Nucleic acid regulation of SNCA

Abstract

The present invention relates to nucleic acids comprising two or more RNA coding sequences, where at least one of the sequences comprises a guide sequence substantially complementary to a portion of the SNCA gene. The present invention also relates to related AAVs, compositions, pharmaceutical compositions and therapeutic uses thereof.

Description

[Technical field]

[0001] The present invention relates to the fields of biotechnology, medicine and gene therapy. In particular, the present invention relates to a nucleic acid comprising two or more RNA coding sequences, wherein the sequences comprise a guide sequence substantially complementary to a portion of the alpha-synuclein (SNCA) gene. The present invention also relates to related AAV, compositions, pharmaceutical compositions and their use in therapy. [Background technology]

[0002] Fibrillar α-synuclein inclusions define two major neurodegenerative disease classes: Lewy body diseases, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and those characterized by Papp-Lantos bodies, including multiple system atrophy (MSA), collectively referred to as synucleinopathies.

[0003] PD is a complex progressive neurodegenerative disorder that includes several motor symptoms, such as tremor, rigidity, bradykinesia and / or postural instability. Non-motor symptoms are also observed in PD patients before and / or after clinical diagnosis. Non-motor symptoms include depression, sleep disorders, pain and / or fatigue in the early stages of the disease, and anxiety, dementia and / or cognitive impairment in the later stages of the disease. Both motor and non-motor symptoms are very debilitating for patients and their caregivers.

[0004] Current treatment focuses on short-term relief of motor symptoms.However, before showing any signs of motor symptoms, PD patients may suffer from non-motor symptoms before diagnosis, which significantly reduces quality of life as described above.Therefore, symptomatic treatment is not a preventive approach to treat and / or prevent PD.

[0005] PD is a complex disorder, the cause of which remains unknown. Nevertheless, a number of genes have been found to be involved in the cause and / or progression of PD. One of these genes is the SNCA gene, which codes for the α-synuclein protein.

[0006] The main feature of PD pathology is neurodegeneration of dopaminergic neurons in the substantia nigra, a brain region in the midbrain with associated dopaminergic projections to the striatum and cortex, which are central to movement-related functions. In addition to loss of nigrostriatal dopaminergic innervation and degeneration in other brain regions, PD is characterized by the presence of cytoplasmic protein aggregates (Lewy bodies) that contain an insoluble protein encoded by the SNCA gene.

[0007] As PD progresses, Lewy bodies spread from initial areas including the olfactory bulb and motor nuclei in the brainstem to locations in the locus ceruleus and substantia nigra at later stages, and finally to cortical regions. Symptoms in different PD stages have been found to be related to the specific brain regions where Lewy bodies spread and / or accumulate. This also means that α-synuclein protein aggregates correlate with PD and may result in loss of normal function and / or toxic effects in neurons, resulting in neurodegeneration and / or neuroinflammation in different brain regions.

[0008] Native α-synuclein protein in the brain is largely unfolded, with no defined tertiary structure. Upon interaction with negatively charged lipids, such as the phospholipids that make up cell membranes, α-synuclein folds into an α-helical structure via its N-terminus. However, in PD, α-synuclein protein adopts an amyloid-like structure rich in β-sheets that is prone to aggregation. The aggregates constitute the majority of Lewy bodies.

[0009] MSA is a progressive adult-onset neurodegenerative disorder of undetermined etiology characterized by argyrophilic glial cytoplasmic inclusions (GCIs) and a distinctive oligodendrogliopathy with selective neurodegeneration. GCIs or Papp-Lantos inclusions / bodies are now accepted as the definitive neuropathological diagnostic feature of MSA and have been suggested to play a central role in the pathogenesis of the disorder. GCIs are composed of hyperphosphorylated α-syn, ubiquitin, LRRK2 (leucine-rich repeat serine / threonine protein) and other proteins. Summary of the Invention [Problem to be solved by the invention]

[0010] Currently, gene therapy for treating and / or preventing diseases is based on completely knocking down genes and / or gene transcripts. The α-synuclein protein encoded by SNCA is thought to be involved in the regulation of dopamine release and transport involved in synaptic transfusion. Therefore, due to the important physiological role of the α-synuclein protein, depletion of SNCA transcripts can have a major impact on the health of patients. Therefore, there is still a need to have an optimized and highly potent treatment that can treat and / or prevent the different stages of PD and other α-synucleinopathies before and / or after clinical diagnosis, while minimizing and / or preventing the risks. [Means for solving the problem]

[0011] The present invention solves this problem by using a nucleic acid that includes at least two RNA coding sequences, both of which include a guide sequence that is substantially complementary to a portion of the SNCA gene.

[0012] The present invention provides a very versatile system that allows the simultaneous use of several guide sequences. Expression of different therapeutic miRNAs from the same vector results in an increased therapeutic efficacy, resulting in an optimized and very potent inhibition of mRNA expression. Thus, when using the nucleic acid or AAV of the present invention in gene therapy, a single dose of treatment is expected. This reduces the risk of toxicity and immunogenicity associated with the use of said nucleic acid of the present invention. Furthermore, guide sequences can target sequences in different SNCA isoforms, whose aggregation properties are qualitatively and quantitatively different, thereby allowing the expression of RNAs encoded by said different isoforms to be substantially simultaneously reduced. Moreover, the cost of treatment based on the nucleic acid or AAV of the present invention is expected to be lower than other gene therapy products, presenting a substantial economic advantage.

[0013] Thus, a first aspect of the invention relates to a nucleic acid comprising a sequence encoding a first RNA and a sequence encoding a second RNA, each of the first RNA and the second RNA comprising a hairpin, the first RNA comprising a first guide sequence of at least 19 nucleotides substantially complementary to a portion of the alpha-synuclein (SNCA) gene, and the second RNA comprising a second guide sequence of at least 19 nucleotides substantially complementary to a portion of the SNCA gene.

[0014] A second aspect of the invention relates to an expression cassette comprising a nucleic acid of the invention, wherein the expression cassette is a DNA molecule.

[0015] A third aspect of the invention relates to an adeno-associated virus (AAV) vector comprising a nucleic acid or expression cassette of the invention (the "AAV vector of the invention").

[0016] A fourth aspect of the invention relates to a pharmaceutical composition comprising a nucleic acid, an expression cassette, or an AAV vector of the invention.

[0017] A fifth aspect of the invention relates to the use of a nucleic acid, an expression cassette, an AAV vector or a pharmaceutical composition of the invention as a medicament.

[0018] A sixth aspect of the invention relates to a kit comprising a nucleic acid, an expression cassette, an AAV vector, or a pharmaceutical composition of the invention.

[0019] A seventh aspect of the invention relates to a cell comprising a nucleic acid, an expression cassette or an AAV vector of the invention.

[0020] Description of the invention definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

[0021] In this specification and the claims, the verb "comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of the elements is present, unless the context clearly requires that one and only one of the element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0022] For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtained". Hereinafter, if for example an antibody is defined as being obtainable from a particular source, this should be understood to also disclose the antibody obtained from this source.

[0023] As used herein, the term "and / or" indicates that one or more of the stated instances may occur alone or in combination with at least one of the stated instances, and up to all of the stated instances.

[0024] As used herein, "at least" a particular value means greater than or equal to the particular value. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15... etc.

[0025] The terms "about" or "approximately" when used in connection with a numerical value (e.g., about 10), preferably mean that the value can be a given value, more or less than 0.1% of the value (10).

[0026] As used herein, "effective amount" refers to the amount of agent required to improve the symptoms of a disease compared to untreated patients. The effective amount of an active agent used to practice the present invention, for example for the therapeutic treatment of cancer, will vary depending on the mode of administration, the age, weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective" amount, determined as genome copies per kilogram (GC / kg) (in the case of viral delivery vectors). Thus, in the context of this disclosure, in the context of administering a drug, substance or pharmaceutical product that is "effective against" a disease or condition indicates that administration in a clinically relevant manner will result in a beneficial effect in at least a statistically significant proportion of patients, such as amelioration of symptoms, cure, reduction of at least one disease sign or symptom, prolongation of life, improvement in quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of a particular type of disease or condition.

[0027] The use of a substance as a medicament as described herein can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. The products for use as medicaments described herein can be used in a method of treatment, such a method of treatment comprising the administration of the product for use.

[0028] The terms "homology", "sequence identity" and the like are used interchangeably herein. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as may be determined by the match between strings of such sequences. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

[0029] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptides or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman). Sequences can be called "substantially identical" or "essentially similar" when they share at least a certain minimum percentage of sequence identity (defined below) (e.g., when optimally aligned by the programs GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. When two sequences have similar lengths, a global alignment is appropriately used to determine sequence identity. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and a gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and score for percent sequence identity can be determined using computer programs such as GCG Wisconsin Package, Version 10.3 available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or open source software such as the programs "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as GAP described above, or using default settings (for both "needle" and "water", and for both protein and DNA alignments, the default gap opening penalty is 10.0, the default gap extension penalty is 0.5, and the default scoring matrix is ​​Blossum62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments such as those using the Smith Waterman algorithm are preferred.

[0030] Alternatively, the percentage of similarity or identity can be determined by searching against public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. 1990 J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present invention. BLAST protein searches can be performed with the BLASTx program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., 1997 Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0031] Within the context of the present invention, when referring to a given sequence (SEQ ID NO:), the term "variant thereof" includes any nucleic acid that retains at least some of the properties of the corresponding naturally occurring nucleic acid, such as reduced RNA expression. The term "variant" may include any nucleic acid that has at least 50; 55; 60; 65; 70; 75; 80; 90; or 95% sequence identity with the naturally occurring nucleic acid.

[0032] In some embodiments, the sequences of the nucleic acids of the invention are codon-optimized. Codon optimization refers to an experimental approach designed to improve the codon composition of a recombinant gene based on various criteria without changing the amino acid sequence. This is possible because most amino acids are coded for by more than one codon. Most codon optimization approaches avoid the use of rare codons. However, different approaches differ in the extent to which other features are considered, including mRNA elements that can inhibit expression, the nucleotide context of the start codon, mRNA secondary structure, sequence repeats, nucleotide composition, internal ribosome entry sites, promoter sequences, and putative splice donor and acceptor sites. In addition, some programs take into account protein structural information, intragenic poly(A) sites, stop codons in alternative reading frames, and dinucleotides that are targets for RNase cleavage, mutation, and methylation-dependent gene silencing. Those skilled in the art understand the requirements necessary to design such codon-optimized nucleic acids.

[0033] As used herein, the terms "selectively hybridizing", "selectively hybridizing" and similar terms are intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 66%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, preferably at least 95%, more preferably at least 98% or more preferably at least 99% homologous to each other typically remain hybridized to each other, i.e., such hybridizing sequences may share at least 45%, at least 50%, at least 55%, at least 60%, at least 65, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, more preferably at least 98% or more preferably at least 99% sequence identity.

[0034] A preferred, non-limiting example of such hybridization conditions is hybridization in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 1x SSC, 0.1% SDS at about 50°C, preferably about 55°C, preferably about 60°C, and even more preferably about 65°C.

[0035] Highly stringent conditions include, for example, hybridization in 5xSSC / 5xDenhardt's solution / 1.0% SDS at about 68°C and washing in 0.2xSSC / 0.1% SDS at room temperature. Alternatively, washing may be performed at 42°C.

[0036] Those of skill in the art will know what conditions to apply for stringent and highly stringent hybridization conditions. Further guidance regarding such conditions can be found, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3 rd Current Protocols in Molecular Biology, (John Wiley & Sons, NY), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, are readily available in the art.

[0037] Of course, polynucleotides that hybridize only to polyA sequences (such as the 3' terminal poly(A) region of an mRNA) or only to complementary stretches of T (or U) will not be included among the polynucleotides of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such polynucleotides will hybridize to any nucleic acid molecule containing a poly(A) stretch or its complement (e.g., substantially any double-stranded cDNA clone).

[0038] A "nucleic acid construct" or "nucleic acid vector" is understood herein to mean an artificial nucleic acid molecule resulting from the use of recombinant DNA technology. Thus, the term "nucleic acid construct" does not include naturally occurring nucleic acid molecules, although a nucleic acid construct may include (parts of) naturally occurring nucleic acid molecules. A "vector" is a nucleic acid construct (typically DNA or RNA) that serves to transfer an exogenous nucleic acid sequence (i.e. DNA or RNA) into a host cell. The vector is preferably maintained in the host by at least one of autonomous replication and integration into the genome of the host cell. The term "expression vector" or "expression construct" refers to a nucleotide sequence that can affect the expression of a gene in a host cell or host organism that is compatible with such a sequence. These expression vectors typically contain at least one "expression cassette", which is a functional unit that can affect the expression of a sequence that codes for an expressed product, the coding sequence being operably linked to a suitable expression control sequence that includes at least a suitable transcription control sequence and optionally a 3' transcription termination signal. Further factors necessary or useful for affecting expression may also be present, such as expression enhancer elements. The expression vector can be introduced into a suitable host cell to affect expression of the coding sequence in an in vitro cell culture of the host cell. Preferred expression vectors are suitable for expressing viral proteins and / or nucleic acids, in particular recombinant AAV proteins and / or nucleic acids.

[0039] As used herein, the term "promoter" or "transcriptional control sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, is located upstream in the direction of transcription of the transcription start site of the coding sequence, and is structurally identified by the presence of binding sites for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequence of nucleotides known to those of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by application of a chemical inducer or biological entity.

[0040] The term "reporter" can be used interchangeably with marker, but is primarily used to refer to a visible marker such as green fluorescent protein (GFP) or luciferase.

[0041] The terms "protein" and "polypeptide" are used interchangeably and refer to a molecule consisting of a chain of amino acids, without reference to a particular mode of action, size, three-dimensional structure, or origin.

[0042] The term "gene" refers to a DNA fragment that contains a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell and is operably linked to appropriate control regions (e.g., a promoter). A gene will usually contain several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) that contains a polyadenylation site. "Expression of a gene" refers to the process by which a DNA region operably linked to appropriate control regions, particularly a promoter, is transcribed into RNA that is biologically active, i.e., can be translated into a biologically active protein or peptide.

[0043] The term "homologous", when used to indicate the relationship of a given (recombinant) nucleic acid or polypeptide molecule to a given host organism or host cell, is understood to mean that in nature, the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably of the same type or strain. When homologous to a host cell, the nucleic acid sequence encoding the polypeptide is typically (but not necessarily) operably linked to another (heterologous) promoter sequence than in its natural environment, and, if applicable, another (heterologous) secretion signal sequence and / or terminator sequence. It is understood that control sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloning" genetically modified organisms (GMOs) (self-cloning is defined herein as in Annex II of the European Directive 98 / 81 / EC). When used to indicate the relationship of two nucleic acid sequences, the term "homologous" means that one single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization will depend on many factors, including the amount of identity between the sequences and the hybridization conditions, such as temperature and salt concentration, as discussed below.

[0044] The terms "heterologous" and "exogenous", when used in reference to a nucleic acid (DNA or RNA) or protein, refer to a nucleic acid or protein that is not naturally occurring as part of the organism, cell, genome or DNA or RNA sequence in which it is found, or that is found in a location in a cell or genome or DNA or RNA sequence different from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell in which they are introduced, but are obtained from another cell or are synthetically or recombinantly produced. Generally, although not necessarily, such nucleic acids code for proteins that are not normally produced by the cell in which the DNA is transcribed or expressed, i.e., exogenous proteins. Similarly, exogenous RNA codes for proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterologous / exogenous nucleic acids and proteins are sometimes referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is encompassed herein by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two of the combined sequences are heterologous to each other.

[0045] As used herein, the term "non-naturally occurring" when used with respect to an organism means that the organism has at least one genetic change that is not normally found in naturally occurring strains of the referenced species, including wild-type strains of the referenced species. Genetic changes include, for example, modifications that introduce expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruptions of the organism's genetic material. Such modifications include, for example, coding regions of heterologous or homologous polypeptides of the referenced species and functional fragments thereof. Further modifications include, for example, non-coding control regions where the modifications alter the expression of a gene or operon. Genetic modifications to nucleic acid molecules encoding enzymes or functional fragments thereof can confer biochemical reaction capabilities or metabolic pathway capabilities to a non-naturally occurring organism that has been altered from its naturally occurring state.

[0046] As used herein, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous, and, where necessary to join two protein-coding regions, contiguous and in reading frame.

[0047] An expression control sequence is "operably linked" to a nucleotide sequence if the expression control sequence controls and regulates the transcription and / or translation of the nucleotide sequence. Thus, expression control sequences can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signals for introns, and stop codons.

[0048] The term "expression control sequence" is intended to include at least a sequence whose presence is designed to affect expression, and may also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term may also include the design of a nucleic acid sequence such that undesirable potential initiation codons in or out of frame are removed from the sequence. It may also include the design of a nucleic acid sequence such that undesirable potential splice sites are removed. This includes polyA signals, i.e. sequences that direct the addition of a series of adenine residues at the 3' end of mRNA, or polyadenylation sequences (pA), sequences called polyA sequences. They may also be designed to increase mRNA stability. Expression control sequences that affect transcriptional and translational stability, such as promoters, as well as sequences that affect translation, such as Kozak sequences, are known in insect cells. Expression control sequences may be of a nature such that they regulate the nucleotide sequence to which they are operably linked, such that lower or higher levels of expression are achieved.

[0049] Detailed Description of the Invention A first aspect of the invention relates to a nucleic acid comprising a sequence encoding a first RNA and a sequence encoding a second RNA, each of the first RNA and the second RNA comprising a hairpin, the first RNA comprising a first guide sequence of at least 19 nucleotides substantially complementary to a portion of the alpha-synuclein (SNCA) gene, and the second RNA comprising a second guide sequence of at least 19 nucleotides substantially complementary to a portion of the SNCA gene.

[0050] nucleic acid The term "nucleic acid" as used herein takes its ordinary meaning in the art. Thus, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" as used herein refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30 or more ribonucleotides), and the term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" as used herein refers to a polymer of deoxyribonucleotides. DNA and RNA can be naturally synthesized (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis, where ribosomes bind to the mRNA.

[0051] As used herein, the term "small interfering RNA" ("siRNA") refers to an RNA (or RNA analog) comprising about 10-50 nucleotides (or nucleotide analogs) capable of directing or mediating RNA interference. Preferably, an siRNA comprises about 15-30 nucleotides or nucleotide analogs, more preferably about 16-25 nucleotides (or nucleotide analogs), even more preferably about 18-23 nucleotides (or nucleotide analogs), and even more preferably about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an RNA (or RNA analog) comprising about 10-50 nucleotides (or nucleotide analogs) capable of directing or mediating RNA interference. Preferably, an siRNA comprises about 15-30 nucleotides or nucleotide analogs, more preferably about 16-25 nucleotides (or nucleotide analogs), even more preferably about 18-23 nucleotides (or nucleotide analogs), and even more preferably about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term refers to siRNAs that contain about 21 nucleotides (or nucleotide analogs), such as 19, 20, 21, or 22 nucleotides. The term "long" siRNAs refers to siRNAs that contain about 24-25 nucleotides, such as 23, 24, 25, or 26 nucleotides. Short siRNAs may contain fewer than 19 nucleotides, such as 16, 17, or 18 nucleotides, in some cases, so long as the shorter siRNA retains the ability to mediate RNAi. Similarly, long siRNAs may contain more than 26 nucleotides in some cases, so long as the longer siRNA retains the ability to mediate RNAi without further processing, such as enzymatic processing, to short siRNAs.

[0052] As used herein, the term "RNA interference" ("RNAi") refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove foreign RNA (e.g., viral RNA). Natural RNAi proceeds through fragments cleaved from free dsRNA that direct the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be induced, for example, to silence the expression of a target gene. Double-stranded RNA structures suitable for inducing RNAi are well known in the art. For example, small interfering RNA (siRNA) can induce RNAi. siRNA comprises two separate RNA strands, one strand comprising a first RNA sequence and the other strand comprising a second RNA sequence, thus comprising a first and a second strand. Frequently used siRNA designs comprise consecutive base pairs with a 3' overhang. The first and / or second strand may comprise a 3'-overhang. The 3'-overhang is preferably a dinucleotide overhang on both strands of the siRNA. Such designs are based on observed Dicer processing of larger double-stranded RNAs resulting in siRNAs with these characteristics. A 3'-overhang may be included in the first strand. A 3'-overhang may be added to the first strand. The length of the duplex from which the siRNA is composed may be 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides or more.

[0053] siRNA can also function as a dicer substrate. For example, the dicer substrate can be a 27-mer consisting of a double strand of RNA with 27 consecutive base pairs. The first strand is located at the 3' end of the 27-mer duplex. As with siRNA, at the 3' end, each or one of the strands can include a two nucleotide overhang. The 3'-overhang can be included in the first strand. The 3' overhang can be added to the first strand. 5' from the first strand, additional sequences can be included that are adjacent or non-adjacent to the target RNA sequence that are complementary. The other end of the siRNA dicer substrate is blunt-ended. This dicer substrate design can favor processing by dicer so that siRNA can be formed like the above siRNA design with 19 consecutive base pairs and two nucleotide overhangs at both 3' ends.

[0054] In either case, the siRNA etc. is composed of two separate RNA strands (Fire et al. 1998, Nature 19;391(6669):806-11), each RNA strand comprising or consisting of a first and second RNA strand or a third and fourth RNA strand according to the invention. Thus, the nucleic acid of the invention may be said to induce a first and second RNA strand which is a first RNA, and a third and fourth RNA strand which is a second RNA. Alternative naming conventions for each of the first, second, third and fourth RNA strands are within the scope of the invention, and may be complementary, substantially complementary or unique to each other, or in any other required arrangement as discussed herein.

[0055] The loop sequence may be a stem-loop sequence, which extends the double-stranded region of the shRNA.Similar to the above siRNA dicer substrate, the shRNA can be processed, for example, by dicer to provide an siRNA with the above siRNA design, for example, with 19 consecutive base pairs and 2 nucleotide overhangs at both 3' ends.When the shRNA is processed by dicer, it is preferred to have a first and second strand at the end of the shRNA, i.e., the putative strands of the siRNA are linked via a stem-loop sequence: 5'-first strand-stem-loop sequence-second strand-optional 2nt overhang sequence-3'.Or conversely, 5'-second strand-stem-loop sequence-first strand-optional 2nt overhang sequence-3'. Another shRNA design can be a shRNA structure that is processed by the RNAi machinery to provide an activated RNA-induced silencing complex (RISC), so-called AgoshRNA or Ago2-processed RNA, that does not require Dicer processing (Liu et al., 2013 Nucleic Acids Res. 41(6):3723-33, incorporated herein by reference), which is based on a structure very similar to the miR-451 scaffold described below. Such shRNA structures contain a portion of the first RNA sequence in their loop sequence. Such shRNA structures can also consist of a first strand followed by a second strand.

[0056] Spacer In a preferred embodiment of the present invention, the sequence coding for the first RNA is followed by a spacer comprising at least 15 nucleotides and a sequence coding for the second RNA. Thus, preferably, in the 5' to 3' direction, the sequence coding for the first RNA is followed by a first spacer comprising at least 15 nucleotides, which is followed by a sequence coding for the second RNA. The 5' to 3' direction is understood to refer to the coding strand in the case of a double-stranded (ds) nucleic acid.

[0057] As above, the nucleic acid may be said to induce a first and second RNA strand, which is a first RNA, and a third and fourth RNA strand, which is a second RNA, where the sequences encoding the first and second RNA strands are followed by a spacer of at least 15 nucleotides, and sequences encoding the third and fourth RNA strands.

[0058] In some embodiments, a nucleic acid of the invention comprises a third sequence that encodes a third RNA.

[0059] In a preferred embodiment of the invention, the sequence encoding the third RNA is followed by a second spacer comprising at least 15 nucleotides, followed by the sequence encoding the first RNA. Thus, preferably, in the 5' to 3' direction, the sequence encoding the third RNA is followed by a second spacer comprising at least 15 nucleotides, which is followed by the sequence encoding the first RNA, which is followed by the first spacer and the sequence encoding the second RNA.

[0060] Thus, the nucleic acid may be said to induce a first and second RNA strand, which is a first RNA, a third and fourth RNA strand, which is a second RNA, and a fifth and sixth RNA strand, which is a third RNA, preferably in the 5' to 3' direction, a sequence encoding the fifth and sixth RNA strand is followed by a spacer of at least 15 nucleotides, which is followed by a sequence encoding the first and second RNA strand, which is followed by a spacer of at least 15 nucleotides, which is followed by a sequence encoding the third and fourth RNA strand.

[0061] In some embodiments of the invention, the spacer comprises at least 25; at least 30; or at least 35 nucleotides.

[0062] In some embodiments of the invention, the spacer comprises at least 15; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 105; 110; 115; 120; 125; 130; 135; 140; 145; 150; 155; 160; 165; 170; 175; 180; 185; 190; 195 or 200 nucleotides.

[0063] In some specific embodiments, a spacer of the invention comprises 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; or 40 nucleotides.

[0064] In some specific embodiments of the invention, the spacer comprises 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; or 105 nucleotides.

[0065] In some embodiments of the invention, the spacer comprises or consists of a sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and variants thereof.

[0066] Thus, in some particular embodiments of the invention, the sequences encoding the first and second RNA strands are followed by a spacer of at least 15 nucleotides and sequences encoding the third and fourth RNA strands.

[0067] In some particular embodiments of the invention, the sequences encoding the first and second RNA strands are followed by a spacer that comprises or consists of SEQ ID NO:3 and sequences encoding the third and fourth RNA strands.

[0068] Thus, in some specific embodiments of the invention, the sequences encoding the first and second RNA strands are followed by a spacer of at least 75 nucleotides and sequences encoding the third and fourth RNA strands.

[0069] In some specific examples of the invention, the sequences encoding the first and second RNA strands are followed by a spacer comprising or consisting of SEQ ID NO:1 and sequences encoding the third and fourth RNA strands.

[0070] In some particular embodiments, the sequences encoding the fifth and sixth RNA strands are followed by a spacer of at least 75 nucleotides, the sequences encoding the first and second RNA strands are followed by a spacer of at least 15 nucleotides, and the sequences encoding the third and fourth RNA strands. In some specific examples of the invention, the sequences encoding the fifth and sixth RNA strands are followed by a spacer comprising or consisting of SEQ ID NO:1, and the sequences encoding the first and second RNA strands are followed by a spacer comprising or consisting of SEQ ID NO:3 and the sequences encoding the third and fourth RNA strands.

[0071] In some embodiments, when one or both RNAs are shRNAs processed by Dicer, the above related shRNA structures are also applicable.In some embodiments, when one or both RNAs are Ago shRNAs or Ago2 processing RNAs, the above related shRNA structures are also applicable.Thus, one or both RNAs can be processed by the same or different RNAi mechanisms.

[0072] RNA scaffolds The double-stranded RNA according to the invention can be incorporated into a pre-miRNA or pri-miRNA scaffold. MicroRNAs, i.e. miRNAs, are guide strands derived from double-stranded RNA molecules that are endogenously expressed, for example, in mammalian cells. miRNAs are processed from pre-miRNA precursor molecules by the RNAi machinery, similar to the processing of shRNAs or extended siRNAs described above, and incorporated into activated RISC (Tijsterman M, Plasterk RH. Dicers at RISC; the mechanism of RNAi. Cell. 2004 Apr2;1 17(1):1-3). Pre-miRNAs are hairpin RNA molecules that can be part of a larger RNA molecule (pri-miRNA), for example contained in an intron, which is first processed by Drosha to form a pre-miRNA hairpin molecule. Pre-miRNA molecules are shRNA-like molecules that can then be processed by Dicer to result in siRNA-like double-stranded RNA duplexes. The miRNA, which is part of the double-stranded RNA duplex, the guide strand, is then incorporated into the RISC.

[0073] The first and second RNAs of the present invention comprise first and second guide sequences of at least 19 nucleotides that are substantially complementary to a portion of the SNCA gene.

[0074] Naturally occurring RNA molecules, i.e., pri-miRNA, pre-miRNA or miRNA duplexes, may be used as scaffolds to generate artificial miRNAs that specifically target selected genes. Based on the predicted RNA structure of naturally occurring RNA molecules, for example, based on the predicted RNA structure of RNA molecules using m-fold software with standard settings (Zuker. Nucleic Acids Res. 31(13), 3406-3415, 2003), the natural miRNA sequence present in the RNA structure (i.e., duplex, pre-miRNA or primiRNA) and the sequence present in the structure that is substantially complementary thereto are removed and replaced with the first and second strands according to the present invention, i.e., the first and second strands of the second RNA, which may also be called the first and second strands of the first RNA, or the third and fourth strands. Thus, when the first and second strands are used merely for illustrative purposes, the first and second strands are preferably selected so that the predicted secondary RNA structure formed, i.e., pre-miRNA, pri-miRNA and / or miRNA duplex, resembles the corresponding predicted original secondary structure of natural RNA sequence. Pre-miRNA, pri-miRNA and miRNA duplex (consisting of two separate RNA strands hybridized through complementary base pairing) are often not perfectly base-paired as found in nature, i.e., not all nucleotides corresponding to the first and second strands as defined above are base-paired, and the first and second strands are often not the same length. How to use miRNA precursor molecules as scaffolds for any selected target RNA sequence and substantially complementary first strands is described, for example, in Liu YP Nucleic Acids Res.2008 36(9):281 1-24.

[0075] Pri-miRNAs can be processed by the RNAi machinery of cells. Pri-miRNAs contain flanking sequences at the 5' and 3' ends of the pre-miRNA hairpin and / or shRNA-like molecule. Such pri-miRNA hairpins can be processed by Drosha to produce pre-miRNAs. The length of the flanking sequences can vary, but can be about 80 nt long (Zeng and Cullen, J Biol Chem. 2005 280(30):27595-603; Cullen, Mol Cell. 2004 16(6):861-5). The minimum length of the single-stranded flanks can be easily determined as if they are too short, the RNA molecule may lose its function, for example because Drosha processing fails and sequence-specific inhibition is reduced or absent. In one embodiment, a pri-miRNA scaffold carrying the first and second strands according to the invention has 5' and 3' sequence flanks to the predicted pre-miRNA structure of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides. Preferably, the flanking sequences (5' and 3') from the pri-miRNA contained in the miRNA scaffold are derived from the same naturally occurring pri-miRNA sequence. Preferably, the flanking sequences (5' and 3') and / or loop sequences from the pre-miRNA and / or pri-miRNA contained in the miRNA scaffold are derived from the same naturally occurring pri-miRNA sequence. Since the (putative) guide strand RNA contained in the endogenous miRNA sequence may be replaced by a sequence comprising (or consisting of) the first strand and the passenger strand sequence may be replaced by a sequence comprising (or consisting of) the second strand, it is understood that the flanking and / or loop sequences of the pri-miRNA sequence or pre-miRNA sequence of the endogenous sequence may contain minor sequence modifications such that the predicted structure (e.g., M-fold predicted structure) of the scaffold miRNA sequence is the same as the predicted structure of the endogenous miRNA sequence.

[0076] The first and second strands and the third and fourth strands are encoded by an expression cassette, for example to form two double-stranded RNAs, i.e., the first and second RNAs of the present invention. Unless otherwise specified, it is understood that any additional RNA contained in the nucleic acid of the present invention, such as the third RNA of the present invention described below, is also encoded by an expression cassette. It is also understood that more than one expression cassette is required when the double-stranded RNA is, for example, two siRNAs, each consisting of two strands. If each double-stranded RNA is contained in a single RNA molecule encoding, for example, an shRNA, a pre-miRNA, or a pri-miRNA, one expression cassette per RNA molecule may be sufficient. The pol II expression cassette may include a promoter sequence that includes a sequence encoding the RNA to be expressed followed by a polyadenylation sequence. If the double-stranded RNA to be expressed includes a pri-miRNA scaffold, the encoded RNA sequence may encode intron and exon sequences as well as 3'-UTR and 5'-UTR. A pol III expression cassette generally comprises a promoter sequence followed by a sequence encoding an RNA (e.g., an shRNA sequence, a pre-miRNA, or a strand of a double-stranded RNA, e.g., contained in an siRNA or five extended siRNAs). A pol I expression cassette may comprise a pol I promoter followed by an RNA coding sequence and a 3'-sequence. Expression cassettes for double-stranded RNA are well known in the art and any type of expression cassette may suffice, e.g., a pol III promoter, a pol II promoter, or a pol I promoter may be used (iater Brake et al., 2008 Mol Ther. Mar; 16(3): 557-64, Maczuga et al., 2012 BMC Biotechnol. Jul 24; 12: 42). In some embodiments, the expression cassette is a DNA molecule, as described further below. In some particular embodiments, the expression cassette comprises a pol II promoter.

[0077] As is clear from the above, the first and second strands contained in the double-stranded RNA can contain additional nucleotides and / or nucleotide sequences. The double-stranded RNA of the present invention can be contained in a single RNA sequence or can be contained in two separate RNA strands. Whatever design is used, the antisense RNA molecule containing the first or second strand is designed from the RNA sequence so that it can be processed in whole or in substantial part by the RNAi machinery, as further described below, and thereby incorporated into the RISC complex to have its effect, i.e., to induce RNAi against the RNA target sequence contained in the RNA encoded by the SNCA gene. The sequence containing or consisting of the first or second strand can have a sequence that specifically targets the RNA encoded by the human SNCA gene in whole or in substantial part. Thus, as long as the double-stranded RNA can induce RNAi, such double-stranded RNA is contemplated in the present invention.

[0078] In some embodiments, the double-stranded RNA according to the present invention is included in a pre-miRNA scaffold, a pri-miRNA scaffold, an shRNA, or an siRNA.Preferably, the first and second strands, or the third and fourth strands, or the fifth and sixth strands, or all strands encoded by the expressed cassette are included in a single transcript.It is understood that the subsequent processing, i.e. cleavage, of the expressed transcript results in the processing of the single transcript into multiple separate RNA molecules.

[0079] The term complementary is defined herein as a nucleotide of a nucleic acid sequence that can bind to another nucleic acid sequence through hydrogen bonds, i.e., nucleotides capable of base pairing. Ribonucleotides, the building blocks of RNA, are composed of monomers (nucleotides) that contain a sugar, a phosphate, and a base that is either a purine (guanine, adenine) or a pyrimidine (uracil, cytosine). Complementary RNA strands form double-stranded RNA. Double-stranded RNA can be formed from two separate complementary RNA strands, or two complementary RNA strands can be included in one RNA strand. In complementary RNA strands, the nucleotides cytosine and guanine (C and G) can form base pairs, guanine and uracil (G and U), and uracil and adenine (U and A) can also form base pairs. The term substantial complementarity means that it is not required that the first and second RNA sequences are completely complementary, or that the first RNA sequence and the one or more target RNA sequences of the RNA encoded by the human SNCA gene are completely complementary.

[0080] Guide Chain The first and second RNAs expressed according to the present invention comprise, in whole or in substantial part, a guide strand.

[0081] In certain embodiments of the present invention, the guide strand is complementary ("anti") to the target RNA sequence in the sense transcript, and since the sense target RNA sequence is contained in the RNA encoded by the SNCA gene, it may also be called the antisense strand. Thus, the first and second RNAs also include a "sense strand" that has substantial sequence identity or may be identical to the target RNA sequence. Thus, the first and second RNAs can be described as hairpins or double-stranded RNAs that are substantially complementary to themselves.

[0082] The double-stranded RNA according to the present invention is to induce RNA interference, thereby reducing the expression of SNCA transcripts, including knockdown of SNCA-derived transcripts.Transcripts that can be targeted can include spliced ​​transcripts, including splice variants, and unspliced ​​RNA transcripts.Thus, the RNA encoded by human SNCA gene is understood to include unspliced ​​mRNA, including 5' untranslated region (UTR), intron and exon sequences, followed by 3' UTR and poly A signal, and its splice variants.The double-stranded RNA according to the present invention can also induce transcription silencing.

[0083] Thus, reducing expression of SNCA transcripts is understood herein as preferably reducing the steady-state level of functional SNCA mRNA in a target cell, such that less mRNA is available in the cell for translation into alpha-synuclein protein, thereby reducing the steady-state level of protein in the target cell. Thus, reducing expression of SNCA transcripts does not necessarily involve reducing de novo transcription of the SNCA gene, but rather involves increasing the degradation of SNCA mRNA and / or its precursors, e.g., unspliced ​​RNA transcripts.

[0084] In some embodiments, the double-stranded RNA according to the present invention comprises a first RNA sequence and a second RNA sequence, i.e., a first and a second or a third and a fourth RNA strand, the first and the second RNA sequences being substantially complementary, the first RNA sequence having a sequence length of at least 19 nucleotides and being substantially complementary to a target RNA sequence of the RNA encoded by the SNCA gene, and the first RNA sequence being capable of inducing RNA interference to reduce the expression of an RNA transcript comprising the target RNA sequence in a sequence-specific manner. In some further embodiments, said induction of RNA interference to reduce the expression of an RNA transcript comprising the target RNA sequence means reducing SNCA gene expression.

[0085] In some embodiments, the double-stranded RNA according to the present invention comprises a first RNA sequence and a second RNA sequence, i.e. a first and a second or a third and a fourth RNA strand, the first and the second RNA sequence being substantially complementary, the second RNA sequence having a sequence length of at least 19 nucleotides and being substantially complementary to a target RNA sequence of the RNA encoded by the SNCA gene, and the second RNA sequence being capable of inducing RNA interference to reduce the expression of an RNA transcript comprising the target RNA sequence in a sequence-specific manner. In some further embodiments, said induction of RNA interference to reduce the expression of an RNA transcript comprising the target RNA sequence means reducing SNCA gene expression.

[0086] Those skilled in the art can easily determine whether this is the case by using standard luciferase reporter assays and appropriate controls as described in the Examples and as known in the art (Zhuang et al. 2006 Methods Mol Biol. 342:181-7). For example, a luciferase reporter containing a target RNA sequence can be used to show that the double-stranded RNA according to the present invention is capable of sequence-specific knockdown. Furthermore, SNCA expression levels can be determined by detecting endogenous SNCA mRNA, α-synuclein protein (soluble, aggregated or phosphorylated form), and / or α-synuclein protein isoforms (SNCA140, SNCA126, SNCA112, SNCA98; SEQ ID NOs: 35, 36, 37 and 38, respectively). SNCA mRNA or α-synuclein protein levels can be determined in different sample types, such as cell lysates, tissue lysates, blood cells and biological fluids such as serum, plasma and cerebrospinal fluid.

[0087] As used herein, the term "RNA silencing" refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules that result in the inhibition or "silencing" of expression of corresponding protein-coding genes (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression). RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0088] "Substantially complementary" in this context is understood to mean that all nucleotides of the guide sequence and the target sequence are base-paired, i.e., perfectly complementary, or that all nucleotides of the guide sequence and the target sequence need not be base-paired. Such substantial complementarity is contemplated according to the present invention, so long as the first, second, and, where applicable, third RNA of the present invention are capable of inducing RNA interference, thereby sequence-specifically targeting a sequence, including the target RNA sequence.

[0089] Substantial complementarity between the strands complementary to the target RNA sequence, also referred to as the part of the SNCA gene, preferably consists of at most two mismatched nucleotides, more preferably has one mismatched nucleotide, and most preferably has no mismatches. One mismatched nucleotide is understood to mean that one nucleotide does not base pair with the target RNA sequence over the entire length of the strand complementary to the target RNA sequence when base paired with the target RNA sequence. No mismatches means that all nucleotides of the strand complementary to the target RNA sequence base pair with the target RNA sequence, and two mismatches means that two nucleotides of the strand complementary to the target RNA sequence do not base pair with the target RNA sequence. The strand complementary to the target RNA may also contain additional nucleotides that do not have complementarity to the target RNA sequence, for example, it may be longer than 21 nucleotides. In such a scenario, substantial complementarity is determined over the entire length of the target RNA sequence. This means that the target RNA sequence in this embodiment has zero, one or two mismatches along its entire length when base-paired with a strand complementary to the target RNA.

[0090] Substantial complementarity between the strand complementary to the target RNA and the target RNA sequence consists of no, one or two mismatches over the entire length of either the strand complementary to the target RNA or the target RNA sequence encoded by the RNA of human SNCA, whichever is shortest.

[0091] As mentioned above, mismatch according to the present invention means that the nucleotides of the first strand (or second strand) and the third strand (strand) do not base pair with the target RNA sequence encoded by the RNA of the first gene or the second gene. The non-base paired nucleotides are A and A, G and G, C and C, U and U, A and C, C and U, or A and G. The mismatch can also be due to a deletion of a nucleotide or an insertion of a nucleotide. When the mismatch is a deletion of a strand sequence, this means that the nucleotide of the target RNA sequence does not base pair with that sequence when compared with the full length of the strand sequence. The nucleotides that can base pair are AU, GC and GU. The GU base pair is also called GU wobble or wobble base pair. In one embodiment, the number of GU base pairs between the strand sequence and the target RNA sequence is 0, 1 or 2.

[0092] In some embodiments, there are no mismatches between the strand RNA sequence and the target RNA sequence, and GU base pairs or GU pairs are allowed. Preferably, there may be no GU base pairs between the strand sequence and the target RNA sequence, or the strand sequence and the target RNA sequence only have AU or GC base pairs. Preferably, there are no GU base pairs, and there are no mismatches between the strand sequence and the target RNA sequence. The strand sequence of the double-stranded RNA according to the present invention is preferably fully complementary to the target RNA sequence, said complementarity consisting of GU, GC and AU base pairs. The strand sequence of the double-stranded RNA according to the present invention is more preferably fully complementary to the target RNA sequence, said complementarity consisting of GC and AU base pairs.

[0093] Thus, in some embodiments, the strand sequence and the target RNA sequence have at least 15, 16, 17, 18 or 19 nucleotides that form base pairs. Preferably, the strand and the target RNA sequence are substantially complementary, said complementarity comprising at least 19 base pairs. In other embodiments, the strand has at least 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides that base pair with consecutive nucleotides of the target RNA sequence. In other embodiments, the strand has at least 19 consecutive nucleotides that base pair with consecutive nucleotides of the target RNA sequence. In other embodiments, the strand comprises at least 19 consecutive nucleotides that base pair with 19 consecutive nucleotides of the target RNA sequence. In other embodiments, the strand has at least 17 nucleotides that base pair with the target RNA sequence and at least 15 consecutive nucleotides that base pair with consecutive nucleotides of the target RNA sequence. The sequence length of the first strand is preferably at most 21, 22, 23, 24, 25, 26 or 27 nucleotides. In other embodiments, the strand has at least 20 contiguous nucleotides that base pair with 20 contiguous nucleotides of the target RNA sequence. In other embodiments, the strand comprises at least 21 contiguous nucleotides that base pair with 21 contiguous nucleotides of the target RNA sequence.

[0094] As mentioned above, it may not be necessary to have perfect complementarity (i.e., perfect base pairing (no mismatches) and no GU base pairing) between the first or second strand of the first RNA and the first strand of the second RNA, and the target RNA sequence as such strand may still allow sufficient suppression of gene expression. Also, not having perfect complementarity may be contemplated, for example, to avoid or reduce off-target RNA sequence-specific gene suppression while maintaining sequence-specific inhibition of transcripts containing the target RNA sequence. However, having perfect complementarity may be preferred, as it may result in more potent inhibition. Without being bound by theory, having perfect complementarity between the first or second strand of the first RNA and the first strand of the second RNA, and the target RNA sequence may allow an activated RISC complex containing said first or second strand of the first RNA or the first strand of the second RNA (or a substantial portion thereof) to cleave the target RNA sequence, while having mismatches may prevent cleavage and may mainly allow inhibition of translation, so that the latter may result in less potent inhibition.

[0095] With respect to the second strand on the first or second RNA, the second strand is substantially complementary to the first strand. The second strand combined with the first strand forms a double-stranded RNA. As mentioned above, this is to form a suitable substrate for the RNA interference mechanism so that the guide sequence from the first strand is included in the RISC complex to sequence to specifically inhibit the expression of its target, i.e. the RNA encoded by the human SNCA gene. The sequence of the second strand has sequence similarity with the target RNA sequence. However, the substantial complementarity of the second strand with the first strand may be selected to be less substantial compared to the substantial complementarity between the first strand and the target RNA sequence. Thus, the second strand may contain 0, 1, 2, 3, 4 or more mismatches, 0, 1, 2, 3 or more GU wobble base pairs, and may contain 0, 1, 2, 3, 4 nucleotide insertions and / or 0, 1, 2, 3, 4 nucleotide deletions. It will be understood that if the guide sequence is contained within the second strand or second strand of the first RNA of the invention, the above description applies to the first strand of the invention.

[0096] Preferably, the first strand and the second strand are substantially complementary, said complementarity comprising 0, 1, 2 or 3 GU base pairs, and / or said complementarity comprising at least 17 base pairs. These mismatches, GU wobble base pairs, insertions and deletions are with respect to the first strand, i.e. the double-stranded region formed between the first strand and the second strand. Such substantial complementarity is permissible according to the present invention, as long as the first and second strands can substantially base pair and induce sequence-specific inhibition of the RNA encoded by the human SNCA gene. It is also understood that the substantial complementarity between the first strand and the second strand may depend on the double-stranded RNA design selected. It may depend, for example, on the miRNA scaffold selected for the double-stranded RNA to be incorporated.

[0097] As is clear from the above, substantial complementarity between the first and second strands of the first and second RNAs may include mismatches, deletions and / or insertions relative to the fully complementary (i.e. fully base-paired) first and second RNA sequences. In some embodiments, the first and second strands of the first and / or second RNA have at least 11 consecutive base pairs. Thus, at least 11 consecutive nucleotides of the first strand and at least 11 consecutive nucleotides of the second strand are fully complementary. In some embodiments, the first and second strands of the first and / or second RNA have at least 15 nucleotides that form base pairs. The base pairing between at least 15 nucleotides of the first strand and at least 15 nucleotides of the second strand may consist of GU, GC and AU base pairs, or may consist of GC and AU base pairs.

[0098] In some embodiments, the first and second RNA sequences have at least 15 nucleotides that form base pairs and have at least 11 consecutive base pairs. In another embodiment, the first and second RNA sequences are substantially complementary, and the complementarity comprises at least 17 base pairs. The 17 base pairs may preferably be 17 consecutive base pairs, and the base pairs consist of GU, GC and AU base pairs, or consist of GC and AU base pairs.

[0099] Thus, the present invention provides an expression cassette encoding a first and second strand of a first and / or second RNA, the first and second strands being substantially complementary, the first strand having a sequence length of at least 19 nucleotides and being substantially complementary to a target RNA sequence contained in an RNA encoded by the human SNCA gene. Suitable target RNA sequences according to the present invention are provided (see, for example, Table 1). Where applicable, the expression cassette may also encode a first and second strand of a third RNA, the first and second strands being substantially complementary.

[0100] In a particular embodiment of the invention, the first RNA comprises in its second strand a guide sequence substantially complementary to the first target sequence, in which case the above embodiments apply to the second and third strands of the invention instead of the first and third strands of the invention.

[0101] [Table 1]

[0102] In some embodiments, an expression cassette is provided encoding a first strand and a second strand, wherein the first and second strands of the first and / or second RNA are substantially complementary, and the first strand has a sequence length of at least 22 nucleotides and is substantially complementary to a target RNA sequence selected from the group listed in Table 1 contained in RNA encoded by the human SNCA gene.

[0103] Similarly, in some embodiments, an expression cassette is provided that encodes a first strand and a second strand, wherein the first and second strands of the first and / or second RNA are substantially complementary, and wherein the second strand of the first RNA and the first strand of the second RNA have a sequence length of at least 22 nucleotides and are substantially complementary to a target RNA sequence selected from the group listed in Table 1. If applicable, the expression cassette may also encode a first strand and a second strand of a third RNA, wherein the first and second strands are substantially complementary.

[0104] In some embodiments of the invention, each of the guide sequences is substantially complementary to a sequence selected from the group consisting of SEQ ID NO:4-SEQ ID NO:10.

[0105] In some embodiments of the invention, the first and second guide sequences of the invention are the same sequence, while in other embodiments, the first and second guide sequences of the invention are different sequences.

[0106] As described above, the RNA of the present invention may be incorporated into a miRNA scaffold. The miRNA scaffold sequence is processed by the RNAi machinery present in the cell. In some specific embodiments of the present invention, processing of the miRNA scaffold sequence results in a guide sequence comprising a first strand of the first RNA or a substantial portion thereof in the range of 21-30 nucleotides; and a guide sequence comprising a first strand of the second RNA or a substantial portion thereof in the range of 21-30 nucleotides. In some other specific embodiments of the present invention, processing of the miRNA scaffold sequence results in a guide sequence comprising a second strand of the first RNA or a substantial portion thereof in the range of 21-30 nucleotides; and a guide sequence comprising a first strand of the second RNA or a substantial portion thereof in the range of 21-30 nucleotides.

[0107] Such a guide strand is capable of reducing SNCA gene transcript expression by targeting a selected target sequence.

[0108] As shown in the examples, the first strand of the first RNA and the first strand of the second RNA, or alternatively, the second strand of the first RNA and the first strand of the second RNA, as encoded by the expression cassette of the present invention, are partially or entirely included in the guide strand when processed by the RNAi machinery of the cell. Thus, the guide strand generated from the RNA encoded by the expression cassette, including the first or second strand of the first RNA and the first strand of the second RNA, will include at least 18 nucleotides of the second RNA sequence.

[0109] Preferably, such guide strand comprises at least 19, 20, 21 or 22 nucleotides. The guide strand may also comprise the first or second strand of the first RNA sequence or the first strand of the second RNA sequence as a whole. When selecting a miRNA scaffold, the first or second strand of the first RNA sequence and the first strand in the second RNA sequence may be selected to replace the original guide strand. However, this does not necessarily mean that the guide strand generated from such an artificial scaffold is identical in length to the first or second strand of the first RNA or the first strand of the selected second RNA, or that the first or second strand of the first RNA or the first strand of the second RNA is found in its entirety in the generated guide strand.

[0110] miR-451 In some embodiments of the invention, each of the first and second RNAs is incorporated into a pre-miRNA or pri-miRNA scaffold derived from miR-451. In some specific embodiments of the invention, each of the first and second RNAs comprises SEQ ID NO: 11 or a variant thereof.

[0111] Thus, as shown in the examples, the first strand, second strand, third strand and fourth strand of the nucleic acid of the present invention can be incorporated into a pre-miRNA scaffold or a pri-miRNA scaffold derived from miR-451. The miR-451 scaffold has been found to be particularly useful in the present invention, since it can induce RNA interference, which can mainly result in guide strand-induced RNA interference. The pri-miR-451 scaffold does not produce a passenger strand, since its processing is different from the canonical miRNA processing pathway (Cheloufi et al. 2010 Nature 465(7298):584-9 and Yang et al., 2010 Proc Natl Acad Sci USA 107(34):15163-8). The scaffold represents a good candidate for developing gene therapy products, since it can largely, if not completely, avoid the potential undesirable off-targeting caused by the passenger strand. Because the passenger strand (corresponding to the second sequence) may result in targeting of transcripts other than SNCA RNA, using such a scaffold may prevent such undesired targeting. Thus, it is preferred that the selected scaffold produces less than 15%; less than 10%; less than 5%; less than 4%; or less than 3% of the passenger strand.

[0112] As shown in the examples, the miRNA 451 scaffold preferably comprises, from 5' to 3', first, 5'-CUUGGGAAUGGCAAGG-3' (SEQ ID NO: 12), followed by a 22 nucleotide sequence comprising or consisting of a first RNA sequence, followed by a 17 nucleotide sequence, which can be considered as a second RNA sequence, which is complementary to nucleotides 2 to 18 of said 22 nucleotide sequence over its entire length, followed by the sequence 5'-SWCUUGCUAUACCCAGA-3' (SEQ ID NO: 13) (wherein S is A or G, or C and W are A or U). Preferably, the first 5'-G / C / A nucleotide of the latter sequence does not base pair with the first nucleotide of the first strand of the first or second RNA.

[0113] Such scaffolds may contain additional flanking sequences as found in the original pri-miR-451 scaffold. Alternatively, the hairpin stem sequences, 5'-CUUGGGAAUGGCAAGG-3' (SEQ ID NO: 12) and 5'-SWCUUGCUAUCCCAGA-3' (SEQ ID NO: 13), may be replaced by hairpin stem sequences of other pri-mRNA structures. As is clear from the above, the sequences of the scaffolds may not only differ with respect to the (presumed) guide strand sequence and the sequence complementary thereto, as present in the wild-type scaffold, but may also contain additional mutations in the 5', loop and 3' sequences, and additional mutations may be required to provide an RNA structure predicted to mimic the secondary structure of the wild-type scaffold. Such scaffolds may be included in larger RNA transcripts, such as pol II expressed transcripts that contain 5'UTR and 3'UTR and polyA. Flanking structures may also be absent.

[0114] Thus, an expression cassette according to the invention expressing an shRNA-like structure having a 22 nucleotide sequence comprises or consists of a first strand of a second RNA followed by a 17 nucleotide sequence which can be considered to be the second strand of the second RNA and is complementary over its entire length to nucleotides 2 to 18 of said 22 nucleotide sequence. The latter shRNA-like structure derived from the miR-451 scaffold can be referred to as a pre-miRNA scaffold derived from miR-451.

[0115] In some embodiments, an expression cassette according to the invention is provided, wherein each of said first strands of the first and second RNAs is substantially complementary to a target RNA sequence contained in an antisense RNA transcript encoded by the human SNCA gene.

[0116] In some embodiments, each of the first strands of the first and second RNAs is substantially complementary to a target sequence selected from the group consisting of SEQ ID NO: 4-10. In some preferred embodiments, each of the first strands of the first and second RNAs has a length of 19, 20, 21 or 22 nucleotides. In some particular embodiments, each of the first strands of the first and second RNAs is fully complementary to a target sequence over its entire length. In some preferred embodiments, each of the first strands of the first and second RNAs has a length of 19, 20, 21 or 22 nucleotides, and the first strands of the first and second RNAs are fully complementary to a target sequence over its entire length. Each of the first strands of the first and second RNAs can be selected from the group consisting of SEQ ID NO: 14-20.

[0117] As described above, the first strand of each of the first and second RNAs is combined with the second strand of the first and second RNAs. As described herein, a person skilled in the art is fully capable of designing and selecting suitable second strands of the first and second RNAs to provide first and second strands for the first and second RNAs that can induce RNA interference when expressed in a cell. Suitable second strands of the first and second RNAs can be selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 27.

[0118] The first strand of the first and second RNA is comprised in a miR-451 scaffold as shown in the examples. A suitable scaffold comprising the first and second strand for the first and second RNA according to the present invention may be a sequence such as SEQ ID NO: 11.

[0119] The first strand of the first and / or second RNA as described above may be included in an expression cassette. The first strand of the first and / or second RNA may also be included in an RNA structure encoded by an expression cassette.

[0120] The first and second strands of the first and second RNA sequences as described above can be included in an expression cassette. The first and second strands of the first and second RNAs can also be included in the RNA structure encoded by the expression cassette. It is understood that the cassette, if present, can further include the first and second strands of a third RNA as described above.

[0121] Targeting the RNA sequences disclosed above using such first and second RNAs has been found to be particularly useful for reducing the expression of RNA transcripts encoded by the human SNCA gene. By targeting human SNCA in this way, the inventors have been able to very efficiently reduce human SNCA gene expression, thus reducing the expression of SNCA RNA and α-synuclein protein, and ultimately reducing the formation of α-synuclein aggregates and Lewy and / or Papp-Lantos bodies. Ultimately, this can reverse, prevent, slow down or completely stop any pathology associated with the SNCA gene.

[0122] In some embodiments, the nucleic acid of the invention comprises a third sequence encoding a third RNA. In some particular embodiments, the third RNA comprises SEQ ID NO: 34 or a variant thereof.

[0123] Thus, a nucleic acid can be said to induce a first and second RNA strand, which is a first RNA, a third and fourth RNA strand, which is a second RNA, and a fifth and second RNA strand, which is a third RNA, where the sequence encoding the fifth and sixth RNA strands is followed by a spacer of at least 15 nucleotides, and the sequence encoding the first and second RNA strands is followed by a spacer of at least 15 nucleotides, and the sequence encoding the third and fourth RNA strands.

[0124] Said third RNA may be incorporated into a pre-miRNA or pri-miRNA scaffold derived from miR-144. In some specific embodiments of the present invention, the third RNA comprises SEQ ID NO: 34 or a variant thereof. In some embodiments, the variant is SEQ ID NO: 28. In other words, as described above, the first and second strands of the third RNA of the present invention are incorporated into a pre-miRNA or pri-miRNA scaffold derived from miR-144. Thus, the third RNA can be described as a hairpin or double-stranded RNA substantially complementary to itself. In some embodiments, the hairpin in the second RNA comprises at least 70 nucleotides.

[0125] In the above embodiments in which the third RNA is incorporated into a pre-miRNA or pri-miRNA scaffold derived from miR-144, the third RNA is processed by Dicer and thus the putative strands of the subsequent siRNA are linked via a stem-loop sequence: 5'-first strand-tip loop sequence-second strand-optional 2 nt overhang sequence-3' or conversely, 5'-second strand-tip loop sequence-first strand-optional 2 nt overhang sequence-3'.

[0126] In some embodiments of the invention where the third RNA is incorporated into a pre-miRNA or pri-miRNA scaffold derived from miR-144, the third RNA is mutated to reduce processing and / or expression of the third RNA. In some particular embodiments, SEQ ID NO: 34 or a variant thereof is mutated to reduce processing and / or expression of the third RNA.

[0127] In some embodiments, the mutation is a single point mutation. In other words, the first RNA comprises a single point mutation to reduce processing and / or expression of the first RNA. Any mismatch, bulge or GU wobble introduced at positions 4-8 of the DROSHA cleavage site may impair the enzymatic activity of DROSHA. Double and triple mismatches, bulges or wobbles within said positions further reduce the activity of DROSHA. Thus, any of the following single nucleotide polymorphisms (SNPs) and their combinations within a 4-8 nucleotide stretch of mir-144 may alter (pre)-mir-144 expression:

[0128] In some embodiments, the third RNA comprises at least one mutation selected from the group consisting of: U>G at position 4; A>U or G at position 5; U>A at position 6; C>G or U at position 7; and A>U or G at position 8.

[0129] In a preferred embodiment, the third RNA comprises a single point mutation, A>U, at position 5. One skilled in the art can readily determine whether this is the case by using standard assays and appropriate controls as described in the Examples and known in the art.

[0130] The particular combination of miR-144 / miR-451 scaffolds in the nucleic acids of the present invention is particularly useful in the context of gene therapy and RNA silencing. miR-144 and miR-451 are examples of trans-regulated clustered miRNAs, with miR-144 controlling the processing of miR-451 by Ago2. Specifically, miR-144 enhances the biogenesis of miR-451 in trans by suppressing Dicer, which in turn suppresses global canonical miRNA processing (Kretov et al., 2020 Molecular Cell 78, 317-328). Thus, when this combination of scaffolds is used, the third RNA of the present invention plays an important role in enhancing the biogenesis of the first and second RNAs of the present invention, and thus the delivery of the guide sequences contained in said first and second RNAs.

[0131] In some embodiments of the invention, the first RNA is incorporated into a miR-144 scaffold.

[0132] In some specific embodiments of the invention, the first RNA is incorporated into a miR-144 scaffold and the second RNA is incorporated into a miR-451 scaffold.

[0133] As described above, the first RNA comprises a first guide sequence. In embodiments of the invention in which the first RNA is incorporated into a miR-144 scaffold, the first guide sequence may be incorporated into the first or second strand of the first RNA.

[0134] In those embodiments in which the first RNA of the invention is incorporated into a miR-144 scaffold, the miRNA-144 scaffold for use in the invention preferably comprises, from 5' to 3', first 5'-UGGGGCCCUGGCUM-3', where M is A or C or G or U (SEQ ID NO:29), followed by a 22 nucleotide sequence comprising or consisting of the first RNA sequence, followed by an apical loop 5'-UUUGCGAUGAGAWMM-3', where W is preferably C or G, but may also be A or U, and the last nucleotide of the first strand of the first RNA. where M is A or C and does not base pair with nucleotide 21 of the first strand of the first RNA) (SEQ ID NO:30), followed by a 20 nucleotide sequence which can be considered to be a second RNA sequence and which is complementary over its entire length to nucleotides 1 and 3-10 and 12-20 of said 22 nucleotide sequence, except for nucleotide 18, which forms a mismatch with nucleotide 2 of the first strand, followed by the sequence 5'-AGUCCGGGCACCCCC-3' (SEQ ID NO:31).

[0135] Preferably, the first 5'-U nucleotide of the latter sequence does not base pair with the first nucleotide of the first strand of the third RNA. Such a scaffold may include the flanking sequences found in the original pri-miR-144 scaffold. Alternatively, the flanking sequences 5'-5'-ATCGGCGCTATGCTTCCTGTGCCCCCAG-3' (SEQ ID NO: 32) and 5'-AGCTCTGGAGCCTGACAAGGAGGACAGGAGAGATGCTGCAAGCCCAAGAAGCTCTCTGCTCAGCCTGTCACAACCTACTGACTGCCAGGGCA-3' (SEQ ID NO: 33) may be replaced by flanking sequences of other pri-mRNA structures. The flanking structures may also be absent.

[0136] In an embodiment in which the first RNA of the present invention comprises a miR-144 scaffold, the guide sequence can be contained within the 5p or 3p arm of the scaffold. DROSHA processing of miR-144, followed by Dicer processing, generates a miRNA duplex that eventually enters the process of miRNA strand selection. Strand selection takes place within RISC and determines which strand will become the active strand (also called the guide strand) and which strand will be degraded (the passenger strand) (Noland and Doudna, 2013 RNA, 19:639-648). Guide strand selection is highly determined by the thermodynamic characteristics of the miRNA duplex, and thus guide strand selection can be influenced by modifying the nucleotide sequence encoding the miRNA. Generally speaking, the strand with the lower thermodynamic stability at its 5' end becomes the guide strand. Another important feature of the human miRNA guide strand is a 5'-terminal U bias associated with an enrichment of A and G nucleotides, whereas the passenger strand shows a 5'-terminal C bias and an enrichment of C and U nucleotides (Hai Yang Hu et al. 2009, BMC Genomics 2009, 10:413).

[0137] Advantageously, the miR-144 construct of the present invention comprising the guide strand may be designed as a 5p- or 3p-guide-containing region (SEQ ID NO: 101) since the guide strand selection is sequence-dependent. Importantly, the probability that the desired guide strand of the present invention is selected in RISC can be adjusted by single nucleotide mutations, such as selecting U as the first nucleotide of the first strand (5p-design) or the first nucleotide of the second strand (3p-design), and selecting C or G as nucleotide 20 of the first strand (5p-design) or nucleotide 19 of the second strand (3p-design).

[0138] Similar to the miR-451 scaffold, the sequence of the miR-144 scaffold may not only differ with respect to the (presumed) guide strand sequence and the sequence complementary thereto, as present in the wild-type scaffold, but may also contain additional mutations in the 5', loop and 3' sequences, which may be required to provide an RNA structure predicted to mimic the secondary structure of the wild-type scaffold. Again, such a scaffold may be included in a larger RNA transcript, such as a pol II expression transcript, including 5'UTR and 3'UTR and polyA. Flanking structures may also be absent. Thus, the expression cassette according to the invention may express an shRNA-like structure having a 22 nucleotide sequence comprising or consisting of a first strand of a first RNA, followed by a 17 nucleotide sequence, which may be considered as a second strand of the first RNA, complementary over its entire length to nucleotides 1 and 3-10 and 11-19 of said sequence of 22 nucleotides, except for nucleotide 18, which forms a mismatch with nucleotide 2 of the first strand. The latter shRNA-like structures derived from the miR-144 scaffold can be referred to as miR-144-derived pre-miRNA scaffolds.

[0139] In some embodiments, an expression cassette according to the invention is provided, wherein the first or second strand of the first RNA is substantially complementary to a target RNA sequence contained in an RNA transcript encoded by the SNCA gene.

[0140] In some preferred embodiments, the first or second strand of the first RNA has a length of 19, 20, 21 or 22 nucleotides. In some specific embodiments, the first or second strand of the first RNA is fully complementary to the first target sequence over its entire length. In some preferred embodiments, the first or second strand of the first RNA has a length of 19, 20, 21 or 22 nucleotides, and said first strand of the first RNA is fully complementary to the target sequence over its entire length.

[0141] As described above, when the first strand of the first RNA comprises a guide sequence, the first strand should be combined with the second strand of the first RNA. As described above, those skilled in the art are fully capable of designing and selecting a suitable second strand of the first RNA.

[0142] Similarly, if the second strand of the first RNA contains a guide sequence, the second strand should be combined with the first strand of the first RNA. As described above, those skilled in the art are fully capable of designing and selecting an appropriate first strand of the first RNA.

[0143] Thus, in some embodiments of the invention, the first RNA comprises SEQ ID NO: 101 or a variant thereof. In some more particular embodiments of the invention, the first RNA comprises SEQ ID NO: 101 or a variant thereof and the second RNA comprises SEQ ID NO: 11 or a variant thereof.

[0144] In some embodiments of the invention where the first RNA comprises SEQ ID NO: 101, the first guide sequence is incorporated into the first strand of the first RNA, while in other embodiments the guide sequence is incorporated into the second strand of the first RNA.

[0145] In some embodiments of the present invention, the guide sequence is selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 20. In some embodiments, the complementary strand sequences to SEQ ID NO: 14 to SEQ ID NO: 20 are SEQ ID NO: 21 to SEQ ID NO: 27, respectively.

[0146] In some embodiments of the invention, the first RNA comprises a sequence selected from the group consisting of SEQ ID NO: 71 to SEQ ID NO: 77. In some embodiments of the invention, the second RNA comprises a sequence selected from the group consisting of SEQ ID NO: 71 to SEQ ID NO: 77.

[0147] In some embodiments of the invention, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO:78-SEQ ID NO:81 and SEQ ID NO:95-SEQ ID NO:98.

[0148] In some embodiments of the invention, the first and second RNAs comprise SEQ ID NO:11, and the sequence encoding the first RNA is followed by a spacer comprising SEQ ID NO:3.

[0149] In some embodiments of the invention, the first RNA comprises SEQ ID NO:101 and the second RNA comprises SEQ ID NO:11, and the sequence encoding the first RNA is followed by a spacer comprising or consisting of SEQ ID NO:1.

[0150] In some embodiments of the invention, the third RNA comprises SEQ ID NO:34, the second and third RNA comprise SEQ ID NO:11, the sequence encoding the third RNA is followed by a spacer that comprises or consists of SEQ ID NO:1, and the sequence encoding the first RNA is followed by a spacer that comprises or consists of SEQ ID NO:3.

[0151] In a second aspect of the present invention, an expression cassette is provided comprising the nucleic acid of the present invention, the expression cassette being a DNA molecule. In some particular embodiments, the nucleic acid contained within the cassette is operably linked to a promoter and optionally a polyA signal. In some embodiments, the expression cassette comprises, in 5' to 3' order, the following elements: at least one promoter, at least a first and a second RNA, and at least a polyA signal.

[0152] The nucleotide sequence comprising the expression cassette(s) as defined herein above for expression in mammalian cells comprises at least one mammalian cell-compatible expression control sequence, e.g., a promoter, operably linked to a sequence encoding a gene product of interest, thus forming an expression cassette for expression of the gene product of interest in a mammalian target cell to be treated by gene therapy with the gene product of interest. Many such promoters are known in the art (see Sambrook and Russel, supra, 2001). Constitutive promoters that are widely expressed in many cell types, such as the CMV promoter, can be used. However, promoters that are inducible, tissue-specific, cell type-specific or cell cycle-specific would be more preferred. Preferably, pol II promoters are used, such as the CAG promoter (ia Miyazaki et al. 1989 Gene 79(2):269-77; Niwa, Gene. 108(2):193-9), the PGK promoter, or the CMV promoter (e.g., as shown in FIG. 2 of WO2016102664). Since any atypical presentation that mimics PD and other types of neurodegenerative brain diseases primarily affects the brain, it may be particularly useful to use neuron-specific promoters.

[0153] Thus, in some embodiments of the present invention, the promoter is a promoter capable of driving transcription in brain cells. Examples of suitable neuron-specific promoters are neuron-specific enolase (NSE), human synapsin 1, CaMKII kinase, natural or engineered chicken beta-actin (CAG) promoter, human synapsin I with minimal CMV sequence (SynI-minCMV), platelet-derived growth factor-beta chain (PDGF), tyrosine hydroxylase (TH), forkhead box A2 (FOXA2) and tubulin alpha (Hioki et al. 2007 Gene Ther. 14(11):872-82). Other suitable promoters that may be envisaged are inducible promoters, i.e. promoters that initiate transcription only when the host cell is exposed to some specific stimulus.

[0154] The expression cassettes comprising the nucleic acids of the invention encode a polyA signal operably linked to the 3' end of the RNA molecule. In some embodiments of the invention, the polyA signal is simian virus 40 polyadenylation (SV40 polyA); or bovine growth hormone polyadenylation (bGH polyA); or human growth hormone polyadenylation (hGH polyA).

[0155] The expression cassette according to the invention can be transferred into the cell, for example, by transfection. Any suitable means may be sufficient to transfer the expression cassette according to the invention. Preferably, the expression cassette according to the invention is included in a viral vector, preferably a gene therapy vector. Preferably, a gene therapy vector is used that stably transfers the expression cassette into the cell, so that stable expression of the double-stranded RNA that induces sequence-specific inhibition of SNCA can be achieved. Suitable vectors may be lentiviral vectors, retrotransposon-based vector systems, or AAV vectors. For example, when the lentiviral vector has an RNA genome, it is understood that the RNA genome codes for the expression cassette, such that after transduction of the cell, the DNA sequence and the expression cassette are formed. Preferably, the gene therapy vector is a viral vector. Preferably, the viral vector is an AAV. Thus, in some embodiments, the expression cassette disclosed herein is flanked by inverted terminal sequences.

[0156] Thus, in a preferred embodiment of the present invention, an expression cassette comprising a nucleic acid of the present invention is flanked by at least one AAV inverted terminal repeat (ITR). In some specific embodiments, the expression cassette is flanked by one 5'ITR and one 3'ITR. In other words, the expression cassette is flanked by an ITR sequence at the 5' end of the cassette and an ITR sequence at the 3' end of the cassette.

[0157] AAV A third aspect of the invention relates to an adeno-associated virus (AAV) comprising a nucleic acid or expression cassette of the invention.

[0158] Recombinant parvoviruses, particularly dependoviruses such as infectious human or simian adeno-associated viruses (AAV), and components thereof (e.g., parvovirus genomes), can be used as vectors for the introduction and / or expression of nucleic acids in mammalian cells, preferably human cells. An "AAV vector" is defined as a recombinantly produced AAV or AAV particle that contains a polynucleotide to be delivered to a host cell either in vivo, ex vivo, or in vitro. As used herein, an AAV vector construct refers to a polynucleotide that contains a viral genome or a portion thereof, typically at least one ITR, and a transgene. As used herein, a transgene refers to a nucleotide sequence of interest, and may include promoter and / or control sequences necessary for expression as well as a sequence encoding a gene of interest.

[0159] A "recombinant parvovirus or AAV vector" (or "rAAV vector") or "parvovirus or AAV vector" herein refers to a parvovirus or AAV virion (i.e., capsid) that contains (or "packages") one or more nucleotide sequences of interest, genes of interest or "transgenes" flanked by at least one parvovirus or AAV inverted terminal repeat (ITR). Preferably, the transgene is flanked by ITRs, one on each side of the transgene. Such (r)AAV vectors can be replicated and packaged into infectious viral particles when present in a suitable host cell expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When a transgene of interest flanked by at least one ITR is incorporated into a larger nucleic acid construct (e.g., in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), it is typically referred to as a "pro-vector," which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.

[0160] Preferably, the AAV vector used is a serotype 5 or 9 AAV vector. Serotype 5 or 9 AAV (also called AAV5 and AAV9) can be particularly useful for transducing human neurons and human astrocytes, as shown in the examples. Thus, in some embodiments, there is an AAV that comprises an expression cassette as disclosed herein. Thus, AAV5 and AAV9 can efficiently transduce various human cell types of the CNS, including FBN, dopaminergic neurons, motor neurons and astrocytes, and are therefore suitable vector candidates for delivering therapeutic genes to the CNS to treat neurodevelopmental diseases (including, but not limited to, treating PD by targeting the SNCA gene as described herein). The production of AAV vectors containing any expression cassette of interest is fully described in WO 2007 / 046703, WO 2007 / 148971, WO 2009 / 014445, WO 2009 / 104964, WO 2011 / 122950, ​​WO 2013 / 036118, which are incorporated herein in their entireties.

[0161] AAV sequences that may be used in the present invention for the production of AAV vectors, for example produced in insect or mammalian cell lines, may be derived from the genome of any AAV serotype. In general, AAV serotypes share significant genomic sequence homology at the amino acid and nucleic acid levels, provide the same set of gene functions, produce essentially physically and functionally equivalent virions, and replicate and assemble by substantially identical mechanisms. For an overview of the genomic sequences and genomic similarities of various AAV serotypes, see, for example, GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716; Chlorini et al. (1997, J. Vir. 71:6823-33); Srivastava et al. (1983, J. Vir. 45:555-64); Chlorini et al. (1999, J. Vir. 73:1309-1319); Rutledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74:8635-47). AAV serotypes 1, 2, 3, 4 and 5 are preferred sources of AAV nucleotide sequences for use in the context of the present invention. Preferably, the AAV ITR sequences for use in the context of the present invention are derived from AAV1, AAV2, and / or AAV5. Similarly, the Rep52, Rep40, Rep78, and / or Rep68 coding sequences are preferably derived from AAV1, AAV2, and AAV5. However, the sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the context of the present invention can be taken from any of the 42 known serotypes, more preferably from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, or from newly developed AAV-like particles obtained, for example, by capsid shuffling technology and AAV capsid libraries. The AAV capsid can be composed of VP1, VP2, and VP3, but also VP1 and VP3.

[0162] In some embodiments, AAV vectors according to the invention comprise AAV5 or AAV9 capsid proteins. In some embodiments, AAV vectors according to the invention comprise AAV5 capsid proteins. In some embodiments, AAV vectors according to the invention comprise AAV9 capsid proteins.

[0163] In another embodiment, a host cell is provided comprising said nucleic acid or said expression cassette according to the invention. For example, said expression cassette or nucleic acid may be comprised in a plasmid contained in a bacterium. Said expression cassette or nucleic acid may also be comprised in a producer cell, for example producing a viral vector. Said expression cassette may also be provided in a baculovirus vector.

[0164] Various modifications of the above-defined nucleotide sequences, including, for example, wild-type AAV sequences, for suitable expression in host cells can be achieved by application of well-known genetic engineering techniques, for example as described in Sambrook and Russell (2001, supra). Various further modifications of the coding region are known to those skilled in the art and can increase the yield of the encoded protein. These modifications are within the scope of the present invention.

[0165] In one embodiment, any mammalian cell can be infected with an AAV vector of the invention, including but not limited to muscle cells, liver cells, neuronal cells, glial cells and epithelial cells, and the mammalian species can be any species, including but not limited to mouse, dog, non-human primate and human, In a preferred embodiment, the infected cell is a human cell.

[0166] It is understood that the capsid amino acid sequences and the nucleotide sequences encoding them can be engineered, e.g., the sequences may be in hybrid form or may be codon optimized, e.g., by AcmNPv or Spodoptera frugiperda codon usage. The capsid proteins can be engineered, e.g., by DNA shuffling, error-prone PCR, bioinformatics rational design or site-saturation mutagenesis. The resulting capsids are based on existing serotypes but contain various amino acid or nucleotide changes that improve the characteristics of such capsids. The resulting capsids can be combinations of various parts of existing serotypes, "shuffled capsids," or can contain entirely novel changes, i.e., additions, deletions, or substitutions of one or more amino acids or nucleotides, organized into groups or spanning the entire length of the gene or protein. See, e.g., Schaffer and Maheshri; Proceedings of the 26th Annual International Conference of the IEEE EMBS San Francisco, CA, USA; September 1-5, 2004, pages 3520-3523; Asuri et al., 2012 Molecular Therapy 20(2):329-3389; Lisowski et al., 2014, Nature 506(7488):382-386, which are incorporated by reference herein.

[0167] In some embodiments, the ITRs and capsid proteins (or portions thereof) in an AAV vector of the invention can be derived from different AAV serotypes. By way of example and not limitation, the ITRs can be derived from AAV2, while the capsid proteins can be derived from a different serotype, such as AAV5 or AAV9.

[0168] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising a nucleic acid, an expression cassette, or an AAV vector of the invention and at least one pharma- ceutical acceptable excipient ("pharmaceutical composition of the invention").

[0169] Suitable excipients include, but are not limited to, buffers and stabilizers, antioxidants, etc. By way of example, a pharmaceutical composition of the present invention may include a physiological buffer, such as PBS, and a stabilizer, such as sucrose.

[0170] The compositions of the present invention are compatible, suitable and intended for use in subsequent intravenous, intrastriatal, intracerebellar, intrathecal, intraparenchymal, intravitreal, subretinal administration, or for use in organ targeted vascular delivery, such as intraportal or intracoronary delivery or isolated limb perfusion.

[0171] In some embodiments, the pharmaceutical composition of the invention may also comprise at least one immunosuppressant compound, which may reduce and / or prevent an immune response induced by administration of the pharmaceutical composition of the invention.

[0172] In some embodiments, these compositions are used to transduce cells in vitro or ex vivo, in which case the excipients must be compatible with cell culture.

[0173] In a fifth aspect, the present invention relates to a nucleic acid according to the invention, an expression cassette according to the invention, an AAV vector according to the invention or a pharmaceutical composition according to the invention for use as a medicament. Suitable definitions are provided elsewhere in this application.

[0174] In some embodiments, the medicament reduces expression of RNA encoded by the human SNCA gene, as described herein above.

[0175] In some embodiments, at least one of the guide sequences is substantially complementary to a portion of an exon contained in the human SNCA gene. The exon may be selected from the group consisting of exon 2; exon 4; and exon 6.

[0176] In some particular embodiments, the medicament reduces expression of a transcript encoded by at least one splice site variant of the SNCA gene, hi some more particular embodiments, the at least one splice site variant is selected from the group consisting of SNCA140, SNCA112, SNCA126, and SNCA98.

[0177] The human SNCA gene encodes different isoforms by alternative splicing. The SNCA isoforms SNCA140 (SEQ ID NO: 35), SNCA126 (SEQ ID NO: 36), SNCA112 (SEQ ID NO: 37), SNCA98 (SEQ ID NO: 38) are encoded by SNCA nucleic acids that contain common exons such as exons 2, 4 and 6. These four isoforms qualitatively and quantitatively differ in their aggregation properties. The canonical isoform SNCA140 is more prone to aggregation than SNCA126, SNCA112 and / or SNCA98. Furthermore, in aggregates, SNCA140 forms relatively straight fibrils, SNCA126 forms shorter fibrils arranged in parallel fibril bundles, and SNCA98 forms ring structures. The pathological aggregation of SNCA protein is a central process in the pathogenesis of PD. Thus, the use of the nucleic acids of the present invention allows the expression of different isoforms to be reduced substantially simultaneously, providing a very powerful method for treating and / or preventing PD.

[0178] Within the context of the present invention, "potency" is understood as the expression of activity of a pharmaceutical or therapeutic substance in terms of the concentration or amount required to produce a defined effect. The potency of a given pharmaceutical can be expressed as the concentration (EC50) or dose (ED50) of drug required to produce 50% of the maximum effect of that pharmaceutical. In certain cases, relative potency can be used, and instead of using units to describe the dose required to achieve a particular endpoint, a ratio of equivalent doses is used. As an example, pharmaceutical A is 10 times more potent than pharmaceutical B, i.e., pharmaceutical A achieves the same effect at 1 / 10 the dose.

[0179] In some particular embodiments, the pharmaceutical agent reduces or knocks down the amount of alpha-syn protein aggregates and / or the amount of Lewy and / or Papp-Lantos bodies.

[0180] In some embodiments of the invention, the medicament is used to treat and / or prevent PD, DLB, MSA, neuropsychiatric symptoms, motor symptoms of PD, cognitive disorders, sleep disorders, autonomic disorders and / or olfactory disorders.

[0181] As used herein, the term "treat" and any of its variations refers to any type of health care intended to alleviate or eliminate the symptoms and / or causes of an illness, injury, mental health issue, etc. The term "prevent" and any of its variations refers to any action taken to reduce the likelihood of acquiring a disease or condition.

[0182] It is understood that treatment of PD, LBD, MSA, neuropsychiatric symptoms, motor symptoms of PD, cognitive disorders, sleep disorders, autonomic disorders, and / or olfactory disorders may be provided to human subjects who are afflicted with any of the above diseases, as well as to human subjects who have a genetic predisposition to developing the disease, which may or may not exhibit symptoms of the disease, including, but not limited to, subjects with a known duplication or triplication of the SNCA gene, or subjects with disease-causing mutations in the SNCA gene, i.e., A30P, E46K, H50Q, G51D, and A53T.

[0183] In certain embodiments, the nucleic acid of the present invention, the AAV of the present invention or the pharmaceutical composition of the present invention needs to be delivered to a target cell for use as a medicine.As mentioned above, PD, DLB, MSA, neuropsychiatric symptoms, motor symptoms of PD, cognitive disorders, sleep disorders, autonomic disorders, and / or olfactory disorders mainly affect the central nervous system (CNS).Therefore, in some embodiments of the present invention, the target cell is a CNS cell.In some embodiments of the present invention, the target cell is a neuron.In some specific embodiments of the present invention, the target cell is a brainstem neuron or a midbrain neuron or a hippocampal neuron or an amygdala neuron and / or a cerebral cortex neuron.

[0184] In a specific embodiment, the nucleic acid of the invention, the AAV of the invention or the pharmaceutical composition of the invention is delivered to the cerebrospinal fluid (CSF).

[0185] In a particular embodiment, the nucleic acid of the invention, the AAV of the invention or the pharmaceutical composition of the invention is delivered to the substantia nigra and / or the striatum and / or the thalamus.

[0186] In certain embodiments of the invention, the AAV of the invention or the pharmaceutical composition of the invention is delivered to the CNS target cells by injection. In certain embodiments, the injection is an intraparenchymal injection. In some embodiments, the injection is an intrathecal injection. In some other embodiments, the injection is a cisternal injection. In some other embodiments, the injection is an intraventricular injection. In some other embodiments, the injection is a leptomeningeal injection. In certain embodiments of the invention, the injection is an MRI-guided injection. In certain embodiments of the intervention, the injection is followed by focused ultrasound (fUS).

[0187] In certain embodiments, the AAV of the present invention or the pharmaceutical composition of the present invention is delivered to CNS target cells by a combination of delivery methods.As a non-limiting example, the combination of delivery methods can include intrathecal or subpial injection combined with intracerebroventricular and / or intrasternal injection, or intrathecal or subpial injection combined with intraparenchymal injection.In some embodiments of the present invention, the AAV of the present invention or the pharmaceutical composition of the present invention is delivered by convection-enhanced delivery.

[0188] Methods for producing the nucleic acids of the invention include any method for producing nucleic acids, including, but not limited to, de novo synthesis of miRNA sequences, cloning and subcloning, construction of plasmids with promoters and replication elements that are part of the plasmid containing the designed miRNA sequence, all of which would be apparent to one of skill in the art.

[0189] A method for producing an AAV vector of the present invention may comprise the steps of: a) culturing a host cell as defined herein above under conditions such that the AAV vector is produced; and b) optionally one or more steps of recovering, purifying and formulating the AAV vector.

[0190] The host cell is a host cell suitable for the production of AAV vectors. Thus, the host cell is preferably a host cell suitable for large-scale in vitro culture. Host cells suitable for the production of AAV vectors are well known in the art and are typically mammalian or insect cell lines. Mammalian cell lines for producing AAV vectors are selected from any mammalian species, including, but not limited to, cells such as A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK293 cells (expressing functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblasts, hepatocytes and myoblasts, derived from mammals including human, monkey, mouse, rat, rabbit and hamster. The choice of mammalian species providing the cells is not a limitation of the present invention, nor are the types of mammalian cells, i.e., fibroblasts, hepatocytes, tumor cells. Mammalian cell lines for producing AAV vectors particularly include the wide range of HEK293 cell lines, of which the HEK293T cell line is preferred.

[0191] The insect cell line for producing the AAV vector can be any cell line suitable for the production of heterologous proteins. Preferably, the insect cell allows the replication of the baculovirus vector and can be maintained in culture, more preferably in suspension culture. In a preferred embodiment, the insect cell allows the replication of the recombinant parvovirus vector, including the rAAV vector. For example, the cell line used can be derived from Spodoptera frugiperda, Drosophila melanogaster, or a mosquito, such as Aedes albopictus, derived cell line. Preferred insect cells or cell lines are cells from insect species susceptible to baculovirus infection, including, for example, S2 (CRL-1963, ATCC), Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, Ha2302, Hz2E5, High Five (Invitrogen, CA, USA) and expresSF+® (U.S. Pat. No. 6,103,526; Protein Sciences Corp., CT, USA).

[0192] Thus, in some embodiments, the expression cassette or construct is an insect cell-compatible vector or a mammalian cell-compatible vector. A "mammalian cell-compatible vector" is understood to be a nucleic acid molecule capable of productive transformation or transfection of a mammalian cell or cell line. Mammalian cell-compatible vectors are well known in the art. An "insect cell-compatible vector" is understood to be a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary insect cell-compatible vectors include plasmids, linear nucleic acid molecules, and recombinant viruses such as baculoviruses. Any vector can be used as long as it is compatible with insect cells. A mammalian or insect cell-compatible vector can be integrated into the genome of the cell, but the presence of the vector in the cell need not be permanent, and also includes transient episomal vectors. The vector can be introduced by any known means, such as chemical treatment, electroporation, or infection of the cell.

[0193] The AAV in the supernatant can be recovered and / or purified using suitable techniques known to those skilled in the art. For example, monolith columns (e.g., in ion exchange, affinity or IMAC mode), chromatography (e.g., capture chromatography, immobilized chromatography, and expanded bed chromatography), centrifugation, filtration and precipitation can be used for purification and concentration. These methods can be used alone or in combination. In one embodiment, a capture chromatography method, including a column-based or membrane-based system, is utilized in combination with filtration and precipitation. For example, a suitable precipitation method utilizing polyethylene glycol (PEG) 8000 and NH3SO4 can be easily selected by the skilled artisan. The precipitate can then be treated with benzonase and purified using suitable techniques. Furthermore, the recovery can preferably include a step of affinity purification of the recombinant parvovirus (rAAV) vector (including virions) using an anti-AAV antibody, preferably an immobilized antibody. The anti-AAV antibody is preferably a monoclonal antibody. Particularly suitable antibodies are single chain camelid antibodies or fragments thereof, which can be obtained, for example, from camels or llamas (see, for example, Muyldermans, 2001, Biotechnol. 74:277-302). Antibodies for affinity purification of rAAV are preferably antibodies that specifically bind to an epitope on an AAV capsid protein, whereby preferably the epitope is present on the capsid protein of more than one AAV serotype. For example, an antibody can be produced or selected based on specific binding to the AAV2 capsid, but can also specifically bind to AAV1, AAV3 and AAV5 capsids.

[0194] In general, suitable methods for producing AAV vectors according to the invention in mammalian or insect host cells, and means therefor (e.g., expression constructs for expression of AAV rep proteins), can be found in Clark et al. (1995, Hum. Gene Ther. 6, 1329-134), Gao et al. (1998, Hum. Gene Ther. 9, 2353-2362), Inoue and Russell (1998, J. Virol. 72, 7024-7031), Grimm et al. (1998, Hum. Gene Ther. 9, 2745-2760), Xiao et al. (1998, J. Virol. 72, 2224-2232) and Judd et al. (Mol Ther Nucleic Acids. 2012;1:e54) for mammalian cells and in Urabe et al. al. (2002, Hum. Gene Ther. 13:1935-1943), WO 2007 / 046703, WO 2007 / 148971, WO 2009 / 014445, WO 2009 / 104964, WO 2011 / 122950, ​​WO 2013 / 036118, WO 2015 / 137802, WO 2019 / 016349, and co-pending applications EP 21177449.2, PCT / EP2021 / 058794 and PCT / EP2021 / 058798.

[0195] Methods for producing a pharmaceutical composition of the invention include any method for producing a pharmaceutical composition, all of which will be apparent to one of skill in the art, and generally involve combining a nucleic acid or AAV of the invention with an excipient under specific conditions.

[0196] Another aspect of the invention relates to a kit comprising a nucleic acid of the invention, an expression cassette, an AAV vector of the invention, or a pharmaceutical composition of the invention, and an immunosuppressant compound.

[0197] In certain embodiments of the invention, the immunosuppressant compound may reduce and / or prevent the immune response induced by administration of a nucleic acid, AAV vector, or pharmaceutical composition of the invention.

[0198] In another aspect, the present invention relates to a cell or a host cell comprising a nucleic acid of the invention, an expression cassette or an AAV of the invention.

[0199] In some embodiments, the cell of the invention is a prokaryotic cell. In some specific embodiments, the cell of the invention is a bacterial cell. In some embodiments, the cell of the invention is a eukaryotic cell. In some embodiments, the cell of the invention is a mammalian cell. In some embodiments, the cell of the invention is an insect cell.

[0200] The nucleic acid of the present invention or the AAV vector of the present invention can be delivered to the cell of the present invention by any suitable method, including but not limited to transfection, transformation, electroporation, nucleofection, transduction and microinjection. For example, the expression cassette or nucleic acid of the present invention can be contained in a plasmid contained in a bacterium. The expression cassette or nucleic acid of the present invention can also be contained in a producer cell, for example, producing a viral vector. The expression cassette can also be provided in a baculovirus vector.

[0201] Further details regarding host cells comprising the AAV vectors according to the invention are provided elsewhere in this application. Any mammalian cell can be infected with the AAV vectors of the invention, including but not limited to muscle cells, hepatic cells, neuronal cells, glial cells or epithelial cells. In some preferred embodiments of the invention, the cells to be infected are human cells.

[0202] In another aspect, the present invention relates to a method for treating or preventing a disorder, the method comprising administering a nucleic acid of the invention or an AAV of the invention to a subject, thereby treating or preventing the disorder.

[0203] In certain embodiments, the disorder is caused by the SNCA gene or a pathological human SNCA gene (duplication, triplication or mutation of A30P, E46K, H50Q, G51D and A53T).

[0204] In certain embodiments, the disorder is PD, DLB, MSA, neuropsychiatric symptoms, motor symptoms of PD, cognitive disorders, sleep disorders, autonomic disorders, and / or olfactory disorders, or any atypical presentation that mimics other types of neurodegenerative brain diseases.

[0205] In another aspect, the invention relates to a nucleic acid, an expression cassette or an AAV of the invention for use in the manufacture of a medicament for the treatment of a disorder. [Brief description of the drawings]

[0206] [Figure 1] FIG. 1 is a diagram of the design strategy of guide sequences and miRNAs of the present invention. [Diagram 2] FIG. 1 is a diagram of the design strategy for isoform-targeting guide sequences. [Diagram 3] FIG. 10. Dual-luciferase SNCA miRNA candidate selection: α-synuclein reporter downregulation. [Figure 4] FIG. 13. In vivo studies in A53T KI rats (A. viral DNA (vDNA) levels; B. miSNCA expression levels; C. SNCA mRNA reduction in the striatum). [Diagram 5] Dual luciferase titration experiment: Degradation of α-synuclein reporter. [Figure 6] FIG. 1. Schematic of candidate scaffold designs. [Figure 7] FIG. 13. Dual luciferase assay results for candidate scaffolds. [Figure 8] FIG. 1 shows the results of endogenous mRNA reduction by candidate scaffolds. [Figure 9] FIG. 1. Diagram of miRNA processing of candidate scaffolds. [Figure 10]FIG. 11. Improvement of motor behavior in C. elegans PD model treated with empty vector (EV), full-length SNCA RNAi (SNCA) or candidate scaffolds (miSNCA 5, 13, 15). [Figure 11] FIG. 1. SNCA mRNA reduction and α-synuclein protein reduction by full-length SNCA RNAi or a candidate scaffold (miSNCA5; also called miRNA5) in a C. elegans PD model. [Figure 12] FIG. 13. Small RNA sequencing results (day 1 and day 4) of C. elegans samples following treatment of C. elegans at the L1 stage (full length SNCA RNAi, miSNCA5 and miSNCA15). [Figure 13] FIG. 1. Diagram of miRNA processing of candidate scaffolds in vitro. [Figure 14-1] FIG. 1. miRNA processing of candidate scaffold 1 in vivo. [Figure 14-2] FIG. 1. miRNA processing of candidate scaffold 1 in vivo. [Figure 15] FIG. 11. vDNA levels in the striatum of an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2). [Figure 16-1] FIG. 1 shows miSNCA levels in the striatum of an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2); (A) miSNCA2; (B) miSNCA5; (C) miSNCA15. [Figure 16-2] FIG. 1 shows miSNCA levels in the striatum of an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2); (A) miSNCA2; (B) miSNCA5; (C) miSNCA15. [Figure 17] FIG. 11. Human SNCA mRNA levels in the striatum of an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2). [Figure 18] FIG. 11. Human α-syn protein levels in the striatum of an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2). [Figure 19]FIG. 13. Dopamine transporter levels in the striatum of an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2) (assessed by ([I]-RTI-121 autoradiography). [Figure 20] FIG. 13. Dopamine and dopamine metabolite levels in the striatum of an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2) (assessed by LC / MS). [Figure 21] FIG. 1. Motor behavior testing (cylinder test, assessment of asymmetry of paw use) in an in vivo proof-of-concept study of the AAV-Syn rat model (Study 2). (A) At baseline (pre-infusion); (B) Day 56 after treatment. [Figure 22] FIG. 14. Results of an in vivo proof-of-concept study in the AAV-Syn rat model (Study 2), assessed by immunohistochemistry for (A) total TH; (B) total human a-syn; and (C) a-syn positive TH neurons in the substantia nigra. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0207] Working Example Within the experimental section, the nucleic acids comprising SEQ ID NOs: 95-98 and SEQ ID NOs: 78-81 are designated "Scaffold 1" and the nucleic acid of the invention comprising at least three RNAs is referred to as "Scaffold 2."

[0208] Materials and Methods Design of SNCA miRNA guide strand. miSNCA (miRNA guide strand) was designed to target the common RNA sequence of the most common SNCA mRNA variants; SNCA140, SNCA126, SNCA112 and SNCA98. To design miRNAs that target all of these variants, miRNAs were designed in regions common to all of the major mRNA variants of SNCA (Figure 1). The target regions of the SNCA mRNA sequence are exon 2, exon 4 and part of exon 6. The most common SNP outside these exons, namely A30P, was avoided from being included in the guide RNA. Each of the conserved sequences was used to generate a number of different guide strands with 22 nts. Seventeen guides targeting SNCA were designed and incorporated into the miR451 scaffold: miSNCA2 (SEQ ID NO: 71), miSNCA5 (SEQ ID NO: 72), miSNCA7 (SEQ ID NO: 73), miSNCA12 (SEQ ID NO: 74), miSNCA13 (SEQ ID NO: 75), miSNCA15 (SEQ ID NO: 76), miSNCA16 (SEQ ID NO: 77), miSNCA1 (SEQ ID NO: 82), miSNCA3 (SEQ ID NO: 83), miSNCA4 (SEQ ID NO: 84), miSNCA6 (SEQ ID NO: 85), miSNCA9 (SEQ ID NO: 86), miSNCA10 (SEQ ID NO: 87), miSNCA11 (SEQ ID NO: 88), miSNCA14 (SEQ ID NO: 89), miSNCA18 (SEQ ID NO: 90), and miSNCA19 (SEQ ID NO: 91). These were tested in vitro for their efficacy in globally reducing the SNCA reporter gene (SEQ ID NO: 92) linked in a dual luciferase reporter plasmid and tested in a dual luciferase assay (Figure 3). Of these 17 miRNAs, 7 showed the potential to reduce SNCA mRNA levels in a dose-dependent manner (SEQ ID NOs: 71-77) (Figure 5).

[0209] miSNCA guides were selected based on the following criteria: miRNA guide sequences should not contain stretches of more than 4 G, more than 4 C, more than 5 A and more than 5 T nt, GC content of 30%-70%, predicted off-target genes of the miRNA seed sequence less than 4000 for SNCA targeting guides by using siSPOTR analysis (https: / / sispotr.icts.uiowa.edu. / sispotr / tools / lookup / evaluate.html) and pre-miRNA sequence folding energies of -44 kcal / mol to -55 kcal / mol, and predicted off-target genes of the miRNA seed sequence less than 5000 for SNCA targeting guides. To generate negative controls, two scrambled guides were selected for in vitro testing (miR144_miSCR(ANG1) (SEQ ID NO: 93) and miR144_miSCR(ANG1)+miSCR(C9O) (SEQ ID NO: 94)).

[0210] The selected miSNCA guides fulfill the following criteria: conservation with the monkey SNCA gene sequence (Rhesus monkey, NCBI accession number NC_041768.1), the miRNA guide sequence should not contain a stretch of >4 G or >4 Cnt, 20%-70% GC content, 40%-70% GC seed content, pre-miRNA sequence folding energy of -45 to -55 kcal / mol, and do not match endogenous miRNA seeds.

[0211] Guide sequences were incorporated into the human pri-miRNA miR-451 scaffold sequence and the mFold program (http: / / unafold.rna.albany.edu / ?q=mfold) with standard settings was used to determine whether the candidates folded into a secondary structure.

[0212] SNCA scaffold 1) consists of a miR-144 hairpin / scaffold combined with one mir-451 downstream hairpin / scaffold. SNCA scaffold 2) consists of a miR-144 hairpin combined with two or more mir-451 downstream scaffolds. The placement of the miR144 hairpin is always the most 5' compared to the miR451 hairpin sequence. Four SNCA scaffolds 1 (SEQ ID NO: 95-98) were generated to target SNCA mRNA, and four SNCA scaffolds 2 were generated to target different or the same regions of SNCA mRNA (SEQ ID NO: 78-81).

[0213] Dual portal luciferase assay For dual luciferase assays, HEK293T cells (1x105 cells / well) were seeded in triplicate in 24-well tissue culture treated plates. Cells were co-transfected with a reporter plasmid (10 ng) carrying the full-length SNCA gene encoding the α-synuclein protein of 140 amino acids in length (full length; SEQ ID NO: 39) and various amounts (0.1-1-10-100 ng) of the miSNCA candidate plasmids (Figure 1) using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were then harvested 2 days after transfection and cell samples were analyzed for Renilla luciferase and firefly luciferase activities using the Dual Luciferase assay kit from Promega. The assay was performed on a GloMax Luminescence reader. α-synuclein reduction was measured as a decrease in the RL / FL activity ratio. Experiments were repeated at least three times.

[0214] Dual luciferase assays for preferentially targeting different mRNA variants of SNCA are performed in a similar manner. HEK293T cells are co-transfected with a reporter plasmid (10 ng) carrying a single gene encoding a shorter mRNA variant encoding an α-synuclein isoform of 126, 112 or 98 amino acids in length and with plasmids carrying various amounts (0.1-1-10-100 ng) of miSNCA candidates (Figure 2). The remainder of the assay is performed as described in the previous paragraph.

[0215] Transfection and reduction of endogenous α-synuclein HEK293T cells were used to assess the reduction of endogenous α-synuclein by miSNCA candidates. For these assays, HEK293T cells (1 or 5x10 5 Cells (1000 ng / well) were plated in 24- or 6-well tissue culture-treated plates, respectively. Cells were transfected with various amounts (50-100-200-1000 ng) of plasmids harboring miSNCA candidates using Lipofectamine2000 or Lipofectamine3000 (Thermo Fisher Scientific). Each transfection was performed in triplicate. Cells were then harvested 2 days after transfection. Cell samples were analyzed for SNCA mRNA levels and α-synuclein protein levels. Experiments were repeated at least three times.

[0216] Transfection of isoforms and reduction of endogenous α-synuclein HEK293T cells are used to evaluate endogenous α-synuclein reduction by miSNCA candidates targeting different isoforms. For these assays, HEK293T cells (1 or 5x105 cells / well) are plated in 24- or 6-well tissue culture-treated plates, respectively. Cells are transfected with various amounts (50-100-200-1000ng) of plasmids carrying miSNCA candidates using Lipofectamine2000 or Lipofectamine3000 (Thermo Fisher Scientific). Each transfection is performed in triplicate. Cells are then harvested 2 days after transfection. Cell samples are analyzed for mRNA levels of SNCA mRNA splicing variants using RT qPCR with exon-spanning primer sets as described in McLean et al., 2012 Mol. and Cell. Neuroscience, 49(2):230-239. Additionally, α-synuclein isoform protein levels are analyzed from the cell samples by Western blot using antibodies targeting different isoforms or antibodies recognizing epitopes present in all isoforms (each isoform can be distinguished by its molecular weight). Experiments are repeated at least three times.

[0217] DNA constructs for baculovirus seed generation Expression cassettes carrying candidate miSNCA constructs were subcloned into pVD1746 (SEQ ID NO: 40), pVD1747 (SEQ ID NO: 41), pVD1748 (SEQ ID NO: 42), pVD1749 (SEQ ID NO: 43), pVD1750 (SEQ ID NO: 44), pVD1751 (SEQ ID NO: 45), and pVD1752 (SEQ ID NO: 46), which contain ITR regions for AAV5 and AAV6 packaging.

[0218] The pVD plasmid containing the Scaffold 1 construct has a constitutive promoter P1 (SEQ ID NO: 47) followed by a sequence encoding an RNA of the invention and an hGH polyA sequence (SEQ ID NO: 48). The pVD plasmid containing the Scaffold 2 construct has a short CAG promoter followed by a sequence encoding an RNA of the invention and an hGH polyA sequence (SEQ ID NO: 48).

[0219] AAV5 vector (HEK material) AAV production by co-transfection of HEK 293T cells with transfer vector and packaging plasmids for the AAV5 serotype. AAV purification was performed by affinity chromatography and iodixanol gradient ultracentrifugation. After formulation and concentration, the titer of purified AAV was determined using QPCR.

[0220] AAV5 vector (baculovirus material) Recombinant AAV5 carrying the expression cassette was produced by infecting SF+ insect cells (Protein Sciences Corporation, Meriden, Connecticut, USA) with two baculoviruses encoding Rep, Cap and the transgene. The titer of purified AAV was determined using QPCR according to standard protein purification procedures on a fast protein liquid chromatography system (AKTA Explorer, GE 30 Healthcare) using AVB Sepharose (GE Healthcare).

[0221] AAV6 vector (HEK material) Recombinant AAV6 carrying the expression cassette was produced by co-transfection of HEK293T cells with packaging plasmids encoding Rep, Cap and the transgene. The titer of purified AAV was determined using QPCR following standard protein purification procedures on a fast protein liquid chromatography system (AKTA Explorer, GE 30 Healthcare) using AVB Sepharose (GE Healthcare).

[0222] In vitro models and transduction assays To measure the effect of AAV5-miSNCA scaffold 1 / 2 on human α-synuclein mRNA and protein levels, control non-patient-derived iPSC-derived forebrain neurons, patient-derived iPSC-derived forebrain neurons, and Lund human midbrain (LUHMES)-derived DA neurons are used. iPSC cell lines (Table 1) are obtained from the NINDS RUCDR repository, and LUHMES cells are obtained from ATCC.

[0223] [Table 2]

[0224] Differentiation of iPSC cells into forebrain neurons is performed using the Forebrain Neuron Differentiation Kit from StemCell Technologies.LUHMES cells were differentiated into DA neurons using the protocol described in Harischandra et al., 2020 BBA Mol.Basis of Disease 1866:165533.

[0225] The in vitro cell models described above are transduced using either HEK cells or baculovirus-produced AAV5-miSNCA scaffold 1 and AAV5-miSNCA scaffold 2 (or HEK293T cell-produced AAV6-miSNCA scaffold 1 / 2) candidates at various multiplicities of infection (MOI) of virus. Cells are transduced at 5x10 5 Plate cells / well onto PDL-laminin or PLO-laminin coated 6-well plates. After 3-4 days of passaging, cells were cultured at 10 3 , 10 4 , 10 5 and 10 6 Transduce at an MOI of 100 / cell. Cells are then harvested 7-15 days post-transduction. Cell samples are used for RNA and DNA isolation to determine vector DNA levels, miSNCA expression, SNCA mRNA and α-syn protein expression.

[0226] RNA Isolation and Small RNA Sequencing Using Next-Generation Sequencing (NGS) a) From HEK293T cells transfected with candidate miSNCA scaffolds 1 and 2 10 ng of miSNCA scaffold 1 or 2-containing plasmid (1x10) was prepared using Direct-zol RNA Microprep Kits (ZymoResearch) with a DNAse treatment step. 5 Total RNA was isolated from HEK293T cells transfected with 10 ng plasmid per cell). Total RNA was checked for quality and quantified using Nanodrop. 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, including BluePippin size selection of the final library coupled with Illumina NovaSeq6000 PE150 sequencing. Data were analyzed using CLC Genomics v20 Suit (Qiagen). Trimmed small RNA reads were mapped against a human database of microRNAs (miRbase v22) and annotated in parallel with the mapping and annotation to a custom database containing synthetic (pre)miRNA sequences. Quantification of miSNCA and miR-144A>T (guide strand and passenger strand, if relevant) sequences was expressed as number of counts / sequence versus total number of small RNA counts per sample. Processing of miSNCA candidates was also examined by aligning reads to miRNA guide sequences and quantified as the number of counts per isomiR sequence versus the total number of isomiR counts per sample.

[0227] b. From a neuronal model transduced with AAV-miSNCA scaffold 1 and 2 candidates RNA is isolated from AAV5 or AAV6 scaffold 1 or 2 (either HEK cells or baculovirus produced) transduced cells (DA neurons, forebrain neurons and / or LUHMES-derived DA neurons) using Zymogen RNA isolation kit. RNA quality is tested using Bioanalyzer and quantified using Nanodrop. Samples are then sent to GenomeScan BV (Leiden, Netherlands) for small RNA sequencing using next generation sequencing methods. Data is analyzed using CLC Genomics Suit (Qiagen) to extract information on expression values ​​of miSNCA candidates and to evaluate the processing of miSNCA candidates expressed from AAV5 or AAV6 packaged scaffold 1 and 2 candidates.

[0228] Small RNA Sequencing (NGS) Data Analysis Analysis of data obtained from neurons transduced with AAV5-scaffold1 / scaffold2 or AAV6-scaffold1 / scaffold2 miSNCA is performed using CLC Genomics Workbench 21 suit. Trimmed small RNA sequence reads are counted and annotated using the miRbase database. miSNCA molecules are annotated by aligning pri-miRNA sequences to these small RNA libraries. Expression values ​​of miSNCA candidates are expressed as counts of miSNCA candidate counts versus total annotated small RNA counts. Mostly expressed miSNCA molecules are analyzed by examining the relative counts of miSNCAs of various sizes aligning pre-miSNCA to the small RNA library and using the RNA counts derived therefrom.

[0229] RNA Sequencing (NGS) and Off-Target Analysis RNA sequencing is performed on samples collected from transduced forebrain neurons or differentiated LUHMES cells.

[0230] a. Data Processing and Quality Control Paired-end RNA-seq data are generated for all samples using an Illumina NovaSeq 6000. Alignment of reads from all samples to the human genome is performed using the STAR aligner. Raw expression data are assessed using several automated outlier tests, including the sum of Euclidean distances, Hoeffding's D statistic, average Pearson correlation, and the Kolmogorov-Smirnov test statistic.

[0231] Generate expression data. Normalization was performed using trimmed mean normalization and data were transformed with voom (https: / / genomebiology.biomedcentral.com / articles / 10.1186 / gb-2014-15-2-r29).

[0232] Principal component analysis (PCA) and hierarchical clustering are performed to identify clustering of samples.

[0233] b.Differential Ten statistical contrasts are performed to determine significantly differentially expressed genes (DEGs) between all sample groups. Significant DEGs are determined using a statistical threshold corrected for multiple testing using a false discovery rate (FDR) adjustment.

[0234] 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 by using TaqMan qPCR assays (Thermo Fisher Scientific) with primers against the polyA region of the vector. Quantification (GC / ugDNA) was performed using linearized pVD plasmid and generating a standard curve with various amounts of this linearized plasmid. Using the standard curve thus generated, vector DNA copy numbers were calculated from DNA isolated from cells transduced with AAV5-miSNCA scaffold 1 and AAV5-miSNCA scaffold 2.

[0235] Isolation of RNA and protein from transfected HEK cells and quantification of mRNA for SNCA and α-synuclein protein levels For RNA isolation, Direct-zol™ RNA Miniprep (Cat. No. R2050) was used. Quick-frozen cell pellets were coated with TRIzol and lysed. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific).

[0236] Protein isolation was performed using RIPA buffer (Sigma) containing PhosSTOP phosphatase inhibitors (Roche) and EDTA-free protease inhibitors (Roche). For protein extraction, buffer was added to the cell pellet and cells were stirred at 400 rpm for 30 min at 4C. The cell extract was then centrifuged at maximum speed. The clarified supernatant was used for α-synuclein and total protein measurements, i.e., HTRF and BCA assays.

[0237] For detection of mSNCA levels, a SYBR Green-based RT-qPCR assay was used using a primer set designed for SNCA (Table 3). Results were displayed as fold change using the ΔΔ cycle threshold (ΔΔCt) of treated samples relative to untreated samples normalized to the average expression of the household genes (Table 3).

[0238] [Table 3]

[0239] For detection of α-synuclein protein levels, a total α-syn HTRF kit (Cisbio) was used. HTRF measurements were then normalized by the total protein added to the HTRF assay. Total protein measurements were performed using a bicinchoninic acid assay (BCA Protein Assay Kit; Pierce™). HTRF results were expressed as HTRF ratio / μg total protein.

[0240] RNA isolation and quantification of miSNCA candidates, GFP mRNA and SNCA mRNA from animal tissues Tissues 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 by Nanodrop and Bioanalyzer.

[0241] For miSNCA expression, the following protocol was used: Total RNA was isolated using the AllPrep DNA / RNA Micro kit (Qiagen). The levels of miRNA expression were measured by RT-qPCR using Taqman stem-loop-miRNA assays (Thermo Fisher) designed to detect miSNCA2, 28 nts, miSNCA5, 23 nts, and miSNCA15, 22 nts. Expression levels were expressed as miRNA molecules / ug total RNA.

[0242] For mRNA expression, the following protocol was used: Total RNA was isolated using the AllPrep DNA / RNA Micro kit (Qiagen). SNCA and GFP mRNA expression was measured using Taqman assays for RT-qPCR. Genes used as housekeeping genes were ACTB, B2M, GAPDH, HPRT. Primer sequences shown in Table 4.

[0243] [Table 4]

[0244] LC-MS / MS from animal tissues Striatal tissue samples were shipped on dry ice to the Vanderbilt Neurochemistry Core Facility (Nashville, TN, USA) for determination of catecholamine levels and data were returned in a blinded manner for analysis.

[0245] Tissue extraction. Brain sections were homogenized in 100-750 μl of 0.1 M TCA containing 10-2 M sodium acetate, 10-4 M EDTA, and 7.5% methanol (pH 3.8) using a tissue dismembrator. 10 μl of homogenate was removed for measurement of protein concentration. Samples were then spun in a microcentrifuge at 10,000 g for 20 min at 4°C. Supernatants were transferred to new microcentrifuge tubes for biogenic amine analysis.

[0246] Biogenic amine analysis. Dopamine, HVA, and DOPAC levels were determined by a sensitive and specific liquid chromatography / mass spectrometry (LC-MS / MS) method after derivatization of the analytes with benzoyl chloride (BZC). 5 μl of the supernatant was treated with 10 μl each of 500 mM NaCO3 (aq) and 2% BZC in acetonitrile. After 4 min, the reaction was stopped by adding 10 μl of internal standard solution (in 20% acetonitrile containing 3% sulfuric acid) containing 200 pg each of 13C6-derivatized dopamine-d4, HVA, and DOPAC. Liquid chromatography was performed using a Waters Acquity UPLC on a 2.0x50 mm, 1.7 μm particle Acquity BEH C18 column (Waters Corporation, Milford, MA, USA). Mobile phase A was 0.15% formic acid in water and mobile phase B was acetonitrile. Samples were separated by a gradient of 98 to 5% mobile phase A over 11 min at a flow rate of 600 μl / min before delivery to a SCIEX 6500+QTrap mass spectrometer (AB Sciex, Framingham, MA, USA). The following MRM transitions were monitored for quantitative purposes: 466 to 105, BZC-dopamine; 488 to 111, 13C6-BZC-dopamine-d4; 304 to 150, BZC-HVA; 310 to 111, 13C6-BZC-HVA; 394 to 105, BZC-DOPAC; 406 to 111, 13C6-BZC-DOPAC. Automated peak integration was performed using SCIEX Multiquant software version 3.0.2. All peaks were visually inspected to ensure proper integration. Calibration curves constructed based on peak area ratio (Panalyte / PI.S.) versus concentration of the internal standard were used to calculate the levels of dopamine, HVA, and DOPAC in the samples by linear regression. Levels were normalized to protein concentration in tissue extracts.

[0247] Protein assay. Protein concentration in tissue homogenates was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA USA) as described in the kit instructions provided. Absorbance was measured using a POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).

[0248] ELISA of transgene-derived human α-syn Dissected striatal tissue from fresh frozen cryosections of all animals was homogenized in lysis buffer containing protease and phosphatase inhibitors (Roche: 11836153001). Samples were stirred for 30 min at 4°C, then centrifuged (135000 rpm for 10 min at 4°C) to generate the supernatant. A portion of the supernatant was used to determine total protein levels (BCA assay, Pierce, Rockford, IL) using a 1:500 dilution at a concentration of 0.001 mg / ml. 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 that quantifies luminescence counts relative to the amount of aSyn. Levels of α-Syn were expressed as pg / mg total protein (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA USA).

[0249] Dopamine transporter (DAT) binding The level of striatal DAT was assessed by [125I]-RTI-121 binding autoradiography in cryostat-cut sections prepared from 20 μm fresh frozen tissue. Briefly, thawed slides were placed in binding buffer containing 50 mM Tris, 120 mM NaCl and 5 mM KCl (2x15 min, room temperature). Sections were then placed in the same buffer containing 50 pM [125I]-RTI-121 (Perkin-Elmer, specific activity 2200 Ci / μmol) for 120 min at 25°C to determine total binding. Non-specific binding was defined as that observed in the presence of 100 μM GBR12909 (Tocris Bioscience). All slides were then washed with ice-cold binding buffer (4x15 min), rinsed with ice-cold distilled water and air-dried. Slides were then mounted on autoradiography film (Kodak) along with [125I] microscale standards (Amersham) and left at room temperature for approximately 7 days before developing. Autoradiograms were then analyzed using MCID software (Image Research Inc., Ontario, Canada). Densitometric analysis of three striatum from each animal was performed and a reference curve of cpm vs. optical density was calculated from the β-emitting [14C] microscale standard and used to quantify the intensity of the signal as nCi / g. Background intensity was subtracted from each reading. Data were then expressed as the mean ± sem signal intensity of each treatment group. Nonspecific binding was calculated in the same way and subtracted from the total to obtain specific binding. Nonspecific binding was typically found to account for less than 1% of the total binding.

[0250] Immunofluorescence and conformation Immunofluorescence: Brains were cryosectioned in the coronal plane at 40 μm thickness on a freezing slide microtome (Leica Microsystems Inc., Richmond Hill, ON), and six series of sections were stored in cryoprotectant (30% glycerol, 30% ethoxyethanol, 40% PBS). A single series of midbrain sections was used to perform double-label immunofluorescence to reveal hemagglutinin (HA)-tagged human aSyn and tyrosine hydroxylase (TH). Briefly, in free-floating sections, the levels and distribution of TH (sheep anti-TH, 1:1000, Pel Freez, P60101; secondary antibody, Alexa fluor donkey anti-sheep, Fisher Scientific, A21099, 1:500) and HA (rabbit anti-HA, 1:1000; Abcam, AB9110; AlexaFluor donkey anti-rabbit, 1:500, FisherScientific, A21206, 1:500) were assessed by double-label immunofluorescence.

[0251] Stereology: Estimation of the number of TH+ve neurons with and without human α-syn colocalization in the substantia nigra pars compacta (SNc) was performed using StereoInvestigator software (MBF Bioscience, Williston, VT) according to stereological principles. Seven or eight sections separated by 240 μm from the anterior to the posterior SN, respectively, were used for counting each case. Stereological analysis was performed using a Zeiss microscope (AxioImager M2 with Apotome, Carl Zeiss, Canada) coupled to a monochrome digital camera for visualization of the 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 tissue section analyzed, the section thickness was empirically assessed and guard zones of approximately 2 μm thickness were used at the top and bottom of each section. The SNc was outlined under low magnification (5x) and TH+ve neurons were counted under 40x magnification. Stereo parameters were empirically determined (i.e., grid size, count frame size, and dissector height) using Stereo Investigator software (MicroBrightfield, VT, USA). The coefficient of error (CE) was calculated according to the procedure of West and colleagues, known as Gunderson CE (m=1). Gunderson values ​​of less than 0.10 were accepted.

[0252] The results of counting stereology yielded absolute numbers of TH+ve neurons in the SNc to assess neuroprotection. The number of remaining TH+ve neurons containing reactivity to human α-synuclein was also produced to provide an indication of the number of human α-syn expressing TH+ve neurons. The ratios of TH+ve / synuclein+ve:TH+ve / synuclein-ve were then calculated.

[0253] Study design In vivo study Study 1. Mechanism of action study in α-synuclein KI rats In this study, the mechanism of action of two AAV-miSNCA candidates was evaluated in human α-synuclein KI rats. A total of three treatment groups were used with N=3 animals per group (total N=9, Envigo, USA). On day 1, all animals received a 3x3ul unilateral injection of AAV5 into the striatum. The contralateral side served as a control and was injected with formulation buffer in the same manner as the AAV5 injection. Groups are shown in Table 5.

[0254] [Table 5]

[0255] Striatal stereotaxic 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: -0.6 mm AP, - / +4.0 ML, -5.5 DV (with the tooth bar set at -3.3). Viral vectors were administered at a rate of 0.5 ul / min and a 5 minute wait period was allowed after each injection.

[0256] On day 43, all rats were given an isoflurane overdose and perfused transcardially with ice-cold 0.9% saline. The brains were then removed as quickly as possible and divided into left and right hemispheres. In all animals in each group, the following regions were freshly dissected from the left and right hemispheres: prefrontal cortex, striatum, hippocampus, hypothalamus, thalamus, posterior cortex, cerebellum, ventral midbrain, and brainstem, frozen on dry ice, and stored at -80°C for molecular analysis.

[0257] Study 2. Proof-of-concept study in the AAV-α-synuclein rat model This study is designed to evaluate the ability of two miRNAs targeting SNCA mRNA (encoding α-synuclein) to protect dopaminergic function in the AAV1 / 2-hA53T-aSyn rat model of Parkinson's disease. On day 1 (D1), AAV1 / 2 human A53T α-synuclein (AAV1 / 2-hA53T-α-Syn) is administered unilaterally into the right substantia nigra following a stereotaxic approach in combination with one of four other AAV5 vectors. Behavioral assessments are performed with the cylinder test to evaluate forelimb asymmetry before surgery (baseline, D-3) and at D14, D21, D42 and D56 (2, 3, 6 and 8 weeks after AAV administration). Groups are shown in Table 6.

[0258] [Table 6]

[0259] On D57, the animals were sacrificed for post-mortem evaluation. Blood samples were collected, processed as required, and stored.

[0260] Primary endpoints of the study included assessment of forelimb asymmetry (by cylinder test); quantification of striatal dopamine and metabolite levels (by LC-MS / MS); quantification of dopamine transporters (by autoradiography); and quantification of striatal transgene-derived αSyn levels (by ELISA).

[0261] Optional endpoints included quantification of tyrosine hydroxylase positive (TH+ve) cells in the substantia nigra with or without co-expression with human aSyn (by double-label immunofluorescence).

[0262] Sacrifice and sampling were performed as follows: animals were deeply anesthetized with isoflurane and then sacrificed by exsanguination via transcardial perfusion with ice-cold 0.9% saline containing 0.2% heparin. Brains were placed ventrally upwards into ice-cold stainless steel rat brain matrix and cut in the coronal plane initially at the level of the hypothalamus. The rostral part of the brain, including the entire striatum, was immediately frozen in isopentane cooled to -42°C and later 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. Tissues were stored in a -80°C lock freezer. Additional regions of interest (including additional striatal dissections) were collected according to Table 11 for molecular assays.

[0263] [Table 7]

[0264] The remaining caudal portion of the brain, including the midbrain, was immersed in 4% paraformaldehyde (PFA) for 48 h for fixation and subsequently cryoprotected in graded sucrose solutions (15-30% sucrose). Tissue prepared in this manner was used for immunohistochemistry of tyrosine hydroxylase and quantification of dopamine neuron numbers in the SNc via unbiased stereochemistry.

[0265] Test 3. Phenotypic rescue of the locomotor phenotype in a C. elegans PD model In this study, we studied the effect of expression of miSNCA candidates on the phenotypic rescue of altered locomotor behavior in a C. elegans PD model (OW40; van Ham et al 2008 PLoS Genet4(3):e1000027). In this model, α-synuclein is overexpressed in the body wall muscles of C. elegans. This α-synuclein overexpression causes a slowing of worm movement when compared to control worms. We used the full-length SNCA gene or our miSNCA constructs to study the effect of reducing SNCA mRNA levels by RNAi, and thereby reducing α-synuclein protein levels. Double-stranded RNA containing one of these constructs was introduced into the organism by feeding. C. elegans OW40 worms were fed with E. coli overexpressing either the empty T444T plasmid as a negative control or the full-length SNCA gene or one of our miSNCA candidates (miSNCA5, miSNCA13 or miSNCA15) at different stages of their lifespan; larval stage 1 (L1), larval stage 4 (L4) and day 1 of their adulthood. Treatment experiments were repeated at 25°C and 15°C. After treatment at days 1, 4 and 8 of their adulthood, the worms were video tracked using a high-throughput tracking setup to measure their movement (speed as μm / s) (Perniet et al. 2018 Journal of Neuroscience Methods,306:57-67).

[0266] Further readout was RT-qPCR of SNCA mRNA and α-synuclein protein levels using Western blot analysis. Primers used for RT-qPCR of SNCA mRNA are shown in Table 7.

[0267] [Table 8]

[0268] Western blot analysis was used to detect α-synuclein protein levels. For this purpose, proteins were extracted using RIPA buffer and Tissuelyser (Qiagen). Similar protein amounts from the different treatment conditions were loaded onto SDS PAGE and Western blot was performed using anti-human α-synuclein antibody (Table 8) to detect α-synuclein levels. For normalization, tubulin was used and tubulin was detected using anti-tubulin antibody (Table 8).

[0269] [Table 9]

[0270] Study 4. Study in PFF rat model This study evaluates the spread of α-synuclein (α-Syn) pathology in transgenic mice (line 83, or line 20 or line 61) following administration of α-Syn preformed fibrils (PFFs) to the striatum. The effect of AAV5-Scaffold 1 or AAV5-Scaffold 2 in reducing the spread of α-Syn pathology is evaluated.

[0271] Animals will receive stereotactic injections of α-Syn-PFF (mouse or human based) or the respective monomer as a control, in combination with a control AAV (non-targeting miRNA) or different doses of AAV5-miSNCA.

[0272] At different time points after administration of α-Syn with or without AAV, animals were sacrificed by intracardial perfusion with ice-cold 0.9% saline followed by 4% PFA. Brains were then removed, post-fixed (overnight) in 4% paraformaldehyde, and cryoprotected in sucrose solution. Forebrains and midbrains were then sectioned on a freeze-slide microtome for histological processing. Qualitative assessment of pSer129α-Syn expression and the extent of pathological α-Syn diffusion in the forebrain (prefrontal cingulate cortex, insular cortex, striatum) and midbrain (substantia nigra; SN) was assessed by immunohistochemistry.

[0273] Study 5. Dose-response study in rodent models of Parkinson's disease In this study, we test several doses of the most potent AAV5-miSNCA candidates determined in previous experiments in relevant rodent (rat or mouse) Parkinson's disease models (transgenic lines) to determine the dose-response relationship between AAV5-miSNCA and phenotypic rescue using molecular, histological and locomotor readouts.

[0274] result In vitro experiments Example 1. In vitro silencing efficacy of artificial miSNCA constructs To evaluate the miSNCA knockdown efficacy of the miSNCA constructs in vitro, HEK293T cells were co-transfected with a Renilla 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 1ng-10ng-50ng or 250ng of each miSNCA construct and the Dual Luc reporter carrying the SNCA gene. From the 17 miSNCA constructs (SEQ ID NO: 71-77 and SEQ ID NO: 82-91) designed to target the SNCA gene, miSNCA2 (SEQ ID NO: 71), miSNCA5 (SEQ ID NO: 72), miSNCA7 (SEQ ID NO: 73), miSNCA12 (SEQ ID NO: 74), miSNCA13 (SEQ ID NO: 75), miSNCA15 (SEQ ID NO: 76) and miSNCA16 (SEQ ID NO: 77) induced the most potent dose-dependent reduction in the RL / FL ratio (Figure 3). To further determine the potency, the above constructs were further used in titration experiments. The constructs were co-transfected into HEK293T cells at different concentrations; 0.1, 1, 10 or 100 ng with 10 ng of SNCA luciferase reporter plasmid. According to these results, transfection with 100 ng of miSNCA plasmid showed at least 50% reduction for all miSNCA candidates used in the titration experiments (Figure 5). For further testing in different models, miSNCA5, miSNCA13 and miSNCA15 were selected due to their relatively great potency in reducing mSNCA levels.

[0275] Example 2. Reduction of endogenous SNCA expression in transfected cells by scaffold 1 and scaffold 2 constructs To test the knockdown of SNCA mRNA expression in cells, miSNCA5, miSNCA15 and miSNCA2 scaffold 1 (SEQ ID NO: 96, 98, 95) and scaffold 2 constructs (miSNCA2+5, miSNCA5+15, miSNCA5+5, miSNCA5+15+2; SEQ ID NO: 78 to SEQ ID NO: 81, respectively) and control miRNAs (single miRNA (CTR1), double scrambled miRNA (CTR1+CTR2) or triple scrambled miRNA (CTR1+CTR2+CTR3)) for scaffolds 1 and 2 (SEQ ID NO: 93, 94 and 99) were selected (Figure 7A and Figure 7B). Knockdown of endogenous SNCA gene expression in HEK293T cells was confirmed by RT-QPCR on transfected cells. Transfection of 50 ng of miRNA plasmid resulted in a reduction of SNCA mRNA expression by 60-40% by all miSNCA scaffold 2 candidates tested (Figure 8). A similar reduction in protein expression would be expected.

[0276] Example 3. Processing of miSNCA constructs upon transfection in cells (NGS data) The processing of miRNAs was examined by alignment of reads to pre-miRNA sequences of miSNCA5, miSNCA15 and miSNCA2 constructs in scaffold 1 and scaffold 2 scaffolds. The results showed that all miSNCAs were processed in the same manner in both scaffold 1 and scaffold 2 scaffolds. The length of the most abundant form for miSNCA5 was 25 nts, followed by 24 nts (Figure 9A), for miSNCA15 it was 24 nts, followed by 25 nts (Figure 9B) and for miSNCA2 it was 28 nts (Figure 9C).

[0277] Example 4. AAV-miSNCA transduction and dose-dependent SNCA mRNA reduction in human cells To examine the ability of AAV5 or AAV6-scaffold 1 or scaffold 2 to transduce and deliver the packaged expression cassettes, DA neurons or forebrain neurons and / or LUHMES-derived DA neurons were infected with AAV5 or AAV6-scaffold 1 or scaffold 2 at various multiplicities of infection (MOI);4 , 10 5 , 10 6 and 10 7 The cells are transduced with 100% ribosomal RNA (100% ribosomal RNA). Vector DNA levels are measured and a dose-dependent increase in vDNA levels is expected in these cells. RNA is isolated from the transduced cells and SNCA mRNA levels are measured using an RT-qPCR assay and a dose-dependent decrease in SNCA mRNA levels is expected in these transduced cells.

[0278] Example 5. AAV5-miSNCA and AAV6-miSNCA transduction in vitro and in vivo studies: evaluation of miSNCA processing To evaluate miRNA processing of miSNCA2, miSNCA5 and miSNCA15 in scaffold 1 and scaffold 2, AAV5-miSNCA2, AAV5-miSNCA5, AAV5-miSNCA15, AAV5-miSNCA5+15 were used to transduce human induced pluripotent stem cell (iPSC)-derived forebrain neurons at MOI 5. Total RNA was isolated from these samples and subjected to small RNA sequencing. Processing of miRNA was examined by alignment of reads to pre-miRNA sequences of miSNCA2, miSNCA5 and miSNCA15 constructs in scaffold 1 and scaffold 2 scaffolds. Results showed that all miSNCAs were processed in the same way in both scaffold 1 and scaffold 2 scaffolds (Figure 13). The length of the most abundant form for miSNCA2 was 27 nts, followed by 25–26 nts, for miSNCA5 it was 24 nts, followed by 25–24 nts, and for miSNCA15 it was 23 nts, followed by 22–24 nts (Figure 13).

[0279] Example 6. AAV5-miSNCA processing from in vivo samples To investigate miRNA processing and isoforms of miRNAs expressed from scaffold 1, RNA isolated from striatal tissue samples from in vivo study 2 was analyzed by small RNA sequencing (next generation sequencing by GenomeScan). Processing of miSNCA2, miSNCA5 and miSNCA15 in scaffold 1 in vivo samples was similar to the in vitro processing of these miRNAs (Figure 13 vs. Figure 14). The length of the most abundant isoforms was 25 and 28 nts for miSNCA2, followed by 27 and 29 nts (Figure 14A), 24 nts for miSNCA5, followed by 23, 25 and 22 nts (Figure 14B), and 22 nts for miSNCA15, followed by 23, 21 and 24 nts (Figure 14C).

[0280] In vivo testing Example 7. AAV5-miSNCA candidates reduced human SNCA mRNA expression in α-synKI rats (Study 1) To evaluate the ability of two of the design candidates (miSNCA5 and miSNCA15) to reduce human SNCA mRNA expression, AAV5-miSCR (miSCR non-targeting scrambled control (SEQ ID NO: 100), AAV5-miSNCA5 or AAV5-miSNCA15 were injected into the left striatum of adult α-syn KI rats. miSNCA13 inhibited the expression of SNCA Since it targets a region outside the humanized portion of the KI rat model and has three mismatches to the wt rat SNCA gene, it was excluded from the in vivo study. As an additional control, the formulation buffer was injected into the right striatum. At the single dose used, vDNA was detected in the AAV5-injected brain hemisphere, whereas in the control hemisphere, vDNA levels were below the lower limit of quantification (LLOQ) (Figure 4A). Transduction led to expression of miSNCA candidates 5 and 15 in a vector-specific manner (Figure 4B): miSNCA5 was detected only in the AAV5-miSNCA5-injected hemisphere, and miSNCA15 was detected only in the AAV5-miSNCA15-injected hemisphere. At the single dose used, AAV5-miSNCA5 significantly increased SNCA expression in the injected striatum compared to the control striatum, as assessed by two different RT-QPCR SNCA assays (primer set SNCA1 and primer set SNCA2). Both miSNCA5 and miSNCA15 were effective in reducing SNCA mRNA expression (Figure 4C). This study indicates that AAV5-miSNCA candidates for the treatment of Parkinson's disease can reduce the expression of human SNCA mRNA and ultimately reduce the associated toxicity of α-synuclein. Furthermore, the combination of miSNCA5 and miSNCA15 is more effective in lowering SNCA mRNA levels.

[0281] Example 8. Proof-of-concept study in the AAV-Syn rat model (Study 2) Different AAV5-miSNCA scaffold 1 and AAV5-miSNCA scaffold 2 candidates were tested in the AAV-Syn rat model. The AAV5-miSNCA vector rescued the disease phenotype in the AAV-Syn rat model in terms of motor phenotype, molecular and neurochemical changes.

[0282] To determine the in vivo proof of concept of lowering SNCA levels and its relationship to motor phenotype improvement, a rat PD model was used (AAV1 / 2-hA53T-aSyn; virus-induced overexpression of human A53T a-syn). The right substantia nigra (SN) was unilaterally injected with a disease-inducing AAV1 / 2-hSNCA vector followed by administration of a different therapeutic vector (AAV5-miSNCA), with the respective control miCTR1 or miCTR1+CTR2 empty vector (EV_CTR) used as a control for the disease model. At the single dose used, vDNA was detected in the striatum of the AAV5-injected brain hemisphere in all injection groups. Dose-dependent vDNA levels were detected in the last three AAV5-miSNCA scaffold 2 groups at low, medium and high doses (Figure 15), whereas in the control hemisphere (left striatum), vDNA levels were below the lower limit of quantification (LLOQ) (not shown). Transduction resulted in specific expression of miSNCA2, miSNCA5, or miSNCA15 in a vector-specific manner (Figures 16A, B, and C): miSNCA2 was detected only in the AAV5-miSNCA2-injected group, miSNCA5 was detected only in the AAV5-miSNCA5 and miSNCA5+15-injected groups, and miSNCA15 was detected only in the AAV5-miSNCA15 and miSNCA5+15-injected groups, but not in other samples from the negative control group. In A53T-aSyn animals, all of the AAV5-miSNCA candidates were effective in reducing SNCA mRNA expression in the striatum of the injected hemisphere as assessed by Taqman RT-qPCR assays (SNCA2 primer and probe combination), comparing AAV5-miSNCA scaffold 1 candidates to control striatum injected with an unrelated miRNA (light grey solid bars; miCTR1) and comparing AAV5-miSNCA scaffold 2 candidates to control striatum injected with an unrelated miRNA (light grey checkered bars; miCTR1+CTR2) (Figure 17). miSNCA expression also resulted in a reduction in protein levels reflected by reduced α-syn protein levels in all AAV5-miSNCA treatment groups compared to controls as measured by ELISA (Figure 18).Dopamine transporter deficits measured by [125I]-RTI-121 autoradiography were evident in A53T-aSyn animals injected with control (non-targeting) miRNA (unrelated miR, groups 2 and 3 (miCTR1 and miCTR1+CTR2 in Table 6), similar to deficits observed in PD patients, and were corrected in miSNCA15 and miSNCA5+15 (low and high dose) treated groups (groups 6, 7 and 9 in Table 6) (Figure 19). In conjunction with metabolite changes in this model, correction of hA53T-aSyn-induced striatal dopamine deficits by miSNCA candidates was observed in groups 5 to 9. Figure 20 shows dopamine levels in the tested groups.

[0283] Motor behavior impairment, as measured by percent asymmetry of left paw use, was significantly rescued in groups treated with miSNCA2, miSNCA5, miSNCA15 or miSNCA5+15 (medium dose) (groups 4, 5, 6 and 8) on day 56 compared to baseline levels (Figures 21A and 21B).

[0284] The molecular, biochemical and motor 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-miSNCA5 and AAV5-miSNCA15 candidates rescued dopaminergic (TH) neuronal cell loss (Figure 22A) and reduced the number of human α-syn positive cells (Figure 22B). This was reflected by a decrease in the percentage of positive dopaminergic (TH) cells that expressed a-syn (Figure 22C).

[0285] Overall, AAV5-miSNCA rescued the disease phenotype, ameliorated the motor phenotype, and rescued molecular and neurochemical alterations in the AAV-Syn rat model.

[0286] Example 9. Phenotypic rescue of locomotor phenotypes in a C. elegans PD model (Test 3) To compare the locomotor speed between C. elegans fed with different plasmid-expressing E. coli (expressing the human SNCA gene in body wall muscles, causing reduced motility), 100 worms per condition were video tracked. Results showed that worms fed with full-length SNCA RNAi-expressing or miSNCA-expressing E. coli showed improved locomotor speed compared to worms fed with E. coli transformed with an empty plasmid. Worms treated with full-length SNCA or miSNCA showed increased speed compared to untreated disease model worms on all days their movement was tracked. These results indicate that reducing the expression of the SNCA gene, and thereby reducing α-synuclein levels, improves the locomotor phenotype in this C. elegans PD model.

[0287] RNAi using miSNCA candidates has been shown to be effective when treated at different stages of C. elegans development; larval stage 1 (L1), larval stage 4 (L4) and adult stage day 1 (Day1). C. elegans locomotor behavior was rescued by treatment at L1, L4 and Day1 stages (Figure 10A-C). SNCA mRNA levels (Figure 11A-C) and α-synuclein protein levels (Figure 11D-F) were also shown to be reduced in these conditions.

[0288] RNAi with full-length SNCA and miSNCA rescued the motor phenotypic behavior caused by SNCA overexpression in muscle cells of a C. elegans PD model. SNCA full-length RNAi always shows a much more effective reduction of SNCA mRNA and α-synuclein protein. This can be explained by SNCA full-length RNAi, resulting in various small RNA pieces that are overall much more effective in reducing SNCA mRNA expression. Furthermore, this can be attributed to the increased dosage of miRNA introduced by full-length SNCA RNAi. Small RNA sequencing results demonstrated this by showing many different SNCA fragments (most of the fragment sizes ranged from 20 to 25 bps). Some of the fragments have sequences overlapping with miSNCA designed within the scope of this study (Figure 12), supporting the beneficial effect of combining different miRNAs to increase efficacy.

[0289] Example 10. Proof-of-concept study in the PFF rat model (Study 4) Different AAV-miSNCA candidates will be tested in the PFF rat model, and a reduction in the pathological spread of α-synPFFs in the rat brain is expected.

[0290] Example 11. Dose-response study in a rodent model of Parkinson's disease (Study 5) Several doses of the most potent AAV5-miSNCA Scaffold 1 and Scaffold 2 candidates will be tested in relevant rodent (rat or mouse) Parkinson's disease models (transgenic lines). AAV5-miSNCA Scaffold 1 and Scaffold 2 are expected to rescue the molecular, histological and motor readout phenotypes.

Claims

1. A nucleic acid comprising a sequence encoding a first RNA and a sequence encoding a second RNA, i. The first and second RNAs each contain a hairpin, ii. The first RNA comprises a first guide sequence of at least 19 nucleotides that is substantially complementary to a portion of the alpha-synuclein (SNCA) gene, iii. The second RNA comprises a second guide sequence of at least 19 nucleotides that is substantially complementary to a portion of the SNCA gene. The sequence encoding the first RNA and the sequence encoding the second RNA each include the sequence of Sequence ID No. 11 or a sequence having at least 70% sequence identity therewith. The nucleic acid further comprises a sequence encoding a third RNA, wherein the sequence encoding the third RNA comprises the sequence of sequence number 34 or a sequence having at least 70% sequence identity therewith.

2. The nucleic acid according to claim 1, wherein in the 5' to 3' direction, the sequence encoding the first RNA is followed by a first spacer containing at least 15 nucleotides, and the sequence encoding the second RNA is followed by the first spacer.

3. The nucleic acid according to claim 2, wherein in the 5' to 3' direction, the sequence encoding the third RNA is followed by a second spacer containing at least 15 nucleotides, the second spacer is followed by the sequence encoding the first RNA, and the first spacer and the sequence encoding the second RNA follow.

4. The nucleic acid according to claim 1, wherein each of the guide sequences is substantially complementary to a sequence selected from the group consisting of sequence numbers 4 to 10.

5. An expression cassette comprising the nucleic acid described in claim 1, wherein the expression cassette is a DNA molecule.

6. The expression cassette according to claim 5, wherein the nucleic acid is operably connected to a promoter and optionally to a poly(A) signal.

7. The expression cassette according to claim 5, wherein the expression cassette is adjacent to at least one AAV inverted terminal sequence (ITR).

8. An adeno-associated virus (AAV) vector comprising the expression cassette described in claim 5.

9. The AAV vector according to claim 8, comprising AAV5 capsid protein or AAV9 capsid protein.

10. A pharmaceutical composition comprising the nucleic acid described in claim 1 and at least one pharmaceutically acceptable excipient.

11. A pharmaceutical composition according to claim 10, for use as a pharmaceutical product.

12. The pharmaceutical composition according to claim 10 for use in a treatment that reduces the expression of RNA encoded by the SNCA gene.

13. The pharmaceutical composition according to claim 12, wherein the treatment reduces or knocks down the amount of α-synuclein aggregates and / or Lewy bodies and / or Papp-Lantos bodies.

14. The pharmaceutical composition according to claim 12, used to treat and / or prevent Parkinson's disease (PD), Lewy body dementia (DLB), multiple system atrophy, neuropsychiatric symptoms, motor symptoms of Parkinson's disease (PD), cognitive impairment, sleep disorders, autonomic nervous system disorders, and / or olfactory disorders.

15. A kit comprising the nucleic acid described in claim 1, further comprising an immunosuppressive compound.

16. Cells transfected with the nucleic acid described in claim 1.

Images