Compositions and methods for the treatment of synucleinopathy

Abstract

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

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 035,297, filed Jun. 5, 2020. The entire contents of the foregoing application are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application incorporates by reference in its entirety a Sequence Listing (68 KB) named "269548 - 493385_ST25.txt", created on Jun. 4, 2021 at 1:09 PM and electronically filed together with this specification.

[0003] Technical Field New fusion proteins comprising one or more of a J domain, an α - synuclein - binding domain, a linker, a targeting reagent, an epitope, a cell - penetrating agent, and combinations thereof are described and claimed. Additionally, the present disclosure describes the use of these novel fusion proteins and claims the recruitment by them of native chaperone mechanisms, such as those of the b, Hsp70 - mediated system, to specifically reduce α - synuclein - mediated protein aggregation and related proteopathies.

Background Art

[0004] All proteins expressed within a cell need to fold correctly into their intended structure in order to function properly. A growing number of diseases and disorders have been shown to be associated with improper protein folding and / or improper deposition and aggregation of proteins and lipoproteins, as well as infectious proteinogenic substances. Examples of diseases caused by misfolding, also known as conformational diseases or proteopathies, include Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTLD). Mutant proteins aggregate in cells, resulting in typical cytotoxic cellular inclusions.

[0005] Various neurodegenerative diseases are pathologically characterized by the accumulation of intracellular or extracellular protein aggregates composed of amyloid fibrils (Forman et al., (2004) Nat Med. 10:1055~1063). For example, the pathogenesis of Alzheimer's disease (AD) is defined by senile plaques and neurofibrillary condensations, which are composed of β-amyloid and microtubule-associated protein tau, respectively.

[0006] Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD). More than 1% of people over 60 years of age have the disease, and there are over one million patients in the United States alone. Approximately 40% of PD patients experience fluctuating manifestations of bradykinesia, rigidity, tremors, balance problems, and dementia, and some patients develop AD-like dementia later in their illness. The main pathological features of Parkinson's disease (PD) are the loss of dopaminergic neurons in the substantia nigra and the presence of abnormal protein aggregates that form fibrous inclusions in the neuronal cytoplasm, called Lewy bodies (LBs) or Lewy neurites (nerve fibers) (LNs) in the PD brain (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). The majority of PD cases are sporadic. Familial forms of Parkinson's disease (PD) account for approximately 10% of all cases and can be either autosomal recessive or autosomal dominant, suggesting a complex cause of the disorder (as outlined by Lang and Lozano) (Lang and Lozano, (1998) N Engl J Med 339, 1044-1053; Lang and Lozano, (1998) N Engl J Med 339, 1130-1143). In addition, environmental factors also play an important role in the development of PD (as outlined by Di Monte, 2003) (Di Monte et al., (2003) Lancet Neurol 2, 531-538).

[0007] The discovery of genetic linkage for PD to several gene loci offers the prospect of identifying genetic factors contributing to the development of the disease. A study of a patient with a rare dominant variant of PD in a family originating from southern Italy linked the disorder to a gene 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 of alpha-synuclein involvement in familial PD was provided by the identification of a missense mutation in the gene resulting 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 at position 30 of the alpha-synuclein gene, changing from alanine to proline, was identified in a German family (Kruger et al., (1998) Nat Genet 18, 106-108). Immunohistochemical analysis of LB showed that LB contains many different proteins. The most frequently and consistently described proteins are nerve filaments, 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 primarily found in Lewy bodies in the brain of Parkinson's disease (PD), as well as in dystrophic Lewy neurites (LNs) (Dickson, (2012) Cold Spring Harb Perspect Med 2; Stefanis, (2012) Cold Spring Harb Perspect Med 2, a009399; Lashuel et al., (2013) Nat Rev Neurosci 14, 38-48), suggesting that α-synuclein misfolding is a major culprit in the development of neurodegenerative diseases associated with aggregated α-synuclein accumulation (Baba et al., (1998) Am J Pathol 152, 879-884; Kalia et al., (2013) Ann Neurol 73, 155-169).

[0008] The α-synuclein gene encodes a 140-amino acid protein of approximately 19 kDa. While the function of α-synuclein is not yet 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 in the mammalian brain, but is concentrated in presynaptic nerve terminals and associated with membrane and vesicular structures. Mice possess a functional homolog of α-synuclein. Homozygous knockout of the mouse α-synuclein gene is viable, reproductive, and nearly normal (Abeliovich et al., (2000) Neuron 25, 239-252). α-synuclein- / - mice exhibit increased DA release in double-stimulation, decreased striatal DA, and attenuation of the DA-dependent gait response to amphetamine, suggesting that α-synuclein is a regulator of DA neurotransmission. Mice with α-synuclein overexpression appeared normal, but showed significant degeneration of DA nerve terminals in mouse strains with high human α-synuclein expression, although no loss of DA neurons was observed (Masliah and Rockenstein, (2000) J Neural Transm Suppl 59, 175-183). Overexpression of the A53T α-synuclein mutant induced substantial 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 wild-type and mutant α-synuclein mediated by adeno-associated virus (AAV) suggests that the PD model can be used to test PD treatments (Kirik et al. (2002) Neuron 35, 219-222).

[0009] Currently, there is no prevention or cure for Parkinson's disease (PD), only symptomatic treatments such as drug therapy and surgical treatment. Degeneration of dopaminergic substantia nigra neurons leads to a loss of dopaminergic projections to the striatum, which represents a primary defect in the neurochemical pathway in PD. Therefore, one treatment for PD is dopamine (DA) replacement, in which case the patient takes the drug levodopa. Levodopa is converted to dopamine by aromatic L-amino acid decarboxylase (AADC). However, its benefits are temporary due to numerous side effects (outlined by Nutt 2003) (Nutt, (2003) Exp Neurol 184, 9-13). In addition, if the disease progresses and as a result the patient may experience increased off-time symptoms such as stiffness, rigidity, and muscle spasms, it is possible to reduce the amount of AADC used. Therefore, there is a substantial need for the development of new treatments for this devastating disease.

[0010] Heat shock 70kDa proteins (referred to herein as "Hsp70s") constitute a ubiquitous class of chaperone proteins in cells of various species (Tavaria et al., (1996) Cell Stress Chaperones 1, 23-28). Hsp70s require assistant proteins called co-chaperone proteins, such as J-domain proteins and nucleotide exchange factors (NEFs), to function (Hartl et al., (2009) Nat Struct Mol Biol 16, 574-581). In the latest model of the Hsp70 chaperone mechanism for protein folding, Hsp70 cycles between ATP-bound and ADP-bound states, and the J-domain protein interacts with the ATP-bound form of Hsp70 (Hsp70-ATP) by binding to another protein (called a "client protein") that requires folding or refolding (Young (2010) Biochem Cell Biol 88, 291-300; Mayer, (2010) Mol Cell 39, 321-331). The binding of the J-domain protein-client protein complex to Hsp70-ATP stimulates ATP hydrolysis, which causes a conformational change in the Hsp70 protein, closing the helix lid, thereby stabilizing the interaction between the client protein and Hsp70-ADP, and also inducing the release of the J-domain protein, which can then freely bind to another client protein.

[0011] Therefore, this model suggests that J-domain proteins play a crucial role in the Hsp70 mechanism by acting as crosslinks and facilitating the capture of various client proteins and their submission to the Hsp70 mechanism, thereby promoting folding or refolding into the appropriate three-dimensional structure (Kampinga & Craig (2010) Nat Rev Mol Cell Biol 11, 579-592). The J-domain family is widely conserved across species ranging from prokaryotes (DnaJ proteins) to eukaryotes (Hsp40 protein family). The J-domain (approximately 60-80aa) consists of four helices: I, II, III, and IV. Helices II and III are connected via a mobile loop containing an "HPD motif," which is highly conserved across the J-domain and is thought to be important for activity (Tsai & Douglas, (1996) J Biol Chem 271, 9347-9354). Mutations within the HPD sequence have been found to cause loss of J-domain function.

[0012] Given the background information provided above regarding proteopathies such as Parkinson's disease (PD), it seems clear that reducing levels of misfolded proteins could be a useful means of treating, preventing, or otherwise improving the symptoms of these devastating disorders, and that mobilizing the cells' innate ability to repair protein misfolding would be a natural choice to pursue. [Overview of the project]

[0013] The inventors have developed a novel class of fusion proteins that mobilize the cell's innate chaperone mechanism, specifically the Hsp70-mediated system, to specifically reduce α-synuclein-mediated protein misfolding. Unlike previous studies by the inventors that used fusion proteins containing a fragment of the Hsp40 protein (also known as the J protein), a co-chaperone that interacts with Hsp70, to enhance protein secretion and expression, this study uses a J-domain-containing fusion protein with the aim of reducing protein misfolding and cytotoxicity caused by the α-synuclein protein. In this regard, the inventors have made the surprising discovery that the J-domain elements required for function are entirely different from the use of the J-domain in enhancing protein expression and secretion, demonstrating a distinct mechanism for the mode of action of this fusion protein. The fusion proteins described herein contain a J-domain and a domain having affinity for α-synuclein. The presence of the α-synuclein-binding domain within the fusion protein results in a specific reduction of α-synuclein protein misfolding.

[0014] E1. Accordingly, in a first embodiment, an isolated fusion protein comprising the J domain and the α-synuclein-binding domain of the J protein is disclosed herein. E2. A fusion protein described in E1, in which the J domain of the J protein is of eukaryotic origin. E3. A fusion protein described in any one of E1-E2, in which the J domain of the J protein is of human origin. E4. A fusion protein described in any one of E1-E3, in which the J domain of the J protein is localized in the cytoplasm. E5. A fusion protein described in any one of E1 to E4, wherein the J domain of the J protein is selected from the group consisting of Sequence IDs 1 to 50. E6. A fusion protein described in any one of E1 to E5, wherein the J domain contains a sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49. E7. A fusion protein described in any one of E1-E6, wherein the J domain contains the sequence of Sequence ID No. 5. E8. A fusion protein described in any one of E1-E6, wherein the J domain contains the sequence of SEQ ID NO: 10. E9. A fusion protein described in any one of E1 to E6, wherein the J domain contains the sequence of Sequence ID No. 16. E10. A fusion protein described in any one of E1 to E6, wherein the J domain contains the sequence of SEQ ID NO: 25. E11. A fusion protein described in any one of E1 to E6, wherein the J domain contains the sequence of Sequence ID No. 31. E12. When the α-synuclein-binding domain is measured using an ELISA assay, the K2 concentration relative to α-synuclein is 1 μM or less, for example, 300 nM or less, 100 nM or less, 30 nM or less, or 10 nM or less. D A fusion protein having any one of the following characteristics: E1 to E11. E13. A fusion protein described in any one of E1 to E12, wherein the α-synuclein-binding domain contains a sequence selected from the group consisting of SEQ ID NOs. 51 to 65. E14. A fusion protein described in any one of E1 to E13, wherein the α-synuclein-binding domain contains the sequence of SEQ ID NO: 51. E15. A fusion protein described in any one of E1 to E13, wherein the α-synuclein-binding domain contains the sequence of SEQ ID NO: 52. E16. A fusion protein described in any one of E1 to E13, wherein the α-synuclein-binding domain contains the sequence of SEQ ID NO: 63. E17. A fusion protein described in any one of E1 to E13, wherein the α-synuclein-binding domain contains the sequence of SEQ ID NO: 64. E18. A fusion protein described in any one of E1 to E17, containing multiple α-synuclein-binding domains. E19. A fusion protein consisting of two α-synuclein-binding domains, as described in any one of E1 to E18. E20. A fusion protein according to any one of E1 to E19, comprising three α-synuclein binding domains. E21. The following constructs: a. DNAJ-X-S, b. DNAJ-X-S-X-S, c. DNAJ-X-S-X-S-X-S, d. S-X-DNAJ, e. S-X-S-X-DNAJ, f. S-X-S-X-S-X-DNAJ, g. S-X-DNAJ-X-S, h. S-X-DNAJ-X-S-X-S, i. S-X-S-X-DNAJ-X-S-X-S-X-S, j. S-X-S-X-S-X-DNAJ-X-S, k. S-X-S-X-S-X-DNAJ-X-S-X-S, l. S-X-S-X-S-X-DNAJ-X-S-X-S-X-S, m. DnaJ-X-DnaJ-X-S-X-S, n. S-X-DnaJ-X-DnaJ, and o. S-X-S-X-DnaJ-X-DnaJ, containing one of the above, where S is an α-synuclein binding domain, DNAJ is the J domain of the J protein, X is an optional linker, a fusion protein according to any one of E1 to E20. E22. A fusion protein according to any one of E1 to E21, comprising the J domain sequence of SEQ ID NO: 5 and the α-synuclein binding domain sequence of SEQ ID NO: 51. E23. A fusion protein according to any one of E1 to E22, comprising two copies of the J domain sequence of SEQ ID NO: 5 and the α-synuclein binding domain sequence of SEQ ID NO: 51. E24. A fusion protein described in any one of E1 to E23, containing a sequence selected from the group consisting of sequence numbers 88, 90-96, and 98-100. E25. A fusion protein described in any one of E1 to E24, containing the sequence of Sequence ID No. 88. E26. A fusion protein described in any one of E1 to E24, containing the sequence of SEQ ID NO: 90. E27. A fusion protein described in any one of E1 to E24, containing the sequence of sequence number 99. E28. A fusion protein described in any one of E1 to E24, containing the sequence of Sequence ID No. 100. E29. A fusion protein described in any one of E1 to E28, further comprising a targeting reagent. E30. A fusion protein described in any one of E1-E29, further containing an epitope. E31. A fusion protein as described in E30, wherein the epitope is a polypeptide selected from the group consisting of SEQ ID NOs. 77-83. E32. A fusion protein described in any one of E1 to E31, further comprising a cell permeabilizing agent. E33. A fusion protein as described in E32, wherein the cell permeabilizer contains a peptide sequence selected from the group consisting of SEQ ID NOs. 84-87. E34. A fusion protein described in any one of E1-E33, further comprising a signal sequence. E35. A fusion protein as described in E34, wherein the signal sequence contains a peptide sequence selected from the group consisting of SEQ ID NOs. 102-104. E36. A fusion protein described in any one of E1-E35 that has the ability to reduce α-synuclein protein misfolding in cells. E37. A fusion protein described in any one of E1-E36 that has the ability to reduce phosphorylated α-synuclein protein in cells. E38. A fusion protein described in any one of E1-E37 that has the ability to reduce the secretion of α-synuclein protein. E39. A fusion protein described in any one of E1-E38 that has the ability to reduce α-synuclein-mediated cytotoxicity. E40. A nucleic acid sequence encoding a fusion protein described in any one of E1 to E39. E41. The nucleic acid sequence described in E40, wherein the nucleic acid is DNA. E42. The nucleic acid sequence described in E41, wherein the nucleic acid is RNA. E43. A nucleic acid sequence according to any one of E40 to E42, wherein the nucleic acid comprises at least one modified nucleic acid. E44. A nucleic acid sequence described in any one of E40-E43, further comprising a promoter region, a 5'UTR, and a 3'UTR such as a poly(A) signal. E45. The nucleic acid sequence described in E44, wherein the promoter region comprises a sequence selected from the group consisting of a CMV enhancer sequence, a CMV promoter, a CBA promoter, a UBC promoter, a GUSB promoter, a NSE promoter, a synapsin promoter, a MeCP2 promoter, and a GFAP promoter. E46. A vector containing one of the nucleic acid sequences listed in E40 to E45. E47. A vector as described in E46, selected from the group consisting of adeno-associated viruses (AAV), adenoviruses, lentiviruses, retroviruses, herpesviruses, poxviruses (vaccinia or myxoma), paramyxoviruses (measles, RSV, or Newcastle disease virus), baculoviruses, reoviruses, alphaviruses, and flaviviruses. E48. Viral particles containing a capsid and the vector described in E46 or E47. E49. A viral particle as described in E48, wherein the capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, pseudotyped AAV, rhesus monkey-derived AAV, AAVrh8, AAVrh10, and AAV-DJan AAV capsid variants, AAV hybrid serotypes, organotropic AAV, cardiacotropic AAV, and cardiacotropic AAVM41 variants. E50. A viral particle as described in E48 or E49, wherein the capsid is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, and AAVrh10. E51. A viral particle listed in any one of E48-E50, whose capsid is AAV2. E52. A viral particle listed in any one of E48-E50, whose capsid is AAV5. E53. A viral particle listed in any one of E48-E50, whose capsid is AAV8. E54. A viral particle listed in any one of E48-E50, whose capsid is AAV9. E55. A viral particle listed in any one of E48-E50, whose capsid is AAVrh10. E56. A pharmaceutical composition comprising a fusion protein described in any one of E1 to E39, a cell expressing the fusion protein described in any one of E1 to E39, a nucleic acid described in any one of E40 to E45, a vector described in any one of E46 to E47, a viral particle described in any one of E48 to E55, and a pharmaceutically acceptable carrier or excipient. E57. A method for reducing the toxicity of α-synuclein protein in cells, comprising the step of contacting the cells with an effective amount of one or more active substances selected from the group consisting of a fusion protein according to any one of E1 to E39, cells expressing a fusion protein according to any one of E1 to E39, a nucleic acid according to any one of E40 to E45, a vector according to any one of E46 to E47, a viral particle according to any one of E48 to E55, and a pharmaceutical composition according to E56. E58. The method described in E57, wherein the cells are within the target area. E59. A method described in any one of E57-E58, wherein the subject is a human. E60. The method described in any one of E57-E59, wherein the cells are central nervous system cells. E61. The method described in any one of E57-E60, wherein the subject has been identified as having alpha-synuclein disease. E62. The method according to E61, wherein α-synuclein disease is selected from the group consisting of PD, Lewy body dementia, multiple system atrophy, and synucleinopathy (diseases associated with abnormal accumulation of aggregated α-synuclein protein). E63. The method according to E61 or E62, wherein α-synuclein disease is PD. E64. The method described in any one of E57-E63, wherein there is a decrease in the amount of misfolded α-synuclein protein in the cells compared to control cells. E65. A method for treating, preventing, or delaying the progression of alpha-synuclein disease in a subject requiring treatment, prevention, or delay of its progression, comprising the step of administering an effective amount of one or more active substances selected from the group consisting of a fusion protein described in any one of E1 to E39, cells expressing a fusion protein described in any one of E1 to E39, a nucleic acid described in any one of E40 to E45, a vector described in any one of E46 to E47, a viral particle described in any one of E48 to E55, and a pharmaceutical composition described in E56. E66. The method according to E65, wherein α-synuclein disease is selected from the group consisting of PD, Lewy body dementia, multiple system atrophy, and synucleinopathy (diseases associated with the abnormal accumulation of aggregated α-synuclein protein). E67. The method described in E65, in which alpha-synuclein disease is PD. E68. Use of one or more of the fusion protein described in any one of E1 to E39, cells expressing the fusion protein described in any one of E1 to E39, nucleic acids described in any one of E40 to E45, vectors described in any one of E46 to E47, viral particles described in any one of E48 to E55, and the pharmaceutical composition described in E56 in preventing or delaying the progression of alpha-synuclein disease in a subject. E69. Use of one or more of the fusion protein described in any one of E1 to E39, cells expressing the fusion protein described in any one of E1 to E39, nucleic acids described in any one of E40 to E45, vectors described in any one of E46 to E47, viral particles described in any one of E48 to E55, and the pharmaceutical composition described in E56 in the preparation of a pharmacopoeia for the treatment or prevention of alpha-synuclein disease in a subject. E70. Use as described in E68 or E69 when α-synuclein disease is PD. [Brief explanation of the drawing]

[0015] [Figure 1A] This figure shows the Clustal Omega sequence alignment of representative human J-domain sequences. Highly conserved HPD domains are indicated within the highlighted rectangles. [Figure 1B] This figure shows the culcal omega sequence alignment of representative human J-domain sequences. [Figure 2] This figure shows several representative fusion protein constructs containing the J domain and α-synuclein-binding domain, as well as control constructs. [Figure 3A]This figure shows the effects of expressing a fusion protein containing a J domain and an α-synuclein-binding domain. Figure 3A: Wild-type (WT) α-synuclein (Syn(WT): bars 1-3) or mutant α-synuclein (Syn(A53T): bars 4-6) was expressed alone or together with a Flag epitope-tagged fusion protein containing the J domain DnaJB1 and the α-synuclein-binding domain (J-SynBP1: bars 2 and 5), or with a control fusion protein construct (J(MT)-SynBP1: bars 3 and 6) containing the mutant J domain (containing a P33Q substitution) and the α-synuclein-binding domain in HEK293 cells. After 2 days, the cells were homogenized in Tris buffer (10 mM Tris, pH 7.4, 140 mM NaCl, 1 mM EDTA with added protease inhibitor cocktail). The level of aggregation of mutant α-synuclein was quantified using sandwich ELISA. In this sandwich ELISA, the aggregated α-synuclein protein was captured by a coated Syn-O4 antibody and subsequently detected by an HRP-conjugated anti-α-synuclein antibody (BioLegend: A15115A). Figure 3B: Solubilizes were prepared from HEK293 cells transfected transiently with wild-type Syn(WT) (lanes 1-3) or mutant Syn(A53T) (lanes 4-6) alone, or co-expressed with a fusion protein construct. After short-term sonication, the cell lysates were subjected to immunoblotting with SDS-PAGE / anti-α-synuclein antibody (Ab-2: upper panel), Flag antibody (center panel), or anti-tubulin antibody (lower panel). [Figure 3B]This figure shows the effects of expressing a fusion protein containing a J domain and an α-synuclein-binding domain. Figure 3A: Wild-type (WT) α-synuclein (Syn(WT): bars 1-3) or mutant α-synuclein (Syn(A53T): bars 4-6) was expressed alone or together with a Flag epitope-tagged fusion protein containing the J domain DnaJB1 and the α-synuclein-binding domain (J-SynBP1: bars 2 and 5), or with a control fusion protein construct (J(MT)-SynBP1: bars 3 and 6) containing the mutant J domain (containing a P33Q substitution) and the α-synuclein-binding domain in HEK293 cells. After 2 days, the cells were homogenized in Tris buffer (10 mM Tris, pH 7.4, 140 mM NaCl, 1 mM EDTA with added protease inhibitor cocktail). The level of aggregation of mutant α-synuclein was quantified using sandwich ELISA. In this sandwich ELISA, the aggregated α-synuclein protein was captured by a coated Syn-O4 antibody and subsequently detected by an HRP-conjugated anti-α-synuclein antibody (BioLegend: A15115A). Figure 3B: Solubilizes were prepared from HEK293 cells transfected transiently with wild-type Syn(WT) (lanes 1-3) or mutant Syn(A53T) (lanes 4-6) alone, or co-expressed with a fusion protein construct. After short-term sonication, the cell lysates were subjected to immunoblotting with SDS-PAGE / anti-α-synuclein antibody (Ab-2: upper panel), Flag antibody (center panel), or anti-tubulin antibody (lower panel). [Figure 4]This figure shows a decrease in the amount of α-synuclein secreted into the culture medium in cells co-expressing a fusion protein containing both a J domain and an α-synuclein-binding domain. Transfection of cells with wild-type (Bar 2) or mutant (A53T; Bar 4) α-synuclein alone results in synuclein secretion detectable by ELISA. Co-expression with a fusion protein construct containing either normal (Bar 3) or a mutant J domain (containing the P33Q substitution; Bar 5) results in the exclusion of secreted α-synuclein to detectable levels. [Figure 5] This figure shows the effects of various cellular process inhibitors on total α-synuclein levels in cells expressing a fusion protein construct. Cells were transfected with either wild-type (lane 2) or mutant (lanes 3-8) α-synuclein, and also with the JB1-SynBP1 fusion protein (lanes 4-8). The cells were also treated with either bafilomycin A1 (a late autophagy inhibitor) (lanes 5 and 6, 10 nM and 100 nM, respectively) or MG132 (a proteasome inhibitor) (lanes 7 and 8, 0.1 μM and 1 μM, respectively). Cell lysates were analyzed with anti-synuclein antibodies. [Figure 6] This figure shows the effects of various cellular process inhibitors on aggregated α-synuclein levels in cells expressing a fusion protein construct. Cells were transfected with either wild-type or mutant α-synuclein, and also with the JB1-SynBP1 fusion protein. The cells were also treated with either bafilomycin A1 (late autophagy inhibitor) (10 nM and 100 nM) or MG132 (proteasome inhibitor) (0.1 μM and 1 μM). Agglutinated synuclein in cell solubles was determined by ELISA using an anti-aggregated synuclein antibody. [Figure 7]This figure shows the improvement of α-synuclein (A53T)-mediated cytotoxicity in U87-MG glioma cells by the JB1-SynBP1 construct. U87MG cells were infected with a lentivirus expressing either wild-type ("SynWT") or mutant ("SynA53T") α-synuclein protein alone or in combination with the JB1-SynBP1 construct. Culture media were collected from U87-MG cells 7 days post-infection. Lactate dehydrogenase (LDH) activity in the culture media was measured using the LDH-Cytox® assay kit (BioLegend) and expressed as a value relative to the LDH level in cells expressing SynWT alone. [Modes for carrying out the invention]

[0016] definition As used herein and in the claims, the singular forms “a,” “an,” and “the” include plural nouns unless the context clearly indicates otherwise. For example, the term “a cell” includes multiple cells, including a mixture thereof.

[0017] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, and may contain modified amino acids, which may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified by any other operation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component.

[0018] As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including, but not limited to, both D- and L-type optical isomers, as well as amino acid analogs and peptide mimes. Standard one-letter or three-letter codes are used to name amino acids.

[0019] "Host cells" include individual cells or cell cultures that may or may have been recipients of the vector of interest. Host cells include offspring of a single host cell. These offspring are not necessarily identical (in morphology or in the genome of the whole DNA complement) to the original parent cell due to natural, accidental, or planned mutations. Host cells include cells transfected in vivo with the vector of this invention.

[0020] Where used to describe the various polypeptides disclosed herein, “isolated” means a polypeptide that has been identified and separated from components of its natural environment, and / or recovered. These contaminating components of its natural environment are typically materials that interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not exist naturally do not require “isolation” to distinguish them from their naturally occurring counterparts. In addition, “concentrated,” “isolated,” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are distinguishable from their naturally occurring counterparts by generally having a higher concentration or number of molecules per unit volume than their naturally occurring counterparts. Generally, polypeptides produced by recombinant means and expressed in host cells are considered “isolated.”

[0021] An "isolated" polynucleotide, or nucleic acid encoding a polypeptide, or any other nucleic acid encoding a polypeptide, is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule that is normally associated with the natural source of the nucleic acid encoding that polypeptide. An isolated polypeptide-encoding nucleic acid molecule takes on a form or configuration other than that which is found in nature. Thus, an isolated polypeptide-encoding nucleic acid molecule is distinguished from the nucleic acid molecule encoding that particular polypeptide as it would be if it were present in a natural cell. However, an isolated polypeptide-encoding nucleic acid molecule is one that is contained in a cell that normally expresses that polypeptide, where, for example, the nucleic acid molecule is located in a chromosomal or extrachromosomal location different from its location in a natural cell.

[0022] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “nucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure may be given before or after the assembly of the polymer, if present. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components.

[0023] As defined herein, the term “alpha-synuclein disorder” or “synucleinopathy” refers to a disorder associated with the formation of intracellular alpha-synuclein aggregates, particularly aggregates of alpha-synuclein mutant proteins. Examples of alpha-synuclein disorders include, but are not limited to, parkinsonism, including PD, Lewy body dementia, multiple system atrophy, and diseases associated with the abnormal accumulation of aggregated alpha-synuclein proteins (synucleopathies).

[0024] A “vector” is a nucleic acid molecule that preferably self-replicates in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for the insertion of DNA or RNA into cells, replication vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. It also includes vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into polypeptides. An “expression system” usually implies a suitable host cell composed of an expression vector capable of functioning to produce a desired expression product.

[0025] The term “operatably ligated” refers to the juxtaposition of the described components, where they are related in a way that allows them to function in their intended manner. A coding sequence and an “operatably ligated” regulatory sequence are ligated so that the expression of the coding sequence is achieved under conditions compatible with the regulatory sequence. “Operatably ligated” sequences may include both expression regulatory sequences that are close to the gene of interest and expression regulatory sequences that act trans or at a distance to control the gene of interest. The term “expression regulatory sequence” refers to a polynucleotide sequence that is necessary for the expression and processing of the coding sequence to which it is ligated. Expression regulatory sequences include appropriate transcription start, terminate, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, where necessary, sequences that enhance protein secretion. The properties of such regulatory sequences vary depending on the host organism; in prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such regulatory sequences generally include promoters and transcription termination sequences. The term “regulatory sequence” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Unless otherwise specified, any description or reference herein to inserting a nucleic acid molecule encoding the fusion protein of the present invention into an expression vector means that the inserted nucleic acid is also operably linked within the vector to a functional promoter and other transcription and translation regulatory elements required for the expression of the encoded fusion protein when the expression vector containing the inserted nucleic acid molecule is introduced into a compatible host cell or a compatible cell of an organism.

[0026] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of in vitro cloning, restriction and / or ligation steps, and various combinations of other procedures that produce a construct that can potentially be expressed in host cells.

[0027] The terms “gene” and “gene fragment” are used interchangeably herein. They refer to polynucleotides containing at least one open reading frame that, after transcription and translation, has the ability to code for a particular protein. A gene or gene fragment can be genomic DNA or cDNA, insofar as its polynucleotides contain at least one open reading frame, and it may encompass an entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterogeneous polynucleotides linked together.

[0028] The terms “disease” and “disorder” are used interchangeably to refer to pathological conditions identified by an acceptable level of medical care and practice in the art.

[0029] As used herein, the term “effective dose” means an amount of treatment that is sufficient to reduce or improve the severity and / or duration of a disease or one or more symptoms; prevent the progression of an adverse or pathological condition; cause regression of a pathological condition; prevent the recurrence, development, onset or progression of one or more symptoms associated with a pathological condition; detect a disorder; or enhance or improve the prophylactic or therapeutic effect of a treatment (e.g., administration of another prophylactic or therapeutic agent).

[0030] As used herein, the term “J domain” refers to the fragment that retains the ability to accelerate the catalytic activity of Hsp70 and its congeners' endogenous ATPases. The J domains of various J proteins have been determined (see, e.g., Kampinga et al. (2010) Nat. Rev., 11:579-592; Hennessy et al. (2005) Protein Science, 14:1697-1709 (each incorporated as a whole by reference)), and are characterized by several hallmarks: four α-helices (I, II, III, IV), and a highly conserved tripeptide sequence motif of histidine, proline, and aspartic acid (called the “HPD motif”), usually between helices II and III. Typically, the J-domain of a J-protein is between 50 and 70 amino acids long, and the site of interaction (binding) of the J-domain with the Hsp70-ATP chaperone protein is thought to be a region extending from within helix II, where the HPD motif is required for stimulation of Hsp70 ATPase activity. As used herein, the term “J-domain” is intended to include native J-domain sequences and their functional variants that retain the ability to accelerate the endogenous ATPase activity of Hsp70, the ability of which can be measured using methods well known in the art (e.g., see Horne et al. (2010) J. Biol. Chem., 285, 21679–21688, incorporated herein by reference in whole). An unrestricted list of human J-domains is provided in Table 1.

[0031] Detailed explanation The inventors have found that contacting cells to some extent with a fusion protein construct containing the J domain and α-synuclein-binding domain of the J protein produces an unexpected effect of reducing α-synuclein protein aggregation. Aggregation of mutant α-synuclein is thought to cause several devastating diseases, including, but not limited to, parkinsonism, Lewy body dementia, multiple system atrophy, and diseases associated with the abnormal accumulation of aggregated α-synuclein protein (synucleinopathy). Therefore, useful compositions and methods for treating α-synuclein disorders, for example in subjects where such treatment is needed, are provided herein.

[0032] To overcome the problems associated with chaperone-based therapy, the inventors investigated whether it was possible to design highly specific artificial chaperone proteins. The inventors designed a series of fusion protein constructs comprising an effector domain (J domain sequence) for Hsp70 binding / activation and a domain conferring specificity to α-synuclein protein. The resulting fusion proteins act to accelerate the catalytic activity of Hsp70 and its related endogenous ATPases, resulting in increased protein folding, reduced aggregation, and / or accelerated clearance.

[0033] I. Fusion protein constructs a. Useful J domains in the present invention The J domains of various J proteins have been determined. See, for example, Kampinga et al., Nat. Rev., 11:579~592 (2010); Hennessy et al., Protein Science, 14:1697~1709 (2005). The J domains useful in preparing the fusion proteins of the present invention are key to defining the features of the J domain that primarily accelerate Hsp70 ATPase activity. Therefore, the isolated J domains useful in the present invention comprise a polypeptide domain characterized by four α-helices (I, II, III, IV) and a tripeptide sequence of histidine, proline, and aspartic acid (called the "HPD motif"), which is usually highly conserved between helices II and III. Typically, the J-domain of a J-protein is between 50 and 70 amino acids long, and the interaction (binding) site of the J-domain with the Hsp70-ATP chaperone protein is thought to be a region extending from within helix II, with the HPD motif forming the basis of primitive activity. Representative J-domains include, but are not limited to, those of DnaJB1, DnaJB2, DnaJB6, DnaJC6, the J-domain of the SV40 large T antigen, and the J-domain of mammalian cysteine ​​string protein (CSP-α). Amino acid sequences for these and other J-domains that can be used in the fusion protein of the present invention are provided in Table 1. Conserved HPD motifs are highlighted in bold.

[0034] [Table 1-1]

[0035] [Table 1-2]

[0036] [Table 1-3]

[0037] In one embodiment, the fusion protein includes a J-domain sequence selected from a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 to 50. The inventors have demonstrated that a J-domain containing a conserved "HPD" motif is active (data not presented). Therefore, in another embodiment, the fusion protein includes a J-domain sequence containing a conserved "HPD" motif. For example, in a particular embodiment, the fusion protein includes a J-domain sequence selected from a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 to 15 and 17 to 50. In another embodiment, the fusion protein includes a J-domain sequence selected from a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49.

[0038] b. α-synuclein-binding domain The fusion protein also contains at least one α-synuclein-binding domain. The α-synuclein-binding domain may be a single-chain polypeptide or a polymer polypeptide linked to the J domain to form the fusion protein.

[0039] Ideally, the α-synuclein-binding domain should have sufficient affinity to bind to α-synuclein protein when it is present at pathological levels within the cell. Therefore, in one embodiment, when the fusion protein is tested by ELISA on a 96-well microtiter plate, it should have K2+, 1+, 500+, 300+, 100+, and 30+ K2+ of α-synuclein. D It contains an α-synuclein-binding domain having . In another embodiment, when the fusion protein is tested by ELISA on a 96-well microtiter plate, it has K to aggregated α-synuclein at concentrations of, for example, ≤2 μM, ≤1 μM, ≤500 nM, ≤300 nM, ≤100 nM, and ≤30 nM. DIt contains an α-synuclein-binding domain. In another embodiment, the α-synuclein-binding domain has selectivity for aggregated α-synuclein; for example, the α-synuclein-binding domain has an affinity for aggregated α-synuclein that is at least twice as high as its affinity for soluble α-synuclein, for example, 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 100 times higher than its affinity for aggregated α-synuclein.

[0040] The α-synuclein-binding domains have been previously identified and characterized (see, for example, U.S. Patent No. 7,605,133, WO2019 / 161386, Abe et al., (2007) BMC Bioinformatics, 8:451; each of these is incorporated herein by reference). Thus, in another embodiment, the fusion protein comprises an α-synuclein-binding domain selected from the group consisting of SEQ ID NOs. 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.

[0041] In 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 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 SEQ ID NO: 65, and the J domain is selected from the group consisting of SEQ ID NOs: 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.

[0042] In another embodiment, the fusion protein comprises a J domain and the target-binding protein of SEQ ID NO: 65. It has been previously shown that fusion proteins containing QBP1 (SEQ ID NO: 65) reduce the mechanical stability and the formation of mechanically highly stable conformational isomers in A53T α-synuclein (Hervas et al., (2012) PLoS Biol. 10(5):e1001335). Therefore, in certain embodiments, the fusion protein construct comprises the α-synuclein-binding domain of SEQ ID NO: 65. In certain embodiments, the fusion protein construct comprises a J domain sequence selected from the group consisting of SEQ ID NOs: 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.

[0043] In another embodiment, the fusion protein also intends to use an α-synuclein-binding domain that is chemically conjugated with the J domain. The α-synuclein-binding domain may be conjugated directly with the J domain, or it may be conjugated with the J domain by a linker. For example, there are numerous chemical crosslinkers known to those skilled in the art that are useful for crosslinking the α-synuclein-binding domain with the J domain, or a targeting domain, with a fusion protein containing the α-synuclein-binding domain and the J domain. For example, crosslinkers are heterobifunctional crosslinkers that can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers offer the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of undesirable side reactions such as homoprotein polymers. Various heterobifunctional crosslinking agents are known in the art, including succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Crosslinking agents having an N-hydroxysuccinimide moiety can generally be obtained as N-hydroxysulfosuccinimide analogs with higher water solubility. In addition, crosslinking agents having disulfide crosslinks in the linking chain can be synthesized instead as alkyl derivatives to reduce the amount of linker cleavage in vivo. Several other crosslinking agents exist, including heterobifunctional crosslinking agents, homobifunctional crosslinking agents, and photoreactive crosslinking agents.Disuccinimidyl sberate (DSS), bismaleimide hexane (BMH), and dimethylpimerimidate·2HCl are examples of useful homobifunctional crosslinking agents used in this disclosure, while bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinking agents. For a recent overview of protein coupling techniques, see Means et al., (1990) Bioconj. Chem. 1:2~12, incorporated herein by reference.

[0044] [Table 2]

[0045] c. Optional linker The fusion proteins described herein may optionally contain one or more linkers. The linkers may be peptidogenic or non-peptidogenic. The purpose of the linkers is, among other things, to provide appropriate distances between functional domains within the protein (e.g., between the J domain and the α-synuclein-binding domain, between tandem arrangements of α-synuclein-binding domains, between the J domain and the α-synuclein-binding domain and any of the optional targeting reagents, or between the J domain and the α-synuclein-binding domain and any of the optional detection domains or epitopes) for the optimal function of each domain. Clearly, the linkers preferably do not interfere with the function of the J domain, the target protein-binding domain, or any of the other domains of the fusion protein of the present invention. If the linkers are present in the fusion protein of the present invention, they are selected to attenuate cytotoxicity caused by the target protein (α-synuclein protein), and they may be omitted if direct attachment achieves the desired effect. The linker present in the fusion protein of the present invention may comprise one or more amino acids encoded by a nucleotide sequence present on the nucleic acid segment per cloning site of an expression vector into which a nucleic acid segment encoding a protein domain or the entire fusion protein, as described herein, is inserted in frame. In one embodiment, the peptide linker is between 1 and 20 amino acids long. In another embodiment, the peptide linker is between 2 and 15 amino acids long. In yet another embodiment, the peptide linker is between 2 and 10 amino acids long.

[0046] Selecting one or more polypeptide linkers to construct the fusion proteins according to the present invention is within the scope of the knowledge and skills of those skilled in the art. See, for example, Arai et al., Protein Eng., 14(8):529-532 (2001); Crasto et al., Protein Eng., 13(5):309-314 (2000); George et al., Protein Eng., 15(11):871-879 (2003); Robinson et al., Proc. Natl. Acad. Sci. USA, 95:5929-5934 (1998) (each incorporated herein by reference as a whole). Examples of linkers of two or more amino acids that may be used in preparing the fusion proteins according to the present invention are, but are not limited to, those provided in Table 3 below.

[0047] [Table 3]

[0048] d. Targeting reagents The fusion proteins disclosed herein may further include a targeting moiety. As used herein, the terms “targeting moiety” and “targeting reagent” refer to a substance associated with a fusion protein that is interchangeable and enhances the binding, transport, accumulation, residence time, bioavailability, or modifies its biological activity or therapeutic effect in cells or in the body of a subject. The targeting moiety may be functional at the tissue, cell, and / or intracellular levels. The targeting moiety can direct the localization or intracellular distribution of the fusion protein to specific cells, tissues, or organs, for example, upon administration of the fusion protein to a subject. In one embodiment, the targeting moiety is located at the N-terminus of the fusion protein. In another embodiment, the targeting moiety is located at the C-terminus of the fusion protein. In yet another embodiment, the targeting moiety is located internally. In yet another embodiment, the targeting moiety attaches to the fusion protein via chemical conjugation. In a further embodiment, the targeting moiety may be an amino acid sequence for intracellular localization, such as a nuclear localization signal or an extranuclear localization signal. The targeting moieties may include, but are not limited to, organic or inorganic molecules, peptides, peptide mimes, proteins, antibodies or fragments thereof, growth factors, enzymes, lectins, antigens or immunogens, viruses or components thereof, viral vectors, receptors, receptor ligands, toxins, polynucleotides, oligonucleotides or aptamers, nucleotides, carbohydrates, sugars, lipids, glycolipids, nucleoproteins, glycoproteins, lipoproteins, steroids, hormones, growth factors, chemoattractants, cytokines, chemokines, drugs, or small molecules.

[0049] In exemplary embodiments of the present invention, the targeting moiety enhances the binding, transport, accumulation, residence time, and bioavailability of the platform, or its associated ligand and / or active substance, or modifies its biological activity or therapeutic effect, in target cells or tissues, such as nerve cells, the central nervous system, and / or the peripheral nervous system. Therefore, the targeting moiety may have specificity for cellular receptors associated with the central nervous system, or otherwise be related to enhanced delivery to the CNS via the blood-brain barrier (BBB). Consequently, such ligands may be both ligands and targeting moieties.

[0050] In some embodiments, the targeting moiety may be a cell-permeable peptide, such as that described in U.S. Patent No. 10,111,965, which is incorporated herein by reference in whole. In other embodiments, the targeting moiety may be an antibody or its antigen-binding fragment or single-chain derivative, such as that described in U.S. Patent Application No. 16 / 131,591, which is incorporated herein by reference in whole. The targeting moiety may be conjugated to a platform for targeted cell delivery by directly or indirectly conjugating to its core. For example, in embodiments in which the core comprises nanoparticles, the conjugation of the targeting moiety with the nanoparticles may utilize similar functional groups used to tether PEG to the nanoparticles. Thus, the targeting moiety may be directly conjugated to the nanoparticles through functionalization of the targeting moiety. Alternatively, the targeting moiety may be indirectly conjugated to the nanoparticles through conjugation of the functionalized PEG of the targeting moiety, as discussed above. The targeting moiety may adhere to the core by covalent, non-covalent, or electrostatic interactions. In one embodiment, the targeting moiety is a peptide. In certain embodiments, the targeting moiety is a peptide that covalently attaches to the N-terminus of the fusion protein.

[0051] e. Epitope In certain embodiments, the fusion protein of the present invention contains an optional epitope or tag that can impart additional properties to the fusion protein. As used herein, the terms “epitope” and “tag” are used interchangeably to refer to an amino acid sequence, typically 300 amino acids or less in length, that typically attaches to the N-terminus or C-terminus of the fusion protein. In one embodiment, the fusion protein of the present invention further includes an epitope used to facilitate purification. Examples of such epitopes useful for purification, provided in Table 4 below, include the human IgG1 Fc sequence (SEQ ID NO: 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 includes an epitope used to increase the half-life of the fusion protein when administered to a subject, e.g., humans. An example of such an epitope useful for increasing half-life is the human Fc sequence. Therefore, in one particular embodiment, the fusion protein includes a human Fc epitope in addition to the J domain and the α-synuclein-binding domain. The epitope is located at the C-terminus of the fusion protein.

[0052] [Table 4]

[0053] f. Cell-permeable peptides In other embodiments, the fusion proteins described herein may further include cell-permeable peptides. Cell-permeable peptides are known to deliver conjugated cargo, whether it be small molecules, peptides, proteins, or nucleic acids, into cells. Non-limiting examples of cell-permeable peptides in the fusion proteins of the present invention include polycationic peptides, e.g., HIV TAT peptides 49-57, polyarginine, and penetratin pAntan (43-58); amphiphilic peptides, e.g., pep-1; hydrophobic peptides, e.g., C405Y; and others of the same kind. See Table 5 below.

[0054] [Table 5]

[0055] Therefore, in one embodiment, the fusion protein comprises a cell-permeable peptide and a fusion protein, wherein the cell-permeable peptide is selected from the group consisting of SEQ ID NOs: 84-87, and the fusion protein is selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. In another embodiment, the fusion protein comprises the cell-permeable peptide of SEQ ID NO: 84 and a fusion protein selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. In yet another embodiment, the fusion protein comprises the cell-permeable peptide of SEQ ID NO: 85 and a fusion protein selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. In yet another embodiment, the fusion protein comprises the cell-permeable peptide of SEQ ID NO: 86 and a fusion protein selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. Cells expressing this fusion protein construct, which contains a cell-permeable peptide, can be administered to subjects, such as human subjects (e.g., patients with or at risk of developing α-synuclein disorders). This fusion protein is secreted from cells and helps reduce α-synuclein-containing protein aggregation and / or related cytotoxicity.

[0056] g. Arrangement of the J domain and α-synuclein-binding domain The fusion proteins described herein can be configured in numerous 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. The α-synuclein-binding domain and the J-domain can optionally be separated via a linker as described above in any of the configurations.

[0057] In some embodiments, the J domain may be attached to multiple α-synuclein-binding domains, for example, two α-synuclein-binding domains, three α-synuclein-binding domains, four α-synuclein-binding domains, or more. The α-synuclein-binding domains may be attached to the N-terminal side of the J domain, or to the C-terminal side of the J domain. In another embodiment, the α-synuclein-binding domains may be attached 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 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).

[0058] In some embodiments, the fusion protein belongs to the following group: a. DNAJ-XS, b. DNAJ-XSXS, c. DNAJ-XSXSXS, d. SX-DNAJ, e. SXSX-DNAJ, f. SXSXSX-DNAJ, g. SX-DNAJ-XS, h. SX-DNAJ-XSXS, i. SXSX-DNAJ-XSXSXS, j. SXSXSX-DNAJ-XS, k. SXSXSX-DNAJ-XSXS, l. SXSXSX-DNAJ-XSXSXS, m. DnaJ-X-DnaJ-XSXS, n. SX-DnaJ-X-DnaJ, and o. SXSX-DnaJ-X-DnaJ, It may include a structure selected from, Here, S is an α-synuclein-binding domain, DNAJ is the J domain of the J protein. X is an optional linker.

[0059] In one embodiment, the fusion protein includes a J domain selected from the group consisting of SEQ ID NOs: 1-15 and 17-50. In another embodiment, the fusion protein includes a J domain selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49. In one particular embodiment, the fusion protein includes the J domain of SEQ ID NO: 5.

[0060] In another embodiment, the α-synuclein-binding domain is selected from the group consisting of SEQ ID NOs: 51 to 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.

[0061] In another embodiment, the fusion protein includes 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 includes at least two copies of the J domain of SEQ ID NO: 5 and the α-synuclein-binding domain of SEQ ID NO: 51.

[0062] Non-limiting examples of fusion protein constructs containing the J domain and the α-synuclein-binding domain, as well as control constructs, are schematically illustrated in Figure 2 and also shown in Table 6 below. In one embodiment, a particular fusion protein construct is selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100.

[0063] [Table 6-1]

[0064] [Table 6-2]

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

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

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

[0068] In certain embodiments, the nucleic acid encoding one or more fusion proteins is an RNA molecule and may be pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. In various embodiments, the nucleic acid is mRNA introduced into a cell to transiently express a desired polypeptide. As used herein, “transient” refers to the expression of an unintegrated transgene for a period of several hours, several days, or several weeks, the duration of expression being shorter than that for polynucleotide expression when integrated into the genome or contained within a stable plasmid replicon in a cell.

[0069] In certain embodiments, the mRNA encoding the polypeptide is in vitro transcribed mRNA. As used herein, “in vitro transcribed RNA” means RNA, preferably mRNA, that is synthesized in vitro. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used to produce in vitro transcribed RNA. In certain embodiments, the mRNA may further include a 5' cap or a modified 5' cap and / or a poly(A) sequence. As used herein, the 5' cap (also called the RNA cap, RNA 7-methylguanosine cap, or RNA m7G cap) is a modified guanine nucleotide attached to the “front” or 5' end of eukaryotic messenger RNA immediately after the start of transcription. The 5' cap contains a terminal group that is ligated to the first transcribed nucleotide, is recognized by ribosomes, and is protected from Rnases. The capping portion may be modified to modulate the functionality of the mRNA, for example, its stability or translational efficiency. In a particular mechanism, mRNA contains poly(A) sequences of between approximately 50 and 5000 adenine bases. In a mechanism, mRNA contains poly(A) sequences between approximately 100 and 1000 bases, between approximately 200 and 500 bases, or between approximately 300 and 400 bases. In a mechanism, mRNA contains poly(A) sequences of approximately 65 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, or 1000 bases or more. Poly(A) sequences may be chemically or enzymatically modified to regulate mRNA functionality, such as localization, stability, or translation efficiency. As used herein, the terms “polynucleotide variant” and “variant” and other similar terms refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes with a reference sequence under the stringent conditions defined below.These terms include polynucleotides in which one or more nucleotides are added, deleted, or replaced with different nucleotides compared to a reference polynucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, and that the thereby modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0070] In a particular embodiment, the nucleic acid sequence contains a nucleotide sequence encoding the gene of interest (e.g., a fusion protein comprising a J domain and a polyglutamine-binding domain) within the nucleic acid cassette. As used herein, the terms “nucleic acid cassette” or “expression cassette” refer to the genetic sequence within a vector capable of expressing RNA and subsequently polypeptides. In one embodiment, the nucleic acid cassette contains the gene of interest, e.g., the polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and the gene of interest, e.g., the polynucleotide of interest. The vector may contain one, two, three, four, five, six, seven, eight, nine, or ten or more nucleic acid cassettes. The nucleic acid cassette is locologically and ordinally oriented within the vector so that the nucleic acids within the cassette can be transcribed to RNA, translated into proteins or polypeptides as needed, undergo appropriate post-translational modifications required for activity in transformed cells, and translocated to appropriate compartments for biological activity by targeting to appropriate intracellular compartments or secretion to extracellular compartments. Preferably, the cassette has 3' and 5' ends adapted for immediate insertion into the vector, for example, it has restriction endonuclease sites at each end. The cassette can be removed and inserted into plasmids or viral vectors as a single unit.

[0071] Exemplary ubiquitous expression regulatory sequences suitable for use in specific embodiments include the cytomegalovirus (CMV) early promoter, the simian virus 40 (SV40) (e.g., early or late), the Moloney's mouse leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, and vaccinia virus-derived H5, P7.5, and Pl. I promoter, elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (b-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477~1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter (Okabe et al. (1997) FEBS let. Examples include, but are not limited to, the β-actin promoter and myeloproliferative sarcoma virus enhancer, and the (MND)U3 promoter in which the negative regulatory region is deleted and replaced with a dl587rev primer binding site (Haas et al., Journal of Virology. 2003;77(l7):9439~9450). In one embodiment, at least one element for enhancing the specificity and expression of the transgene target, such as a promoter (see, for example, Powell et al. (2015) Discovery Medicine 19(102):49~57 (the contents of which are incorporated herein by reference in their entirety)), may be used in conjunction with the polynucleotides described herein.Promoters that promote expression in most tissues include, but are not limited to, human elongation factor la-subunit (EFla), early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements are used to restrict expression to certain cell types, and include, but are not limited to, nervous system promoters that can be used to restrict expression to neurons, astrocytes, or oligodendrocytes. Non-exclusive examples of tissue-specific expression elements for neurons include, but are not limited to, promoters for neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl CpG-binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH, ηβ2, PPE, Enk, and EAAT2. Non-limiting examples of tissue-specific expression elements for astrocytes include the promoters of glial fibrillary acidic protein (GFAP) and EAAT2. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include the myelin basic protein (MBP) promoter. Yu et al. (incorporated collectively by reference in (2011) Molecular Pain, 7:63) evaluated eGFP expression under CAG, EFIa, PGK, and UBC promoters in rat DRG cells and primary DRG cells using lentiviral vectors and found that UBC showed weaker expression than the other three promoters, with only 10-12% glial expression for all promoters. Soderblom et al. (incorporated collectively by reference in E. Neuro 2015) evaluated eGFP expression in AAV8 with CMV and UBC promoters and AAV2 with CMV promoter after injection into the motor cortex.Intranasal administration of plasmids containing the UBC or EFIa promoter resulted in higher sustained airway expression than expression with the CMV promoter (see, e.g., Gill et al., (2001) Gene Therapy, vol. 8, 1539-1546, incorporated as a whole by reference). Husain et al. (incorporated as a whole by reference, (2009) Gene Therapy) evaluated ΗβΗ constructs with the hGUSB promoter, HSV-1LAT promoter, and NSE promoter and found that the ΗβΗ constructs showed weaker expression than NSE in the mouse brain. Passini and Wolfe (incorporated as a whole by reference, J. Virol. 2001, 12382-12392) evaluated the long-term effects of ΗβΗ vectors after intracerebroventricular injection in neonatal mice and found sustained expression for at least one year. Xu et al. (incorporated collectively by reference, (2001) Gene Therapy, 8, 1323-1332) found low expression in all brain regions when NF-L and NF-H promoters were used in comparison 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(1.8kb), EF, NSE(0.3kb), GFAP, CMV, hENK, PPE, NFL, and NFH. NFL is a 650-nucleotide promoter and NFH is a 920-nucleotide promoter; neither is present in the liver, but NFH is abundant in proprioceptive neurons, the brain, and the spinal cord, and NFH is present in the heart. Scn8a is a 470-nucleotide promoter expressed in the DRG, spinal cord, and brain, with particularly high expression in hippocampal neurons and cerebellar Purkinje cells, the cortex, thalamus, and hypothalamus. (See, for example, Drews et al. 2007 and Raymond et al. 2004, incorporated as a whole by reference).

[0072] III. Vectors containing nucleic acids encoding fusion proteins Vectors containing nucleic acids according to the present invention are also provided. Such vectors preferably include additional nucleic acid sequences such as elements necessary for the transcription / translation of the nucleic acid sequence encoding a phosphatase (e.g., promoter and / or terminator sequences). The vector may also include a nucleic acid sequence encoding a selection marker (e.g., an antibiotic) for selecting or maintaining host cells transformed with the vector. The term “vector” refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally ligated to the vector nucleic acid molecule, for example, by insertion therein. The vector may include sequences that direct self-replication in a cell, or may include sequences sufficient to enable integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides intended herein to infected cells (e.g., nerve cells). In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a fusion protein comprising a J domain and an α-synuclein-binding domain. Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0073] Examples of vectors include, but are not limited to, plasmids, self-replicating sequences, and transposition factors such as piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives. Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or Pl-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of viruses useful as vectors include, but are not limited to, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus (VIMS)), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V 5-DEST®, pLenti6 / V 5-DEST®, and pLenti6.2 / V 5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of polypeptides disclosed herein may be ligated into such expression vectors for polypeptide expression in mammalian cells.

[0074] In certain embodiments, the vector is an episomal vector, or a vector maintained outside the chromosome. As used herein, the term “episomal” means a vector that can replicate without integration into the host's chromosomal DNA and without being gradually lost from a dividing host cell; that is, the vector replicates outside the chromosome or episomalally.

[0075] A vector may contain one or more recombination sites for any of a variety of site-specific recombinases. It should be understood that the target sites for site-specific recombinases are in addition to any sites required for the incorporation of the vector, e.g., a retroviral vector or a lentiviral vector. As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombinant site” refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0076] For example, one recombination site for Cre recombinase is loxP, a 34-base pair sequence containing two 13-base pair reverse repeats (which act as recombinase binding sites) adjacent to an 8-base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521~527 (1994)). Appropriate recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod et al., 1996), FI, F2, F3 (Schlake and Bode, 1994), FyFs (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0077] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme pC3l. (pC3l SSR mediates recombination only between heterotype sites, attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). AttB and attP, named in relation to the attachment site for phage integrase in bacterial and phage genomes, respectively, both contain incomplete reverse repeats, possibly linked by a φ031 homodimer (Groth et al., 2000). The generation sites, attL and attR, are effectively further tpQA It is inactive to 1-mediated recombination (Belteki et al., 2003), making the reaction irreversible. Regarding the catalysis of insertion, it has been found that attB-containing DNA is more easily inserted into genomic attP sites than attP sites are inserted into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to position the attP-containing "docking site" at a limited gene locus via homologous recombination, and then that attP-containing "docking site" is partnered with the sequence into which the attB-containing insertion will enter.

[0078] As used herein, “intrasequence ribosome entry site” or “IRES” refers to an element that facilitates direct intrasequence ribosome entry into a start codon, such as ATG, of a cistron (protein-coding region), thereby resulting in cap-independent translation of the gene. See, for example, Jackson et al., 1990 Trends Biochem Sci 15(12):477-83, and Jackson and Kaminski 1995 RNA 1(10):985-1000. In certain embodiments, the vector comprises one or more polynucleotides of interest encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is an EMCV IRES.

[0079] As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome and increases translation (Kozak, 1986, Cell 44(2):283-92, and Kozak, 1987, Nucleic Acids Res. 15(20):8125-48). In certain embodiments, the vector comprises a polynucleotide having a consensus Kozak sequence, as well as a polynucleotide encoding a fusion protein containing a J domain and an α-synuclein-binding domain. Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector comprises a polyadenylation sequence on the 3' side of the polynucleotide encoding the polypeptide to be expressed.

[0080] Examples of viral vector systems suitable for use in the specific embodiments envisioned herein include, but are not limited to, vectors of adeno-associated viruses (AAV), retroviruses, herpes simplex viruses, adenoviruses, and vaccinia viruses.

[0081] In various embodiments, one or more polynucleotides encoding a fusion protein containing a J domain and a polyglutamine-binding domain are introduced into cells, such as nerve cells, by transduction of recombinant adeno-associated virus (rAAV) containing one or more polynucleotides into the cells. AAV is a small (about 26 nm), replication-deficient, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the host cell's genome. Recombinant AAV (rAAV) typically consists minimally of a transgene, as well as its regulatory sequence, and 5' and 3' AAV terminal inversion sequences (ITRs). The ITR sequence is about 145 bp long. In certain embodiments, rAAV comprises an ITR and capsid sequence isolated from AAV1, AAV2 (e.g., US6962815B2, incorporated herein by reference as a whole), AAV3, AAV4, AAV5 (e.g., US7479554B2, incorporated herein by reference as a whole), AAV6, AAV7, AAV8 (e.g., US7282199B2, incorporated herein by reference as a whole), AAV9 (e.g., US9737618B2, incorporated herein by reference as a whole), AAV rh10 (e.g., US9790472B2, incorporated herein by reference as a whole), or AAV10. In one embodiment, the vector of the present invention is encapsulated within a capsid selected from the group consisting of AAV2, AAV5, AAV8, AAV9, and AAV rh10. In one embodiment, the vector is encapsulated within AAV2. In one embodiment, the vector is encapsulated within AAV5. In one embodiment, the vector is encapsulated within AAV8. In one embodiment, the vector is encapsulated within AAV9. In one embodiment, the vector is encapsulated within AAV rh10.

[0082] In some embodiments, chimeric rAAVs are used in which the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from a different AAV serotype. For example, an rAAV having an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6 is called AAV2 / AAV6. In certain embodiments, the rAAV vector may contain an ITR derived from AAV2 and a capsid protein derived from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In preferred embodiments, the rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In preferred embodiments, the rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0083] In some embodiments, the manipulation and selection methods can be applied to the AAV capsid to increase the likelihood of transducing the cells of interest.

[0084] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Patents 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated herein by reference as a whole.

[0085] IV. Delivery In certain embodiments, one or more polynucleotides encoding a fusion protein containing a J domain and an α-synuclein-binding domain are introduced into cells by non-viral or viral vectors. Exemplary methods for non-viral delivery of polynucleotides as intended in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, particle guns, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene guns, and heat shock.

[0086] Examples of polynucleotide delivery systems suitable for use in specific embodiments as envisioned in particular embodiments include, but are not limited to, those offered by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in the literature, e.g., Liu et al., (2003) Gene Therapy 10:180-187; and Balazs et al., (20W) Journal of Drug Delivery 2011:1-12. Delivery based on antibody-targeted, bacterial, non-viable nanocells is also envisioned in particular embodiments.

[0087] Viral vectors containing polynucleotides as intended in a particular embodiment may be delivered in vivo by administration to individual patients, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection), subarachnoid injection, intracerebroventricular injection, or topical application, as described below. Alternatively, the vector may be delivered ex vivo to cells explanted from individual patients (e.g., mobilized peripheral blood, lymphocytes, bone marrow fluid, tissue biopsy, etc.) or to hematopoietic stem cells from a pluripotent donor, and then re-implanted into the patient.

[0088] In one embodiment, a viral vector comprising a polynucleotide encoding the fusion protein disclosed herein is administered directly to a living organism for in vivo cell transduction.

[0089] Appropriately packaged and formulated viral vectors can be delivered to the central nervous system (CNS) via subarachnoid delivery. For example, an adeno-associated virus vector can be delivered using the method described in U.S. Patent Application No. 15 / 771,481, which is incorporated herein by reference as a whole.

[0090] Alternatively, naked DNA may be administered. Administration is by one of the routes commonly used to introduce the molecule to final contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Appropriate methods for administering such nucleic acids are available and well known to those skilled in the art, and more than one route may be used to administer a particular composition, although a particular route may provide a more rapid and effective response than another.

[0091] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into cells, such as nerve cells or neural stem cells, by transduction of a retrovirus, such as a lentivirus, containing one or more polynucleotides into the cells. As used herein, the term “retrovirus” refers to an RNA virus that transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in particular embodiments include, but are not limited to, Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses. As used herein, the term “lentivirus” refers to a group (or genus) of complex lentiviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and HIV 2); Visna-Maedivirus (VMV); Caprine arthritis encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Monkey immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., an HIV cis-acting sequence element) is preferred.

[0092] Lentiviral vectors preferably include several safety enhancements as a result of modifying the LTR. “Self-inactivating” (SIN) vectors refer to vectors in which the replication-deficient vector, for example, the right-side (3') LTR enhancer-promoter region known as the U3 region, is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter that drives transcription of the viral genome during viral particle production. Examples of heterologous promoters that may be used include, for example, promoters for simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., very early), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (vims) (HSV) (thymidine kinase). In certain embodiments, lentiviral vectors are prepared according to known methods. For example, see Kutner et al., BMC Biotechnol. 2009;9:10 Doi:10.1186 / 1472-6750-9-10; and Kutner et al., Nat. Protoc. 2009;4(4):495~505 Doi:l0.l038 / nprot.2009.22.

[0093] According to certain embodiments contemplated herein, the majority or all of the viral vector backbone sequence is derived from a lentivirus, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences may be used, or that numerous combined substitutions and modifications in certain parts of the lentiviral sequence may be acceptable without impairing the transfer vector's ability to perform the functions described herein. Furthermore, various lentiviral vectors are known in the art; see Naldini et al. (1996a, 1996b, and 1998); Zufferey et al. (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and No. 5,994,136 (many of which can be adapted to construct the viral vectors or transfer plasmids contemplated herein).

[0094] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into target cells by transduction of an adenovirus containing one or more polynucleotides into the cells. Adenovirus-based vectors have a very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. These vectors can be produced in large quantities in relatively simple systems. Most adenovirus vectors are engineered so that the transgene replaces the Ad Ela, Elb, and / or E3 genes, and then the replication-deficient vector is grown in human 293 cells that transduce the deleted gene function. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing differentiated cells such as those found in the liver, kidney, and muscle. Typical Ad vectors have high transport capacity.

[0095] The current production and transmission of replication-deficient adenovirus vectors can utilize a unique helper cell line named 293, which is transformed from human embryonic kidney cells with an Ad5 DNA fragment and constitutively expresses the El protein (Graham et al., 1977). Since the E3 region does not need to be from the adenovirus genome (Jones & Shenk, 1978), current adenovirus vectors with the help of 293 cells have foreign DNA in either the El, E3, or both regions (Graham & Prevec, 1991). Adenovirus vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies involving the administration of recombinant adenovirus to different tissues include tracheal infusion (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Examples of Ad vector use in clinical trials include polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083~9 (1998)).

[0096] In various embodiments, one or more polynucleotides encoding the fusion protein of the present invention are introduced into target cells by transduction of a herpes simplex virus containing one or more polynucleotides, such as HSV-1 or HSV-2, into the cells.

[0097] A mature HSV virion consists of an enveloped icosahedral capsid containing a viral genome consisting of a linear double-stranded DNA molecule of 152 kb. In one embodiment, an HSV-based viral vector is deficient in one or more essential or non-essential HSV genes. In one embodiment, an HSV-based viral vector is replication-deficient. Most replication-deficient HSV vectors contain deletions that remove one or more very early, early, or late HSV genes to prevent replication. For example, an HSV vector may be deficient in very early genes selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter a latent period, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA insertion fragments up to 25 kb. Vectors based on HSV are described, for example, in U.S. Patents 5,837,532, 5,846,782, and 5,804,413, and in International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637, and WO99 / 06583 (each of which is incorporated herein by reference as a whole).

[0098] V. Cells expressing fusion proteins In yet another embodiment, the present invention provides cells expressing the fusion proteins described herein. The cells can be transfected with a vector encoding the fusion protein as described above. In one embodiment, the cells are prokaryotic cells. In another embodiment, the cells are eukaryotic cells. In yet another embodiment, the cells are mammalian cells. In a particular embodiment, the cells are human cells. In yet another embodiment, the cells are human cells derived from a patient who has or is at risk of developing an α-synuclein-mediated disorder, such as ALS, FTD, and Alzheimer's disease. The cells may be nerve cells or muscle cells.

[0099] Cells expressing the fusion protein may be useful in producing the fusion protein. In this embodiment, cells are transfected with a vector that overexpresses the fusion protein. The fusion protein may optionally contain an epitope, for example, a human Fc domain or a FLAG epitope as described herein, which facilitates purification (using a protein A column or an anti-FLAG antibody column, respectively). The epitope may be linked to the rest of the fusion protein via a linker or a protease substrate sequence so that the epitope can be removed from the fusion protein during or after purification.

[0100] Cells expressing fusion proteins may also be useful in therapeutic applications. In one embodiment, cells are collected from a patient in need of treatment (e.g., a patient with or at risk of developing alpha-synuclein-mediated disorders). In one embodiment, the cells are nerve cells. The collected cells are then transfected with a vector expressing the fusion protein. The transfected cells may then be processed to enrich or select for transfected cells. The transfected cells may also be treated to differentiate into different cell types, such as nerve cells. After processing, the transfected cells may be administered to the patient. In one embodiment, the cells are administered by directed injection into the central nervous system via subarachnoid injection, intracranial injection, or intracerebroventricular injection.

[0101] In an alternative embodiment, cells expressing the secreted form of the fusion protein may be used. For example, the fusion protein construct may be designed to have a signal sequence at its N-terminus. Typical signal sequences are shown in Table 7 below.

[0102] [Table 7]

[0103] Therefore, in one embodiment, the fusion protein comprises a signal sequence and a fusion protein, wherein the signal sequence is selected from the group consisting of SEQ ID NOs: 102-104, and the fusion protein is selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. In another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 102 and a fusion protein selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 103 and a fusion protein selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. In yet another embodiment, the fusion protein comprises the signal sequence of SEQ ID NO: 104 and a fusion protein selected from the group consisting of SEQ ID NOs: 88, 90-96, and 98-100. Cells expressing a fusion protein construct containing a signal sequence may be administered to a subject, such as a human subject (e.g., a patient with or at risk of developing α-synuclein disorder). This fusion protein is secreted by cells and helps reduce α-synuclein protein aggregation and / or related cytotoxicity.

[0104] As described above, in certain embodiments, the fusion protein may further contain a cell-permeable peptide. Cells expressing a fusion protein containing a signal sequence and a cell-permeable peptide have the ability to secrete a fusion protein lacking the signal sequence. The secreted fusion protein, also containing the cell-permeable peptide, then has the ability to enter nearby cells and, in those cells, has the potential to reduce α-synuclein protein-mediated aggregation and / or cytotoxicity.

[0105] VI. How to Use In another embodiment, the present invention provides a method for achieving beneficial effects in and / or in alpha-synuclein disorders, disorders, or conditions mediated by alpha-synuclein aggregation. Alpha-synuclein disorders are selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), parkinsonism, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, and Lewy body dementia.

[0106] In some embodiments, the present invention provides a method for treating a subject, such as a human, having an alpha-synuclein disease, disorder, or condition, comprising the step of administering to the subject a therapeutically or prophylactically effective amount of a fusion protein, a nucleic acid encoding such a fusion protein, or a viral vector encoding such a fusion protein as described herein, wherein the administration results in an improvement of one or more biochemical or physiological parameters or clinical endpoints related to the alpha-synuclein disease, disorder, or condition.

[0107] In other embodiments, the present invention provides a method for reducing alpha-synuclein aggregation in cells. The cells may be cultured cells or isolated cells. The cells may also be derived from a subject, e.g., a human subject. In one embodiment, the cells are located within the central nervous system of a human subject. In another embodiment, the human subject has or is at risk of developing an alpha-synuclein disorder, which may include, but is not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. In one particular embodiment, the alpha-synuclein disorder is amyotrophic lateral sclerosis.

[0108] Agglutination of α-synuclein protein can be detected by several methods. For example, agglutinated α-synuclein protein can be detected in an ELISA using an antibody that specifically recognizes the α-synuclein structure. A decrease in α-synuclein protein agglutination can also be directly detected in cells, for example, using immunofluorescence microscopy with a labeling reagent that detects α-synuclein protein (see, e.g., Ding et al., (2015) Oncotarget, 6:24178~24191; Chou et al., (2015) Hum.Mol.Genet. 24:5154~5173, and Example 1). In certain embodiments, a greater decrease in α-synuclein polypeptide levels compared to a control indicates higher efficacy.

[0109] Therefore, in one embodiment, the method includes the step of contacting cells with an amount of a fusion protein or nucleic acid, vector, or viral particle encoding the fusion protein that is effective in reducing α-synuclein protein aggregation by at least 10%, for example, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to untreated or control cells.

[0110] As shown in Example 1 below, it has been found that expression of a fusion protein containing a J domain and an α-synuclein-binding domain reduces the overall level of an α-synuclein-containing reporter construct. Therefore, in another embodiment, the method includes the step of contacting cells with an amount of a fusion protein, cells expressing the fusion protein, or nucleic acid, vector, or viral particles encoding the fusion protein that is effective in reducing the level of 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%, compared to untreated or control cells.

[0111] VII. Pharmaceutical Compositions The compositions envisioned herein may include one or more fusion proteins containing a J domain and an α-synuclein-binding domain, polynucleotides encoding such fusion proteins, vectors containing them, genetically modified cells, etc. The compositions include, but are not limited to, pharmaceutical compositions. “Pharmaceutical composition” means a composition formulated as a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals, either alone or in combination with one or more other therapeutic modalities. It will also be understood that, where necessary, the composition may be administered in combination with other active agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active substances. There are virtually no limitations on other components that may also be included in the composition, provided that the additional active agents do not adversely affect the composition’s ability to deliver the intended treatment. As used herein, the phrase “pharmaceutically acceptable” refers to a compound, material, composition, and / or dosage form that, in proportion to a reasonable benefit / risk ratio, is suitable, within the bounds of sound medical judgment, for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications. As used herein, “pharmaceutically acceptable carrier,” “diluent,” or “excipient” includes, but is not limited to, any adjuvant, carrier, excipient, flow enhancer, sweetener, diluent, preservative, colorant, flavoring, surfactant, humectant, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that is approved by the U.S. Food and Drug Administration as acceptable for use in human or animal husbandry.Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn (com) starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, wax, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn (com) oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and any other suitable substances used in pharmaceutical formulations.

[0112] VIII. Dosage The dosage of the compositions described herein (e.g., compositions comprising fusion protein constructs, nucleic acids, or gene therapy virus particles) may vary depending on many factors, including the pharmacodynamic properties of the compound; the mode of administration; the recipient's age, health, and weight; the nature and severity of symptoms; the frequency of treatment, and, if any, the type of concurrent treatment; and the clearance rate of the compound in the animal to be treated. The compositions described herein may be administered initially at an appropriate dosage, which may be adjusted as needed depending on the clinical response. In some embodiments, the dosage of the composition is a prophylactic or therapeutically effective dose.

[0113] IX. Kit A kit is intended to include (a) a pharmaceutical composition comprising a fusion protein construct, a nucleic acid encoding such a fusion protein, or a viral particle containing such nucleic acid, which reduces the aggregation of α-synuclein protein in cells or subjects as described herein, and (b) a package insert containing instructions for use to carry out any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising the composition described herein that reduces the aggregation of α-synuclein protein in cells or subjects as described herein, (b) an additional therapeutic agent, and (c) a package insert containing instructions for use to carry out any of the methods described herein. [Examples]

[0114] To test whether the J domain can be specifically manipulated to promote the proper folding of aggregated proteins, we designed and tested several fusion protein constructs designed to target α-synuclein protein.

[0115] Example 1 Fusion protein design A. Method General technologies and materials The invention will be carried out using the usual techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, unless otherwise indicated, which are within the capabilities of those skilled in the art. Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3rd edition, Cold Spring Harbor Laboratory Press, 2001; "Current protocols in molecular biology," FM Ausubel et al. (eds.), 1987; "Methods in Enzymology" series, Academic Press, San Diego, Calif.; "PCR 2: a practical approach," MJ MacPherson, B.D. Hames, and GR. Taylor (eds.), Oxford University Press, 1995; "Antibodies, a laboratory manual," Harlow, E. and Lane, D. (eds.), Cold Spring Harbor Laboratory, 1988; "Goodman & Gilman's The Pharmacological Basis of Therapeutics," 11th edition, McGraw-Hill, 2005; and Freshney, RI, "Culture of Animal Cells: A Manual of Basic Technique," 4th edition, John Wiley & See Sons, Somerset, NJ, 2000 (their contents are incorporated herein by reference in their entirety). HEK-293 cells (human embryonic kidney cells) were purchased from the American Type Culture Collection (Manassas, VA). Anti-FLAG antibody was purchased from Thermo Fisher Scientific. Rabbit anti-GFP antibody was purchased from GenScripts (Piscataway, NJ).To facilitate purification and characterization, some of the fusion protein constructs used in this Example 1 contain the FLAG epitope of SEQ ID NO: 78, in addition to the sequences provided in SEQ ID NOs: 88-101, at either the C-terminus or N-terminus of the protein, in addition to a short linker sequence.

[0116] Protein expression and detection in HEK293 cells Expression vector plasmids encoding various protein constructs were transfected into HEK293 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Cell solubles were analyzed for expressed proteins using an immunoblot assay. Before analysis, the culture medium samples were centrifuged to remove debris. Cells were lysed in a lysis buffer containing 2 mM PMSF and a protease cocktail (complete protease inhibitor cocktail; Sigma) (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 2% SDS). After short sonication, the samples were analyzed for expressed proteins using an immunoblot assay. For immunoblot analysis, the samples were boiled in SDS sample buffer and subjected to polyacrylamide electrophoresis. The separated protein bands were then transferred to a PVDF membrane.

[0117] The expressed proteins were detected using chemiluminescent signals. Briefly, the blot was reacted with a primary antibody capable of binding to a specific epitope (e.g., anti-α-synuclein antibody). After washing away the unreacted primary antibody, an enzyme-conjugated secondary antibody (e.g., HRP-conjugated anti-IgG antibody) was reacted with the primary antibody molecules bound to the blot. After rinsing, a chemiluminescent reagent was added, and the resulting chemiluminescent signal in the blot was captured on X-ray film.

[0118] BFA / MG132 Incubation 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 bafilomycin a1 (BFA) or MG132, and the cells were incubated for a further 48 hours. The 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 protease inhibitor cocktail (Complete Protease Inhibitor Cocktail; Sigma) and 2 mM PMSF. After brief sonication, the cell lysates were analyzed by ELISA or immunoblotting assay.

[0119] ELISA (Enzyme-linked immunosorbent assay) The aggregation state of expressed α-synuclein was monitored using the α-synuclein aggregate-specific antibody Syn-O4 (BioLegend). Syn-O4 is a structure-specific monoclonal antibody that specifically recognizes α-synuclein aggregates but does not recognize the soluble monomeric form of the protein. 58 The microtiter plate wells were coated with Syn-O4 overnight at 4°C. After coating, the wells were washed three times with PBS, blocked for 1 hour with PBS containing 1% BSA, and washed again with PBS.

[0120] After expression, cultured cells were dissolved in a lysis buffer containing 2 mM PMSF and a protease cocktail (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40), and then briefly sonicated. After removing debris by centrifugation, the cell solubilates were added to the wells and incubated overnight at 4°C. The wells were then washed three times with PBS, incubated with HRP-conjugated anti-alpha-synuclein antibody in PBS containing 1% BSA for 1 hour, and washed three more times with PBS. Finally, peroxidase activity was assayed 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. After adding an equal volume of 1 M HCl, the optical density was determined at 450 nm.

[0121] Reporter structure The inventors first investigated whether a fusion protein targeting α-synuclein improves its aggregation in cultured cells. To achieve this objective, the inventors created two constructs expressing wild-type α-synuclein and a mutant (containing the A53T substitution) known to be associated with familial Parkinson's disease (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.

[0122] [Table 8]

[0123] Fusion protein constructs To determine whether the J domain could be used to reduce α-synuclein aggregation, initial experiments were performed by co-expression of wild-type or mutant synuclein with a fusion protein (see construct 1 in Table 9 below) containing a J domain sequence derived from the Hsp40 J domain protein (derived from human DnaJB1), conjugated with the synuclein-binding peptide SynBP1. As shown in Figure 3A, expression of either wild-type or mutant (A53T) α-synuclein in HEK293 cells resulted in the appearance of aggregated α-synuclein detectable by ELISA in cell solubles (see bars 1 and 4). Co-expression of construct 1 resulted in a dramatic decrease in the amount of detectable aggregated α-synuclein (bars 2 and 5). However, co-expression of a mutant variant of construct 1 containing the P33Q mutation within the highly conserved HPD motif in the J domain resulted in higher total aggregation (see bars 3 and 6), suggesting that the mechanism of aggregation reduction is through the Hsp70 system. To determine whether the fusion protein affects the total level of α-synuclein, Western blot analysis of cell solubles was performed using an anti-α-synuclein antibody. As shown in Figure 3B, all cells had relatively similar levels of α-synuclein, suggesting that the decrease in aggregated α-synuclein is not solely due to α-synuclein degradation.

[0124] [Table 9]

[0125] Alpha-synuclein is known to be secreted. Therefore, we investigated whether the fusion protein construct is effective in reducing the amount of secreted alpha-synuclein. Culture media were collected from cells expressing wild-type (bars 2 and 3) or mutant (bars 4 and 5) alpha-synuclein alone, or also expressing construct 1 (JB1-SynBP1; bars 3 and 5), and the levels of secreted alpha-synuclein were tested by ELISA analysis. As shown in Figure 4, both wild-type and mutant (A53T) alpha-synuclein are secreted when expressed without the fusion protein construct (bars 2 and 4, respectively). However, cells co-expressing construct 1 do not produce detectable alpha-synuclein secreted into the culture medium when measured by ELISA (bars 3 and 5 for wild-type and mutant alpha-synuclein, respectively).

[0126] The inventors then investigated the mechanism by which the fusion protein reduces aggregation. HEK293 cells were transfected with either wild-type or mutant (A53T) α-synuclein alone or together with construct 1 (JB1-SynBP1), and either BFA (bafilomycin A1, a late-stage autophagy inhibitor) or MG132 (a proteasome inhibitor) was also added. Figures 5 and 6 show the results, which are also summarized in Table 10 below (values ​​are standardized against wild-type alone control). As can be seen, expression of construct 1 in cells also expressing mutant (A53T) α-synuclein resulted in a moderate decrease in total α-synuclein (from 134.9% to 99%), but a more significant decrease in aggregated α-synuclein (from 111.5% to 53.9%). Treatment of these cells with BFA at either 10 nM or 100 nM resulted in the restoration of both whole and aggregated α-synuclein to levels consistent with cells expressing mutant α-synuclein alone (i.e., not transfected with fusion protein construct 1). Bafilomycin A1 is a potent inhibitor of late autophagy, suggesting that the fusion protein construct exerts its effects via chaperone-mediated autophagy.

[0127] In contrast, treatment of cells with MG132 (a proteasome inhibitor) had little effect on either the overall level or the aggregated level of α-synuclein.

[0128] [Table 10]

[0129] Additional constructs were tested for their ability to reduce mutant (A53T) synuclein aggregation. Table 11 below shows that several other constructs containing different synuclein-binding domains were able to reduce α-synuclein aggregation. Interestingly, construct 10, a fusion protein construct containing QBP1, which had previously been shown to bind to polyglutamine repeats, also showed a potent ability to reduce α-synuclein aggregation.

[0130] [Table 11]

[0131] Additional constructs were tested to further optimize the arrangement of the J domain and the α-synuclein-binding domain. Table 12 below shows the capabilities of constructs using either an alternative J domain (construction 6 using a J domain derived from DnaJB6; construction 7 using a J domain derived from DnaJB13) or a different linker between the J domain and the α-synuclein-binding domain.

[0132] [Table 12]

[0133] 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 phosphorylated mutant (A53T) α-synuclein at Ser129.

[0134] [Table 13]

[0135] Finally, experiments were conducted to determine whether the expression of various fusion protein constructs could improve the cytotoxicity induced by the expression of mutant (A53T) α-synuclein. While neither wild-type (WT) nor mutant (A53T) α-synuclein expression induced significant cytotoxicity in HEK293 cells (data not shown), the cytotoxic effect induced by α-synuclein was observed in U-87 MG cells, as evidenced by the increased LDH activity in the culture medium collected from U87-MG cells at post-infection day 7, as measured by the LDH-Cytotox® assay kit (BioLegend, San Diego, CA, USA) (Figure 7). Co-expression with JB1-SynBP1 (construct 1) strongly suppressed the cytotoxic effect of α-synuclein. We interpret these results to mean that fusion protein constructs have the ability to reduce α-synuclein-mediated cytotoxicity.

[0136] Example 2 AAV vector encoding a fusion protein construct Exemplary gene therapy vectors are constructed using an AAV9 vector containing codon-optimized cDNAs encoding the fusion protein constructs shown 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 beta-actin promoter. The cDNA encoding JB1-SynBP1 is located downstream of the Kozak sequence and is polyadenylated by the bovine growth hormone polyadenylation (BGHpA) signal. The entire cassette is flanked by two non-coding terminal inversion sequences of AAV-9.

[0137] Recombinant AAV vectors are prepared using a baculovirus expression system similar to that described above (Urabe et al., 2002; Unzu et al., 2011 (reviewed in Kotin, 2011)). Briefly, three recombinant baculoviruses are used to infect SF9 insect cells: one encoding REP for replication and packaging, one encoding CAP-5 for the AAV9 capsid, and one having an expression cassette. Purification is performed using AVB Sepharose rapid affinity medium (GE Healthcare Life Sciences, Piscataway, NJ). The vectors are titrated using QPCR with primer-probe combinations for the transgene, and the titer is expressed as genome copy number per ml (GC / ml). The titer of the vector is approximately 8 × 10⁶. 13 ~2×10 14 It is between GC / ml.

[0138] Example 3 Efficacy testing of PD in a mouse model The vectors prepared above are tested in a mouse model of PD. A useful model is the mouse model described by Fares et al., (2006) Proc. Natl. Acad. Sci. USA, 113:E912~E921, which is based on SNCA - / -In the background, human α-synuclein expression generates this protein, which in turn produces highly phosphorylated (at S129) and ubiquitin-positive LB-like inclusion bodies in primary neurons. These inclusion bodies enlarge and take up soluble proteins.

[0139] In animal models, different AAV virus particles containing a fusion protein and a vector encoding a corresponding control are administered to transgenic animals to test the effects of the test compounds described in this application or known compounds. In one embodiment, the virus particles are administered by tail vein injection. In another embodiment, the virus particles are administered by intramuscular injection. In yet another embodiment, the particles are administered by intracranial injection, for example, as described by Stanek et al., (2014) Hum. Gene. Ther. 25:461-474.

[0140] After administration, disease progression is monitored and compared to control injected mice. In one embodiment, the development of PD-like inclusions is evaluated in animals. Several other animal models that reproduce PD-like pathology can also be used.

[0141] Other embodiments All publications, patents, and patent applications referenced in this specification are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually referenced and incorporated as a whole. If any term in this application is found to be defined differently from a term in any document incorporated herein by reference, the definition provided herein shall be the definition of that term.

[0142] While the present invention is described in relation to its particular aspects, it is intended and understood that this application is intended to cover any variations, uses, or adaptations of the present invention, including departures from this disclosure, that can be further modified and generally applied to the essential features set forth above, falling within the scope of known or customary practices in the art and generally following the principles of the present invention.

Claims

1. a) The J domain of the J protein, selected from the group consisting of amino acid sequences described in either SEQ ID NOs. 5 and 10, b) An α-synuclein-binding domain selected from the group consisting of amino acid sequences described in any of SEQ ID NOs. 51 to 65, Includes, An isolated fusion protein that has the ability to reduce α-synuclein aggregation in cells.

2. The fusion protein according to claim 1, wherein the J domain of the J protein is selected from the group consisting of amino acid sequences described in either SEQ ID NOs. 5 and 10.

3. The fusion protein according to claim 1, comprising a sequence selected from the group consisting of amino acid sequences described in any of SEQ ID NOs: 88, 93, 95-100.

4. The fusion protein according to claim 1, further comprising a targeting reagent or an epitope selected from the group consisting of amino acid sequences described in any of SEQ ID NOs: 77 to 83.

5. The fusion protein according to claim 1, further comprising a cell-permeable peptide sequence selected from the group consisting of amino acid sequences described in any one of Sequence IDs 84 to 87.

6. The fusion protein according to claim 1, further comprising a signal sequence, wherein the signal sequence comprises a peptide sequence selected from the group consisting of amino acid sequences described in any of SEQ ID NOs: 102 to 104.

7. a) It has the ability to reduce α-synuclein protein misfolding in cells. b) It has the ability to reduce the level of phosphorylated α-synuclein protein in cells. c) It has the ability to reduce the secretion of α-synuclein protein, and, d) It has the ability to reduce α-synuclein-mediated cytotoxicity. A fusion protein according to any one of claims 1 to 6.

8. A nucleic acid encoding the fusion protein described in claim 7.

9. The nucleic acid according to claim 8, comprising a promoter region, a 5'UTR, an 'UTR, and a control domain selected from the group consisting of a combination thereof, wherein the promoter region is selected from the group consisting of a CMV enhancer, a CMV promoter, a CBA promoter, a UBC promoter, a GUSB promoter, a NSE promoter, a synapsin promoter, a MeCP2 promoter, and a GFAP promoter.

10. A vector comprising nucleic acid according to claim 9, wherein the vector is selected from the group consisting of adeno-associated viruses, adenoviruses, lentiviruses, retroviruses, herpesviruses, poxviruses, paramyxoviruses, baculoviruses, reoviruses, alphaviruses, and flaviviruses.

11. A viral particle comprising a capsid and the adeno-associated virus vector according to claim 10, wherein the capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, pseudotyped AAV, rhesus monkey-derived AAV, AAVrh8, AAVrh10, and AAV-DJan AAV capsid variants, AAV hybrid serotypes, organotropic AAV, cardiacotropic AAV, and cardiacotropic AAVM41 variants.

12. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 7, a cell expressing the fusion protein according to any one of claims 1 to 7, a nucleic acid according to claim 8 or 9, a vector according to claim 10, an active substance selected from the group consisting of viral particles according to claim 11, and a pharmaceutically acceptable carrier or excipient.

13. A method for reducing the toxicity of α-synuclein protein in cells, comprising the step of contacting the cells with an effective amount of one or more active substances selected from the group consisting of a fusion protein according to any one of claims 1 to 7, a cell expressing the fusion protein according to any one of claims 1 to 7, a nucleic acid according to claim 8 or 9, a vector according to claim 10, a viral particle according to claim 11, and a pharmaceutical composition according to claim 12, wherein the method is not a therapeutic method performed on a human or animal body.

14. One or more active substances selected from the group consisting of a fusion protein according to any one of claims 1 to 7, a cell expressing the fusion protein according to any one of claims 1 to 7, a nucleic acid according to claim 8 or 9, a vector according to claim 10, a viral particle according to claim 11, and a pharmaceutical composition according to claim 12, for use in reducing α-synuclein aggregation in cells.

15. The active substance according to claim 14, wherein the cells are derived from a subject diagnosed with an α-synuclein disease selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and multiple system atrophy.

16. The use of a fusion protein according to any one of claims 1 to 7, cells expressing the fusion protein according to any one of claims 1 to 7, the nucleic acid according to claim 8 or 9, the vector according to claim 10, or the viral particle according to claim 11, in the preparation of a pharmaceutical for reducing alpha-synuclein aggregation in cells derived from a subject diagnosed with alpha-synuclein disease, wherein the alpha-synuclein disease is selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's disease, Huntington's disease, Alzheimer's disease, hippocampal sclerosis, Lewy body dementia, and multiple system atrophy.

17. The use according to claim 16, wherein the α-synuclein disease is Parkinson's disease.

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