5, 6, 10, 24, and 31. In a particular embodiment, the fusion protein includes the J domain of SEQ ID NO: 5.

[0175] In another embodiment, the TDP-43 binding domain is selected from SEQ ID NO:51-55. In a particular embodiment, the TDP-43 binding domain is selected from SEQ ID NO:51-53.

[0176] In another embodiment, the fusion protein comprises 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 comprises at least two copies of the J domain of SEQ ID NO: 5 and the TDP-43 binding domain of SEQ ID NO: 53.

[0177] Non-limiting examples of fusion protein constructs containing a J domain and a TDP-43 binding domain are provided. Figure 2 The details are schematically depicted and also shown in Table 6 below. In another embodiment, the specific fusion protein construct is selected from SEQ ID NO: 80-85 and 89-96.

[0178] Table 6: Fusion protein constructs and control constructs

[0179]

[0180]

[0181]

[0182] II. Nucleic acids encoding the fusion protein construct

[0183] According to another aspect of the invention, an isolated nucleic acid is provided comprising a polynucleotide sequence selected from: (a) a polynucleotide encoding a fusion protein of any of the foregoing embodiments, or (b) a complement of the polynucleotide of (a). The invention provides an isolated nucleic acid encoding a fusion protein comprising a J domain and a TDP-43 binding domain, and a sequence complementary to such a nucleic acid molecule encoding the fusion protein, including its homologous variants. In another aspect, the invention includes a method for generating a nucleic acid encoding a fusion protein disclosed herein, and a sequence complementary to a nucleic acid molecule encoding the fusion protein, including its homologous variants. The nucleic acid according to this aspect of the invention can be a pre-messenger RNA (mRNA precursor), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0184] In another aspect, a method for producing a fusion protein is disclosed, comprising (a) synthesizing and / or assembling nucleotides encoding the fusion protein, (b) incorporating the encoding gene into an expression vector suitable for host cells, (c) transforming the expression vector into appropriate host cells, and (d) culturing the host cells under conditions that induce or allow expression of the fusion protein in the transformed host cells, thereby producing a biologically active fusion protein, which is recovered as an isolated fusion protein by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology are used to prepare the polynucleotides and expression vectors of the present invention.

[0185] According to the present invention, a nucleic acid sequence (or its complement) encoding the fusion protein disclosed herein is used to generate 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 generate a construct containing a gene encoding the fusion protein of the present invention or its complement. In some embodiments, the cloning strategy is used to generate a gene encoding the fusion protein of the present invention or its complement.

[0186] In some implementations, the nucleic acid encoding one or more fusion proteins is an RNA molecule, and may be a pre-messenger RNA (mRNA precursor), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0187] In various implementations, the nucleic acid is mRNA introduced into the cell for transient expression of the desired polypeptide. As used herein, “transient” refers to a period of time during which non-integrated transgene expression occurs over hours, days, or weeks, wherein the time of expression is shorter than the time of expression when the polynucleotide is integrated into the genome or contained within a stable plasmid replicon in the cell.

[0188] In a particular embodiment, the mRNA encoding the polypeptide is an in vitro transcribed mRNA. As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA that has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated by an in vitro transcription vector. The in vitro transcription vector contains a template for generating the in vitro transcribed RNA.

[0189] In certain embodiments, the mRNA may further comprise a 5' cap or a modified 5' cap and / or a poly(A) sequence. As used herein, a 5' cap (also known as an RNA cap, RNA7-methylguanosine cap, or RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of the eukaryotic messenger RNA shortly after transcription initiation. The 5' cap contains a terminal group that is linked to the first nucleotide transcribed and is recognized by the ribosome and protected from RNases. The capped portion may be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency. In certain embodiments, the mRNA comprises a poly(A) sequence of about 50 to about 5000 adenine. In one embodiment, the mRNA comprises a poly(A) sequence of about 100 to about 1000 bases, about 200 to about 500 bases, or about 300 to about 400 bases. In one embodiment, the mRNA comprises a poly(A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1000 bases or more. The poly(A) sequence can be modified chemically or enzymatically to regulate mRNA functionality, such as localization, stability, or translation efficiency.

[0190] As used herein, the terms “polynucleotide variant” and “variant” etc. refer to a polynucleotide that exhibits basic sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms include polynucleotides in which one or more nucleotides have been added, deleted, or substituted with different nucleotides compared to a reference polynucleotide. In this regard, it is well known in the art that certain alterations can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, such altered polynucleotides retaining the biological function or activity of the reference polynucleotide.

[0191] In some embodiments, the nucleic acid sequence comprises a nucleotide sequence encoding a target gene within the nucleic acid cassette (e.g., a fusion protein comprising a J domain and a polyglutamine-binding domain). As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence within a vector that can express RNA and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains a target gene, such as a target polynucleotide. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, such as promoters, enhancers, poly(A) sequences, and a target gene, such as a target polynucleotide. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are oriented positionally and sequentially within the vector such that the nucleic acids within the cassettes can be transcribed into RNA and, if necessary, translated into proteins or polypeptides, undergo appropriate post-translational modifications required for activity in transformed cells, and translocate to appropriate compartments for biological activity by targeting appropriate intracellular compartments or secreting into extracellular compartments. Preferably, the cassette has its 3' and 5' ends adapted for easy insertion into a vector, for example, it has a restriction endonuclease site at each end. The cassette can be removed as a single unit and inserted into a plasmid or viral vector.

[0192] Illustrative ubiquitous expression control sequences applicable to specific implementations include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5 and P11 promoters from vaccinia virus, elongation factor 1-α (EF1a) 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 protein 70 kDa 5 (HSPA5), heat shock protein 90 kDa β, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinin (b-KIN), and human ROSA. Locus 26 (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 let.407: 313-9), β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al., Journal of Virology. 2003; 77 (l7): 9439-9450).

[0193] In one implementation, at least one element may be used in conjunction with the polynucleotide described herein to enhance transgenic target specificity and expression (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, for example promoters. Promoters that promote expression in most tissues include, but are not limited to, human elongation factor 1a-subunit (EF1a), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression for certain cell types, such as, but not limited to, nervous system promoters, which can be used to restrict expression for neurons, astrocytes, or oligodendrocytes. Non-limiting examples of tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synaptic protein (Syn), methyl-CpG-binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH, ηβ2, PPE, Enk, and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements in astrocytes include the glial fibrillary acidic protein (GFAP) and the EAAT2 promoter. Non-limiting examples of tissue-specific expression elements in oligodendrocytes include the myelin basic protein (MBP) promoter. (Yu et al., 2011) Molecular Pain (7:63, incorporated herein by reference in its entirety) Lentiviral vectors were used to assess eGFP expression in rat DRG cells and primary DRG cells under the CAG, EFIa, PGK, and UBC promoters, and UBC was found to show weaker expression than the other three promoters, with only 10–12% glial cell expression visible for all promoters. Soderblom et al. (E. Neuro 2015, incorporated herein by reference in its entirety) reported eGFP expression in AAV8 with CMV and UBC promoters and AAV2 with CMV promoters after injection into the motor cortex. Intranasal administration of plasmids containing UBC or EFIa promoters showed greater sustained airway expression than expression with CMV promoters (see, for example, Gill et al., (2001)). Gene TherapyVolume 8, 1539–1546; incorporated in its entirety by reference). Husain et al. (2009) Gene Therapy Passini and Wolfe (J. Virol. 2001, 12382-12392, cited in its entirety) evaluated the HβH constructs with the hGUSB, HSV-1LAT, and NSE promoters and found that the HβH constructs showed weaker expression in the mouse brain than NSE. Passini and Wolfe (J. Virol. 2001, 12382-12392, cited in its entirety) evaluated the long-term effects of the HβH vector following intraventricular injection in newborn mice and found sustained expression for at least one year. Compared with CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE + wpre, NSE (0.3 kb), NSE (1.8 kb), and NSE (1.8 kb + wpre), when using the NF-L and NF-H promoters, according to Xu et al. (2001)... Gene Therapy Low expression was found in all brain regions (see, 8, 1323-1332; cited in its entirety). Xu et al. found promoter activity in descending order as follows: NSE (1.8 kb), EF, NSE (0.3 kb), GFAP, CMV, hENK, PPE, NFL, and NFH. NFL is a 650 nucleotide promoter, while NFH is a 920 nucleotide promoter; neither is present in the liver, but NFH is abundant in sensory proprioceptive neurons, the brain, and the spinal cord, while NFH is present in the heart. Scn8a is a 470 nucleotide promoter, expressed in the DRG, spinal cord, and brain, with particularly high expression observed in hippocampal neurons and cerebellar Purkinje cells, the cortex, thalamus, and hypothalamus (see, for example, Drews et al. 2007 and Raymond et al. 2004; cited in its entirety).

[0194] III. Vectors containing nucleic acids encoding fusion proteins

[0195] Vectors containing nucleic acids according to the invention are also provided. Such vectors preferably contain additional nucleic acid sequences, such as elements necessary for transcription / translation (e.g., promoter and / or terminator sequences) encoding nucleic acid sequences of phosphatases. The vectors may also contain nucleic acid sequences encoding selection markers (e.g., antibiotics) to select or maintain host cells transformed with the vector. The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, for example, inserted into, the vector nucleic acid molecule. The vector may include sequences that guide autonomous replication in the cell, or may include sequences sufficient to allow integration into the host cell DNA. In certain embodiments, nonviral vectors are used to deliver one or more polynucleotides considered herein to affected cells (e.g., neuronal 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. Illustrative examples of nonviral vectors include, but are not limited to, mRNA, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, kinases, and bacterial artificial chromosomes.

[0196] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposon elements, such as piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives. Further illustrative examples of vectors include, but are not limited to, plasmids, phage particles, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacterial bacteriophages such as λ phage or M13 phage, and animal viruses. Illustrative examples of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Illustrative examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V 5-DEST™, pLenti6 / V 5-DEST™, and pLenti6.2 / V 5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequence of the polypeptide disclosed herein may be ligated into such expression vectors for expression of the polypeptide in mammalian cells.

[0197] In certain embodiments, the vector is a free vector or a vector maintained outside the chromosome. As used herein, the term "free" refers to a vector that is capable of replicating without integrating into the host's chromosomal DNA and is not gradually lost from dividing host cells, which also means that the vector replicates outside the chromosome or in a free manner.

[0198] Vectors may contain one or more recombination sites for any of a wide variety of site-specific recombinases. It should be understood that the target site of the site-specific recombinase, plus any sites required for vector integration, refers to the vector itself, such as a retroviral or lentiviral vector. As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombination site” refer to a specific nucleic acid sequence that the recombinase recognizes and binds to.

[0199] For example, one recombination site for Cre recombinase is loxP, a 34-base-pair sequence containing two 13-base-pair inverted repeats flanking an 8-base-pair core sequence (acting as the recombinase binding site) (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Suitable recognition sites for FLP recombinase include, but are not limited to: FRT (McLeod et al., 1996), FI, F2, F3 (Schlake and Bode, 1994), FyFs (Schlake and Bode, 1994), FRT (LE) (Senecoff et al., 1988), and FRT (RE) (Senecoff et al., 1988).

[0200] Other examples of recognized sequences are attB, attP, attL, and attR sequences, which are recognized by recombinase integrase such as phi-c3l. (pC3l SSR mediates recombination only between heterologous sites attB (34 bp) and attP (39 bp) (Groth et al., 2000). AttB and attP, named after the attachment sites of phage integrase on the bacterial and phage genomes, respectively, both contain incomplete inverted repeats that are likely to be bound by the φ031 homodimer (Groth et al., 2000). The product sites attL and attR are effectively inert to further tpQA1-mediated recombination (Belteki et al., 2003), making the reaction irreversible. For catalytic insertion, it has been found that inserting DNA carrying attB into the genomic attP site is easier than inserting attP into the genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to locate the "docking site" carrying attP into a defined locus via homologous recombination, which then pairs with the entry sequence carrying attB for insertion.)

[0201] As used herein, “internal ribosome entry site” or “IRES” refers to an element that facilitates direct internal ribosome entry into the start codon, such as ATG, of a cistron (protein-coding region), 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 a particular embodiment, the vector comprises one or more target polynucleotides encoding one or more polypeptides. In a particular embodiment, in order to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequence may be separated by one or more IRES sequences or a polynucleotide sequence encoding a self-cleaving polypeptide. In one embodiment, the IRES used in the polynucleotides considered herein is an EMCV IRES.

[0202] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates 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 a particular embodiment, the vector contains a polynucleotide having a shared Kozak sequence and encoding a fusion protein comprising a J domain and a TDP-43 binding domain. Elements that guide 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 a particular embodiment, the vector contains a polyadenylated 3' sequence of a polynucleotide encoding the polypeptide to be expressed.

[0203] Illustrative examples of viral vector systems applicable to the specific implementations considered herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0204] In various implementations, one or more polynucleotides encoding a fusion protein containing a J domain and a polyglutamine-binding domain are introduced into cells, such as neurons, by transducing cells with recombinant adeno-associated virus (rAAV) containing one or more polynucleotides. AAV is a small (~26 nm) replication-deficient, primarily free, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can incorporate its genome into the host cell's genome. Recombinant AAV (rAAV) typically consists at a minimum of transgenes and their regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequence is approximately 145 bp in length. In certain embodiments, rAAV comprises an ITR and capsid sequence separated from: AAV1, AAV2 (e.g., described in US6962815B2, which is incorporated herein by reference in its entirety), AAV3, AAV4, AAV5 (e.g., described in US7479554B2, which is incorporated herein by reference in its entirety), AAV6, AAV7, AAV8 (e.g., described in US7282199B2, which is incorporated herein by reference in its entirety), AAV9 (e.g., described in US9737618B2, which is incorporated herein by reference in its entirety), AAV rh10 (e.g., described in US9790472B2, which is incorporated herein by reference in its entirety), or AAV10. In one embodiment, the vector of the present invention is encapsulated within a capsid selected from AAV2, AAV5, AAV8, AAV9, and AAV rh10. In one embodiment, the vector is encapsulated in AAV2. In one embodiment, the vector is encapsulated in AAV5. In one embodiment, the carrier is encapsulated in an AAV8. In another embodiment, the carrier is encapsulated in an AAV9. In yet another embodiment, the carrier is encapsulated in an AAV10.

[0205] In some embodiments, chimeric rAAVs are used, where the ITR sequence is isolated from one AAV serotype, while 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 referred to as AAV2 / AAV6. In certain embodiments, the rAAV vector may contain an ITR from AAV2 and a capsid protein from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In another preferred embodiment, the rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0206] In some implementations, modification and selection methods can be applied to the AAV capsid to make it more likely to transduce target cells.

[0207] The construction, production and purification of rAAV vectors have been disclosed in, for example, U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated herein by reference in its entirety.

[0208] IV. Delivery

[0209] In certain embodiments, one or more polynucleotides encoding a fusion protein comprising a J domain and a TDP-43 binding domain are introduced into cells via a non-viral or viral vector. Exemplary methods for non-viral delivery of polynucleotides considered in certain embodiments include, but are not limited to: electroporation, sonoporation, lipid transfection, microinjection, bioballistics, virions, liposomes, immunoliposomes, nanoparticles, polycationic or lipid nucleic acid conjugates, naked DNA, artificial viral particles, DEAE-dextran-mediated transfer, gene gun, and heat shock.

[0210] Illustrative examples of multinucleotide delivery systems considered in specific embodiments include, but are not limited to, those provided 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 effective receptor recognition of multinucleotides via lipid transfection have been described in the literature. See, for example, Liu et al., (2003) Gene Therapy. 10:180-187; and Balazs et al., (20W) Journal of Drug Delivery. 2011:1-12. Antibody-targeted, bacterial-derived, nanocell-based delivery is also considered in specific embodiments.

[0211] In certain implementations, the multinucleotide-containing viral vectors considered can be delivered in vivo by administration to individual patients, typically via systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion), intrathecal injection, intraventricular injection, or local application, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by re-entrapment of the cells into the patient.

[0212] In one embodiment, a viral vector containing a polynucleotide encoding the fusion protein disclosed herein is directly administered to a organism for in vivo cell transduction.

[0213] Properly packaged and formulated viral vectors can be delivered into the central nervous system (CNS) via intrathecal delivery. For example, adeno-associated virus vectors can be delivered using the method described in U.S. Serial No. 15 / 771,481, which is incorporated herein by reference in its entirety.

[0214] Alternatively, naked DNA may be applied. Application is by any route commonly used to introduce molecules into final contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation. Suitable methods for applying such nucleic acids are available and well known to those skilled in the art, and although more than one route may be used for applying a particular composition, a particular route often facilitates a more direct and efficient response than another.

[0215] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into cells, such as neuronal cells or neuronal stem cells, by transducing cells with a retrovirus containing one or more polynucleotides, such as a lentivirus. As used herein, the term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Illustrative retroviruses applicable to specific embodiments include, but are not limited to: Moloney mouse leukemia virus (M-MuLV), Moloney mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses. As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); caprine arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence element) is preferred.

[0216] As a result of LTR modification, lentiviral vectors preferably contain several safety enhancements. "Self-inactivating" (SIN) vectors refer to replication-defective vectors, for example, in which the right (3') LTR enhancer-promoter region (referred to as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the lentiviral vector is generated according to known methods. See, for example, Kutner et al., BMC Biotechnol. 2009; 9:10. Doi: 10.1186 / 1472-6750-9-10; Kutner et al., Nat. Protoc. 2009; 4 (4):495-505. Doi: l0.l038 / nprot.2009.22.

[0217] According to certain specific embodiments considered herein, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, and numerous substitutions and alterations can be adapted in certain lentiviral sequences without impairing the transfer vector's ability to perform the functions described herein. Furthermore, a variety of 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 Nos. 6,013,516; and 5,994,136, many of which can be adapted to produce the viral vectors or transfer plasmids considered herein.

[0218] In various embodiments, one or more polynucleotides encoding the fusion protein disclosed herein are introduced into target cells by transducing cells with an adenovirus containing one or more polynucleotides. Adenovirus-based vectors can achieve 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 modified such that the Ad E1a, E1b, and / or E3 genes are replaced with transgenes; subsequently, the replication-deficient vector is propagated in human 293 cells, which trans-supply the missing gene function. Ad vectors can be transduced in vivo into multiple tissue types, including non-dividing differentiated cells, such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0219] The generation and propagation of replication-deficient current adenoviral vectors can be achieved using a unique helper cell line designated 293, which is transformed from human embryonic kidney cells via the Ad5 DNA fragment and constitutively expresses the E1 protein (Graham et al., 1977). Since the E3 region is optional in the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors, with the aid of 293 cells, carry foreign DNA in the E1, D3, or both regions (Graham & Prevec, 1991). Adenoviral vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies of recombinant adenovirus administration 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 implantation into the brain (Le Gal LaSalle et al., 1993). Examples of the use of Ad vectors in clinical trials involve intramuscular injection for polynucleotide therapy in antitumor immunotherapy (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)).

[0220] In various embodiments, one or more polynucleotides encoding the fusion protein of the present invention are introduced into the target cells of a subject by transducing cells with a herpes simplex virus containing one or more polynucleotides, such as HSV-1 or HSV-2.

[0221] Mature HSV viral particles consist of an enveloped icosahedral capsid containing a viral genome, which is a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-deficient. Most replication-deficient HSV vectors contain deletions to remove one or more immediate early, early, or late HSV genes to prevent replication. For example, an HSV vector may lack an immediate early gene selected from ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors lie in their ability to enter latent phases that can lead to long-term DNA expression and their large viral DNA genomes capable of accommodating exogenous DNA inserts up to 25 kb. HSV-based carriers are described, for example, in U.S. Patent Nos. 5,837,532, 5,846,782, and 5,804,413, and international patent applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0222] V. Cells expressing fusion proteins

[0223] In another aspect, the present invention provides cells expressing the fusion protein described herein. 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 another embodiment, the cells are human cells derived from a patient who has or is at risk of having a TDP-43-mediated condition, including but not limited to ALS, FTD, and Alzheimer's disease. The cells can be neuronal cells or muscle cells.

[0224] Cells expressing the fusion protein can be used to produce the fusion protein. In this embodiment, cells are transfected with a vector overexpressing the fusion protein. The fusion protein may optionally contain an epitope, such as a human Fc domain or a FLAG epitope, as described above, which will facilitate purification (using protein A or anti-FLAG antibody columns, respectively). The epitope may be attached to the remainder of the fusion protein via a linker or protease substrate sequence, such that the epitope can be removed from the fusion protein during or after purification.

[0225] Cells expressing fusion proteins can also be used in a therapeutic context. In one embodiment, cells are collected from a patient requiring treatment (e.g., a patient with or at risk of having a TDP-43-mediated condition). In one embodiment, the cells are neurons. The collected cells are then transfected with a vector expressing the fusion protein. The transfected cells can then be processed to enrich or select for transfected cells. The transfected cells can also be processed to differentiate into different cell types, such as neurons. After processing, the transfected cells can be administered to the patient. In one embodiment, the cells are administered by direct injection into the central nervous system via intrathecal, intracranial, or intraventricular injection.

[0226] In another implementation, cells expressing a secretory form of the fusion protein can be used. For example, the fusion protein construct can be designed to have a signal sequence at its N-terminal end. Representative signal sequences are shown in Table 7 below.

[0227] Table 7: Representative signal sequences

[0228] SEQ ID NO: sequence 98 MGVKVLFALICIAVAEA 99 MAPVQLLGLLVLFLPAMRC 100 MAVLGLLFCLVTFPSCVLS

[0229] Therefore, in one embodiment, the fusion protein comprises a signal sequence and a fusion protein, wherein the signal sequence is selected from SEQ ID NO: 98-100, and the fusion protein comprises a J domain and a TDP-43 binding domain. In one embodiment, the signal sequence is selected from SEQ ID NO: 98-100, and the fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 98, and the fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 99, and the fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 100, and the fusion protein is selected from SEQ ID NO: 80-85, 89-90, and 92-96. Cells expressing a fusion protein construct with a signaling sequence can be administered to subjects, such as human subjects (e.g., patients with or at risk of developing TDP-43 syndrome). The fusion protein is secreted from the cells, which helps reduce TDP-43 protein aggregation and / or associated cytotoxicity.

[0230] As described above, in some embodiments, the fusion protein may further comprise a cell-penetrating peptide. Cells expressing a fusion protein comprising both a signaling sequence and a cell-penetrating peptide will be able to secrete a fusion protein lacking the signaling sequence. The secreted fusion protein, also comprising the cell-penetrating peptide, can then enter nearby cells and has the potential to reduce TDP-43 protein-mediated aggregation and / or cytotoxicity in these cells.

[0231] VI. How to Use

[0232] In another aspect, the present invention provides a method for achieving beneficial effects in conditions and / or TDP-43 conditions, conditions or conditions mediated by TDP-43 aggregation. TDP-43 conditions are selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and age-related TDP-43 encephalopathy primarily involving the limbic lobe.

[0233] In some embodiments, the present invention provides a method for treating a subject, such as a human, with TDP-43 disease, condition, or status, comprising the steps of: administering to the subject a therapeutically or preventively effective amount of a fusion protein, a nucleic acid encoding such a fusion protein, or a viral vector encoding the fusion protein described herein, wherein the administration results in an improvement in one or more biochemical or physiological parameters or clinical endpoints associated with TDP-43 disease, condition, or status.

[0234] In other embodiments, the present invention provides a method for reducing TDP-43 accumulation in cells. The cells may be cultured cells or isolated cells. The cells may also be derived from a subject, such as a human subject. In one embodiment, the cells are in the central nervous system of a human subject. In another embodiment, the human subject has or is at risk of having a TDP-43 disease, including but not limited to amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. In a particular embodiment, the TDP-43 disease is amyotrophic lateral sclerosis.

[0235] The aggregation of TDP-43 protein can be detected in a variety of ways. In one instance, for example, aggregated TDP-43 protein can be distinguished from free (i.e., soluble) TDP-43-containing protein based on solubility by passing cell lysates through a selective filter to trap insoluble aggregates. Non-aggregated proteins pass through these filters, while aggregates remain on the filters and can be detected using any number of reagents, including antibodies against TDP-43 protein. The amount of aggregated protein trapped in lysates of cell samples treated with fusion proteins as described herein, cells expressing fusion proteins, or nucleic acids, vectors, or viral particles encoding fusion proteins can be compared with lysates from untreated or control-treated cells, wherein a reduction in the amount of aggregated TDP-43 protein in the treated sample, compared to a control sample, indicates the efficacy 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 reduction in aggregated TDP-43 protein compared to the control indicated higher potency. The reduction in TDP-43 protein aggregation can also be detected directly in cells, for example, by examining the labeling reagents accompanying the detection of TDP-43 protein using immunofluorescence microscopy (see, for example, Ding et al., (2015)). Oncotarget, 6: 24178–24191; Chou et al., (2015) Hum. Mol. Genet (24:5154-5173 and Example 1). In some embodiments, a greater reduction in TDP-43 peptide levels compared to a control indicates higher potency.

[0236] Therefore, in one embodiment, the method includes contacting cells with an amount of fusion protein or nucleic acid, vector, or viral particle encoding the fusion protein, said amount effectively reducing TDP-43 protein aggregation by at least 10%, such as 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 cells or control cells.

[0237] 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 a reporter construct containing TDP-43. Similarly, in another embodiment, the method includes contacting cells with an amount of the fusion protein, cells expressing the fusion protein, nucleic acid, vector, or viral particle encoding the fusion protein, said amount effectively reducing the level of TDP-43 protein by at least 10%, such as 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 cells or control cells.

[0238] VII. Pharmaceutical Compositions

[0239] The compositions considered herein may include one or more fusion proteins comprising a J domain and a TDP-43 binding domain, polynucleotides encoding such fusion proteins, vectors comprising such fusion proteins, genetically modified cells, etc., as considered herein. Compositions include, but are not limited to, pharmaceutical compositions. “Pharmaceutical composition” means a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration alone or in combination with one or more other therapeutic modalities to cells or animals. It should also be understood that, when necessary, the composition may also be administered in combination with other pharmaceutical agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There are no practical limitations on other components that may also be included in the composition, provided that the additional pharmaceutical agents do not adversely affect the composition’s ability to deliver the intended therapy.

[0240] The phrase “pharmaceutically acceptable” in this document refers to compounds, materials, compositions, carriers, and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0241] As used herein, “pharmaceutically acceptable carrier,” “diluent,” or “excipient” includes, but is not limited to, any adjuvant, carrier, excipient, gliding agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the United States Food and Drug Administration as acceptable for use in humans or domesticated animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragali gum; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin wax, silicone, bentonite, silicic acid, and zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical preparations.

[0242] VIII. Dosage

[0243] The dosage of the compositions described herein (e.g., compositions comprising fusion protein constructs, nucleic acid or gene therapy viral particles) can vary depending on a number of factors, such as the pharmacodynamic properties of the compound; the administration method; the recipient's age, health, and weight; the nature and severity of symptoms; the frequency of treatment and the type of concurrent treatment (if present); and the clearance rate of the compound in the treated animal. The compositions described herein may initially be administered at an appropriate dosage, which may be adjusted as needed based on the clinical response. In some respects, the dosage of the composition is a prophylactic or therapeutically effective amount.

[0244] IX. Reagent Kit

[0245] Kits considered include: (a) pharmaceutical compositions comprising the fusion protein constructs described herein, nucleic acids encoding such fusion proteins, or viral particles containing such nucleic acids, which reduce TDP-43 protein aggregation in cells or subjects; and (b) a package insert with instructions for performing any of the methods described herein. In some aspects, a kit may include (a) a pharmaceutical composition comprising the compositions described herein, which reduces TDP-43 protein aggregation in cells or subjects, (b) additional therapeutic agents; and (c) a package insert with instructions for performing any of the methods described herein. Example

[0246] To test whether the J domain could be specifically modified to promote proper folding of aggregate proteins, we designed and tested a number of fusion protein constructs designed to target the TDP-43 protein.

[0247] Example 1: Fusion Protein Design

[0248] A. Method

[0249] General techniques and materials

[0250] Unless otherwise stated, the practice of this invention employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope of the art. See Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3rd ed., Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology,” FM Ausubel et al., eds., 1987; series “Methods in Enzymology,” Academic Press, San Diego, Calif.; “PCR 2: a practical approach,” MJ MacPherson, BD 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 ed., McGraw-Hill, 2005; and Freshney, RI, “Culture of Animal Cells: A Manual of Basic Technique,” ​​4th ed., John Wiley & Sons, Somerset, NJ, 2000, the contents of which 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). For ease of purification and characterization, some fusion protein constructs used in Example 1, in addition to the sequences provided in SEQ ID NO: 80-85 and 89-96, also contain the FLAG epitope of SEQ ID NO: 68 at the C-terminus or N-terminus of the protein, along with a short linker sequence.

[0251] Protein expression and detection in HEK293 cells

[0252] Expression vector plasmids encoding various protein constructs were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Cell lysates were analyzed for expressed proteins using Western blotting. Samples of culture medium were centrifuged to remove debris prior to analysis. Cells were lysed in lysis buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA, 2% SDS) containing 2 mM PMSF and a complete protease inhibitor cocktail (Sigma). After brief sonication, samples were analyzed for expressed proteins using Western blotting. For Western blotting analysis, samples were boiled in SDS sample buffer and run on polyacrylamide gel electrophoresis. Subsequently, the separated protein bands were transferred to a PVDF membrane.

[0253] The expression of proteins is detected using chemiluminescent signals. In short, the blot is reacted with a primary antibody capable of binding to a specific epitope (e.g., GFP). After washing away the unreacted primary antibody, an enzyme-linked secondary antibody (e.g., an HRP-linked anti-IgG antibody) is allowed to react with the primary antibody molecule bound to the blot. After washing, a chemiluminescent reagent is added, and the resulting chemiluminescent signal in the blot is captured on an X-ray film.

[0254] Fluorescence microscopy examination

[0255] In some cases, aggregations of the TDP-43 (full-length C-terminal fragment) GFP reporter construct (described below) were detected in vivo using fluorescence microscopy. Cultured cells expressing the reporter construct and the fusion protein containing the J domain and the TDP-43 binding domain were washed with PBS and fixed with PBS solution containing 4% paraformaldehyde for 5 minutes. After three 5-minute washes with PBS, nuclear DNA was stained with DAPI. The percentage of transfected cells containing TDP-43 (GFP lesions) was counted.

[0256] Grading determination

[0257] Transfected HEK293 cells were 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 mixture, 2 mM PMSF, 10 mM NaF, and 2 mM Na3VO4. Protein concentration was measured using a BCA assay kit (Pierce) after a brief sonication. Equal volumes of protein were fractionated into soluble fractions (supernatant) and insoluble fractions (clumps) by centrifugation at 16,000 xg for 30 min at 4 °C. The insoluble fractions were further dissolved in SDS lysis buffer (10 mM Tris pH 8.0, 150 mM NaCl, 2% SDS). Both soluble and insoluble fractions were subjected to SDS-PAGE under reducing conditions, followed by Western blotting with an anti-GFP antibody.

[0258] B. Report Subconstruct

[0259] We first investigated whether the TDP-43-targeting fusion molecule of the present invention improved its aggregation in cultured cells. To this end, we generated GFP-based reporter constructs GFP-TDP43 and GFP-TDP43, wherein GFP is fused at its C-terminus to either full-length human TDP-43 protein or a C-terminal fragment of TDP-43, the latter known to cause cytoplasmic aggregation and cytotoxicity (see Table 8 below). HEK293 cells were cultured and transfected with plasmids encoding full-length TDP43 or a C-terminal fragment of TDP43 [GFP-TDP43FL (SEQ ID NO: 101) or GFP-TDP43CTF (SEQ ID NO: 102)], which contain the C-terminal 207 amino acids of TDP-43 (amino acids 208-414 of human TDP-43). We found that most of the expressed GFP-TDP43FL was localized in the cell nucleus (…). Figure 3 (See Figure 1), while GFP-TDP43CTF prepared obvious cytoplasmic inclusions ( Figure 3 ,Small Figure 5 (Zhang et al., (2009) Proc Natl Acad SciU S A., 106 (18):7607-12).

[0260] Table 8: TDP-43 Report Substructs

[0261]

[0262]

[0263] C. Fusion protein construct

[0264] To determine whether the fusion protein of this invention can be used to reduce TDP-43 aggregation, initial experiments were conducted by co-expressing a fusion protein containing a J domain sequence derived from a human Hsp40 J domain protein, conjugated to a single-stranded variable fragment (scFv) that recognizes GFP (data not shown). Surprisingly, when GFP-TDP43CTF was expressed with this construct, most of the aggregation disappeared, while no significant effect was observed when GFP-TDP43CTF was expressed as GFP scFv (without the J domain sequence). This suggests that an HSP70-mediated pathway (not shown) can be used to resolve TDP-43 aggregation.

[0265] We then designed a series of fusion protein constructs, as depicted in Table 9.

[0266] Table 9. Fusion protein constructs and controls

[0267] Builder number SEQ ID NO: Builder Name Remark 1 78 J-domain only In contrast, it contains the J domain from human DnaJB1. 2 79 scFv(3B12A) Control only for scFv(3B12A); combined with TDP-43 3 80 JB1-scFv(3B12A) DnaJB1 J domain fused with scFv(3B12A) 4 81 JB1(P33Q)-scFv(3B12A) Similar to construct 3, it has the P33Q mutation in the conserved HPD motif. 5 82 scFv(3B12A)-JB1 The J domain from construct 3 and the reverse arrangement of TDP-43 6 83 scFv(3B12A)-JB1- scFv(3B12A) J-domain sandwiched between two scFvs combining TDP-43 7 84 JB6-scFv(3B12A) J domain from DnaJB6 8 85 JC6-scFv(3B12A) J domain from DnaJC6 9 86 DnaJB1 Full-length J protein control 10 87 Hsp70 HSP70 reference 11 88 Hsp110 HSP110 comparison 12 89 JB1-scFv(51C1) J domain from DnaJB1 fused with an alternative scFV combining TDP-43 13 90 JB1-scFv(3F10) J domain from DnaJB1 fused with an alternative scFV combining TDP-43 14 91 JB1-2XQBP1 J-domain from DnaJB1 fused with two concatenated copies of QBP1

[0268] Initial experiments were conducted to test the ability of the fusion protein constructs to reduce TDP-43 aggregation. Construct 3 (JB1-scFv(3B12A)), along with chaperone control construct 2 (TDP-43 binding domain only) and construct 4 (identical to construct 3 but containing the mutant P33Q within the conserved HPD motif in the J domain), were transfected into cells expressing either the GFP-TDP43FL or GFP-TDP43CTF reporter constructs. Figure 3 Cells expressing the GFP-TDP43CTF reporter showed greater aggregation of the GFP construct than cells expressing GFP-TDP43FL. Cells expressing construct 3 showed substantially reduced aggregation, while expression of controls (constructs 2 and 4) did not reduce aggregation. Figure 4 (See also Table 10 below). The absence of activity in construct 4 containing P33Q strongly suggests that the ability to reduce aggregation is driven by the J domain acting via the Hsp70 pathway.

[0269] Table 10: Efficacy of fusion protein constructs in reducing aggregation

[0270]

[0271] Cell extracts from these cells were analyzed using immunoblotting, and the levels of GFP-containing reporter constructs or FLAG-containing fusion proteins were determined using either anti-GFP or anti-FLAG antibodies. Figure 5When probed with an anti-GFP antibody, cell extracts expressing GFP-TDP43FL showed the presence of a dominant ~70 kDa band, while cells expressing GFP-TDP43CTF showed the presence of a ~50 kDa band. Interestingly, cells containing the GFP-TDP43CTF reporter and also expressing construct 3 (JB1-scFv(3B12A)) showed a significant reduction in the amount of reporter protein compared to the negative control, the scFv control (construct 2), or the P33Q mutant (construct 4). No difference was observed in the level of the full-length reporter construct (GFP-TDP43FL).

[0272] To investigate whether the reduction in TDP-43 levels was dependent on protein aggregation, extracts from cells expressing 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 reporter fractions was detected using an anti-GFP antibody probe. Figure 6 As shown, in cells expressing the full-length reporter (GFP-TDP43FL), there were no significant changes in reporter levels in either the soluble or insoluble fractions. In contrast, in cells expressing the GFP-TDP43CTF reporter, a moderate reduction in reporter levels 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, a significant reduction in reporter levels was observed, with almost complete disappearance of reporters in the insoluble fraction (presumably in aggregate form).

[0273] In summary, these data strongly suggest that the fusion protein can reduce TDP-43 levels in cells and may act to preferentially accelerate the clearance of aggregated TDP43 protein.

[0274] Based on the results above, several additional constructs (constructs 5-7) were generated, containing different configurations of the TDP-43 binding domain associated with the J domain. These constructs were tested for their ability to reduce aggregation. These new constructs were compared with construct 3 (JB1-scFv(3B12A)), and cells expressing no scFv (negative control) or expressing only scFv (construct 2). Figure 7 The results of these experiments are shown (and summarized in Table 11 below).

[0275] Table 11: Other constructs and controls

[0276]

[0277] like Figure 7As observed, when compared with negative control cells and cells expressing construct 2 (scFv alone), constructs 3 (JB1-scFv(3B12A)), 5 (scFv(3B12A)-JB1), 6 (scFv(3B12A)-JB1-scFv(3B12A)), and 7 (JB6-scFv(3B12A)) showed a modest reduction in aggregation levels in cells expressing the GFP-TDP43FL reporter construct. In contrast, cells expressing the GFP-TDP43CTF construct showed higher overall levels of protein aggregation, consistent with earlier observations, in negative control cells and cells expressing construct 2. Furthermore, cells expressing constructs 3, 5, 6, and 7 all exhibited a sharp reduction in protein aggregation levels, confirming the effectiveness of the following configuration of the fusion protein: DNAJ-T, T-DNAJ, T-DNAJ-T (where DNAJ is the J domain and T is the TDP-43 binding domain). Additionally, multiple J domains (e.g., from DnaJB1 and DnaJB6) were found to be active in the fusion protein. Further constructs were then generated and tested, such as... Figure 8 As shown in Figure 9 (see also Table 12 below). Interestingly, expression of full-length DnaJB1 without the TDP-43 binding domain reduced TDP-43 aggregation by ~43% compared to a >90% reduction by construct 3 (JB1-scFv(3B12A)). Additionally, expression of construct 14, containing two tandem copies of the QBP1 peptide, also showed a significant reduction in aggregation (~71% reduction). QBP1 has previously been shown to interact with TDP-43 (see, for example, 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 placed in the fusion protein. Furthermore, when the levels of the reporter constructs were quantified by immunoblotting of cell extracts using an anti-GFP antibody (…),… 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 reporter constructs.

[0278] Table 12: Generation and testing of other fusion protein constructs

[0279]

[0280] Other builds were tested, as shown in Tables 13 and 14 below.

[0281] Table 13: Generation and testing of other fusion protein constructs

[0282]

[0283] Table 14: Generation and testing of other fusion protein constructs

[0284]

[0285] As shown above, numerous constructs, including those using alternative J domains (see, for example, JB6 and JC7 domains), effectively reduced GFP-TDP43CTF aggregation. Other fusion protein constructs indicating J domains from DNAJC6 and from SV40 or bacterial J domain proteins (DnaJ) were also found to effectively reduce aggregation of other reporter constructs (data not shown). However, construct 16, containing a J domain without a shared HPD sequence (see Table 1, SEQ ID NO: 16), did not reduce GFP-TDP43CTF reporter construct aggregation.

[0286] Other constructs were tested, as shown in Table 15 below. Construct 13 (JB1-scFv(3F10), SEQ ID NO: 90), which binds TDP-43 to scFv 3F10, was fused with the J domain of DNAJB1. Construct 20 (JB1-scFv(3B12A)-DD), SEQ ID NO: 97, contained a dimerizing domain from human DnaJA1. As shown below, construct 13 showed a modest ability to reduce GFP-TDP43CTF aggregation. Expression of construct 20 (construct 3 containing the dimerizing domain) showed a strong reduction in GFP-TDP43CTF aggregation. The further enhancement of the effect through the dimerizing domain is likely due to the enhanced interaction between dimerizing construct 3 and GFP-TDP43CTF, consistent with the domain configuration found in some native J domain proteins (Sha (2000)). Structure 8 (8), 799-807).

[0287] Table 15: Generation and Testing of Additional Fusion Protein Constructs

[0288]

[0289] We then investigated the mechanism by which the fusion protein reduces aggregation. HEK293 cells were transfected with either GFP-TDP43FL or GFP-TDP43CTF reporter constructs, either alone or with construct 3 (JB1-scFv(3B12A)) and either BFA (bafloxacin A1, an inhibitor of late autophagy) or MG132 (a proteasome inhibitor). Figure 10 As shown, treatment of cells with 10 nM or 100 nM BFA resulted in the dose-dependent re-emergence of pathogenic TDP43. In contrast, treatment with 0.1 µM or 1.0 µM MG132 had little to no effect on the accumulation of pathogenic TDP43 forms. Overall, these results suggest that the fusion protein construct exerts its effects via chaperone-mediated autophagy.

[0290] Example 2: AAV vector encoding fusion protein construct

[0291] The exemplary gene therapy vector was constructed from an AAV9 vector carrying codon-optimized cDNA encoding the fusion protein constructs listed in Table 6, specifically constructs 2, 4, 6, 7, 17, and 20-31, as well as control construct 1 (DnaJB1 J domain only) and GFP (negative control), under the control of a CAG promoter containing a cytomegalovirus (CMV) early enhancer element and a chicken β-actin promoter. The cDNA encoding the constructs was located downstream of the Kozak sequence and polyadenylated via bovine growth hormone polyadenylation (BGHpA) signaling. The entire cassette is flanked by two non-coding reversed-end sequences of AAV-2.

[0292] Recombinant AAV vectors were prepared using a baculovirus expression system similar to that described above (Urabe et al., 2002; Unzu et al., 2011 (reviewed in Kotin, 2011)). In short, three recombinant baculoviruses (one encoding a REP for replication and packaging, one encoding a CAP-5 about the AAV9 capsid, and one with an expression cassette) were used to infect SF9 insect cells. Purification was performed using AVB Sepharose high-speed affinity media (GE Healthcare Life Sciences, Piscataway, NJ). The vectors were titrated using qPCR accompanied by primer-probe combinations for transgenesis, and the titer is expressed as genome copies per ml (GC / ml). The vector titer was approximately 8 x 10⁻⁶. 13 Up to 2 x 10 14 GC / ml.

[0293] Example 3: Expression and efficacy testing in a mouse model of ALS

[0294] First, experiments were conducted in wild-type C57BL / 6J mice to confirm construct 3 expression and determine whether its expression had any harmful effects on the animals. (6x10) 10 As described above, the AAV rh10 capsid containing either the control or construct 3 was injected intrathecally or intraventricularly, as shown in Table 16 below.

[0295] Table 16: IT and ICV injections of AAV rh10 into expression vectors containing encoding construct 3

[0296] Group N gender compound Dosage (vg / mouse) route of administration Dosage frequency and age 1 6 male AAV (empty) <![CDATA[1x10 11 ]]> Intrasheath 1 x at 5 weeks 2 6 male AAV (Constructor-3) <![CDATA[1x10 11 ]]> Intrasheath 1 x at 5 weeks 3 6 male AAV (empty) <![CDATA[6x10 10 ]]> ICV 1xP1 4 6 male AAV (Constructor-3) <![CDATA[6x10 10 ]]> ICV 1xP1

[0297] Observe mice for ataxia, hind limb weakness, or dragging feet. Body weight and clinical observation were performed weekly, one week after AAV injection. Mice (n=3) were humanely euthanized via CO2 at 3 weeks to confirm AAV expression. No difference in body weight was observed during the three-week period following ICV or IT injection (data not shown). As shown in Figure 15, via intrathecal ( Figure 15B ) or ICV ( Figure 15C Injection resulted in construct 3 expression in mice, as detected by immunoblotting of brain extract, but was not detected in control mice. Furthermore, expression was found to be significantly higher at 8 weeks post-ICV injection than at 3 weeks.

[0298] The vector prepared above was tested for TDP-43-related pathology in a novel AAV NEFH-tTA × hTDP-43ΔNLS bigenic mouse (rNLS mouse). This model has a doxycycline (DOX)-repressible construct expressing pathogenic TDP-43 (human TDP-43ΔNLS). Following DOX removal, TDP-43ΔNLS expression leads to rapid and progressive deterioration in the animals, resulting in severe weight loss and death generally within 6–8 weeks. We tested the efficacy of construct 3 (JB1-scFv(3B12A), SEQ ID NO: 80) in slowing TDP-43ΔNLS-mediated pathological progression. As shown in Table 17 below, AAV rh10 containing either control or construct 3 was administered unilaterally via ICV at P1 / P2 in 2 μL (maximum 4 μL) volumes in different groups. DOX removal occurred at week 5, except for control group 1. Control mice in the groups.

[0299] Table 17: ICV injection in ΔNLS8 mice

[0300] Group number mouse strains gender deal with dose Application time 1 4 rNLS8 mice (Dox+) 4 males AAV (control) <![CDATA[6x10 10 vg]]> P1 2 8 rNLS8 mice (Dox-) 5M / 3F AAV (control) <![CDATA[6x10 10 vg]]> P1 3 10 rNLS8 mice (Dox-) 4M / 6F AAV (Constructor 3) <![CDATA[6x10 10 vg]]> P1

[0301] Weight was measured twice weekly after weaning. Samples were collected from all surviving mice after testing. The entire brain was collected and dissected into two hemispheres. One hemisphere was weighed and frozen on dry ice. The second hemisphere was post-fixed in 4% PFA for histological evaluation.

[0302] like Figure 16B As shown, after DOX removal, the control mice in group 2 began to lose significant weight over the following weeks, resulting in a statistically significant weight difference compared to group 1. Surprisingly, group 3, which expressed construct 3, also showed a statistically significant weight increase when compared to group 2 (since group 1 contained only males, the results for groups 2 and 3 only show male weight to account for sex differences).

[0303] When examining survival, we found that animals in control group 2 (DOX off) rapidly deteriorated in health, resulting in only 37.5% of mice surviving at week 10 (0% males). However, mice expressing construct 3 showed 100% survival at week 10, with no difference from the control group 1 (DOX on).

[0304] Other aspects

[0305] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication, patent, or patent application specifically and individually indicates its inclusion by reference in its entirety. Where a term in this application is found to be defined differently in a document incorporated herein by reference, the definition provided herein shall serve as the definition of that term.

[0306] While the invention has been described in conjunction with its specific aspects, it should be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention, generally following the principles of the invention and including such deviations from the scope of this disclosure, which are within the known or customary practices in the field to which the invention pertains, and which may be applied to the basic features set forth above and in accordance with the scope of the claimed protection.

Claims

1. An isolated fusion protein selected from SEQ ID NO: 80, 82, 83 and 94-97.

2. The fusion protein of claim 1, wherein the fusion protein comprises the sequence of SEQ ID NO:

80.

3. The fusion protein of claim 1, wherein the fusion protein comprises the sequence of SEQ ID NO:

94.

4. The fusion protein of claim 1, wherein the fusion protein comprises the sequence of SEQ ID NO:

95.

5. The fusion protein of claim 1, wherein the fusion protein comprises the sequence of SEQ ID NO:

96.

6. A nucleic acid encoding a fusion protein of any one of claims 1-5.

7. The nucleic acid of claim 6, wherein the nucleic acid is DNA.

8. The nucleic acid of claim 6, wherein the nucleic acid is RNA.

9. The nucleic acid of claim 6, further comprising a promoter region, a 5' UTR or a 3' UTR, or a combination thereof.

10. The nucleic acid of claim 9, wherein the promoter region comprises a sequence selected from CMV enhancer, CMV promoter, CBA promoter, UBC promoter, GUSB promoter, NSE promoter, synaptophysin promoter, MeCP2 promoter and GFAP promoter.

11. A vector comprising the nucleic acid of any one of claims 6-10.

12. The vector of claim 11, wherein the vector is selected from adeno-associated virus, adenovirus, retrovirus, herpesvirus, poxvirus, paramyxovirus, baculovirus, reovirus, alphavirus, and flavivirus.

13. The carrier of claim 12, wherein the carrier is AAV.

14. A viral particle comprising a capsid and a vector of any one of claims 11-13.

15. The viral particle of claim 14, wherein the capsid is selected from 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 mutants, AAV heterozygous serotypes, organophilic AAV, cardophilic AAV and cardophilic AAVM41 mutants.

16. The viral particle of claim 15, wherein the capsid is selected from AAV2, AAV5, AAV8, AAV9 and AAVrh10.

17. The viral particle of claim 16, wherein the capsid is AAV9.

18. The viral particle of claim 16, wherein the capsid is AAV rh10.

19. A pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier or excipient selected from the following: a fusion protein of any one of claims 1-5, a cell expressing a fusion protein of claims 1-5, a nucleic acid of any one of claims 6-10, a carrier of any one of claims 11-13, and a viral particle of any one of claims 14-18.

20. Use of one or more of the following to prepare a medicament for preventing or delaying the progression of TDP-43 disease in a subject: a fusion protein of any one of claims 1-5, a cell expressing a fusion protein of claims 1-5, a nucleic acid of any one of claims 6-10, a vector of any one of claims 11-13, a viral particle of any one of claims 14-18, and a pharmaceutical composition of claim 19, wherein the TDP-43 disease is selected from ALS, FTD, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, and Lewy body dementia.

21. The use of claim 20, wherein the TDP-43 disease is ALS.

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