Compositions and methods for treating synucleinopathies

Abstract

Disclosed is a novel class of fusion proteins to recruit the innate chaperone machinery of cells, in particular the Hsp70-mediated system, to specifically reduce alpha-synuclein-mediated protein aggregation and associated protein conformational diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application 63 / 035,297, filed June 5, 2020, pursuant to 35 USC §119(e). The entire contents of the foregoing application are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application references the sequence list, which is incorporated in its entirety into the name “269548-493385_ST25.txt” (68KB), which was created on June 4, 2021 at 1:09 p.m. and submitted electronically. Technical Field

[0005] This disclosure describes and claims protection for novel fusion proteins comprising one or more J domains, α-synuclein-binding domains, adaptors, targeting agents, epitopes, cell-penetrating agents, and combinations thereof. Additionally, this disclosure describes uses of these novel fusion proteins and similarly describes and claims protection for their systematic recruitment by innate molecular chaperone mechanisms, such as Hsp70-mediated fusion, to specifically reduce α-synuclein-mediated protein aggregation and related proteases. Background Technology

[0006] 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. Examples of diseases caused by misfolding, also known as conformational disorders or protein conformational diseases, 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.

[0007] A wide variety of neurodegenerative diseases are pathologically characterized by the accumulation of intracellular or extracellular protein aggregates composed of amyloid fibrils (Forman et al., (2004) Nat Med. 10:1055-1063). For example, the pathology of Alzheimer's disease (AD) is defined by senile plaques and neurofibrillary tangles composed of β-amyloid protein and microtubule-associated protein tau, respectively.

[0008] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). More than 1% of people over the age of 60 have the disease, with over 1 million patients in the United States alone. PD patients experience bradykinesia, rigidity, tremor, balance difficulties, and various forms of dementia in about 40% of PD patients, some of whom develop AD-like dementia later in the disease. The main pathological features of PD are the loss of dopaminergic neurons in the substantia nigra and the presence of abnormal protein aggregates that form filamentous inclusions in the neuronal cytoplasm, known in the PD brain as Lewy bodies (LBs) or Lewy neurites (neural fibers) (LNs) (Galvin et al., (2001) Arch Neurol 58, 186-190; Lang and Lozano, (1998) N Engl J Med 339, 1044-1053; Lang and Lozano, (1998) N Engl J Med 339, 1130-1143). Most PD cases are sporadic. Familial PD accounts for approximately 10% of all cases and can be inherited in an autosomal recessive or autosomal dominant manner, suggesting a complex etiology of the disease (as reviewed by Lang and Lozano, (1998) N Engl J Med 339, 1044-1053; Lang and Lozano, (1998) N Engl J Med 339, 1130-1143). Furthermore, environmental factors also play an important role in the development of PD (as reviewed by Di Monte, 2003) (Di Monte et al., (2003) Lancet Neurol 2, 531-538).

[0009] The discovery of genetic linkages between PD and several loci offers hope for identifying genetic factors that contribute to the pathogenesis of the disease. Studies of patients with a rare, dominant variant of PD from a family in southern Italy have linked the condition to a locus (park 1) on chromosome 4q21-23, where a previously identified gene called α-synuclein (SNCA) is located (Jakes et al., (1994) FEBS Lett 345, 27-32; Shibazaki et al., (1995) Cytogenet Cell Genet 71, 54-55; Polymeropoulos et al., (1996) Science 274, 1197-1199). Direct evidence regarding the involvement of α-synuclein in familial PD was provided by the identification of missense mutations in the gene that resulted in an AT substitution at amino acid 53 in this Italian-American family and three subsequent unrelated Greek families (Polymeropoulos et al., (1997) Science 276, 2045-2047). Subsequently, a second mutation was identified in a German family, changing alanine to proline at position 30 of the α-synuclein gene (Kruger et al., (1998) Nat Genet 18, 106-108). Immunohistochemical examination of LB revealed that LB contains many different proteins. The most consistently described proteins are neurofilaments, ubiquitin, and alpha-synuclein (α-synuclein) (Takeda et al., (1998) Am J Pathol 152, 367-372; Gai et al., Brain 118(Pt 6), 1447-1459). Large, near-nuclear aggregates of misfolded α-synuclein are predominantly found in the Lewy body and dystrophic Lewy neurites (LNs) of the brains of patients with PD (Dickson, (2012) Cold Spring Harb Perspect Med 2; Stefanis, (2012) Cold Spring Harb Perspect Med 2, a009399; Lashuel et al., (2013) NatRev Neurosci 14, 38-48), suggesting that misfolding of α-synuclein is a key factor in the pathogenesis of neurodegenerative diseases associated with the accumulation of aggregated α-synuclein (Baba et al., (1998) Am J Pathol 152, 879-884; Kalia et al., (2013) Ann Neurol 73, 155-169).

[0010] The α-synuclein gene encodes a 140-amino acid protein of ~19 kDa. While the function of α-synuclein is not fully understood, there is evidence suggesting its role in neurotransmitter release (Liu et al., (2004) EMBO J 23, 4506-4516; Chandra et al., (2005) Cell 123, 383-396; Fortin et al., (2005) J Neurosci 25, 10913-10921). α-synuclein is highly expressed throughout the mammalian brain, but is enriched in presynaptic nerve endings associated with membrane and vesicle structures. Mice possess functional homologs of α-synuclein. Homozygous knockout of the mouse α-synuclein gene is viable, fertile, and nearly normal (Abeliovich et al., (2000) Neuron 25, 239-252). α-synuclein- / - mice exhibited increased dopamine release with paired stimulation, decreased striatal dopamine, and diminished dopamine-dependent motor responses to amphetamine, suggesting that α-synuclein is a regulator of dopamine neurotransmission. Mice with α-synuclein overexpression appeared normal, and although significant degeneration of dopamine nerve endings was observed in mouse strains with high human α-synuclein expression, no loss of dopamine neurons was observed (Masliah and Rockenstein, (2000) J Neural Transm Suppl 59, 175-183). Overexpression of the A53T α-synuclein mutant induced extensive neurodegeneration (Lee et al., (2002) Proc Natl Acad Sci USA 99, 8968-8973; Giasson et al., (2002) Neuron 34, 521-533; Dawson et al., (2002) Neuron 35, 219-222). Progressive loss of DA neurons in rats overexpressing adeno-associated virus (AAV)-mediated wild-type and mutant α-synuclein suggests that PD models could be used to test PD treatment (Kirik et al., (2002) Neuron 35, 219-222).

[0011] Currently, there is no cure or preventative treatment for Parkinson's disease (PD), only symptomatic treatments such as medication and surgery. Degeneration of dopaminergic substantia nigra neurons leads to the loss of dopaminergic projections to the striatum, representing a major defect in the neurochemical pathways of PD. Therefore, one treatment for PD is dopamine (DA) replacement, in which patients take the drug levodopa. Levodopa is converted to dopamine by aromatic L-amino acid decarboxylases (AADCs). However, this benefit is temporary due to numerous side effects (as reviewed by Nutt 2003) (Nutt, (2003) Exp Neurol 184, 9-13). Furthermore, as the disease progresses, fewer AADCs become available, potentially leading to increased off-time, such as stiffness, rigidity, and spasticity. Therefore, there is a significant need to develop new treatments for this devastating disease.

[0012] Heat shock 70 kDa proteins (referred to as “Hsp70” 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, such as J domain proteins and nucleotide exchange factors (NEFs) (Hartl et al., (2009) Nat Struct Mol Biol 16, 574-581), to function. In current models of the Hsp70 molecular chaperone mechanism for folding proteins, Hsp70 cycles between ATP- and ADP-bound states, and the J domain protein binds to another protein (referred to as 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) MolCell 39, 321-331). The binding of the J domain protein-client 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, as well as triggering the release of the J domain protein, which then freely binds to another client protein.

[0013] Therefore, according to this model, J-domain proteins play a crucial role in 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 connected via a flexible loop containing the “HPD motif,” which is highly conserved in the J-domain 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.

[0014] Given the background provided above on protein conformation disorders such as PD, 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

[0015] The inventors have developed a novel class of fusion proteins to recruit cellular innate molecular chaperone mechanisms, particularly Hsp70-mediated systems, to specifically reduce α-synuclein-mediated protein misfolding. Unlike previous studies that used fusion proteins containing fragments of the co-chaperone Hsp40 protein (also known as the J protein) that interacts with Hsp70 to enhance protein secretion and expression, this study employs fusion proteins containing a J domain to reduce protein misfolding and cytotoxicity caused by α-synuclein proteins. In this context, the inventors unexpectedly discovered that the J domain element required for this function is quite different from that used in enhancing protein expression and secretion, confirming a different mechanism regarding the mode of action of this fusion protein. The fusion protein described herein comprises a J domain and a domain with affinity for α-synuclein. The presence of the α-synuclein-binding domain within the fusion protein results in a specific reduction in α-synuclein protein misfolding.

[0016] E1. Therefore, in the first aspect, this article discloses an isolated fusion protein comprising the J domain of the J protein and the α-synuclein binding domain.

[0017] The E2.E1 fusion protein, wherein the J domain of the J protein is of eukaryotic origin.

[0018] A fusion protein of any one of E3, E1-E2, wherein the J domain of the J protein is of human origin.

[0019] A fusion protein of any one of E4, E1-E3, wherein the J domain of the J protein is cytoplasm-localized.

[0020] A fusion protein of any one of E5, E1-E4, wherein the J domain of the J protein is selected from SEQ ID No:1-50.

[0021] A fusion protein of any one of E6, E1-E5, wherein the J domain comprises a sequence selected from SEQ ID NO:1, 5, 6, 10, 16, 24, 25, 31 and 49.

[0022] A fusion protein of any one of E7, E1-E6, wherein the J domain comprises the sequence of SEQ ID NO:5.

[0023] A fusion protein of any one of E8, E1-E6, wherein the J domain comprises the sequence of SEQ ID NO:10.

[0024] A fusion protein of any one of E9, E1-E6, wherein the J domain comprises the sequence of SEQ ID NO:16.

[0025] A fusion protein of any one of E10, E1-E6, wherein the J domain comprises the sequence of SEQ ID NO:25.

[0026] A fusion protein of any one of E11, E1-E6, wherein the J domain comprises the sequence of SEQ ID NO:31.

[0027] A fusion protein of any one of E12-E11, wherein, when measured using an ELISA assay, the α-synuclein binding domain has a K+ of 1 μM or less for α-synuclein, for example, 300 nM or less, 100 nM or less, 30 nM or less, or 10 nM or less. D .

[0028] A fusion protein of any one of E13, E1-E12, wherein the α-synuclein binding domain comprises a sequence selected from SEQ ID NO:51-65.

[0029] A fusion protein of any one of E14, E1-E13, wherein the α-synuclein binding domain comprises the sequence of SEQ ID NO:51.

[0030] A fusion protein of any one of E15, E1-E13, wherein the α-synuclein binding domain comprises the sequence of SEQ ID NO:52.

[0031] A fusion protein of any one of E16, E1-E13, wherein the α-synuclein binding domain comprises the sequence of SEQ ID NO:63.

[0032] A fusion protein of any one of E17, E1-E13, wherein the α-synuclein binding domain comprises the sequence of SEQ ID NO:64.

[0033] A fusion protein of any one of E18, E1-E17, which contains multiple α-synuclein binding domains.

[0034] A fusion protein of any one of E19, E1-E18, which consists of two α-synuclein binding domains.

[0035] A fusion protein of any one of E20, E1-E19, which consists of three α-synuclein binding domains.

[0036] A fusion protein of any one of E21, E1-E20, comprising one of the following constructs:

[0037] a.DNAJ-XS,

[0038] b.DNAJ-XSXS,

[0039] c.DNAJ-XSXSXS,

[0040] dS-X-DNAJ,

[0041] eS-XSX-DNAJ,

[0042] fS-XSXSX-DNAJ,

[0043] gS-X-DNAJ-XS,

[0044] hS-X-DNAJ-XSXS,

[0045] iS-XSX-DNAJ-XSXSXS,

[0046] jS-XSXSX-DNAJ-XS,

[0047] kS-XSXSX-DNAJ-XSXS,

[0048] lS-XSXSX-DNAJ-XSXSXS,

[0049] m.DnaJ-X-DnaJ-XSXS,

[0050] nS-X-DnaJ-X-DnaJ, and

[0051] oS-XSX-DnaJ-X-DnaJ,

[0052] in,

[0053] S is the α-synuclein binding domain.

[0054] DNAJ is the J domain of the J protein, and

[0055] X is an optional connector.

[0056] A fusion protein of any one of E22.E1-E21, wherein the fusion protein comprises the J domain sequence of SEQ ID NO:5 and the α-synuclein binding domain sequence of SEQ ID NO:51.

[0057] A fusion protein of any one of E23, E1-E22, wherein the fusion protein comprises two copies of the J domain sequence of SEQ ID NO:5 and the α-synuclein binding domain sequence of SEQ ID NO:51.

[0058] The fusion protein of any one of E24, E1-E23, wherein the fusion protein comprises a sequence selected from SEQ ID NO:88, 90-96, 98-100.

[0059] A fusion protein of any one of E25, E1-E24, wherein the fusion protein comprises the sequence of SEQ ID NO:88.

[0060] A fusion protein of any one of E26.E1-E24, wherein the fusion protein comprises the sequence of SEQ ID NO:90.

[0061] A fusion protein of any one of E27.E1-E24, wherein the fusion protein comprises the sequence of SEQ ID NO:99.

[0062] A fusion protein of any one of E28, E1-E24, wherein the fusion protein comprises the sequence of SEQ ID NO:100.

[0063] A fusion protein of any one of E29, E1-E28, which further contains a targeting agent.

[0064] A fusion protein of any one of E30, E1-E29, which further contains an epitope.

[0065] The fusion protein of E31 and E30, wherein the epitope is a polypeptide selected from SEQ ID NO:77-83.

[0066] A fusion protein of any one of E32, E1-E31, which further includes a cell penetrant.

[0067] The fusion protein of E33 and E32, wherein the cell penetrant comprises a peptide sequence selected from SEQ ID NO:84-87.

[0068] A fusion protein of any one of E34, E1-E33, which further includes a signal sequence.

[0069] The fusion protein of E35 and E34, wherein the signal sequence comprises a peptide sequence selected from SEQ ID NO:102-104.

[0070] A fusion protein of any one of E36, E1-E35, which can reduce the misfolding of α-synuclein proteins in cells.

[0071] A fusion protein of any one of E37, E1-E36, which can reduce phosphorylated α-synuclein proteins in cells.

[0072] A fusion protein of any one of E38, E1-E37 can reduce the secretion of α-synuclein protein.

[0073] A fusion protein of any one of E39, E1-E38 can reduce α-synuclein-mediated cytotoxicity.

[0074] E40. A nucleic acid sequence that encodes a fusion protein of any one of E1-E39.

[0075] The nucleic acid sequences of E41 and E40, wherein the nucleic acid is DNA.

[0076] The nucleic acid sequence of any of E42 and E41, wherein the nucleic acid is RNA.

[0077] The nucleic acid sequence of any one of E43, E40-E42, wherein the nucleic acid comprises at least one modified nucleic acid.

[0078] The nucleic acid sequence of any one of E44, E40-E43, further includes a promoter region, 5'UTR, 3'UTR such as poly(A) signal.

[0079] The 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.

[0080] E46. A vector containing a nucleic acid sequence of any one of E40-E45.

[0081] Vectors of E47 and E46, wherein the vectors are selected from adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, poxvirus (vaccinia or myxoma), paramyxovirus (measles, RSV or Newcastle disease virus), baculovirus, reovirus, alphavirus and flavivirus.

[0082] E48. A viral particle comprising a capsid and a vector of E46 or E47.

[0083] Viral particles of E49 and E48, 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-DJanAAV capsid mutants, AAV heterozygous serotypes, organophilic AAV, cardophilic AAV and cardophilic AAVM41 mutants.

[0084] Viral particles of E50, E48, or E49, wherein the capsid is selected from AAV2, AAV5, AAV8, AAV9, and AAVrh10.

[0085] Viral particles of any of E51, E48-E50, wherein the capsid is AAV2.

[0086] Viral particles of any of E52, E48-E50, wherein the capsid is AAV5.

[0087] Viral particles of any of E53, E48-E50, wherein the capsid is AAV8.

[0088] Viral particles of any of E54, E48-E50, wherein the capsid is AAV9.

[0089] Viral particles of any of E55, E48-E50, wherein the capsid is AAV rh10.

[0090] E56. 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 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-E47, and a viral particle of any one of E48-E55.

[0091] E57. A method for reducing the toxicity of α-synuclein proteins 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, 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-E47, a viral particle of any one of E48-E55, and a pharmaceutical composition of E56.

[0092] The methods of E58 and E57, wherein the cells are in the subject.

[0093] The method of any of E59, E57-E58, wherein the subject is a human being.

[0094] The method of any one of E60, E57-E59, wherein the cell is a cell of the central nervous system.

[0095] The method of any one of E61, E57-E60, wherein the subject is identified as having α-synuclein disease.

[0096] The methods of E62 and E61, wherein the α-synuclein disease is selected from PD, Lewy body dementia, multiple system atrophy, and diseases associated with the abnormal accumulation of aggregated α-synuclein proteins (synucleinopathy).

[0097] Methods E63, E61, or E62, wherein the α-synuclein disease is PD.

[0098] The method of any one of E64, E57-E63, wherein, when compared with control cells, the amount of misfolded α-synuclein protein present in the cells is reduced.

[0099] E65. A method for treating, preventing, or delaying the progression of α-synuclein disease in subjects in need of it, 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-E47, a viral particle of any one of E48-E55, and a pharmaceutical composition of E56.

[0100] The methods of E66 and E65, wherein the α-synuclein disease is selected from PD, Lewy body dementia, multiple system atrophy, and diseases associated with the abnormal accumulation of aggregated α-synuclein proteins (synucleinopathy).

[0101] The method of E67.E65, wherein the α-synuclein disease is PD.

[0102] E68. Use of one or more of the following in subjects for the prevention of α-synuclein disease or for delaying the progression of α-synuclein disease: fusion protein of any one of E1-E39, cells expressing fusion proteins of E1-E39, nucleic acid of any one of E40-E45, vector of any one of E46-E47, viral particles of any one of E48-E55, and pharmaceutical composition of E56.

[0103] E69. Use of one or more of the following in the preparation of a medicament for the treatment or prevention of α-synuclein 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-E47, a viral particle of any one of E48-E55, and a pharmaceutical composition of E56.

[0104] The use of E70, E68, or E69, wherein the α-synuclein disease is PD. Attached Figure Description

[0105] 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.

[0106] Figure 1B The Clustal Omega sequence alignment of a representative human J domain sequence is shown.

[0107] Figure 2 The images show some representative fusion protein constructs containing the J domain and the α-synuclein binding domain, as well as control constructs.

[0108] Figure 3 shows the effect of expressing a fusion protein containing a J domain and an α-synuclein binding domain. Figure 3AWild-type (WT) α-synuclein (Syn(WT): strips 1-3) or mutant α-synuclein (Syn(A53T): strips 4-6) were expressed alone in HEK293 cells, or co-expressed with a Flag-tagged fusion protein (J-SynBP1: strips 2 and 5) or a control fusion protein construct (J(MT)-SynBP1: strips 3 and 6). The Flag-tagged fusion protein contained the J domain DnaJB1 and the α-synuclein-binding domain, and the control fusion protein construct contained a mutant J domain (containing a P33Q substitution) and the α-synuclein-binding peptide. Two days later, cells were homogenized in Tris buffer (10 mM Tris, pH 7.4, 140 mM NaCl, 1 mM EDTA supplemented with a mixture of protease inhibitors). Aggregation of mutant α-synuclein levels was quantified using a sandwich ELISA in which aggregated α-synuclein was captured by a Syn-O4-coated antibody and subsequently detected with an HRP-conjugated anti-α-synuclein antibody (BioLegend: A15115A). Figure 3B Lysates were prepared from HEK293 cells, which were transiently transfected alone with wild-type Syn(WT) (lanes 1-3) or mutant Syn(A53T) (lanes 4-6) or co-expressed with fusion protein constructs. After brief sonication, the cell lysates were subjected to SDS-PAGE / Western blotting with anti-α-synuclein antibody (Ab-2: top), Flag antibody (middle), or anti-microtubule antibody (bottom).

[0109] Figure 4 This study demonstrates a reduction in the amount of α-synuclein secreted into the culture medium in cells co-expressing a fusion protein containing both the J domain and the α-synuclein-binding domain. Transfection of cells with wild-type (strip 2) or mutant (A53T; strip 4) α-synuclein alone resulted in synuclein secretion, detectable by ELISA. Co-expression with a fusion protein construct containing either the normal (strip 3) or mutant J domain (containing a P33Q substitution; strip 5) resulted in the elimination of detectable levels of secreted α-synuclein.

[0110] Figure 5The effects of various inhibitors of cellular processes on total α-synuclein levels in cells expressing the fusion protein construct were demonstrated. Cells were transfected with wild-type (lane 2) or mutant (lanes 3-8) α-synuclein, and also with the JB1-SynBP1 fusion protein (lanes 4-8). Cells were also treated with the late autophagy inhibitor bafloxacin A1 (10 nM and 100 nM in lanes 5 and 6, respectively) or the proteasome inhibitor MG132 (0.1 μM and 1 μM in lanes 7 and 8, respectively). Cell lysates were detected using an anti-synuclein antibody.

[0111] Figure 6 The effects of various inhibitors of cellular processes on the levels of aggregated α-synuclein in cells expressing fusion protein constructs were demonstrated. Cells were transfected with wild-type or mutant α-synuclein and also with the JB1-SynBP1 fusion protein. Cells were also treated with the late autophagy inhibitor bafloxacin A1 (10 nM and 100 nM) or the proteasome inhibitor MG132 (0.1 μM and 1 μM). Aggregated synuclein in cell lysates was determined by ELISA using an anti-aggregated synuclein antibody.

[0112] Figure 7 This study demonstrates how the JB1-SynBP1 construct improves α-synuclein (A53T)-mediated cytotoxicity in U87-MG glioma cells. U87MG cells were infected with lentivirus alone or together with the JB1-SynBP1 construct to express wild-type (“SynWT”) or mutant (“SynA53T”) α-synuclein. Culture medium was collected from U87-MG cells 7 days post-infection. Lactate dehydrogenase (LDH) activity in the culture medium was measured using LDH-Cytox. TM The assay kit (BioLegend) was used to measure and the results are expressed as values ​​of LDH levels relative to cells expressing SynWT alone.

[0113] definition

[0114] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0115] 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 (e.g., conjugation with a labeled component).

[0116] 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 indicate amino acids.

[0117] "Host cell" includes a single cell or cell culture that may be, or has been, a recipient of the subject vector. Host cell includes 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 cell includes cells transfected in vivo with the vector of the present invention.

[0118] When used to describe the various peptides disclosed herein, "isolated" means a peptide 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 would typically interfere with the diagnostic or therapeutic use of the peptide 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, peptides prepared by recombinant means and expressed in host cells are considered "isolated."

[0119] "Isolated" polynucleotides or nucleic acids encoding polypeptides, or other nucleic acids encoding polypeptides, 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 nucleic acid molecule encoding the polypeptide is in a form or environment different from that found in nature. Therefore, the isolated nucleic acid molecule encoding the polypeptide is different from a specific nucleic acid molecule encoding the polypeptide present in natural cells. However, the isolated nucleic acid molecule encoding the polypeptide includes nucleic acid molecules encoding the polypeptide contained in cells that typically express the polypeptide, in cells, for example, in a chromosomal or extrachromosomal location different from that in natural cells.

[0120] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length, which are 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, one or more loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be conferred before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. After polymerization, polynucleotides can be further modified, for example, by conjugation with labeled components.

[0121] As defined herein, the term “α-synuclein disorder” or “synucleinopathy” refers to a condition associated with the formation of intracellular α-synuclein aggregates, particularly aggregates of mutant α-synuclein proteins. Examples of α-synuclein disorders include, but are not limited to, Parkinson’s disease (including PD), Lewy body dementia, multiple system atrophy, and diseases associated with the abnormal accumulation of aggregated α-synuclein proteins (synucleinopathy).

[0122] A “vector” is a nucleic acid molecule that transfers an inserted nucleic acid molecule into a host cell and / or between host cells, preferably replicating autonomously in a suitable host. The 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 function. An “expression vector” is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into one or more polypeptides. An “expression system” generally means a suitable host cell containing an expression vector that can function to produce the desired expression product.

[0123] The term "operably linked" refers to the juxtaposition of the described components, wherein the components are in a relationship that allows them to function in their intended manner. A control sequence "operably linked" to a coding sequence is connected in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. An "operably linked" sequence may include both an expression control sequence adjacent to the target gene and an expression control sequence that acts trans- or at a distance to control the expression of the target gene. The term "expression control sequence" refers to a polynucleotide sequence that is essential to influencing 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 the insertion of nucleic acid molecules encoding the fusion proteins of the present invention into expression vectors mean that, when an expression vector containing the inserted nucleic acid molecules is introduced into a compatible host cell or compatible cell of an organism, the inserted nucleic acid is also operatively linked within the vector to a functional promoter and other transcriptional and translational control elements required for the expression of the encoded fusion protein.

[0124] 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 can potentially be expressed in a host cell.

[0125] The terms "gene" and "gene segment" are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a specific protein after transcription and translation. A gene or gene segment can be genomic DNA or cDNA, provided that the polynucleotide contains at least one open reading frame that 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.

[0126] 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.

[0127] As used herein, the term “effective amount” means 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 the condition; or enhance or improve one or more preventive or therapeutic effects of a therapy (e.g., the administration of another preventive or therapeutic agent).

[0128] 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 a highly conserved tripeptide motif (referred to as the "HPD motif") between helices II and III. Typically, the length of the J domain of J proteins is between 50 and 70 amino acids, and the interaction (binding) site of the J domain with Hsp70-ATP chaperone proteins is believed 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 is incorporated herein by reference in its entirety). A non-limiting list of human J domains is provided in Table 1. Detailed Implementation

[0129] The inventors have discovered that contacting cells with a fusion protein construct containing the J domain of the J protein and the α-synuclein binding domain has a surprisingly effective effect in reducing the aggregation of mutant α-synuclein proteins. Aggregation of mutant α-synuclein is believed to lead to many devastating diseases, including but not limited to Parkinson's disease, Lewy body dementia, multiple system atrophy, and diseases associated with the abnormal accumulation of aggregated α-synuclein proteins (synucleinopathy). Accordingly, this article provides useful compositions and methods for treating α-synucleinopathy, for example, in subjects who require them.

[0130] To overcome the challenges associated with molecular chaperone-based therapies, we investigated the possibility of designing highly specific artificial chaperone proteins. We designed a series of fusion protein constructs containing an effector domain (J domain sequence) for Hsp70 binding / activation and a domain conferring specificity for α-synuclein proteins. The resulting fusion proteins function to accelerate the intrinsic ATPase catalytic activity of Hsp70 and its homologs, leading to increased protein folding, decreased aggregation, and / or accelerated clearance.

[0131] I. Fusion protein construct

[0132] a. The J-structure field that can be used in this invention

[0133] 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 of histidine, proline, and aspartic acid (referred to as the “HPD motif”) between helices II and III. Typically, the length of the J domain of a J protein is between 50 and 70 amino acids, and the interaction (binding) site of the J domain with the Hsp70-ATP chaperone protein is believed to be a region extending from helix II, and the HPD motif is fundamental for native 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.

[0134] Table 1. Representative sequences of human J-domain structures

[0135]

[0136]

[0137]

[0138]

[0139]

[0140] In one embodiment, the fusion protein comprises a J domain sequence selected from the group consisting of polypeptide sequences selected from SEQ ID NO:1-50. The inventors have demonstrated that the J domain containing the conserved “HPD” motif is active (data not shown). Therefore, in another embodiment, the fusion protein comprises a J domain sequence containing the conserved “HPD” motif. For example, in a particular embodiment, the fusion protein comprises a J domain sequence selected from the group consisting of polypeptide sequences selected from SEQ ID NO:1-15 and 17-50. In another embodiment, the fusion protein comprises a J domain sequence selected from the group consisting of polypeptide sequences selected from SEQ ID NO:1, 5, 6, 10, 16, 24, 25, 31, and 49.

[0141] b. α-synuclein binding domain

[0142] The fusion protein also contains at least one α-synuclein binding domain. The α-synuclein binding domain can be a single-chain polypeptide or a multimeric polypeptide linked to the J domain to form the fusion protein.

[0143] Ideally, the α-synuclein binding domain possesses sufficient affinity to bind to α-synuclein proteins (when present at pathological levels within the cell). Therefore, in one embodiment, the fusion protein comprises an α-synuclein binding domain that, when tested by ELISA on a 96-well microtiter plate, has a Kc for α-synuclein, 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 In another embodiment, the fusion protein comprises an α-synuclein binding domain, which, when tested by ELISA on a 96-well microtiter plate, has a Kc for aggregated α-synuclein, 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 In yet another embodiment, the α-synuclein binding domain is selective for aggregated α-synuclein; for example, the α-synuclein binding domain has an affinity for aggregated α-synuclein that is at least two times higher, such as at least three times higher, at least four times higher, at least five times higher, at least ten times higher, at least thirty times higher, or at least one hundred times higher than that for soluble α-synuclein.

[0144] The α-synuclein binding domain has been previously identified and characterized (see, for example, U.S. Patent No. 7,605,133, WO 2019 / 161386, Abe et al., (2007) BMC Bioinformatics, 8:451, each of which is incorporated herein by reference). Therefore, in another embodiment, the fusion protein comprises an α-synuclein binding domain selected from SEQ ID NO:51-64 (see, for example, Table 2). In one particular embodiment, the fusion protein comprises the α-synuclein binding domain of SEQ ID NO:51. In another embodiment, the fusion protein comprises the α-synuclein binding domain of SEQ ID NO:63.

[0145] In yet another embodiment, the fusion protein comprises a J domain and an α-synuclein binding domain, wherein the α-synuclein binding domain does not contain the sequence of SEQ ID NO:65. In yet another embodiment, the fusion protein comprises a J domain and an α-synuclein binding domain, wherein the fusion protein does not contain the sequence of SEQ ID NO:65. In a particular embodiment, the fusion protein comprises a J domain and an α-synuclein binding domain, wherein the fusion protein does not contain the sequence of SEQ ID NO:65, and the J domain is selected from SEQ ID NO:1, 5, 6, 10, 16, 24, 25, 31, and 49, and the α-synuclein binding domain is attached to the C-terminus of the J domain, with or without an optional linker.

[0146] In another aspect, the fusion protein comprises a J domain and a target-binding protein SEQ ID NO:65. Fusion protein constructs comprising QBP1 (SEQ ID NO:65) have previously been shown to reduce the formation of mechano- and hypermechanical conformational isomers in A53T α-synuclein (Hervás et al., (2012) PLoS Biol. 10(5):e1001335). Therefore, in a particular embodiment, the fusion protein comprises the α-synuclein-binding domain of SEQ ID NO:65. In a particular embodiment, the fusion protein construct comprises a J domain sequence selected from SEQ ID NO:1, 5, 6, 10, 16, 24, 25, 31, and 49, and the α-synuclein-binding domain of SEQ ID NO:65. In one embodiment, the fusion protein construct comprises the J domain sequence of SEQ ID NO:5 and the α-synuclein-binding domain of SEQ ID NO:65.

[0147] In another embodiment, the fusion protein also considers the use of an α-synuclein binding domain chemically conjugated to the J domain. The α-synuclein 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, known to those skilled in the art, are available and can be used to cross-link the α-synuclein binding domain to the J domain, or to cross-link the targeting domain to a fusion protein comprising both the α-synuclein 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 the occurrence of unwanted side reactions such as homopolymerization. Various heterobifunctional crosslinking agents 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). Crosslinking agents having an N-hydroxysuccinimide moiety can be obtained as N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. Additionally, crosslinking agents with disulfide bridges within the linker chain can be synthesized alternatively as alkyl derivatives to reduce the amount of linker cleavage in the bulk. Besides heterobifunctional crosslinking agents, many other crosslinking agents exist, including homobifunctional crosslinking agents and photoreactive crosslinking agents. Disuccinimide octanoate (DSS), bismaleimide hexane (BMH), and dimethyl heptamethine ester 2HCl (Forbes-Cori) are examples of useful homobifunctional crosslinking agents, and bis-[B-(4-azidosalicylic acid)ethyl] disulfide (BASED) and N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinking agents used in this disclosure. For a recent review of protein coupling techniques, see Means et al., (1990) Bioconj. Chem. 1:2-12, which are incorporated herein by reference.

[0148] Table 2: Examples of α-synuclein binding domains

[0149]

[0150]

[0151] c. Optional connector

[0152] 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 α-synuclein-binding domain, between tandem arrangements of the α-synuclein-binding domains, between the J domain and the α-synuclein-binding domain and optional targeting agents, or between the J domain and the α-synuclein-binding domain and optional detection domains or epitopes) for optimal function of each domain. Clearly, linkers preferably do not interfere with the function of the J domain and the target protein-binding domain of the fusion protein according to the invention, respectively. If a linker is present in the fusion protein of the invention, it is chosen to attenuate cytotoxicity caused by the target protein (α-synuclein protein), and the linker 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 of an expression vector containing a nucleotide segment encoding the protein domains as described herein or the entire fusion protein. In one embodiment, the peptide linker is between 1 and 20 amino acids in length. In another embodiment, the peptide linker is between 2 and 15 amino acids in length. In yet another embodiment, the peptide linker is between 2 and 10 amino acids in length.

[0153] Choosing 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, 95:5929-5934 (1998), each of which is incorporated herein by reference in its entirety. Examples of linkers having two or more amino acids that can be used to prepare the fusion protein according to the invention include, but are not limited to, those provided in Table 3 below.

[0154] Table 3: Connector Sequence

[0155] SEQ ID NO: length sequence 66 2 SR 67 4 GTGS 68 5 GLESR 69 4 GGSG 70 5 GGGGS 71 5 DIAAA 72 5 EAAAK 73 15 GGGGSGGGGSGGGGS 74 11 AEAAAKEAAAK 75 15 SGGGSGGGGSGGGGS 76 25 DIGGGGSGGGGSGGGGSGGGGSAAA

[0156] d. Targeted reagents

[0157] 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 the binding, transport, accumulation, residence time, bioavailability, or modify the biological activity or therapeutic efficacy of the fusion protein 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 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 another embodiment, the targeting moiety is attached to the fusion protein via chemical conjugation. In a further embodiment, the targeting moiety may be an amino acid sequence for subcellular localization, such as a nuclear localization signal or nuclear export signal. 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, etc.

[0158] In one exemplary embodiment of the invention, a targeting portion enhances the binding, transport, accumulation, residence time, bioavailability, 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.

[0159] 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 its antigen-binding fragment or single-chain derivative, such as that described in, U.S. Serial No. 16 / 131,591, which is incorporated herein by reference in its entirety. The targeting portion may be coupled to a platform for targeted cell delivery by binding directly or indirectly to a core. For example, in embodiments in which the core comprises nanoparticles, the conjugation of the targeting portion to the nanoparticles may utilize similar functional groups for tethering PEG to the nanoparticles. Thus, the targeting portion may bind directly to the nanoparticles by functionalization of the targeting portion. Alternatively, as discussed above, the targeting portion may bind indirectly to the nanoparticles by conjugation of the targeting portion to functionalized PEG. The targeting portion may attach to the core by 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.

[0160] e. Epitope

[0161] 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 the human IgG1 Fc sequence (SEQ ID NO:77), the FLAG epitope (DYKDDDDK, SEQ ID NO:78), the His6 epitope (SEQ ID NO:79), c-myc (SEQ ID NO:80), HA (SEQ ID NO:81), the V5 epitope (SEQ ID NO:82), or glutathione S-transferase (SEQ ID NO:83). In another embodiment, the fusion protein of the present invention further comprises epitopes that are used to increase the half-life of the fusion protein when administered to a subject (e.g., a human). 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 α-synuclein binding domain. The epitope is located at the C-terminus of the fusion protein.

[0162] Table 4: Representative Examples of Epitopes

[0163]

[0164]

[0165] f. Cell-penetrating peptides

[0166] 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 HIV TAT peptides 49-57, polyarginine, and penetrating peptide pAntan(43-58); amphiphilic peptides, such as pep-1; hydrophobic peptides, such as C405Y; and so on. See Table 5 below.

[0167] Table 5: Examples of cell-penetrating peptides

[0168] SEQ ID NO: length sequence 84 9 RKKRRQRRR 85 15 RQIKWFQNRRMKWKK 86 21 KETWWETWWTEWSQPKKKRKV 87 17 CSIPPEVKFNKPFVYLI

[0169] 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:84-87, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. In another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO:84, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. In yet another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO:85, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. In yet another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO:86, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. In yet another embodiment, the fusion protein comprises the cell-penetrating peptide of SEQ ID NO:87, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. Cells expressing a fusion protein construct with a cell-penetrating peptide can be administered to subjects, such as human subjects (e.g., patients with or at risk of developing α-synucleinemia). The fusion protein is secreted from the cells, which helps reduce the aggregation of α-synuclein-containing proteins and / or associated cytotoxicity.

[0170] Arrangement of gJ domain and α-synuclein binding domain

[0171] The fusion protein described herein can be arranged in several ways. In one embodiment, the α-synuclein binding domain is attached to the C-terminal side of the J domain. In another embodiment, the α-synuclein binding domain is attached to the N-terminal side of the J domain. In either configuration, the α-synuclein binding domain and the J domain can optionally be separated via a linker as described above.

[0172] In some embodiments, the J domain may attach to multiple α-synuclein-binding domains, such as two, three, four, or more α-synuclein-binding domains. The α-synuclein-binding domains may attach to the N-terminal side of the J domain. Alternatively, the α-synuclein-binding domain may attach to the C-terminal side of the J domain. In yet another embodiment, the α-synuclein-binding domain may attach to both the N-terminal and C-terminal sides of the J domain. Each of the multiple α-synuclein-binding domains may be the same α-synuclein-binding domain. In yet another embodiment, each of the multiple α-synuclein-binding domains in the fusion protein may be a different α-synuclein-binding domain (i.e., a different sequence).

[0173] In some implementations, the fusion protein may comprise structures selected from the group consisting of:

[0174] a.DNAJ-XS,

[0175] b.DNAJ-XSXS,

[0176] c.DNAJ-XSXSXS,

[0177] dS-X-DNAJ,

[0178] eS-XSX-DNAJ,

[0179] fS-XSXSX-DNAJ,

[0180] gS-X-DNAJ-XS,

[0181] hS-X-DNAJ-XSXS,

[0182] iS-XSX-DNAJ-XSXSXS,

[0183] jS-XSXSX-DNAJ-XS,

[0184] kS-XSXSX-DNAJ-XSXS,

[0185] lS-XSXSX-DNAJ-XSXSXS,

[0186] m.DnaJ-X-DnaJ-XSXS,

[0187] nS-X-DnaJ-X-DnaJ, and

[0188] oS-XSX-DnaJ-X-DnaJ,

[0189] in,

[0190] S is the α-synuclein binding domain.

[0191] DNAJ is the J domain of the J protein, and

[0192] X is an optional connector.

[0193] In one embodiment, the fusion protein comprises a J domain selected from SEQ ID NO:1-15 and 17-50. In another embodiment, the fusion protein comprises a J domain selected from SEQ ID NO:1, 5, 6, 10, 16, 24, 25, 31, and 49. In a particular embodiment, the fusion protein comprises the J domain of SEQ ID NO:5.

[0194] In another embodiment, the α-synuclein binding domain is selected from SEQ ID NO:51-64. In one particular embodiment, the α-synuclein binding domain is SEQ ID NO:49. In another embodiment, the α-synuclein binding domain is SEQ ID NO:63. In yet another embodiment, the α-synuclein binding domain is SEQ ID NO:64.

[0195] In yet another embodiment, the fusion protein comprises the J domain of SEQ ID NO:5 and the α-synuclein 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 α-synuclein binding domain of SEQ ID NO:51.

[0196] Non-limiting examples of fusion protein constructs containing a J domain and an α-synuclein-binding domain, as well as control constructs, are presented in [the literature / concept]. Figure 2 The diagram is schematically illustrated and also shown in Table 6 below. In one embodiment, the specific fusion protein construct is selected from SEQ ID NO:88, 90-96, 98-100.

[0197] Table 6: Fusion protein constructs and control constructs

[0198]

[0199]

[0200]

[0201] II. Nucleic acids encoding fusion protein constructs

[0202] 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 an α-synuclein-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 producing 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 (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0203] 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 suitable host cells with the expression vector, 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.

[0204] 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 constructs containing a gene encoding the fusion protein of the present invention or its complement. In some embodiments, cloning strategies are used to generate a gene encoding the fusion protein of the present invention or its complement.

[0205] In some embodiments, the nucleic acid encoding one or more fusion proteins is an RNA molecule and may be a pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. In various embodiments, the nucleic acid is mRNA introduced into the cell for transient expression of the desired polypeptide. As used herein, “transient” means a period of time during which non-integrated transgene expression occurs over hours, days, or weeks, wherein such expression period is shorter than the expression period of the polynucleotide when it is integrated into the genome or contained within a stable plasmid replicon in the cell.

[0206] 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 synthesized in vitro. Generally, in vitro transcribed RNA is generated by an in vitro transcription vector. The in vitro transcription vector contains a template used to generate the in vitro transcribed RNA. In a particular embodiment, the mRNA may further contain a 5' cap or a modified 5' cap and / or a poly(A) sequence. As used herein, a 5' cap (also referred to as an RNA cap, RNA 7-methylguanosine cap, or RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5' end of a eukaryotic messenger RNA shortly after transcription initiation. The 5' cap contains a terminal group that is linked to the first transcribed nucleotide and is recognized by the ribosome and protected from RNases. The capping portion may be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency. In a particular embodiment, the mRNA contains a poly(A) sequence of between about 50 and 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 another embodiment, the mRNA comprises a poly(A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1000 or more bases. The poly(A) sequence may be chemically or enzymatically modified to modulate mRNA functionality, such as localization, stability, or translation efficiency. 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, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0207] 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 one or more target genes, such as one or more target polynucleotides. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, such as promoters, enhancers, poly(A) sequences, and one or more target genes (e.g., one or more target polynucleotides). 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 insertion into a vector, for example, it has restriction endonuclease sites at each end. The cassette can be removed and inserted as a single unit into a plasmid or viral vector.

[0208] 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, Raul's 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. 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter (Okabe et al. (1997) FEBS let. 407: 313-9), β-actin promoter and myeloproliferative sarcoma virus enhancer (negative control region deletion), dl587rev primer binding site replaced (MND)U3 promoter (Haas et al., Journal of Virology. 2003; 77(l7): 9439-9450). In one embodiment, at least one element may be used with the polynucleotides 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), such as 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 in certain cell types, such as, but not limited to, nervous system promoters, which can be used to restrict expression in neurons, astrocytes, or oligodendrocytes.Non-limiting examples of tissue-specific expression elements for neurons include promoters for 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. Non-limiting examples of tissue-specific expression elements for astrocytes include promoters for glial fibrillary acidic protein (GFAP) and EAAT2. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include promoters for myelin basic protein (MBP). Yu et al. (2011, Molecular Pain, 7:63, incorporated herein by reference) used lentiviral vectors to evaluate eGFP expression in rat DRG cells and primary DRG cells under the CAG, EFIa, PGK, and UBC promoters, and found that UBC showed 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) 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 that with CMV promoters (see, for example, Gill et al., (2001) Gene Therapy, Vol. 8, 1539–1546; incorporated herein by reference). Husain et al. (2009, Gene Therapy, incorporated herein by reference) evaluated HβH constructs with hGUSB, HSV-1LAT, and NSE promoters and found that the HβH construct showed weaker expression than NSE in the mouse brain. Passini and Wolfe (J. Virol. 2001, 12382-12392, incorporated herein by reference) evaluated the long-term effects of HβH vectors after intraventricular injection in newborn mice and found sustained expression for at least one year. Xu et al. (2001, Gene Therapy, 8, 1323-1332; incorporated herein by reference in its entirety) found that, when using the NF-L and NF-H promoters, expression was lower in all brain regions compared to CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE+wpre, NSE (0.3kb), NSE (1.8kb), and NSE (1.8kb+wpre).Xu et al. found that promoter activity, in descending order, was 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, and 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; incorporated herein by reference in their entirety).

[0209] III. Vectors containing nucleic acids encoding fusion proteins

[0210] 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 direct 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 an α-synuclein-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.

[0211] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, FrogPrince, 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 phages 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 multivoid 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 the pLenti4 / V 5-DEST vector for lentivirus-mediated gene transfer and expression in mammalian cells. TM pLenti6 / V 5-DEST TM and pLenti6.2 / V 5-GW / lacZ (Invitrogen). In a particular embodiment, the coding sequence of the polypeptide disclosed herein can be ligated into such an expression vector for expression of the polypeptide in mammalian cells.

[0212] In certain implementations, the vector is a free vector or an extrachromosomally maintained vector. 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 extrachromosomally or in a free manner.

[0213] 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 a site-specific recombinase is any site other than that required for vector integration, such as a retroviral vector or a 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.

[0214] For example, one recombination site for Cre recombinase is loxP, which is a 34-base sequence consisting of two 13-base-pair inverted repeats of the core sequence flanked by 8 base pairs (acting as recombinase binding sites) (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).

[0215] Other examples of recognized sequences are attB, attP, attL, and attR sequences, which are recognized by recombinase integrase (e.g., phi-c3l). pC3l SSRs mediate recombination only between the heterologous sites attB (34 bp) and attP (39 bp) (Groth et al., 2000). attB and attP, named as attachment sites of phage integrase on the bacterial and phage genomes respectively, both likely contain sequences that are recognized by integrase integrase. Incomplete inverse repeats of homodimer binding (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 the attP site into the genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to locate the attP-carrying “docking site” into the defined locus via homologous recombination, which then pairs with the attB-carrying entry sequence for insertion.

[0216] As used herein, an "internal ribosome entry site" or "IRES" refers to an element that facilitates direct internal ribosome entry into the start codon of a cistron (protein-coding region), such as ATG, thereby leading to 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.

[0217] 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 comprises a polynucleotide having a shared Kozak sequence and encoding a fusion protein containing a J domain and an α-synuclein-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 comprises a polyadenylated sequence at the 3' of a polynucleotide encoding the polypeptide to be expressed.

[0218] 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.

[0219] 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-defective, predominantly 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 vector is encapsulated in AAV8. In another embodiment, the vector is encapsulated in AAV9. In yet another embodiment, the vector is encapsulated in AAVrh10.

[0220] 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 a particular embodiment, 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 a preferred embodiment, the rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0221] In some implementations, modification and selection methods can be applied to the AAV capsid to make it more likely to transduce target cells.

[0222] 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.

[0223] IV. Delivery

[0224] In certain embodiments, one or more polynucleotides encoding a fusion protein comprising a J domain and an α-synuclein-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.

[0225] Illustrative examples of multinucleotide delivery systems considered in specific implementations include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipid transfection reagents are commercially available (e.g., Transfectam). TM and Lipofectin TM Cationic and neutral lipids suitable for transfection with effective receptor-recognition lipids for polynucleotides 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, non-living nanocell-based delivery has also been considered in specific embodiments.

[0226] 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 to ex vivo 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.

[0227] 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.

[0228] 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.

[0229] Alternatively, naked DNA may be applied. Application is performed via 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 to apply a particular composition, a particular route often provides a more direct and efficient response than another.

[0230] 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 (e.g., a lentivirus) containing one or more polynucleotides. As used herein, the term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Illustrative retroviruses 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 Raul's 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); viscammavirus (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.

[0231] 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. Further 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), Raul's sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, lentiviral vectors are 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.

[0232] According to certain specific embodiments considered herein, most or all of the viral vector backbone sequence is 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 in certain lentiviral sequences can be permitted without impairing the transfer vector's ability to perform the functions described herein. Furthermore, various lentiviral vectors are known in the art; see Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516; and 5,994,136, many of which are suitable for producing the viral vectors or transfer plasmids considered herein.

[0233] 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 expression levels 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 by transgenes; subsequently, replication-deficient vectors are propagated in human 293 cells supplying the missing gene function. Ad vectors can be transduced in multiple tissue types in vivo, including non-dividing differentiated cells, such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0234] 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 an 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 antitumor immunotherapy with polynucleotides (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0235] 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 (e.g., HSV-1, HSV-2) containing one or more polynucleotides.

[0236] Mature HSV viral particles consist of an enveloped icosahedral capsid containing a viral genome composed of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is 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 genome, which can accommodate foreign DNA insertions 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.

[0237] V. Cells expressing fusion proteins

[0238] In yet another aspect, the present invention provides cells expressing the fusion proteins described herein. Cells can be transfected using vectors encoding the fusion proteins as described above. In one embodiment, the cells are prokaryotic cells. In another embodiment, the cells are eukaryotic cells. In yet another embodiment, the cells are mammalian cells. In a particular embodiment, the cells are human cells. In another embodiment, the cells are human cells derived from a patient who has or is at risk of having an α-synuclein-mediated condition, including but not limited to ALS, FTD, and Alzheimer's disease. The cells can be neuronal cells or muscle cells.

[0239] 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 epitopes, such as human Fc domains or FLAG epitopes, as described above, which will facilitate purification (using protein A or anti-FLAG antibody columns, respectively). The epitopes may be linked to the remainder of the fusion protein via linkers or protease substrate sequences, allowing the epitopes to be removed from the fusion protein during or after purification.

[0240] Cells expressing fusion proteins can also be used in a therapeutic context. In one embodiment, cells are collected from patients requiring treatment (e.g., patients with α-synuclein-mediated conditions or at risk of developing them). 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 via intrathecal injection, intracranial injection, or intraventricular injection to inject directly into the central nervous system.

[0241] In an alternative 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.

[0242] Table 7: Representative signal sequences

[0243] SEQ ID NO: length sequence 102 17 MGVKVLFALICIAVAEA 103 19 MAPVQLLGLLVLFLPAMRC 104 19 MAVLGLLFCLVTFPSCVLS

[0244] 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:102-104, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO:102, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO:103, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO:104, and the fusion protein is selected from SEQ ID NO:88, 90-96, 98-100. Cells expressing the fusion protein construct having the signal sequence can be administered to subjects, such as human subjects (e.g., patients with α-synucleinemia or at risk of having it). The fusion protein is secreted from the cells, which helps reduce the aggregation of α-synuclein proteins and / or associated cytotoxicity.

[0245] 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, will then be able to enter nearby cells and has the potential to reduce aggregation and / or cytotoxicity mediated by α-synuclein proteins in these cells.

[0246] VI. How to Use

[0247] In another aspect, the present invention provides methods for achieving beneficial effects in conditions and / or α-synuclein disorders, conditions or states mediated by α-synuclein aggregation. α-synuclein disorders are selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, and Lewy body dementia.

[0248] In some embodiments, the present invention provides a method for treating a subject (e.g., a human) with an α-synuclein 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 a fusion protein described herein, wherein the administration results in an improvement in one or more biochemical or physiological parameters or clinical endpoints associated with an α-synuclein disease, condition, or status.

[0249] In other embodiments, the present invention provides a method for reducing α-synuclein aggregation 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 an α-synuclein disorder, including but not limited to amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. In a particular embodiment, the α-synuclein disorder is amyotrophic lateral sclerosis.

[0250] α-synuclein aggregation can be detected in a variety of ways. In one example, aggregated α-synuclein proteins can be detected in an ELISA using an antibody that specifically recognizes the conformation of α-synuclein. A reduction in α-synuclein protein aggregation can also be detected directly in cells, for example, using immunofluorescence microscopy with labeling reagents accompanying the detection of α-synuclein proteins (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 α-synuclein peptide levels compared to a control indicates greater potency.

[0251] Therefore, in one embodiment, the method includes contacting cells with an amount of fusion protein or nucleic acid, vector, or viral particles encoding the fusion protein, said amount effectively reducing the aggregation of α-synuclein 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.

[0252] As shown in Example 1 below, it has been found that the expression of a fusion protein containing a J domain and an α-synuclein binding domain reduces the overall level of a reporter gene construct containing α-synuclein. Therefore, 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, when compared to untreated cells or control cells, effectively reducing the level of α-synuclein protein by at least 10%, for example, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0253] VII. Pharmaceutical Composition

[0254] The compositions considered herein may include one or more fusion proteins comprising a J domain and an α-synuclein-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, where 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 such additional pharmaceutical agent does not adversely affect the composition’s ability to deliver the intended therapy. The phrase “pharmaceutically acceptable” herein refers to a compound, material, composition, carrier, and / or dosage form that, to the extent of reasonable medical judgment, is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. As used herein, “pharmaceuticalally 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 for acceptable 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.

[0255] VIII. Dosage

[0256] 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.

[0257] IX. Reagent Kit

[0258] Kits considered include: (a) pharmaceutical compositions described herein comprising fusion protein constructs, nucleic acids encoding such fusion proteins, or viral particles containing such nucleic acids, which reduce the aggregation of α-synuclein proteins in cells or subjects; and (b) a package insert containing instructions for performing any of the methods described herein. In some aspects, the kit includes (a) a pharmaceutical composition described herein comprising the compositions described herein, which reduces the aggregation of α-synuclein proteins in cells or subjects; (b) an additional therapeutic agent; and (c) a package insert containing instructions for performing any of the methods described herein.

[0259] Example

[0260] To test whether the J domain could be specifically modified to facilitate proper folding of aggregate proteins, we designed and tested numerous fusion protein constructs designed to target α-synuclein proteins.

[0261] Example 1: Fusion Protein Design

[0262] A. Method

[0263] General techniques and materials

[0264] 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,” edited by F.M. Usubel et al., 1987; series “Methods in Enzymology,” Academic Press, San Diego, Calif.; “PCR2: a practical approach,” edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor, Oxford University Press, 1995; “Antibodies, a laboratory manual,” edited by Harlow, E., and Lane, D., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman’s The Pharmacological Basis of Therapeutics,” 11th ed., McGraw-Hill, 2005; and Freshney, RI, “Culture of Animal Cells: A Manual of Basic Technique,” ​​4th ed., John Wiley & Sons, Somerset, NJ, 2000, the contents of 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:88-101, also contain the FLAG epitope of SEQ ID NO:78 at the C-terminus or N-terminus of the protein, along with a short linker sequence.

[0265] Protein expression and detection in HEK293 cells

[0266] Expression vector plasmids encoding various protein constructs were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). The expressed proteins were analyzed from cell lysates using Western blotting. Samples of the culture medium were centrifuged to remove debris prior to analysis. Cells were lysed in a 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, the expressed proteins were analyzed from the samples using Western blotting. For Western blotting analysis, samples were boiled in SDS sample buffer and run on polyacrylamide gel electrophoresis. The separated protein bands were then transferred to a PVDF membrane.

[0267] The expression of proteins is detected using chemiluminescence signals. In short, the blot is reacted with a primary antibody (e.g., an anti-α-synuclein antibody) capable of binding to a specific epitope. 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 is captured on an X-ray film.

[0268] BFA / MG132 Wenyu

[0269] Expression vector plasmids encoding various protein constructs were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). One day after transfection, the culture medium was replaced with fresh medium containing bafloxacin a1 (BFA) or MG132, and the cells were incubated for another 48 hours. Cells were lysed in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40) supplemented with a complete protease inhibitor cocktail (Sigma) and 2 mM PMSF. Cell lysates were analyzed by ELISA or Western blotting after a brief sonication.

[0270] ELISA (Enzyme-Linked Immunosorbent Assay)

[0271] The aggregation state of expressed α-synuclein was monitored using the α-synuclein aggregate-specific antibody Syn-O4 (BioLegend). Syn-O4 is a conformation-specific monoclonal antibody that specifically recognizes α-synuclein aggregates, rather than the soluble monomeric form of the protein. 58 The wells of the microtiter plate were coated with Syn-O4 overnight at 4°C. After coating, the wells were washed three times with PBS, blocked with PBS containing 1% BSA for 1 hour, and washed again with PBS.

[0272] Following expression, cultured cells were lysed in a lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40) containing a mixture of 2 mM PMSF and protease, followed by brief sonication. After removing debris by centrifugation, cell lysates were added to wells and incubated overnight at 4°C. The wells were then washed three times with PBS, incubated for 1 hour with HRP-linked anti-α-synuclein antibody in PBS containing 1% BSA, and washed three times again with PBS. Finally, peroxidase activity was measured using 0.01 mg / ml 3',3',5',5'-tetramethylbenzidine (TMB) and 0.01% H2O2 in 0.1 M sodium acetate (pH 6.0) buffer. Density was determined at 450 nm after adding an equal volume of 1 M HCl. Reporter construct

[0273] We first investigated whether fusion proteins targeting α-synuclein improved its aggregation in cultured cells. To this end, we generated two constructs: one expressing wild-type α-synuclein and the other a mutant form known to be associated with familial Parkinson's disease (containing an A53T substitution) (see Table 8 below). HEK293 cells were cultured and transfected with plasmids encoding either wild-type (SEQ ID NO: 105) or mutant (SEQ ID NO: 106) α-synuclein.

[0274] Table 8: Synuclein constructs

[0275]

[0276] Fusion protein constructs

[0277] To determine whether the J domain could be used to reduce α-synuclein aggregation, initial experiments were conducted by co-expression of wild-type or mutant synuclein with a fusion protein containing a J domain sequence derived from the Hsp40 J domain protein (from human DnaJB1), which was conjugated to the synuclein-binding peptide SynBP1 (see Construct 1 in Table 9 below). Figure 3AAs shown, expression of wild-type or mutant (A53T) α-synuclein in HEK293 cells resulted in the appearance of aggregated α-synuclein, as detected by ELISA of cell lysates (see Items 1 and 4). Co-expression of construct 1 resulted in a sharp reduction in the amount of detectable aggregated α-synuclein (Items 2 and 5). However, co-expression of a mutant variant of construct 1 containing a P33Q mutation within the highly conserved HPD motif in the J domain resulted in higher overall aggregation (see Items 3 and 6), suggesting that the mechanism of reduced aggregation is through the HSP70 system. Western blot analysis of cell lysates was performed using an anti-α-synuclein antibody to determine whether the fusion protein affected the overall level of α-synuclein. Figure 3B As shown, all cells had relatively similar α-synuclein levels, suggesting that the reduction in aggregated α-synuclein was not solely due to α-synuclein degradation.

[0278] Table 9. Fusion protein constructs and controls

[0279]

[0280]

[0281] Alpha-synuclein is known to be secreted. Therefore, we investigated whether the fusion protein construct effectively reduced the amount of secreted α-synuclein. Culture media were collected from cells expressing wild-type (strips 2 and 3) or mutant (strips 4 and 5) α-synuclein alone, or also expressing construct 1 (JB1-SynBP1; strips 3 and 5), and the level of secreted α-synuclein was measured by ELISA analysis. Figure 4 As shown, when expressed in the absence of the fusion protein construct, both wild-type and mutant (A53T) α-synuclein forms were secreted (strips 2 and 4, respectively). However, cells co-expressing construct 1 did not detect any α-synuclein secreted into the culture medium, as measured by ELISA (strips 3 and 5, respectively, for wild-type and mutant α-synuclein).

[0282] We then investigated the mechanism by which the fusion protein reduces aggregation. HEK293 cells were transfected with wild-type or mutant (A53T) α-synuclein alone or with construct 1 (JB1-SynBP1) and with BFA (bafomycin A1, an inhibitor of late autophagy) or MG132 (a proteasome inhibitor). Figure 5 and Figure 6The results are shown and summarized in Table 10 below (values ​​normalized relative to the wild-type control alone). As can be seen, expression of construct 1 in cells also expressing mutant (A53T) α-synuclein resulted in a modest reduction in total α-synuclein (from 134.9% to 99%), but a more significant reduction in aggregated α-synuclein (from 111.5% to 53.9%). Treatment of these cells with 10 nM or 100 nM BFA resulted in the restoration of total and aggregated α-synuclein to levels matching those in cells expressing mutant α-synuclein alone (i.e., not also transfected with fusion construct 1). Bafloromycin A1 is a potent inhibitor of late autophagy, suggesting that the fusion construct exerts its effects via chaperone-mediated autophagy.

[0283] In contrast, treatment of cells with the proteasome inhibitor MG132 had little effect on the total or aggregated levels of α-synuclein.

[0284] Table 10. Effects of inhibitors on the reduction of α-synuclein aggregation mediated by fusion proteins

[0285]

[0286] The ability to reduce α-synuclein aggregation was tested on other constructs. Table 11 below shows that many other constructs containing different synuclein-binding domains can reduce α-synuclein aggregation. Interestingly, construct 10, a fusion protein construct containing QBP1, which was previously shown to bind to polyglutamine repeats, also showed a strong ability to reduce α-synuclein aggregation.

[0287] Table 11. Effects of fusion protein constructs containing various α-synuclein binding domains on reducing aggregation.

[0288]

[0289]

[0290] To further optimize the arrangement of the J domain and the α-synuclein binding domain, additional constructs were tested. Table 12 below shows the capabilities of constructs using alternative J domains (construction 6, using a J domain derived from DnaJB6; construct 7, using a J domain derived from DnaJB13) or different linkers between the J domain and the α-synuclein binding domain.

[0291] Table 12. Effects of fusion protein constructs containing various α-synuclein binding domains on reducing aggregation.

[0292]

[0293] Next, experiments were conducted to determine whether the expression of various fusion protein constructs had an effect on the level of phosphorylated α-synuclein. Table 13 below shows that the expression of various fusion protein constructs had a significant effect on the level of mutant (A53T) α-synuclein phosphorylated at Ser129.

[0294] Table 13. Effects of co-expression of various fusion protein constructs on phosphorylated α-synuclein (Ser129) levels.

[0295]

[0296]

[0297] Finally, experiments were conducted to determine whether the expression of various fusion protein constructs could improve cytotoxicity through the expression of mutant (A53T) α-synuclein. Although expression of wild-type (WT) or mutant (A53T) α-synuclein did not induce significant cytotoxicity in HEK293 cells (data not shown), α-synuclein-induced cytotoxic effects were observed in U-87MG cells, as evidenced by increased LDH activity in the culture medium collected from U87-MG cells 7 days post-infection, as demonstrated by LDH-Cytotox. TM The assay kit (BioLegend, San Diego, CA, USA) measures ( Figure 7 Co-expression with JB1-SynBP1 (constructor 1) strongly suppressed the cytotoxic effects of α-synuclein. We interpret these results as indicating that the fusion protein construct can reduce α-synuclein-mediated cytotoxicity.

[0298] Example 2: AAV vector encoding fusion protein construct

[0299] The exemplary gene therapy vector is constructed from an AAV9 vector carrying codon-optimized cDNA encoding the fusion protein constructs listed in Table 6, specifically constructs 1, 3, 4, and 5, under the control of a CAG promoter containing a cytomegalovirus (CMV) early enhancer element and a chicken β-actin promoter. The cDNA encoding JB1-SynBP1 is located downstream of the Kozak sequence and is polyadenylated via bovine growth hormone polyadenylation (BGHpA) signaling. The entire cassette is flanked by two AAV-9 non-coding reversed-end sequences.

[0300] Recombinant AAV vectors were prepared using a baculovirus expression system similar to that described above (Urabe et al., 2002; Unzu et al., 2011 (reviewed in Kotin, 2011)). In short, three recombinant baculoviruses were used to infect SF9 insect cells: one encoding REP for replication and packaging, one encoding CAP-5 for the AAV9 capsid, and one containing an expression cassette. 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 was expressed as genome copies per ml (GC / ml). The vector titer was approximately 8 x 10⁻⁶. 13 Up to 2x 10 14 GC / ml.

[0301] Example 3: Efficacy testing in a mouse model of PD

[0302] The vector prepared above was tested in a mouse model of PD. Useful models include the mouse model described by Fares et al., (2006) Proc. Natl. Acad. Sci. USA, 113: E912-E921, which is obtained through SNCA... - / - The expression of human α-synuclein in the background leads to the generation of hyperphosphorylation (at S129) and ubiquitin-positive LB-like inclusion bodies in primary neurons, which are enlarged in size and incorporated with soluble proteins.

[0303] To test the effects of the test compounds or known compounds described in this application in animal models, different AAV viral particles containing vectors encoding fusion proteins and corresponding controls were administered to transgenic animals. In one embodiment, the viral particles were administered via tail vein injection. In another embodiment, the viral particles were administered via intramuscular injection. In yet another embodiment, the particles were administered via intracranial injection, as described, for example, as in Stanek et al., (2014) Hum. Gene. Ther. 25:461-474.

[0304] Following administration, disease progression is monitored and compared with control mice. In one implementation, the development of PD-like inclusion bodies is assessed in animals. Several other animal models that replicate PD-like pathology may also be used.

[0305] Other aspects

[0306] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated herein by reference in its entirety. Where a term in this application is found to be defined differently in documents incorporated herein by reference, the definition provided herein shall serve as the definition of that term.

[0307] 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 can be applied to the essential features set forth above and within the scope of the claimed protection. sequence list <110> Sola Biosciences Ltd. <120> Compositions and methods for treating synucleinosis <130> 269548-493385 <150> US 63 / 035,297 <151> 2020-06-05 <160> 106 <170> PatentIn version 3.5 <210> 1 <211> 63 <212> PRT <213> artificial <220> <223> DNAJA1 <400> 1 Thr Tyr Tyr Asp Val Leu Gly Val Lys Pro Asn Ala Thr Gln Glu Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Tyr His Pro Asp Lys 20 25 30 Asn Pro Asn Glu Gly Glu Lys Phe Lys Gln Ile Ser Gln Ala Tyr Glu 35 40 45 Is Asp Served By Lys Gly Gly 50 55 60 <210> 2 <211> 63 <212> PRT <213> household <220> <223> DNAJA2 <400> 2 Lys Leu Tyr Asp Ile Leu Gly Val Pro Pro Gly Ala Ser Glu Asn Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Lys Leu Lys Glu Tyr His Pro Asp Lys 20 25 30 Asn Pro Asn Ala Gly Asp Lys Phe Lys Glu Ile Ser Phe Ala Tyr Glu 35 40 45 Val Leu Ser Asn Pro Glu Lys Arg Glu Leu Tyr Asp Arg Tyr Gly 50 55 60 <210> 3 <211> 66 <212> PRT <213> household <220> <223> DNAJA3 <400> 3 Asp Tyr Tyr Gln Ile Leu Gly Val Pro Arg Asn Ala Ser Gln Lys Glu 1 5 10 15 Ile Lys Lys Ala Tyr Tyr Gln Leu Ala Lys Lys Ala Tyr His Pro Asp Thr 20 25 30 Asn Lys Asp Asp Pro Lys Ala Lys Glu Lys Phe Ser Gln Leu Ala Glu 35 40 45 Ala Tyr Glu Val Leu Ser Asp Glu Val Lys Arg Lys Gln Tyr Asp Ala 50 55 60 Tyr Gly 65 <210> 4 <211> 67 <212> PRT <213> Artificial <220> <223> DNAJA4 <400> 4 Glu Thr Gln Tyr Tyr Asp Ile Leu Gly Val Lys Pro Ser Ala Ser Pro 1 5 10 15 Glu Glu Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Tyr His Pro 20 25 30 Asp Lys Asn Pro Asp Glu Gly Glu Lys Phe Lys Leu Ile Ser Gln Ala 35 40 45 Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg Asp Val Tyr Asp Gln Gly 50 55 60 Gly Glu Gln 65 <210> 5 <211> 69 <212> PRT <213> Artificial <220> <223> DNAJB1 <400> 5 Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser Asp 1 5 10 15 Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His Pro 20 25 30 Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile Ala 35 40 45 Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe Asp 50 55 60 Arg Tyr Gly Glu Glu 65 <210> 6 <211> 70 <212> PRT <213> household <220> <223> DNAJB2 <400> 6 Wing Ser Tyr Tyr Glu Ile Leu Asp Val Pro Arg Ser Wing Ser Ala Asp 1 5 10 15 Asp With Tyr Arg Lys Only Leu Gln Trp His Pro Asp 20 25 30 Lys Asn Pro Asp Asn Lys Glu Phe Ala Glu Lys Phe Lys Glu Val 35 40 45 Only Glu Only Tyr Glu Will Be Asp Lys Lys Arg Glu Ile Tyr 50 55 60 Asp Arg Tyr Gly Arg Glu 65 70 <210> 7 <211> 69 <212> PRT <213> household <220> <223> DNAJB3 <400> 7 Met Val Asp Tyr Tyr Glu Val Leu Asp Val Pro Arg Gln Ala Ser Ser 1 5 10 15 Glu Ala Ile Lys Lys Ala Tyr Arg Lys Ala Leu Lys Trp His Pro 20 25 30 Asp Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Arg Phe Lys Gln 35 40 45 Only Glu Only Tyr Glu Will Be Asp Only Only Lys Only Arg Asp Ile 50 55 60 Tyr Asp Arg Tyr Gly 65 <210> 8 <211> 69 <212> PRT <213> household <220> <223> DNAJB4 <400> 8 Gly Lys Asp Tyr Tyr Cys Ile Leu Gly Ile Glu Lys Gly Ala Ser Asp 1 5 10 15 Glu Asp Ile Lys Lys Ala Tyr Arg Lys Gln Ala Leu Lys Phe His Pro 20 25 30 Asp Lys Asn Lys Ser Pro Gln Ala Glu Glu Lys Phe Lys Glu Val Ala 35 40 45 Glu Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg Glu Ile Tyr Asp 50 55 60 Gln Phe Gly Glu Glu 65 <210> 9 <211> 65 <212> PRT <213> Artificial <220> <223> DNAJB5 <400> 9 Asp Tyr Tyr Lys Ile Leu Gly Ile Pro Ser Gly Ala Asn Glu Asp Glu 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Met Ala Leu Lys Tyr His Pro Asp Lys 20 25 30 Asn Lys Glu Pro Asn Ala Glu Glu Lys Phe Lys Glu Ile Ala Glu Ala 35 40 45 Tyr Asp Val Leu Ser Asp Pro Lys Lys Arg Gly Leu Tyr Asp Gln Tyr 50 55 60 Gly 65 <210> 10 <211> 68 <212> PRT <213> Artificial <220> <223> DNAJB6 <400> 10 Val Asp Tyr Tyr Glu Val Leu Gly Val Gln Arg His Ala Ser Pro Glu 1 5 10 15 Asp Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Trp His Pro Asp 20 25 30 Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys Phe Lys Gln Val 35 40 45 Ala Glu Ala Tyr Glu Val Leu Ser Asp Ala Lys Lys Arg Asp Ile Tyr 50 55 60 Asp Lys Tyr Gly 65 <210> 11 <211> 67 <212> PRT <213> Artificial <220> <223> DNAJB7 <400> 11 Asp Tyr Tyr Glu Val Leu Gly Leu Gln Arg Tyr Ala Ser Pro Glu Asp 1 5 10 15 Ile Lys Lys Ala Tyr His Lys Val Ala Leu Lys Trp His Pro Asp Lys 20 25 30 Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys Phe Lys Glu Val Ala 35 40 45 Glu Ala Tyr Glu Val Leu Ser Asn Asp Glu Lys Arg Asp Ile Tyr Asp 50 55 60 Light Tyr Gly 65 <210> 12 <211> 67 <212> PRT <213> Artificial <220> <223> DNAJB8 <400> 12 Asn Tyr Tyr Glu Val Leu Gly Val Gln Ala Ser Ala Ser Pro Glu Asp 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Arg Trp His Pro Asp Lys 20 25 30 Asn Pro Asp Asn Lys Glu Glu Ala Glu Lys Lys Phe Lys Leu Val Ser 35 40 45 Glu Ala Tyr Glu Val Leu Ser Asp Ser Lys Lys Arg Ser Leu Tyr Asp 50 55 60 Arg Ala Gly 65 <210> 13 <211> 65 <212> PRT <213> Artificial <220> <223> DNAJB9 <400> 13 Ser Tyr Tyr Asp Ile Leu Gly Val Pro Lys Ser Ala Ser Glu Arg Gln 1 5 10 15 Ile Lys Lys Ala Phe His Lys Leu Ala Met Lys Tyr His Pro Asp Lys 20 25 30 Asn Lys Ser Pro Asp Ala Glu Ala Lys Phe Arg Glu Ile Ala Glu Ala 35 40 45 Tyr Glu Thr Leu Ser Asp Ala Asn Arg Arg Lys Glu Tyr Asp Thr Leu 50 55 60 Gly 65 <210> 14 <211> 66 <212> PRT <213> Artificial <220> <223> DNAJB11 <400> 14 Asp Phe Tyr Lys Ile Leu Gly Val Pro Arg Ser Ala Ser Ile Lys Asp 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Gln Leu His Pro Asp Arg 20 25 30 Asn Pro Asp Asp Pro Gln Ala Gln Glu Lys Phe Gln Asp Leu Gly Ala 35 40 45 Ala Tyr Glu Val Leu Ser Asp Ser Glu Lys Arg Lys Gln Tyr Asp Thr 50 55 60 Tyr Gly 65 <210> 15 <211> 65 <212> PRT <213> Artificial <220> <223> DNAJB12 <400> 15 Tyr Glu Ile Leu Gly Val Ser Arg Gly Ala Ser Asp Glu Asp Leu Lys 1 5 10 15 Lys Ala Tyr Arg Arg Leu Ala Leu Lys Phe His Pro Asp Lys Asn His 20 25 30 Pro Gly Ala Thr Glu Ala Phe Lys Ala Ile Gly Thr Ala Tyr Ala 35 40 45 Val Leu Ser Asn Pro Glu Lys Arg Lys Gln Tyr Asp Gln Phe Gly Asp 50 55 60 Asp 65 <210> 16 <211> 65 <212> PRT <213> household <220> <223> DNAJB13 <400> 16 Asp Tyr Tyr Ser Val Leu Gly Ile Thr Arg Asn Ser Glu Asp Ala Gln 1 5 10 15 Ile Lys Gln Ala Tyr Arg Leu Ala Leu Lys His Pro Leu Lys 20 25 30 Ser Asn Glu Pro Ser Ser Ala Glu Ile Phe Arg Gln Ile Ala Glu Ala 35 40 45 Tyr Asp Val Leu Serves Asp Pro Met Lys Arg Gly Ile Tyr Asp Lys Phe 50 55 60 Gly 65 <210> 17 <211> 65 <212> PRT <213> household <220> <223> DNAJB14 <400> 17 Asn Tyr Tyr Glu Val Leu Gly Val Thr Lys Asp Ala Gly Asp Glu Asp 1 5 10 15 Leu Lys Ala Tyr Arg Lys Leu Ala Leu Lys Phe His Pro Asp Lys 20 25 30 Asn His Ala Pro Gly Ala Thr Asp Ala Phe Lys Lys Ile Gly Asn Ala 35 40 45 Tyr Ala Val Served Asn Pro Glu Lys Arg Lys Gln Tyr Asp Leu Thr 50 55 60 Gly 65 <210> 18 <211> 65 <212> PRT <213> household <220> <223> DNAJC1 <400> 18 Asn Phe Tyr Gln Phe Leu Gly Val Gln Gln Asp Ala Ser Ser Ala Asp 1 5 10 15 With Arg Lys Only Tyr Arg Lys Is Pro Asp Lys 20 25 30 Asn Lys Asp Glu Asn Ala Glu Thr Gln Phe Arg Gln Leu Val Ala Ile 35 40 45 Tyr Glu Val Leu Lys Asp Asp Glu Arg Arg Gln Arg Tyr Asp Asp Ile 50 55 60 Leu 65 <210> 19 <211> 74 <212> PRT <213> household <220> <223> DNAJC2 <400> 19 Asp His Tyr Ala Val Leu Gly Leu Gly His Val Arg Tyr Lys Ala Thr 1 5 10 15 Gln Arg Gln Ile Lys Ala Ala His Lys Ala Met Val Leu Lys His His 20 25 30 Pro Asp Lys Arg Lys Ala Ala Gly Glu Pro Ile Lys Glu Gly Asp Asn 35 40 45 Asp Tyr Phe Thr Cys Ile Thr Lys Ala Tyr Glu Met Serves Asp Pro 50 55 60 Val Lys Arg Ala Phe Asn Ser Val Asp 65 70 <210> 20 <211> 69 <212> PRT <213> household <220> <223> DNAJC3 <400> 20 Asp Tyr Tyr Lys Ile Leu Gly Val Lys Arg Asn Ala Lys Gln Glu 1 5 10 15 Ile Ile Lys Only Tyr Arg Lys Only Only Only On Gln Trp His Pro Asp Asn 20 25 30 Phe Gln Asn Glu Glu Glu Lys Lys Ala Glu Lys Lys Phe Ile Asp 35 40 45 Ile Ala Ala Ala Lys Glu Val Leu Ser Asp Pro Glu Met Arg Lys Lys 50 55 60 Phe Asp Asp Gly Glu 65 <210> 21 <211> 66 <212> PRT <213> household <220> <223> DNAJC4 <400> 21 Thr Tyr Tyr Glu Leu Leu Gly Val His Pro Gly Ala Serving Thr Glu Glu 1 5 10 15 Val Lys Arg Ala Phe Phe Ser Lys Ser Lys Glu Leu His Pro Asp Arg 20 25 30 Asp Pro Gly Asn Pro Ser Leu His Ser Arg Phe Val Glu Leu Ser Glu 35 40 45 Only Tyr Arg Val Leu Arg Ser Glu Gln Ser Arg Arg Ser Tyr Asp Asp 50 55 60 Lion's Gln 65 <210> 22 <211> 70 <212> PRT <213> household <220> <223> DNAJC5 <400> 22 Gly Glu Ser Leu Tyr His Val Leu Gly Leu Asp Lys Asn Ala Thr Ser 1 5 10 15 Asp Asp Ile Lys Ser Tyr Arg Lys Ala Leu Lys Tyr His Pro 20 25 30 Asp Lys Asn Pro Asp Asn Pro Glu Ala Ala Asp Lys Phe Lys Glu Ile 35 40 45 Asn Asn Only His Only With Thr Asp Only Thr Lys Arg Only With Tyr 50 55 60 Asp Lys Tyr Gly Ser Lion 65 70 <210> 23 <211> 66 <212> PRT <213> household <220> <223> DNAJC5B <400> 23 Only Tyr Glu Only Gly Only His Gly Only Ser Asn Glu Glu 1 5 10 15 Ile Lys Lys Thr Tyr Arg Lys Leu Ala Leu Lys His Pro Asp Lys 20 25 30 Asn Pro Asp Asp Pro Ala Ala Thr Glu Lys Phe Lys Glu Ile Asn Asn 35 40 45 Only His Only With Thr Asp With Lys Arg Only With Tyr Asp Lys 50 55 60 Tyr Gly 65 <210> 24 <211> 65 <212> PRT <213> Artificial <220> <223> DNAJC6 <400> 24 Thr Lys Trp Lys Pro Val Gly Met Ala Asp Leu Val Thr Pro Glu Gln 1 5 10 15 Val Lys Lys Val Tyr Arg Lys Ala Val Leu Val Val His Pro Asp Lys 20 25 30 Ala Thr Gly Gln Pro Tyr Glu Gln Tyr Ala Lys Met Ile Phe Met Glu 35 40 45 Leu Asn Asp Ala Trp Ser Glu Phe Glu Asn Gln Gly Gln Lys Pro Leu 50 55 60 Taurus 65 <210> 25 <211> 71 <212> PRT <213> Artificial <220> <223> DNAJC7 <400> 25 Asp Tyr Tyr Lys Ile Leu Gly Val Asp Lys Asn Ala Ser Glu Asp Glu 1 5 10 15 Ile Lys Lys Ala Tyr Arg Lys Arg Ala Leu Met His His Pro Asp Arg 20 25 30 His Ser Gly Ala Ser Ala Glu Val Gln Lys Glu Glu Glu Lys Lys Phe 35 40 45 Lys Glu Val Gly Glu Ala Phe Thr Ile Leu Ser Asp Pro Lys Lys Lys 50 55 60 Thr Arg Tyr Asp Ser Gly Gln 65 70 <210> 26 <211> 68 <212> PRT <213> Artificial <220> <223> DNAJC8 <400> 26 Asn Pro Phe Glu Val Leu Gln Ile Asp Pro Glu Val Thr Asp Glu Glu 1 5 10 15 Ile Lys Lys Arg Phe Arg Gln Leu Ser Ile Leu Val His Pro Asp Lys 20 25 30 Asn Gln Asp Asp Ala Asp Arg Ala Gln Lys Ala Phe Glu Ala Val Asp 35 40 45 Lys Ala Tyr Lys Leu Leu Leu Asp Gln Glu Gln Lys Lys Arg Ala Leu 50 55 60 Asp Val Ile Gln 65 <210> 27 <211> 68 <212> PRT <213> Artificial <220> <223> DNAJC9 <400> 27 Asp Leu Tyr Arg Val Leu Gly Val Arg Arg Glu Ala Ser Asp Gly Glu 1 5 10 15 Val Arg Arg Gly Tyr His Lys Val Ser Leu Gln Val His Pro Asp Arg 20 25 30 Val Gly Glu Gly Asp Lys Glu Asp Ala Thr Arg Arg Phe Gln Ile Leu 35 40 45 Gly Lys Val Tyr Ser Val Leu Ser Asp Arg Glu Gln Arg Ala Val Tyr 50 55 60 Asp Glu Gln Gly 65 <210> 28 <211> 66 <212> PRT <213> Artificial <220> <223> DNAJC10 <400> 28 Asp Phe Tyr Ser Leu Leu Gly Val Ser Lys Thr Ala Ser Ser Arg Glu 1 5 10 15 Ile Arg Gln Ala Phe Lys Lys Leu Ala Leu Lys Leu His Pro Asp Lys 20 25 30 Asn Pro Asn Asn Pro Asn Ala His Gly Asp Phe Leu Lys Ile Asn Arg 35 40 45 Ala Tyr Glu Val Leu Lys Asp Glu Asp Leu Arg Lys Lys Tyr Asp Lys 50 55 60 Tyr Gly 65 <210> 29 <211> 69 <212> PRT <213> people <220> <223> DNAJC11 <400> 29 Asp Tyr Tyr Ser Leu Leu Asn Val Arg Arg Glu Ala Ser Ser Glu Glu 1 5 10 15 Leu Lys Ala Ala Tyr Arg Arg Leu Cys Met Leu Tyr His Pro Asp Lys 20 25 30 His Arg Asp Pro Glu Leu Lys Ser Gln Ala Glu Arg Leu Phe Asn Leu 35 40 45 His Gln Ala Tyr Glu Val Leu Ser Asp Pro Gln Thr Arg Ala Ile 50 55 60 Tyr Asp and Tyr Gly 65 <210> 30 <211> 66 <212> PRT <213> people <220> <223> DNAJC12 <400> 30 Asp Tyr Tyr Thr To Gly Cys Asp Glu To Ser Ser Val Glu Gln 1 5 10 15 Ile Leu Ala Glu Phe Lys Val Arg Ala Leu Glu Cys His Pro Asp Lys 20 25 30 His Pro Glu Asn Pro Lys Ala Val Glu Thr Phe Gln Lys Leu Gln Lys 35 40 45 Ala Lys Glu Ile Leu Thr Asn Glu Glu Ser Arg Ala Arg Tyr Asp His 50 55 60 Trp Arg 65 <210> 31 <211> 66 <212> PRT <213> people <220> <223> DNAJC13 <400> 31 Asp Ala Tyr Glu Val Leu Asn Leu Pro Gln Gly Gln Gly Pro His Asp 1 5 10 15 Glu Ser Lys Ile Arg Lys Ala Tyr Phe Arg Leu Ala Gln Lys Tyr His 20 25 30 Pro Asp Lys Asn Pro Glu Gly Arg Asp Met Phe Glu Lys Val Asn Lys 35 40 45 Ala Tyr Glu Phe Leu Cys Thr Lys Ser Ala Lys Ile Val Asp Gly Pro 50 55 60 Asp Pro 65 <210> 32 <211> 65 <212> PRT <213> people <220> <223> DNAJC14 <400> 32 Asn Pro Phe His Val Leu Gly Val Glu Ala Thr Ala Ser Asp Val Glu 1 5 10 15 Leu Lys Lys Ala Tyr Arg Gln Leu Ala Val Met Val His Pro Asp Lys 20 25 30 Asn His His Pro Arg Ala Glu Glu Ala Phe Lys Val Leu Arg Ala Ala 35 40 45 Trp Asp Ile Will Serve Asn Ala Glu Lys Arg Lys Glu Tyr Glu Met Lys 50 55 60 Arg 65 <210> 33 <211> 55 <212> PRT <213> people <220> <223> DNAJC15 <400> 33 Glu Ala Gly Leu Ile Leu Gly Val Ser Pro Ser Ala Gly Lys Ala Lys 1 5 10 15 His Arg Arg Val Met Ile Leu Asn His Pro Asp Lys 20 25 30 Gly Gly Ser Pro Tyr Val Ala Ala Lys Ile Asn Glu Ala Lys Asp Leu 35 40 45 Leu Glu Thr Thr Thr Lys His 50 55 <210> 34 <211> 65 <212> PRT <213> people <220> <223> DNAJC16 <400> 34 Asp Pro Tyr Arg Val Leu Gly Val Ser Arg Val Ser Gln Val Asp 1 5 10 15 Ile Lys Lys Ala Tyr Lys Leu Ala Arg Glu Trp His Pro Asp Lys 20 25 30 Asn Lys Asp Pro Gly Ala Glu Asp Lys Phe Ile Gln Ile Ser Lys Ala 35 40 45 Tyr Glu Ile Leu Ser Asn Glu Glu Lys Arg Ser Asn Tyr Asp Gln Tyr 50 55 60 Gly 65 <210> 35 <211> 66 <212> PRT <213> household <220> <223> DNAJC17 <400> 35 Asp Leu Tyr Ala Leu Gly And Glu Glu Lys Ala Ala Asp Lys Glu 1 5 10 15 Val Lys Lys Ala Tyr Arg Gln Lys Ala Leu Ser Cys His Pro Asp Lys 20 25 30 Asn Pro Asp Asn Pro Arg Ala Ala Glu Leu Phe His Gln Leu Ser Gln 35 40 45 Only Leu Glu Val Only Thr Asp Only Only Only Only Only Arg Only Only Tyr Asp Lys 50 55 60 Val Arg 65 <210> 36 <211> 65 <212> PRT <213> people <220> <223> DNAJC18 <400> 36 Asn Tyr Tyr Glu Ileu Gly Val Ser Arg Asp Ala Ser Asp Glu Glu 1 5 10 15 Leu Lys Ala Tyr Arg Lys Leu Ala Leu Lys Phe His Pro Asp Lys 20 25 30 Asn Cys Ala Pro Gly Ala Thr Asp Ala Phe Lys Ala Ile Gly Asn Ala 35 40 45 Phe Ala Val Leu Ser Asn Pro Asp Lys Arg Leu Arg Tyr Asp Glu Tyr 50 55 60 Gly 65 <210> 37 <211> 55 <212> PRT <213> people <220> <223> DNAJC19 <400> 37 Glu Ala Ala Leu Ile Leu Gly Val Ser Pro Thr Ala Asn Lys Gly Lys 1 5 10 15 With Arg Asp Only His Arg With His Pro Asp Lys 20 25 30 Gly Gly Ser Pro Tyr Ile Ala Lys Ile Asn Glu Ala Lys Asp Leu 35 40 45 Leu Glu Gly Gln Ala Lys Lys 50 55 <210> 38 <211> 72 <212> PRT <213> people <220> <223> DNAJC20 <400> 38 Asp Tyr Phe Ser Leu Met Asp Cys Asn Arg Ser Phe Arg Val Asp Thr 1 5 10 15 Only Lys Leu Gln His Arg Tyr Gln Gln Leu Gln Arg Leu Val His Pro 20 25 30 Asp Phe Ser Gln Arg Ser Gln Thr Glu Lys Asp Phe Ser Glu Lys 35 40 45 His Ser Thr Leu Val Asn Asp Ala Tyr Lys Thr Thr Leu Ala Pro Leu 50 55 60 Ser Arg Gly to Tyr to Lys 65 70 <210> 39 <211> 67 <212> PRT <213> people <220> <223> DNAJC21 <400> 39 Cys His Tyr Glu Ala Leu Gly Val Arg Arg Asp Ala Ser Glu Glu Glu 1 5 10 15 Leu Lys Lys With Tyr Arg Lys Leu Lys With Leu Lys Trp His Pro Asp Lys 20 25 30 Asn Leu Asp Asn Ala Ala Glu Ala Ala Glu Gln Phe Lys Leu Ile Gln 35 40 45 Ala Ala Tyr Asp Val Leu Ser Asp Pro Gln Glu Arg Ala Trp Tyr Asp 50 55 60 Asn His Arg 65 <210> 40 <211> 65 <212> PRT <213> Artificial <220> <223> DNAJC22 <400> 40 Leu Ala Tyr Gln Val Leu Gly Leu Ser Glu Gly Ala Thr Asn Glu Glu 1 5 10 15 Ile His Arg Ser Tyr Gln Glu Leu Val Lys Val Trp His Pro Asp His 20 25 30 Asn Leu Asp Gln Thr Glu Glu Ala Gln Arg His Phe Leu Glu Ile Gln 35 40 45 Ala Ala Tyr Glu Val Leu Ser Gln Pro Arg Lys Pro Trp Gly Ser Arg 50 55 60 Arg 65 <210> 41 <211> 62 <212> PRT <213> Artificial <220> <223> DNAJC23 <400> 41 Asn Pro Tyr Glu Val Leu Asn Leu Asp Pro Gly Ala Thr Val Ala Glu 1 5 10 15 Ile Lys Lys Gln Tyr Arg Leu Leu Ser Leu Lys Tyr His Pro Asp Lys 20 25 30 Gly Gly Asp Glu Val Met Phe Met Arg Ile Ala Lys Ala Tyr Ala Ala 35 40 45 Leu Thr Asp Glu Glu Ser Arg Lys Asn Trp Glu Glu Phe Gly 50 55 60 <210> 42 <211> 72 <212> PRT <213> Artificial <220> <223> DNAJC24 <400> 42 Asp Trp Tyr Ser Ile Leu Gly Ala Asp Pro Ser Ala Asn Ile Ser Asp 1 5 10 15 Leu Lys Gln Lys Tyr Gln Lys Leu Ile Leu Met Tyr His Pro Asp Lys 20 25 30 Gln Ser Thr Asp Val Pro Ala Gly Thr Val Glu Glu Cys Val Gln Lys<着 35 40 45 Phe Ile Glu Ile Asp Gln Ala Trp Lys Ile Leu Gly Asn Glu Glu Thr 50 55 60 Lys Arg Glu Tyr Asp Leu Gln Arg 65 70 <210> 43 <211> 76 <212> PRT <213> Artificial <220> <223> DNAJC25 <400> 43 Asp Cys Tyr Glu Val Leu Gly Val Ser Arg Ser Ala Gly Lys Ala Glu 1 5 10 15 Ile Ala Arg Ala Tyr Arg Gln Leu Ala Arg Arg Tyr His Pro Asp Arg 20 25 30 Tyr Arg Pro Gln Pro Gly Asp Glu Gly Pro Gly Arg Thr Pro Gln Ser 35 40 45 Ala Glu Glu Ala Phe Leu Leu Val Ala Thr Ala Tyr Glu Thr Leu Lys 50 55 60 Asp Glu Glu Thr Arg Lys Asp Tyr Asp Tyr Met Leu 65 70 75 <210> 44 <211> 65 <212> PRT <213> Artificial <220> <223> DNAJC26 <400> 44 Ser Arg Trp Thr Pro Val Gly Met Ala Asp Leu Val Ala Pro Glu Gln 1 5 10 15 Val Lys Lys His Tyr Arg Arg Ala Val Leu Ala Val His Pro Asp Lys 20 25 30 Ala Ala Gly Gln Pro Tyr Glu Gln His Ala Lys Met Ile Phe Met Glu 35 40 45 Leu Asn Asp Ala Trp Ser Glu Phe Glu Asn Gln Gly Ser Arg Pro Leu 50 55 60 Phe 65 <210> 45 <211> 57 <212> PRT <213> Artificial <220> <223> DNAJC27 <400> 45 Asp Ser Trp Asp Met Leu Gly Val Lys Pro Gly Ala Ser Arg Asp Glu 1 5 10 15 Val Asn Lys Ala Tyr Arg Lys Leu Ala Val Leu Leu His Pro Asp Lys 20 25 30 Cys Val Ala Pro Gly Ser Glu Asp Ala Phe Lys Ala Val Val Asn Ala 35 40 45 Arg Thr Ala Leu Leu Lys Asn Ile Lys 50 55 <210> 46 <211> 65 <212> PRT <213> Artificial <220> <223> DNAJC28 <400> 46 Glu Tyr Tyr Arg Leu Leu Asn Val Glu Glu Gly Cys Ser Ala Asp Glu 1 5 10 15 Val Arg Glu Ser Phe His Lys Leu Ala Lys Gln Tyr His Pro Asp Ser 20 25 30 Gly Ser Asn Thr Ala Asp Ser Ala Thr Phe Ile Arg Ile Glu Lys Ala 35 40 45 Tyr Arg Lys Val Leu Ser His Val Ile Glu Gln Thr Asn Ala Ser Gln 50 55 60 Looking 65 <210> 47 <211> 88 <212> PRT <213> Artificial <220> <223> DNAJC29 <400> 47 Ile Leu Lys Glu Val Thr Ser Val Val Glu Gln Ala Trp Lys Leu Pro 1 5 10 15 Glu Ser Glu Arg Lys Lys Ile Ile Arg Arg Leu Tyr Leu Lys Trp His 20 25 30 Pro Asp Lys Asn Pro Glu Asn His Asp Ile Ala Asn Glu Val Phe Lys 35 40 45 His Leu Gln Asn Glu Ile Asn Arg Leu Glu Lys Gln Ala Phe Leu Asp 50 55 60 Gln Asn Ala Asp Arg Ala Ser Arg Arg Thr Phe Ser Thr Ser Ala Ser 65 70 75 80 Arg Phe Gln Ser Asp Lys Tyr Ser 85 <210> 48 <211> 66 <212> PRT <213> artificially <220> <223> DNAJC30 <400> 48 Ala Leu Tyr Asp Leu Leu Gly Val Pro Ser Thr Ala Thr Gln Ala Gln 1 5 10 15 Ile Lys Ala Ala Tyr Tyr Arg Gln Cys Phe Leu Tyr His Pro Asp Arg 20 25 30 Asn Ser Gly Ser Ala Glu Ala Ala Glu Arg Phe Thr Arg Ile Ser Gln 35 40 45 Ala Tyr Val Val Leu Gly Ser Ala Thr Leu Arg Arg Lys Tyr Asp Arg 50 55 60 Gly Leu 65 <210> 49 <211> 64 <212> PRT <213> artificially <220> <223> SV40 J structure <400> 49 Gln Leu Met Asp Leu Leu Gly Leu Glu Arg Ser Ala Trp Gly Asn Ile 1 5 10 15 Pro Leu Met Arg Lys Ala Tyr Leu Lys Lys Cys Lys Glu Phe His Pro 20 25 30 Asp Lys Gly Gly Asp Glu Glu Lys Met Lys Lys Met Asn Thr Leu Tyr 35 40 45 Lys Lys Met Glu Asp Gly Val Lys Tyr Ala His Gln Pro Asp Phe Gly 50 55 60 <210> 50 <211> 70 <212> PRT <213> Artificial <220> <223> Bacterial J - domain <400> 50 Lys Gln Asp Tyr Tyr Glu Ile Leu Gly Val Ser Lys Thr Ala Glu Glu 1 5 10 15 Arg Glu Ile Arg Lys Ala Tyr Lys Arg Leu Ala Met Lys Tyr His Pro 20 25 30 Asp Arg Asn Gln Gly Asp Lys Glu Ala Glu Ala Lys Phe Lys Glu Ile 35 40 45 Lys Glu Ala Tyr Glu Val Leu Thr Asp Ser Gln Lys Arg Ala Ala Tyr 50 55 60 Asp Gln Tyr Gly His Ala 65 70 <210> 51 <211> 10 <212> PRT <213> Artificial <220> <223> SynBP1 <400> 51 Lys Asp Gly Ile Val Asn Gly Val Lys Ala 1 5 10 <210> 52 <211> 7 <212> PRT <213> artificial <220> <223> SynBP2 <400> 52 Trp Arg Gln Thr Arg Lys Asp 1 5 <210> 53 <211> 7 <212> PRT <213> artificial <220> <223> SynBP3 <400> 53 His Tyr Ala Lys Asn Pro Ile 1 5 <210> 54 <211> 7 <212> PRT <213> artificial <220> <223> SynBP4 <400> 54 Ala Thr Ile Asn Lys Ser Leu 1 5 <210> 55 <211> 7 <212> PRT <213> artificial <220> <223> SynBP5 <400> 55 Arg Arg Arg Gly Met Ala Ile 1 5 <210> 56 <211> 7 <212> PRT <213> artificial <220> <223> SynBP6 <400> 56 Thr Lys His Gly Pro Arg Lys 1 5 <210> 57 <211> 7 <212> PRT <213> artificial <220> <223> SynBP7 <400> 57 Ser Leu Lys Arg Leu Pro Lys 1 5 <210> 58 <211> 7 <212> PRT <213> artificial <220> <223> SynBP8 <400> 58 Arg Leu Arg Gly Arg Asn Gln 1 5 <210> 59 <211> 7 <212> PRT <213> artificial <220> <223> SynBP9 <400> 59 Trp Pro Phe His His His Arg 1 5 <210> 60 <211> 7 <212> PRT <213> artificial <220> <223> SynBP10 <400> 60 His Leu Tyr His His Lys Thr 1 5 <210> 61 <211> 7 <212> PRT <213> artificial <220> <223> SynBP11 <400> 61 Thr His Ile His His Pro Ser 1 5 <210> 62 <211> 7 <212> PRT <213> Artificial <220> <223> SynBP12 <400> 62 Met Met Met Met Met Arg Leu 1 5 <210> 63 <211> 248 <212> PRT <213> Artificial <220> <223> NAC32 <400> 63 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Phe Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Ala Ser Val Lys Ser Arg Ile Thr Ile Asp Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Thr Arg Gln Asn Leu Ala Gly Pro Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ile Leu Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val 130 135 140 Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 145 150 155 160 Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn Tyr Leu Ala 165 170 175 Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Arg Leu Leu Ile Ser Gly 180 185 190 Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp 210 215 220 Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Thr Ala Phe Gly 225 230 235 240 Pro Gly Thr Lys Val Asp Ile Lys 245 <210> 64 <211> 247 <212> PRT <213> Artificial <220> <223> Syn_scFv <400> 64 Leu Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly 1 5 10 15 Gln Arg Val Ala Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Thr 20 25 30 Gly Tyr Asp Val Asn Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Arg 35 40 45 Leu Leu Ile Tyr Gly Asn Thr Tyr Arg Pro Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Ala Ser Thr Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Val 65 70 75 80 Ile Thr Asp Leu Gln Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser 85 90 95 Phe Asp Asn Ser Leu Arg Gly Ser Val Phe Gly Gly Gly Thr Lys Val 100 105 110 Thr Val Leu Gly Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys 115 120 125 Ala Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Lys Pro 130 135 140 Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ile Leu Ser 145 150 155 160 Asp Tyr Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175 Trp Leu Ala Val Ile Asp Ile Thr Ser Ser Tyr Thr Asn Tyr Ala Asp 180 185 190 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 195 200 205 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 210 215 220 Tyr Cys Ala Arg Leu Glu Ser Gly Phe Phe Asp Tyr Trp Gly Gln Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser 245 <210> 65 <211> 11 <212> PRT <213> Artificial <220> <223> QBP1 <400> 65 Ser Asn Trp Lys Trp Trp Pro Gly Ile Phe Asp 1 5 10 <210> 66 <400> 66 000 <210> 67 <211> 4 <212> PRT <213> artificial <220> <223> GTGS <400> 67 Gly Thr Gly Ser 1 <210> 68 <211> 5 <212> PRT <213> artificial <220> <223> GLESR <400> 68 Gly Leu Glu Ser Arg 1 5 <210> 69 <211> 4 <212> PRT <213> artificial <220> <223> GGSG <400> 69 Gly Gly Ser Gly 1 <210> 70 <211> 5 <212> PRT <213> artificial <220> <223> GGGGS <400> 70 Gly Gly Gly Gly Ser 1 5 <210> 71 <211> 5 <212> PRT <213> artificial <220> <223> DIAAA <400> 71 Asp Ile Ala Ala Ala 1 5 <210> 72 <211> 5 <212> PRT <213> Artificial <220> <223> EAAAK <400> 72 Glu Ala Ala Ala Lys 1 5 <210> 73 <211> 15 <212> PRT <213> Artificial <220> <223> GGGGSGGGGSGGGGS <400> 73 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 74 <211> 11 <212> PRT <213> Artificial <220> <223> AEAAAKEAAAK <400> 74 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 <210> 75 <211> 15 <212> PRT <213> Artificial <220> <223> SGGGSGGGGSGGGGS <400> 75 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 76 <211> 25 <212> PRT <213> Artificial <220> <223> DIGGGGSGGGGSGGGGSGGGGSAAA <400> 76 Asp Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Ala Ala Ala 20 25 <210> 77 <211> 232 <212> PRT <213> Artificial <220> <223> Human IgG1 Fc domain <400> 77 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 78 <211> 8 <212> PRT <213> Artificial <220> <223> FLAG tabletop <400> 78 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 79 <211> 6 <212> PRT <213> artificial <220> <223> His6 <400> 79 His His His His His His 1 5 <210> 80 <211> 10 <212> PRT <213> artificial <220> <223> c-myc <400> 80 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 81 <211> 9 <212> PRT <213> artificial <220> <223> HA <400> 81 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 82 <211> 14 <212> PRT <213> artificial <220> <223> V5 Tablet <400> 82 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 83 <211> 220 <212> PRT <213> Artificial <220> <223> Glutathione-S-transferase <400> 83 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser [[ID=3​​​​​​​​ Gly Asp His Val Thr His Pro Asp Phe Met Leu Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe was synthesized by Arg and Glu synthesized by Pro Gln and Asp synthesized by Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp 210 215 220 <210> job <211> 9 <212> PRT <213> people <220> <223> RKKRRQRRR <400> job Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 <210> 85 <211> 15 <212> PRT <213> people <220> <223> RQIKWFQNRRMKWKK <400> 85 Arg Gln With Lys Trp Phe Gln Asn Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 86 <211> 21 <212> PRT <213> Artificial <220> <223> KETWWETWWTEWSQPKKKRKV <400> 86 Light Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Light 1 5 10 15 Light Light Arg Light Val 20 <210> 87 <211> 17 <212> PRT <213> Artificial <220> <223> CISPPEVKFNKPFVYLI <400> 87 Cys Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Tyr Leu 1 5 10 15 Ile <210> 88 <211> 110 <212> PRT <213> Artificial <220> <223> JB1‑SynBP1 <400> 88 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Lys Asp Gly Ile Val Asn Gly Val Lys Ala 100 105 110 <210> 89 <211> 110 <212> PRT <213> Artificial <220> <223> J(P33Q)-SynBP1 <400> 89 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 1​​​​​​​​​​​​​​ 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Lys Asp Gly Ile Val Asn Gly Val Lys Ala 100 105 110 <210> 90 <211> 120 <212> PRT <213> Artificial <220> <223> JB1-2XSynBP1 <400> 90 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Lys Asp Gly Ile Val Asn Gly Val Lys Ala Lys Asp 100 105 110 Gly Ile Val Asn Gly Val Lys Ala 115 120 <210> 91 <211> 123 <212> PRT <213> Artificial <220> <223> SynBP1‑JB1‑SynBP1 <400> 91 Met Lys Asp Gly Ile Val Asn Gly Val Lys Ala Glu Phe Met Gly Lys 1 5 10 15 Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser Asp Glu Glu 20 25 30 Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His Pro Asp Lys 35 40 45 Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile Ala Glu Ala 50 55 60 Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe Asp Arg Tyr 65 70 75 80 Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly Gly Ser Gly 85 90 95 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala 100 105 110 Ala Lys Asp Gly Ile Val Asn Gly Val Lys Ala 115 120 <210> 92 <211> 90 <212> PRT <213> Artificial <220> <223> SynBP2‑JB1 <400> 92 Met Trp Arg Gln Thr Arg Lys Asp Glu Phe Met Gly Lys Asp Tyr Tyr 1 5 10 15 Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser Asp Glu Glu Ile Lys Arg 20 25 30 Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His Pro Asp Lys Asn Lys Glu 35 40 45 Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile Ala Glu Ala Tyr Asp Val 50 55 60 Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe Asp Arg Tyr Gly Glu Glu 65 70 75 80 Gly Leu Lys Gly Ser Asp Ile Ala Ala Ala 85 90 <210> 93 <211> 111 <212> PRT <213> Artificial <220> <223> JB6‑SynBP1 <400> 93 Met Val Asp Tyr Tyr Glu Val Leu Gly Val Gln Arg His Ala Ser Pro 1 5 10 15 Glu Asp Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Trp His Pro 20 25 30 Asp Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys Phe Lys Gln 35 40 45 Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Ala Lys Lys Arg Asp Ile 50 55 60 Tyr Asp Lys Tyr Gly Lys Glu Gly Leu Asn Gly Gly Asp Ile Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 85 90 95 Gly Ser Ala Ala Ala Lys Asp Gly Ile Val Asn Gly Val Lys Ala 100 105 110 <210> 94 <211> 110 <212> PRT <213> Artificial <220> <223> JB13-SynBP1 <400> 94 Met Gly Gln Asp Tyr Tyr Ser Val Leu Gly Ile Thr Arg Asn Ser Glu 1 5 10 15 Asp Ala Gln Ile Lys Gln Ala Tyr Arg Arg Leu Ala Leu Lys His His 20 25 30 Pro Leu Lys Ser Asn Glu Pro Ser Ser Ala Glu Ile Phe Arg Gln Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Met Lys Arg Gly Ile Tyr 50 55 60 Asp Lys Phe Gly Glu Glu Gly Leu Lys Gly Gly Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Lys Asp Gly Ile Val Asn Gly Val Lys Ala 100 105 110 <210> 95 <211> 348 <212> PRT <213> Artificial <220> <223> JB1-NAC32 <400> 95 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val 100 105 110 Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser 115 120 125 Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser 130 135 140 Arg Gly Leu Glu Trp Leu Gly Arg Thr Phe Tyr Arg Ser Lys Trp Tyr 145 150 155 160 Asn Asp Tyr Ala Ala Ser Val Lys Ser Arg Ile Thr Ile Asp Pro Asp 165 170 175 Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu 180 185 190 Asp Thr Ala Val Tyr Tyr Cys Thr Arg Gln Asn Leu Ala Gly Pro Phe 195 200 205 Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ile Leu 210 215 220 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 245 250 255 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 260 265 270 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Arg Leu 275 280 285 Leu Ile Ser Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe 290 295 300 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 305 310 315 320 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser 325 330 335 Thr Ala Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 340 345 [[ID=​​​​​​​​​<223> JB1‑Syn_scFv <400> 96 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Leu Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser 100 105 110 Gly Ala Pro Gly Gln Arg Val Ala Ile Ser Cys Thr Gly Thr Ser Ser 115 120 125 Asp Ile Gly Thr Gly Tyr Asp Val Asn Trp Tyr Gln His Leu Pro Gly 130 135 140 Thr Ala Pro Arg Leu Leu Ile Tyr Gly Asn Thr Tyr Arg Pro Ser Gly 145 150 155 160 Val Pro Asp Arg Phe Ser Ala Ser Thr Gly Ser Lys Ser Gly Thr Ser 165 170 175 Ala Ser Leu Val Ile Thr Asp Leu Gln Ala Glu Asp Glu Gly Asp Tyr 180 185 190 Tyr Cys Gln Ser Phe Asp Asn Ser Leu Arg Gly Ser Val Phe Gly Gly 195 200 205 Gly Thr Lys Val Thr Val Leu Gly Glu Gly Lys Ser Ser Gly Ser Gly 210 215 220 Ser Glu Ser Lys Ala Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly 225 230 235 240 Val Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly 245 250 255 Phe Ile Leu Ser Asp Tyr Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly 260 265 270 Lys Gly Leu Glu Trp Leu Ala Val Ile Asp Ile Thr Ser Ser Tyr Thr 275 280 285 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 290 295 300 Ala Lys Asn Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 305 310 315 320 Thr Ala Val Tyr Tyr Cys Ala Arg Leu Glu Ser Gly Phe Phe Asp Tyr 325 330 335 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 340 345 <210> 97 <211> 111 <212> PRT <213> Artificial <220> <223> JB1-QBP1 <400> 97 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Asp Ile Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Ala Ala Ala Ser Asn Trp Lys Trp Trp Pro Gly Ile Phe Asp 100 105 110 <210> 98 <211> 90 <212> PRT <213> Artificial <220> <223> JB1GGGS_SynBP1 <400> 98 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Lys Asp Gly Ile Val Asn Gly Val Lys Ala 85 90 <210> 99 <211> 90 <212> PRT <213> Artificial <220> <223> JB1EAAAK‑SynBP1 <400> 99 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Glu Ala Ala Ala Lys 65 70 75 80 Lys Asp Gly Ile Val Asn Gly Val Lys Ala 85 90 <210> 100 <211> 85 <212> PRT <213> Artificial <220> <223> JB1SynBP1 <400> 100 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Lys Asp Gly Ile Val 65 70 75 80 Asn Gly Val Lys Ala 85 <210> 101 <211> 91 <212> PRT <213> Artificial <220> <223> JB1GGGGS-QBP1 <400> 101 Met Gly Lys Asp Tyr Tyr Gln Thr Leu Gly Leu Ala Arg Gly Ala Ser 1 5 10 15 Asp Glu Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His 20 25 30 Pro Asp Lys Asn Lys Glu Pro Gly Ala Glu Glu Lys Phe Lys Glu Ile 35 40 45 Ala Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe 50 55 60 Asp Arg Tyr Gly Glu Glu Gly Leu Lys Gly Ser Gly Gly Gly Gly Ser 65 70 75 80 Ser Asn Trp Lys Trp Trp Pro Gly Ile Phe Asp 85 90 <210> 102 <211> 17 <212> PRT <213> artificial <220> <223> MGVKVLFALICIAVAEA <400> 102 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala <210> 103 <211> 19 <212> PRT <213> artificial <220> <223> MAPVQLLGLLVLFLPAMRC <400> 103 Met Ala Pro Val Gln Leu Leu Gly Leu Leu Val Leu Phe Leu Pro Ala 1 5 10 15 Met Arg Cys <210> 104 <211> 19 <212> PRT <213> artificial <220> <223> MAVLGLLFCLVTFPSCVLS <400> 104 Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser <210> 105 <211> 140 <212> PRT <213> artificial <220> <223> α-synuclein <400> 105 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135 140 <210> 106 <211> 140 <212> PRT <213> Artificial <220> <223> α-Synuclein (A53T) <400> 106 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Thr Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135 140

Claims

1. An isolated fusion protein selected from SEQ ID NO: 88, 93, 95-100.

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

88.

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

98.

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

99.

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

100.

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 and 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. A viral particle comprising a capsid and a vector of claim 11 or 12.

14. The viral particle of claim 13, wherein the vector is an adeno-associated virus and the capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAVrh10.

15. The viral particle of claim 14, wherein the capsid is selected from AAV2, AAV5, AAV8, AAV9 and AAVrh10.

16. The viral particle of claim 15, wherein the capsid is AAV9.

17. The virus particle of claim 15, wherein the capsid is AAV rh10.

18. 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 any one of claims 1-5, a nucleic acid of any one of claims 6-10, a vector of claim 11 or 12, and a viral particle of any one of claims 13-17.

19. Use of one or more of the following in the preparation of a medicament for reducing α-synuclein aggregation in cells of subjects diagnosed with amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, or multiple system atrophy: a fusion protein of any one of claims 1-5, cells expressing a fusion protein of any one of claims 1-5, a nucleic acid of any one of claims 6-10, a vector of claim 11 or 12, viral particles of any one of claims 13-17, and a pharmaceutical composition of claim 18.

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