Genetic constructs for silencing α-synuclein and their use

Abstract

The present invention relates to nucleic acids, compositions and medical uses of said compositions in the treatment and / or prevention of Parkinson's disease (PD), multiple system atrophy (MSA) and / or other alpha-synucleopathies.

Description

[Technical Field]

[0001] The present invention relates to nucleic acids, particularly in the context of gene therapy, for reducing and / or knocking down transcripts of alpha-synuclein (α-syn) genes (SNCAs) to treat and / or prevent Parkinson's disease (PD) and other alpha-synucleopathies, and to the use of such nucleic acids. [Background technology]

[0002] Fibrous α-synuclein inclusions define two major classes of neurodegenerative diseases: Lewy body diseases, e.g., PD, Lewy body dementia (LBD), e.g., PD with dementia (PDD), and dementia with Lewy bodies (DLB), as well as multiple system atrophy (MSA), all characterized by Papp-Lantos bodies. These are collectively called synucleinopathy.

[0003] Parkinson's disease (PD) is a complex, progressive neurodegenerative disorder that can cause both motor and non-motor symptoms. Typical clinical features of PD include bradykinesia, resting tremor, rigidity, and / or postural instability that develops later in life. Non-motor symptoms can occur before and / or after clinical diagnosis. These include depression, sleep disturbances, pain, and fatigue in the early stages of the disease, and anxiety, dementia, and cognitive impairment in the later stages. Both motor and non-motor symptoms are debilitating for patients and burdensome for caregivers.

[0004] Parkinson's disease (PD) is a complex disorder whose cause remains unknown, although numerous genes have been identified as being involved in its cause and / or development. The main feature of PD pathology is the neurodegeneration of dopaminergic neurons in the substantia nigra, a midbrain region with dopaminergic projections associated with the striatum and cortex, which are central to motor-related functions. In addition to the loss of dopaminergic innervation in the substantia nigra and striatum and degeneration in other brain regions, PD is characterized by the presence of cytoplasmic protein aggregates (Lewy bodies) containing insoluble α-syn protein.

[0005] Native α-syn proteins in the brain lack a defined tertiary structure and are mostly unfolded. When interacting with negatively charged lipids such as phospholipids that make up the cell membrane, α-syn folds into an α-helical structure via its N-terminus. However, in PD, α-syn adopts an amyloid-like structure rich in easily aggregated β-sheets. These aggregates constitute the majority of Lewy bodies.

[0006] Massive neurodegenerative disorders (MSA) are progressive, adult-onset neurodegenerative disorders of unknown etiology characterized by argyrophilic glial cytoplasmic inclusions (GCIs) and distinctive oligodendrogliosis with selective neurodegeneration. GCIs, or Papp-Lantos inclusions / bodies, are now accepted as definitive neuropathological features of MSA and are suggested to play a central role in the pathogenesis of this disorder. GCIs are composed of hyperphosphorylated α-syn, ubiquitin, LRRK2 (leucine-rich repeat serine / threonine protein), and other proteins.

[0007] Generally speaking, α-syn proteins tend to form aggregates, and these aggregates can lead to loss of normal function and / or toxic effects in neurons, resulting in neurodegeneration and / or neuroinflammation in different brain regions. Furthermore, mutations or duplication / triplication of the α-syn gene are known to be associated with α-synucleopathy.

[0008] Currently, therapies for treating and / or preventing disease are based on the complete knockdown of genes and / or gene transcripts. However, due to the crucial physiological role of α-syn, depletion of the α-syn protein may raise patient safety concerns, such as attenuation of synaptic transmission in the central nervous system (CNS).

[0009] Therefore, there remains a need for therapies that can treat and / or prevent PD and / or other synucleinopathy while reducing and / or preventing undesirable safety risks. [Overview of the project] [Means for solving the problem]

[0010] A first aspect of the present invention relates to a nucleic acid ("the nucleic acid of the present invention") comprising a nucleic acid sequence encoding RNA ("the RNA of the present invention"), wherein the RNA sequence contained in the RNA is substantially complementary to the target sequence of the α-synuclein (α-syn) gene (SNCA), the RNA sequence has at least 15 nucleotides, and the RNA contains a hairpin.

[0011] A second aspect of the present invention relates to the nucleic acid of the present invention, which is a DNA molecule ("the DNA molecule of the present invention").

[0012] A third aspect of the present invention relates to an adeno-associated virus (AAV) vehicle containing a DNA molecule ("the AAV (vehicle) of the present invention").

[0013] Further aspects of the present invention relate to compositions comprising the AAV vehicle of the present invention and at least one pharmaceutically acceptable excipient; methods for producing the AAV vehicle of the present invention; and, in the case of the present invention, kits comprising the AAV vehicle, further comprising an immunosuppressive compound. [Modes for carrying out the invention]

[0014] The present invention relates to gene therapy, and more particularly to the use of RNA interference (RNAi) in gene therapy to target RNA encoded by the α-syn gene, preferably the human α-syn gene.

[0015] nucleic acid According to the present invention, a nucleic acid ("the nucleic acid of the present invention") is provided which comprises a nucleic acid sequence encoding RNA ("the RNA of the present invention"), wherein the RNA sequence contained in the RNA is substantially complementary to the target sequence of the α-syn gene, the RNA sequence has at least 15 nucleotides, and the RNA comprises a hairpin.

[0016] As used herein, the term "substantially complementary" refers to two nucleic acid sequences that are complementary to each other, whereby the two nucleic acid sequences bind to each other. The term "substantially" means that the complementarity between the two sequences is sufficient to allow binding to each other for a time sufficient to have at least a partial inhibitory effect. Of course, complete complementarity (full complementarity) is preferred, but several gaps and / or mismatches may be tolerated. The number of mismatches must be 10% or less. An important feature is that the complementarity is sufficient to allow the binding of the two strands in situ. The binding must be of sufficient strength to exert the inhibitory effect.

[0017] The nucleic acid sequence encoding the above RNA may optionally have a maximum of 4 nucleotides; 5 nucleotides; or 6 nucleotides that differ from the complementary ("anti") sequence of the target sequence. The nucleic acid sequence encoding the RNA may have 1 nucleotide, 2 nucleotides, or 3 nucleotides that differ from the complementary sequence of the target sequence encoded by the α-syn gene. Preferably, the nucleic acid sequence as described above is identical to the complementary sequence of the target sequence.

[0018] As used herein, the term "α-syn gene" refers to the α-synuclein gene or the SNCA gene. As described herein, the α-syn gene is preferably a mammalian α-syn gene, more preferably a mouse or rat α-syn gene, still more preferably a NHP α-syn gene, and most preferably a human α-syn gene. All SNPs of an α-syn gene can be further included in the present invention.

[0019] As used herein, the term "α-syn protein" refers to a protein encoded by an α-syn gene.

[0020] Typically, the nucleic acids according to the present invention are intended to reduce the expression of disease-related genes. According to the present invention, such nucleic acids can be delivered to target cells by, for example, a gene delivery vehicle, particularly a viral gene delivery vehicle, preferably an adeno-associated virus (AAV) vehicle, as described below. The nucleic acids can then be transcribed to RNA. In the course of RNA intervention (RNAi), the RNA is cleaved by Drosha (i.e., a class II ribonuclease III enzyme) in the nucleus of the target cell into short hairpin RNA (shRNA) or long hairpin RNA (lhRNA) without facultative regions at the 5' and 3' ends of the RNA. The cleaved RNA is then transported to the cytoplasm of the cell, where it is not further cleaved by the endoribonuclease Dicer. The cleaved RNA is further cleaved by Argonaute-2 (AGO-2) of the RNA-induced silencing complex (RISC), and the passenger RNA sequence of the cleaved RNA is trimmed (i.e., cleaved) by poly(A)-specific ribonuclease (PARN). The other strand of the cleaved RNA is called the guide strand (i.e., the guide sequence). As described above, the guide strand containing a sequence substantially complementary to the target RNA sequence is not processed and / or cleaved by AGO-2.

[0021] In situations where the passenger strand of cleaved RNA remains intact without being cleaved, the passenger strand may be partially complementary to off-target sequences and / or even target sequences. Therefore, the passenger strand can bind to off-target sequences and / or even compete with the guide strand of the cleaved RNA to bind to the target sequence. Such “off-target issues” can affect the accuracy of gene editing interventions and therefore must be reduced and / or eliminated.

[0022] This prevents and / or suppresses "off-target problems" by cleaving the passenger sequence. Consequently, the binding specificity of the guide sequence to the target mRNA is improved, and "off-target" events are reduced. This is a preferred embodiment of the present invention.

[0023] The present invention includes RNA containing two complementary strands, in which one of the strands (passenger strand) is cleaved during RNAi. For example, double-stranded RNA (dsRNA), small interfering RNA (siRNA), and microRNA (miRNA) are included in the present invention.

[0024] As used herein, the terms “RNA hairpin” or “hairpin” refer to a secondary structure of RNA comprising two complementary strands and a loop connecting the two strands. One strand is called the passenger strand (i.e., the passenger sequence), and the other strand is called the guide strand (i.e., the guide sequence). RNA hairpins can guide RNA folding, determine interactions in ribozymes, protect messenger RNA (mRNA) from degradation, and function as recognition motifs for RNA-binding proteins.

[0025] Other RNAs having two strands are also included in the present invention, preferably such that one strand is degraded (i.e., trimmed) in RNA interference (RNAi) and the other strand remains undegraded, thereby improving the “off-target problem.” lhRNA and / or shRNA can also be included in the present invention. In certain embodiments, the hairpin may be shRNA or lhRNA.

[0026] Preferably, the hairpin described above has a sequence of at least 39 nucleotides; at least 44 nucleotides; at least 49 nucleotides; at least 54 nucleotides; or at least 59 nucleotides. In some embodiments of the present invention, the hairpin described above has a sequence of at least 39 nucleotides. Therefore, preferably, the nucleic acid sequence encoding RNA has a sequence of at least 39 nucleotides.

[0027] The hairpin described above may optionally have an RNA sequence of up to 80 nucleotides, optionally up to 78 nucleotides, optionally up to 76 nucleotides, optionally up to 74 nucleotides, optionally up to 72 nucleotides, optionally up to 70 nucleotides, optionally up to 68 nucleotides, optionally up to 66 nucleotides, and optionally up to 64 nucleotides. Preferably, the hairpin described above has an RNA sequence of 72 nucleotides.

[0028] miRNA scaffolding A nucleic acid sequence encoding the hairpin having the sequence length described above can be easily incorporated into an AAV and delivered to a target organ such as the central nervous system. Furthermore, the sequence length allows the hairpin to fold correctly, and as a result, the passenger strand can be cleaved by RNAi as described above. Therefore, as described above, the sequence having the sequence length can reduce and / or prevent the off-target problem. Furthermore, the off-target problem can be further reduced and / or prevented by RNA having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and variants of SEQ ID NO: 1 and SEQ ID NO: 2.

[0029] Therefore, in a preferred embodiment, the RNA (RNA of the present invention) includes SEQ ID NO: 1, SEQ ID NO: 2, or a variant of SEQ ID NO: 1 or SEQ ID NO: 2.

[0030] Accordingly, in a preferred embodiment, the present invention provides a nucleic acid comprising a nucleic acid sequence encoding RNA, wherein the RNA sequence is substantially complementary to the target sequence of an α-syn gene, the RNA sequence has at least 15 nucleotides, the RNA comprises a hairpin, and the RNA comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant of SEQ ID NO: 1 or SEQ ID NO: 2.

[0031] Sequence ID 1 refers to the miR451 scaffold or hairpin. The scaffold preferably contains, from 5' to 3', first (i) 5'-CUUGGGAAUGGCAAGG-3' (Sequence ID 46), followed by (ii) a 22-nucleotide sequence containing or derived from a first RNA sequence, followed by (iii) a 17-nucleotide sequence that can be considered a second RNA sequence complementary to nucleotides 2-18 of the first 22-nucleotide sequence over its entire length, followed by (iv) the sequence 5'-MWCUUGCUAUACCCAGA-3' (where M is G or C and W is A or U) (Sequence ID 47). Preferably, the first 5'-A / C nucleotide of the latter sequence does not base pair with the first nucleotide of the first strand of the first or second RNA.

[0032] Such scaffolds may contain adjacent sequences, as found in the original pri-miR451 scaffold. Alternatively, the adjacent sequences may be replaced by adjacent sequences of other pri-mRNA structures. Table 3 shows pri-mRNA sequences of exemplary scaffolds of the present invention.

[0033] The miR451 scaffold enables the induction of RNA interference (RNAi); in particular, RNAi is induced by the guide strand of this scaffold. The pri-miR451 scaffold does not produce a passenger strand because its processing differs from the canonical miRNA processing pathway (Cheloufi, S. et al., 2010 and Yang, J. Set. al., 2010). Thus, the use of miR-451 can prevent or reduce the possibility of undesirable potential off-targeting due to the passenger strand.

[0034] Sequence ID 2 refers to the miR-144 scaffolding combined with the miR451 scaffolding described above.

[0035] The nucleic acid can be transcribed into the RNA as described above. Preferably, the RNA contains a hairpin of miR-451 containing SEQ ID NO: 1. The use of miR451 prevents and / or reduces the off-target problem because the passenger strand is cleaved and not present in the final miR451. More preferably, the RNA contains SEQ ID NO: 2 and has a double hairpin structure. The structure includes a hairpin miR144 and the subsequent hairpin miR451 from the 5' end to the 3' end of the RNA. It has been found that when the RNA contains SEQ ID NO: 2, the off-target problem is prevented and / or reduced. Furthermore, the biosynthesis of the hairpin miR451 is improved, thereby increasing the amount of the guide strand. Therefore, inhibition and / or knock-off of the target RNA transcript can be enhanced.

[0036] The RNA variant of SEQ ID NO: 1 or SEQ ID NO: 2 is defined as having substantially the same function as the RNA containing SEQ ID NO: 1 or SEQ ID NO: 2, respectively. The RNA containing the variant of SEQ ID NO: 1 or SEQ ID NO: 2 has the function of preventing and / or reducing the off-target problem as described above. The variants of SEQ ID NO: 1 and SEQ ID NO: 2 also have substantially the same function as SEQ ID NO: 1 and SEQ ID NO: 2, respectively, for folding into RNA secondary structures. Furthermore, the RNA containing the variant of SEQ ID NO: 2 can not only reduce and / or prevent the off-target problem as described above, but can also improve the biosynthesis of the hairpin.

[0037] When the “off-target problem” is described as reduced / improved as described herein, it means that the off-target problem is prevented, reduced, and / or eliminated.

[0038] Optionally, the variant of SEQ ID NO: 1 described above is substantially the same as SEQ ID NO: 1 and has substantially the same function as SEQ ID NO: 1 described above. Optionally, the variant contains at least one nucleotide different from SEQ ID NO: 1, or optionally up to five nucleotides. In some cases, the variant of SEQ ID NO: 1 contains up to 30 nucleotides; up to 25 nucleotides; up to 20 nucleotides; up to 15 nucleotides; or up to 10 nucleotides different from SEQ ID NO: 1.

[0039] Optionally, the variant of SEQ ID NO: 2 described above is substantially the same as SEQ ID NO: 2 and has substantially the same function as SEQ ID NO: 2 described above. Optionally, the variant may contain at least one nucleotide different from SEQ ID NO: 2, or optionally up to five nucleotides. In some cases, the variant of SEQ ID NO: 2 may contain up to 30 nucleotides; up to 25 nucleotides; up to 20 nucleotides; up to 15 nucleotides; or up to 10 nucleotides different from SEQ ID NO: 2.

[0040] Preferably, the RNA sequence substantially complementary to the target RNA sequence encoded by the α-syn gene has at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, or at least 24 nucleotides. Preferably, the RNA sequence described herein has at least 18 nucleotides.

[0041] Depending on the case, the RNA sequence may have up to 32 nucleotides, up to 31 nucleotides, up to 30 nucleotides, up to 29 nucleotides, up to 28 nucleotides, up to 27 nucleotides, up to 26 nucleotides, or up to 25 nucleotides. In some embodiments of the present invention, the RNA sequence may have up to 32 nucleotides. Therefore, the nucleic acid sequence encoding the RNA may have up to 32 nucleotides.

[0042] The RNA sequence having the sequence length described above constitutes the guide strand of the hairpin described above. As described above, the length of the guide strand is designed to form the guide strand of the hairpin and enable the formation of the RNA secondary structure (i.e., the hairpin). Furthermore, the length of the guide strand is selected to provide sufficient binding specificity to the target RNA. These contribute to reducing the off-target problem.

[0043] An RNA sequence substantially complementary to the target sequence of the α-syn gene (the sequence contained in the DNA) is designed based on one of the conserved regions of the α-syn gene, as described below.

[0044] Preferably, the conserved region is located in mammalian α-syn genes, more preferably in non-human primates (NHPs) and / or human α-syn genes.

[0045] Preferably, the target RNA is encoded by a portion of the exons contained in the α-syn gene. Since exons are not removed by RNA splicing, they are useful to take into consideration when designing the target RNA.

[0046] The term "(a) portion" as defined herein refers to a partial sequence. The term "exon" as defined herein refers to a region contained in the α-syn gene that codes for a portion of mRNA without being removed by RNA splicing. An exon may contain at least one conserved sequence. Exons contained in the NHP and human α-syn genes were aligned to design the target RNA and the guide strand. For example, the NHPα-syn gene consists of the NHPα-syn gene (gene ID: 706985, https: / / www.ncbi.nlm.nih.gov / gene / 706985). For example, the human α-syn gene consists of the human α-syn gene (gene ID: 6622 (https: / / www.ncbi.nlm.nih.gov / gene / 6622)).

[0047] The term "at least one" as used herein means that there are one, two, three or more indicated objects, such as the conserved sequences described herein.

[0048] As used herein, the terms “conserved sequence” or “conserved region” refer to short sequences that may be found in various species exhibiting a high degree of similarity. Conserved sequences can be identified by aligning numerous nucleic acid sequences from different species to encode RNA or proteins with similar functions, so that some or most of the sequences are identical.

[0049] Each of exons 2, 3, 4, 5, and 6 in the α-syn gene contains at least one conserved region for designing a target RNA to which the guide strand can bind. Preferably, the exons are selected from the group consisting of exons 2, 4, and 6. Multiple conserved sequences have been found to be present in exons 2, 4, and 6 of the NHPα-syn gene and / or the human α-syn gene. Therefore, these exons are useful for designing the RNA.

[0050] Preferably, the guide strand binds to the target RNA encoded by a portion of exon 2 or exon 4, more preferably by a portion of exon 4. In other words, the target RNA sequence is a portion of exon 2, exon 4, or exon 6; preferably a portion of exon 2 or 4; more preferably a portion of exon 4.

[0051] The transcript of the target RNA, designed based on the conserved sequences in exon 2, exon 4, and / or exon 6, may be reduced and / or knocked down by the guide strand as described below.

[0052] As used herein, the term “transcript” refers to mRNA, protein, and / or protein aggregates encoded by the α-syn gene. As used herein, the terms “α-syn aggregate,” “α-syn protein aggregate,” and / or other variants refer to aggregates composed of the α-syn protein.

[0053] Any exon included in the α-syn gene that contains at least a conserved sequence, such as exon 3 and / or exon 5, is also included in the present invention.

[0054] More preferably, the portion of the exon described above is selected from the group consisting of SEQ ID NOs: 3-9 (Table 1) and variants of SEQ ID NOs: 3-9, preferably from the group consisting of SEQ ID NOs: 4, 7, and 8, and more preferably from the group consisting of SEQ ID NOs: 4 and 8, and more preferably from the group consisting of variants of SEQ ID NOs: 4 and 8. In other words, the portion of the exon consists of sequences selected from the group consisting of SEQ ID NOs: 3-9 and variants of SEQ ID NOs: 3-9.

[0055] [Table 1]

[0056] The variants of SEQ ID NOs. 3-9 each have substantially the same sequence and function as SEQ ID NOs. 3-9. The variants may be linked by a guide strand as described below, after which the target RNA and its transcript, e.g., protein, are reduced and / or knocked down. Each variant of SEQ ID NOs. 3-9 has at least one nucleotide and up to five nucleotides that are different from SEQ ID NOs. 3-9.

[0057] The term “variant” as used herein refers to a variant of the target RNA sequence having substantially the same function as the target sequence. Furthermore, the variants of the guide strand described below have substantially the same function as the guide strand described below. That is, the variants of the guide strand can still bind to the target RNA or the variant of the target RNA to further inhibit and / or reduce the transcript encoded by the α-syn gene. In some cases, the variants of the target RNA sequence each contain up to 4 nucleotides, up to 3 nucleotides, up to 2 nucleotides, or at least 1 nucleotide different from the target RNA sequence.

[0058] Preferably, the RNA sequence substantially complementary to the target RNA is selected from the group consisting of SEQ ID NOs: 10-16 (Table 2) and variants of SEQ ID NOs: 10-16, preferably from the group consisting of SEQ ID NOs: 11, 14, and 15 and variants of SEQ ID NOs: 11, 14, and 15, more preferably from the group consisting of SEQ ID NOs: 11 and 15 and variants of SEQ ID NOs: 11 and 15. Therefore, the RNA sequence includes one sequence selected from the group consisting of SEQ ID NOs: 10-16 and variants of SEQ ID NOs: 10-16.

[0059] [Table 2]

[0060] As described above, the RNA sequence (i.e., the guide strand) which is substantially complementary to the target RNA sequence is designed so that the RNA sequence binds to the target RNA sequence. This makes it possible to reduce and / or knock down transcripts and / or proteins such as mRNA of the α-syn gene.

[0061] The variants of SEQ ID NOs. 10-16 have substantially the same sequence as SEQ ID NOs. 10-16, and each has the same function and substantially the same binding to the target DNA as SEQ ID NOs. Optionally, the variants of SEQ ID NOs. 10-16 each have at least one nucleotide and up to five nucleotides that are different from SEQ ID NOs. Optionally, the variants of SEQ ID NOs. 10-16 each contain up to four nucleotides, up to three nucleotides, up to two nucleotides, or at least one nucleotide that are different from SEQ ID NOs. 10-16.

[0062] Table 3 shows exemplary sequences of the pri-miRNA scaffold of the present invention, including sequence numbers 10-16.

[0063] [Table 3]

[0064] DNA molecule and expression cassette A second aspect of the present invention relates to the nucleic acid of the present invention, which is a DNA molecule ("the DNA molecule of the present invention"). The present invention preferably provides a DNA molecule, the DNA molecule having a sequence in one of its strands corresponding to the above-mentioned nucleic acid sequence.

[0065] The DNA molecule may be useful for maintaining the nucleic acid sequence as described above, and may be included in AAV for transduction in the target organ as described above.

[0066] Preferably, the DNA molecule comprises a DNA expression cassette, the DNA expression cassette comprising the nucleic acid sequence described above, a promoter and a poly-A tail, and the 3' and 5' ends of the nucleic acid sequence being adjacent to an inverse terminal repeat (ITR). In other words, the DNA molecule is contained in a DNA expression cassette, the DNA expression cassette further comprising a promoter and a poly-A tail, and the nucleic acid being adjacent to an inverse terminal repeat (ITR).

[0067] As used herein, the term “DNA expression cassette” refers to a DNA nucleic acid sequence comprising a gene or nucleic acid sequence encoding an RNA molecule, a promoter, and a nucleic acid sequence encoding a poly(A) tail. The DNA expression cassette is adjacent to the ITR, contained within a viral vehicle, and subsequently delivered to a target organ, such as the brain and / or other organs of the CNS.

[0068] As used herein, the term “promoter” typically refers to a DNA sequence located at the 5' end of a transcription start site for driving or initiating the transcription of a ligated nucleic acid sequence. In some embodiments of the present invention, the promoter is a constitutive promoter or ubiquitous promoter; a neuron-specific promoter; and / or a glial-specific promoter.

[0069] The constitutive promoter may be selected from the group consisting of the pol II promoter, the natural or engineered chicken beta-actin promoter (CBA), the CAG promoter, the PGK promoter, and the CMV promoter (for example, as shown in Figure 2 of International Publication No. 2016102664, which is incorporated herein by reference).

[0070] As used herein, the term “glia-specific promoter” refers to a promoter that can be appropriately used to increase the expression of exogenous nucleic acids and / or genes in glial cells such as astrocytes, oligodendrocytes, or microglia. GFAP may be used for expression in astrocytes. MBP, PLP, CNP, or MAG may be used for expression in oligodendrocytes. CD68 or Hexb may be used for expression in microglia. In some preferred embodiments of the present invention, the glia-specific promoter is an oligodendrocyte promoter selected from the group consisting of MBP, PLP, CNP, and MAG. Preferably, the promoter is a neuron-specific promoter. As used herein, the term “neuron-specific promoter” refers to a promoter that can be appropriately used to increase the expression of foreign nucleic acids and / or genes in neuronal cells, such as brain cells.

[0071] Preferably, the neuron-specific promoter is selected from the group consisting of synapsin, neuron-specific enolase (NSE), human synapsin 1, CaMKII kinase, tubulin α (Hioki et al. Gene Ther. 2007 Jun;14(11):872-82), and platelet-derived growth factor β chain (PDGF). More preferably, the promoter includes a dopaminergic neuron-specific promoter. Preferably, the dopaminergic neuron-specific promoter is selected from TH (tyrosine hydroxylase) or Forkhead Box A2 (FOXA2).

[0072] By using a neuron-specific promoter in the DNA expression cassette, the expression of the nucleic acid in the CNS is induced and / or enhanced, which is preferable for reducing and / or knocking down the transcript of the α-syn gene, as the transcript of the α-syn gene is expressed mainly in the CNS, e.g., the brain and spinal cord, and more primarily in the brain, and more primarily in neurons.

[0073] Other suitable promoters that may be included in the present invention are inducible and / or repressive promoters, i.e., promoters that initiate transcription only when the host cell is exposed to a specific stimulus.

[0074] In some cases, the DNA expression cassette includes at least two promoters, including the promoter described above.

[0075] As used herein, the term "poly-A tail" refers to a long chain of adenine nucleotides added to an mRNA molecule to enhance the stability of the RNA molecule. Preferably, the poly-A tail is Simian virus 40 polyadenylation (SV40 poly-A; SEQ ID NO: 44), bovine growth hormone (BGH) polyadenylation (BGH poly-A; SEQ ID NO: 45), human growth hormone polyadenylation (hGH poly-A; SEQ ID NO: 79), or synthetic polyadenylation. More preferably, the poly-A tail is BGH poly-A (SEQ ID NO: 45) or hGH poly-A; SEQ ID NO: 79.

[0076] Preferably, as described above, the poly(A) tail included in the DNA expression cassette is operably ligated to the 3' end of the RNA molecule.

[0077] As used herein, the term “reverse end repeat (ITR)” refers to the 5' and 3' end sequences of the DNA expression cassette that function cis as the origin of DNA replication and as a viral packaging signal. The ITR is preferably selected from the group consisting of adeno-associated virus (AAV) ITR sequences. More preferably, the ITR sequences are both AAV1, both AAV2, both AAV5, both AAV6, both AAV7, both AAV8, or both AAV9 ITR sequences. Furthermore, more preferably, the ITR sequence at the 5' end of the DNA expression cassette is different from the ITR sequence at the 3' end of the DNA expression cassette, and the ITR sequence is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9 ITR sequences.

[0078] The ITRs are positioned at the left and right ends (i.e., the 5' and 3' ends, respectively) of the nucleic acid sequence as described above. Preferably, the ITRs are selected from the group consisting of adeno-associated virus (AAV) ITR sequences. More preferably, the ITR sequences include AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9 ITR sequences. In some cases, the two ITR sequences include both AAV1, both AAV2, both AAV5, both AAV6, both AAV7, both AAV8, or both AAV9 ITR sequences. Also in some cases, the ITR sequence at the 5' end of the nucleic acid sequence is different from the ITR sequence at the 3' end of the nucleic acid sequence, and the ITR sequence is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9 ITR sequences.

[0079] AAV According to the present invention, an AAV vehicle containing the above-described DNA ("the AAV vehicle of the present invention") is provided.

[0080] A viral vehicle used to deliver the aforementioned nucleic acid or other foreign genetic material such as DNA is part of the present invention. Examples of such viral vehicles include alphaviruses, flaviviruses, herpes simplex virus (HSV), measles virus, rhabdovirus, retrovirus, Newcastle disease virus (NDV), poxvirus, picornavirus, lentivirus, adenovirus vector, and preferably an AAV gene delivery vehicle.

[0081] The term “AAV vehicle” as used herein refers to wild-type or recombinant AAVs that act as vehicles for delivering genetic material, such as foreign nucleic acids, genes of interest, nucleic acids of interest, vectors containing the foreign nucleic acids, vectors containing the genes of interest, the aforementioned DNA expression cassettes, and / or vectors containing the aforementioned DNA expression cassettes, to target cells, organs, and / or tissues.

[0082] As described above, the AAV vehicle has been found to be a useful viral vehicle for the delivery of nucleic acid or DNA expression cassettes to mammals. The AAV vehicle has the ability to efficiently infect both dividing and non-dividing human cells. Furthermore, the AAV vehicle is not associated with any disease. Therefore, the AAV vehicle is useful in the present invention, as described below, and for treating and / or preventing diseases involving the α-syn gene.

[0083] According to the present invention, the AAV vehicle comprises a nucleic acid comprising a nucleic acid sequence encoding RNA, wherein the RNA sequence is substantially complementary to a target RNA sequence encoded by the α-syn gene, the RNA sequence has at least 15 nucleotides, and the RNA comprises a hairpin comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a variant of SEQ ID NO: 1 or 2. The RNA sequence substantially complementary to the target RNA sequence is selected from the group consisting of SEQ ID NOs: 10-16 and variants of SEQ ID NOs: 10-16, preferably SEQ ID NOs: 11, 14, and 15 and variants of SEQ ID NOs: 11, 14, and 15, more preferably SEQ ID NOs: 11 and 15 and variants of SEQ ID NOs: 11 and 15.

[0084] Furthermore, according to the present invention, the AAV vehicle may also include a further nucleic acid comprising a nucleic acid sequence encoding RNA, wherein the RNA sequence is substantially complementary to the target RNA sequence encoded by the α-syn gene, the RNA sequence has at least 15 nucleotides, and the RNA comprises a hairpin containing SEQ ID NO: 1 or SEQ ID NO: 2, or a variant of SEQ ID NO: 1 or 2. The RNA sequence substantially complementary to the target RNA sequence is selected from the group consisting of SEQ ID NOs: 10-16 and variants of SEQ ID NOs: 10-16, preferably SEQ ID NOs: 11, 14, and 15 and variants of SEQ ID NOs: 11, 14, and 15, more preferably SEQ ID NOs: 11 and 15 and variants of SEQ ID NOs: 11 and 15.

[0085] As described above, the AAV vehicle for delivering the DNA expression cassette can modify and / or reduce the (over)expression level of the product encoded by the α-syn gene. Preferably, the AAV vehicle is used to reduce and / or knock down α-syn aggregates, which typically contain the protein encoded by the α-syn gene.

[0086] As used herein, the term “decrease” means a decrease or reduction in the level and / or quantity of the indicated subject. As used herein, the term “knockdown” means a level and / or quantity in which the indicated subject is substantially depleted or removed.

[0087] Optionally, the AAV vehicle is used to reduce and / or knock down the transcript encoded by the mutant SNCA gene. Studies of families with a history of Parkinson's disease have led to the identification of a set of familial mutations that result in early-onset (A30P, E46K, A53T, G51D) or late-onset (H50Q) forms of the disease.

[0088] Preferably, the AAV vehicle is used to reduce and / or knock down at least one isoform, including but not limited to the α-syn isoform encoded by SEQ ID NO: 36 (SNCA140), SEQ ID NO: 76 (SNCA126), SEQ ID NO: 77 (SNCA112), or SEQ ID NO: 78 (SNCA98). More preferably, the AAV vehicle is used to reduce and / or knock down at least one isoform encoded by an α-syn nucleic acid sequence including exons 2, 4, and / or 6.

[0089] More preferably, as described above, at least two of the RNAs intended to reduce and / or knock down transcripts of different target RNAs can be combined in a single AAV vehicle to further enhance the inhibitory effect on the transcript of the α-syn gene. Thus, the treatment and / or prevention of the diseases described below is further improved.

[0090] In some embodiments of the present invention, a combination of RNA intended to reduce and / or knock down the target RNA having SEQ ID NO: 4 and RNA intended to reduce and / or knock down the target RNA having SEQ ID NO: 8 can be combined in a single AAV vehicle to further enhance the inhibitory effect on the α-syn gene transcript, as described below.

[0091] Preferably, the AAV vehicle is an AAV5, AAV8, or AAV9 vehicle. More preferably, the AAV vehicle is an AAV5 or AAV9 vehicle or a hybrid thereof. The AAV vehicle of the present invention may also be an AAV2 / AAV5 or AAV2 / AAV9 hybrid capsid.

[0092] In some embodiments of the present invention, the AAV vehicle is an AAV5 vehicle. AAV5 is useful in the present invention because the prevalence of anti-AAV5 neutralizing antibody (Nab) is lower than the prevalence of Nab against other serotypes. Furthermore, existing antibodies (Ab) or low levels of existing antibodies against AAV5 typically do not affect transduction by the AAV gene therapy vehicle and / or the expression of the nucleic acid in the target organ. In addition, cytotoxic T cell responses to AAV5 have not been reported in clinical trials.

[0093] In some embodiments of the present invention, the AAV vehicle is an AAV9 vehicle. The AAV9 is useful for delivering foreign nucleic acids to neuronal cells and glial cells, such as oligodendrocytes.

[0094] In some cases, the AAV vehicle may contain capsids derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, and their variants (e.g., capsid variants or hybrid capsids involving amino acid insertion, addition, and substitution).

[0095] Preferably, the AAV vehicle of the present invention comprises a capsid containing AAV5 and / or AAV9 and / or a hybrid capsid protein sequence.

[0096] AAV capsids typically contain the VP1 protein and two shorter proteins called VP2 and VP3, which are essentially amino-terminal cleavages of VP1. The three capsid proteins VP1, VP2, and VP3 are typically present in the capsid in a ratio approximately 1:1:10, but this ratio, particularly the ratio of VP3, can vary and should not be considered limiting.

[0097] The present invention further includes other hybrid capsids having an optimized VP1:2:3 stoichiometry. The optimized VP1:2:3 stoichiometry can improve the AAV vehicle in terms of infectivity to target organs and correct virion assembly.

[0098] AAV vehicles possessing capsid proteins VP1, VP2, and VP3 in a ratio approximating 1:1:10 or an optimized VP 1:2:3 stoichiometry are useful for delivering foreign nucleic acid sequences to human subjects and / or transducing target organs, such as those involved in PD.

[0099] As described herein and above, an AAV vehicle may be defined as a “hybrid,” meaning that the viral ITR and viral capsid are derived from different AAV serotypes. The viral ITR is preferably derived from AAV2, and the capsid is preferably derived from a different one, which may be AAV5 or AAV9. Other hybrids, such as hybrids containing combinations of different serotypes of capsid and ITR, and possibly with even more ITRs, can also be used in the present invention, including capsid elements from different serotypes.

[0100] Preferably, the AAV vehicle of the present invention is a gene therapy vehicle.

[0101] As used herein, the term “gene therapy” refers to a therapy that has a more stable and / or longer-lasting effect than existing therapies for treating and / or preventing diseases involving the α-syn gene. A preferred method for achieving a stable therapeutic effect is by a single dose of the AAV gene therapy vehicle. The stability of the therapy can be measured by common techniques known to scientists in the art. The long-lasting effect can be measured by the length of time the therapeutic effect persists and / or by the amount and / or frequency of injections required to maintain such therapeutic effect.

[0102] As used herein, the terms “to treat” or “to cure” refer to any means that can stop, alleviate, delay, slow down and / or improve the disease and / or preferably at least one symptom caused by the disease, such as a neurological progressive disorder, as described below. Such means may include, but are not limited to, delaying and / or slowing the progression of a neurological progressive disorder, stopping the onset of at least one symptom, reducing the disease caused by the disease, and / or improving the patient’s health. As used herein, the terms “to prevent” or “prevent” refer to any means to stop the onset of the disease, including but not limited to the onset of new symptoms of the disease. The terms “disease” and “disorder” can be used interchangeably in this invention.

[0103] The AAV gene therapy vehicle can provide a consistent effect on the expression level and / or activity level of the transcript. Therefore, in some embodiments of the present invention, the AAV gene therapy vehicle can be used to provide a consistent and / or long-term therapeutic effect for the treatment and / or prevention of the diseases and / or symptoms described below. This also allows for an improvement in the quality of life of patients suffering from the diseases and / or symptoms by administering the AAV vehicle.

[0104] The long-term effects of the AAV gene therapy vehicle can be evaluated by measuring improved outcomes of disease parameters over a long period compared to existing treatments for the disease and / or for the disease.

[0105] composition The present invention provides a composition comprising the AAV vehicle described above and at least one pharmaceutically acceptable excipient. Preferably, the composition comprises the AAV gene therapy vehicle.

[0106] The composition may be in solid or liquid form. In some embodiments of the present invention, the composition is a pharmaceutical formulation.

[0107] The terms “additive” or “excipient” as used above and herein refer to substances further added to the composition to impart at least one function to the composition. Such functions include, but are not limited to, complementing the properties of the composition, stabilizing the composition for easier storage and / or extended shelf life, inhibiting side effects such as immune responses, improving the transduction effectiveness of the AAV vehicle to a target organ, and / or improving blood-brain barrier (BBB) ​​bypass. Additives or excipients acting as fillers without altering and / or affecting the properties of the composition may further be included in the present invention.

[0108] Pharmacokinetically acceptable excipients for administering AAV gene delivery vehicles are well known to those skilled in the art and may be as simple as water for injection. They may also include surfactants, osmotic agents, antioxidants, and the like.

[0109] In some cases, the composition further comprises an immunosuppressive compound. Immunosuppressive compounds that can reduce and / or prevent the immune response induced by injection of a viral vehicle may be included in the present invention. Immunosuppressive compounds may also be administered separately from the AAV composition. Such combinations are included in the present invention as a kit.

[0110] Depending on the circumstances, compounds for improving the in vivo distribution of the RNA in the brain, such as the hairpin, may further be included in the composition.

[0111] In some cases, the composition further comprises at least one additive selected from the group consisting of aqueous liquids, organic solvents, buffers, and excipients. In some cases, the aqueous liquid is water. In some cases, the buffer is selected from the group consisting of acetates, citrates, phosphates, tris, histidine, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In some cases, the organic solvent is selected from the group consisting of ethanol, methanol, and dichloromethane. In some cases, the excipient is a salt, a sugar, cholesterol, or a fatty acid. In some cases, the salt is selected from the group consisting of sodium chloride and potassium chloride, as described above. In some cases, the sugar is sucrose, mannitol, trehalose, and / or dextran, as described above.

[0112] The term “target organ” as used herein refers to an organ in which the α-syn gene transcript accumulates. For example, the target organ is the brain of a human subject. Other organs in the CNS (i.e., the brain and spinal cord) may be further included in the present invention, provided that the α-syn transcript, such as aggregates, is present in the said organ.

[0113] Purpose The present invention provides the use of the AAV (vehicle) and / or the AAV gene therapy vehicle as pharmaceuticals. Accordingly, the present invention also provides the use of the composition as a pharmaceutical.

[0114] The terms “AAV” and “AAV vehicle” are used interchangeably herein. The AAV vehicle (and / or the composition comprising the AAV vehicle) as described above can reduce and / or knock down the transcript of the α-syn gene. Thereafter, the AAV vehicle is useful for treating and / or preventing diseases caused by the transcript of the α-syn gene, typically caused by overexpression of the transcript encoded by the α-syn gene and / or by aggregated proteins encoded by the α-syn gene.

[0115] The transcript is mRNA and / or protein, preferably mRNA. Thus, the AAV vehicle and the composition are useful (i.e., have therapeutic effects) for the treatment and / or prevention of diseases involving the α-syn gene. Therefore, according to the present invention, the AAV vehicle and / or composition described above are intended for use as pharmaceuticals in the treatment and / or prevention of diseases involving the α-syn gene.

[0116] Diseases and / or symptoms caused by the α-syn gene or by transcripts encoded by the α-syn gene (for example, by overexpression of the transcripts) are preferably diseases caused by overexpression of the α-syn protein and / or aggregates of the α-syn protein.

[0117] In some embodiments of the present invention, the AAV vehicle and / or the AAV gene therapy vehicle and / or composition described above is used to treat and / or prevent a disease by reducing and / or knocking down the transcript encoded by the α-syn gene. Accordingly, the present invention also provides the use of the AAV vehicle and / or the AAV gene therapy vehicle and / or composition described above as a pharmaceutical, wherein the pharmaceutical reduces and / or knocks down the transcript encoded by the α-syn gene.

[0118] The aforementioned diseases may further include diseases involving at least one single nucleotide polymorphism (SNP) of the α-syn gene. For example, the disease is caused by a protein and / or aggregate encoding at least one SNP of the α-syn gene.

[0119] Preferably, the α-syn protein (SEQ ID NO: 35) expression level is reduced by at least 30% and / or up to 70% compared to the α-syn protein expression level without administration of the AAV vehicle and / or the composition. More preferably, the protein encoded by the SNCA gene is reduced by up to 50% compared to the endogenous α-syn protein expression level without administration of the AAV vehicle and / or the composition. Complete knockdown may be undesirable due to the central role of the α-syn gene.

[0120] Preferably, the AAV vehicle and / or composition described above can reduce the transcripts described above by about at least 30% and / or up to 70%, more preferably up to 50%, compared to when the AAV vehicle and / or composition is not administered to human subjects.

[0121] Preferably, the expression level of the transcript is reduced by at least 30% and up to 70%, more preferably up to 50%, compared to the expression level without administration of the AAV vehicle and / or the composition. More preferably, the α-syn protein is reduced by at least 30% and up to 70%, even more preferably up to 50%, compared to the α-syn protein level without administration of the AAV vehicle and / or the composition.

[0122] Preferably, the expression level of the transcript is reduced by at least 30% and at most 70%, more preferably up to 50%, compared to the expression level when the AAV vehicle and / or the composition is not administered to human subjects. More preferably, the α-syn protein is reduced by at least 30% and at most 70%, and even more preferably up to 50%, compared to the α-syn protein level when the AAV vehicle and / or the composition is not administered to human subjects.

[0123] By using the AAV vehicle, the level of the α-syn gene transcript is reduced, but not completely and substantially depleted. Therefore, diseases caused by overexpression of the transcript and / or aggregates of the α-syn protein are at least partially treated and / or prevented, and diseases and / or symptoms caused by complete knockdown of the transcript are also at least partially treated and / or prevented.

[0124] In some embodiments of the present invention, the AAV vehicle and / or the composition is used to reduce and / or knock down α-syn protein aggregates, which typically contain the protein encoded by the α-syn gene.

[0125] Preferably, the amount of α-syn aggregates is reduced by at least 30% and / or up to 70%, more preferably up to 50%, compared to the amount of α-syn aggregates in a patient / human subject not administered the AAV vehicle and / or the composition.

[0126] Although reducing and / or knocking down the transcript encoded by the aforementioned α-syn gene may be beneficial, complete depletion (i.e., complete knockdown) may result in reduced synaptic transmission and / or neurodegeneration in the CNS, potentially endangering the patient.

[0127] As a result, the AAV vehicle and / or the composition is useful in treating and / or preventing diseases involving the α-syn gene, and in reducing and / or preventing diseases and / or symptoms, without causing substantially complete knockdown, thereby reducing and / or avoiding the risks caused by the complete depletion of the transcript encoded by the α-syn gene.

[0128] The AAV vehicle and / or composition described above can be used to treat and / or prevent the disease caused by the formation and / or presence of oligomer α-syn, fibrillary α-syn, aggregated α-syn, phosphorylated α-syn, Lewy bodies and / or Papp-Lantos bodies.

[0129] Preferably, the AAV vehicle and / or composition described above are used to reduce and / or knock down the amount of Lewy bodies and / or Papp-Lantos bodies.

[0130] α-syn protein / aggregates form the majority of Lewy bodies and Papp-Lantos bodies (also known as Papp-Lantos inclusions). Thus, by reducing and / or knocking down α-syn protein, the amount (and progression) of Lewy bodies and / or Papp-Lantos bodies can be reduced and / or depleted. Thus, by using the AAV vehicle and / or the composition, the accumulation of Lewy bodies and / or Papp-Lantos bodies can be reduced to achieve the treatment and / or prevention of disease and / or symptoms, as described below.

[0131] Therefore, the AAV vehicle and / or the composition can be used as a pharmaceutical to reduce the amount of total α-syn, oligomeric α-syn, aggregated α-syn, and phosphorylated α-syn, and thus the levels of Lewy bodies and Paplando bodies, thereby halting the progression of the disease and / or improving the symptoms of the disease.

[0132] Such diseases and / or symptoms include, but are not limited to, the clinical symptoms of PD, LBD, MSA, motor symptoms of PD, cognitive impairment, sleep disorders, autonomic dysfunction, and / or olfactory dysfunction. Motor symptoms of PD include rigidity, tremor, and / or loss of balance and / or coordination of the limbs, or at least two of these symptoms.

[0133] Clinical symptoms of Parkinson's disease (PD) include, but are not limited to, resting tremor, bradykinesia, rigidity and loss of postural reflexes, secondary motor symptoms (hypomania, dysarthria, dysphagia, salivary gland disorders, micrographia, shuffling gait, fasting, orthostatic hypotension, dystonia, and / or glabellar reflex) and / or non-motor symptoms (e.g., autonomic dysfunction, cognitive / neurobehavioral disorders, sleep disorders, anosmia, paresthesia, and / or pain).

[0134] Clinical symptoms of LBD include, but are not limited to, rigidity of the limbs, tremors and / or impairment of balance and / or coordination, intellectual disability, hallucinations, dysregulation of bodily functions (autonomic nervous system), sudden changes in attention and mood, cognitive problems, sleep disturbances, attentional fluctuations, and motor disorders typical of PD, such as depression and apathy.

[0135] Clinical symptoms of MSA include, but are not limited to, sexual dysfunction, urinary dysfunction, REM sleep behavior disorder, orthostatic hypotension, gait, parkinsonism, cerebellar features, multi-domain autonomic dysfunction, pyramidal tract signs, and / or motor disorders typical of Parkinson's disease, such as frontal executive dysfunction.

[0136] Therefore, AAV vehicles or compositions for use as pharmaceuticals are used to treat and / or prevent the clinical symptoms of PD, LBD, MSA, neurological symptoms, motor symptoms of PD, cognitive impairment, sleep disorders, autonomic disorders, and / or olfactory disorders. Preferably, the AAV vehicles and / or compositions are used to treat and / or prevent PD, MSA, and / or LBD. Preferably, the disease is PD and / or MSA.

[0137] Overexpression of the α-syn gene, aggregation of the α-syn protein, and / or formation and / or presence of Lewy bodies are indicators of a patient suffering from PD. By using the AAV vehicle and / or composition as described, the transcript of the α-syn gene can be reduced and / or knocked down. Thus, the AAV vehicle and / or composition is useful for treating and / or preventing PD.

[0138] Preferably, the AAV vehicle and / or composition is used to treat and / or prevent PD patients in the presymptomatic or symptomatic stage.

[0139] As used herein, the term “presymptomatic stage” refers to the stage of a neuronal progressive disease, such as Parkinson’s disease, before the onset of clinical symptoms.

[0140] As used herein, the term “symptomatic stage” refers to the stage of a neuronal progressive disease, such as Parkinson’s disease, following the clinical diagnosis of the disease.

[0141] Patients with Parkinson's disease (PD) typically become aware they have PD when at least one of the aforementioned symptoms appears. However, because a significant portion of neurons are lost before the onset of symptoms, it may be too late to treat and / or prevent disease progression. Therefore, having therapeutic methods, such as the use of the aforementioned compositions, is useful in treating and / or preventing disease progression before at least one symptom of PD, such as motor symptoms, appears.

[0142] As described above, the AAV vehicle and / or the composition can lower α-syn protein levels, thereby making the AAV vehicle and / or the composition useful for treating and / or preventing at least one PD symptom. The symptom can be selected from the group consisting of depression, sleep disturbances, pain and fatigue in the early stages of the disease, and anxiety, dementia and cognitive impairment in the later stages of the disease.

[0143] Similarly, Lewy body deposits can cause a form of dementia called Lewy body dementia or LBD. In fact, LBD causes some or all of the motor symptoms of Parkinson's disease. Therefore, the AAV vehicle and / or the composition may also prove useful in treating and / or preventing at least one PD symptom.

[0144] Furthermore, overexpression of the α-syn gene and / or the aggregated protein encoded by the α-syn gene may increase the risk of MSA, a progressive brain disorder that affects and / or impairs motor and balance and / or disrupts the function of the autonomic nervous system. The disease was initially known as Scheidlager syndrome. Currently, MSA is considered “sporadic,” meaning there is no established genetic or environmental factor causing the disease.

[0145] While many clinical symptoms exist in patients with Parkinson's disease, patients with MSA typically show symptom onset at a young age, with the average onset being in their early 50s. Many patients are initially diagnosed with Parkinson's disease, but over time, the degree, severity, and type of symptoms change, increasing the likelihood of a diagnosis of MSA.

[0146] A key difference distinguishes MSA from Parkinson's disease in terms of its symptoms and course. Specifically, MSA affects several areas of the brain, including the cerebellum, the brain's balance and coordination center, and the autonomic nervous system, as described above. Furthermore, while Parkinson's disease affects dopamine-producing neurons in the motor control region of the brain known as the substantia nigra and striatum, MSA affects both neurons and glial cells.

[0147] In MSA, hyperphosphorylated α-syn is found in Papp-Lantos inclusions (or GCIs). Therefore, the AAV vehicle and / or composition of the present invention may also be useful in reducing and / or inhibiting the amount of Papp-Lantos inclusions, and in treating and / or preventing MSA.

[0148] Other diseases, such as CNS diseases, can be treated and / or prevented by a similar approach using AAV vehicles and / or compositions comprising AAV vehicles, said diseases are caused by the overexpression of genes, but complete knockout of the transcript of said genes is less desirable.

[0149] Furthermore, at different stages (i.e., phases) of progressive neurological disorders caused by the accumulation of α-syn gene transcripts, patients may develop different symptoms and / or different disease levels. The AAV vehicle and / or the composition provides a solution for treating and / or preventing the different symptoms and / or different disease levels without constantly modifying the treatment regimen.

[0150] method According to the present invention, a method for manufacturing the AAV vehicle described above is provided.

[0151] In some cases, the AAV vehicle may be prepared using mammalian cells. In some cases, the AAV vehicle may be prepared using insect cells, preferably baculoviruses. Suitable methods for preparing an AAV gene therapy vehicle containing such a DNA expression cassette are described in International Publication Nos. 2007 / 046703, International Publication Nos. 2007 / 148971, International Publication Nos. 2009 / 014445, International Publication Nos. 2009 / 104964, International Publication Nos. 2011 / 122950, ​​and International Publication Nos. 2013 / 036118, which are incorporated herein in whole and referred to in particular to their preparation methods. In some cases, the composition further comprises the immunosuppressive compound.

[0152] According to the present invention, a method for producing the above-described composition is provided.

[0153] kit For the purpose of treating and / or preventing the above-mentioned diseases or disorders, the above-mentioned AAV vehicle and at least one of the above-mentioned additives can be combined into a kit. The kit may optionally include means for holding and / or housing the AAV vehicle and at least one of the above-mentioned additives.

[0154] In some embodiments of the present invention, as described above, the kit comprises the AAV vehicle of the present invention and the immunosuppressive compound described above. Healthcare professionals and patients may easily apply the AAV vehicle to human subjects according to the label and / or instructions.

[0155] It is understood that a kit is also provided comprising a composition comprising the AAV vehicle of the present invention and at least one pharmaceutically acceptable excipient. Therefore, optionally, the kit further comprises at least one additive selected from the group consisting of an aqueous liquid, an organic solvent, a buffer, and an excipient. optionally, the aqueous liquid is water. optionally, the buffer is selected from the group consisting of acetates, citrates, phosphates, tris, histidine, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). optionally, the organic solvent is selected from the group consisting of ethanol, methanol, and dichloromethane. Furthermore, the excipient is a salt, a sugar, cholesterol, or a fatty acid. optionally, the salt is selected from the group consisting of sodium chloride and potassium chloride, as described above. optionally, the sugar is sucrose, mannitol, trehalose, and / or dextran, as described above. [Brief explanation of the drawing]

[0156] [Figure 1] Other splicing variants of SNCA mRNA (SNCA140, SNCA126, SNCA112, SNCA98), and regions within the region targeted by the miSNCA candidate sequences (candidates 2, 5, 7, 12, 13, 15, 16). [Figure 2] Vector maps for (A) the original expression cassette containing only the miR451 skeleton, and (B) the improved expression cassette containing miR144 in its skeleton. [Figure 3] Titration of miSNCA candidate double luciferase assay [Figure 4] Dual luciferase assay: Original and improved tTitration of miSNCA5 and miSNCA15. [Figure 5] Dose-dependent endogenous α-syn reduction in HEK293T cells at both mRNA and protein levels. A significant correlation was observed between SNCA mRNA and α-syn protein levels. [Figure 6A]miSNCA5 expression level relative to endogenous miRNA. [Figure 6B] Expression level of miSNCA15 relative to endogenous miRNA. [Figure 7] miRNA processing of miSNCA5 and miSNCA15. [Figure 8] Plasmid pVD1502 plasmid ap. [Figure 9A] Administration pathway in wild-type (wt) rats. AAV5-GFP vDNA level. [Figure 9B] Administration study pathway in wild-type (wt) rats. AAV5-GFP mRNA expression for GAPDH as a housekeeping gene. [Figure 10-1] Mechanism of action in a-syn KI rats. (A) vDNA level; (B) miSNCA5 level. [Figure 10-2] Mechanism of action in a-syn KI rats. (C) miSNCA15 levels; (D) Decreased SNCA mRNA in the striatum. [Figure 11] Rescue of motor phenotype in a nematode PD model using miSNCA candidates. [Figure 12] Processing of miRNAs extracted from rat brain tissue from in vivo study #2 and pooled samples from group 4 of this study. [Figure 13] This figure shows the vDNA levels in the striatum during in vivo study 3 (AAV1 / 2-ha53T-aSyn rat model for Parkinson's disease). The vDNA levels were comparable in all groups treated with the same dose of either AAV5-unrelated miR or AAV5-miSNCA. [Figure 14] miSNCA levels in the striatum in in vivo study 3 (AAV1 / 2-ha53T-aSyn rat model for Parkinson's disease). (A) miSNCA5 levels were higher in the group injected with AAV5-miSNCA5; (B) miSNCA15 levels were higher in the group injected with AAV5-miSNCA15. [Figure 15]In vivo study 3 (AAV1 / 2-hA53T-aSyn rat model for Parkinson's disease), human SNCA mRNA levels in the striatum were highest in the AAV1 / 2-hA53T-aSyn group, which was injected concurrently or sequentially with an AAV5-unrelated miR. Human SNCA mRNA levels were significantly lower in the AAV1 / 2-hA53T-aSyn group, which was injected concurrently or sequentially with AAV5-miSNCA5 or AAV5-miSNCA15. [Figure 16] Human α-syn protein levels in the striatum during in vivo study 3 (AAV1 / 2-Ha53T-aSyn rat model for Parkinson's disease). Human α-syn protein levels were highest in the AAV1 / 2-Ha53T-aSyn group, which was injected concurrently or sequentially with AAV5-unrelated miR. Human α-syn protein levels were significantly lower in the AAV1 / 2-HA53T-aSyn group, which was injected concurrently or sequentially with AAV5-miSNCA5 or AAV5-miSNCA15. [Figure 17] In in vivo study 3 (AAV1 / 2-hA53T-aSyn rat model of Parkinson's disease), striatal dopamine transporter levels were assessed by [125I]-RTI-121 autoradiography; in the AAV1 / 2-hA53T-aSyn group, which received serial injections of AAV5-unrelated miRs, dopamine transporter levels were significantly reduced (similar to those observed in PD patients). Serial injections of AAV5-miSNCA5 or AAV5-miSNCA15 rescued dopamine transporter loss. [Figure 18]In vivo study 3 (AAV1 / 2-hA53T-aSyn rat model of Parkinson's disease), dopamine and dopamine metabolite levels in the striatum (assessed by LC / MS): (A) Dopamine levels were significantly reduced in the ipsilateral striatum of the AAV1 / 2-hA53T-aSyn group that received sequential injections of AAV5-unrelated miRs (similar to those observed in PD patients). Sequential injections of AAV5-miSNCA5 or AAV5-miSNCA15 rescued dopamine loss. (B) The ratio of dopamine metabolites to dopamine was significantly increased in the ipsilateral striatum of the AAV1 / 2-hA53T-aSyn group that received sequential injections of AAV5-unrelated miRs, indicating a deficiency in dopamine turnover (similar to those observed in PD patients). Sequential injections of AAV5-miSNCA5 or AAV5-miSNCA15 rescued the loss of dopamine turnover. [Figure 19] Motor behavior tests (cylinder test, assessment of foot asymmetry) in in vivo study 3 (AAV1 / 2-hA53T-aSyn rat model for Parkinson's disease). (A) At baseline (before injection), there was no foot asymmetry in either treatment group; (B) In the AAV1 / 2-hA53T-aSyn group that received serial injections of AAV5-unrelated miRs, contralateral foot underuse was observed at 56 days post-treatment (similar to that observed in PD patients). Serial infusions of AAV5-miSNCA5 or AAV5-miSNCA15 rescued this motor deficit. [Figure 20-1]Immunohistochemistry was used to evaluate only the substantia nigra (A) TH and (B) human a-syn in the serial injection group in in vivo study 3 (AAV1 / 2-hA53T-aSyn rat model for Parkinson's disease). (A) Substantia nigra TH-positive cells were significantly reduced in AAV1 / 2-hA53T-aSyn animals that received serial injections of AAV5-unrelated miRs compared to AAV1 / 2-empty vector animals that received serial injections of AAV5-unrelated miRs (similar to what is observed in PD patients). Serial infusions of AAV5-miSNCA5 or AAV5-miSNCA15 rescued TH neuron loss. (B) Substantia nigra a-syn-positive cells were reduced by AAV5-miSNCA5 or AAV5-miSNCA15, confirming target involvement. [Figure 20-2] (C) a-syn-positive TH neurons in the substantia nigra of in vivo study 3 (AAV1 / 2-hA53T-aSyn rat model for Parkinson's disease), evaluated by immunohistochemistry; only the group receiving continuous injections was evaluated. (C) TH-positive cells in the substantia nigra showed significantly lower a-syn expression in the AAV5-miSNCA5 or AAV5-miSNCA15 treatment groups. [Figure 21-1] Decreased SNCA mRNA and α-syn protein expression in a C. elegans PD model induced by miSNCA candidates, as evaluated by RT-qPCR and Western blotting, respectively. (A) SNCA mRNA levels, measured at L1 stage and on day 1; (B) SNCA mRNA levels, measured at L4 stage and on days 1, 4, 8, and 11; (C) SNCA mRNA levels, measured at day 1 and on days 1, 4, 8, and 11. Both SNCA mRNA levels and α-syn protein levels were decreased by miSNCA candidates compared to the EV-treated group. [Figure 21-2]Decreased SNCA mRNA and α-syn protein expression in a nematode (C. elegans) PD model by miSNCA candidates, as evaluated by RT-qPCR and Western blotting, respectively. (D) α-syn protein levels, measured after treatment in L1 stage and on day 1; (B) α-syn protein levels, measured after treatment in L4 stage and on days 1, 4, 8, and 11; (C) α-syn protein levels, measured after treatment on day 1 and on days 1, 4, 8, and 11. Both SNCA mRNA levels and α-syn protein levels were decreased by miSNCA candidates compared to the EV-treated group. [Figure 22] (A)Motor phenotypic rescue in a nematode (C. elegans) PD model using miSNCA candidates after treatment at the L1, L4, or day 1 stage. [Figure 23-1] Low-molecular-weight RNA sequencing results of nematode (C. elegans) samples treated with full-length SNCA RNAi, miSNCA5, and miSNCA15 miRNA at the L1 stage, and collected on day 1 and day 4 of the adult stage after treatment. (A) miSNCA5 and (B) miSNCA15 are correctly processed and can be found in the relevant samples. (C) miSNCA5 and miSNCA15 sequences in full-length SNCA-treated nematode (C. elegans) samples, as well as all other designed miSNCAs (miSNCA2, miSNCA7, miSNCA12, miSNCA13, miSNCA16). [Figure 23-2] This is a continuation of Figure 23-1.

[0157] Examples of the present invention Materials and methods Design of SNCA miRNA guide strands. miSNCA (miRNA guide strands) were designed to target the common RNA sequences of the most common SNCA mRNA variants: SNCA140, SNCA126, SNCA112, and SNCA98. The miRNAs were designed in regions common to all major SNCA mRNA variants (Figure 1) (McLean et al. 2012 Mol and Cell Neuroscience 49(2)230-239). The target regions of the SNCA mRNA sequences are parts of exons 2, 4, and 6. The most common SNP outside these exons (A30P) was avoided in the guide RNA. Using each of the conserved sequences, several different guide strands with 22 nucleotides (nt) were generated. Seventeen guides targeting SNCA were designed and incorporated into the miR451 scaffold: miSNCA2 (SEQ ID NO: 24), miSNCA5 (SEQ ID NO: 25), miSNCA7 (SEQ ID NO: 26), miSNCA12 (SEQ ID NO: 27), miSNCA13 (SEQ ID NO: 28), miSNCA15 (SEQ ID NO: 29), miSNCA16 (SEQ ID NO: 30), miSNCA1 (SEQ ID NO: 80), miSNCA3 (SEQ ID NO: 81), miSNCA4 (SEQ ID NO: 82), miSNCA6 (SEQ ID NO: 83), miSNCA9 (SEQ ID NO: 84), miSNCA10 (SEQ ID NO: 85), miSNCA11 (SEQ ID NO: 86), miSNCA14 (SEQ ID NO: 87), miSNCA18 (SEQ ID NO: 88), and miSNCA19 (SEQ ID NO: 89). These structures contained miR451 only as a framework, and not as a miR144 helper. These were tested in vitro using a double luciferase assay to assess their effectiveness in reducing the expression of the linked SNCA reporter gene (SEQ ID NO: 34) in a double luciferase reporter plasmid. Of these 17 miRNAs, seven miRNAs encoded in ITR-containing vectors (SEQ ID NOs: 24-30) (pri-miRNAs of miSNCA2, miSNCA5, miSNCA7, miSNCA12, miSNCA13, miSNCA15, and miSNCA16; SEQ ID NOs: 17-23) showed the potential to reduce SNCA mRNA levels in a dose-dependent manner (Figure 3).

[0158] miSNCA guides were selected based on the following criteria: miRNA guide sequences should not contain stretches of >4G, >4C, >5A, and >5T; have a GC content of 30% to 70%; for exon 1a targeting guides, they should have fewer than 4000 predicted off-target genes in the miRNA seed sequence, and for intron 1 targeting guides, fewer than 5000 predicted off-target genes in the miRNA seed sequence, by using siSPOTR analysis (https: / / sispotr.icts.uiowa.edu. / sispotr / tools / lookup / evaluate.html); and have a pre-miRNA sequence folding energy of -44kcal / mol to -55kcal / mol. To create a negative control, a scrambled guide was designed for in vitro testing and named miSCR (SEQ ID NO: 90).

[0159] The selected miSNCA guide meets the following criteria: conservation by monkey SNCA gene sequence (rhesus macaque, NCBI accession number NC_041768.1); the miRNA guide sequence does not contain stretches of >4 G or >4 C nt; GC content is 20% to 70%; GC seed content is 40% to 70%; pre-miRNA sequence folding energy is -45 kcal / mol to -55 kcal / mol; and there is no agreement with endogenous miRNA seeds.

[0160] The guide sequence was incorporated into the human pri-miRNA miR-451 scaffold sequence, and the mFold program (http: / / unafold.rna.albany.edu / ?q=mfold) was used with standard settings to determine whether the candidate structures were folded into secondary structures.

[0161] The original SNCA scaffold (Scaffold 1) consists of only one miR451 as the scaffold. The improved SNCA scaffold (Scaffold 2) consists of a miR-144 hairpin and one miR-451 downstream scaffold. Scaffold 2 is an improved version of the original construct and contains miR144, which is a helper for the processing of miSNCA. The placement of the miR144 hairpin is always at the 5' end of the miR451 hairpin sequence (compared to most miR451 hairpin sequences). Seven SNCA constructs (Scaffold 1) (SEQ ID NOs. 37-43) were constructed to target SNCA mRNA, and two improved SNCA constructs (Scaffold 2) (SEQ ID NOs. 91 and 92) were generated to target different parts of SNCA mRNA.

[0162] Dual reporter luciferase assay HEK293T cells were used for the dual luciferase assay and endogenous α-syn reduction. For the dual luciferase assay, HEK293T cells (1 × 10⁶) were used. 5 Cells were triple-seed in 24-well tissue culture plates. Cells were co-transfected with a reporter plasmid (SEQ ID NO: 33) containing a ligated SNCA reporter sequence (SEQ ID NO: 34) (10 ng) and plasmids containing various amounts (0.1–1–10–100 ng) of miSNCA candidates using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were then collected two days after transfection, and cell samples were analyzed for sea urchin luciferase and firefly luciferase activity using the Promega Dual Luciferase Assay Kit. The assay was performed using a GloMax Luminescence reader. α-syn decrease was measured as a decrease in the RL / FL activity ratio. The experiment was repeated an average of three times.

[0163] Transfection and endogenous α-syn depletion HEK293T cells were used to evaluate the reduction of endogenous α-syn by candidate miSNCAs. For these assays, HEK293T cells (5 × 10⁶) were used.5 Cells were triple-seed in 6-well tissue culture plates. Cells were transfected with plasmids containing various amounts (50-200-1000 ng) of miSNCA candidates using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were then harvested 2 days after transfection. Cell samples were analyzed for SNCA mRNA and α-syn protein levels. The experiment was repeated an average of 3 times.

[0164] DNA construct for baculovirus seed production Expression cassettes containing different miSNCA constructs were subcloned into plasmid-containing ITRs to produce pVD1496 (SEQ ID NO: 37), pVD1497 (SEQ ID NO: 38), pVD1498 (SEQ ID NO: 39), pVD1499 (SEQ ID NO: 40), pVD1500 (SEQ ID NO: 41), pVD1501 (SEQ ID NO: 42), and pVD1502 (SEQ ID NO: 43; Figure 8).

[0165] All of these pVD plasmids carry the CAG promoter and the introns necessary for promoter activity, followed by the miR451 backbone (SEQ ID NOs. 24-30) and the miSNCA construct in the bGH poly-A sequence (Figure 2A).

[0166] Improved constructive versions of miSNCA5 and miSNCA15 were also constructed by incorporating the miSNCA5 guide sequence or the miSNCA15 guide sequence into miR451, which is downstream of the miR144 helper miRNA (scaffolds containing miR144 and miR451; Figure 2B), thereby producing miR144-miSNCA5 (SEQ ID NO: 31) and miR144-miSNCA15 (SEQ ID NO: 32). These expression cassettes were subcloned into pVD1587 (SEQ ID NO: 91) and pVD1588 (SEQ ID NO: 92), which contain the ITR region for AAV5 packaging.

[0167] AAV5 Vector Recombinant AAV5 with an expression cassette was produced by infecting SF+ insect cells (Protein Sciences Corporation, Meriden, Connecticut, USA) with two baculoviruses encoding Rep, Cap, and Transgene. The titer of purified AAV was determined by QPCR following a standard protein purification procedure using a high-performance protein liquid chromatography system (AKTA Explorer, GE 30 Healthcare) with AVB Sepharose (GE Healthcare).

[0168] in vitro models and transduction assays To measure the effects of AAV5-miSNCA on human α-syn mRNA and protein levels, patient-derived iPSC-derived dopaminergic neurons (DA neurons) were used. The iPSC cell lines (Table 4) were obtained from the NINDS RUCDR repository.

[0169] [Table 4]

[0170] We differentiated iPSC cells into DA neurons using Thermo Fisher Scientific's PSC Dopaminergic Neuron Differentiation Kit.

[0171] The above in vitro cell models were transduced using baculovirus-produced AAV5-miSNCA candidates at various MOIs (Multiple Infection Intensities) of the virus. The cells were 5 × 10⁶ 5 Cells were seeded at a rate of one cell / well in 6-well plates coated with PDL-laminin or PLO-laminin. After subculturing for 3-4 days, the cells were passed through 10 4 , 10 5 , 10 6 and 10 7Cells were transduced at the specified MOI. Cells were then harvested 7–15 days after transduction. Cell samples were used for RNA and DNA isolation to determine vector DNA levels, miSNCA expression, SNCA mRNA, and a-syn protein expression.

[0172] RNA isolation and small RNA sequencing using next-generation sequencing (NGS) a. HEK-produced AAV5-miSNCA candidate RNA was transduced into AAV5-miSNCA (HEK-producing) DA neurons (MOI is 10) using the Allprep DNA / RNA Microkit (Qiagen). 6 RNA was isolated from cells. RNA integrity was determined using a Bioanalyzer, and RNA was quantified using Nanodrop. The samples were then sent to GenomeScan BV (Leiden, Netherlands) for small RNA sequencing. Small RNA sequencing was performed by GenomeScan using the NebNext small RNA library preparation method, which includes BluePippin size selection of the final library combined with Illumina NovaSeq 6000 PE 150 sequencing. The data were analyzed using CLC Genomics Suit (Qiagen). The expression levels of miSNCA candidates were expressed as the RNA count of miSNCA candidates relative to the total annotated miRNA sequence count. Processing of miSNCA candidates was analyzed by aligning the miSNCA pri-miRNA sequence to the sequenced RNA molecule. MiSNCA molecules of various sizes and their counts were obtained.

[0173] b. Derived from a baculovirus-produced AAV5-miSNCA candidate. RNA was isolated from AAV5-miSNCA candidate (baculovirus-produced) transduced cells (DA neurons, forebrain neurons, and / or LUHMES-derived DA neurons) using the Zymogen RNA isolation kit. RNA quality was determined using Bioanalyzer, and RNA was quantified using Nanodrop. The samples were then sent to GenomeScan BV (Leiden, Netherlands) for small RNA sequencing using next-generation sequencing. Data were analyzed using CLC Genomics Suit (Qiagen) to extract information about the expression levels of the miSNCA candidate and to identify the processing of the miSNCA candidate expressed from the baculovirus-produced AAV5-miSNCA candidate.

[0174] RNA was isolated from rat striatum samples from in vivo study #2, group #4 (described below). Both AAV5-miSNCA5 and AAV5-miSNCA15 were injected into the striatum of these animals, and their processed sequences were identified by sequencing analysis of the samples. RNA was isolated using the AllPrep DNA / RNA isolation kit. RNA quality was tested using a Bioanalyzer and quantified using Nanodrop. The samples were then sent to GenomeScan BV (Leiden, Netherlands) for small RNA sequencing using next-generation sequencing. Data were analyzed using the CLC Genomics Suit (Qiagen) to evaluate the processing of miSNCA candidates expressed from baculovirus-produced AAV5-miSNCA candidates.

[0175] Analysis of Small RNA Sequencing (NGS) Data Data analysis was performed using the CLC Genomics Workbench 10 suite. Trimmed small RNA sequence reads were counted and annotated using the miRbase database. miSNCA molecules were annotated by aligning pri-miRNA sequences to these small RNA libraries. The expression level of miSNCA candidates was expressed as the number of miSNCA candidate counts relative to the total annotated small RNA counts. The most expressed miSNCA molecules were analyzed by examining the relative counts of miSNCAs of various sizes, aligning pre-miSNCAs to small RNA libraries, and using the resulting RNA counts.

[0176] Isolation and quantification of vector DNA from cells and animal tissues DNA extraction was performed using the AllPrep DNA / RNA Mini Kit (Qiagen) according to the manufacturer's instructions. Vector genome copies were quantified using a TaqMan qPCR assay (Thermo Fisher Scientific) with primers targeting the poly-A region of the vector. Quantification (GC / ug DNA) was performed using a linearized pVD plasmid, and standard curves were created using various amounts of this plasmid. Using these standard curves, the vector DNA copy number was calculated from DNA isolated from cells transduced with AAV5-miSNCA.

[0177] Isolation of RNA and proteins from transfected HEK cells, and quantification of mSNCA and α-syn protein levels. For RNA isolation, Direct-zol® RNA Miniprep (catalog number R2050) was used. Rapidly frozen cell pellets were lysed with TRIzol. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) for RT-qPCR.

[0178] For protein isolation, a RIPA buffer (Sigma) containing the PhosSTOP phosphatase inhibitor (Roche) and the EDTA-free protease inhibitor (Roche) was used. For protein extraction, the buffer was added to the cell pellet, and the cells were stirred at 4C and 400 rpm for 30 minutes. The cell extract was then centrifuged at maximum speed. The clarified supernatant was used for α-syn and total protein assays, i.e., HTRF and BCA assays.

[0179] For mSNCA level detection, a SYBR Green-based RT-qPCR assay was used with a primer set designed for SNCA (Table 5). Results were presented as a magnification change using the ΔΔ cycle threshold (ΔΔCt) of the treated sample compared to the untreated sample, normalized to the mean expression of housekeeping genes: UBE, CYC 1, and ACTB (Table 5).

[0180] [Table 5]

[0181] The Total α-syn HTRF Kit (Cisbio) was used to detect α-syn protein levels. The HTRF measurements were then normalized by the total protein added to the HTRF assay. Total protein was measured using the bicinchoninate assay (BCA Protein Assay Kit; Pierce®). HTRF results are expressed as HTRF ratio / total protein μg.

[0182] RNA isolation and quantification of miSNCA candidate, GFP mRNA, and SNCA mRNA from animal tissues. Tissue samples were homogenized using the Tissue Lyser System (Qiagen) and the AllPrep DNA / RNA Mini Kit (Qiagen) according to the manufacturer's instructions. The quantity and integrity of DNA and RNA were determined using Nanodrop and Bioanalyzer.

[0183] For miSNCA expression, the following protocol was used: Total RNA was isolated using the AllPrep DNA / RNA Microkit (Qiagen). RT-qPCR was performed using the Taqman stem-loop-miRNA assay (Thermo Fisher), designed to detect 23 nt miSNCA5 and 22 nt miSNCA15. Expression levels were expressed as miRNA molecules / ug total RNA.

[0184] The assay IDs for these Taqman assays (Thermo Fisher) are CTNKRV7 for miSNCA5_23nt and CTTZ9KY for miSNCA15_22nt. Total RNA was isolated using the AllPrep DNA / RNA Micro kit (Qiagen) for mRNA expression.

[0185] We measured SNCA and GFP mRNA expression using two different RT-qPCR assays: 1. SNCA or GFP mRNA expression was measured using a SYBR Green-based RT-qPCR assay. The housekeeping genes used were ACTB, B2M, GAPDH, and HPRT. Primer sequences are shown in Table 6. 2. Taqman assay for SNCA mRNA: Table 7 shows primer and probe sequences designed for SNCA mRNA expression and Taqman ID for immediate-use Taqman assays for housekeeping genes.

[0186] [Table 6]

[0187] [Table 7]

[0188] LC-MS / MS from animal tissues Striatal tissue samples were sent on dry ice to the Vanderbilt Neurochemistry Core Facility (Nashville, TN, USA), where catecholamine levels were determined, and the data were sent back to Atuka in a blinded state for analysis.

[0189] Tissue-extracted brain sections were processed using a tissue dismembrator in 10 -2 M sodium acetate, 10 -4 M EDTA and 7.5% methanol (pH 3.8), homogenized in 100 to 750 μl of 0.1 M TCA. 10 μl of homogenate was removed for protein concentration measurement. The samples were then centrifuged at 10,000 g for 20 minutes at 4°C in a microcentrifuge. The supernatant was transferred to a new microcentrifuge tube for biogenic amine analysis.

[0190] Biogenic amine analysis: Dopamine, HVA, and DOPAC levels were determined by a highly sensitive and specific liquid chromatography / mass spectrometry (LC-MS / MS) method after derivatization of analytes with benzoyl chloride (BZC). 5 μl of the supernatant was treated with 10 μl each of 500 mM NaCO3 (aqueous solution) and 2% BZC in acetonitrile. After 4 minutes, 13 the reaction was stopped by adding 10 μl of internal standard solution (in 20% acetonitrile containing 3% sulfuric acid) containing 200 pg each of C6-derivatized dopamine-d4, HVA and DOPAC. Liquid chromatography was performed on a 2.0 × 50 mm, 1.7 μm particle Acquity BEH C18 column (Waters Corporation, Milford, MA, USA) using a Waters Acquity UPLC system. Mobile phase A was 0.15% formic acid aqueous solution, and mobile phase B was acetonitrile. Before samples were delivered to a SCIEX 6500+ QTrap mass spectrometer (AB Sciex, Framingham, MA, USA), they were separated by a gradient of 98% to 5% mobile phase A over 11 minutes at a flow rate of 600 μl / min. The following MRM transitions were monitored for quantitative purposes: 466 to 105 for BZC-dopamine; 488 to 111, 13C6-BZC-dopamine-d4; 304 to 150, BZC-HVA; 310 to 111, 13 C6-BZC-HVA; 394 to 105, BZC-DOPAC; 406 to 111, 13 C6-BZC-DOPAC. Automated peak integration was performed using SCIEX Multiquant software version 3.0.2. All peaks were visually inspected to ensure proper integration. Peak area ratio (P 分析物 / P I.S. Using a calibration curve constructed based on concentrations against an internal standard, levels of dopamine, HVA, and DOPAC in the samples were calculated by linear regression. The levels were normalized against protein concentrations in tissue extracts.

[0191] Protein concentrations in tissue homogenates were determined using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific, MA USA) as described in the kit instructions. Absorbance was measured using a POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).

[0192] ELISA for human α-syn derived from transgenes Dissected striatal tissue from fresh frozen sections of all animals was homogenized in a lysis buffer containing protease and phosphatase inhibitors (Roche: 11836153001). The samples were stirred at 4°C for 30 minutes, then centrifuged (4°C at 135,000 rpm for 10 minutes) to produce the supernatant. Total protein levels were determined using a portion of the supernatant with a 1:500 dilution at a concentration of 0.001 mg / ml (BCA assay, Pierce, Rockford, IL). Another portion of the supernatant was subjected to an ELISA procedure according to the manufacturer's instructions (BioLegend: 844101). Samples were analyzed using a CLARIOstar system to quantify luminescence counts relative to the amount of aSyn. αSyn levels were expressed as pg / mg total protein (Pierce® BCA Protein Assay Kit, Thermo Fisher Scientific, MA USA).

[0193] Dopamine transporter (DAT) binding Striatal DAT levels were assessed by [125I]-RTI-121-bound autoradiography in cryostat sections prepared from 20 μm fresh frozen tissue. Briefly, thawed slides were placed in a binding buffer containing 50 mM Tris, 120 mM NaCl, and 5 mM KCl (2 × 15 min, room temperature). The sections were then placed in the same buffer containing 50 pM [125I]-RTI-121 (Perkin-Elmer, specific activity 2200 Ci / μmol) at 25°C for 120 minutes to determine total binding. Nonspecific binding was defined as that observed in the presence of 100 μM GBR 12909 (Tocris Bioscience). All slides were then washed in ice-cold binding buffer (4 × 15 min), rinsed with ice-cold distilled water, and air-dried. Next, the slides were mounted on autoradiography film (Kodak) along with a [125 I] microscale standard (Amersham), left at room temperature for approximately 7 days, and then developed. Autoradiograms were then analyzed using MCID software (Image Research Inc, Ontario, Canada). Densitometry analysis was performed on three striatum from each animal, and a baseline curve of cpm versus optical density was calculated from a β-emitting [14C] microscale standard. This was used to quantify the signal intensity as nCi / g. Background intensity was subtracted from each reading. The data were then expressed as the mean ± standard error signal intensity for each treatment group. Non-specific binding was calculated in the same manner and subtracted from the total to obtain specific binding. Non-specific binding was typically found to account for less than 1% of total binding.

[0194] Immunofluorescence and three-dimensional structure Immunofluorescence: Brain was frozen into 40 μm thick sections in the coronal plane using a freeze-slide microtome (Leica Microsystems Inc., Richmond Hill, ON), and six series of sections were stored in a cryoprotective solution (30% glycerol, 30% ethoxyethanol, 40% PBS). Dual-labeled immunofluorescence was performed using a single series of midbrain sections to identify hemagglutinin (HA)-tagged human aSyn and tyrosine hydroxylase (TH). In short, the levels and distribution of TH (sheep anti-TH, 1:1000, Pel Freez, P 60101; secondary antibody, Alexa fluor donkey anti-sheep, Fisher Scientific, A 21099, 1:500) and HA (rabbit anti-HA, 1:1000; Abcam, AB 9110; Alexa Fluor donkey anti-rabbit, 1:500, Fisher Scientific, A 21206, 1:500) were evaluated in free suspension sections by dual-labeled immunofluorescence.

[0195] Three-dimensional structure: TH with and without human α-syn colocalization in substantia nigra compacta (SNc) +veNeuron counts were estimated using Stereo Investigator software (MBF Bioscience, Williston, VT) according to stereochemical principles. Seven or eight sections, separated 240 μm from anterior SN to posterior SN, were used for counting in each case. Stereoscopic analysis was performed using a Zeiss microscope (AxioImager M2 and Apotome, Carl Zeiss, Canada) coupled to a monochrome digital camera for visualization of tissue sections. The total number of TH+ve neurons, with and without human α-syn inclusions, was estimated from coded slides using optical fractionation. For each analyzed tissue section, the section thickness was empirically assessed, and guard zones approximately 2 μm thick were used at the top and bottom of each section. SNc were outlined at low magnification (5x), and TH+ve neurons were counted at 40x magnification. Stereo parameters (i.e., grid size, count frame size, and dissector height) were empirically determined using Stereo Investigator software (MicroBrightfield, VT, USA). The tolerance coefficient (CE) was calculated according to the procedure of West et al., known as Gunderson CE (m=1). Gunderson values ​​less than 0.10 were observed.

[0196] Based on the results of counting stereochemistry, TH within the SNC is used to evaluate neuroprotection. +ve The absolute number of neurons was obtained. The number of remaining TH+ve neurons, including those responsive to human α-sinculaneum, was also obtained. +ve It was produced to provide an indicator of the number of human α-syn expressing neurons. Subsequently, TH +ve / Sinukrain +ve :TH +ve / Sinukrain -ve The ratio was calculated.

[0197] in vivo testing Study 1. Administration route study in wild-type (WT) rats In this study, the distribution of transgene expression (GFP) was evaluated 14 days after administration of AAV5-GFP to either the substantia nigra (SN), striatum, or cisterna magna. Two treatment groups were used, with N=5 animals per group (total N=10, female Sprague-Dawley rats, Envigo, USA). On day 1, animals received either a bilateral stereotactic injection of 4 µl of AAV5-GFP into the SN, a 3 × 3 µl (bilateral) stereotactic injection of AAV5-GFP into the striatum, or a 25 µl injection of AAV5-GFP into the cisterna magna. The groups are shown in Table 8.

[0198] [Table 8]

[0199] For intrasitu injection, the stereotactic coordinates were -5.2 mm AP, - / +2 mm ML relative to the bregma, the needle was lowered to -7.5 mm below the skull, and the basil was set to -3.3. Striatal stereotactic injection coordinates were site 1: +1.3 mm AP, - / +2.8 ML, -4.5 DV; site 2: +0.2 mm AP, - / +3.0 ML, -5.0 DV; site 3: site 2: -0.6 mm AP, - / +4.0 ML, -5.5 DV; the basil was set to -3.3. The viral vector was administered at a rate of 0.5 ul / min, with a 5-minute waiting period allowed after each injection. ICM administration was performed according to a method adapted from Chen et al. 2013 Acta Neurobiol Exp(Wars) 73(2):304-11.

[0200] On day 14, rats were administered an excess of isoflurane and sacrificed by transcardiac perfusion with ice-cold 0.9% saline. The brains were then removed, and the right hemisphere was post-fixed in 4% paraformaldehyde (overnight) and cryoprotected in sucrose solution. The forebrain and midbrain of the right hemisphere were then dissected with a freeze-slide microtome for histological procedures. The left hemisphere was dissected fresh into the region of interest and individually frozen for molecular analysis.

[0201] Study 2. Mechanism of action study in a-syn KI rats In this study, the mechanisms of action of two AAV-miSNCA candidates were evaluated in human α-synuclein KI rats. A total of three treatment groups were used, each containing N=3 animals (total N=9, Envigo, iUSA). On day 1, all animals received a unilateral injection of 3 × 3 µl of AAV5 into the striatum. The other side was used as a control and injected with the formulation buffer using the same method as the AAV5 injection. The groups are shown in Table 9.

[0202] [Table 9]

[0203] The stereotactic injection coordinates for the striatum were: Site 1: +1.3 mm AP, - / +2.8 ML, -4.5 DV; Site 2: +0.2 mm AP, - / +3.0 ML, -5.0 DV; Site 3: Site 2: -0.6 mm AP, - / +4.0 ML, -5.5 DV; the basil was set to -3.3. The viral vector was administered at a rate of 0.5 ul / min, with a 5-minute waiting period allowed after each injection.

[0204] On day 43, all rats were administered an excess of isoflurane and perfused transcardially with ice-cold 0.9% saline. Subsequently, the brains were removed as quickly as possible, and the left and right hemispheres were separated. In all animals in each group, the following regions—prefrontal cortex, striatum, hippocampus, hypothalamus, thalamus, posterior cortex, cerebellum, ventral midbrain, and brainstem—were newly dissected, separated into left and right hemispheres, frozen on dry ice, and stored at -80°C for molecular analysis.

[0205] Study 3. Study in the AAV-Syn rat model This study was designed to evaluate the ability of two artificial miRNAs (encoding aSyn) that target SNCA mRNA to protect dopaminergic function in an AAV1 / 2-hA53T-aSyn rat model of Parkinson's disease. The model involved injecting one side of wild-type rats with either AAV1 / 2 human A53Tα-syn (AAV1 / 2-hA53T-aSyn) or AAV5-miRNA (either miSNCA or an unrelated (control) miRNA). Two groups were used for the injection of these two viruses: a co-injection group and a sequential injection group. In the co-injection group, both viruses were injected on day 1 (groups 1-4 in Table 10), while in the sequential injection group, AAV1 / 2-Ha53T-aSyn was injected on day 1, followed by AAV5-miSNCA on day 14 (groups 5-8 in Table 10). On the days shown in Table 10, a single virus or combination was administered unilaterally to the right substantia nigra using stereotactic techniques. To assess forelimb asymmetry, behavioral assessments were performed using the cylinder test preoperatively (baseline, -3 days) and on days 14, 21, 42, and 56 (2, 3, 6, and 8 weeks after AAV administration). The groups are shown in Table 10.

[0206] [Table 10]

[0207] [Table 11]

[0208] On day 57, the animals were slaughtered for post-mortem evaluation. Blood and samples were collected, processed as necessary, and preserved.

[0209] The primary outcome measures of this study included the following: • Assessment of forelimb asymmetry (using the cylinder test) • Quantitative determination of striatal dopamine and metabolite levels (by LC-MS / MS) • Quantification of dopamine transporters (by autoradiography) • Quantification of aSyn levels derived from striatal transgenes (by ELISA)

[0210] Any endpoints included: Quantification of tyrosine hydroxylase-positive (TH+ve) cells in the substantia nigra with or without co-expression of human aSyn (by dual-labeled immunofluorescence). • Qualitative evaluation of the activation state of microglia in the substantia nigra by Iba-1 immunoreactivity (immunofluorescence).

[0211] Slaughter and sampling were performed as follows: Animals were deeply anesthetized with isoflurane and then killed by transcardiac perfusion with ice-cold 0.9% saline containing 0.2% heparin. The brains were placed ventrally superiorly in an ice-cold stainless steel rat brain matrix and first cut at the hypothalamic level in the coronal plane. The rostral portion of the brain, including the entire striatum, was immediately frozen in isopentane cooled to -42°C and subsequently sectioned for DAT autoradiography and dissected for quantification of dopamine and dopamine metabolites (HVA and DOPAC) levels by LC-MS / MS and human aSyn levels by ELISA. The tissues were stored in a lock freezer at -80°C. For molecular assays, additional regions of interest (including additional striatal dissections) were collected according to Table 11.

[0212] [Table 12]

[0213] The remaining portion of the brain's tail, including the midbrain, was immersed in 4% paraformaldehyde (PFA) for 48 hours for fixation, followed by cryoprotection in graded sucrose solutions (15 to 30% sucrose). Tissues prepared in this manner were used for quantification of dopamine neurons in the SNc via tyrosine hydroxylase immunohistochemistry and unbiased stereochemistry.

[0214] Experiment 4. Phenotype rescue of the motility phenotype in the nematode (C. elegans) PD model. This study evaluated the effect of miSNCA candidate expression on phenotypic rescue of altered motility behavior in the nematode (C. elegans) PD model (OW 40; van Ham et al 2008 PLoS Genet 4(3):e 1000027). In this model, human α-syn is overexpressed in the body wall muscle of C. elegans. This α-syn overexpression slows the movement of helminths compared to control helminths. The effect of reducing SNCA mRNA levels, and thereby reducing RNAi-mediated α-syn protein levels, was studied using the full-length SNCA gene or our miSNCA constructs. Double-stranded RNA containing one of these constructs was introduced into organisms by feeding. OW 40 nematodes (C. elegans) were fed with either an empty T 444T plasmid as a negative control, or E. coli overexpressing either the full-length SNCA gene or one of our miSNCA candidates (miSNCA5, miSNCA13, or miSNCA15) at different life stages: larval stage 1 (L1), larval stage 4 (L4), and day 1 of the adult stage. Treatment experiments were repeated at 25°C and 15°C. After treatment on days 1, 4, and 8 of the adult stage, the helminths were video-tracked using a high-throughput tracking setup configured to measure their movement (velocity as μm / s) (Perni et al 2018 Journal of Neuroscience Methods 306 57-67).

[0215] Further analysis involved RT-qPCR at the SNCA mRNA and α-syn protein levels using Western blot analysis. The primer sequences used for RT-qPCR of SNCA mRNA are shown in Table 12.

[0216] [Table 13]

[0217] Western blotting was used to detect α-syn protein levels. For this purpose, the protein was extracted using RIPA buffer and a tissue lyzer (Qiagen). Similar protein amounts from different processing conditions were loaded onto SDS-PAGE and Western blotting was performed using an anti-human α-syn antibody (Table 12) to detect α-syn levels. Tubulin was used for normalization and detected using an anti-tubulin antibody (Table 13).

[0218] [Table 14]

[0219] result: in vitro experiments In vitro silencing efficacy of artificial miSNCA constructs To evaluate the miSNCA knockdown efficacy of miSNCA constructs in vitro, HEK293T cells were co-transfected with a sea urchin luciferase reporter encoding the SNCA gene. The firefly luciferase (FL) gene was expressed from the same reporter vector and used as an internal control to correct for transfection efficiency. In the initial screening, HEK cells were co-transfected with 1 ng–10 ng–50 ng or 250 ng of each miSNCA construct and a Dual Luc reporter carrying the SNCA gene. Of the 17 miSNCA constructs designed to target the SNCA gene, miSNCA2, miSNCA5, miSNCA7, miSNCA12, miSNCA13, miSNCA15, and miSNCA16 induced a dose-dependent decrease in the RL / FL ratio. To further determine efficacy, the aforementioned constructs were further used in titration experiments. The constructs were co-transfected into HEK293T cells with 10 ng of SNCA luciferase reporter plasmid at different concentrations: 0.1, 1, 10, or 100 ng. These results indicate that transfection with 100 ng of miSNCA plasmid resulted in at least a 50% reduction for all miSNCA candidates used in titration experiments (Figure 3). miSNCA5, miSNCA13, and miSNCA15 were selected for further testing in different models due to their relatively large potency in reducing mSNCA levels.

[0220] To improve the efficacy of the miSNCA candidate, i.e., the natural companion of miR451, a modified miR144 was added to the scaffolds of miSNCA5 and miSNCA15 (Figure 2B) (SEQ ID NOs. 32-33). These constructs were designed so that the modified miR144 was added to the 5' end scaffold of miR451 containing the miSNCA candidate. Dual luciferase assays were performed to evaluate the efficacy of the original and improved miSNCA candidates. Constructs (miSNCA5 (SEQ ID NO: 25), miSNCA15 (SEQ ID NO: 29), miSNCA5+miR144 (SEQ ID NO: 31), miSNCA15+miR144 (SEQ ID NO: 32) and control miRNAs) were co-transfected into HEK293T cells with 10 ng of SNCA luciferase reporter plasmid at different concentrations per 24 wells: 0.1, 1, 10, or 100 ng. These results showed that the potency of miSNCA5 and miSNCA15 was improved by at least 2-3 times (Figure 4).

[0221] Decreased endogenous SNCA expression in transfected cells miSNCA5, miSNCA13, and miSNCA15 constructs were selected to test the knockdown of SNCA mRNA expression in cells. Knockdown of endogenous SNCA gene expression in HEK293T cells was confirmed by RT-QPCR on transfected cells. Transfection with 50ng–200ng–1000ng of miRNA plasmids resulted in a reduction of less than 40% of SNCA mRNA expression for all tested miSNCA candidates. The results were consistent at the protein level, with a dose-dependent decrease in α-syn levels as measured by HTRF (Figure 5).

[0222] miRNA expression levels in transduced cells (low-molecular-weight RNA sequencing data) The expression levels of mature miRNAs were quantified based on the total number of reads annotated using miRBase and the target pre-miRNA sequence. 10 6Expression levels of the top 30 and 35 most expressed miRNAs were obtained in DA neurons transduced with HEK-producing AAV5-miSNCA at the / cell MOI (Figures 6A and 6B), and the expression levels of miSNCA5 (Figure 6A) and miSNCA15 (Figure 6B) were well within the range of endogenous miRNA levels.

[0223] Processing of miSNCA constructs during transfection in cells (NGS data) miRNA processing was also investigated by read alignment to pre-miRNA sequences. For miSNCA5, the most abundant morphology length was 24 nt, followed by 23 nt and 25 nt (Figure 7A); for miSNCA15, it was 22 nt, followed by 24 nt and 23 nt (Figure 7B).

[0224] AAV5-miSNCA transduction in human cells To investigate the ability to transduce and deliver expression cassettes containing AAV5-miSNCA5 and AAV5-miSNCA15, DA neurons or forebrain neurons and / or LUHMES-derived DA neurons were transduced to various infection multiplicities (MOI);10 4 , 10 5 , 10 6 and 10 7 Transduction is performed. Vector DNA levels are measured, and a dose-dependent increase in vDNA levels should be observed in these cells. RNA is isolated from the transduced cells, and SNCA mRNA levels are measured using the RT-qPCR Syber Green assay. A dose-dependent decrease in SNCA mRNA levels is expected in these transduced cells.

[0225] Processing of miSNCA constructs from baculovirus-producing constructs (NGS data) The processing of miRNAs extracted from rat brain tissue in in vivo experiment #2, group #4 was also investigated to evaluate the processing of baculovirus-produced AAV5-miSNCA. Small RNA sequencing was performed on these samples, and the data were aligned with reads to pre-miRNA sequences. For miSNCA5, the most abundant morphology was 23 nt, followed by 24 nt and 25 nt; for miSNCA15, it was 22 nt, followed by 24 nt and 23 nt (Figure 12).

[0226] in vivo testing Study 1. Different administration routes of AAV5-GFP in WT rats showed good coverage in the target region affected by Parkinson's disease. To address the suitability of the AAV5 vector for delivering miSNCA candidates to target brain regions, coverage of brain regions (brainstem, midbrain, and cortex) exhibiting significant α-syn pathology in Parkinson's disease was evaluated after AAV5 administration. Different administration routes—substantia nigra (SN), striatum, or cisterna magna—were tested. GFP was used as the reporter gene. AAV5-GFP injected into the SN at the two tested doses, or into the striatum at the tested single dose, showed adequate in vivo distribution in the target regions, as assessed by AAV-GFP vDNA levels in the brain (Figure 9A) and corresponding GFP mRNA expression (Figure 9B). AAV5-GFP injected into the cisterna magna (directly into the cerebrospinal fluid) provided, to a lesser degree, equivalent coverage of all brain regions examined. Therefore, it was concluded that AAV5 is a suitable vector for delivering miSNCA candidates to target brain regions for the treatment of Parkinson's disease.

[0227] Test 2. The AAV5-miSNCA candidate reduced human SNCA mRNA expression in a-syn KI rats. To evaluate the ability of two design candidates (miSNCA5 and miSNCA15) to reduce human SNCA mRNA expression, AAV5-miSCR (non-targeted scrambled control), AAV5-miSNCA5, or AAV5-miSNCA15 were injected into the left striatum of adult a-syn KI rats. One group received an equivalent dose of a combination of AAV5-miSNCA5 and AAV5-miSNCA15. miSNCA13 was excluded from the in vivo study because it targets the region outside the humanized portion of the SNCA KI rat model and has three mismatches with the WT rat SNCA gene. The right striatum was injected with a formulation buffer and used as a further control. With the single dose used, vDNA was detected in the AAV5-injected hemisphere, but vDNA levels were below the limit of quantification (LLOQ) in the control hemisphere (Figure 10-1A). Transduction resulted in the expression of miSNCA candidates 5 and 15 or a combination thereof in vector-specific manner (Figure 10-1B and Figure 10-2C): miSNCA5 was detected only in the AAV5-miSNCA5 injected hemisphere, miSNCA15 was detected only in the AAV5-miSNCA15 injected hemisphere, and both miSNCA5 and miSNCA 15 were detected in the AAV5-miSNCA5+AAV5-miSNCA15 injected group. At the single dose used, AAV5-miSNCA5 and AAV5-miSNCA5+AAV5-miSNCA15 were effective in reducing SNCA mRNA expression in the injected striatum compared to the control striatum (Figure 10-2D), as evaluated by two different RT-QPCR SNCA assays (primer set SNCA1 and primer set SNCA2). This study supports the mechanism of action of AAV5-miSNCA candidate for reducing human SNCA mRNA expression and α-syn toxicity in the treatment of Parkinson's disease.

[0228] Study 3. Study in the AAV-Syn rat model Different AAV5-miSNCA candidates were tested in the AAV-Syn rat model.

[0229] To demonstrate in vivo proof of the concept that reducing SNCA levels improves the motor phenotype of the α-syn virus-overexpressing human A53T mutant, a rat PD model (AAV1 / 2-Ha53T-aSyn) was used. Both AAV1 / 2-hSNCA and AAV5-miSNCA viruses were injected unilaterally into the right substantia nigra (SN). In both the concurrent and sequential injection groups, the single dose used resulted in the detection of vDNA in the AAV5-injected hemisphere in the striatum (Figure 13), while vDNA levels in the control hemisphere (left striatum) were below the limit of quantification (LLOQ) (not shown). Transduction resulted in the expression of miSNCA5 and miSNCA15 in a vector-specific manner (Figure 14A and B): miSNCA5 was detected only in the AAV5-miSNCA5 injected hemisphere, and miSNCA15 was detected only in the AAV5-miSNCA15 injected hemisphere; neither was detected in other samples from the negative control group. In A 53 T-aSyn animals, evaluation by Taqman RT-qPCR assay (SNCA 2 primer and probe combination) compared to a control striatum injected with an unrelated miRNA (black solid line) (Figure 15) showed that AAV5-miSNCA5 and AAV5-miSNCA15 were effective in reducing SNCA mRNA expression in the injection site striatum with the single dose used. miSNCA expression also showed a decrease at the protein level, reflected by reduced α-syn protein levels measured by ELISA (Figure 16). Dopamine transporter deficiency, as measured by [125 I]-RTI-121 autoradiography, was evident in A 53 T-aSyn animals sequentially injected with control miRNA (irrelevant miR, group 6 in Table 10), similar to the case in PD patients, and was corrected in the miSNCA-treated groups (groups 7 and 8 in Table 10) (Figure 17). In relation to metabolite changes in this model, correction of Ha53T-aSyn-induced striatal dopamine deficiency with candidate miSNCAs was observed. Figure 18A shows dopamine levels in the test group, and Figure 18B shows (DOPAC+HVA) / DA levels measured by LC / MS.

[0230] Locomotor behavior, measured by percent left foot asymmetry, was significantly improved on day 56 in the continuous injection group that received miSNCA5 treatment or miSNCA15 treatment compared with baseline levels (Figure 19A and Figure 19B).

[0231] Molecular, biochemical and locomotor behavioral results were supported by histological observations. Immunostaining and quantification of dopaminergic (TH-positive) and α-syn-positive neurons in the substantia nigra showed that both AAV5-miSNCA candidates (continuous injection group) rescued dopaminergic (TH) neuronal cell loss (Figure 20-1A) and reduced the number of human α-syn cells (Figure 20-1B). This was reflected by a reduction in the proportion of positive dopaminergic (TH) cells expressing α-syn (Figure 20-2C). A reduction in inflammation in this model, assessed by Iba1 immunoreactivity in the substantia nigra, is also expected.

[0232] Overall, AAV5-miSNCA reversed the disease phenotype, improved the locomotor phenotype and rescued molecular and neurochemical changes in the AAV-Syn rat model, demonstrating that miSNCA treatment is an effective therapy for reducing α-syn toxicity.

[0233] Test 4. Phenotype rescue in *C. elegans* PD model using miSNCA candidate sequences To compare the movement speed between nematodes supplied by different plasmid-expressing *E. coli*, 100 *C. elegans* nematodes were video-tracked for each condition. According to the results, worms fed with *E. coli* expressing full-length SNCA or miSNCA showed improved movement speed compared with worms fed with *E. coli* transformed with the empty plasmid (Figure 11). Worms treated with full-length SNCA or miSNCA had increased speed compared with untreated worms on all days when their movement was tracked. These results indicate that reducing SNCA gene expression and thereby decreasing α-syn levels improves the motor phenotype in this *C. elegans* PD model. Furthermore, the results demonstrate that worms can be treated at different life stages.

[0234] Treatment with miSNCA reduced SNCA mRNA levels (A of Figure 21-1 to C of Figure 21-1) and α-synuclein protein levels (D of Figure 21-2 to F of Figure 21-2) in the *C. elegans* PD model when nematodes were treated at the larval stage or adult stage. Consistent with this, as shown in Figure 22, miSNCA treatment rescued the motor phenotypic behavior in this model as an improvement in swimming speed in miSNCA-treated worms compared with negative control worms (EV-treated worms).

[0235] Small RNA sequencing performed on *C. elegans* samples collected from nematodes treated with miSNCA5, miSNCA15 and full-length SNCA confirms the presence of correctly processed miSNCA candidates in the samples (Figure 23-1 and Figure 23-2). The miSNCA5 sequence and miSNCA15 sequence, as well as other miSNCA sequences (e.g., miSNCA 7, miSNCA12 and miSNCA13), were detected from full-length SNCA-treated samples.

Claims

1. A nucleic acid comprising a nucleic acid sequence encoding RNA, wherein the RNA sequence contained in the RNA is substantially complementary to the target sequence of the α-synuclein (α-syn) gene (SNCA), the RNA sequence has at least 15 nucleotides, the RNA contains a hairpin, the RNA contains SEQ ID NO: 1 and SEQ ID NO: 2, and the target sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 3 to 9.

2. The nucleic acid according to claim 1, wherein the RNA sequence has at least 18 nucleotides.

3. The nucleic acid according to claim 1, wherein the RNA sequence has a maximum of 32 nucleotides.

4. The nucleic acid according to claim 1, wherein the RNA sequence includes one sequence selected from the group consisting of sequence numbers 10 to 16.

5. The nucleic acid according to claim 1, which is a DNA molecule.

6. A DNA molecule contained in a DNA expression cassette, wherein the DNA expression cassette further comprises a promoter and a polyA tail, and the nucleic acid is adjacent to an inverse terminal repeat sequence (ITR), as described in claim 5.

7. The DNA molecule according to claim 6, wherein the promoter is a ubiquitous promoter; a neuron-specific promoter; or a glial-specific promoter.

8. An adeno-associated virus (AAV) vehicle comprising the DNA molecule described in claim 5.

9. The AAV vehicle according to claim 8, comprising a capsid containing an AAV5 or AAV9 capsid protein sequence.

10. The AAV vehicle according to claim 8, wherein the AAV vehicle is a gene therapy vehicle.

11. A composition comprising the AAV vehicle according to claim 10 and at least one pharmaceutically acceptable excipient.

12. The AAV vehicle according to claim 8 for use as a pharmaceutical.

13. The composition according to claim 11 for use as a pharmaceutical.

14. The AAV vehicle according to claim 12, wherein the pharmaceutical agent reduces and / or knocks down the transcript encoded by the α-syn gene.

15. The composition according to claim 13, wherein the pharmaceutical agent reduces and / or knocks down a transcript encoded by the α-syn gene.

16. The AAV vehicle according to claim 12, wherein the pharmaceutical agent reduces the amount of Lewy bodies and / or Papp-Lantos bodies.

17. The composition according to claim 13, wherein the pharmaceutical agent reduces the amount of Lewy bodies and / or Papp-Lantos bodies.

18. The AAV vehicle according to claim 12, wherein the pharmaceutical is used to treat and / or prevent clinical symptoms of Parkinson's disease (PD), Lewy body dementia (LBD), multiple system atrophy (MSA), neurological symptoms, motor symptoms of PD, cognitive impairment, sleep disorders, autonomic disorders, and / or olfactory disorders.

19. The composition according to claim 13, wherein the pharmaceutical is used to treat and / or prevent clinical symptoms of Parkinson's disease (PD), Lewy body dementia (LBD), multiple system atrophy (MSA), neurological symptoms, motor symptoms of PD, cognitive impairment, sleep disorders, autonomic nervous system disorders, and / or olfactory disorders.

20. The AAV vehicle according to claim 12, wherein the pharmaceutical is used to treat and / or prevent PD and / or MSA.

21. The composition according to claim 13, wherein the pharmaceutical is used to treat and / or prevent PD and / or MSA.

22. A method for manufacturing the AAV vehicle described in claim 8.

23. A kit comprising the AAV vehicle according to claim 8, further comprising an immunosuppressive compound.

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