Double stranded RNA and uses thereof

Abstract

To provide double stranded RNA and methods of use thereof for Machado Joseph disease (MJD).SOLUTION: The disclosure uses RNA silencing technology, e.g., RNA interference, against exonic single nucleotide polymorphism (SNP) in ataxin-3 gene encoding dominant gain-of-function mutant ataxin-3 protein. For that purpose, provided is a highly-target specific gene silencing RNA having an anti-sense sequence that is complementary to a SNP that is in linkage disequilibrium with the disease-causing expansion. Furthermore, the disclosure provides a selected adeno-associated viral vector, in particular serotype 9 (AAV9) as a gene delivery vector, upon which the double stranded RNA can be delivered into the central nervous system (CNS).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to allele-specific, non-invasive treatments, particularly for Macado-Joseph Disease (MJD). The present disclosure relates to an ataxin-3 protein encoding a dominant gain-of-function mutant ataxin-3 protein. RNA silencing in response to exonic single nucleotide polymorphisms (SNPs) in the taxin-3 gene and the use of RNA-binding technologies (e.g., RNA interference), thereby resulting in an effective treatment of MJD. For this purpose, we have identified SNPs that are in linkage disequilibrium with the disease-causing expansions. Highly target-specific gene silencing RNA with complementary antisense sequences was designed and tested.

[0002] Further, the present disclosure provides an adeno-associated virus vector selected as a vector for gene delivery. - and in particular the adeno-associated viral vector serotype 9 (AAV9), but this particular The serotypes efficiently cross the blood-brain barrier (BBB) ​​and therefore can be delivered via minimally invasive routes (e.g. The double-stranded RNA can be delivered to the central nervous system (CNS) by intravenous administration. Cut. [Background technology]

[0003] Mackay-Joseph disease (MJD) is a neurodegenerative disorder characterized by cerebellar dysfunction and loss of motor coordination. It is a chromosomal overt neurodegenerative disorder. It is the most common type of spinocerebellar ataxia worldwide. This corresponds to ataxin-3 gene (MJD1 / ATXN3 gene) mutation. It is caused by a genetic mutation in the chromosome 1 (CAG) triplet region. It requires DNA segments of the ataxin-3 gene known as nucleotide repeats. Usually, the CAG segment in the human ataxin-3 gene occurs multiple times, i.e., approximately It is repeated 10 to 42 times. People who develop MJD have at least one allele The number of CAG repeats is increased in individuals with the disorder. The allele is inherited. People with more than 51 CAG repeats may develop signs and symptoms of MJD. Although most people with 60 or more repeats may develop symptoms, those with 60 or more repeats almost always develop the disorder. Increasing the size of the CAG repeat results in the formation of elongated (mutated) ataxin-3 proteins. This protein is processed intracellularly and accumulates and aggregates within neurons. This leads to multiple pathogenic mechanisms, ultimately resulting in smaller cytotoxic fragments. Ultimately, this leads to neurodegeneration in several brain regions, which is responsible for the symptoms and progression of MJD. and the underlying cause of symptoms.

[0004] One of the most direct, specific and effective solutions to correct MJD is By inhibiting mutant ataxin-3 expression using RNA interference (RNAi), The goal of RNAi is to target the primary cause of the disease. It is a naturally occurring mechanism involving sequence-specific downregulation of mRNA. RNAi is a process that reduces the amount of protein expressed by double-stranded RNA. A (dsRNA) induces the transcription factor A (TFA) in the target mRNA. This strand is substantially or completely complementary to the NA. This strand is called the guide strand or antisense strand. The RNAi mechanism involves guidance to the RNA-induced silencing complex (RISC). In this process, RISC assembles the 5' end of the target RNA into a DNA fragment that resembles its complement. RISC prefers the more loosely paired strand. It binds to the target mRNA by complementary base pairing. Once bound to the target mRNA, RI The SC can cleave the mRNA or reduce the translation efficiency. It can cleave the mRNA between residues paired to nucleotides 10 and 11 of the Since its discovery, RNAi has been widely used to knock down specific target genes. The triggers that have been used to induce RNAi are small interfering RNAs (SMRNAs). In addition, the use of RNA cleavage may involve the use of small interfering RNA (siRNA) or short hairpin RNA (shRNA). Molecules that can naturally induce i, so-called microRNAs (miRNAs), are naturally occurring These have been used to create artificial miRNAs that mimic their counterparts found in The strategy involves preparing dsRNA molecules designed to target a gene of choice. Commonalities: RNAi-based therapeutics exploit the sequence-specific mode of RNAi are in development, with several currently in clinical trials.

[0005] RNA interference targets both mutated and non-mutated ataxin-3 genes. The latter has been used for targeting (WO2005105995, Alves et al., 2010). In this case, knockdown of normal ataxin-3 protein in rats has no clear effect. However, the researchers found that the mutant AT-1 had no obvious adverse effects on human brain cells. Tolerance of long-term silencing of both mutant and non-mutated ataxin-3 genes It is not known whether MJD can be cured. Therefore, treatment may be required for decades. Therefore, efforts to control silencing or inhibit only mutant alleles are being considered. It should be.

[0006] One of the most specific and effective solutions is the coding for the ataxin-3 gene. SNPs located within the region, particularly SNP-based nucleotide sequences that are in linkage disequilibrium with disease alleles For example, the targeting of the nucleotide sequence at the 3' end of the expanded CAG tract may be SNP(C 987 GG / G 987 Cytosine (C) in GG:rs12895357 It has been described as being in linkage disequilibrium with the above diseases and is found in 7 of MJD patients worldwide. 0% are associated with abnormal CAG repeat expansions.

[0007] Allele-specific suppression of mutant ataxin-3 gene is associated with cytosine at rs12895357 (C) siRNA or shRNA was used to target the cells (see US10072264 B2) and rodent models of MJD (Alves et al., 2008a; Nobrega et al., 2009). 013). However, in these previous studies, The sequence was not able to achieve complete allele-specific silencing of the mutant allele. Furthermore, the toxicity of silencing sequences in the central nervous system (CNS) of rodent models has been shown to be persistent. The effects of shRNAs on long-term treatment or in wild-type animals were not evaluated. It has recently been reported that in the case of treatment or when high doses are used, it can lead to severe brain toxicity. Toxic side effects are due to saturation of the cellular RNAi machinery and the expression of endogenous miRNAs. Furthermore, previous studies have shown that mutations in the gene for IL-16 are associated with altered expression of IL-16. Allele-specific viral silencing of IFN-3 was achieved by craniotomy and intracerebral viral injection. Previously, direct administration of a viral vector was required, but this direct administration of a viral vector is an invasive procedure. This is sequential and associated with potential adverse effects, and the vector is distributed throughout the brain. However, this does not target all areas affected by MJD. Put away. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2005105995 [Non-patent literature]

[0009] [Non-Patent Document 1] Alves et al., 2010 [Non-Patent Document 2] Alves et al., 2008a [Non-Patent Document 3] Nobrega et al., 2013 Summary of the Invention [Problem to be solved by the invention]

[0010] The above facts are disclosed to illustrate the technical problem addressed by the present disclosure. [Means for solving the problem]

[0011] MJD is associated with the expression of mutant ataxin-3 protein, RNAi reduces the expression of the ataxin-3 gene, because accumulation of ataxin leads to disease. This offers the possibility of treating diseases because it allows The theoretical framework is to preserve normal ataxin-3 mRNA while suppressing the expression of mutant ataxin-3 By reducing the levels of mRNA, the toxicity caused by mutant ataxin-3 protein was reduced. and reducing the sexual effects of MJD to reduce and / or delay symptoms of MJD or to completely eliminate symptoms of MJD. It is completely preventative.

[0012] The present disclosure relates to a method for producing a nucleic acid sequence comprising the steps of: The RNA sequence has a sequence length of at least 19 nucleotides, preferably 19 to 23 nucleotides in length and is complementary to SEQ ID NO: 1, 7, 13 or 19 The present invention provides a dsRNA for targeting a SNP, the dsRNA comprising a first RNA sequence and a second RNA sequence. The dsRNA induces target-specific RNAi against the human mutant ataxin-3 gene. This document is intended to be used to guide

[0013] The SNP-targeting dsRNA of the present disclosure targets two disease allele-encoding regions, i.e., rs 12895357 (exon 10) and rs1048755 (exon 8) (Figure 1). Such dsRNAs may be used alone or in combination to target SNPs present in the Direct delivery into cells by transfection or delivery of DNA (e.g., transfection) or a vector capable of expressing the dsRNA. Indirectly deliver rs12895357( C 987 GG / G 987 GG) (exon 10) to cytosine (C) (SEQ ID NOs: 2, 3, 4 , 5 and 6) or guanine (G) (SEQ ID NOs: 8, 9, 10, 11 and 12); or rs1048755(A 669 T / G669 TG) (exon 8) adenine (A) ( SEQ ID NOs: 14, 15, 16, 17 and 18) or guanine (G) (SEQ ID NOs: 20, 2 Specifically targeting expression of mutated ataxin-3 genes, including those in the 1, 22, 23, and 24 Alternatively, SNP-targeting dsRNAs can be used in combination. to target both the non-mutated and mutated ataxin-3 genes. It is also possible.

[0014] In particular, the first strand / sequence of the designed SNP-targeting dsRNA of the present disclosure is SEQ ID NO:2. One of A is rs1289 when prepared in a miRNA scaffold. By targeting the C nucleotide at 5357, mutant ataxin-3 mRNA and This dsRNA was able to reduce the levels of ribosomal and protein. This represents an improvement over previous SNP-targeting dsRNAs and targets the mutant ataxin-3 gene. AAV9 in a miRNA cassette targets genes more specifically. Delivered to the Striatum of a Lentivirus-Based Mouse Model of MJD via IL-1-Mediated Expression When combined with α-amyloides, it was possible to reduce neuronal cell death and mutant ataxin-3 aggregates. Furthermore, in a transgenic mouse model of very severe MJD, when administered i.v. Functional biomarkers of motor behavior disorders, cerebellar neuropathology, and magnetic resonance spectroscopy It was also possible to reduce the total.

[0015] The dsRNA according to the present disclosure may be an siRNA, an shRNA, a pre-miRNA, or a pri Such dsRNA can be prepared as, for example, a transmembrane-like RNA (miRNA). Direct delivery to target cells via intracellular uptake using transfection techniques. Preferably, the delivery is accomplished using a gene therapy vector, such as a si Expression cassettes for RNA, shRNA, pre-miRNA or pri-miRNA In this way, by supplying cells with a constant amount of dsRNA, it is possible to Thus, sustained ataxin-3 gene suppression can be achieved without the need for Preferably, the selected viral vector is AAV9 or an AAV9 derivative. The reason is that this particular AAV serotype efficiently crosses the BBB, allowing for intravenous administration. AAV9, AAVrh10, or PHP.B or PHP.eB or Derivatives such as PHP.S are available at https: / / www.addgene.org / vira It can be obtained from l-service / aav-prep / .

[0016] Thus, the present disclosure provides methods for the use of dsRNA according to the present disclosure for medical purposes, such as for the treatment of MJD. The medical use of the present invention can be achieved by expressing the dsRNA of the present disclosure. It may also comprise an expression cassette or a viral vector such as AAV9.

[0017] The present disclosure relates to The first strand of RNA and the second strand of RNA are substantially complementary to each other, and preferably Alternatively, the first and second strands of RNA are at least 90% complementary to each other. ; the first strand of RNA has a sequence length of at least 19 nucleotides; The first strand of the RNA is at least 86% complementary to SEQ ID NO: 1, 7, 13, or 19. and; the first strand of the RNA is different from SEQ ID NO:26; the first nucleotide of the first strand of RNA is different from cytosine; It relates to a double-stranded RNA comprising a first strand of RNA and a second strand of RNA.

[0018] In one embodiment, the first strand of RNA is a nucleic acid sequence of at least 19 nucleotides. The first strand of RNA may have a sequence length of up to 23 nucleic acids. Preferably, the first strand of RNA has a sequence length of It may have a sequence length of 20 to 22 nucleotides, and more preferably, a better To obtain the result, the first strand of RNA has a sequence length of 21-22 nucleotides. Even more preferably, for better results, the first strand of RNA is , may have a sequence length of 22 nucleotides.

[0019] In one embodiment, the first strand of RNA is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 8, 9, 1 For 0, 11, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23 or 24 90% identical to the sequence of SEQ ID NO: 2, 3, 4, 5, 6, 8, 9, 10 , 11, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23 or 24 More preferably, the sequences are SEQ ID NOs: 2, 3, 4, 5, 6, 8, 9, 1 For 0, 11, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23 or 24 may be 100% identical.

[0020] Identity was determined as summarized in the table below. [Table 1]

[0021] In one embodiment, the first strand of the RNA has a low affinity to SEQ ID NO: 1, 7, 13 or 19. At least 90% complementary, preferably to SEQ ID NO: 1, 7, 13 or 19. 95% complementary to SEQ ID NO: 1, 7, 13 or 19, more preferably 99% complementary, and even more preferably, the first strand of RNA is SEQ ID NO:1, 100% complementary to 7, 13 or 19.

[0022] In one embodiment, the first strand of RNA is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 8, 9, 1 From 0, 11, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23 or 24 You can choose.

[0023] In one embodiment, for better results, the first strand of RNA is SEQ ID NO:1. and the first strand of RNA may be complementary to SEQ ID NO: 2, 3, 4, 5 or 6. You can also select from the following.

[0024] In one embodiment, for better results, the first strand of RNA is SEQ ID NO:2 Or it may be SEQ ID NO:3.

[0025] In one embodiment, the first strand of the RNA may be complementary to SEQ ID NO: 7, The first strand of the RNA can be selected from SEQ ID NO:8, 9, 10, 11 or 12.

[0026] In one embodiment, the first strand of the RNA may be complementary to SEQ ID NO:13. The first strand of the RNA may be selected from SEQ ID NO: 14, 15, 16, 17 or 18. Cut.

[0027] In one embodiment, the first strand of the RNA may be complementary to SEQ ID NO:19. The first strand of the RNA may be selected from SEQ ID NO: 20, 21, 22, 23 or 24. Cut.

[0028] In one embodiment, in order to obtain better results, the first nucleotide of the first strand of RNA is The oxidase may be uracil.

[0029] In one embodiment, the double-stranded RNA is a pre-miRNA scaffold, -miRNA scaffold, miRNA scaffold, shRNA or siRNA A may be included in A, and preferably, it is included in the miRNA scaffold or shRNA. It may be contained in the miRNA, more preferably in the miRNA.

[0030] In one embodiment, the double-stranded RNA is contained within a miRNA scaffold. Preferably, the miRNA sequencing method disclosed by Chung et al. (2006) is used. The miRNA scaffold derived from miR-155 is included in the More preferably, the miR155 based scaffold comprises the sequence Includes Nos. 27, 28 and 29.

[0031] The present disclosure provides isolated DNA sequences encoding the double-stranded RNA disclosed herein. The present invention also relates to an expression cassette comprising said isolated DNA sequence or said double-stranded RNA.

[0032] The present disclosure relates to the isolated DNA or double-stranded RNA or expression cassettes disclosed herein. Preferably, the vector is an adeno-associated virus vector. vector, lentivirus vector, adenovirus vector, or non-viral vector more preferably, the adeno-associated viral vector is AAV9 or AAVrh10 or is PHP.B or PHP.eB or PHP.S.

[0033] The present disclosure relates to the isolated DNA sequences or double-stranded RNA or expression vectors disclosed herein. The present invention also relates to a host cell comprising the cassette or vector, preferably said host cell being a eukaryotic cell. Preferably, the host cell is a human cell, and more preferably, the host cell is a mammalian cell.

[0034] The present disclosure relates to the isolated DNA or double-stranded RNA or expression cassettes disclosed herein. The present invention further relates to a composition comprising the vector or host cell.

[0035] The present disclosure relates to the isolated DNA sequences or double-stranded RNA or expression vectors disclosed herein. It further relates to a kit comprising the cassette or the vector or the host cell or the composition.

[0036] Further, the present disclosure provides a double-stranded RNA for use in medicine, A vector comprising an isolated DNA sequence for loading a nucleic acid sequence of a nucleic acid fragment containing said isolated DNA sequence. It also relates to expression cassettes.

[0037] The present disclosure relates to a method for treating or preventing a neurodegenerative disease or for treating the cytotoxic effects of said neurodegenerative disease. Double-stranded RNA for use in treatment or prevention, isolated double-stranded RNA encoding said double-stranded RNA A vector comprising the isolated DNA sequence, or an expression cassette comprising the isolated DNA sequence. Preferably, the neurodegenerative disease is a trinucleotide repeat disease. More preferably, the neurodegenerative disease may be a CAG trinucleotide repeat disease; Even more preferably, the double-stranded RNA, vector or expression cassette is is administered to regulate the levels of neurometabolites, preferably Increases N-acetylaspartate, myoinositol, and glycerophosphocholine and is administered to reduce phosphocholine.

[0038] In one embodiment, the neurodegenerative disease is Macadamia-Joseph disease.

[0039] In one embodiment, the double-stranded RNA is administered systemically, intravenously, or intratumorally. It can be administered orally, intranasally, intraperitoneally, intramuscularly, or intraspinally. , intracerebral, intraventricular, intracisternal, intrathecal, intraocular, cardiac It may be administered intraviscerally, intradermally or subcutaneously, preferably intravenously and intracisternally. The drug may be administered intrathecally, intrathecally, or in situ into the brain.

[0040] In this disclosure, the term complementary refers to binding to another nucleic acid sequence via hydrogen bonds. The nucleotides of a nucleic acid sequence that can form base pairs are Complementary RNA strands form double-stranded RNA. Double-stranded RNA is a set of two The nucleic acid sequence may be formed from independent complementary RNA strands, or the two complementary RNA strands may be In one RNA strand, the complementary RNA strand may contain a nucleotide C and guanine (C and G) can form base pairs, while guanine and uracil (G and U) can also form base pairs, and uracil and adenine (U and A) can also form base pairs. can be done.

[0041] In this disclosure, the term substantial complementarity refers to a first RNA sequence and a second RNA sequence. or a first RNA sequence and a second RNA sequence and a sequence identical to SEQ ID NO: 1, 7, 13 or 1 This means that it does not have to be the exact complement of 9.

[0042] Further, in the present disclosure, between the first RNA sequence and SEQ ID NO: 1, 7, 13 or 19 Substantial complementarity of has no mismatches, has one mismatched nucleotide, having two mismatched nucleotides or having three mismatched nucleotides For example, when considering the first RNA sequence and SEQ ID NO: 1, one amino acid sequence is The matched nucleotides are the nucleotides of the first RNA sequence that form base pairs with SEQ ID NO:1. It is noted that not a single nucleotide forms a base pair with SEQ ID NO:1 throughout the entire length. Having no mismatches is understood to mean that all nucleotides are the same as SEQ ID NO: 1 means that there is a base pair between the two. This means that the nucleotide does not form a base pair with SEQ ID NO: 1. The presence of a nucleotide sequence in the sequence of SEQ ID NO:1 means that three nucleotides do not form a base pair with SEQ ID NO:1. The same applies to the first RNA sequence and SEQ ID NO: 7, the first RNA sequence and SEQ ID NO: No. 13, or the first RNA sequence and SEQ ID NO: 19.

[0043] In the present disclosure, the first RNA sequence may be longer than 19 nucleotides; In this case, substantial complementarity is determined over the entire length of SEQ ID NO:1. However, in this embodiment, when base paired with the first RNA sequence, the sequence No. 1 has no mismatches, one mismatch, or two mismatches over the entire length of the sequence. It means that it has a match. The table below illustrates what is explained in the paragraph above. [Table 2]

[0044] The following drawings provide preferred embodiments for illustrating the present specification, but do not limit the scope of the disclosure. should not be considered as limiting. [Brief description of the drawings]

[0045] [Figure 1] Schematic diagram of the MJD1 gene and the exonic single nucleotide polymorphisms rs1048755 and rs12895357. The MJD1 gene is composed of 11 exons (grey boxes). The CAG repeat is located in exon 10, and MJD can be caused by more than 51 repeats. A SNP was identified (rs12895357) immediately after the CAG repeat expansion (nucleotide 987). The non-mutated allele generally represents a guanine (G) at this position, whereas the mutated allele represents a cytosine (C) in 70% of MJD patients. Another SNP was identified (rs1048755) in exon 8 (nucleotide 669). In this case, the non-mutated allele generally represents a guanine (G) at this position, whereas the mutated allele represents an adenine (A) in 70% of MJD patients. [Diagram 2]FIG. 1 shows that miR-ATXN3 mediates efficient allele-specific silencing of mutant ataxin-3 in vitro. (a) Diagram of artificial microRNA (miR:microRNA) and short hairpin (sh) type vector construct. An artificial microRNA construct was designed based on the silencing sequence SEQ ID NO: 2 disclosed herein to specifically silence mutant ataxin-3 (miR-ATXN3). A control miRNA (miR-control) was also designed with a sequence that does not silence any mammalian RNA. Both the artificial microRNA construct and the control miRNA were inserted into AAV2 plasmid vector backbone together with EGFP reporter gene under the control of U6 promoter. A plasmid encoding an shRNA known in the art (Alves et al., 2008a) specifically targeting mutant ataxin-3 was also used (sh-mutATXN3); b, c) Neuro2a cells (a mouse neural crest-derived cell line) pre-infected with a lentiviral vector encoding human mutant ataxin-3 with 72Q (b) or human wild-type ataxin-3 with 27Q (c) were transfected with plasmids encoding miR-control (control condition), miR-ATXN3 and sh-ATXN3. miR-ATXN3 induced a 42.03±6.26% decrease in human mutant ataxin-3 mRNA levels, but did not affect wild-type human ataxin-3 mRNA levels. These results were confirmed by Western blotting in d) and e), respectively. Data represent the mean±sem; NS P>0.05, *P<0.05 and **P<0.01. b, c, d, e) One-way analysis of variance (ANOVA) with Bonferroni post-hoc test. miR-control n=5; miR-ATXN3 n=5; sh-ATXN3 n=5. Internal controls for normalization were chosen according to the analysis by GeNex and correspond to endogenous mouse ataxin-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels.CMV, cytomegalovirus enhancer; CBA, chicken β-actin promoter; EGFP, enhanced green fluorescent protein; ITR, inverted terminal repeat. [Diagram 3] Diagram of SEQ ID NO: 3 mediating efficient allele-specific silencing of mutant ataxin-3 in vitro similar to SEQ ID NO: 2 (miR-ATXN3). a, b) Neuro2a cells pre-infected with lentiviral vectors encoding human mutant ataxin-3 with 72Q (a) or human wild-type ataxin-3 with 27Q (b) were transfected with plasmids encoding miR-control, SEQ ID NO: 2 (miR-ATXN3), SEQ ID NO: 3 or sh-ATXN3. The artificial miR155-based construct encoding SEQ ID NO: 3 induced a similar reduction in human mutant ataxin-3 mRNA levels as the construct encoding SEQ ID NO: 2 (miR-ATXN3), but did not affect wild-type mRNA levels. Data represent mean ± sem; NS P>0.05, *P<0.05, **P<0.01 and ***P<0.001. (a, b) One-way analysis of variance (ANOVA) with Bonferroni post-hoc test. miR-control n=5; miR-ATXN3 n=5; SEQ ID NO:3 n=5 and sh-ATXN3 n=5. Internal controls for normalization were chosen according to the analysis by GeNex and correspond to endogenous mouse ataxin-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. [Figure 4]Figure 1. miR-ATXN3 treatment does not induce changes in endogenous mouse ataxin-3 mRNA levels in vitro. (a, b) Neuro2a cells infected with (a) human mutant ataxin-3 (72Q) or (b) human wild-type ataxin-3 (27Q) were transfected with plasmids encoding miR-control, miR-ATXN3 and sh-ATXN3. Relative expression levels of mouse ataxin-3 mRNA were determined by quantitative reverse transcription PCR. Data show relative mean mRNA levels ± sem; ns = p > 0.05 when compared to miR-control. One-way analysis of variance (ANOVA) with Bonferroni post-hoc test. miR-control n = 5; miR-ATXN3 n = 5; sh-ATXN3 n = 5. [Diagram 5]FIG. 1. miR-ATXN3 reduces levels of mutant ataxin-3 mRNA and aggregated mutant ataxin-3 and prevents striatal degeneration when injected intracranially in a lentivirus-based mouse model of MJD. (a) Schematic of the strategy used to generate a striatal lentivirus-based mouse model of MJD and silence mutant ataxin-3 using AAV9. Ten-week-old mice were bilaterally co-injected into the striatum with a lentivirus vector encoding human mutant ataxin-3 with 72Q (LV-Atx3-MUT) and AAV9 vectors encoding miR-ATXN3 for the right hemisphere (AAV9-miR-ATXN3) and miR-control for the left hemisphere (AAV9-miR-control). Five weeks after surgery, mice were euthanized. (b) Confocal microscopy images showing effective transduction of the mouse striatum of the striatal lentiviral model of MJD by both rAAV9 vectors. (c) Quantitative reverse transcription PCR analysis demonstrated that miR-ATXN3 induced a 63.75±2.25% decrease in mutant ataxin-3 mRNA levels compared to the control left hemisphere. (d) Western blot analysis also confirmed that miR-ATXN3 expression significantly reduced mutant ataxin-3 aggregates. (e) Ubiquitin immunoreactivity in the striatum of the striatal lentiviral-based mouse model of MJD co-injected with miR-control or mir-ATXN3. In (f), the total number of mutant ataxin-3 inclusions was counted and quantified. Scale bar, 50 μm. (g) Staining for DARPP-32 revealed a strong reduction in DARPP-32 immunoreactivity in the striatum of hemispheres co-infected with human mutant ataxin-3 and miR-control. Scale bar, 200 μm. In (h), this was quantified as a reduction in the volume of DARPP-32 staining. (i) Cresyl violet staining showing pycnotic nuclei in both hemispheres. A higher number of pycnotic nuclei were visible in the control hemisphere. In (j), this was quantified. Scale bar, 20 μm.Data show mean ± sem; ns p>0.05, *p<0.05, ***p<0.001 compared to control hemisphere. (c,d) Paired Student's t test. n=5. (f,h) Paired Student's t test. n=8. (j) Paired Student's t test. n=4. [Figure 6] FIG. 13. Intravenously injected rAAV9 vectors mediate efficient transduction throughout the brains of wild-type and transgenic MJD mice. Representative images of GFP immunohistochemistry (grey) in the brains of 3-month-old mice: A) transgenic mice without injection; B) transgenic mice that received rAAV9-miR-ATXN3 IV injection at postnatal day 1; C) wild-type mice subjected to the same procedure. Images show rAAV9 transduction of the whole brain, cerebellum (CB), hippocampus (HIP), pontine nuclei (PN) and medulla / spinal cord (Md / SC) obtained at 5x and 20x magnification. [Figure 7] FIG. 1 shows that rAAV9 vectors efficiently transduce the cerebellum of transgenic mice. Representative images of visible GFP immunohistochemistry (gray) in the cerebellum of a 3-month-old mouse that received a neonatal IV injection of rAAV9-miR-ATXN3. Images were obtained at 20x magnification and show cerebellar regions of particularly efficient transduction, including the deep cerebellar nuclei (DCN), the 10th, 9th, 7th, and 6th lobules, and the choroid plexus cells of the fourth ventricle (4V). [Figure 8] FIG. 1: rAAV9 targets major areas of mutant ataxin-3 accumulation in the cerebellum of transgenic mice. Representative images show immunofluorescence directed against HA and GFP in the cerebellum of a transgenic mouse receiving rAAV9-miR-ATXN3 injection at P1. Images were obtained with a confocal microscope at 20x magnification. a) Representative image of a rAAV9-positive Purkinje cell showing colocalization of HA and GFP signals (white). DCN deep cerebellar nuclei; PCL Purkinje cell layer. [Figure 9]Figure 1. Silencing of mutant ataxin-3 improves rotarod performance in MJD transgenic mice. a) Experimental design of MJD transgenic mice divided into three key tasks: 1) AAV9 intravenous injection at PN1; 2) behavioral assessment at three different time points, and 3) sacrifice and neuropathological analysis. b) Rotarod performance at constant speed (5 rpm). c) Rotarod performance at accelerating speed. Data are presented as mean latency to fall ± SEM for control mice (miR-control, n=11) and mice injected with miR-ATXN3 (n=8). Statistical analysis was performed using unpaired Student's t-test (*P≦0.05, **p<0.01). [Figure 10] Figure 1. miR-ATXN3 treatment improves swimming, beam walking performance and gait ataxia in MJD transgenic mice. a) Animals were scored based on the time it took to complete the pool and climb the platform. Data are presented as mean latency ± SEM. b) Animals were scored based on their performance when walking on a 9 mm circular beam. Each animal was given a score considering the total time it took to traverse the beam and motor coordination. Gait patterns were analyzed by measuring c) posterior stride width, d) anterior stride width and e) footprint overlap (cm). Data are presented as mean performance score ± SEM. Statistical analysis was performed using unpaired Student's t-test (*p<0.05) comparing the performance of control mice (miR-control, n=11) with that of mice injected with rAAV9-miR-ATXN3 (n=8). [Figure 11]Figure 1: miR-ATXN3 treatment efficiently reduces the number of mutant ataxin-3 aggregates and efficiently preserves the molecular layer thickness. a) Representative images of immunofluorescence labeling mutant ataxin-3 (white HA) in the 10th lobule of control (miR-control) and treated (miR-ATXN3) transgenic mice. Images were obtained with a confocal microscope at 20x magnification. b) Quantification of mutant ataxin-3 aggregates per area in the 10th, 9th and 6th lobules. c) Representative images of cresyl violet staining of the 10th lobule of treated and control transgenic mice obtained at 20x magnification. d) Quantification of molecular layer thickness in the 10th, 9th and 6th lobules. Values ​​correspond to the mean ± SEM of three specific sections for each animal (miR-control, n=11; miR-ATXN3, n=8). Statistical analysis was performed using unpaired Student's t-test (*p<0.05, **p<0.01, ***p<0.001). ML molecular layer thickness; Lob10 10th lobule [Figure 12] Schematic diagram of the possible mechanism underlying the therapeutic effect of AAV9-miR-ATXN3 in the present disclosure: i) intravenous injection of rAAV9 vector encoding miR-ATXN3 into neonatal MJD transgenic mice ii) led to silencing of mutant ataxin-3 in the cerebellum, which in turn iii) reduced neuropathological and behavioral deficits. Although rAAV9 vectors efficiently transduced Purkinje cells (PCs) in the 10th and 9th lobules, other mechanisms may also enhance the transduction levels of PCs and / or enhance beneficial effects, such as 1) transfer of viral vectors from blood to CSF ​​and / or secretion of miR constructs into CSF ​​by transduced epithelial cells in the choroid plexus; 2) retrograde transport of rAAV9 from DCN to PC layer and / or transfer of miR from transduced cells in DCN to PC processes; 3) transfer of miR between adjacent PCs; and 4) neuroprotective effects induced by rAAV9-positive PCs. CSF, cerebrospinal fluid; DCN, deep cerebellar nuclei; PC, Purkinje cells. [Figure 13] Different rAAV9 transduction levels correlate with neuropathological and behavioral parameters of treated mice. a) Linear regression graph of GFP mean intensity (AU = arbitrary units) in the 9th and 10th lobule versus number of aggregates / mm2 in the same area for rAAV9-miR-ATXN3 treated animals (n = 8) (p = 0.0309, R2 = 0.5675). b) Linear regression graph of GFP integrated intensity (AU = arbitrary units) in all cerebellar lobules versus mean latency to fall on the accelerating rotarod for rAAV9-miR-ATXN3 treated animals (n = 8) taking into account all time points (days 35, 55 and 85). According to residual analysis, one animal was considered to be an outlier of the predictive linear regression model. The analysis was performed without including this animal (p = 0.0123, R2 = 0.7457). Statistical analysis was performed using Pearson's correlation (two-tailed p-values). Dotted lines represent 95% confidence intervals. [Figure 14] Figure 1. rAAV9-miR-ATXN3 IV injection does not affect rotarod performance in wild-type mice. a) Rotarod performance at constant speed (5 rpm). b) Rotarod performance at accelerating speed. Data are presented as mean latency to fall ±_SEM for wild-type mice (miR-control, n=5) and mice injected with AAV9-miR-ATXN3 (n=5). Statistical analysis was performed using unpaired Student's t-test (ns=not significant). [Figure 15]Figure 1. miR-ATXN3 treatment improves the levels of key metabolites in the cerebellum. a) Magnetic resonance spectroscopy: Cerebellar neurochemical profiles of miR-control, miR-ATXN3 and WT mice at day 75. NAA, tChol and Ins metabolites were significantly deregulated in transgenic MJD compared to WT mice. Compared to control mice, mice injected with rAAV9 miR-ATXN3 presented higher levels of NAA (a neuronal marker) and lower levels of Ins and tCho (markers of cell death), demonstrating the efficacy of miR-ATXN3 treatment. b) The NAA / Ins, NAA / tCho and NAA / (Ins+tCho) ratios were used to evaluate the efficacy of this gene-based therapy. All values ​​are presented as mean ± SEM and statistical analysis was performed using one-way ANOVA. miR-control (n=8), miR-ATXN3 (n=7) and WT (n=8). Asterisks indicate statistically significant differences between groups, *p<0.05, ****p<0.0001. Ins: myo-inositol, NAA: N-acetylaspartate, tCho: glycerophosphocholine + phosphocholine. [Figure 16] FIG. 1 is an example of a dsRNA of the present disclosure targeting ataxin-3 mRNA at rs12895357 (cytosine) embedded in an artificial miRNA scaffold using pri-miR-155. The first RNA sequence / strand of the dsRNA (SEQ ID NO: 2) is depicted in the rectangle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present disclosure relates to a method for producing a nucleic acid sequence comprising the steps of: Preferably, the first strand of RNA and the second strand of RNA are at least 90% complementary to the first RNA sequence / strand, and the first RNA sequence / strand is at least 19 nucleotides long. The sequence length is preferably 19 to 23 nucleotides, and the sequence is represented by SEQ ID NO: 1, 7 , 13 or 19, The present invention provides a dsRNA for targeting a SNP comprising an RNA sequence / strand of the following structure: The first strand is different from SEQ ID NO:26; the first nucleotide of the first strand of the RNA is Unlike Shin.

[0047] In the present disclosure, a method for enhancing gene silencing efficiency in mammalian cells is described. All SNP-targeting dsRNAs designed contained, without exception, i) one uracil residue at the 5' end; ii) at least 5 U in the first 8 nucleotides of the 5' end iii) a length of nucleoside in the first strand (antisense strand); No GC interval exceeds 5 leutions.

[0048] The allele-specific gene silencing disclosed herein is achieved by first cleavage of the first allele close to the cleavage site. Precise matching of the RNA sequence / strand (i.e., antisense) outside the seed region, More precisely, exact matching at position 12 is achieved. Antisense (positions 2-8) The 5'-terminal "seed" sequence of Therefore, all selected SNP-targeting dsRNAs are complementary to the target A non-target mRNA that is perfectly complementary to the mRNA containing the target SNP allele Since it forms a mismatch at position 12 with NA, it enables characteristic silencing. do.

[0049] According to the above rationale, first, a silencing sequence (SEQ ID NO: 2) was inserted into ataxin A single nucleotide polymorphism (SNP) located at the 3' end of the expanded CAG tract in exon 10 of the -3 gene (C 987 GG / G 987 It was designed to target cytosine (C) in the GG:rs12895357 Exon 10 of the ataxin-3 gene contains more than 51 CAG lipids when mutated. 70% of MJD patients also have a C nucleotide after the CAG overexpansion, Nonmutated ataxin-3 alleles generally have a G at this position (Figure 1). SEQ ID NO:2, like SEQ ID NO:3, 4, 5 or 6, is an allele-specific sequence for mutant ataxin-3. SEQ ID NO: 8, 9, 10, 11 or 12 can promote selective silencing of G In the rare cases where a nucleotide is present at this position and is associated with a mutant allele, In addition, rs12895357 (SEQ ID NO: 2, 3, 4, Any silencing sequence targeting C at position 5 or 6) will be a subsequence of any sequence targeting G at this position. When combined with isolating (SEQ ID NO: 8, 9, 10, 11 or 12), the mutant Silencing both taxin-3 and non-mutated ataxin-3 and suppressing ataxin-3 expression can be completely knocked down.

[0050] Following the same rationale, the exonic SNP rs1048755 located in exon 8 (A 669 T / G 669 TG) is an allele-specific silencing agent of mutant ataxin-3 genes. For example, SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 4 7 or 18 are also in linkage disequilibrium with disease-causing expansions in 70% of MJD kindreds. Targeting an adenine (A) at this position in SEQ ID NO: 20, 21, 22, 23 or 24 indicates that in rare circumstances a G nucleotide at this position is associated with a mutant allele. It can be used in the following cases.

[0051] The rationale employed in this disclosure for the design of allele-specific silencing sequences To evaluate the evidence, the inventors first performed in vitro studies to confirm that SEQ ID NO:2 The therapeutic potential of SEQ ID NO:2 was then evaluated in two different mouse models of MJD, The lentivirus-based mouse model of MJD and the transgenic mouse model of MJD The study was carried out in a mouse model.

[0052] In vitro studies In one embodiment, miRNA-based RNAi plasmids are prepared as follows: Based on SEQ ID NO: 2 or SEQ ID NO: 3, rs12895357 (miR A miR155-based artificial miR targeting ataxin-3 mRNA (ATXN3) A control RNA was designed with a sequence that does not silence any mammalian mRNA. We also designed an iRNA (miR-control). Sincerely, Miguel Sena-Esteves (UMass Medical Sch. ool, Gene Therapy Center, Worcester, MA, USA. The self-complementary adeno-associated virus serotype 2 backbone (scAAV2-U) provided by 6-miRempty-CBA-eGFP plasmid), but this The self-complementary adeno-associated virus serotype 2 backbone expresses an enhanced green fluorescent reporter gene (EGFR). FP), in which the artificial miRNA is driven by a U6 promoter (Figure 2 A).

[0053] In one embodiment, a method is provided comprising administering to the subject a taxin-3 mutant ataxin-3 antibody that is known in the art to specifically target mutant ataxin-3. A plasmid encoding the known shRNA (sh-ATXN3) was prepared as previously described. The sh-ATXN was synthesized as described in SEQ ID NO:2 (Alves et al., 2008a) (FIG. 2A). 3 (SEQ ID NO: 26) is an expanded CAG tract in exon 10 (rs12895357) The shRN is intended to target the C nucleotide in the SNP located at the 3' end. A expression is driven by the H1 promoter.

[0054] In one embodiment, the 27Q-linked A lentiviral vector encoding human wild-type ataxin-3 (LV-WT-AT XN3) and a lentiviral vector encoding a mutant ataxin-3 with 72Q. (LV-Mut-ATXN3) was transformed into HEK293T cells using a system consisting of four plasmids. Lentiviral particles were pre-produced in cells and were then added to phosphate-buffered saline (PBS) The viral particle content of the batch was: HIV-1 p24 antigen levels (RETROtek, Gentaur, Paris, France) The virus stocks were stored at -80°C until use. was stored in.

[0055] In one embodiment, mouse neural crest-derived cell line (Neuro2a cells) cultures were cultured as follows: The mouse neural crest-derived cell line was obtained from the American Type C culture collection cell biology bank(CCL- 131) and incubated at 5% CO 2 / 10% fetal bovine serum at 37°C in air, 100 U / ml penicillin and 100 mg / ml streptomycin (Gibco) The cells were maintained in DMEM medium supplemented with (complete medium).

[0056] In one embodiment, Neuro2a cell infection is carried out as follows: As described above, mutant or non-mutant (i.e., wild-type) ataxin-3 was stably expressed. To obtain a neuronal cell line that expresses the gene rs12895357 in Neuro2a cells, (exon 10) full-length human mutant ataxin-3 (72Q) with C or the same SNP The lentiviral vector encoding the wild-type form (27Q) with G at the end of the vector was then infected. Briefly, Neuro2a cells were cultured in the presence of polybrene for 10 ng p24 antigen / 10 5 The cells were incubated with each vector at a ratio of 1:1.

[0057] In one embodiment, transfection of Neuro2a cells is carried out as follows: The day before transfection, the mutant attenuated DNA was transfected using lentiviral vectors. Neuro2 cells pre-infected with ataxin-3 or wild-type ataxin-3 a Cells were seeded in 12-well plates (180.000 cells / well). Linear polyethyleneimine (PEI) with Mw 40,000 was used as a transfection reagent. ) (Polysciences, inc., Warrington, PA, USA). Each AAV plasmid: miR-control, miR-ATXN3 and sh- Briefly, the formation of DNA:PEI complexes was confirmed by 1 Mix 0 μL of DMEM, 4 μL of PEI (1 mg / ml) and 800 ng of DNA. After 10 min of incubation at room temperature, 500 μL of DMEM was added. Complete medium was added to the mixture. Finally, after removing half of the medium, the Neuro2a cells were , and incubate with 500 μL of transfection solution per well. 48 hours after transfection, Neuro2a cells were washed with PBS1X. , treated with trypsin, separated by centrifugation, and stored at -80°C.

[0058] In one embodiment, RNA extraction, DNase treatment and cDNA synthesis are carried out as follows: The RNA was analyzed using the Nucleospin RNA Kit (Matrix) according to the manufacturer's instructions. Total RNA was isolated using a PCR kit (Chery Nagel, Duren, Germany). Briefly, after cell lysis, total RNA was adsorbed onto a silica matrix and diluted with the recommended buffer. The RNA was washed with PBS and eluted with RNAase-free water by centrifugation. The total amount was measured using a Nanodrop 2000 Spectrophotometer (Ther Optical density (OD) was measured using a 100% chromatographic microscope (MSI) (Molecular Weight 100,000, 10 ... Purity is assessed by measuring the ratio of OD at 260 nm and 280 nm. was evaluated by.

[0059] In one embodiment, the manufacturer's handling instructions are used to avoid genomic DNA contamination and co-amplification. Use Qiagen RNAase-Free DNase Set (Qiagen DNase treatment was performed using the DNase enzyme ELISA kit (Faculty of Engineering, Hilden, Germany). In short, the final volume of the reaction is 0.6 μL DNase buffer, 0.25 μL D The volume was 6 μL, including Nase and 500 ng of RNA. Incubate at 37°C for 30 minutes. After incubation, 0.5 μL of 20 mM EDTA pH=8 was added to stop the reaction. The final step was a 10 min incubation at 65°C.

[0060] In one embodiment, the iScript Se is then run according to the manufacturer's instructions. lect cDNA Synthesis Kit (Bio-Rad, Hercules cDNA was obtained by converting 420 ng of total RNA using a PCR kit (Mitsubishi Microbial Cells, Inc., USA). 2 μL of reaction mixture (5x), 0.5 μL of iScript reverse transcriptase and the appropriate volume Prepare a complete mixture with 10 µL total volume using RNA template and nuclease-free water. The entire reaction mixture was incubated at 25°C for 5 min, followed by incubation at 42°C for 30 min. The reaction was incubated for 1 min at 85°C and for 5 min at 85°C. After the reaction, the mixture was stored at -20°C.

[0061] In one embodiment, quantitative real-time PCR (qPCR) is performed as follows: All qPCR was performed in 96-well microtiter plates according to the manufacturer's instructions. Plates and SsoAdvanced SYBR Green Supermix (Bi o-Rad, Hercules, USA) using Applied Biosystems ems StepOnePlus Real-Time PCR system (Lif The study was conducted at e technologies, USA.

[0062] In one embodiment, 10 μL of SsoAdvan ced SYBR Green Supermix (Bio-Rad, Hercules , USA), 10 ng of DNA template, and human ataxin-3, mouse ataxin- 3. Mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and mouse human 500n for poxanthine guanine phosphoribosyltransferase (HPRT) Reactions with a final volume of 20 μL containing M pre-validated specific primers The reaction was carried out in the mixture. The PCR protocol consisted of a denaturing program (95°C for 30 seconds) The reaction was started by denaturing at 95° C. for 5 s, followed by two steps: denaturation at 95° C. for 5 s and denaturation at 56° C. for 5 s. Two steps of annealing / extension at 20 °C for 10 s were performed for 40 cycles. After the amplification cycle, the temperature was gradually increased from 65°C to 95°C at a heating rate of 0.5°C / 5s. The melting curve protocol was initiated by increasing the temperature to 0.

[0063] In one embodiment, the cycle threshold (Ct) is determined using the StepOnePlus software. The data were automatically determined by the software (Life Technologies, USA). Standard curves were generated for each gene, and quantitative PCR efficiency was determined by software. Relative quantification of mRNA relative to control samples was determined by the Pfaff method. The ideal reference genes were determined using GeNex software.

[0064] In one embodiment, proteins are extracted from neuro2a cells and subjected to protease inhibitors. The RIPA lysis buffer was mixed with the drug mixture and 2 mM dithiothreitol. The lysate was further sonicated and the protein concentration was determined by blood phosphatase. The protein content was estimated by the Bio-Rad Protein Assay (Bio-Rad). ad). Micrograms of the total denatured protein are then subjected to electrophoretic separation. The proteins were then loaded onto a 4% stacking and 10% separating polyacrylamide gel. The membrane was a polyvinylidene difluoride (PVDF) membrane (Merck Millipor re) and blocked in 5% skim milk. Monoclonal anti-ataxin-3 antibody (1H9, 1:1000; Chemicon) and β-tubulin were used for immunofluorescence. Blotting was performed. Mutant and non-mutant human ataxin-3 and Quantification of endogenous mouse ataxin-3 was performed relative to β-tubulin protein. It was.

[0065] In vivo studies In one embodiment, the production of an adeno-associated virus serotype 9 (AAV9) vector is carried out as follows: Briefly, rAAV9-miR-ATXN3 was transfected as described above. and the AAV construct (m iR-ATXN3 and miR-control), pFΔ6 (adenovirus helper protein plasmid) and AAV9 rep / cap plasmid were eluted by calcium phosphate precipitation. Vector stocks were transfected into HEK293T cells by triple transfection. The AAV9 vector was then purified by iodixanol gradient centrifugation. This was followed by concentration and dialysis as previously described. Quantitative real-time PCR (PCR) using specific primers and probe (pBGH) for The vector titer was measured by qPCR.

[0066] In one embodiment, the lentivirus (LV:le) of MJD when administered intracranially. The functionality and efficacy of the AAV9-based strategy in an antiviral-based mouse model Efficacy was evaluated (Figure 5A). This particular mouse model allows for rapid testing of therapeutics, Neuropathology induced by mutant ataxin-3 expression (Alves et al., 2008b) Allows for quantitative analysis of failures.

[0067] In one embodiment, thirteen 10-week-old mice are anesthetized and the striatum of these mice is Human mutant ataxin-3(72Q)(3×10 5 lentivirus encoding p24 of ng The mice were then co-transduced with a rRNA vector and an artificial miR targeting mutant ataxin-3 mRNA for the right hemisphere. The rAAV9 vector (AAV9-miR-ATAX3) (7 × 10 9 Virus miR) and a rAAV9 vector encoding a control miR for the left hemisphere (AAV-miR C ontrol)(7×10 9 The virus genome was injected bilaterally into the mice (Figure 5A).

[0068] In one embodiment, Western blotting was performed on both hemispheres of three animals. The injected striatum was dissected and incubated with a protease inhibitor mixture and 2 mM dithiothreitol. The lysates were then homogenized using RIPA lysis buffer mixed with 100% ethanol. The protein concentration was estimated by the Bradford method (Bio-Rad Laboratories, Inc.). d Protein Assay, Bio-Rad). Total denatured protein was then Sixty micrograms of the mixture was added to 4% polyacrylamide gel for concentration and 10% polyacrylamide gel for separation by electrophoresis. The proteins were then loaded onto a polyvinylidene difluoride (P The cells were transferred to a VDF membrane (Merck Millipore) and blotted in 5% nonfat milk. Monoclonal anti-ataxin 3 antibody (1H9, 1:1000; Chemical on) and anti-actin (clone AC-74, 1:5000; Sigma) Immunoblotting was performed using the densitometric analysis of aggregated mutant ataxin- Quantification of 3 was based on β-actin protein.

[0069] In one embodiment, complete rostrocaudal sampling of the striatum (12 slices / animal) Coronal sections showing 10x CT images were taken using a Zeiss Axio Imager Z2 microscope at 20x magnification. The striatal analysis area was clearly stained with anti-ubiquitin antibody. As revealed by the results, the entire region of the ubiquitin inclusion was included. The area of ​​these inclusion bodies was determined using an automated image analysis software package (Image J software). Counting was performed using a 3D scanner (Ware, USA).

[0070] In one embodiment, 12 stained sections (200 μm apart) were taken per animal. The sections (25 μm thick, spaced apart) were digitized to obtain a complete rostrocaudal sampling of the striatum. The extent of DARPP-32 reduction in the striatum was analyzed by performing To calculate the RPP-32 reduction, the tile function of Zen software (Zeiss) was used. Sections were imaged using the CT scan. The area of ​​the reduced striatum was estimated using the following formula: Volume = d(a + a2 + a3 + ...) (where d is the distance between successive sections (200 μm ), and a1, a2, and a3 are the areas in which DARPP-32 was decreased in each serial section. product. ).

[0071] In one embodiment, a quantitative analysis of the number of condensed pyknosis nuclei in the striatum is performed. Three stained sections (close to the needle track) were taken per animal. Quantification was performed by analyzing the 3D images of the 3D images at 200 μm intervals. This was done manually using Hotoshop software.

[0072] In one embodiment, polyQ69 transgenic MJD mice were also used. This model is a novel siRNA-based recombinant siRNA that contains an N-terminal 69 polyglutamine tract under the control of the L7 promoter. A truncated form of human ataxin-3 is specifically expressed in cerebellar Purkinje cells. The mutant protein displays a hemagglutinin (HA) epitope at the amino terminus. The transgene was inserted downstream of the CAG repeat expansion (rs12895357) and It contains a previously identified SNP and, as a result, shows complementation with miR-ATXN3. Transgenic mice express mutant atakine in the Purkinje cell layer and deep cerebellar nuclei. The transgenic mice were characterized by accumulation of syn-3 and marked cerebellar atrophy after birth. Beginning at day 21 (P21), they display an ataxic phenotype.

[0073] In one embodiment, the colony of transgenic mice (C57BL / 6 background) The Ce was then isolated by backcrossing heterozygous males to C57BL / 6 females. ntre for Neuroscience and Cell Biology o f The animals were maintained in the animal housing facility at Coimbra (CNC). The animals were kept under 12-h light The mice were housed in a temperature-controlled room where a 12-h light / 12-h dark cycle was maintained. Food and water Genotyping was performed by PCR at 4 weeks of age.

[0074] In one embodiment, the experiment is conducted at the European Commission on the Care and Use of Laboratory Animals. a Community Council Directive(86 / 609 / EE C) The researchers had sufficient training (FELASA certified course) The Portuguese authorities (Direccao Geral de Veterinar ia) to conduct the experiment.

[0075] In one embodiment, the experimental design was carried out as follows: P1) was transfected with AAV9 encoding miR-ATXN3 (n=8) and miR-control l (n = 11) (Fig. 9A), 19 female heterozygotes were injected with AAV9 encoding MJD mice were used.

[0076] In one embodiment, the control and treated MJD mice are then The animals were evaluated based on behavioral performance and neuropathological changes. A battery of behavioral tests was , 35, 55 and 85 days. Mice were sacrificed on postnatal day 95 (P95). Then, brain pathology analysis was performed.

[0077] In one embodiment, AAV9 neonatal injections were performed as follows: intravenous injections were performed as follows: , was performed into the facial vein of newborn MJD mice and wild-type littermates (P1). In the protocol, the neonates were first anesthetized with ice for approximately 1 minute. Then, the total is 3.5 × 10 11 vg of AAV9 vector in a total volume of 50 μL. A 30-gauge syringe (Hamilton, Reno, NV, USA) was used to The injection was performed into the facial vein. Correct injection was confirmed by noting the blanching of the vein. This was confirmed by

[0078] In one embodiment, behavioral testing was performed as follows: MJD transgenic Mice were housed in a temperature-controlled, quiet, dark, uniform room at 35, 55 and 85 days of age. After 1 h of acclimation indoors, a battery of behavioral tests was performed.

[0079] In one embodiment, by measuring the average latency (seconds) required to fall To evaluate motor coordination and balance in MJD mice, we used the rotarod apparatus (Letic a Scientific Instruments, Panlab) was used. The fixed speed rotarod adopts a constant speed of 5 rpm, and the fixed speed rotarod adopts a speed range of 4 rpm to 40 rpm. The study was carried out on an accelerating rotarod and a rotarod with gradually increasing speed, both of which showed a maximum At each time point (days 35, 55, and 85), the study was A total of four trials per day were conducted on three consecutive days. The mice were given a rest period of at least 20 min. For statistical analysis, the number of falls at each time point was calculated. The mean latency to respond to the test was calculated considering all consecutive days and consecutive trials.

[0080] In one embodiment, rAAV9-m is administered to assess potential toxicity of the treatment. iR-ATXN3 (n=5) and rAAV9-miR-control (n=5) IV injection A group of wild-type mice receiving injections was also subjected to the rotarod test. In this case, the test was performed on the first day. A total of four trials were performed per day, with the final time point (day 85) being the only time point on two consecutive days. For statistical analysis, the mean latency to fall was calculated considering the second day. It was calculated.

[0081] In one embodiment, limb coordination in MJD mice was also assessed using a glass tank (70 cm long, 18.5 cm wide). The swimmers were assessed based on their swimming performance in a swimming pool (12.5 cm wide with a 19.5 cm high wall). The board has a visible platform at its end, the height of which is 8.5 cm. The tank was filled with water up to 100 mm. The mouse was then placed at one end of the tank and placed at the opposite end. At each time point, the animals were prompted to swim to a certain escape platform. Four trials were performed, with two complete swims and at least a 20-minute break between trials. The performance of these animals was measured by the time required to swim the total distance and to climb the platform using all four limbs. Statistical analysis was based on the mean scores of trials 2, 3, and 4. Ta.

[0082] In one embodiment, motor coordination and balance of MJD mice are assessed by passing a series of elevated beams. The test was based on an assessment of the ability of the device to support a long wooden beam with a pad attached at each end. The mice were placed horizontally, 20 cm above a fixed surface. Two consecutive trials were performed on each beam, proceeding from the easiest to the most difficult. Above were carried out: i) 18mm wide square beam, ii) 9mm wide square beam and ii i) Circular beams with a diameter of 9 mm. In all of these beams, mice were placed in a safe enclosed area. You have to cross 40cm to reach the safety platform. The latency to cross the beam and motor performance were evaluated according to pre-defined criteria. The responses were recorded and scored according to a value scale.

[0083] In one embodiment, footprints of MJD mice were collected to compare various gait parameters. The patterns were analyzed. The front and hind legs were painted with non-toxic red and blue paint, respectively. After coating, the animals were placed on a narrow walkway covered with paper, 50 cm long and 10 cm wide. The subjects were asked to walk in a straight line and enter the box. Five consecutive steps on the side of the run, preferably in the middle of the run, are selected for analysis. Stride length values, corresponding to the distance between successive left and right forelimbs and between hindlimbs, were measured. The hind stride width and the front stride width were measured by measuring the distance between the right and left hind legs and the front legs, respectively. To assess the uniformity of step alternation, overlap was determined by At each time point, the distance between the hind paw and the same side was measured. The average values ​​obtained from steps 1 and 2 were used for statistical analysis.

[0084] In one embodiment, the Institute for Nuclear Science ces Applied to Health (ICNAS), University In Coimbra, the volume coil for excitation (inner diameter / outer diameter are 86mm / 112mm) and a quadrature mouse surface for signal detection. Scoil (Bruker Biospin, Ettlingen, Germany) The standard Bruker cross coil configuration used allows for a 9.4T magnetic resonance small animal scanner. In vivo images were acquired using a 1H scanner (BioSpec94 / 20). -MRS was performed.

[0085] In one embodiment, tissue preparations are performed after an overdose of pentobarbital, and mice are , and then perfused intracardially with cold PBS 1X, followed by 4% cold paraformaldehyde (PFA The brains were then removed and fixed in 4% paraformaldehyde for 24 hours at 4°C. Post-fix in aldehyde and 25% sucrose / PBS for 48 h at 4°C. The mixture was cryoprotected by incubation in

[0086] In one embodiment, each animal is cultured in a cryostat (LEICA) at -20°C. Ninety-six 30 μm sagittal sections were cut using a 30-mm microscope (CM3050S, Germany). The entire brain hemisphere was then cut into sagittal sections, which were then washed with 0.05% sodium azide. Two 48-well plates were prepared as free-floating sections in 1X PBS supplemented with thorium. The wells were stored at 4°C in well plates.

[0087] In one embodiment, the immunohistochemistry protocol was performed as previously described (Alves et al., 2010). For each animal, eight sagittal sections with a cross distance of 240 μm were selected. I chose.

[0088] In one embodiment, the procedure involves the use of 0.1% phenylhydrazine (Merck, USA). Endogenous peptides were isolated by incubating sections in PBS1X containing IgG for 30 min at 37°C. The procedure started with peroxidase inhibition. Then, tissue blocking and permeabilization were performed with PBMC. 0.1% Triton X-100 10% NGS (normal goat serum) prepared in S1X , Gibco) for 1 hour at room temperature. The primary antibody rabbit anti-GFP (Invitrogen) was pre-prepared on the blocking solution and then incubated for 3 h. The sections were incubated with 5% CO2 at 4°C overnight. After three washes, the brain sections were 100 μg / mL of anti-rabbit biotinylated secondary antibody (Ve The plates were then incubated at room temperature for 2 hours in a 30-well plate (Vol. 10, No. 10, 150-ml) at room temperature. Free-floating sections were rinsed and stained with Vectastain ABC kit (Vectastain ABC kit). The mixture was treated with avidin / biotin ELISA kit (Davidson Laboratories, NY) for 30 minutes at room temperature. The formation of the oxidized peroxidase complex was induced. The sections were then treated with peroxidase substrate:3 ,3'-Diaminobenzidine tetrahydrochloride (DAB Substrate Kit t, Vector Laboratories) and incubated with After optimal staining was achieved, wash the sections in PBS1X. The reaction was stopped by incubation at 4°C for 10 min at 20°C for 24 h. The brain sections were then transferred to gelatin-coated slides. The slides were mounted and dehydrated in an ascending ethanol series (75%, 95% and 100%). Then, the sections were cleared with xylene, and finally, the sections were mounted in Eukitt mounting medium. ) (Sigma-Aldrich) was used to attach coverslips.

[0089] In one embodiment, images of sagittal brain sections subjected to GFP immunohistochemistry were obtained from Zeiss Images were acquired on an Axio Imager Z2 microscope. Whole brain images were acquired on an EC Plan-Neo microscope. Images of specific areas were acquired using a fluar 5× / 0.16 objective lens. Images were obtained using a lan-Apochromat 20× / 0.8 objective.

[0090] In one embodiment, immunofluorescence was also performed. Eight sagittal sections at 0 μm were selected. Briefly, the protocol consisted of blocking and permeabilization. The experiment started with a perfusion step, in which the free-floating sections were 0.1% Tris in PBS1X supplemented with 0% NGS (normal goat serum, Gibco) The brain slices were then placed in a blocking solution and incubated at room temperature for 1 hour. The mixture was dissolved and diluted in PBS (10% NGS, 0.1% Triton X-100). Incubated overnight at 4°C with the following primary antibodies: mouse anti-HA (1:1000 , Invivo Gen) and rabbit anti-GFP (1:1000, Invitrogen). After three washing steps in PBS1X, the free-floating sections were incubated at 4°C for 1 h in the appropriate dilution: Place chamber in fluorophore-conjugated secondary antibody prepared in blocking solution to ensure isolation. The antibodies were incubated at room temperature for 2 hours. Anti-mouse and anti-rabbit antibodies were used, respectively, for Alexa Flu. or594 and 488 (1:200, Life Technologies After three rinsing steps in PBS1X, DAPI (4',6-diamidino-2 Nuclear staining was performed using 1-phenylindole. Brain sections were then washed and gelatinized. Mount onto tin-coated microscope slides and finally, fill with Dako fluorescent mounting medium ( S3023) and a coverslip was attached.

[0091] In one embodiment, eight sagittal sections with a crossing distance of 240 μm are used per animal. Selected brain sections were then stained with cresyl violet. The slides were pre-mounted and dried at room temperature. After washing in water, Sections were dehydrated (using 96% and 100% ethanol) and then washed (using xylene substitutes). The cells were then delipidated (using 75% ethanol and water) and rehydrated. To stain the Nissl substances present in the smear, slides were immersed in cresyl violet for 5 min. Finally, the sections were washed in water, differentiated in 70% ethanol, and then in 96% and 1% ethanol. The sections were dehydrated by passing them through a 100% ethanol solution. After a clearing step in xylene, Sections were mounted using Eukitt (Sigma-Aldrich).

[0092] Quantitative analysis by immunofluorescence After GFP and HA immunofluorescence, select specific sagittal sections to obtain images of the entire cerebellum. Serial z-stack images (interval = 0.9 μm) were taken using a confocal microscope (Zeiss Ce By using the Il Observer Spinning Disk Microscope Images were captured using solid-state laser lines (561 nm or 488 nm) for excitation. Acquired using a Plan-Apochromat 20× / 0.8 objective It was done.

[0093] In one embodiment, quantification of the mean GFP fluorescence intensity and the integrated GFP fluorescence intensity is performed on the treated The results were obtained from animals that had been treated with 0.48 mm, 0.72 mm, and 0.96 mm lateral to the midline. Sagittal Plane: Sagittal diagrams 105 and 107 in (Franklin and Paxinos) The study was performed on three specific sagittal sections (cut at 109 and 110). The whole cerebellum was then imaged using confocal microscopy. Maximum intensity projections were obtained for each section using 2012 software.

[0094] In one embodiment, the mean GFP fluorescence intensity is measured to identify viral load in specific cerebellar lobules. The transduction level was quantified. The mean GFP fluorescence intensity was calculated for each section. The results were determined by Zen software and calculated after background subtraction. Average values ​​correspond to the mean intensity considering three analyzed sections per animal.

[0095] In one embodiment, the total viral transduction levels in different animals are To compare the effect of cerebellar lobules, we measured the integrated GFP fluorescence intensity of the cerebellar lobules as a group. In this case, the average GFP fluorescence intensity was measured including all cerebellar lobules, and this value was multiplied by The final value was calculated from three analyzed cuts per animal. Strips were considered and correspond to the average integrated intensity.

[0096] In one embodiment, quantitative analysis of hemagglutinin (HA) tagged aggregates is performed as follows: Three specific sections were selected per animal, the 10th, 9th and 10th The number of aggregates in the six lobules was quantified (Franklin and Paxinos ) according to the 9th and 10th lobules, the midlines are 0.48 mm, 0.72 mm, and 0.96 mm lateral sagittal plane; for the 6th lobule, 0.72 mm relative to the midline, 0.96 mm and 1.68 mm lateral sagittal plane).

[0097] Images were acquired using a confocal microscope as previously described. Zen Black The mean intensity projection was obtained for each section using the 2012 software. The number of aggregates in the leaves was quantified manually and then the value was determined with Zen software. Final values ​​were normalized to the area of ​​each lobule selected. Average number of aggregates / mm2 in a piece 2 This analysis includes both the treatment and control groups. was included.

[0098] In one embodiment, quantification of molecular layer thickness was performed as follows: per animal Three specific sections were selected and stained with cresyl violet, and the 10th, 9th and 6th sections were The molecular layer thickness in the leaflets was quantified (Franklin and Paxinos) According to the above, the 9th and 10th lobules were 0.48 mm, 0.72 mm, and 0. .96 mm lateral sagittal plane; for the 6th lobule, 0.72 mm, 0.96 mm relative to the midline m and 1.68 mm lateral sagittal plane).

[0099] In one embodiment, images of the entire cerebellum were taken using a Plan-Apochromat 20x / Obtained on a Zeiss Axio Imager Z2 microscope with a 0.8 objective lens, Zen Analysis was performed by Blue software.

[0100] In one embodiment, each section is subjected to a predetermined specific region. Triplicate measurements were used to calculate molecular layer thickness in the 10th, 9th, and 6th leaflets separately. Final values ​​are the average for each lobule, examining three selected sections per animal. This corresponds to the molecular layer thickness. Both treatment and control groups were included in the analysis.

[0101] In one embodiment, Prism GraphPad software is used to perform statistical analysis. Data are presented as mean ± standard error of the mean (SEM) and external Values ​​were excluded according to the Grubb test (α = 0.05). A paired t-test was performed to compare the control and treatment groups, whereas a one-way ANOVA test The mean correlation coefficient was used for multiple comparisons. Correlations between parameters were calculated using the Pearson correlation coefficient. The significance was determined according to the following criteria: p>0.05= not significant (ns); * p<0.05, ** p<0.01, *** p<0.001 and * *** p<0.0001.

[0102] The present disclosure provides a method for increasing levels of human mutant ataxin-3 protein while maintaining levels of the non-mutated form. The present invention relates to SNP-targeting dsRNA that can specifically target and reduce the level of SNPs.

[0103] This disclosure provides evidence that 70% of MJD patients worldwide have abnormal CAG repeat expansions. The expanded CAG tract in exon 10 of the ataxin-3 gene has been reported Precisely targeting the C nucleotide of the SNP (rs12895357) located at the 3' end In particular, the present invention relates to a dsRNA sequence (SEQ ID NO:2) that is designed to bind to the .alpha.-aminobutyric acid of

[0104] In one embodiment, SEQ ID NO:2 is incorporated into the miR-155 scaffold to produce a human In parallel, we have produced engineered microRNAs that can silence any mammalian mRNA. A non-singling control sequence (SEQ ID NO: 25) was also used and was incorporated into the miR-155 scaffold. Both artificial miRNAs were transduced into reporter genes under the control of the U6 promoter. The GFP-binding domain was cloned into the self-complementary AAV2 backbone together with EGFP as a target vector (m iR-control and miR-ATXN3) (Figure 2A).

[0105] To confirm the silencing ability and specificity of this novel silencing sequence, The R-ATXN3 plasmid i) detects human serotype 1 with a C nucleotide at rs12895357; ii) ataxin-3 with a G nucleotide at rs12895357; A lentiviral vector stably expressing human wild-type ataxin-3 (27Q) carrying the In mouse neural crest-derived cell line (Neuro2a) pre-infected with The miR-control plasmid was used as a negative control. It has been reported in the art that silencing human mutant ataxin-3 (SEQ ID NO: 26) A lentiviral plasmid encoding sh-ATXN3, known to be effective in treating glaucoma, was used as a positive control. was used (Figure 2A).

[0106] Quantitative reverse transcription PCR (qPCR) results showed that the miR-ATXN3 plasmid Transfection of the mutants resulted in mutations similar to those that occur in the presence of sh-ATXN3. A 42.03% ± 6.26% decrease in ataxin-3 mRNA levels occurred (Figure 2B ). However, in stark contrast to sh-ATXN3, the mut-ATXN3 plasmid No change in wild-type ataxin-3 mRNA levels was detected after transfection with (FIG. 2C), which indicates that SEQ ID NO:2 is the C Precisely targeted nucleotide silencing of human ataxin-3 allows allele-specific silencing Similar results were obtained with SEQ ID NO:3, as shown in FIG. This was obtained when an artificial miRNA-155 construct encoding

[0107] In relation to protein levels, miR-ATXN3 was as potent as sh-ATXN3. However, it was effective in reducing mutant ataxin-3 protein levels (Fig. 2D). miR-ATXN: 50.66±8.34% vs. sh-ATXN3: 55.65±6 miR-ATXN3 was much more selective. No changes in wild-type protein levels were detected following plasmid transfection. However, sh-ATXN3 suppressed the expression of the wild-type morphology in Neuro2a cells. It induced a significant decrease in human wild-type ataxin-3 mRNA levels (Fig. 2E).

[0108] Generally, this makes the miR-based strategies disclosed herein a first in the art. Mutations of previously reported sequences retained their ataxin-3 silencing ability but were much more potent than previously reported sequences. It has been suggested that the effect of miR-ATXN3 is selective. The researchers were able to distinguish between the wild-type and ataxin-3 transcripts, thereby preserving normal ataxin-3 function. This means that it will be possible to adapt this therapeutic approach to human patients. In this case, this is a significant advantage.

[0109] Furthermore, no changes in the levels of endogenous mouse ataxin-3 mRNA were detected ( Fig. 4), which indicates that the silencing effect is specific to human ataxin-3 mRNA. It proved to be true.

[0110] To investigate the therapeutic potential of SEQ ID NO:2 in vivo, miR-ATXN3 A The AAV plasmid and miR-control AAV plasmid were transformed into rAAV9 capsid. AAV vectors packaged in IgG4- and IgG5-specific IgG4-specific vectors have been shown to be effective for efficient neuronal transduction, long-term Given the favorable transgene expression and safety profile, In particular, AAV serotype 9 (AAV9) is considered to be a promising platform for Ability to bypass the BBB in live rodents, cats, non-human primates, and humans This allows for intravenous (IV) injection.

[0111] miR-ATXN3 was identified in two different mouse models of MJD: In a virion-based mouse model and a transgenic mouse model of MJD, Both intraparenchymal and intravenous administration were tested.

[0112] First, we demonstrated that lentivirus (L) induces MJD when administered intracranially (IC). V) We evaluated the functionality and efficacy of miR-ATXN3 in a mouse model based on the The mouse model allows rapid testing of therapeutic approaches and is induced by mutant ataxin-3 expression. This allows for accurate quantitative analysis of the neuropathological damage (Alves et al., 2008b) that occurs during brain injury. Thus, the striatum of 13 10-week-old mice contained a human mutant 72 CAG repeats. A lentiviral vector encoding ataxin-3 (LV) (LV-Atx3-MUT ) and a rAAV9 vector encoding miR-ATXN3 for the right hemisphere and m rAAV9 vectors encoding iR-control were co-injected bilaterally (Figure 5 A).

[0113] Five weeks after injection, five mice were sacrificed and analyzed for the presence of mutant ataxin-3 (by qPCR). Expression levels of mRNA and aggregated mutant ataxia (by Western blotting) Assessment of EGFR-3 protein levels was performed by perfusing eight mice and immunohistochemical analysis (EGFR-3). FP, anti-ubiquitin; DARPP-32, cresyl violet).

[0114] As shown in Figure 5B, fluorescence microscopy confirmed that intracranial administration of AAV9 vectors was effective in both hemispheres as evidenced by strong expression of the reporter gene EGFP. It has been shown to be effective.

[0115] qPCR (Figure 5C) and Western blotting (Figure 5D) confirmed the expression of mir-AT Expression of XN3 significantly increased striatal mRNA levels of mutant ataxin-3 compared to control left hemisphere. A 63.75±2.25% decrease in the expression of mutated ataxin 3 and 37.64±4% in the aggregated form of mutant ataxin 3. It was observed that they induced a 0.52% decrease in β-lactamase activity, respectively.

[0116] The presence of neuronal intranuclear inclusions containing aggregated ataxin-3 is one of the key features of MJD. Therefore, we next investigated the effect of IC administration on the total number and size of ubiquitin-positive inclusion bodies. We assessed the potential of miR-ATXN3 treatment to reduce cerebral infarction in the left hemisphere compared to the control left hemisphere. The number of aggregates in the hemisphere injected with rAAV9 encoding miR-ATXN3 was Clearance was observed, demonstrating the efficacy of the treatment (FIGS. 5E and 5F).

[0117] We then used DARPP-3 to assess whether this strategy could mediate striatal neuroprotection. Immunohistochemistry was performed against 2, and DARPP-32 was found to be a dopamine receptor signaling This is a regulator of LV-based signaling in MJD (Alves et al., 2008b). It is a sensitive marker for detecting early neurological dysfunction in a mouse model of miR-ATX is also a sensitive marker for neuronal dysfunction, as previously demonstrated. Intracranial administration of N3 reduced DARP-32-depleted lesions compared to miR-control. It was decreased (80.87%) (Figures 5G and 5H).

[0118] Finally, cresyl violet staining was performed to confirm cell injury due to mutant ataxin-3 expression. When assessing toxicity, a clear reduction in hyperchromatic nuclei was observed in the treated right hemisphere. (approximately 30%) (Figures 5I and 5J).

[0119] Overall, the above results demonstrate the feasibility of the AAV9-based strategy for the expression of mutant ataxin-3. Leukocyte-specific silencing of mutant ataxin-3 mRNA when injected IC and has been shown to be effective in reducing levels of aggregated mutant proteins. This promotes the clearance of ubiquitin-positive inclusions, preventing cell injury and striatal alterations. Prevented sexual intercourse.

[0120] Next, the inventors investigated whether the developed rAAV9 vector has the ability to translocate the BBB and Transgenic mice with severe MJD were produced when injected intravenously into the P1 PolyQ69 transgenic mice and their transgenic The ability of the mouse model to transduce neurons in wild-type littermates was investigated. This transgenic mouse model expresses a human attenuated 69 glutamine repeat gene. A truncated form of oxime-3 was specifically expressed in cerebellar Purkinje cells (PCs) and induced early onset (P21) leads to a severe pathological phenotype. Furthermore, truncated human ataxin-3 , with the C variant of the rs12895357 SNP, present in 70% of MJD patients Therefore, this MJD transgenic mouse model allows for the evaluation of allele-specific strategies. to.

[0121] In fact, to elicit a therapeutic effect in PolyQ69 transgenic mice, IV injected rAAV9 vectors bypass the BBB and efficiently transduce the brain. As a result, after sacrifice at 95 days of age, MJD transgenic mice An investigation of rAAV9 distribution in the mouse brain was carried out. To this end, AAV-plus The reporter gene, green fluorescent protein (GFP), was detected in the GFP-GFP-GFP vector. Immunohistochemistry of sagittal brain sections was performed using rAAV9-injected transgenic mice. In addition to analyzing sections from M. cerevisiae mice, we also analyzed non-injected M. cerevisiae mice as a negative control. JD mice and rAAV9-injected wild-type (WT) mice to compare rAAV9 distribution ) mice were also used (Figure 6).

[0122] The pattern of GFP expression in transgenic mice receiving rAAV9 IV injection The expression levels of the vector in the cerebellum, brainstem, spinal cord, and cerebellum were very similar in both control and treated groups. Efficiently throughout the brain, including areas commonly affected in MJD, such as the medulla and striatum In particular, the pontine nuclei, which are the primary site of degeneration in MJD, showed a significant Other areas where the transgene was efficiently transduced included the cerebral cortex, The olfactory bulb and hippocampus were included. rAAV9 IV into the tail vein of adult transgenic animals injection also mediated effective transduction of mouse brain. Transgenic animals and The main difference observed between wild-type and cerebellar GFP expression corresponds to the cerebellar GFP expression. D animals show spatially localized transgene expression compared to active transgene expression throughout the cerebellum of WT mice. These findings indicate a localized and weaker GFP signal (Figures 6B and 6C). , previously reported angiogenesis in the cerebellum in this particular transgenic animal model. This can be explained by a defect in

[0123] Expression of human mutant ataxin-3 increases the risk of developing small bowel disease in polyQ69 MJD transgenic mice Given that rAAV9-miR-ATXN3 is restricted to brain PCs, the therapeutic effect of rAAV9-miR-ATXN3 may be small. The brain, in particular, will depend heavily on the vector's ability to transduce this cell subtype. Therefore, the distribution of GFP signals in this region was further examined after immunohistochemical processing. We then conducted a more detailed analysis.

[0124] As shown in Figure 7, GFP expression was evenly distributed throughout the cerebellum. In particular, the 10th lobule of the cerebellum, followed by the deep cerebellar nuclei (DCN, probably in MJD) The transduction is evident in the 9th lobule (the area most affected in the first place) and the 10th lobule. Pre-treated isolated neurons were also detected in the 6th and 7th lobules, but the remaining Detection was reduced in the lobules. Importantly, choroid plexus cells in the fourth ventricle were also The GFP distribution pattern was consistent across the rA This was observed in all transgenic animals that received AV9 IV injection.

[0125] The preferential cerebellar transduction in the 9th and 10th lobules described above suggests that this area This may be due to better vascularization in the brain, or possibly because this region is the fourth brain. This may be due to the location of the choroid plexus in the ventricles. The choroid plexus (CP) is a membrane that contains cerebrospinal fluid (CSF). It is responsible for the formation of a barrier between blood and CSF (the blood-CSF barrier (BCSFB)). ) is composed of a single layer of epithelial cells that constitute the CP. The rAAV9 vectors that reach the CSF can eventually bypass the BCSFB and migrate to the CSF. The 10th lobule is close to the CP and is in the CSF flow path, allowing for rAAV delivery to the PC. 9 arrival occurs preferentially in this cerebellar region.

[0126] Finally, we demonstrated that rAAV9 inhibits the predominantly affected cell population in this mouse model, i.e. We therefore aimed to determine whether the IL-16 / IL-16 gene could target PCs expressing mutant ataxin-3. Both GFP and hemagglutinin (HA) were co-immunofluorescently labeled. Finally, HA signals were detected in the PC cell layer, where mutant ataxin-3 was distributed in the form of aggregates throughout the cell body with diffuse staining (Fig. 8). Furthermore, mutant ataxin-3 aggregates were also expressed in the axon terminals of PCs and in the deep cerebellar nuclei (DCN). Comparing the distribution of GFP and HA signals, the mutant ataxia Colocalization was observed in the two major areas of phenotypic acylcholine-3 accumulation, the PC layer and the DCN (Fig. 8). This latter finding suggests that AAV vectors can be retrogradely transported from DCN to PCs and contribute to their therapeutic effect. Furthermore, DCN rAAV9 transduction has been shown to be able to mediate the expression of IL-1 in this region. This is also beneficial in MJD patients, as these regions are severely affected in the context of the disease. This pattern was consistent across all rAAV9-I groups, including the control and treatment groups. The results were similar in V-injected mice.

[0127] In the present disclosure, rAAV9-miR ATXN3 injection ameliorates MJD-associated behavioral disorders. The most typical MJD symptom is impaired motor coordination and balance. PolyQ69 transgenic mice have been shown to have these characteristics: These cases mimic the symptoms of glaucoma and display a highly severe ataxic phenotype with early onset (P21). The behavioral impairment in mice is due to the impairment of PCs, a subpopulation of neurons that play an important role in motor coordination and learning. In fact, the PC is fragile and can be easily damaged, causing motor control problems. Ability is impaired.

[0128] To investigate the effects of miR-ATXN3 treatment on the behavior of transgenic mice, To this end, both treated and control animals, i.e., miR-ATXN3 or miR- Animals received a P1 intravenous injection of rAAV9 vectors encoding control The mice were then tested in one battery at three different ages: 35, 55 and 85 days of age. These tests were performed on the constant speed rotarod and accelerating rotarod, as well as on the rats' hind legs (Figure 9A). The beam walking test was included in the study because it assesses balance and motor coordination. In addition, the swimming test allows for further evaluation of athletic performance and strength. On the other hand, footprint analysis allowed us to evaluate MJD-related gait disorders. Ta.

[0129] Rotarod performance was measured by measuring the speed of the rotarod apparatus in mice, both at constant and accelerating speeds. The mean latency to fall was measured when walking through a pool of water. In the theoretical framework, beneficial effects were found at all time points (Figure 1). 9B and 9C). The most consistent results were obtained at day 85 because of the improved The improvement was statistically significant in both the constant speed rotarod and the accelerating rotarod. (a 1.7-fold and 1.5-fold increase in waiting time before falling, respectively).

[0130] In the swimming test, mice were placed at one end of a glass tank filled with water and allowed to swim across the pool. The time it took each animal to swim the full distance and climb the platform was calculated. The results showed that treated animals performed better at day 55. (Figure 10A).

[0131] In the beam walking test, mice were placed on i) an 18 mm wide square elevated beam; ii) a 9 mm wide square elevated beam; iii) a 9 mm diameter square elevated beam and iv) a 9 mm diameter circular elevated beam. The participants were assessed based on the time it took them to complete the task and their motor coordination. The subjects were scored according to a set of evaluation scales, with higher scores indicating better balance and This analysis showed that the difference between the control and treated groups was 18 mm wide and However, no detection was found for the 9 mm square beam. The results were different for a circular beam with a diameter of 9 mm, which is considered to be the most suitable for the purpose (Figure 10B). A gradual decrease in performance scores over time was observed in the control group, whereas the treated mice The results showed that rAAV9-miR-ATXN3-injected mice retained their ability to cross the callosum. Animals treated with serotonin-dependent agonists showed significantly better beam walking performance at the final time point (mean a 2.2-fold increase in score) (Figure 10B).

[0132] To determine whether treatment can reduce the limb and gait ataxia characteristic of MJD. To determine whether ataxic gait is related to step alternation, we analyzed footprint patterns in both experimental groups. This reflects a decrease in uniformity, i) wider stride length; ii) shorter stride length; and iii) Analysis of the gait patterns of treated animals revealed distinct At various time points, there were some improvements compared to the control group, mainly at days 55 and 60, respectively. A significant decrease in posterior stride width and anterior stride width was observed on the 35th day. At later time points (day 85), a significant decrease in the distance of footprint overlap was detected (Figure 10 C, Fig. D and Fig. E).

[0133] Overall, treated animals showed an overall improvement in motor skills across all behavioral tests. The study showed better performance with significant results in rotarod, swimming, beam walking and footprint analysis. 9 and 10. This indicates that AAV-mediated ataxin-3 silencing This is the first report of significant behavioral improvement after injection, and rAAV9-IV injection was This is the first time that a positive impact on behavior in yQ disorder has been demonstrated.

[0134] Generally, this is likely due to the selection of the SNP-targeting dsRNA of the present disclosure, the selected This suggests a superior therapeutic effect of our strategy due to the serotype and delivery route. do.

[0135] Effects of rAAV9-miR-ATXN3 injection on MJD-associated neuropathological changes One of the major features of MJD is the occurrence of mutations in the atakine kinase pathway that reflect the progression of the disease. In selected mouse models, these aggregates are C, but after starting to form at P40, the number and size of the foci increased significantly over time. Increase the number to.

[0136] Therefore, hemagglutinin (Hb) in sagittal sections from treated and control MJD mice Immunofluorescence directed against lutamine (HA) was performed because this tag is a mutated This is because it is present in the N-terminus of ataxin-3 (Figure 11A). We quantified the number of mutant ataxin-3 aggregates per area in the 1st and 9th lobule. The reason is that these mutant ataxin-3 aggregates have higher levels of transduction rAAV9 treatment in areas with low transduction efficiency The sixth lobule was also analyzed to assess the effect of. miR-ATXN3 treatment reduced aggregation in all three analyzed lobules ( 10, 9, and 6 lobule, respectively, with a 35%, 18%, and 20% decrease; This contributed to the attenuation of neuropathology (Figure 11B).

[0137] Another important feature of MJD patients is the result of neurodegeneration in this region, usually In this particular mouse model, 3 Significant cerebellar atrophy was detected as early as 3 weeks of age. The physiological / morphological changes may affect other cerebellar regions due to the strong interconnections between different cell types. In particular, Q69 transgenic mice show reduced dendritic branching in PCs. is insufficient, which results in a thin molecular layer thickness. Sagittal sections from both experimental groups were stained with cresyl violet to identify the cerebellar layers. Staining was performed (Figure 11C). Molecular layer analysis revealed that miR-ATXN3-treated mice Significantly increased thickness was observed in the 10th and 9th lobules of the mouse ( The incidence rates were 21% and 15%, respectively, and a strong tendency was also observed in the 6th lobule (13% , p=0.0587) (Figure 11D).

[0138] Overall, the results disclosed herein demonstrate the efficacy and safety of rAAV9 IV injection for the prevention and treatment of mutated AAVs. Silencing of xinoxime-3 is an efficient treatment in transgenic MJD mice It is an approach that has been demonstrated to reduce both behavioral and neuropathological disorders. Importantly, these positive effects are already evident at birth, when neurological and vascular defects are eliminated. Obtained in a very severe model with early onset, which could show defects in angiogenesis Therefore, this strategy can be applied to other MJD models that exhibit late-onset and milder phenotypes. When tested, even more significant effects may be expected.

[0139] Initial findings regarding rAAV9 distribution in MJD transgenic mice were reported in the first Although the results suggested a treatment response limited to the cerebellum, a highly generalized effect was observed in the entire cerebellum. The highly efficient transduction in the 10th lobule was detected in the 10th lobule and in the behavior of the mice. Since this lobule is part of the vestibular system and is important for balance, It could be speculated that this could be sufficient to elicit an improvement in the rotarod. However, an overall beneficial effect alone would not necessarily affect the general outcome of the treated mice, particularly Explain that the results are better in tests that examine motor coordination, strength and gait One possible explanation is that transduced nuclei in other lobules may have been involved. Although PC is poor, it is sufficient to induce positive effects in each domain. This may be due to the fact that, for example, rAAV9-positive P C-induced neuroprotection releases neurotrophic factors and inhibits neuroinflammation Alternatively, transduced PCs may express miR-ATXN3 They can also transfer their offspring to neighboring cells. PCs may be mediated by the transfer of potential solo miRs and / or miR- Using extracellular vesicles encapsulating ATXN3, we investigated the signal transduction between rAAV9-negative neurons. A similar mechanism can be used to transduce the DCN. The cells were also able to release the miR-ATXN3 constructs, and these miRs The -ATXN3 construct is then delivered to the prominences of PCs. Therefore, the transduction of CP epithelial cells themselves also contributes to our findings. Therefore, gene therapy targeting CP may be a promising treatment for lysosomal storage disorders. In this case, gene therapy directed at CP has been investigated to produce Therapeutic proteins can be secreted continuously to provide beneficial effects. CP epithelial cells can secrete miRNAs that are packaged in extracellular vesicles. Based on these results, rAAV9-positive CP cells in the fourth ventricle were subsequently silenced in the cerebellum. The miR-ATXN3-containing extracellular vesicles, which exert their anti-inflammatory effects, are transported to the CSF. FIG. 12 shows the rAAV9-miR-ATXN3 Possible mechanisms underlying the therapeutic effect are summarized.

[0140] The therapeutic effect in treated mice depends on the level of rAAV9 cerebellar transduction. It was also determined whether certain animals showed more pronounced behavioral improvements or attenuation of neuropathology. What was shown was that the dose of vector used to transduce PCs was increased. I was able to explain that.

[0141] For this purpose, GFP mean fluorescence was analyzed in the 10th and 9th lobules, as well as In addition, the number of aggregates per area in each region was also analyzed. These two parameters There was an inverse correlation between the 10th and 9th lobes, with higher transduction in the 10th and 9th lobes. It was concluded that the increased levels of IL-1 were accompanied by improved aggregate clearance (Figure 13A). However, the same relationship could not be established in the case of the sixth lobule, which suggests that the vascular endothelial cells in this particular region These results suggest that the beneficial effects in nausea and vomiting may depend on other parameters.

[0142] Furthermore, mice with better cerebellar transduction showed better athletic performance. In this regard, we also determined whether the GFP integration in all cerebellar lobules corresponds to A good relationship was observed between strength and mean performance on the accelerating rotarod (Figure 1 3B).

[0143] Taking all the above into consideration, the results of the study were analyzed with regard to behavioral tests and neuropathological signs of treated animals. We conclude that the variability observed in the transduction of This was discussed in relation to intrafacial administration to neonatal mice, combined with the large injection volume. Due to technical requirements or because some animals receive different vector doses This may be because the virus may have been able to reach the cerebellum. The amount of vesicles also differed, possibly due to differences in angiogenesis or AAV receptor levels. This may vary between animals.

[0144] In summary, a higher vector concentration in the cerebellum corresponds to a stronger therapeutic effect, so r AAV9-miR-ATXN3 injection induces a dose-dependent response. Based on the results of et al., the therapeutic effect is potentially dependent on the injected vector dose, i.e., It can be concluded that the efficacy of the antibody can be maximized by increasing the number of viral particles per animal. can.

[0145] In addition to the proven efficacy, the safety profile of the treatment strategy supports its potential clinical applicability. Recent studies have demonstrated that rAAV9 induces inflammatory responses in the brain following delivery. Induced immune responses and toxicity due to miR-induced off-target silencing The present inventors have not investigated all these parameters in detail. However, the therapeutic strategies disclosed herein demonstrate that this treatment is successful in wild-type animals. To determine whether the animals were tolerable, the constant speed rotarod and accelerating rotarod tests were performed at 85 days. The results were based on the results of the rAAV9-miR-control or rAAV9-miR-control. The rotarod performance of wild-type mice intravenously injected with AV9-miR-ATXN3 was No differences were detected (Figures 14A and 14B). These findings suggest that the therapeutic sequence These results suggest that acetaminophen does not induce any toxic effects on the immune system.

[0146] Finally, animals were subjected to magnetic resonance imaging using a 9.4 Tesla scanner at PN75. The treated MJD translocations were also subjected to magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Both transgenic and control MJD mice, as well as wild-type littermates, Morphological and metabolic changes in the cerebellum were evaluated.

[0147] In the cerebellum, these neurochemicals, namely N-acetylaspartate (N AA), myo-inositol (Ins) and glycerophosphocholine + phosphocholine (tCh o) is highly deregulated in transgenic MJD compared to wild-type mice Interestingly, the levels of these three neurometabolites were significantly increased in the miR-100 mice (Fig. 15A). The MJD profile was improved in MJD mice injected with R-ATAX3, which was comparable to that in control MJD mice. Compared to D mice, the levels of NAA (a neuronal marker) were higher, and This means lower levels of ns and tCho (markers of cell death) (Figure 15B). .

[0148] The neurochemical ratios NAA / Ins and NAA / total choline and NAA / (Ins+ The tCho ratio was also used to evaluate the efficacy of the above treatments. All three ratios The values ​​were significantly higher in treated MJD mice compared to control MJD mice. , demonstrating the efficacy of miR-ATAX3 treatment (Figure 15B).

[0149] Overall, this allows the detection of neurochemical biomarkers, especially NAA, Ins and tCho. These gene vectors can be used as important non-invasive therapeutic biomarkers during preclinical trials. The efficacy of therapies from this study can be monitored and subsequently adapted for human clinical trials. It has been proven that this is the case.

[0150] In conclusion, the present disclosure provides a method for encoding an artificial miRNA based on miR155 comprising the steps of: A single intravenous injection of rAAV9, which transmits the rAAV gene to P1, i) translocates the blood-brain barrier; ii) precisely silence mutant ataxin-3 mRNA; and iii) MJD. Convincing evidence that shrimp can reduce neuropathological changes and motor impairments caused by shrimp. Provide density.

[0151] Furthermore, the present disclosure provides significant behavioral evidence in polyglutamine disorders following intravenous administration of rAAV9. reported improvements in the efficacy and safety of ataxin-3 using a non-invasive system. This constitutes the first MJD therapeutic approach that can induce silencing of

[0152] Whenever used in this specification, the term "comprises" refers to the features described. ,indicates the presence of an integer, step, or component, but not one or more other features, integers, steps, or components. It is not intended to exclude the presence or addition of any component or group thereof.

[0153] Those skilled in the art will appreciate that the described steps are consistent with the principles of the present invention unless otherwise indicated herein. The particular order of the steps is for illustrative purposes only and may be changed without departing from the present disclosure. Therefore, unless otherwise stated, The steps are not ordered as described above, which means that they can be It is understood that the present invention can be practiced in any convenient or desirable order, provided that the steps can be performed in any convenient or desirable order.

[0154] This disclosure should in no way be construed as being limited to the described embodiments. Those skilled in the art can envision numerous possibilities for modifying these described embodiments. The above described embodiments can be combined.

[0155] The following claims further describe certain embodiments of the present disclosure. Sequence Listing: 1) Target sequence and double-stranded RNA sequence targeting the ATXN3 common SNP 1.1 Target sequence and SNP in exon 10 (rs12895357) Each double stranded RNA 1.1.1 Target sequence and ataxin-3 m of rs12895357 (cytosine) Antisense sequences of double-stranded RNA targeting RNA SEQ ID NO: 1: Target sequence of exon 10 (rs12895357) (C): agcagca gcagcgggaccuauca SEQ ID NO: 2: (miR357C.22): 5'-ugauaggucccgcugcug cugc-3'(22nt) SEQ ID NO:3: (miR357C.21): 5'-ugauaggucccgcugcug cug-3'(21nt) SEQ ID NO: 4: (miR357C.19): 5'-ugauaggucccgcugcug c-3'(19nt) SEQ ID NO: 5: (miR357C.20): 5'-ugauaggucccgcugcug cu-3'(20nt) SEQ ID NO: 6: (miR357C.23): 5'-ugauaggucccgcugcug cugcu-3'(23nt) 1.1.2. Target sequence and ataxin-3 at rs12895357 (guanine) Antisense sequences of double-stranded RNA targeting mRNA SEQ ID NO: 7: Target sequence of exon 10 (rs12895357) (G): agcagca gcagggggaccuauca SEQ ID NO:8 (miR357G.22): 5'-ugauaggucccccugcugc ugc-3'(22nt) SEQ ID NO:9 (miR357G.19): 5'-ugauaggucccccugcugc -3'(19nt) SEQ ID NO: 10 (miR357G.20): 5'-ugauaggucccccugcug cu-3'(20nt) SEQ ID NO: 11 (miR357G.21): 5'-ugauaggucccccugcug cug-3'(21nt) SEQ ID NO: 12 (miR357G.23): 5'-ugauaggucccccugcug cugcu-3'(23nt) 1.2 Target sequence of exon 8 (rs1048755) and two targeting SNPs double-stranded RNA 1.2.1 Target sequence and ataxin-3 allele at rs1048755 (adenine) Antisense sequence of double-stranded RNA targeting SEQ ID NO: 13: Target sequence of exon 8 (rs1048755) (A): accuggaa cgaauguuagaagca SEQ ID NO: 14 (miR755A.22): 5'-ugcuucuaacauucguuc cagg-3'(22nt) SEQ ID NO: 15 (miR755A.19): 5'-ugcuucuaacauucguuc c-3'(19nt) SEQ ID NO: 16 (miR755A.20): 5'-ugcuucuaacauucguuc ca-3'(20nt) SEQ ID NO: 17 (miR755A.21): 5'-ugcuucuaacauucguuc cag-3'(21nt) SEQ ID NO: 18 (miR755A.23): 5'-ugcuucuaacauucguuc caggu-3'(23nt) 1.2.2 Target sequence and ataxin-3 allele at rs1048755 (guanine) Antisense sequence of double-stranded RNA targeting SEQ ID NO: 19: Target sequence of exon 8 (rs1048755) (G): accuggaa cgaguguuagaagca SEQ ID NO: 20 (miR755G.22): 5'-ugcuucuaacacucguuc cagg-3'(22nt) SEQ ID NO: 21 (miR755G.19): 5'-ugcuucuaacacucguuc c-3'(19nt) SEQ ID NO: 22 (miR755G.20): 5'-ugcuucuaacacucguuc ca-3'(20nt) SEQ ID NO: 23 (miR755G.21): 5'-ugcuucuaacacucguuc cag-3'(21nt) SEQ ID NO: 24 (miR755G.23): 5'-ugcuucuaacacucguuc caggu-3'(23nt) Other Arrays SEQ ID NO: 25 (miR-control): 5'-caacaagaugaagagca ccaa-3'(21nt) SEQ ID NO: 26 (sh-ATXN3): 5'-gauaggucccgcugcugcu- 3'(19nt) SEQ ID NO: 27 (miR155-5' arm): 5'-cuggaggcuugcugaa ggcuguaugcug-3' SEQ ID NO: 28 (miR loop): 5'-guuuuggccacugacugac-3' (19nt) SEQ ID NO: 29 (miR155-3' arm): 5'-caggacaaggccuguu acuagcacucacauggaacaaauggcc-3'(43nt) References US10072264B2 WO2005105995 Alves, S., Nascimento-Ferreira, I., Auregan, G., Hassig, R., Dufour, N., Brouillet, E., Pedr. oso de Lima,MC,Hantraye,P.,Pereira de Almeida,L.,and Deglon,N.(2008a).Allele-s specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model Machado-Joseph disease.PloS one 3,e3 341 Alves, S., Nascimento-Ferreira, I., Dufour, N. .,Hassig,R.,Auregan,G.,Nobrega,C.,Brouil let,E.,Hantraye,P.,Pedroso de Lima,MC, Deglon,N.,et al.(2010).Silencing ataxin- 3 mitigates degeneration in a rat model of Machado-Joseph disease: no role for w ild-type ataxin-3? Human molecular geneti cs 19,2380-2394 Nobrega,C.,Birth-Ferreira,I.,Onofre ,I.,Albuquerque,D.,Hirai,H.,Deglon,N.,an d de Almeida,LP(2013).Silencing mutant ataxin-3 recovers motor and neu deficits pathology in Machado-Joseph disease tr ansgenic mice.PloS a 8.e52396 Alves, S., Regulier, E., Nascimento-Ferreira ,I.,Hassig,R.,Dufour,N.,Koeppen,A.,Carva lho,A.L.,Simoes,S.,de Lima,M.C.,Brouille t,E.,et al.(2008b).Striatal and nigral p athology in a lentiviral rat model of Ma chado-Joseph disease.Human molecular gen etics 17,2071-2083 K.H.Chung,C.C.Hart,S.Al-Bassam et al.,“P olycistronic RNA polymerase II expressio n vectors for RNA interference based on BIC / miR-155,”Nucleic Acids Research,vol. 34,no.7,article e53,2006

Claims

1. Antisense base-paired with a substantially complementary sense ribonucleotide sequence Ribonucleotide sequence A ribonucleic acid (RNA) molecule comprising: Each ribonucleotide of the antisense RNA sequence corresponds to a primer set of the mutant human ataxin-3 gene. A corresponding single nucleotide polymorphism that is in linkage disequilibrium with the Kadow-Joseph disease (MJD) allele is complementary to a ribonucleotide of a mutant human ataxin-3 Antisense RNA complementary to a single nucleotide polymorphism in mutant human ataxin-3 mRNA The ribonucleotides of the sequence are 10 ribonucleotides apart, A ribonucleic acid (RNA) molecule.

2. The base-paired sense ribonucleotide sequence is an antisense ribonucleotide sequence. The RNA molecule of claim 1, which is not perfectly complementary to

3. The antisense ribonucleotide sequence is at least The RNA molecule of claim 1, wherein said RNA molecule is at least 90% complementary to said first sequence.

4. The antisense ribonucleotide sequence is complementary to SEQ ID NO: 1 or 13.

2. The RNA molecule of claim 1.

5. The antisense ribonucleotide sequence is selected from SEQ ID NOs: 2, 3, 4, 5, 6, 14, 15, 1 The RNA molecule of claim 1, wherein the amino acid sequence is 6, 17 or 18.

6. The RNA molecule according to claim 1, wherein the antisense ribonucleotide sequence is SEQ ID NO:

2. child.

7. The ribonucleotides at the 5' and 3' ends of the antisense RNA sequence are at least The RNA molecule of claim 1 , wherein the two sequences are separated by 17 polynucleotides.

8. The ribonucleotides at the 5' and 3' ends of the antisense RNA sequence are ribonucleoside. The RNA molecule of claim 1, wherein the RNA molecule is separated by 17 to 21 base pairs.

9. The RNA molecule of claim 1 , wherein the RNA molecule is a single RNA molecule.

10. The RNA molecule of claim 7, wherein the RNA molecule is a miRNA.

11. A single nucleotide polymorphism (SNP) at rs1048755 (exon rs1048755) in the human ataxin-3 gene 8) allele or rs12895357 (exon 10) allele. The RNA molecule according to claim 1.

12. The RNA molecule is a mutant human ataxin-3 allele that is not a wild-type human ataxin-3 allele. Selectively silencing expression of the MJD allele of the gene , an RNA molecule according to claim 1 which is effective from a therapeutic point of view.

13. The RNA of claim 1, comprising a miRNA scaffold derived from miR-155. A molecule, wherein the antisense ribonucleotide is SEQ ID NO:2 and the sense ribonucleotide is The nucleic acid sequence of claim 1.

14. An adeno-associated vector comprising an isolated DNA sequence operably linked to a promoter. A virus vector, comprising an isolated DNA sequence encoding the RNA molecule of claim 1. An adeno-associated virus vector that delivers

15. Linkage disequilibrium relationship between mutant human ataxin-3 and MJD alleles Selectively silencing the expression of mutant human ataxin-3 alleles carrying a single nucleotide polymorphism A method for infecting an adeno-associated virus vector according to claim 12, administering to a subject in need thereof.

16. A single nucleotide polymorphism (SNP) identified rs1048755 (exon) in the mutant human ataxin-3 gene.

13. The method according to claim 12, further comprising the step of: The method described above.

17. Adeno-associated virus vectors can be administered systemically, intravenously, intratumorally, or intraperitoneally. Orally, intranasally, intraperitoneally, intramuscularly, intraspinally, intracerebrally Intraventricular, intracisternal, intrathecal, intraocular, intracardiac The drug may be administered intradermally or subcutaneously, preferably intravenously or intracisternally.

15. The method of claim 14, wherein the compound is administered intrathecally or in situ into the brain. 。

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