Gene therapy for treating synucleinopathies

Abstract

The present invention relates to a gene therapy for treating synucleinopathies. The present invention provides a novel α-Syn peptide variant and inhibits the formation of amyloid fibrils and degrades α-Syn aggregates, thereby exhibiting excellent preventive, ameliorative, and therapeutic effects on synucleinopathies.

Description

Gene therapy for the treatment of synucleinopathy

[0001] The present invention relates to gene therapy for treating synucleinopathies.

[0002] Synucleinopathies are neurodegenerative diseases characterized by the pathogenic accumulation of α-synuclein (α-Syn) in a subset of neurons and glial cells, including Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (LBD), and multiple system atrophy (MSA).

[0003] The above α-Syn refers to an unstructured soluble protein consisting of 140 amino acids that is mainly expressed in neural tissue, and it is known that familial, early-onset Parkinson's disease and Lewy body dementia are caused when abnormal α-Syn aggregation occurs due to multiplication and missense mutation of SNCA, the gene encoding α-Syn.

[0004] Phenomena that play a crucial role in the onset and progression of synucleinopathies include the spontaneous aggregation of intracellular α-Syn into amyloid fibrils, as well as the prion-like spreading of α-Syn aggregates. Typically, α-Syn aggregates propagate from neuronal axon terminals to subsequent neurons innervated by α-Syn-expressing axon terminals, and these α-Syn aggregates can act as seeds to induce further propagation of α-Syn aggregates.

[0005] In light of the above, despite the need for the development of therapeutic agents capable of inhibiting α-Syn aggregation for the treatment of synucleinopathy, research in this area has made little progress to date. Accordingly, the present inventors provide a genetic construct capable of expressing an α-Syn peptide variant that inhibits amyloid fibril formation and disintegrates α-Syn aggregates, a recombinant expression vector, and uses thereof.

[0006] The present invention aims to provide a novel α-Syn peptide variant.

[0007] The present invention aims to provide novel genetic constructs encoding α-Syn peptide variants and uses thereof.

[0008] The present invention also aims to provide a recombinant expression vector encoding an α-Syn peptide variant and uses thereof.

[0009] The present invention also aims to provide a method for preventing or treating synucleinopathies, comprising administering an α-Syn peptide variant to a subject in need thereof.

[0010] The present invention also aims to provide the use of α-Syn peptide variants in the manufacture of medicaments for the treatment of synucleinopathies.

[0011] The present invention also aims to provide a composition comprising an α-Syn peptide variant for use in the prevention or treatment of synucleinopathies.

[0012] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0013] The present invention provides novel α-Syn peptide variants.

[0014] The present invention also provides a polynucleotide encoding the peptide.

[0015] The above peptide exhibits excellent preventive, improvement and therapeutic effects on synucleinopathy by inhibiting amyloid fibril formation and decomposing α-Syn aggregates.

[0016] In the present invention, "α-Synuclein (α-Syn)" means an unstructured soluble protein composed of 140 amino acids that is mainly expressed in nervous tissue and is mainly present in the central nervous system. α-Synuclein is a key protein associated with Parkinson's disease, and accumulation and aggregation of α-Syn in Lewy bodies and Lewy neurites found in brain lesions are known to be one of the pathological signs of Parkinson's disease.

[0017] In the present invention, “peptide” means a molecule formed by amino acid residues being linked to each other by peptide bonds.

[0018] In the present invention, “peptide variants” refers to peptides in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence of the peptide, and which exhibit almost the same biological function as a peptide composed of the original amino acids.

[0019] Amino acids appearing in the various amino acid sequences presented in the present invention are identified by their known three-letter or one-letter abbreviations (Table 1). Nucleotides appearing in the various nucleic acid fragments are indicated by their standard one-letter designations commonly used in the art.

[0020] As used herein, "amino acid" refers to an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes of the present invention, amino acids include the 20 naturally occurring amino acids, unnatural amino acids, and amino acid analogs (i.e., amino acids in which the α-carbon contains a side chain).

[0021] All sequences of amino acid residues represented in conventional phrases herein represent the conventional amino-terminal to carboxy-terminal direction in left-to-right orientation.

[0022] As used herein, “naturally occurring amino acid” refers to the 20 L-amino acids that appear in peptides.

[0023] SYMBOL1-Letter3-LetterAmino acidsYTyrTyrosineGGlyGlycineFPhePhenylalanineMMetMethionineAAlaAlanineSSerSerineIIleIsoleucineLLeuLeucineTThrThreonineVValValinePProProlineKLysLysineHHisHistidineQGlnGlutamineEGluGlutamic acidZGlxGlu and / or GlnWTrpTryptophanRArgArginineDAspAspartic acidNAsnAsparagineBAsxAsn and / or AspCCysCysteine

[0024] The α-Syn peptide variant of the present invention has the advantage of being expressed like an intracellular protein, and thus can be used without problems such as rapid protein degradation in the bloodstream and reduced delivery through the blood-brain barrier, unlike existing α-Syn aggregation inhibitors. Preferably, the α-Syn peptide variant according to the present invention may be a variant formed based on the wild-type α-Syn protein (SEQ ID NO: 1). The numbering of the peptide sequence according to the present invention is based on the peptide of SEQ ID NO: 1 below.

[0025] α-Syn AA sequence (SEQ ID NO: 1):

[0026] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA*

[0027] The wild-type α-Syn protein mentioned above can be produced from any polynucleotide capable of transcribing it. For example, the wild-type α-Syn protein can be transcribed from the following polynucleotide sequence.

[0028] α-Syn DNA sequence (SEQ ID NO: 2):

[0029] ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGG TGACGGGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

[0030] The α-Syn protein consists of 140 amino acids and is divided into three regions. The N-terminal region, consisting of amino acids 1 to 60, is the region where familial mutations lead to early-onset Parkinson's disease. The non-amyloid component region, consisting of amino acids 61 to 95, is hydrophobic and prone to aggregation, and is primarily involved in α-Syn fibrilization found in Lewy bodies in Parkinson's disease. The C-terminal region, consisting of amino acids 96 to 140, is the region where many post-translational modifications occur, including phosphorylation at Serine-129 (S129).

[0031] The α-Syn peptide variant according to the present invention may include an amino acid mutation based on the full-length sequence of the wild-type α-Syn protein (SEQ ID NO: 1).

[0032] According to one embodiment of the present invention, the α-Syn peptide variant may be a peptide comprising mutations of E46G, G47Q, V48T, V49Y, H50V, G51L, V52P, and A53G, as specifically described in SEQ ID NO: 3.

[0033] The above-mentioned α-Syn peptide variants can be prepared from any polynucleotide capable of transcribing them. As an example, the corresponding DNA sequence is specifically set forth in SEQ ID NO: 4.

[0034] α-Syn peptide variant 1 AA sequence

[0035] (E46G / G47Q / V48T / V49Y / H50V / G51L / V52P / A53G)(SEQ ID NO: 3):

[0036] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKGQTYVLPGTVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA*

[0037] α-Syn peptide variant 1 DNA sequence (SEQ ID NO: 4):

[0038] ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGGCCAGACATATGTGCTGCCTGGAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGG TGACGGGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

[0039] Alternatively, the α-Syn peptide variant may be a peptide comprising mutations V70K, V71I, T72S, G73V, V74R, and T75V, as specifically set forth in SEQ ID NO: 5.

[0040] The above-mentioned α-Syn peptide variants can be prepared from any polynucleotide capable of transcribing them. As an example, the corresponding DNA sequence is specifically set forth in SEQ ID NO: 6.

[0041] α-Syn peptide variant 2 AA sequence

[0042] (V70K / V71I / T72S / G73V / V74R / T75V)(SEQ ID NO: 5):

[0043] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAKISVRVAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA*

[0044] α-Syn peptide variant 2 DNA sequence (SEQ ID NO: 6):

[0045] ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAAAGA TCTCCGTGCGGGTGGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

[0046] Alternatively, the α-Syn peptide variant preferably comprises a substitution at positions 69 and 74 of amino acid residues based on the wild-type α-Syn peptide set forth in SEQ ID NO: 1.

[0047] Specifically, it may be an α-Syn peptide variant comprising amino acid substitutions at A69 and V74 based on the wild-type α-Syn peptide described in SEQ ID NO: 1. Such substitutions may be, for example, any one substitution selected from the group consisting of D, P, E, K, R, N, Q, and H for A69. Additionally, it may be any one substitution selected from the group consisting of P, N, D, E, G, Q, R, H, K, and S for V74.

[0048] Specifically, it comprises any combination of substitutions of the above-mentioned amino acid residues at positions A69 and V74. More specifically, the amino acid substitution comprises any one substitution selected from the group consisting of A69D / V74P, A69P / V74P, A69E / V74P, A69K / V74P, A69R / V74P, A69N / V74P, A69Q / V74P, A69H / V74P, A69D / V74N, A69D / V74D, A69D / V74E, A69D / V74G, A69D / V74Q, A69D / V74R, A69D / V74H, A69D / V74K and A69D / V74S.

[0049] Substitution of amino acids as described above can effectively inhibit the aggregation of α-Syn.

[0050] The above-mentioned sequences are specifically shown in sequence numbers 7 to 23 as shown in Table 2 below.

[0051]

[0052] Preferably, the α-Syn peptide variant may be a peptide comprising mutations A69D and V74P, as specifically described in SEQ ID NO: 7, and the corresponding DNA sequence is specifically described in SEQ ID NO: 24.

[0053] α-Syn peptide variant 3 AA sequence (A69D / V74P) (SEQ ID NO: 7):

[0054] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGDVVTGPTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA*

[0055] α-Syn peptide variant 3 DNA sequence (SEQ ID NO: 24):

[0056] ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGATGTGG TGACGGGTCCGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

[0057] Alternatively, the α-Syn peptide variant may be a peptide comprising mutations E46G, G47Q, V48T, V49Y, H50V, G51L, V52P, A53G, V70K, V71I, T72S, G73V, V74R, and T75V, as specifically set forth in SEQ ID NO: 25.

[0058] The above-mentioned α-Syn peptide variants can be prepared from any polynucleotide capable of transcribing them. As an example, the corresponding DNA sequence is specifically set forth in SEQ ID NO: 26.

[0059] α-Syn peptide variant 4 AA sequence

[0060] (E46G / G47Q / V48T / V49Y / H50V / G51L / V52P / A53G / V70K / V71I / T72S / G73V / V74R / T75V)(SEQ ID NO: 25):

[0061] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKGQTYVLPGTVAEKTKEQVTNVGGAKISVRVAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA*

[0062] α-Syn peptide variant 4 DNA sequence (SEQ ID NO: 26):

[0063] ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGGCCAGACATATGTGCTGCCTGGAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAAAGA TCTCCGTGCGGGTGGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

[0064] Alternatively, the α-Syn peptide variant may be a peptide comprising mutations V40P, V52D, A69D, and V74P, as specifically described in SEQ ID NO: 27.

[0065] The above-mentioned α-Syn peptide variants can be prepared from any polynucleotide capable of transcribing them. As an example, the corresponding DNA sequence is specifically set forth in SEQ ID NO: 28.

[0066] α-Syn peptide variant 5 AA sequence

[0067] (V40P / V52D / A69D / V74P)(SEQ ID NO: 27):

[0068] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYPGSKTKEGVVHGDATVAEKTKEQVTNVGGDVVTGPTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA*

[0069] α-Syn peptide variant 5 DNA sequence (SEQ ID NO: 28):

[0070] ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATCCAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGATGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGATGTGG TGACGGGTCCGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

[0071] Preferably, the α-Syn peptide variant of the present invention may be at least one selected from the group consisting of SEQ ID NOs: 3, 5, 7 to 23, 25, and 27.

[0072] The variants according to the present invention may further include additional mutations, as long as the "biological activity" remains equivalent. Such additional mutations may be conservative amino acid substitutions. Conservative substitutions of suitable amino acids are well known in the art and are generally made without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Other substitutions are also permissible and can be determined experimentally or based on known conservative substitutions. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well defined and known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Even with conservative amino acid substitutions, amino acids can still retain the activity to interfere with the formation of amyloid fibrils and disaggregate α-Syn aggregates. It is expected that there will be.

[0073] In addition, the present invention can utilize peptides having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with respect to each sequence and having substantially the same function and / or effect as the peptide according to the present invention.

[0074] In addition, the encoding sequence for the mutant peptide is also included in the scope of the present invention as a similar mutant sequence related to the α-Syn peptide mutant produced above, as long as the biological activity thereof is maintained at an equivalent level.

[0075] The above α-Syn peptide variant can interfere with the formation of amyloid fibrils and disassemble α-Syn aggregates.

[0076] According to the present invention, the α-Syn peptide variant may include amino acid mutations based on the full-length sequence of the wild-type α-Syn protein (SEQ ID NO: 1). However, mutant peptides in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are truncated at the C-terminus and / or N-terminus are also included within the scope of the present invention.

[0077] The present invention provides a genetic construct comprising a polynucleotide encoding an α-Syn peptide variant and a promoter operably linked thereto.

[0078] Using the construct of the present invention, gene therapy for synucleinopathy can be achieved with a small number of administrations (single or multiple administrations) without the need for direct injection of the recombinant protein. In particular, the α-Syn peptide variant can exhibit excellent disease improvement and therapeutic effects by preventing α-Syn aggregation.

[0079] In the present invention, a polynucleotide encoding an α-Syn peptide variant comprises any sequence encoding the aforementioned α-Syn peptide variant.

[0080] More specifically, it may be a polynucleotide encoding any one or more α-Syn peptide variants selected from the group consisting of SEQ ID NOs: 3, 5, 7 to 23, 25, and 27. Such polynucleotides may be optimized to achieve high expression of the desired α-Syn peptide variant within a genetic construct. The invention is not limited thereto, and exemplary polynucleotides for the production of the variants are shown in SEQ ID NOs: 4, 6, 24, 26, or 28, respectively.

[0081] In the present invention, the term "promoter" refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked to it in a specific host cell, and refers to an untranslated nucleic acid sequence upstream of a coding region that includes a binding site for a polymerase and has transcription initiation activity into mRNA of a gene downstream of the promoter.

[0082] As used herein, the term "operably linked" refers to a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter, signal sequence, or an array of transcription factor binding sites) and another nucleotide. The regulatory sequence can regulate the transcription and / or translation of the other nucleic acid sequence.

[0083] The genetic construct according to the present invention is capable of expressing an α-Syn peptide variant under the control of one or more promoters.

[0084] In the present invention, the promoter sequence may include a promoter sequence generally used in vector expression, a promoter sequence known to be specific to neural cells, or a promoter sequence for overexpression. For example, general promoter sequences that may be used include a CMV (cytomegalovirus) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, an RSV promoter, an EF1 alpha promoter, a metallothionine promoter, a beta-actin promoter, a promoter of a human IL-2 gene, a promoter of a human IFN gene, a promoter of a human IL-4 gene, a promoter of a human lymphotoxin gene, a promoter of a human GM-CSF gene, and the like.

[0085] In addition, for example, neuron-specific promoters such as human synapsin I (hSyn) promoter, mouse calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, rat tubulin alpha I (Tuba1a) promoter, rat neuron-specific enolase (NSE), human platelet-derived growth factor-beta chain (PDGF) promoter, myelin basic protein (MBP) promoter, cluster of differentiation 68 (CD68) promoter, beta-hexosaminidases (HEXB) promoter, and glial fibrillary acidic protein (GFAP) promoter can be used, and overexpression promoters such as EF-1α promoter, CAG promoter, CMV promoter, etc. can be used. Preferably, it can be a CAG promoter.

[0086] The genetic construct according to the present invention may contain one or more suitable transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals that stabilize mRNA in the cytoplasm, e.g., Kozak sequences; sequences that enhance translation efficiency or WPRE; sequences that enhance mRNA stability; and, if desired, sequences that enhance secretion of the encoded product.

[0087] For example, the genetic construct according to the present invention may also comprise an enhancer. Such enhancers are viral enhancers, including but not limited to a CMV enhancer, a WPRE enhancer, an HPRE enhancer, a CTE enhancer, or derivatives or hybrids thereof.

[0088] Additionally, the genetic construct according to the present invention may comprise a Kozak sequence.

[0089] The packaging signal may be a 5' inverted terminal repeat (ITR) and a 3' ITR. For example, the genetic construct comprises AAV ITR sequences for use in an AAV vector. In one embodiment, the ITR is derived from a different AAV than the one supplying the capsid. In a preferred embodiment, the ITR sequence is derived from AAV2, or a deleted version thereof (ITR), which may be used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources may also be selected. When the source of the ITR is AAV2 and the AAV capsid is derived from another AAV source, the resulting vector may be designated a pseudotype. Typically, an AAV vector genome comprises an AAV 5' ITR, any coding sequence according to the invention, any regulatory sequence, and an AAV 3' ITR. However, other arrangements of the elements may also be suitable. A shortened version of the 5' ITR, termed ITR, has been described, which lacks the D-sequence and terminal resolution site (trs). In other embodiments, full-length AAV 5' and 3' ITRs are used.

[0090] In some embodiments, the regulatory sequence comprises a polyadenylation (polyA) signal. In some embodiments, the polyA signal is a bovine growth hormone polyadenylation (bGH polyA) signal, a small polyA (SPA) signal, a human growth hormone polyadenylation (hGH polyA) signal, an SV40 polyA signal, an SV40 late polyA signal, or a derivative or hybrid thereof.

[0091] The genetic constructs according to the present invention can be modified as long as the identity of the construct is maintained. That is, the term "identical" or percent "identity", in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or partial sequences that have a specified percentage (i.e., preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity) of the same or identical amino acid residues or nucleotides over a specified region (e.g., any of the modified ORFs provided herein when compared and aligned for maximum match over a comparison window or a designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website). That is, modifications of the genetic construct are included within the scope of the present invention as long as they maintain the desired functional effect of the α-Syn peptide variant.

[0092] The genetic construct according to the present invention is intended for expression and / or administration in neurons. Specifically, it is intended for expression in cells expressing in neural tissue, more specifically in the brain, including neurons, astrocytes, microglia, oligodendrocytes, and ependymal cells.

[0093] The present invention provides a recombinant expression vector comprising the genetic construct described above. The genetic construct and expression vector described herein can be used for the treatment and improvement of synuclein disease.

[0094] In the present invention, the term "recombinant expression vector" refers to a construct that is capable of expressing a target protein or target RNA in a suitable host cell and includes essential regulatory elements operably linked to enable expression of a gene insert.

[0095] A recombinant expression vector refers to a plasmid, viral vector, or other vector known in the art into which a nucleic acid encoding a genetic construct can be inserted and which can express said nucleic acid in a host cell. Preferably, it may be a viral vector. Examples of such viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, avipoxvirus vectors, lentivirus vectors, and the like. In particular, a method using lentivirus or adeno-associated virus (AAV) is preferred.

[0096] Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles containing an AAV protein capsid that encapsulates a nucleic acid sequence for delivery to target cells. The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the selected AAV. The AAV capsid can be selected from those known in the art, including variants.

[0097] Recombinant AAV (rAAV) can be a naturally occurring vector or a vector having a hybrid AAV serotype.

[0098] "rAAV" refers to a viral particle comprised of an encapsidated polynucleotide rAAV vector comprising at least one AAV capsid protein and a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene to be delivered into a mammalian cell). The rAAV particle may have any AAV serotype, including any modification, derivative, or pseudotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 or derivatives / modifications / pseudotypes thereof). Such AAV serotypes and derivatives / modifications / pseudotypes and methods for producing such serotypes / derivatives / modifications / pseudotypes are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012)). In some embodiments, the rAAV particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, A capsid protein from an AAV capsid serotype selected from AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and AAV.HSC16.In some embodiments, the rAAV particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, A capsid protein that is a derivative, variant, or pseudotype of the AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid protein.

[0099] The rAAV particles of the present disclosure can have any serotype or any combination of serotypes (e.g., a population of rAAV particles comprising two or more serotypes, e.g., two or more of rAAV2, rAAV8, and rAAV9 particles). In some embodiments, the rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAAV particles, or a combination of two or more thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles. In some embodiments, the rAAV particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particle comprises capsid proteins from two or more serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.A capsid protein that is a derivative, variant or pseudotype of two or more serotypes selected from Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and AAV.HSC16 capsid proteins.

[0100] In some embodiments, the rAAV particle has an AAV capsid protein of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, or a derivative, variant, or pseudotype thereof. In some embodiments, the rAAV particle has an AAV capsid protein of the serotype AAV8, AAV9, or a derivative, variant, or pseudotype thereof. In some embodiments, the rAAV particle has an AAV capsid protein of a serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, and AAV.7m8.

[0101] Preferably, the rAAV is any one selected from the group consisting of AAV2, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHP.B, AAV.PHP.eB and AAV.7m8. More preferably, it may be AAV9.

[0102] Adeno-associated virus (AAV) is suitable as a gene delivery system for the present invention because it can infect non-dividing cells and has the ability to infect a variety of cell types. Detailed descriptions of the production and use of AAV vectors are disclosed in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0103] Preferably, the recombinant AAV2 elicits a minimal immune response in the host organism and mediates long-term transgene expression that can persist for at least 1 year after vector administration.

[0104] As used herein, the term "recombinant AAV (rAAV) vector" refers to a recombinant AAV-derived nucleic acid containing at least one terminal repeat sequence.

[0105] A preferred embodiment of a vector comprising the above genetic construct is shown in Fig. 1.

[0106] Specifically, according to FIG. 1, a genetic construct can be constructed so that an α-Syn peptide variant can be expressed by a single promoter, and an example of a polynucleotide expressing each α-Syn peptide variant is shown in SEQ ID NO: 4, 6, 24, 26 or 28. As an example, a genetic construct is constructed by operably linking the above α-Syn peptide variant to a CAG overexpression promoter.

[0107] Optionally, the genetic construct of the present invention may further comprise additional expression cassettes in addition to the α-Syn peptide variants, which may reduce the toxicity of the genetic construct (particularly when loaded onto AAV).

[0108] Such expression cassettes are intended to alleviate toxicity in dorsal root ganglion neurons and may comprise at least one target sequence specific for, for example, miR-183, miR-182, or miR-96. Exemplary sequence information thereof is provided below as SEQ ID NOs: 29 to 33.

[0109] AGTGAATTCTACCAGTGCCATA (SEQ ID NO: 29)

[0110] AGCAAAAATGTGCTAGTGCCAAA (SEQ ID NO: 30)

[0111] AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO: 31)

[0112] AGGGATTCCTGGGAAAACTGGAC (SEQ ID NO: 32)

[0113] AGTGAATTCTCAACGTGCCATA (SEQ ID NO: 33)

[0114] Preferably, it may include an expression cassette including a target sequence of miR-183 to alleviate toxicity of dorsal root ganglion neurons induced upon vector delivery to the central nervous system (DRG inhibition). More preferably, the target sequence of miR-183 may be a sequence in which the sequence described in SEQ ID NO: 33 is repeated four times, i.e., AGTGAATTCTCAACGTGCCATAAGTGAATTCTCAACGTGCCATAAGTGAATTCTCAACGTGCCATAAGTGAATTCTCAACGTGCCATAAGTGAATTCTCAACGTGCCATA (SEQ ID NO: 34).

[0115] Based on the vector information described in FIG. 1, a person skilled in the art can easily produce a genetic construct containing a gene for expression by utilizing the information of the α-Syn peptide variants, promoter information, and expression cassette information mentioned in Tables 2 and 3.

[0116] The present invention provides a pharmaceutical composition comprising the α-Syn peptide variant, the genetic construct or the recombinant expression vector, and a pharmaceutically acceptable carrier.

[0117] The present invention provides a pharmaceutical composition for preventing or treating synucleinopathy comprising the α-Syn peptide variant, genetic construct and / or recombinant expression vector.

[0118] In the present invention, “synucleinopathies” refers to neurodegenerative diseases characterized by pathogenic accumulation of α-synuclein (α-Syn) in a subset of neurons and glial cells.

[0119] Although not limited thereto, the synucleinopathy may be at least one selected from the group consisting of Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (LBD), and multiple system atrophy (MSA).

[0120] Specifically, the synuclein disease may be Parkinson's disease.

[0121] In the present invention, "Parkinson's disease (PD)" refers to a degenerative disease of the brain and nervous system that causes motor and non-motor disorders. The motor disorders include tremor, hypokinesia (e.g., bradykinesia, akinesia, rigidity), postural instability, abnormal gait, or swallowing disorders. Non-motor disorders include autonomic and neuropsychiatric disorders, such as anosmia or sleep disorders. In the context of the present invention, Parkinson's disease includes, but is not limited to, any of the above symptoms.

[0122] In the present invention, “prevention” means any act of inhibiting or delaying the onset of synucleinopathy by administering a composition.

[0123] In the present invention, “treatment” means any action by which the symptoms of the disease are improved or beneficially changed by administration of the composition.

[0124] In the present invention, “improvement” means any action that at least reduces a parameter related to the condition being treated, for example, the degree of a symptom.

[0125] The present invention provides a method for treating, preventing, or alleviating a synucleinopathy in a subject, or for promoting nerve regeneration and / or survival in a subject. The method comprises administering to a subject in need of such treatment a therapeutically effective amount of the α-Syn peptide variant, genetic construct, or recombinant expression vector.

[0126] It will be appreciated that the above α-Syn peptide variants, genetic constructs, or recombinant expression vectors can be used in pharmaceuticals, which can be used as a monotherapy to treat, alleviate, or prevent synucleinopathy, or to promote nerve regeneration and / or survival.

[0127] Alternatively, the α-Syn peptide variants, genetic constructs or recombinant expression vectors according to the present invention may be used in addition to or in combination with known therapies for treating, alleviating or preventing synucleinopathies.

[0128] The present invention also provides an α-Syn peptide variant, genetic construct or recombinant expression vector for use in the treatment of synucleinopathy.

[0129] The present invention also provides the use of an α-Syn peptide variant, genetic construct or recombinant expression vector in the manufacture of a medicament for use in the treatment of a synucleinopathy.

[0130] The α-Syn peptide variants, genetic constructs, or recombinant expression vectors according to the present invention can be combined in compositions having a variety of different forms, particularly depending on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposomal suspension, or any other suitable form that can be administered to a human or animal in need of treatment. It will be appreciated that the carrier of the medicament according to the present invention must be well tolerated by the subject to which it is administered.

[0131] In a preferred embodiment, the agent according to the present invention can be administered to a subject by injection into the bloodstream, into a nerve, or directly into the site requiring treatment. For example, the agent is configured to cross the blood-brain barrier. The injection can be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intrathecal, intracisternal, or intracerebroventricular.

[0132] It will be understood that the amount of α-Syn peptide variant, genetic construct, or recombinant expression vector required will be determined by its biological activity and bioavailability, which in turn will depend on the route of administration, the physicochemical properties of the peptide variant, genetic construct, or recombinant expression vector, and whether it is used as a monotherapy or in a combination therapy. The frequency of administration will also be influenced by the half-life of the circulating polypeptide within the subject being treated. The optimal dosage to be administered can be determined by one of skill in the art and will vary with the particular peptide variant, genetic construct, or recombinant expression vector being used, the strength of the pharmaceutical composition, the route of administration, and the progression or stage of the disorder. Depending on the particular subject being treated, additional factors including subject age, weight, sex, diet, and timing of administration may necessitate dosage adjustment.

[0133] Typically, depending on the α-Syn peptide variant, genetic construct or recombinant expression vector used, a daily dose of 0.001 μg / kg to 10 mg / kg of body weight, or 0.01 μg / kg to 1 mg / kg of body weight, of the peptide variant, construct or vector according to the present invention can be used to treat, alleviate or prevent synucleinopathy.

[0134] The α-Syn peptide variant, genetic construct or recombinant expression vector can be administered before, during or after the onset of the disease and / or disorder.

[0135] Known procedures, such as those commonly employed in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to formulate specific formulations of the genetic constructs or recombinant expression vectors according to the present invention and to formulate precise treatment regimens (e.g., daily dosage and frequency of administration of the formulation).

[0136] In the present invention, the "subject" may be a vertebrate, mammal, or domestic animal. Therefore, the compositions and agents of the present invention may be used to treat any mammal, such as livestock (e.g., horses), pets, or in other veterinary applications. However, most preferably, the subject is a human.

[0137] A “therapeutically effective amount” of an α-Syn peptide variant, genetic construct, recombinant expression vector or pharmaceutical composition is any of the aforementioned amounts that, when administered to a subject, is necessary to treat a synucleinopathy or to produce a desired effect, such as promoting nerve regeneration and / or survival.

[0138] For example, a therapeutically effective amount of the α-Syn peptide variant, genetic construct, recombinant expression vector, or pharmaceutical composition used may be from about 0.01 mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg. It is preferred that the amount of the genetic construct, recombinant expression vector, or pharmaceutical composition be from about 0.1 mg to about 250 mg.

[0139] A "pharmaceutically acceptable carrier" as referred to herein is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0140] The pharmaceutical carrier may be liquid, and the pharmaceutical composition is in the form of a solution. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The genetic constructs or recombinant expression vectors according to the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid carrier may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colors, viscosity modifiers, stabilizers, or tonicity modifiers. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives such as those described above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. Liquid carriers for pressurized compositions may be halogenated hydrocarbons or other pharmaceutically acceptable propellants.

[0141] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be used, for example, by intracerebral, intramuscular, intrathecal, epidural, intrathecal, intraperitoneal, intravenous, and subcutaneous injection. Genetic constructs or recombinant expression vectors can be prepared as sterile solid compositions that can be dissolved or suspended in sterile water, saline, or other suitable sterile injectable media for administration.

[0142] The α-Syn peptide variants, genetic constructs, recombinant expression vectors, and pharmaceutical compositions of the present invention may be administered orally in the form of sterile solutions or suspensions containing other solutes or suspending agents (e.g., saline or glucose sufficient to render the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (the oleate ester of sorbitol and its anhydride copolymerized with ethylene oxide), or the like. The α-Syn peptide variants, genetic constructs, recombinant expression vectors, or pharmaceutical compositions of the present invention may also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0143] The present invention provides a method for preventing or treating synucleinopathies, comprising administering the above-mentioned α-Syn peptide variant to a subject in need thereof.

[0144] The present invention provides the use of the above-mentioned α-Syn peptide variant in the manufacture of a medicament for the treatment of synucleinopathies.

[0145] The present invention provides a composition comprising the above-mentioned α-Syn peptide variant for use in the prevention or treatment of synucleinopathies.

[0146] Any feature described in this application (including all attached claims, abstract and drawings) and / or any step of any method or process so disclosed may be combined with any of the above embodiments in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0147] The present invention provides a novel α-Syn peptide variant that exhibits excellent preventive, improvement and therapeutic effects on synucleinopathy by inhibiting amyloid fibril formation and decomposing α-Syn aggregates.

[0148] FIG. 1 is a diagram illustrating an AAV vector comprising a polynucleotide encoding an α-Syn peptide variant and a CAG promoter operably linked thereto, as an example of an AAV vector.

[0149] Figure 2 is a diagram confirming that α-Syn aggregation inhibitors (αSyn Inh1, 2, 3, 4, 5) do not aggregate on their own and do not form fibrils.

[0150] Figure 3 is a diagram confirming that wild-type (WT) α-Syn aggregation was inhibited by α-Syn aggregation inhibitors (αSyn Inh1, 2, 3, 4, 5).

[0151] Figure 4 is a diagram confirming that αSyn Inh3 inhibits aggregation of wild-type (WT) α-Syn and is effective in disassembling aggregates.

[0152] Figure 5 is a diagram confirming that when wild-type (WT) α-Syn and αSyn Inh3 were cultured together, β-sheet structures and amyloid fibrils were not observed in wild-type (WT) α-Syn.

[0153] Figure 6 is a diagram showing a cell culture model transfected in the presence of αSyn Inh3, confirming that αSyn Inh3 inhibits seeding of α-Syn aggregates.

[0154] Figure 7 is a diagram showing a cell culture model transfected in the presence of αSyn Inh3, confirming that αSyn Inh3 inhibits the propagation of α-Syn aggregates.

[0155] Figure 8 shows that administration of AB103 has a positive effect on motor ability and cognitive memory ability of the mThy1-αSyn mouse model through the Rotarod Test, Beam Walking Test, and Novel Object Recognition Test.

[0156] Figure 9 is a diagram confirming that administration of AB103 reduces pS129 α-Syn levels and alleviates neuroinflammation in the mThy1-αSyn mouse model.

[0157] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0158] Example 1. Screening of α-Syn aggregation-inhibiting mutant candidates

[0159] To screen for α-Syn aggregation-inhibiting mutant candidates, the TANGO and AGGRESCAN programs were used to identify candidates in silico.

[0160] It was confirmed that improved efficacy can be exhibited when A69 has any one substitution selected from the group consisting of D, P, E, K, R, N, Q, and H, or when V74 has any one substitution selected from the group consisting of P, N, D, E, G, Q, R, H, K, and S.

[0161] In particular, A69D / V74P, A69P / V74P, A69E / V74P, A69K / V74P, A69R / V74P, A69N / V74P, A69Q / V74P, A69H / V74P, A69D / V74N, A69D / V74D, A69D / V74E, A69D / V74G, A69D / V74Q, A69D / V74R, A69D / V74H, A69D / V74K and A69D / V74S were identified as examples of suitable mutant forms. The relevant sequences are individually shown in SEQ ID NOs: 3, 5, 7 to 23, 25, and 27.

[0162] Example 2. Preparation of α-Syn aggregation-inhibiting mutant candidate

[0163] Considering that amino acids 61 to 95 of monomeric α-Syn are important for fibril formation, five mutant candidates capable of preventing wild-type α-Syn aggregation were prepared through amino acid substitutions in that region.

[0164] Each mutant candidate was designated as αSyn Inh1 (SEQ ID NO: 3), Inh2 (SEQ ID NO: 5), Inh3 (SEQ ID NO: 7), Inh4 (SEQ ID NO: 25), and Inh5 (SEQ ID NO: 27).

[0165] More specifically, the αSyn Inh1 (SEQ ID NO: 3) was designed to have mutations of E46G / G47Q / V48T / V49Y / H50V / G51L / V52P / A53G. The Inh2 (SEQ ID NO: 5) was designed to have mutations of V70K / V71I / T72S / G73V / V74R / T75V. The Inh3 (SEQ ID NO: 7) was designed to have mutations of A69D / V74P. The Inh4 (SEQ ID NO: 25) was designed to have mutations of E46G / G47Q / V48T / V49Y / H50V / G51L / V52P / A53G / V70K / V71I / T72S / G73V / V74R / T75V. The above Inh5 (SEQ ID NO: 27) was designed to have mutations of V40P / V52D / A69D / V74P.

[0166] The peptide having the above mutant sequence was synthesized and purified by Abrain Co., Ltd. and used in the following experiments.

[0167] Analysis of fibril formation was performed using Thioflavin-T (ThT) fluorescence analysis. As a result of Thioflavin-T (ThT) fluorescence analysis, it was confirmed that none of the five mutants self-aggregated or formed fibrils (Fig. 2).

[0168] As above, it was confirmed that the manufactured mutant did not form self-aggregation and whether it could competitively inhibit the fibrillation of wild-type (WT) α-Syn.

[0169] Specifically, before the fibrillation reaction of wild-type (WT) α-Syn, WT and each mutant were mixed in an equimolar ratio and Thioflavin-T (ThT) fluorescence analysis was performed. As a result, all five mutants were found to reduce the fibrillation of wild-type (WT) α-Syn (Fig. 3).

[0170] Among them, αSyn Inh3 and αSyn Inh4 showed excellent effects, and in particular, αSyn Inh3 completely prevented the aggregation of wild-type (WT) α-Syn, and prevented aggregation very effectively even at a low concentration (Fig. 4a).

[0171] To determine whether αSyn Inh3 can degrade wild-type (WT) α-Syn after fibril formation, aggregated α-Syn and αSyn Inh3 at different concentrations (5, 25, and 50 μM) were mixed in equimolar ratios and Thioflavin-T (ThT) fluorescence analysis was performed. αSyn Inh3 degraded wild-type (WT) α-Syn in a concentration-dependent manner, with >60% loss of ThT signal when 50 μM αSyn Inh3 was mixed ( Fig. 4b ).

[0172] That is, it was confirmed that the most suitable mutant as an α-Syn aggregation inhibitor is αSyn Inh3, which is effective in inhibiting aggregation and degrading wild-type (WT) α-Syn without causing its own aggregation.

[0173] Example 3. Confirmation of the effect of α-Syn aggregation inhibitor (αSyn Inh3)

[0174] 3-1. Secondary structural changes in wild-type (WT) α-Syn

[0175] Three groups (wild-type (WT) α-Syn, αSyn Inh3, and a mixture of wild-type (WT) α-Syn and αSyn Inh3) were cultured sequentially. Considering that amyloid fibrils mainly have a β-sheet structure, the structural characteristics of wild-type (WT) α-Syn aggregates were analyzed using circular dichroism (CD) spectroscopy. As a result, when wild-type (WT) α-Syn and αSyn Inh3 were cultured as an equimolar mixture, no β-sheet structure was observed in wild-type (WT) α-Syn (Fig. 5a).

[0176] Furthermore, the three groups cultured sequentially were imaged using transmission electron microscopy (TEM). As a result, amyloid fibrils were formed in wild-type (WT) α-Syn, but no amyloid fibrils were observed in the mixture of αSyn Inh3, wild-type (WT) α-Syn, and αSyn Inh3 (Fig. 5b).

[0177] This suggests that αSyn Inh3 effectively inhibits the formation of amyloid fibrils in α-Syn.

[0178] 3-2. Inhibition of α-Syn aggregate seeding

[0179] N2a (neuroblastoma) cell lines stably expressing YFP-tagged full-length wild-type (WT) α-Syn (αSyn WT-YFP) were transfected with α-Syn aggregates in the presence or absence of an α-Syn aggregation inhibitor (αSyn Inh3) (Fig. 6a).

[0180] Subsequently, YFP-labeled proteins were identified by the number of fluorescent dots. In the cell culture model transfected in the absence of αSyn Inh3, dots were observed in approximately 20%. In contrast, in the cell culture model transfected in the presence of αSyn Inh3, almost no dots were observed (Fig. 6b).

[0181] This indicates that αSyn Inh3 can inhibit the seeding of α-Syn aggregates and thereby prevent the occurrence of aggregation itself.

[0182] 3-3. Propagation suppression

[0183] A plasmid that simultaneously expresses the α-Syn aggregation inhibitor (αSyn Inh3) gene and RFP was transfected into an N2a cell line stably expressing YFP-tagged full-length wild-type (WT) α-Syn (αSyn WT-YFP), and the N2a cell line was then transfected with α-Syn aggregates in the presence or absence of the α-Syn aggregation inhibitor (αSyn Inh3).

[0184] In the cell model in the absence of αSyn Inh3, the size and number of fluorescent spots continued to increase over time, whereas in the cell model in the presence of αSyn Inh3, the number of fluorescent spots remained constant and the increase in spot size was also suppressed (Fig. 7).

[0185] This suggests that αSyn Inh3 has the effect of inhibiting the propagation of α-Syn aggregates.

[0186] That is, through the above experimental results, it was confirmed that αSyn Inh3 can strongly inhibit the seeding and propagation of α-Syn aggregates.

[0187] Example 4. Production of AAV expressing α-Syn aggregation-inhibiting mutants

[0188] An AAV vector containing the nucleotide sequence of an α-Syn aggregation-inhibiting mutant was prepared, and a schematic diagram thereof is shown in Figure 1.

[0189] Specifically, to express the αSyn aggregation-inhibiting mutant, pAAV-CAG-WPRE-DRG inhibition-polyA was constructed, which contains a CMV enhancer and chicken β-actin promoter (CAG promoter) that increase the transcription rate, a Kozak sequence, which is a nucleic acid motif that functions as a protein translation initiation site in eukaryotic mRNA transcripts, WPRE linked to the 3' end of the Kozak sequence to stabilize the mRNA, a dorsal root gaglion (DRG) inhibition sequence to prevent expression in the spinal cord, a bGH poly A sequence, and AAV2 ITR.

[0190] In addition, DNA expressing αSyn Inh1, αSyn Inh2, αSyn Inh3, αSyn Inh4, or αSyn Inh5 was amplified, cut using restriction enzymes HindIII and NheI, and cloned into pAAV-CAG-WPRE-DRG inhibition-polyA to produce pAAV-CAG-αSyn Inh1-WPRE-DRG inhibition-polyA, pAAV-CAG-αSyn Inh2-WPRE-DRG inhibition-polyA, pAAV-CAG-αSyn Inh3-WPRE-DRG inhibition-polyA, pAAV-CAG-αSyn Inh4-WPRE-DRG inhibition-polyA, or pAAV-CAG-αSyn Inh5-WPRE-DRG inhibition-polyA, respectively.

[0191] For DRG, it was designed to include sequence number 33.

[0192] Similar to the manufacturing process in the above steps, an AAV vector was designed that includes an AAV vector containing the nucleotide sequence of an additional α-Syn aggregation-inhibiting variant as shown in Table 3.

[0193] Polynucleotide sequence of promoter α-Syn aggregation suppressor mutants CAG promoter E46G / G47Q / V48T / V49Y / H50V / G51L / V52P / A53G mutation (SEQ ID NO: 4) CAG promoter V70K / V71I / T72S / G73V / V74R / T75V mutation (SEQ ID NO: 6) CAG promoter A69D / V74P mutation (SEQ ID NO: 24) CAG promoter E46G / G47Q / V48T / V49Y / H50V / G51L / V52P / A53G / V70K / V71I / T72S / G73V / V74R / T75V mutation (SEQ ID NO: 26) CAG promoter V40P / V52D / A69D / V74P mutation (Sequence number 28)

[0194] Additionally, HEK293T cells were transfected with three plasmids (pHelper, pAAV2 / 9n, and ssAAV-αSyn Inh3 gene, or pHelper, pAAV2 / 9n, and scAAV-αSyn Inh3 gene) using the polyethyleneimine (PEI) transfection method. After 24 hours, the medium used for the infection was replaced with fresh medium, and rAAV vectors were harvested at approximately 72 hours after infection.

[0195] All rAAV vectors in cell lysates and media were precipitated with 40% PEG 8000, 2.5 M NaCl. The precipitates were treated with benzonase at 37°C for 1 h, purified and concentrated by centrifugation using an iodixanol density gradient, and the buffer was exchanged using a 100 kDa cutoff concentrator. The rAAV vector titer was measured using RT PCR, and the purity of the rAAV vector was assessed by loading the rAAV vector onto a 10% SDS acrylamide gel.

[0196] Example 5. Confirmation of the effectiveness of AB103 (AAV expressing αSyn Inh3)

[0197] Experimental Example 1. Rotarod Test

[0198] A training trial was conducted for 5 minutes at 4 rpm on a rotarod. After 1 hour, two consecutive accelerating trials were conducted for 6 minutes at varying speeds from 0 to 40 rpm.

[0199] Experimental Example 2. Challenging Beam Tranversal Test

[0200] The beam was made of Plexiglas and consisted of four sections (each 25 cm long, for a total length of 1 m), each with a different width. The beam started at 3.5 cm and narrowed in 0.5 x 1 cm increments, and a 1 cm wide ledge was placed beneath the upper surface of the beam. Mice were trained for 2 days to traverse the beam, starting from the widest section and ending with the narrowest and most difficult section. All training was performed without a mesh grid.

[0201] On the day of the experiment, to increase the difficulty, a mesh grid (1 cm X 1 cm square) was placed on the beam surface, leaving a space of ~1 cm between the grid and the beam surface. During a total of five trials, the mouse was videotaped crossing the beam and replayed in slow motion to evaluate escape latency and the number of error steps. Escape latency was measured as the time it took for the animal to reach the platform and touch the beam with all four paws, and the number of error steps was calculated based on the time when one leg (forelimb or hindlimb) passed the grid and became visible between the grid and the beam surface as the mouse moved forward.

[0202] Experimental Example 3. Novel Object Recognition Test

[0203] On days 1 and 2, place the mouse in an empty test box (54 X 38 X 35 cm) once a day. 3 ) for 5 minutes to eliminate potential neophobic responses. On the third day, two identical objects were placed at opposite locations at the same distance from each other in each corner of the test box, and the mouse was placed into the box to explore for 5 minutes. After 1 hour, the two objects were replaced with new objects, and the mouse was placed again for observation.

[0204] Experimental Example 4. Immunofluorescence

[0205] Samples were cut to a thickness of 20 μm using a Leica CM1950 (Leica Microsystems, Germany), and the sections were stored in a preservation solution at 4°C until staining. The slides were washed in phosphate-buffered saline (PBS) and blocked in a solution containing PBS, 5% normal goat serum, and 0.3% Triton X-100 for 1 h at room temperature. The sections were incubated with primary antibodies (GFAP, pS129) overnight at 4°C. The sections were then washed in PBS and incubated with secondary antibodies for 1 h at room temperature. Finally, the sections were washed in PBS, completely dried, and mounted in a mounting solution containing DAPI.

[0206] 5-1. Confirmation of improved motor function and cognitive memory ability

[0207] An mThy1-αSyn mouse model (tg mouse) overexpressing full-length human wild-type (WT) α-Syn under the Thy-1 promoter was prepared, and AB103 (AAV expressing αSyn Inh3) was administered to the mouse model via intracerebroventricular injection.

[0208] First, to evaluate motor function, the Rotarod Test and the Challenging Beam Tranversal Test were performed 28 weeks after administration.

[0209] As a result of the Rotarod Test, the tg mouse group (G3) administered AB103 showed a significantly higher average speed on the Rotarod compared to the control group (G2) (Fig. 8a).

[0210] In addition, the results of the challenging beam traversal test showed that the escape latency was reduced and the number of error steps was significantly reduced in the tg mouse group (G3) administered AB103 compared to the control group (G2) (Figures 8b and 8c).

[0211] This suggests that AB103 can improve the impaired motor ability of the mThy1-αSyn mouse model.

[0212] Next, the Novel Object Recognition Test was conducted to evaluate cognitive memory ability. As a result, the discrimination index (DI) of the tg mouse group injected with AB103 (G3) was significantly higher than that of the control group (G2) (Fig. 8d).

[0213] This suggests that AB103 can improve impaired cognitive memory ability in the mThy1-αSyn mouse model.

[0214] 5-2. Confirmation of α-Syn levels and neuroinflammation reduction effects

[0215] α-Syn phosphorylation at Serine-129 (pS129) is the most commonly used biomarker for synucleinopathies, including Parkinson's disease.

[0216] Accordingly, when pS129 α-Syn in the hippocampus was confirmed using immunofluorescence, the pS129 α-Syn level in the tg mouse group (G3) administered with AB103 was found to be significantly reduced compared to the control group (G2) (Fig. 9a).

[0217] Next, to determine whether neuroinflammation, a key pathological feature of Parkinson's disease, was improved, glial fibrillary acidic protein (GFAP)-positive glial cells were examined. Analysis using immunofluorescence revealed a significant decrease in these glial cells in the hippocampus of the tg mouse group (G3) administered AB103 (Fig. 9b).

[0218] That is, the above experimental results confirmed that AB103 can treat synucleinopathies by reducing the pS129 α-Syn level and alleviating neuroinflammation in the mThy1-αSyn mouse model.

[0219] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An α-Syn peptide variant comprising amino acid substitutions at A69 and V74 based on the wild-type α-Syn peptide described in SEQ ID NO:

1.

2. An α-Syn peptide variant according to claim 1, wherein the amino acid substitution comprises any one substitution selected from the group consisting of A69D / V74P, A69P / V74P, A69E / V74P, A69K / V74P, A69R / V74P, A69N / V74P, A69Q / V74P, A69H / V74P, A69D / V74N, A69D / V74D, A69D / V74E, A69D / V74G, A69D / V74Q, A69D / V74R, A69D / V74H, A69D / V74K and A69D / V74S.

3. An α-Syn peptide variant according to claim 1, wherein the peptide is any one selected from the group consisting of SEQ ID NOs: 7 to 23.

4. A genetic construct comprising a polynucleotide encoding an α-Syn peptide variant; and a promoter operably linked thereto.

5. A genetic construct according to claim 4, wherein the polynucleotide encoding the α-Syn peptide variant is a polynucleotide encoding a peptide selected from the group consisting of SEQ ID NOs: 3, 5, 7 to 23, 25, and 27.

6. A genetic construct according to claim 4, wherein the promoter is any one selected from the group consisting of human synapsin I (SYN) promoter, mouse calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, rat tubulin alpha I (Ta1) promoter, rat neuron-specific enolase (NSE), human platelet-derived growth factor-beta chain (PDGF) promoter, myelin basic protein (MBP) promoter, cluster of differentiation 68 (CD68) promoter, beta-hexosaminidases (HEXB) promoter, glial fibrillary acidic protein (GFAP) promoter, EF-1α promoter, CAG promoter, and CMV promoter.

7. A genetic construct according to claim 6, wherein the promoter is a CAG promoter.

8. A genetic construct according to claim 4, wherein the genetic construct further comprises at least one selected from the group consisting of an enhancer sequence, a polyadenylation sequence, and a Kozak sequence.

9. A genetic construct according to claim 4, wherein the polynucleotide encoding the α-Syn peptide variant is any one selected from the group consisting of SEQ ID NOs: 4, 6, 24, 26, and 28.

10. A recombinant expression vector comprising a genetic construct according to any one of claims 4 to 9.

11. In the 10th paragraph, the recombinant expression vector is any one selected from the group consisting of an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, an avipoxvirus vector, and a lentivirus vector.

12. In the 11th paragraph, the recombinant expression vector is an adeno-associated virus (AAV) vector, and is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, A recombinant expression vector, any one selected from the group consisting of AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and AAV.HSC16.

13. A recombinant expression vector according to claim 12, wherein the adeno-associated virus (AAV) vector is any one selected from the group consisting of AAV2, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, and AAV.7m8.

14. A pharmaceutical composition for preventing or treating synucleinopathies, comprising a genetic construct according to any one of claims 4 to 9.

15. A pharmaceutical composition according to claim 14, wherein the synucleinopathies are at least one selected from the group consisting of Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (LBD), and multiple system atrophy (MSA).

16. A pharmaceutical composition according to claim 15, wherein the synucleinopathies are Parkinson's disease (PD).

17. A pharmaceutical composition for preventing or treating synucleinopathies, comprising an α-Syn peptide variant according to any one of claims 1 to 3.

18. A pharmaceutical composition according to claim 17, wherein the synucleinopathies are at least one selected from the group consisting of Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (LBD), and multiple system atrophy (MSA).

19. A pharmaceutical composition according to claim 18, wherein the synucleinopathies are Parkinson's disease (PD).

20. A method for preventing or treating synucleinopathies, comprising administering to a subject in need thereof a therapeutically effective amount of an α-Syn peptide variant comprising amino acid substitutions at A69 and V74 based on the wild-type α-Syn peptide described in SEQ ID NO:

1.

21. A method according to claim 20, wherein the synucleinopathies are at least one selected from the group consisting of Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (LBD), and multiple system atrophy (MSA).

22. A method according to claim 21, wherein the synucleinopathies are Parkinson's disease (PD).

23. Use of an α-Syn peptide variant comprising amino acid substitutions at A69 and V74 based on the wild-type α-Syn peptide set forth in SEQ ID NO: 1 for the manufacture of a medicament for the prevention or treatment of synucleinopathies.

24. The use according to claim 23, wherein the synucleinopathies are at least one selected from the group consisting of Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (LBD), and multiple system atrophy (MSA).

25. The use according to claim 24, wherein the synucleinopathies are Parkinson's disease (PD).

26. A pharmaceutical composition comprising an α-Syn peptide variant comprising amino acid substitutions at A69 and V74 based on the wild-type α-Syn peptide set forth in SEQ ID NO: 1 for use in the prevention or treatment of synucleinopathies.

27. A pharmaceutical composition according to claim 26, wherein the synucleinopathies are at least one selected from the group consisting of Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (LBD), and multiple system atrophy (MSA).

28. A pharmaceutical composition according to claim 27, wherein the synucleinopathies are Parkinson's disease (PD).

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