HK40043583A

HK40043583A - Oligonucleotides for modulating atxn2 expression

Info

Publication number: HK40043583A
Application number: HK62021033101.8A
Authority: HK
Inventors: 海迪‧赖依‧胡德布施; 吕克‧彼泽森; 索伦‧韦斯特加德‧拉斯穆森; 彼得‧哈哲多恩; 梅特‧拉迪福吉德
Original assignee: 豪夫迈·罗氏有限公司
Priority date: 2018-06-05
Filing date: 2019-06-03
Publication date: 2021-09-17

Description

Oligonucleotides for modulating expression of ATXN2

Technical Field

The present invention relates to oligonucleotides (oligomers) complementary to a nucleic acid encoding spinocerebellar ataxia protein 2(ataxin 2) (ATXN2), which result in reduced expression of ATXN 2. Reduction of ATXN2 expression is beneficial for many medical disorders, such as neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), alzheimer's frontotemporal dementia (FTD), parkinson's syndrome, and conditions with TDP-43 proteinopathies.

Background

The enlarged glutamine repeat of the ataxin 2(ATXN2) protein, resulting from 31 or more CAG repeats in the ATXN2 gene, leads to spinocerebellar ataxia 2 (SCA2), a rare neurodegenerative disorder. Furthermore, the expanded CAG repeats are a genetic risk factor for Amyotrophic Lateral Sclerosis (ALS) through RNA-dependent interaction with TAR DNA binding protein 43 (TDP-43). Other neurodegenerative diseases associated with TDP-43 proteinopathies are, for example, Alzheimer's frontotemporal dementia (FTD) and Parkinson's syndrome. Recently, TDP-43 transgenic mice (TDP-43) as ALS-related mouse model^T/Tg) Cross breeding with Atxn2 negative mice resulted in TDP-43^T/TgAtxn2^-/Significant extension of mouse LifeThe motor function of the mice was long and improved (Becker et al 2017 Nature 544: 367-371.) in the same article it was shown that TDP-43 treated with antisense oligonucleotides targeting ATXN2^T/TgThe survival of the mice is prolonged and the motor capacity is improved.

Antisense oligonucleotides targeting ATXN2 are also described in US 2017/175113, WO 2015/143246 and WO 2017/117496, where WO 2017/117496 is particularly concerned with the treatment of ALS. Scoles et al 2017 Nature 544:362 evaluated an antisense oligonucleotide for its ability to reduce ATXN2 in the cerebellum and showed its localization to Purkinje cells, suggesting a potential for treatment of SCA 2.

Technical purpose

The present invention provides antisense oligonucleotides that modulate ATXN2, both in vivo and in vitro. The present invention identifies a specific target sequence present in intron 9 of the human ATXN2 precursor mRNA that can be targeted by antisense oligonucleotides to give effective ATXN2 inhibition. In particular, for reducing ATXN2, SEQ ID NO: the target position 83118-83146 of 1 is advantageous. The invention also provides effective antisense oligonucleotide sequences and compounds capable of inhibiting ATXN2, and their use in treating diseases or disorders such as neurodegenerative diseases including spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia alzheimer's (FTD), parkinson's syndrome, and conditions with TDP-43 proteinopathies.

Disclosure of Invention

The present invention relates to oligonucleotides targeting nucleic acids encoding ATXN2, capable of modulating ATXN2 expression, and the use of such oligonucleotides in the treatment or prevention of diseases associated with the function of ATXN 2.

Accordingly, the present invention provides an oligonucleotide comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary, such as fully complementary, to a human ATXN2 target nucleic acid.

The present invention provides oligonucleotides comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that are at least 90% complementary, such as fully complementary, to an intron region of a human ATXN2 precursor mRNA target nucleic acid.

The present invention provides oligonucleotides comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that are at least 90% complementary, such as fully complementary, to an intron 9 region of a human ATXN2 precursor mRNA target nucleic acid.

The present invention provides oligonucleotides comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length, which have at least 90% complementarity, such as being fully complementary, with nucleotides 81429-83313 of SEQ ID NO 1.

The invention provides an oligonucleotide comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length, which has at least 90% complementarity, such as being fully complementary, with a nucleotide of SEQ ID NO 6.

The oligonucleotide may be an antisense oligonucleotide, advantageously with a gapmer design. Advantageously, the oligonucleotide is capable of inhibiting the expression of ATXN2 by cleaving the target nucleic acid, for example, by recruiting RNaseH 1.

In another aspect, the invention provides a pharmaceutical composition comprising an oligonucleotide of the invention and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.

In another aspect, the invention provides in vivo or in vitro methods of modulating the expression of ATXN2 in target cells expressing ATXN2 by administering to said cells an effective amount of an oligonucleotide or composition of the invention.

In another aspect, the invention provides a method for treating or preventing a disease, disorder or dysfunction associated with the in vivo activity of ATXN2, comprising administering to a subject suffering from or susceptible to such a disease, disorder or dysfunction a therapeutically or prophylactically effective amount of an oligonucleotide of the invention.

In another aspect, the oligonucleotides or compositions of the invention are used to treat or prevent a neurodegenerative disease, such as a neurodegenerative disease selected from the group consisting of spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), alzheimer's frontotemporal dementia (FTD), parkinson's syndrome, and a disorder with TDP-43 proteinopathy.

In another aspect, the oligonucleotides or compositions of the invention are used to treat or prevent spinocerebellar ataxia type 2 (SCA2) or Amyotrophic Lateral Sclerosis (ALS).

In some embodiments, the antisense oligonucleotide is in the form of a pharmaceutically acceptable salt.

In some embodiments, the antisense oligonucleotide is in the form of a pharmaceutically acceptable sodium salt.

In some embodiments, the antisense oligonucleotide is in the form of a pharmaceutically acceptable potassium salt.

The invention provides a conjugate comprising an antisense oligonucleotide according to the invention and at least one conjugate moiety covalently attached to said oligonucleotide. In other words, in some embodiments, the antisense oligonucleotides of the invention are in the form of conjugated oligonucleotides. In some embodiments, the oligonucleotide is not conjugated.

The invention provides pharmaceutical compositions comprising an antisense oligonucleotide or conjugate of the invention and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

In some embodiments, the composition comprises a pharmaceutically acceptable diluent, such as sterile phosphate buffered saline.

In some embodiments, the antisense oligonucleotide is formulated in a pharmaceutically acceptable diluent at a concentration of 50-300 μ M solution. The diluent may be phosphate buffered saline.

In some embodiments, the antisense oligonucleotide is formulated in a pharmaceutically acceptable diluent at a concentration of 1-100mg/mL, such as 2-30 or 2-50mg/mL, or such as 4-30 mg/mL. The diluent may be phosphate buffered saline.

The present invention provides a method of modulating the expression of ATXN2 in a target cell expressing ATXN2, said method comprising administering to said cell an antisense oligonucleotide or conjugate or pharmaceutical composition of the invention in an effective amount. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method. In some embodiments, the cell is a neuronal cell, such as a cerebellar cell, such as a purkinje cell or a cortical cell.

The invention provides an oligonucleotide, a conjugate or a pharmaceutical composition of the invention for use in medicine.

The present invention provides the oligonucleotide, conjugate or pharmaceutical composition of the invention for use in the treatment of a disease selected from the group consisting of a neurodegenerative disease selected from the group consisting of spinocerebellar ataxia 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia alzheimer (FTD), parkinson's syndrome and a disorder with TDP-43 proteinopathy.

The present invention provides the use of an oligonucleotide, a conjugate or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment or prevention of a neurodegenerative disease, such as a disease selected from the group consisting of: spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), Alzheimer frontotemporal dementia (FTD), Parkinson's syndrome, and disorders with TDP-43 proteinopathies.

The invention provides a method for treating or preventing a disease, the method comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of an antisense oligonucleotide, conjugate, or pharmaceutical composition of the invention, wherein the disease is selected from the group consisting of a neurodegenerative disease selected from the group consisting of spinocerebellar ataxia 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), alzheimer frontotemporal dementia (FTD), parkinson's syndrome, and a disorder with TDP-43 proteinopathy.

In some embodiments, the disease is spinocerebellar ataxia type 2 (SCA 2).

In some embodiments, the disease is Amyotrophic Lateral Sclerosis (ALS).

Suitably, for example, for therapeutic use, the subject is a human suffering from, or susceptible to, the disease.

In another aspect, the invention provides a method for treating or preventing a disease, disorder or dysfunction associated with the in vivo activity of ATXN2, comprising administering to a subject suffering from or susceptible to the disease, disorder or dysfunction a therapeutically or prophylactically effective amount of an oligonucleotide targeting ATXN2 or a conjugate thereof or a pharmaceutical composition, such as an antisense oligonucleotide of the invention or an siRNA targeting ATXN2, wherein at least said method comprises administering at least two consecutive doses of the oligonucleotide targeting ATXN2, wherein the time interval between at least two consecutive doses is at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least one month, such as at least 6 weeks, such as at least 8 weeks, such as at least two months. Thus, administration may be, for example, once a week, once every two weeks, once a month, or once every two months.

In another aspect, the invention provides a method for treating or preventing a neurodegenerative disease, comprising administering to a subject suffering from or susceptible to a neurodegenerative disease a therapeutically or prophylactically effective amount of an oligonucleotide targeting ATXN2 or a conjugate or pharmaceutical composition thereof, such as an antisense oligonucleotide of the invention or an siRNA targeting ATXN2, wherein at least the method comprises administering at least two consecutive doses of the oligonucleotide targeting ATXN2, wherein the time interval between at least two consecutive doses is at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least one month, such as at least 6 weeks, such as at least 8 weeks, such as at least two months. Thus, administration may be, for example, once a week, once every two weeks, once a month, or once every two months. In another aspect, the invention provides an oligonucleotide targeting ATAXN2 for use in the treatment or prevention of a neurodegenerative disease in a subject, wherein the oligonucleotide is administered in at least two consecutive doses, wherein the time interval between the at least two consecutive doses is at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least one month, such as at least 6 weeks, such as at least 8 weeks, such as at least two months. Thus, administration may be, for example, once a week, once every two weeks, once a month, or once every two months.

In another aspect, the oligonucleotides or compositions of the invention are used to treat or prevent spinocerebellar ataxia type 2 (SCA 2).

In another aspect, the oligonucleotide or composition of the invention is used to treat or prevent Amyotrophic Lateral Sclerosis (ALS).

Drawings

FIG. 1 Compound 7_1 (nucleobase sequence shown in SEQ ID NO 7)

FIG. 2 Compound 13_1 (nucleobase sequence shown in SEQ ID NO 13)

FIG. 3 Compound 17_1 (nucleobase sequence shown in SEQ ID NO 17)

FIG. 4 Compound 18_1 (nucleobase sequence shown in SEQ ID NO 18)

FIG. 5 Compound 15_4 (nucleobase sequence shown in SEQ ID NO 15)

The compounds shown in figures 1, 2,3 and 4 are shown in protonated form (i.e., the S atom on the phosphorothioate linkage is protonated), it being understood that the presence of a proton will depend on the acidity of the molecular environment, as well as the presence of other cations (e.g., when the oligonucleotide is in salt form). The protonated phosphorothioates exist in tautomeric forms.

FIG. 6 screening in human cell lines targeting human spinocerebellar ataxia protein 2 precursor mRNA sequences of 1500 various compounds. Compound 7_1 is represented as an open diamond.

Figure 7 according to figure 6, only compounds targeting SEQ ID NO 6 in the hot spot region. Compound 7_1 is represented as an open diamond.

Figure 8 in vitro potency assessment of compounds 7_1 and 15_4 compared to compound ASO 7.

Figure 9 in vivo mouse study-knock out (mRNA) comparison of 11 selected compounds, summary data from three experiments, study 1 being a solid point, study 2 being a hollow point, and study 3 being a semi-solid point.

Figure 10 in vivo mouse studies-knockdown of compound 7_1 at protein and mRNA levels and exposure to cortical, cerebellar regions. Only cortical protein data is shown.

Figure 11 in vivo mouse studies-knock-out of compound 15_4 at protein and mRNA levels and exposure to cortical, cerebellar regions. Only cortical protein data is shown.

Figure 12 in vivo study in mice, time course after ICV administration of 150 μ g of compound 7_1 and compound 15_4 (measured only on 7 days).

Fig. 13NHP in vivo PK/PD study-mRNA and protein expression levels in critical tissues were measured 14 days after treatment after administration of 4, 8 or 24mg compound 7_1 and 8mg compound 15_ 4.

Fig. 14NHP in vivo PK/PD study-mRNA and protein expression levels in critical tissues were measured 14 days after treatment after administration of 4, 8 or 24mg compound 7_1 and 8mg compound 15_ 4. The data are provided to illustrate the relative specific activity of the two compounds.

Definition of

Oligonucleotides

As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more covalently linked nucleosides as is commonly understood by a skilled artisan. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually prepared in the laboratory by solid phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, reference is made to the nucleobase portion of a covalently linked nucleotide or nucleoside or a modified sequence or order thereof. The oligonucleotides of the invention are artificial and chemically synthesized and are usually purified or isolated. The oligonucleotides of the invention may comprise one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides.

Antisense oligonucleotides

The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, particularly to a contiguous sequence on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded and are therefore not sirnas or shrnas. Preferably, the antisense oligonucleotides of the invention are single stranded. It is understood that single stranded oligonucleotides of the invention may form hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide) provided that the self-complementarity within or with each other is less than 50% of the full length of the oligonucleotide.

Advantageously, the single stranded antisense oligonucleotides of the invention do not comprise RNA nucleosides (2' -OH unmodified ribose).

Advantageously, the antisense oligonucleotides of the invention comprise one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides. Furthermore, advantageously, the unmodified nucleoside is a DNA nucleoside.

Continuous nucleotide sequence

The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. The term is used herein interchangeably with the term "contiguous nucleobase sequence" and the term "oligonucleotide motif sequence". In some embodiments, all nucleotides of an oligonucleotide comprise a contiguous nucleotide sequence. In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence, such as a F-G-F' gapped poly region, and may optionally comprise other nucleotides, such as a nucleotide linker region that may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

Rnai agents

The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," used interchangeably herein, refer to an agent that comprises an RNA nucleoside herein and mediates targeted cleavage of an RNA transcript by the RNA-induced silencing complex (RISC) pathway. irnas direct sequence-specific degradation of mRNA through the process of RNA interference (RNAi). iRNA modulates, e.g., inhibits, expression of a target nucleic acid in a cell, e.g., a cell in a subject, such as a mammalian subject. RNAi agents include single-stranded RNAi agents and double-stranded sirnas, as well as short hairpin rnas (shrnas). The oligonucleotide of the invention or a contiguous nucleotide sequence thereof may be in the form of, or part of, an RNAi agent, such as an siRNA or shRNA. In some embodiments of the invention, an oligonucleotide of the invention or a contiguous nucleotide sequence thereof is an RNAi agent, such as an siRNA.

siRNA

The term siRNA refers to small interfering ribonucleic acid RNAi agents, a class of double-stranded RNA molecules, also known in the art as short interfering or silencing RNAs. siRNA typically comprise a sense strand (also referred to as passenger strand) and an antisense strand (also referred to as guide strand), wherein each strand is 17-30 nucleotides, typically 19-25 nucleotides in length, wherein the antisense strand is complementary to the target nucleic acid such as fully complementary (suitably a mature mRNA sequence) and the sense strand is complementary to the antisense strand such that the sense strand and the antisense strand form a duplex or duplex region. The siRNA strands may form blunt-ended duplexes, or advantageously, the 3 'ends of the sense and antisense strands may form 3' overhangs, e.g., 1, 2, or 3 nucleosides. In some embodiments, both the sense and antisense strands have 2 nt 3' overhangs. Thus, the duplex region may be, for example, 17-25 nucleotides in length, such as 21-23 nucleotides in length.

Once inside the cell, the antisense strand is incorporated into the RISC complex, which mediates target degradation or target inhibition of the target nucleic acid. sirnas typically comprise a modified nucleoside in addition to an RNA nucleoside, or in some embodiments, all nucleotides of the siRNA strand may be modified (sense. 2 'sugar modified nucleosides such as LNA (see, e.g., WO2004083430, WO2007085485), 2' fluoro, 2 '-O-methyl, or 2' -O-methoxyethyl) may be incorporated into the siRNA. In some embodiments, the passenger strand of the siRNA may be discontinuous (see, e.g., WO 2007107162). Incorporation of heat labile nucleotides present in the seed region of the antisense strand of an siRNA has been reported to be useful in reducing off-target activity of an siRNA (see, e.g., WO 18098328).

In some embodiments, the dsRNA agent, such as an siRNA of the invention, comprises at least one modified nucleotide. In some embodiments, substantially all of the nucleotides of the sense strand comprise a modification; substantially all nucleotides of the antisense strand comprise modifications, or substantially all nucleotides of the sense strand comprise modifications and substantially all nucleotides of the antisense strand comprise modifications. In other embodiments, all nucleotides of the sense strand comprise a modification; all nucleotides of the antisense strand comprise modifications, or all nucleotides of the sense strand comprise modifications and all nucleotides of the antisense strand comprise modifications.

In some embodiments, the modified nucleotides may be independently selected from the group consisting of: deoxynucleotides, 3' -terminal deoxythymine (dT) nucleotides, 2' -0-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -hydroxy modified nucleotides, 2' -methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases comprising nucleotides, unlinked nucleotides, tetrahydropyran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxy modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -hydroxy modified nucleotides, 2' -methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases comprising nucleotides, unlinked nucleotides, cyclohexenyl-modified nucleotides, phosphorothioate group-containing nucleotides, methylphosphonate group-containing nucleotides, 5-phosphate-containing nucleotides, 5' -phosphate mimetic-containing nucleotides, ethylene glycol-modified nucleotides, and 2-0- (N-methylacetamide) -modified nucleotides, and combinations thereof. Suitable sirnas comprise a 5' phosphate group or 5' -phosphate mimetic at the 5' end of the antisense strand. In some embodiments, the 5' terminal nucleoside of the antisense strand is an RNA nucleoside.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

Phosphorothioate or methylphosphonate internucleoside linkages may be located at the 3' -terminus of one or both strands (e.g., the antisense strand; or the sense strand); or the phosphorothioate or methylphosphonate internucleoside linkage may be located at the 5' end of one or both strands (e.g., the antisense strand; or the sense strand); or the internucleoside linkage of the phosphorothioate or methylphosphonate may be located at the 5 '-and 3' -ends of one or both strands (e.g., the antisense strand; or the sense strand). In some embodiments, the remaining internucleoside linkages are phosphodiester linkages.

The dsRNA agent may further comprise a ligand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand.

For biological distribution, for example, siRNA can be conjugated to a targeting ligand and/or formulated into lipid nanoparticles.

Other aspects of the invention relate to pharmaceutical compositions comprising these dsrnas suitable for therapeutic use, such as siRNA molecules, and methods of inhibiting expression of a target gene by administering dsRNA molecules, such as the sirnas of the invention, e.g., for treating various disease conditions disclosed herein.

Nucleotide, its preparation and use

Nucleotides are constituents of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides. In practice, nucleotides such as DNA nucleotides and RNA nucleotides comprise a ribose moiety, a nucleobase moiety and one or more phosphate groups (not present in the nucleoside). Nucleosides and nucleotides can also be interchangeably referred to as "units" or "monomers".

Modified nucleosides

As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that is modified by the introduction of one or more modifications of a sugar moiety or a (nucleobase) moiety as compared to an equivalent DNA or RNA nucleoside. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the term "nucleoside analog" or modified "unit" or modified "monomer". Nucleosides having unmodified DNA sugar moieties or nucleosides having RNA sugar moieties are referred to herein as DNA or RNA nucleosides. If Watson Crick base pairing is allowed, the modified nucleoside in the DNA nucleobase region or the RNA nucleobase region is still generally referred to as DNA or RNA.

Modified internucleoside linkages

As generally understood by the skilled person, the term "modified internucleoside linkage" is defined as a linkage other than a Phosphodiester (PO) linkage, which covalently couples two nucleosides together. Thus, the oligonucleotides of the invention may comprise modified internucleoside linkages. In some embodiments, the modified internucleoside linkage increases nuclease resistance of the oligonucleotide compared to phosphodiester linkage. For naturally occurring oligonucleotides, internucleoside linkages include phosphate groups that create phosphodiester linkages between adjacent nucleosides. Modified internucleoside linkages are useful for stabilizing oligonucleotides for use in vivo, and for preventing nuclease cleavage of DNA or RNA nucleoside regions in oligonucleotides of the invention, e.g., within gap region G of a gapmer oligonucleotide and in modified nucleoside regions, e.g., regions F and F'.

In one embodiment, the oligonucleotide comprises one or more internucleoside linkages modified from a natural phosphodiester. In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide or the contiguous nucleotide sequence thereof are modified, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90% of the internucleoside linkages in the oligonucleotide or the contiguous nucleotide sequence thereof are modified. In some embodiments, all internucleoside linkages in the oligonucleotide or a contiguous nucleotide sequence thereof are modified. It will be appreciated that in some embodiments, the nucleoside linking the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be a phosphodiester. In some embodiments, all of the internucleoside linkages of the oligonucleotide or a contiguous nucleotide sequence thereof are nuclease-resistant internucleoside linkages.

For the oligonucleotides of the invention, it is advantageous to use phosphorothioate internucleoside linkages.

Phosphorothioate internucleoside linkages are particularly beneficial due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide or the contiguous nucleotide sequence thereof are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90% of the internucleoside linkages in the oligonucleotide or the contiguous nucleotide sequence thereof are phosphorothioate. In some embodiments, all internucleoside linkages of the oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate.

Advantageously, all internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages, or all internucleoside linkages of the oligonucleotide are phosphorothioate linkages.

It will be appreciated that the antisense oligonucleotide may comprise other internucleoside linkages (besides phosphodiesters and phosphorothioates), such as alkylphosphonate/methylphosphonate internucleosides, which may be otherwise tolerated in the gap region of DNA phosphorothioates, for example, according to EP 2742135, as disclosed in EP 2742135.

Nucleobases

The term nucleobase includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases, which may differ from naturally occurring nucleobases, but which play a role during nucleic acid hybridization. In this context, "nucleobase" refers to naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research, volume 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry supply.371.4.1.

In some embodiments, the nucleobase moiety is modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolocyytosine, 5-propynylcytosine, 5-propynyluracil, 5-bromouracil, 5-thiazolyluracil, 2-thiouracil, 2' thiothymine, inosine, diaminopurine, 6-aminopurine, 2, 6-diaminopurine, and 2-chloro-6-aminopurine.

Nucleobase moieties may be represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C or U, wherein each letter may optionally include modified nucleobases with equivalent functionality. For example, in the exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C and 5-methylcytosine. Optionally, for LNA spacer, 5-methylcytosine LNA nucleosides can be used.

Modified oligonucleotides

The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar modified nucleosides and/or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term that has been used in the literature to describe oligonucleotides having modified nucleosides.

Complementarity

The term "complementarity" describes the ability of a nucleoside/nucleotide to undergo Watson-Crick base pairing. Watson-Crick base pairs are guanine (G) -cytosine (C) and adenine (A) -thymine (T)/uracil (U). It is to be understood that oligonucleotides may comprise nucleosides with modified nucleobases, e.g., 5-methylcytosine is often used instead of cytosine, and thus the term complementarity encompasses Watson Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al (2012) Accounts of Chemical Research, volume 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry supply.371.4.1).

As used herein, the term "percent complementarity" refers to the proportion of nucleotides (expressed as a percentage) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is complementary to a reference sequence (e.g., a target sequence or sequence motif) across the contiguous nucleotide sequence. Thus, the percent complementarity is calculated by counting the number of aligned nucleobases between two sequences that are complementary (forming Watson Crick base pairs) when aligned to the oligonucleotide sequences 5 '-3' and 3 '-5' of the target sequence, dividing this by the total number of nucleotides in the oligonucleotide, and then multiplying by 100. In this comparison, the misalignment (forming base pairs) of nucleobases/nucleotides is called mismatch. Insertions and deletions are not allowed when calculating the percent complementarity of a contiguous nucleotide sequence. It is understood that chemical modification of nucleobases is not considered in determining complementarity (e.g., 5' -methylcytosine is considered the same as cytosine in calculating percent complementarity) so long as the functional ability of the nucleobases to form Watson Crick base pairing is retained.

The term "fully complementary" refers to 100% complementarity.

The following are examples of oligonucleotides that are fully complementary to the target sequence.

The following is an example of an oligonucleotide (SEQ ID NO:15) that is fully complementary to the target sequence (SEQ ID NO: 6).

5’ttaaggaggttaaagtaaaatgtgaattt 3’(SEQ ID NO:6)

3’ctccaatttcattttacact 5’(SEQ ID NO:15)

Identity of each other

As used herein, the term "identity" refers to the proportion of nucleotides (expressed as a percentage) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is identical to a reference sequence (e.g., a sequence motif) across the contiguous nucleotide sequence. Thus, percent identity is calculated by counting the number of aligned nucleobases of two sequences (in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence) that are identical (matched), dividing this number by the total number of nucleotides in the oligonucleotide and multiplying by 100. Thus, percent identity is (number of matches x 100)/length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed when calculating the percent identity of consecutive nucleotide sequences. It is understood that chemical modifications of nucleobases are not considered in determining identity, as long as the functional ability of the nucleobases to form Watson Crick base pairing is retained (e.g., 5-methylcytosine is considered identical to cytosine when calculating percent identity).

Hybridization of

As used herein, the term "hybridization" should be understood as the formation of hydrogen bonds between base pairs on opposing strands to form duplexesThe two nucleic acid strands of (e.g., the oligonucleotide and the target nucleic acid). The affinity of the binding between two nucleic acid strands is the strength of hybridization. Usually by melting temperature (T)_m) Which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. Under physiological conditions, T_mNot strictly proportional to affinity (Mergny and Lacreox, 2003, Oligonucleotides 13: 515-. The gibbs free energy Δ G ° in the standard state more accurately represents binding affinity, and by Δ G ° — RTln (K)_d) Dissociation constant (K) with reaction_d) Where R is the gas constant and T is the absolute temperature. Thus, the very low Δ G ° of the reaction between the oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and the target nucleic acid. Δ G ° is the energy associated with a reaction with a water concentration of 1M, pH of 7 and a temperature of 37 ℃. Hybridization of the oligonucleotide to the target nucleic acid is a spontaneous reaction, with a Δ G ° of less than zero. Δ G ° can be measured experimentally, for example, by using the Isothermal Titration Calorimetry (ITC) method as described in Hansen et al, 1965, chem.Comm.36-38 and Holdgate et al, 2005, Drug Discov Today. The skilled person will know that commercial equipment can be used for Δ G ° measurement. Δ G ° can also be numerically evaluated using the nearest neighbor model described in Santa Lucia,1998, Proc Natl Acad Sci USA, 95: 1460-. In order to have the possibility of modulating its intended nucleic acid target by hybridization, for oligonucleotides of 10-30 nucleotides in length, the oligonucleotides of the invention hybridize with the target nucleic acid with an estimated Δ G ° value of less than-10 kcal. In some embodiments, the degree or intensity of hybridization is measured by the gibbs free energy Δ G ° in the standard state. For oligonucleotides 8-30 nucleotides in length, the oligonucleotide may hybridize to the target nucleic acid with an estimated Δ G ° value of less than-10 kcal, such as less than-15 kcal, such as less than-20 kcal, such as less than-25 kcal. In some embodiments, the oligonucleotide hybridizes to the target nucleic acid at an estimated Δ G ° value of about-10 to-60 kcal, such as-12 to-40, such as-15 to-30 kcal or-16 to-27 kcal, such as-18 to-25 kcal.

Target nucleic acid

According to the invention, the target nucleic acid is a nucleic acid encoding mammalian ATXN2 and can be, for example, a gene, RNA, mRNA and pre-mRNA, mature mRNA or cDNA sequence. This target may therefore be referred to as the ATXN2 target nucleic acid. The oligonucleotides of the invention may, for example, target the exon region of mammalian ATXN2, or may, for example, target the intron region in the ATXN2 pre-mRNA (see table 1).

TABLE 1 human ATXN2 exons and introns

Suitably, the target nucleic acid encodes an ATXN2 protein, particularly a mammalian ATXN2, e.g. human ATXN2 (see e.g. tables 2 and 3), which provides mRNA and pre-mRNA sequences of human, monkey, rat and porcine ATXN 2.

In some embodiments, the target nucleic acid is selected from SEQ ID NOs: 1. 2,3, 4 and 5 or naturally occurring variants thereof (e.g., sequences encoding mammalian Ataxin2 proteins).

If the oligonucleotides of the invention are used in research or diagnosis, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

For in vivo or in vitro applications, the oligonucleotides of the invention are generally capable of inhibiting the expression of the ATXN2 target nucleic acid in cells expressing the ATXN2 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotides of the invention is typically complementary to the ATXN2 target nucleic acid, as measured over the length of the oligonucleotide, optionally except for one or two mismatches, and optionally excluding nucleotide-based linker regions that can link the oligonucleotide with optional functional groups, such as conjugates or other non-complementary terminal nucleotides (e.g., regions D' or D "). In some embodiments, the target nucleic acid can be RNA or DNA, such as messenger RNA, e.g., mature mRNA or precursor mRNA.

In some embodiments, the target nucleic acid is RNA or DNA encoding a mammalian Ataxin2 protein (such as human ATXN2), for example a human ATXN2 mRNA sequence, such as disclosed in SEQ ID NO 1. Tables 2 and 3 provide more information about exemplary target nucleic acids.

Table 2 genome and assembly information for ATXN2 for various species.

Fwd is the forward chain. The genomic coordinates provide the precursor mRNA sequence (genomic sequence). The NCBI reference provides mRNA sequences (cDNA sequences).

National center for biotechnology information reference sequence database is a comprehensive, complete, non-redundant, unequivocal set of annotated reference sequences, including genomes, transcripts and proteins. Its home page is www.ncbi.nlm.nih.gov/refseq.

TABLE 3 sequence details of ATXN2 for various species.

Target sequence

As used herein, the term "target sequence" refers to a nucleotide sequence present in a target nucleic acid that comprises a nucleobase sequence that is complementary to an oligonucleotide of the invention. In some embodiments, the target sequence consists of a region on the target nucleic acid having a nucleobase sequence complementary to a contiguous nucleotide sequence of the oligonucleotide of the invention. This region of the target nucleic acid may be interchangeably referred to as the target nucleotide sequence, target sequence, or target region.

In some embodiments, the target sequence is longer than the complement of a single oligonucleotide and may, for example, represent a preferred region of the target nucleic acid, which may be targeted by several oligonucleotides of the invention. Preferred regions of the target nucleic acid may also be referred to as "hot spots" indicating that the one or more oligonucleotides are capable of effectively reducing the target nucleotide sequence of the target nucleic acid towards, e.g., reducing the target nucleic acid to at least 50% of a control (see, e.g., fig. 1). Hot spots are usually identified with a scanning library of oligonucleotides designed to cover most target nucleic acids. The hot spot is usually identified by other oligonucleotide libraries targeting the hot spot.

In some embodiments, the target sequence is a sequence selected from any region (R _1-R _2421) in table 4. In particular, the target sequence may be selected from the group consisting of R _1-R _13, R _15-R _874, R _876-R _894, R _896-R _902, R _906-R _1151, R _1153-R _1338, R _1341-R _1420, R _1422-R _1435, R _1437-R _1465, R _1468-R _1495, R _1499-R _1542, R _1545-R _1592, R1595-R _1602, R _1604-R _1643, R _1646-R _1869, R _1873-R _1905, R _1907-R _1921, R _1923-R _1929, R _1931-R _2145, R _2147-R _2152, R _ 2235-R _ 1906, R _ 1908-R _ 236, R _ 2158-R _ 2354, R _2404-R _ 2403, R _ 2401-R _ 2353, R _ 2403, R _ 2353, R _ 2401-R _ 2353, R _ 2403, R _ R, R _2409-R _2415 and R _ 2421.

Table 4. SEQ ID NO: region on 1 (reg.)

In some embodiments, the target sequence is a sequence selected from any region (W1-W115) in table 5. Specifically, the target sequence may be selected from one of the regions consisting of W1, W4, W5, W6, W7, W8, W9, W10, W11, W12, W13, W14, W15, W16, W17, W18, W19, W20, W21, W22, W23, W24, W25, W26, W.

Table 5. SEQ ID NOs: region on 1 (reg.)

In some embodiments, the target sequence is a sequence selected from any region (S1-S46) in table 6. In particular, the target sequence may be selected from one of the regions of the group of regions consisting of S1, S2, S3, S5, S6, S9, S10, S11, S12, S14, S15, S16, S19, S21, S22, S25, S26, S27, S28, S29, S30, S31, S33, S36, S41, S43, S45 and S46.

In one embodiment, the target sequence is the sequence of region W54 or S19.

Table 6. SEQ ID NO: region on 1 (reg.)

In one embodiment of the invention, the target sequence is selected from the group consisting of

In one embodiment of the invention, the target sequence is selected from the group consisting of R _274, R _893, R _895, R _1496, R _1992 and R _ 2420.

In some embodiments, the target sequence is a sequence selected from human ATXN2 mRNA intron 1, 3, 5, 9, 10, 11, 1, 18, 20, or 21 (see table 1 above), e.g., selected from intron 1, 3, 9, or 18.

In some embodiments, the target sequence is a sequence selected from human ATXN2 mRNA exons 4,5, or 25 (see table 1 above), e.g., selected from exon 25.

In some embodiments, the target sequence is a sequence spanning the intron 4/exon 5 or exon 9/intron 10 region selected from human ATXN2 mRNA exon/intron.

In one embodiment of the invention, the target sequence is SEQ ID NO: 6.

the oligonucleotides of the invention comprise a contiguous nucleotide sequence that is complementary to or hybridizes to a target nucleic acid, such as a target sequence described herein.

The target sequence to which the oligonucleotide is complementary or hybridizes generally comprises a contiguous nucleobase sequence of at least 10 nucleotides. The contiguous nucleotide sequence is between 10 and 400 nucleotides, such as 10 to 150 nucleotides, such as 10 to 100, such as 10 to 60 nucleotides, such as 10 to 50 nucleotides, such as 12 to 40 nucleotides, such as 12 to 30 nucleotides, such as 14 to 25 nucleotides, such as 15 to 18 contiguous nucleotides.

Target cell

As used herein, the term "target cell" refers to a cell that expresses a target nucleic acid. In some embodiments, the target cell may be in vivo or in vitro. In some embodiments, the target cell is a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell, such as a monkey cell or a human cell.

In some embodiments, the target cell may be a purkinje neuron, such as a purkinje cell. Other relevant target cells are motor neurons, such as upper and lower motor neurons.

For in vitro evaluation, the target cells may be established cell lines, such as A431 or U2-OS cells. Alternatively, motoneurons derived from human induced pluripotent stem cells (iPCs) (see, e.g., Sances et al 2016Nat Neurosci.19(4): 542-.

In preferred embodiments, the target cell expresses ATXN2 mRNA, such as ATXN2 precursor mRNA or ATXN2 mature mRNA. For antisense oligonucleotide targeting, the poly A tail of ATXN2 mRNA is generally not considered.

Naturally occurring variants

The term "naturally occurring variant" refers to a variant of the ATXN2 gene or transcript that originates from the same genetic locus as the target nucleic acid, but may differ, for example, due to the degeneracy of the genetic code leading to the diversity of codons encoding the same amino acid, or due to alternative splicing of precursor mrnas, or the presence of polymorphisms such as Single Nucleotide Polymorphisms (SNPs) and allelic variants. The oligonucleotides of the invention can thus target nucleic acids and naturally occurring variants thereof, based on the presence of a sequence sufficiently complementary to the oligonucleotide.

In some embodiments, the naturally occurring variant has at least 95%, such as at least 98% or at least 99% homology to a mammalian ATXN2 target nucleic acid, such as a nucleic acid selected from the group consisting of SEQ ID NOs: 1. 2,3, 4 and 5. In some embodiments, the naturally occurring variant differs from SEQ ID NO:1 has at least 99% homology to the human ATXN2 target nucleic acid.

Modulation of expression

As used herein, the term "modulation of expression" is understood to be a generic term for the ability of an oligonucleotide to alter the amount of ATXN2 as compared to the amount of ATXN2 prior to administration of the oligonucleotide. Alternatively, modulation of expression may be determined by reference to control experiments. It is generally understood that a control is an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mimetic).

One type of modulation is the ability of the oligonucleotide to inhibit, down-regulate, reduce, repress, remove, stop, block, prevent, reduce, diminish, avoid, or terminate expression of ATXN2, for example, by degrading mRNA or preventing transcription. Another type of modulation is the ability of the oligonucleotide to restore, increase or enhance expression of ATXN2, for example by repairing splice sites or preventing splicing or removing or blocking inhibitory mechanisms (such as microrna repression). The antisense oligonucleotides of the invention are advantageously capable of inhibiting the expression of mammalian ATXN2, such as human ATXN 2.

High affinity modified nucleosides

High affinity modified nucleosides are modified nucleotides that, when incorporated into an oligonucleotide, enhance the affinity of the oligonucleotide for its complementary target, as measured, for example, by the melting temperature (Tm). The high affinity modified nucleosides of the present invention preferably increase the melting temperature of each modified nucleoside by between +0.5 and +12 ℃, more preferably by between +1.5 and +10 ℃, and most preferably by between +3 and +8 ℃. Many high affinity modified nucleosides are known in the art and include, for example, many 2' substituted nucleosides as well as Locked Nucleic Acids (LNA) (see, e.g., Freeer & Altmann; nucleic acid Res.,1997,25, 4429-.

Sugar modification

Oligomers of the invention may comprise one or more nucleosides having a modified sugar moiety, i.e., a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

Many modified nucleosides have been prepared with ribose moieties, the primary purpose being to improve certain properties of the oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those in which the ribose ring structure is modified, for example, by replacing the ribose ring structure with a hexose ring (HNA) or a bicyclic ring, which typically has a diradical bridge between the C2 and C4 carbon atoms of the ribose ring (LNA), or an unconnected ribose ring that typically lacks a bond between the C2 and C3 carbon atoms (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example in the case of Peptide Nucleic Acid (PNA) or morpholino nucleic acid.

Sugar modifications also include modifications by changing the substituent groups on the ribose ring to groups other than hydrogen or to 2' -OH groups naturally present in DNA and RNA nucleosides. For example, substituents may be introduced at the 2', 3', 4 'or 5' positions.

2' sugar modified nucleosides

A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or-OH at the 2' position (a 2' substituted nucleoside) or comprising a 2' linking diradical capable of forming a bridge between the 2' carbon and the second carbon atom in the ribose ring, such as a LNA (2' -4' diradical bridged) nucleoside.

In fact, much effort has been expended to develop 2 'sugar substituted nucleosides, and many 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2' modified sugars can provide enhanced binding affinity and/or increased nuclease resistance to oligonucleotides. Examples of 2 'substituted modified nucleosides are 2' -O-alkyl-RNA, 2 '-O-methyl-RNA, 2' -alkoxy-RNA, 2 '-O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2 '-fluoro-RNA and 2' -F-ANA nucleosides. For further examples, see, e.g., Freier and Altmann; nucleic acids res, 1997,25,4429-4443 and Uhlmann; opinion in Drug Development,2000,3(2),293-213 and Deleavey and Damha, Chemistry and Biology 2012,19, 937. The following are schematic representations of some 2' substituted modified nucleosides.

For the present invention, 2 'substituted sugar modified nucleosides do not include 2' bridged nucleosides like LNA.

Locked nucleic acid nucleosides (LNA nucleosides)

An "LNA nucleoside" is a 2' -modified nucleoside comprising a diradical of C2' and C4' linking the ribose ring of the nucleoside (also referred to as a "2 ' -4' bridge"), which constrains or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or Bicyclic Nucleic Acids (BNA). When LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule, the locking of the ribose conformation is associated with an enhanced affinity for hybridization (duplex stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide/complementary duplex.

Non-limiting exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al, Bioorganic & Med.Chem.Lett.12,73-76, Seth et al J.org.Chem.2010, Vol 75(5) pp.1569-81 and Mitsuoka et al, Nucleic Acids Research 2009,37(4),1225-1238 and Wan and Seth, J.medical Chemistry 2016,59, 9645-9667.

Other non-limiting exemplary LNA nucleosides are disclosed in scheme 1.

Scheme 1:

specific LNA nucleosides are β -D-oxy-LNA, 6 '-methyl- β -D-oxy-LNA such as (S) -6' -methyl- β -D-oxy-LNA (scet) and ENA.

One particularly advantageous LNA is a β -D-oxy-LNA.

The compounds described herein may contain several asymmetric centers and may exist as optically pure enantiomers, mixtures of enantiomers such as racemates, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates.

The term "asymmetric carbon atom" means a carbon atom having four different substituents. The asymmetric carbon atoms may be in either the "R" or "S" configuration according to the Cahn-Ingold-Prelog convention.

Pharmaceutically acceptable salts

The term "pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the free base or free acid, neither of which is biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, in particular hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine. Alternatively, these salts may be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, tertiary, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The compounds of formula (I) may also be present in zwitterionic form. Particularly preferred pharmaceutically acceptable salts of the compounds of formula (I) are salts of hydrochloric, hydrobromic, sulfuric, phosphoric and methanesulfonic acids.

Protecting group

The term "protecting group" used alone or in combination denotes a group that selectively blocks a reactive site in a polyfunctional compound, thereby allowing a chemical reaction to be selectively performed at another unprotected reactive site. The protecting group may be removed. Exemplary protecting groups are amino protecting groups, carboxyl protecting groups, or hydroxyl protecting groups.

Nuclease-mediated degradation

Nuclease-mediated degradation refers to an oligonucleotide that is capable of mediating degradation of a complementary nucleotide sequence when it forms a duplex with this sequence.

In some embodiments, the oligonucleotides can function by nuclease-mediated target nucleic acid degradation, wherein the oligonucleotides of the invention are capable of recruiting nucleases, particularly endonucleases, preferably endoribonucleases (rnases) such as RNase H. Examples of oligonucleotide designs that function by nuclease-mediated mechanisms are oligonucleotides that typically comprise regions of at least 5 or 6 contiguous DNA nucleosides and flanked on one or both sides by affinity-enhancing nucleosides, e.g., gapmers, headmers, and tailmers.

Activity and recruitment of RNase H

The RNase H activity of the antisense oligonucleotide refers to its ability to recruit RNase H when it forms duplexes with complementary RNA molecules. WO01/23613 provides in vitro methods for determining RNaseH activity, which can be used to determine the ability to recruit RNaseH. An oligonucleotide is considered to be capable of recruiting RNase H if it has an initial rate in providing a complementary target nucleic acid sequence which is at least 5%, such as at least 10% or more than 20%, of the initial rate of an oligonucleotide having the same base sequence as the modified oligonucleotide tested but containing only DNA monomers having phosphorothioate linkages between all monomers in the oligonucleotide, measured in pmol/l/min using the method provided in WO01/23613 (incorporated herein by reference) examples 91 to 95. For use in determining the RRNase H activity, recombinant human RNase H1 was obtained from Lubio Science GmbH, Lucerne, Switzerland.

Spacer polymers

The antisense oligonucleotides of the invention or contiguous nucleotide sequences thereof may be gapmer, also referred to as gapmer oligonucleotides or gapmer designs. Antisense gapmers are commonly used to inhibit target nucleic acids by RNase H mediated degradation. A gapmer oligonucleotide comprises at least three different structural regions, respectively 5' flank, gap and 3' flank in the "5- > 3" direction, F-G-F '. The "gap" region (G) comprises a contiguous DNA nucleotide which enables the oligonucleotide to recruit RNase H. The notch region is flanked by a 5' flanking region (F) comprising one or more sugar-modified nucleosides (advantageously high affinity sugar-modified nucleosides) and a 3' flanking region (F ') comprising one or more sugar-modified nucleosides (advantageously high affinity sugar-modified nucleosides). One or more sugar modified nucleosides in regions F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., the affinity-enhanced sugar modified nucleosides). In some embodiments, the one or more sugar modified nucleosides in regions F and F 'are 2' sugar modified nucleosides, e.g., high affinity 2 'sugar modifications, such as independently selected from LNA and 2' -MOE.

In the gapmer design, the 5' and 3' endmost nucleosides of the gapped region are DNA nucleosides, located near the sugar-modified nucleosides of the 5' (F) or 3' (F ') regions, respectively. A flap may be further defined as having at least one sugar modified nucleoside at the end furthest from the notch region, i.e., at the 5 'end of the 5' flap and the 3 'end of the 3' flap.

The region F-G-F' forms a contiguous nucleotide sequence. The antisense oligonucleotides of the invention or contiguous nucleotide sequences thereof may comprise a gapmer region of the formula F-G-F'.

The total length of the gapmer design F-G-F' may be, for example, 12 to 32 nucleosides, such as 13 to 24 nucleosides, such as 14 to 22 nucleosides, such as 14 to 17 nucleosides, such as 16 to 18 nucleosides.

For example, the gapmer oligonucleotides of the invention can be represented by the formula:

F_1-8-G_6-16-F’_1-8such as

F_1-8-G_8-16-F’_2-8

Provided that the total length of the gapmer region F-G-F' is at least 12, such as at least 14 nucleotides.

In one aspect of the invention, the antisense oligonucleotide or a contiguous nucleotide sequence thereof consists of or comprises a gapmer of the formula 5'-F-G-F' -3', wherein regions F and F' independently comprise or consist of 1-8, such as 2-6, such as 3-4, 2 'sugar modified nucleosides, wherein at least one 2' sugar modified nucleoside is located at the 3 'end of region F (adjacent to the DNA nucleosides of region G) and at least one 2' sugar modified nucleoside is located at the 5 'end of region F' (adjacent to the DNA nucleosides of region G), and G is a region between 6 and 16 nucleosides capable of recruiting RNaseH, such as 6-16 DNA nucleosides, such as 10-15 contiguous DNA nucleosides, such as 10-14 contiguous DNA nucleotides, such as 11-15 contiguous DNA nucleotides, such as a region of 13-15 contiguous DNA nucleotides.

LNA spacer polymers

An LNA gapmer is one in which one or both of regions F and F' comprise or consist of LNA nucleosides. A β -D-oxygapmer is a gapmer in which one or both of regions F and F' comprise or consist of β -D-oxylna nucleosides.

In some embodiments, the LNA gapmer has the formula: [ LNA]_1-5- [ region G]-[LNA]_1-5Wherein region G is as defined in the definition of gapmer region G.

MOE gapped mers

A MOE gapmer is one in which regions F and F' consist of MOE nucleosides. In some embodiments, the MOE gapmer is designed as [ MOE]_1-8- [ region G]-[MOE]_1-8Such as [ MOE]_2-7- [ region G]_5-16-[MOE]_2-7Such as [ MOE]_3-6- [ region G]-[MOE]_3-6Wherein region G is as defined in the definition of gapmer. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.

Mixed-wing spacer polymers

The mixed aerofoil gapmers are LNA gapmers wherein one or both of regions F and F 'comprise a 2' substituted nucleoside, such as a MOE nucleoside, independently selected from the group consisting of a2 '-O-alkyl-RNA unit, a 2' -O-methyl-RNA, a2 '-amino-DNA unit, a 2' -fluoro-DNA unit, a2 '-alkoxy-RNA, a MOE unit, an arabinonucleic acid (ANA) unit, and a 2' -fluoro-ANA unit. In some embodiments, wherein at least one of regions F and F ' or both regions F and F ' comprise at least one LNA nucleoside, the remaining nucleosides of regions F and F ' are independently selected from the group consisting of MOE and LNA. In some embodiments, wherein at least one of regions F and F ' or both regions F and F ' comprise at least two LNA nucleosides, the remaining nucleosides of regions F and F ' are independently selected from the group consisting of MOE and LNA. In some hybrid airfoil embodiments, one or both of regions F and F' may further comprise one or more DNA nucleosides.

Hybrid airfoil gapmer designs are disclosed in WO2008/049085 and WO 2012/109395.

Alternating flanking gapmer

The flanking regions may contain both LNA and DNA nucleosides and are referred to as "alternating flanks" because they contain alternating motifs of LNA-DNA-LNA nucleosides. Notch polymers comprising such alternating flanks are referred to as "alternating flank notch polymers". Thus, an "alternative flanking gapmer" is an LNA gapmer oligonucleotide, wherein at least one flank (F or F') comprises a DNA nucleoside in addition to an LNA nucleoside. In some embodiments, at least one of regions F or F 'or both regions F and F' comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking regions F or F ', or both F and F ', comprise at least three nucleosides, wherein the 5' and 3' endmost nucleosides of the F and/or F ' region are LNA nucleosides.

The alternating flanking regions may comprise up to 3 consecutive DNA nucleosides, for example 1 to 2 or 1 or 2 or 3 consecutive DNA nucleosides.

Region D 'or D' in the oligonucleotide "

In some embodiments, the oligonucleotides of the invention may comprise or consist of a contiguous nucleotide sequence of oligonucleotides complementary to the target nucleic acid, such as gapmer F-G-F ', and additional 5' and/or 3' nucleosides. Additional 5 'and/or 3' nucleosides can be fully complementary or not fully complementary to the target nucleic acid. Such additional 5' and/or 3' nucleosides may be referred to herein as regions D ' and D ".

The addition of region D' or D "may be used for the purpose of linking a contiguous nucleotide sequence, such as a gapmer, to a conjugate moiety or another functional group. When used to join a contiguous nucleotide sequence to a conjugate moiety, it can serve as a biocleavable linker. Alternatively, it may be used to provide exonuclease protection or to ease synthesis or manufacture.

The regions D ' and D ″ can be attached to the 5' end of region F or the 3' end of region F ', respectively, to yield the following formulas D ' -F-G-F ', F-G-F ' -D ″, or

Designing D ' -F-G-F ' -D '. In this case, F-G-F 'is the gapmer portion of the oligonucleotide, and region D' or D "constitutes a separate part of the oligonucleotide.

The regions D' or D "may independently comprise or consist of 1, 2,3, 4 or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to region F or F' are not sugar modified nucleotides such as DNA or RNA or base modified versions of these. The region D 'or D' may be used as a nuclease-sensitive, bio-cleavable linker (see definition of linker). In some embodiments, the additional 5 'and/or 3' terminal nucleotide is phosphodiester-liganded and is DNA or RNA. Nucleotide-based bio-cleavable linkers suitable for use as regions D' or D "are disclosed in WO2014/076195, including, for example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/113922, where they are used to ligate multiple antisense constructs (e.g. gapmer regions) within a single oligonucleotide.

In one embodiment, the oligonucleotide of the invention comprises the region D' and/or D "in addition to the contiguous nucleotide sequence constituting the gapmer.

In some embodiments, the oligonucleotides of the invention may be represented by the formula:

F-G-F'; in particular F_1-8-G_6-16-F’_2-8

D ' -F-G-F ', in particular D '_1-3-F_1-8-G_6-16-F’_2-8

F-G-F '-D', in particular F_1-8-G_6-16-F’_2-8-D”_1-3

D '-F-G-F' -D ', especially D'_1-3-F_1-8-G_6-16-F’_2-8-D”_1-3

In some embodiments, the internucleoside linkage between region D' and region F is a phosphodiester linkage. In some embodiments, the internucleoside linkage between region F' and region D "is a phosphodiester linkage.

Conjugates

The term conjugate, as used herein, refers to an oligonucleotide covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

Conjugation of the oligonucleotides of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments, the conjugate moiety modifies or enhances the pharmacokinetic properties of the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular, the conjugates can target the oligonucleotide to a particular organ, tissue, or cell type, thereby enhancing the effectiveness of the oligonucleotide in that organ, tissue, or cell type. Also, the conjugates can be used to reduce the activity of the oligonucleotide in a non-target cell type, tissue or organ, such as off-target activity or activity in a non-target cell type, tissue or organ.

In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a carbohydrate, a cell surface receptor ligand, a drug, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

In some embodiments, the conjugate is an antibody or antibody fragment having specific affinity for transferrin receptor, for example as disclosed in WO 2012/143379, incorporated herein by reference. In some embodiments, the non-nucleotide moiety is an antibody or antibody fragment, such as an antibody or antibody fragment that facilitates delivery across the blood brain barrier, particularly an antibody or antibody fragment that targets the transferrin receptor.

Joint

A bond or linker is a connection between two atoms that links one target chemical group or segment to another target chemical group or segment via one or more covalent bonds. The conjugate moiety may be attached to the oligonucleotide directly or through a linking moiety (e.g., a linker or tether). The linker is used to covalently link the third region, such as a conjugate moiety (region C), to the first region, e.g., an oligonucleotide or contiguous nucleotide sequence (region a) complementary to the target nucleic acid.

In some embodiments of the invention, a conjugate or oligonucleotide conjugate of the invention may optionally comprise a linker region (second region or region B and/or region Y) between the oligonucleotide or contiguous nucleotide sequence (region a or first region) complementary to the target nucleic acid and the conjugate moiety (region C or third region).

Region B refers to a biocleavable linker comprising or consisting of a physiologically labile bond that is cleavable under conditions typically encountered in the mammalian body or similar thereto. Conditions under which the physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions, such as pH, temperature, oxidizing or reducing conditions or agents, and salt concentrations encountered in, or similar to, mammalian cells. Mammalian intracellular conditions also include enzymatic activities typically present in mammalian cells, such as enzymatic activities from proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biologically cleavable linker is susceptible to cleavage by S1 nuclease. In a preferred embodiment, the nuclease-sensitive linker comprises 1 to 10 nucleosides, such as 1, 2,3, 4,5, 6, 7, 8, 9 or 10 nucleosides, more preferably 2 to 6 nucleosides, most preferably 2 to 4 linked nucleosides comprising at least two consecutive phosphodiester linkages, such as at least 3 or 4 or 5 consecutive phosphodiester linkages. Preferably, the nucleoside is DNA or RNA. The phosphodiester-containing biocleavable linker is described in detail in WO2014/076195 (incorporated herein by reference), see also regions D' or D "herein.

Region Y refers to a linker that is not necessarily bio-cleavable but is primarily used to covalently link the conjugate moiety (region C or third region) to the oligonucleotide (region a or first region). The region Y linker may comprise a chain structure or repeating units such as ethylene glycol, amino acid units or oligomers of aminoalkyl groups. The oligonucleotide conjugates of the invention may be composed of the following regional unit elements: A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments, the linker (region Y) is an aminoalkyl group, such as a C2-C36 aminoalkyl group, including, for example, a C6 to C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

Treatment of

The term "treatment" as used herein refers to the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention or prophylaxis of a disease. Thus, it will be appreciated that in some embodiments, the treatment referred to herein may be prophylactic.

In some embodiments, a patient who has been diagnosed with a neurological disorder, such as a neurological disorder selected from the group consisting of neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), alzheimer's frontotemporal dementia (FTD), parkinson's syndrome, and conditions with TDP-43 proteinopathies, is treated.

In some embodiments, the compounds of the invention are used to treat spinocerebellar ataxia type 2 (SCA2) or Amyotrophic Lateral Sclerosis (ALS).

Detailed Description

Oligonucleotides of the invention

The present invention relates to oligonucleotides capable of modulating ATXN2 expression, such as inhibiting (down-regulating) ATXN 2. Modulation is achieved by hybridization to a target nucleic acid encoding Ataxin 2. The target nucleic acid can be a mammalian ATXN2 sequence, such as a sequence selected from the group consisting of SEQ ID NOs: 1. 2,3, 4 and 5.

The oligonucleotides of the invention are antisense oligonucleotides targeted to ATXN 2. It is advantageous if the antisense oligonucleotide is complementary to a target sequence selected from one of the regions listed in tables 4 to 6. In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is at least 90% complementary, such as fully complementary, to the selected target sequence of R1-R2421 (table 4). In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is at least 90% complementary, such as fully complementary, to the selected target sequence of W1-W115 (table 5). In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is at least 90% complementary, such as fully complementary, to the selected target sequence of S1-S46 (table 6).

In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is at least 90% complementary, such as fully complementary, to an intron region of the ATAXN2 target nucleic acid sequence, such as the selected target sequence of i1-i24 of SEQ ID NO1 (table 1).

In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is at least 90% complementary, such as fully complementary, to intron 1, 3, 5, 9, 10, 11, 14, 18, 20, or 21 of a human ATAXN2 precursor mRNA, such as i1, i3, i9, or i18 (table 1) of SEQ ID NO 1.

In embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is at least 90% complementary, such as fully complementary, to exon 4,5, or 25 of a human ATAXN2 precursor mRNA, such as e25 (table 1) from SEQ ID NO 1.

In embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is identical to an exon/intron spanning region of intron 4/exon 5 or exon 9/intron 10 of the human ATAXN2 pre-mRNA such as SEQ ID NO:1 is at least 90% complementary, such as fully complementary.

In some embodiments, the antisense oligonucleotides of the invention are capable of modulating the expression of a target by inhibiting or down regulating the expression of the target. Preferably, such modulation results in at least 20% inhibition of expression compared to the normal expression level of the target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% inhibition compared to the normal expression level of the target. In some embodiments, the oligonucleotides of the invention may be capable of inhibiting the expression level of ATXN2 mRNA in vitro by at least 60% or 70% after applying 25 μ M oligonucleotide to a431 or U2-OS cells. In some embodiments, the compounds of the invention are capable of inhibiting the expression level of ATXN2 protein in vitro by at least 50% using 5 μ M oligonucleotides applied to a431 or U2-OS cells. Suitably, these examples provide assays useful for measuring inhibition of ATXN2 mRNA or protein (e.g., examples 1 and 2). Target modulation is triggered by hybridization between a contiguous nucleotide sequence of an oligonucleotide and a target nucleic acid. In some embodiments, the oligonucleotides of the invention comprise a mismatch between the oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization to the target nucleic acid may be sufficient to show the desired modulation of ATXN2 expression. The reduction in binding affinity caused by mismatches may advantageously be compensated by an increase in the number of nucleotides in the oligonucleotide and/or an increase in the number of modified nucleotides capable of increasing binding affinity to the target, such as 2' sugar modified nucleotides present in the oligonucleotide sequence, including LNA.

One aspect described herein relates to an antisense oligonucleotide 10 to 30 nucleotides in length, comprising a contiguous nucleotide sequence 10 to 30 nucleotides in length, and having a sequence identical to SEQ ID NO:1 have at least 90% complementarity, such as 100% complementarity. It is generally understood that the contiguous nucleotide sequence is the same length as or shorter than the antisense oligonucleotide.

In some embodiments, the oligonucleotide sequence or contiguous nucleotide sequence of the invention is identical to SEQ ID NO:1 and SEQ ID NO:2 has 100% complementarity. In some embodiments, the oligonucleotide sequence or contiguous nucleotide sequence is identical to SEQ ID NO:1 and SEQ ID NO:5 has 100% complementarity. In some embodiments, the oligonucleotide sequence or contiguous nucleotide sequence is identical to SEQ ID NO: 1. the corresponding target nucleic acid regions present in2 and 3 have 100% complementarity. In some embodiments, the oligonucleotide sequence or contiguous nucleotide sequence is identical to SEQ ID NO: 1. 3 and 5 have 100% complementarity. In some embodiments, the oligonucleotide sequence or contiguous nucleotide sequence is identical to SEQ ID NO: 1. 2,3, 4 and 5 have 100% complementarity.

In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary, such as 100% complementary, to a corresponding target sequence (R _1-R _2421) selected from any region in table 4. In particular, the target sequence may be selected from the group consisting of R _1-R _13, R _15-R _874, R _876-R _894, R _896-R _902, R _906-R _1151, R _1153-R _1338, R _1341-R _1420, R _1422-R _1435, R _1437-R _1465, R _1468-R _1495, R _1499-R _1542, R _1545-R _1592, R1595-R _1602, R _1604-R _1643, R _1646-R _1869, R _1873-R _1905, R _1907-R _1921, R _1923-R _1929, R _1931-R _2145, R _2147-R _2152, R _ 2235-R _ 1906, R _ 1908-R _ 236, R _ 2158-R _ 2354, R _2404-R _ 2403, R _ 2401-R _ 2353, R _ 2403, R _ 2353, R _ 2401-R _ 2353, R _ 2403, R _ R, R _2409-R _2415 and R _ 2421.

In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary, such as 100% complementary, to a corresponding target sequence (R _1-R _2421) selected from any region in table 4. In particular, the target sequence may be selected from one of the regions within the group of regions consisting of R _274, R _560, R _596, R _716, R _893, R _895, R _903, R _905, R _1033, R _1421, R _1467, R _1496, R _1498, R _1537, R _1554, R _1690, R _1992, R _2185, and R _ 2420.

In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary, such as 100% complementary, to a corresponding target sequence (R _1-R _2421) selected from any region in table 4. In particular, the target sequence may be selected from one of the regions within the group of regions consisting of R _274, R _893, R _895, R _1496, R _1992 and R _ 2420.

In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length having at least 90% complementarity, such as 100% complementarity, to a corresponding target sequence selected from any region of table 5 (W1-W115). Specifically, the target sequence may be selected from one of the regions consisting of W1, W4, W5, W6, W7, W8, W9, W10, W11, W12, W13, W14, W15, W16, W17, W18, W19, W20, W21, W22, W23, W24, W25, W26, W.

In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length having at least 90% complementarity, such as 100% complementarity, to a corresponding target sequence, which is a sequence selected from any region in table 5 (S1-S46). In particular, the target sequence may be selected from one of the regions of the group consisting of S1, S2, S3, S5, S6, S9, S10, S11, S12, S14, S15, S16, S19, S21. One of the regions of the group of regions consisting of S22, S25, S26, S27, S28, S29, S30, S31, S33, S36, S41, S43, S45 and S46.

In one embodiment, the oligonucleotide comprises or consists of a contiguous nucleotide sequence of 10 to 22 nucleotides in length that is at least 90% complementary, such as 100% complementary, to the target sequence of region S19.

One aspect of the invention relates to an antisense oligonucleotide 10 to 30 nucleotides in length comprising a contiguous nucleotide sequence of 10 to 22 nucleotides in length having at least 90% complementarity, such as 100% complementarity, with SEQ ID NOs 6, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, and 1525.

In some embodiments, the oligonucleotide comprises a contiguous sequence of 10 to 30, such as 10-22 nucleotides in length that is identical to a sequence selected from the group consisting of SEQ ID NOs: 6. 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524 and 1525 have at least 90% complementarity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementarity.

In some embodiments, the oligonucleotide comprises a contiguous sequence of 10 to 30, such as 10-22 nucleotides in length that is identical to a sequence selected from the group consisting of SEQ ID NOs: 1505. 1509, 1510, 1516, 1522 and 1525 have at least 90% complementarity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementarity.

It is advantageous if the oligonucleotide of the invention or a contiguous nucleotide sequence thereof is fully complementary (100% complementary) to the target sequence, or in some embodiments may comprise one or two mismatches between the oligonucleotide and the target nucleic acid.

In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 22 nucleotides in length that hybridizes to SEQ ID NO:1 such as the target nucleic acid region at positions 83118 to 83146 of SEQ ID NO: positions 83122 to 83143 of 1 have at least 90% complementarity, such as full (or 100%) complementarity.

In some embodiments, the oligonucleotide of the invention comprises or consists of nucleotides of 10 to 30 nucleotides in length, such as 11 to 28, such as 10 to 22, such as 12 to 22, such as 14 to 20, such as 15 to 20, such as 16 to 18, such as 17 to 20 or 18 to 20 consecutive nucleotides in length. In a preferred embodiment, the oligonucleotide comprises or consists of nucleotides of length 17 to 20.

In some embodiments, the oligonucleotide or contiguous nucleotide sequence thereof comprises or consists of 24 or fewer nucleotides, such as 22 or fewer nucleotides, such as 20 or fewer nucleotides, such as 17, 18, 19, or 20 nucleotides. It should be understood that any range given herein includes the end of the range. Thus, if an oligonucleotide is said to comprise 10 to 30 nucleotides, both 10 and 30 nucleotides are included.

In some embodiments, the contiguous nucleotide sequence comprises or consists of contiguous nucleotides 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 in length. In a preferred embodiment, the oligonucleotide comprises or consists of 17, 18, 19 or 20 nucleotides in length.

In some embodiments, the oligonucleotide or contiguous nucleotide sequence comprises or consists of a sequence selected from table 7.

In some embodiments, the oligonucleotide or contiguous nucleotide sequence comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34 (table 7 "materials and methods" section).

In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises 10 to 30 nucleotides, such as 10-22 nucleotides in length, that hybridize to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7. 13, 14, 15, 17, 18105, 154, 161, 162, 238, 385, 388, 391, 398, 399, 401, 423, 468, 477, 534, 843, 844, 845, 847, 848, 849, 850, 851, 852, 853, 854, 906, 974, 1003, 1004, 1045, 1054, 1180, 1246, 1247, 1248, 1361, 1408 and 1504 have at least 90% identity, preferably 100% identity.

In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises 10 to 30, such as 10-22, nucleotides that hybridize to a sequence selected from the group consisting of SEQ ID NOs: 7. 13, 14, 15, 17, 18, 105, 385, 388, 391, 1246, 1247, 1248 and 1504 have or consist of at least 90% identity, preferably 100% identity.

It will be appreciated that the contiguous nucleobase sequence (motif sequence) may be modified, for example, to increase nuclease resistance and/or binding affinity to a target nucleic acid.

The mode of incorporation of modified nucleosides (e.g., high affinity modified nucleosides) into oligonucleotide sequences is commonly referred to as oligonucleotide design.

Modified nucleosides and DNA nucleosides are used to design oligonucleotides of the invention. Advantageously, high affinity modified nucleosides are used.

In one embodiment, the oligonucleotide comprises at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In one embodiment, the oligonucleotide comprises 1 to 10 modified nucleosides, such as 2 to 9 modified nucleosides, such as 3 to 8 modified nucleosides, such as 4 to 7 modified nucleosides, such as 6 or 7 modified nucleosides. Suitable modifications are described in the "modified nucleosides", "high affinity modified nucleosides", "sugar modifications", "2' sugar modifications" and "Locked Nucleic Acids (LNAs)" of the "definitions" section.

In one embodiment, the oligonucleotide comprises one or more sugar modified nucleosides, for example, 2' sugar modified nucleosides. Preferably, the oligonucleotide of the invention comprises one or more 2 'sugar modified nucleosides independently selected from the group consisting of 2' -O-alkyl-RNA, 2 '-O-methyl-RNA, 2' -alkoxy-RNA, 2 '-O-methoxyethyl-RNA, 2' -amino-DNA, 2 '-fluoro-DNA, arabinonucleic acid (ANA), 2' -fluoro-ANA, and LNA nucleosides. It is advantageous if the one or more modified nucleosides are Locked Nucleic Acids (LNA).

In another embodiment, the oligonucleotide or contiguous nucleotide sequence comprises at least one modified internucleoside linkage, such as at least one phosphorothioate internucleoside linkage. Suitable internucleoside modifications are described in the "modified internucleoside linkages" of the "definitions" section. It is advantageous if at least 75% such as all internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate internucleoside linkages. In one embodiment, all internucleoside linkages in the contiguous sequence of the oligonucleotide are phosphorothioate linkages and are then strong.

In some embodiments, the oligonucleotide of the invention comprises at least one LNA nucleoside, such as 1, 2,3, 4,5, 6, 7 or 8 LNA nucleosides, such as 2 to 6 LNA nucleosides, such as 3 to 7 LNA nucleosides, 4 to 8 LNA nucleosides or 3, 4,5, 6, 7 or 8 LNA nucleosides. In some embodiments, at least 75% of the modified nucleosides in the oligonucleotide are LNA nucleosides, such as 80%, such as 85%, such as 90% of the modified nucleosides are LNA nucleosides. In another embodiment, all modified nucleosides in the oligonucleotide are LNA nucleosides. In another embodiment, the oligonucleotide may comprise both β -D-oxy-LNA and one or more of the following LNA nucleosides in either the β -D or α -L configuration: thio-LNA, amino-LNA, oxy-LNA, ScET and/or in ENA or combinations thereof. In another embodiment, all LNA cytosine units are 5-methylcytosine. For nuclease stability of an oligonucleotide or a contiguous nucleotide sequence, it is advantageous to have at least 1 LNA nucleotide at the 5 'end of the nucleotide sequence and at least 2 LNA nucleotides at the 3' end of the nucleotide sequence.

In one embodiment of the invention, the oligonucleotide of the invention is capable of recruiting RNase H, such as human RNase H1.

In the context of the present invention, advantageous structural designs are the gapmer designs as described in the section "definitions", for example in "gapmers", "LNA gapmers", "MOE gapmers" and "mixed-wing gapmers", "alternating flanking gapmers". The notch-mer design includes notch-mers with uniform flanks, mixed-wing flanks, alternating flanks, and notch breaker designs. In the present invention, it is advantageous if the oligonucleotide of the invention is a gapmer with a F-G-F 'design, wherein the regions F and F' independently comprise 1-8 nucleosides, of which 1-5 are modified with a2 'sugar and define the 5' and 3 'ends of the regions F and F', G being a region between 6 and 16 nucleosides capable of recruiting RNaseH. In one embodiment, the G region consists of 6-16 contiguous DNA nucleosides. In another embodiment, regions F and F' each comprise at least one LNA nucleoside.

Table 7 ("materials and methods" section) lists the preferred design of each motif sequence.

In all cases, the F-G-F ' design may also include regions D ' and/or D ", as described in region D ' or D" "in the" definitions "section" oligonucleotides. In some embodiments, the oligonucleotides of the invention have 1, 2, or 3 phosphodiester linked nucleoside units, such as DNA units, at the 5 'or 3' end of the gapmer region.

For some embodiments of the invention, the oligonucleotide is selected from the group of oligonucleotide compounds having CMP-ID-NO:7_1, 8_1, 9_1, 10_1, 11_1, 12_1, 13_1, 14_1, 15_1, 16_1, 17_1, 18_1, 19_1, 20_1, 21_1, 22_1, 23_1, 24_1, 25_1, 26_1, 27_1, 28_1, 29_1, 30_1, 31_1, 32_1, 33_1, and 34_1 (see Table 7 of the materials and methods section).

For certain embodiments of the invention, the oligonucleotide is selected from the group of oligonucleotides having CMP-ID-NO 7_1, 13_1, 14_2, 15_1, 16_1, 17_1, 18_2, 105_1, 154_1, 161_1, 162_1, 238_1, 385_1, 388_1, 391_1, 398_1, 399_1, 401_2, 423_1, 468_1, 477_1, 534_1, 843_1, 844_1, 845_1, 847_1, 848_1, 849_1, 850_1, 851_1, 852_1, 853_1, 854_1, 906_1, 974_1, 1003_1, 1004_1, 1045_1, 1054_1, 1180_1, 1408_ 6_1, 1247_1, 1248_1, 1241, 1361_ 1504_1, and 1504_1 (see Table 7).

In the context of the present invention, it is particularly advantageous that the antisense oligonucleotide is a compound selected from the group consisting of:

wherein the capital letters are β -D-oxy LNA nucleosides, the lowercase letters are DNA nucleosides, all LNA Cs are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages.

The invention provides a conjugate comprising an oligonucleotide or an antisense oligonucleotide according to the invention, and at least one conjugate moiety covalently attached to said oligonucleotide. In some embodiments, the conjugate moiety is a conjugate that facilitates delivery across the blood brain barrier, such as an antibody or antibody fragment that targets the transferrin receptor.

Manufacturing method

In another aspect, the invention provides a method for making an oligonucleotide of the invention, comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al,1987, Methods in Enzymology, Vol.154, p.287-313). In another embodiment, the method further comprises reacting the contiguous nucleotide sequence with a conjugate moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In another aspect, there is provided a method for the manufacture of a composition of the invention, comprising mixing an oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

Pharmaceutically acceptable salts

The compounds according to the invention may be present in the form of their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to conventional acid addition salts or base addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid addition salts include, for example, those derived from inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, phosphoric and nitric acids, and those derived from organic acids such as p-toluenesulfonic, salicylic, methanesulfonic, oxalic, succinic, citric, malic, lactic, fumaric, and the like. Base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides such as tetramethylammonium hydroxide. Chemical modification of pharmaceutical compounds into salts in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of the compounds is a well-known technique among pharmaceutical chemists. For example, Basin Organic Process Research & Development, No. 4, pages 427 and 435 or Ansel are described in the following articles: pharmaceutical Dosage Forms and Drug Delivery Systems,6th ed. (1995) pages 196 and 1456-. For example, a pharmaceutically acceptable salt of a compound provided herein can be a sodium salt.

In another aspect, the invention provides pharmaceutically acceptable salts of antisense oligonucleotides or conjugates thereof. In a preferred embodiment, the pharmaceutically acceptable salt is a sodium or potassium salt.

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. Pharmaceutically acceptable diluents include Phosphate Buffered Saline (PBS), while pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used in a pharmaceutically acceptable diluent at a concentration of 50-300 μ M solution.

Suitable formulations for use in the present invention may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed., 1985. For a brief review of drug delivery methods, see, e.g., Langer (Science 249: 1527) -1533, 1990). WO2007/031091 (incorporated herein by reference) provides other suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants. Suitable doses, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations are also provided in WO 2007/031091.

The oligonucleotide or oligonucleotide conjugate of the invention may be mixed with a pharmaceutically acceptable active or inert substance to prepare a pharmaceutical composition or formulation. The composition and method of formulation of the pharmaceutical composition depends on a number of criteria including, but not limited to, the route of administration, the extent of the disease or the dosage administered.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, and the lyophilized formulation combined with a sterile aqueous carrier prior to administration. The pH of the formulation is typically from 3 to 11, more preferably from 5 to 9 or from 6 to 8, most preferably from 7 to 8, such as from 7 to 7.5. The resulting composition in solid form may be packaged in a plurality of single dose units, each unit containing a fixed amount of one or more of the agents described above, such as in a sealed package of tablets or capsules. Compositions in solid form may also be packaged in flexible quantities in containers, such as squeezable tubes for creams or ointments designed for topical use.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular for oligonucleotide conjugates, the conjugate moiety is cleaved from the oligonucleotide once the prodrug is delivered to the site of action, such as a target cell.

Applications of

The oligonucleotides of the invention are useful as research reagents, e.g., for diagnosis, treatment and prevention.

In research, such oligonucleotides can be used to specifically modulate the synthesis of Ataxin2 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or assessing its availability as a target for therapeutic intervention. Typically, target modulation is achieved by degradation or inhibition of the mRNA producing the protein, thereby preventing protein formation, or by degradation or inhibition of a modulator of the gene or mRNA producing the protein.

The invention provides an in vivo or in vitro method of modulating the expression of ATXN2 in target cells expressing ATXN2 comprising administering to said cells an effective amount of an oligonucleotide of the invention.

In some embodiments, the target cell is a mammalian cell, particularly a human cell. The target cell may be an in vitro cell culture or an in vivo cell forming part of a mammalian tissue. In preferred embodiments, the target cell is present in the brain or central nervous system, including the brainstem and spinal cord. In particular, cells in the cerebellum are relevant target cells, such as purkinje neurons or purkinje cells, in particular in individuals with spinocerebellar ataxia type 2 (SCA 2).

Other relevant target cells are motor neurons located in the cerebral cortex and spinal cord. Upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord are the target cells of the present invention. In particular, motor neurons in individuals affected by Amyotrophic Lateral Sclerosis (ALS) are relevant target cells.

In diagnostics, oligonucleotides can be used to detect and quantify ATXN2 expression in cells and tissues by northern blotting, in situ hybridization, or similar techniques.

For treatment, the oligonucleotide may be administered to an animal or human suspected of having a disease or disorder, which may be treated by modulating the expression of ATXN 2.

The invention provides a method for treating or preventing a disease, the method comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention.

The invention also relates to an oligonucleotide, composition or conjugate as defined herein for use as a medicament.

The oligonucleotide, oligonucleotide conjugate or pharmaceutical composition according to the invention is generally administered in an effective amount.

The invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the invention as described in the manufacture of a medicament for the treatment of a disorder as described herein, or in a method of treatment of a disorder as described herein.

As referred to herein, the disease or disorder is associated with the expression of ATXN 2. In some embodiments, the disease or disorder may be associated with a mutation in the ATXN2 gene, such as an extended CAG repeat region. The disease or disorder may be associated with a gene whose protein product is associated with or interacts with ATXN 2. In particular, reduction of ATXN2 may have beneficial effects in diseases associated with TDP-43 proteinopathies, for example, in Amyotrophic Lateral Sclerosis (ALS), alzheimer frontotemporal dementia (FTD), and parkinsonism.

The methods of the invention are preferably used to treat or prevent diseases caused by abnormal levels and/or activity of ATXN 2.

The invention further relates to the use of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of ATXN 2.

In one embodiment, the invention relates to an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition for use in the treatment of a disease or disorder selected from the group consisting of neurodegenerative diseases including spinocerebellar ataxia 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia alzheimer (FTD), parkinson's syndrome and a disease with TDP-43 protein. In particular, the use of the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention for the treatment of spinocerebellar ataxia type 2 (SCA2) or Amyotrophic Lateral Sclerosis (ALS) is advantageous.

Administration of

The oligonucleotide or pharmaceutical composition of the invention may be administered parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intracerebroventricularly, intraocularly or intrathecally).

In some embodiments, the administering is by intrathecal administration.

Advantageously, for example for the treatment of neurological disorders, the oligonucleotide or pharmaceutical composition of the invention is administered intrathecally or intracranially, for example intracerebrally or intraventricularly.

The invention also provides the use of an oligonucleotide or a conjugate thereof, such as a pharmaceutically acceptable salt or composition of the invention, in the manufacture of a medicament, wherein the medicament is in a dosage form for subcutaneous administration.

The invention also provides the use of an oligonucleotide of the invention or a conjugate thereof, such as a pharmaceutically acceptable salt or composition of the invention, in the manufacture of a medicament, wherein the medicament is in a dosage form for intrathecal administration.

The invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the invention as described in the manufacture of a medicament, wherein the medicament is in a dosage form for intrathecal administration.

Combination therapy

In some embodiments, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention is used in combination therapy with another therapeutic agent. The therapeutic agent may be, for example, the standard of care for the above-mentioned disease or disorder.

Example (b):

the following embodiments of the invention may be used in conjunction with any of the other embodiments described herein.

1. An antisense oligonucleotide 10 to 50 nucleotides in length comprising a contiguous nucleotide sequence 10 to 30 nucleotides in length, having at least 90% complementarity, such as 100% complementarity, with any of the target sequences in table 4 (R _1-R _ 2421).

2. The oligonucleotide of example 1, wherein the target sequence is selected from the group consisting of R _1-R _13, R _15-R _874, R _876-R _894, R _896-R _902, R _906-R _1151, R _1153-R _1338, R _1341-R _1420, R _1422-R _1435, R _1437-R _1465, R _1468-R _1495, R _1499-R _1542, R _1545-R _1592, R1595-R _1602, R _1604-R _1643, R _1646-R _1869, R _ 3-R _1905, R _1907-R _1921, R _1923-R _1929, R _1931-R _2145, R _2147-R _2147, R _ 2235-R _2236, R _2238-R _ 2156, R _ 2156-R _ 2153-R _ 23573, R _ 2153-R _ 2353, R _ 2353-R _ 2353, R _1931-R _ 2153, R _ H, One of the regions within R _2404-R _2407, R _2409-R _2415, and R _ 2421.

3. The oligonucleotide of embodiment 1, wherein the target sequence is selected from one of the regions within the group R _274, R _560, R _596, R _716, R _893, R _895, R _903, R _905, R _1033, R _1421, R _1467, R _1496, R _1498, R _1537, R _1554, R _1690, R _1992, R _2185 and R _ 2420.

4. The oligonucleotide of example 1, wherein the target sequence is selected from one of the regions within the group R _274, R _893, R _895, R _1496, R _1992, and R _ 2420.

5. An oligonucleotide as described in example 1, wherein the target sequence is selected from one of the groups W1, W4, W5, W6, W7, W8, W9, W10, W11, W12, W13, W14, W15, W16, W17, W18, W19, W20, W21, W22, W23, W24, W25, W26.

6. The oligonucleotide of example 1, wherein the target sequence is selected from one of the regions of the group consisting of S1, S2, S3, S5, S6, S9, S10, S11, S12, S14, S15, S16, S19, S21, S22, S25, S26, S27, S28, S29, S30, S31, S33, S36, S41, S43, S45 and S46 (see table 6).

7. The oligonucleotide of embodiments 1 to 6, wherein the contiguous nucleotide sequence is identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1526. 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543 and 1544.

8. The oligonucleotide of embodiments 1 to 6, wherein the contiguous nucleotide sequence is identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 1526. 1530, 1531, 1537, 1542 and 1544

9. The oligonucleotide of embodiments 1 to 6, wherein the contiguous nucleotide sequence is identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 6. 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524 and 1525.

10. The oligonucleotide of embodiments 1 to 6, wherein the contiguous nucleotide sequence is identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 6. 1505, 1509, 1510, 1516, 1522 and 1525.

11. The antisense oligonucleotide of any one of embodiments 1 to 10, wherein the contiguous nucleotide sequence is identical to SEQ ID NO: positions 83118 to 83151 of 1 are complementary.

12. The oligonucleotide of examples 1 to 6, wherein the contiguous nucleotide sequence is identical to SEQ ID NO:6 is complementary to the target sequence.

13. The antisense oligonucleotide of any one of embodiments 1 to 12, wherein the contiguous nucleotide sequence is identical to SEQ ID NO: position 83122 to 83143 of 1 are complementary

14. The oligonucleotide of embodiments 1 to 13, wherein the antisense oligonucleotide is capable of modulating expression of ATXN2, such as reducing expression of ATXN 2.

15. The oligonucleotide of examples 1 to 14, wherein the antisense oligonucleotide is capable of hybridizing to the target sequence at a Δ G ° of less than-10 kcal.

16. The oligonucleotide of embodiments 1 to 15, wherein the target nucleic acid in which the target sequence is located is RNA.

17. The oligonucleotide of embodiment 16, wherein the RNA is mRNA.

18. The oligonucleotide of embodiment 17, wherein the mRNA is a precursor RNA or a mature RNA.

19. The oligonucleotide of embodiment 18, wherein the precursor mRNA is selected from the group consisting of SEQ ID NO: 1. 2,3, 4 or 5.

20. The oligonucleotide of embodiments 1 to 19, wherein a contiguous nucleotide sequence comprises or consists of at least 10 contiguous nucleotides, in particular 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 contiguous nucleotides.

21. The oligonucleotide of embodiments 1 to 20, wherein a contiguous nucleotide sequence comprises or consists of 10 to 22 nucleotides.

22. The oligonucleotide of embodiment 21, wherein a contiguous nucleotide sequence comprises or consists of 12 to 22 nucleotides.

23. The oligonucleotide of embodiment 21, wherein the contiguous nucleotide sequence comprises or consists of 14-20 nucleotides.

24. The oligonucleotide of embodiments 1 to 23, wherein the oligonucleotide comprises or consists of nucleotides of 10 to 30 in length.

25. The oligonucleotide of embodiment 24, wherein oligonucleotide comprises or consists of nucleotides of 12 to 22 in length.

26. The oligonucleotide of embodiment 24 or 25, wherein oligonucleotide comprises or consists of nucleotides of 14 to 20 in length.

27. The oligonucleotide of embodiments 1 to 26, wherein the oligonucleotide or contiguous nucleotide sequence is single-stranded.

28. The oligonucleotide of embodiments 1 to 27, wherein the oligonucleotide is neither siRNA nor self-complementary.

29. The oligonucleotide of embodiments 1 to 28, wherein a contiguous nucleotide sequence comprises or consists of a sequence in table 7.

30. The oligonucleotide of embodiments 1 to 29, wherein a contiguous nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34.

31. The oligonucleotide of embodiments 1 to 28, wherein a contiguous nucleotide sequence comprises or consists of a sequence selected from 7, 13, 14, 15, 17, 18, 105, 154, 161, 162, 238, 385, 388, 391, 398, 399, 401, 423, 468, 477, 534, 843, 844, 845, 847, 848, 849, 850, 851, 852, 853, 854, 906, 974, 1003, 1004, 1045, 1054, 1180, 1246, 1247, 1248, 1361, 1408, and 1504.

32. The oligonucleotide of embodiments 1-29, wherein a contiguous nucleotide sequence has zero to three mismatches compared to the target nucleic acid to which it is complementary.

33. The oligonucleotide of example 32, wherein the contiguous nucleotide sequence has one mismatch compared to the target nucleic acid.

34. The oligonucleotide of example 32, wherein the contiguous nucleotide sequence has two mismatches compared to the target nucleic acid.

35. The oligonucleotide of embodiment 32, wherein the contiguous nucleotide sequence is fully complementary to the target nucleic acid sequence.

36. The oligonucleotide of embodiments 1 to 35, comprising one or more modified nucleosides.

37. The oligonucleotide of embodiment 36, wherein the one or more modified nucleosides are high affinity modified nucleosides.

38. The oligonucleotide of embodiment 36 or 37, wherein the one or more modified nucleosides is a 2' sugar modified nucleoside.

39. The oligonucleotide of embodiment 38, wherein one or more 2' sugar modified nucleosides is independently selected from the group consisting of: 2 '-O-alkyl-RNA, 2' -O-methyl-RNA, 2 '-alkoxy-RNA, 2' -O-methoxyethyl-RNA, 2 '-amino-DNA, 2' -fluoro-ANA and LNA nucleosides.

40. The oligonucleotide of example 38 or 39, wherein one or more 2' sugar modified nucleosides is a LNA nucleoside.

41. The antisense oligonucleotide of embodiment 40, wherein the modified LNA nucleoside is selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA, cET and ENA.

42. The antisense oligonucleotide of embodiment 40 or 41, wherein the modified LNA nucleoside is a nucleoside having the following 2'-4' bridge-O-CH₂-oxy-LNA of (a).

43. The antisense oligonucleotide of embodiment 42, wherein the oxy-LNA is β -D-oxy-LNA.

44. The antisense oligonucleotide of embodiment 40 or 41, wherein the modified LNA nucleoside is a nucleoside having the following 2'-4' bridge-O-CH (CH)₃) cET of (E).

45. The antisense oligonucleotide of embodiment 44, wherein cET is (S) cET, i.e., 6' (S) methyl- β -D-oxy-LNA.

46. The antisense oligonucleotide of embodiment 40 or 41, wherein LNA is ENA, having the following 2'-4' bridge-O-CH₂-CH₂-。

47. The oligonucleotide of any one of embodiments 1 to 46, wherein oligonucleotide comprises at least one modified internucleoside linkage.

48. The oligonucleotide of embodiment 47, wherein the modified internucleoside linkage is nuclease resistant.

49. The oligonucleotide of embodiment 47 or 48, wherein at least 50% of the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate internucleoside linkage internucleoside linkages.

50. The oligonucleotide of embodiment 47 or 48, wherein all internucleoside linkages within a contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

51. The oligonucleotide of examples 1 to 50, wherein the oligonucleotide is capable of recruiting RNase H.

52. The oligonucleotide of embodiment 51, wherein the oligonucleotide or contiguous nucleotide sequence is a gapmer.

53. The oligonucleotide of embodiment 52, wherein the gapmer has the formula 5' -F-G-F ' -3', wherein the F and F ' wing regions independently comprise or consist of 1-8 2' sugar modified nucleosides, and G is between 6 and 16 regions capable of recruiting RNaseH

54. The oligonucleotide of example 53, wherein regions F and F' consist of the same LNA nucleosides.

55. The oligonucleotide of example 53 or 54, wherein all 2' sugar modified nucleosides in regions F and F are oxy-LNA nucleosides.

56. The oligonucleotide of embodiment 53, wherein at least one of region F or F 'further comprises at least one 2' substituted modified nucleoside independently selected from the group consisting of 2 '-O-alkyl-RNA, 2' -O-methyl-RNA, 2 '-alkoxy-RNA, 2' -O-methoxyethyl-RNA, 2 '-amino-DNA, and 2' -fluoro-DNA.

57. The oligonucleotide of examples 53 to 56, wherein RNaseH in region G recruits nucleosides independently selected from the group consisting of DNA, α -L-LNA, C4 'alkylated DNA, ANA, and 2' F-ANA and UNA.

58. The oligonucleotide of example 57, wherein the nucleosides in region G are DNA and/or alpha-L-LNA nucleosides.

59. The oligonucleotide of embodiment 57 or 58, wherein region G consists of at least 75% DNA nucleosides.

60. The antisense oligonucleotide of embodiments 57-59, wherein region G consists of 6 to 16 DNA nucleosides.

61. The oligonucleotide of embodiments 1 to 60, wherein the oligonucleotide is selected from the group consisting of CMP ID NO 7_1, 8_1, 9_1, 10_1, 11_1, 12_1, 13_1, 14_1, 15_1, 16_1, 17_1, 18_1, 19_1, 20_1, 21_1, 22_1, 23_1, 24_1, 25_1, 26_1, 27_1, 28_1, 29_1, 30_1, 31_1, 32_1, 33_1, and 34_ 1.

62. The oligonucleotide of embodiments 1 to 60, wherein the oligonucleotide is selected from the group consisting of CMP ID NO 7_1, 13_1, 14_2, 15_1, 16_1, 17_1, 18_2, 105_1, 154_1, 161_1, 162_1, 238_1, 385_1, 388_1, 391_1, 398_1, 399_1, 401_2, 423_1, 468_1, 477_1, 534_1, 843_1, 844_1, 845_1, 847_1, 848_1, 849_1, 850_1, 851_1, 852_1, 853_1, 854_1, 906_1, 974_1, 1003_1, 1004_1, 1045_1, 1054_1, 1180_1, 1246_1, 8_ 7_ 1241, 8_1, 1408_1, 1361_ 1361, 1361_ 1504_1, and 1504_ 1.

63. The oligonucleotide of any one of embodiments 1 to 60, wherein oligonucleotide is a compound selected from the group consisting of

Wherein the capital letters are beta-D-oxyLNA nucleosides, the lowercase letters are DNA nucleosides, all LNA C are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages

64. A conjugate comprising an oligonucleotide according to any one of embodiments 1 to 63, and at least one conjugate moiety covalently attached to the oligonucleotide.

65. The oligonucleotide conjugate of embodiment 64, wherein the conjugate moiety is selected from the group consisting of a carbohydrate, a cell surface receptor ligand, a drug, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin, a vitamin, a viral protein, or a combination thereof.

66. The oligonucleotide conjugate of embodiment 64 or 65, wherein the conjugate facilitates delivery across the blood-brain barrier.

67. The oligonucleotide conjugate of embodiment 66, wherein the conjugate is an antibody or antibody fragment targeting transferrin receptor.

68. A pharmaceutical composition comprising an oligonucleotide as described in examples 1 to 64 or a conjugate as described in examples 63 to 67 and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.

69. A method of making an oligonucleotide as described in examples 1 to 63, comprising reacting nucleotide units, thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide.

70. The method of embodiment 69, further comprising reacting the contiguous nucleotide sequence with a non-nucleotide conjugate moiety.

71. A method for making the composition of example 68, comprising mixing an oligonucleotide with a pharmaceutically acceptable diluent, carrier, salt, and/or adjuvant.

72. An in vivo or in vitro method for modulating the expression of ATXN2 in target cells expressing ATXN2, said method comprising administering to said cells the oligonucleotide of examples 1 to 63 or the conjugate of examples 64 to 67 or the pharmaceutical composition of example 68 in an effective amount.

73. A method of treating or preventing a disease, the method comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of an oligonucleotide as described in examples 1 to 63 or a conjugate as described in examples 64 to 67 or a pharmaceutical composition as described in example 68.

74. An oligonucleotide as described in examples 1 to 63 or a conjugate as described in examples 64 to 67 or a pharmaceutical composition as described in example 68 for use as a medicament for treating or preventing a disease in a subject.

75. Use of an oligonucleotide as described in examples 1 to 63 or a conjugate as described in examples 64 to 67 in the manufacture of a medicament for treating or preventing a disease in a subject.

76. The method, oligonucleotide or use of embodiments 73 to 75, wherein the disease is associated with the in vivo activity of ATXN 2.

77. The method, oligonucleotide or use of embodiments 73 to 76, wherein the disease is associated with overexpression of ATXN2 and/or abnormal levels of ATXN 2.

78. The method, oligonucleotide or use of embodiment 77, wherein ATXN2 is reduced by at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or at least 95% compared to expression without the use of the oligonucleotide of embodiments 1 to 63 or the conjugate of embodiments 64 to 67 or the pharmaceutical composition of embodiment 68.

79. The method, oligonucleotide or use of embodiments 73 to 77, wherein the disease is selected from spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia Alzheimer's (FTD), Parkinson's syndrome and having TDP-43 proteinopathy.

80. The method, oligonucleotide or use of embodiments 73-79, wherein the subject is a mammal.

81. The method, oligonucleotide or use of embodiment 80, wherein the mammal is a human.

Examples of the invention

Materials and methods

Oligonucleotide motif sequence and oligonucleotide compound

Table 7: list of oligonucleotide motif sequences (indicated by SEQ ID NO), design of these sequences and specific oligonucleotide compounds (indicated by CMP ID NO) designed based on motif sequences.

Motif sequences represent a contiguous sequence of nucleobases present in an oligonucleotide.

Design refers to gapmer design F-G-F ', where each number represents the number of consecutive modified nucleosides, e.g., 2' modified nucleosides (first number 5' flanking), followed by the number of DNA nucleosides (second number 3' flanking), followed by the number of modified nucleosides, e.g., 2' modified nucleosides, optionally before or after other repeated regions of DNA and LNA, which regions are not necessarily part of a consecutive nucleotide sequence complementary to the target nucleic acid.

Oligonucleotide compounds represent a specific design of motif sequences. Capital letters represent β -D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and 5-methylDNA cytosine is denoted by "e" and all internucleoside linkages are phosphorothioate internucleoside linkages.

Oligonucleotide synthesis

Oligonucleotide synthesis is well known in the art. The following are applicable schemes. The oligonucleotides of the invention can be produced by slightly varying methods, as far as the equipment, support and concentration are used.

Oligonucleotides were synthesized on a 1 μmol scale on a uridine universal support using the phosphoramidite method on an Oligomaker 48. At the end of the synthesis, the oligonucleotides were cleaved from the solid support using ammonia at 60 ℃ for 5-16 hours. The oligonucleotides were purified by reverse phase HPLC (RP-HPLC) or by solid phase extraction, characterized by UPLC, and further confirmed for molecular weight by ESI-MS.

Extension of the oligonucleotide:

coupling of β -cyanoethylphosphonite (DNA-A (Bz), DNA-G (ibu), DNA-C (Bz), DNA-T, LNA-5-methyl-C (Bz), LNA-A (Bz), LNA-G (dmf) or LNA-T) was carried out by using a 0.1M solution of 5' -O-DMT protected imide in acetonitrile and DCI (4, 5-dicyanoimidazole) as an activator in acetonitrile (0.25M). For the last cycle, phosphoramidites with the desired modification can be used, for example, a C6 linker for attachment of a conjugate group or such a conjugate group. Thiolation was performed by using hydroxanthatin (0.01M in acetonitrile/pyridine 9: 1) to introduce phosphorothioate linkages. The phosphate diester linkage can be introduced using a 0.02M solution of iodine in THF/pyridine/water 7:2: 1. The remaining reagents are those commonly used in oligonucleotide synthesis.

For conjugation after solid phase synthesis, a commercially available C6 amino linker phosphoramidite can be used in the last cycle of solid phase synthesis, and after deprotection and cleavage from the solid support, the amino linked deprotected oligonucleotide is isolated. The conjugates are introduced by activation of the functional groups using standard synthetic methods.

Purification by RP-HPLC:

the crude compound was purified by preparative RP-HPLC on a Phenomenex Jupiter C1810 μ 150X10 mm column. 0.1M ammonium acetate pH 8 and acetonitrile were used as buffers at a flow rate of 5 mL/min. The collected fractions were lyophilized to give the purified compound, usually as a white solid.

Abbreviations:

DCI: 4, 5-dicyanoimidazole

DCM: methylene dichloride

DMF: dimethyl formamide

4, 4' -Dimethyltrimethyl DMT

THF: tetrahydrofuran (THF)

Bz: benzoyl radical

Ibu: isobutyryl radical

RP-HPLC: reversed phase high performance liquid chromatography

T_mAnd (3) determination:

oligonucleotide and RNA target (phosphate-linked, PO) duplexes were diluted to 3mM in 500ml RNase-free water and combined with 500ml 2x T_mBuffer (200 mM)NaCl, 0.2mM EDTA, 20mM sodium phosphate, pH 7.0). The solution was heated at 95 ℃ for 3 minutes and then annealed at room temperature for 30 minutes. The duplex melting temperatures (T.sub.m) were measured using PE Templab software on a Lambda 40UV/VIS spectrophotometer (Perkin Elmer) equipped with a Peltier temperature programmer PTP6_m). The temperature was raised from 20 ℃ to 95 ℃ and then lowered to 25 ℃ and the absorbance at 260nm was recorded. Evaluation of duplex T Using first derivative and local maxima of melting and annealing_m。

Cell lines

TABLE 8 details relating to the cell lines used in examples 1 and 2

All media and additives were purchased from Sigma Aldrich

Example 1: testing of LNA oligonucleotides for in vitro efficacy in A431, NCI-H23 and ARPE19 cell lines at 25 and 5. mu.M

Oligonucleotide screening was performed in three human cell lines using the LNA oligonucleotides in Table 7 (CMP ID NO:7-35_1) targeting the sequences shown in SEQ ID NO: region 83121 to 83144 of 1. Human cell lines A341, NCI-H23, and ARPE19 were purchased from the suppliers listed in Table 8 and maintained in a humidified incubator at 37 ℃ and 5% CO2 as recommended by the supplier. For the screening assay, cells were seeded in 96-well plates using the supplier's recommended media (see table 8 in the "materials and methods" section). The cell number/well has been optimized for each cell line (see table 8 in the "materials and methods" section).

Cells were incubated for 0 to 24 hours and then oligonucleotide (dissolved in PBS) was added at a concentration of 5 or 25 μ M. Cells were harvested 3-4 days after addition of the oligonucleotide (incubation time for each cell line is indicated in table 8 of the "materials and methods" section).

RNA was extracted using Qiagen RNeasy 96 kit (74182) according to the manufacturer's instructions. cDNA synthesis and qPCR were performed using qScript XLT one-step RT-qPCR Toughmix Low ROX, 95134-100(Quanta Biosciences). Target transcript levels were quantified in multiplex reactions with VIC-tagged GUSB controls using FAM-tagged TaqMan assay from Thermo Fisher Scientific. TaqMan primer assays were performed on target transcripts of ATXN2 of interest (Hs01002833_ m1(FAM-MGB)) and the housekeeping gene GUSB (4326320E VIC-MGB probe). Using the technique double repeat pattern setup, n-1 biological replicates.

The relative ATXN2 mRNA expression levels are shown in table 9 as a percentage relative to the control (PBS treated cells), i.e. the lower the values, the greater the inhibition.

Table 9: in vitro efficacy of anti-ATXN 2 compounds (single experiment using dual repeat mode qPCR). ATXN2 mRNA levels were normalized to GUSB and shown as a percentage relative to control (PBS treated cells).

Example 2: selected compounds from example 1 were tested for in vitro EC50 and efficacy in A431, NCI-H23, U251 and U2-OS cell lines.

The EC50 and efficacy (KD) of the oligonucleotides of example 1 were determined using the assay described in example 1 at oligonucleotide concentrations of 50, 15.81, 5.00, 1.58, 0.50, 0.16, 0.05 and 0.016 μ M (half-log dilution, 8 points from 50 μ M) and n ═ 1-2 biological replicates, which showed less than 20% residual ATXN2 mRNA in NCI-H23 cells at 5 μ M.

The conditions for the two other cell lines are shown in Table 8 in the materials and methods section, the TaqMan primer assay for cell line U2-OS is ATXN2, Hs00268077_ m1(FAM-MGB) and housekeeping GAPDH, 4326137E (VIC-MGB). The TaqMan primers for the cell line used in example 1 were the same in this example.

EC50 values were calculated using GraphPad Prism6 and the maximum reduction (maximum KD) of ATXN2 mRNA at 50 μ M is shown in the table as a percentage relative to the control (PBS treated cells). The results for each cell line are listed in tables 10-13.

Table 10: EC50 and maximal efficacy of anti-ATXN 2 compounds in vitro in a431 cells. ATXN2 mRNA levels were normalized to GUSB, shown as a percentage relative to control (PBS treated cells). The experiment was performed in a double-repeat mode (samples A and B)

Table 11: EC50 and maximal efficacy of anti-ATXN 2 compounds in NCI-H23 cells in vitro. ATXN2 mRNA levels were normalized to GUSB, shown as a percentage relative to control (PBS treated cells). The experiment was performed in a double-repeat mode (samples A and B)

Table 12: EC50 and maximal efficacy of anti-ATXN 2 compounds in U251 cells in vitro. ATXN2 mRNA levels were normalized to GUSB, shown as a percentage relative to control (PBS treated cells). The experiment was performed in a double-repeat mode (samples A and B)

Table 13: EC50 and maximal efficacy of anti-ATXN 2 compounds in vitro in US-O2 cells. ATXN2 mRNA levels were normalized to GAPDH and shown as a percentage relative to control (PBS treated cells).

Example 3: testing of LNA oligonucleotides for in vitro efficacy in A431 and U2-OS cell lines at 0.5. mu.M

Scanning libraries were generated and tested for their efficacy in two human cell lines. The oligonucleotides used are shown in table 7. Some of the oligonucleotides tested in example 1 are also included in this example. Human cell lines A341 and U2-OS were purchased from the suppliers listed in Table 8 and maintained at 37 ℃ in a humidified incubator with 5% CO2 as recommended by the supplier. For the screening assay, cells were seeded in 96-well plates using the supplier's recommended media (see table 8 in the "materials and methods" section). The cell number/well has been optimized for each cell line (see table 8 in the "materials and methods" section).

Cells were seeded immediately before the addition of oligonucleotides (dissolved in PBS) at a concentration of 0.5 μ M (this is a 24 hour change described in table 8). Cells were harvested 3 days after the addition of the oligonucleotide.

RNA was extracted using Qiagen RNeasy 96 kit (74182) according to the manufacturer's instructions. cDNA synthesis and qPCR were performed using qScript XLT one-step RT-qPCR Toughmix Low ROX, 95134-100(Quanta Biosciences). Target transcript levels were quantified in multiplex reactions with VIC-labeled housekeeping gene controls using FAM-labeled TaqMan assay from Thermo Fisher Scientific. The TaqMan primer assay used was

Using the technique double repeat pattern setup, n-1 biological replicates.

The relative ATXN2 mRNA expression levels are shown in table 14 as a percentage relative to the control (PBS treated cells), i.e. the lower the values, the greater the inhibition.

Table 14: in vitro efficacy of anti-ATXN 2 compound at 0.5 μ M (single experiment using double repeat mode qPCR). ATXN2 mRNA levels were normalized to the indicated housekeeping gene and shown as a percentage relative to control (PBS treated cells).

The data in table 14 are shown in fig. 1A (a431 cells) and B (U2-OS cells), and fig. 2 shows a good correlation between the screening results in both cell lines.

Example 4: identification of hotspot regions from measurement of ATXN2 mRNA levels in A431 and U2OS cell lines treated with 0.5. mu.M oligonucleotides

The hot spot regions on ATXN2 precursor mRNA were identified by ATXN2 mRNA levels in a431 and U2OS cell lines after treatment with each of the 1483 oligonucleotides listed in table 7, as shown below.

First, oligonucleotide hits that resulted in more than a 50% reduction in mRNA in one or both of the a431 and U2OS cell lines were identified and 48 oligonucleotides were generated.

Second, for each of these 48 hits, any other oligonucleotides with a starting position within 10nt of the hit are grouped with the hit. Any combination comprising one or more identical oligonucleotides is combined into one set. This resulted in 21 different oligonucleotide sets, where each set included one or more oligonucleotides capable of reducing ATXN2 mRNA by at least 50%.

For each of the 21 sets, the hot spot region was defined as the region on the ATXN2 precursor mRNA covered by all oligonucleotides in that set and is shown in Table 15 below.

Table 15: ATXN2 hotspot region, start position and end position were compared to SEQ ID NO:1, the number of gapmers indicates the number of compounds tested in the hot spot region.

FIG. 1C shows the location of the hot spot on the ATXN2 precursor mRNA in gray dots. As can be seen, most of the hot spots are located in the intron region of the target transcript (SEQ ID NO: 1).

Example 5: in vitro EC50 and efficacy of selected compounds from example 3 were tested in A431 and U2-OS cell lines

The EC50 and efficacy (KD, residual mRNA level at 10 μ M) of the selected oligonucleotides from example 3 were determined at oligonucleotide concentrations of 10, 3.2, 1.0, 0.32, 0.10, 0.32, 0.010 and 0.032 μ M (half-log dilution, 8 points from 10 μ M) and n ═ 2 biological replicates using the assay described in example 3.

Specifically, the data were fitted by a least squares method to a 4-parameter logistic model to estimate EC50 values. The model fit is constrained such that the minimum asymptote at high concentration is greater than or equal to 0. The percentage of residual ATXN2 mRNA at 10 μ M relative to the control (PBS treated cells) is shown in table 16.

Table 16: EC50 and maximal efficacy of ATXN2 compound in vitro. ATXN2 mRNA levels were normalized to housekeeping genes and shown as a percentage relative to control (PBS treated cells).

Example 6: comparison of Compounds targeting "Domain 12" with Compounds targeting human AXTN2

1500 LNA gapmer oligonucleotides were designed on ATXN2 precursor mRNA sequence (SEQ ID NO1) and evaluated for their in vitro potency in a431 and U2OS cells at low doses of 0.5 μ M. The results are summarized in FIG. 6. The data confirm that the hot spot region (SEQ ID NO 6) represented by filled circles provides a highly potent compound. Figure 7 shows only selected hot spot zone compounds.

Example 7: testing of LNA oligonucleotides for in vitro efficacy in U2OS and A431 cell lines at 0.5. mu.M

Oligonucleotide screening was performed in three human cell lines using the LNA oligonucleotides in table 17, which also target the sequences of SEQ ID NO: region 83121 to 83144 of 1. As described in the above embodiments. The relative ATXN2 mRNA expression levels, expressed as a percentage relative to the control (PBS treated cells), are shown in table 17, i.e. the lower the values, the greater the inhibition.

TABLE 17

For compounds, the capital letters represent β -D-oxy LNA nucleosides, the lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages.

Example 8: testing selected Compounds for in vitro EC50 and efficacy

The EC50 was determined for the compound tested in example 3 using the method described in example 2, with 10mM as the starting concentration. EC50 values were calculated as follows:

watch 18

Example 9: selected compounds 7_1 and 15_4 were evaluated in mouse primary cortical neuronal cells and compared to the prior art compound ASO 7.

Compound ASO7 ═gtgggatacaaattctaggc(SEQ ID NO M) wherein the bold underline represents a 2' -O-MOE nucleoside and the non-bold underline represents a DNA nucleoside, all internucleoside linkages being phosphorothioate (as disclosed in Scholes et al, Nature vol. 544, pp. 362-366 (20/4.2017)).

Preparation of mouse Primary cortical neuronal cell cultures

Primary cortical neuron cultures were prepared from 15 day old mouse embryonic brains according to standard procedures. Briefly, plates were coated with poly-L-lysine (50. mu.g/ml poly-L-lysine, 10mM sodium tetraborate, pH 8 buffer) for 2-3 hours at 37 ℃ in a humidified incubator with 5% CO 2. Before use, plates were washed with 1 xPBS. Harvested mouse embryonic brains were dissected and homogenized with a razor and then immersed in 38ml of dissection medium (HBSS, 0.01M Hepes, penicillin/streptomycin). 2ml trypsin was added and the cells were incubated at 37 ℃ for 30 minutes. After incubation, 4ml of trypsin terminator was added and the cells were centrifuged.

Cells were dispersed in 20ml DMEM (+ 10% FBS) and further homogenized by syringe with 13g needle. Followed by centrifugation at 500rpm for 15 minutes. The supernatant was removed, the cells were dispersed in DMEM (+ 10% FBS) and seeded in 96-well plates (0.1X 10^6 cells/well, 100. mu.l). The inoculated neuron cell culture can be directly used.

Screening of oligonucleotides in mouse Primary cortical neuronal cell cultures

The following day, the medium was changed to growth medium (Gibco Neurobasal medium, B27 supplement, glutamine, penicillin-streptomycin) and 5 μ M FdU, placed in 96-well plates, and incubated with oligonucleotide at the desired concentration for 6 days. Total RNA was isolated from the cells and knockdown efficacy was measured by qPCR analysis. For one-step qPCR (cDNA synthesis and qPCR), each sample was run in duplicate, with one ATXN2 probe set (IDT, Leuven, Belgium) (ATXN2_ assoy 1, mm.pt.58.7178341) run in duplicate mode (RPL4, mm.pt.58.17609218 or RPS29, mm.pt.58.21577577). To each reaction was added 4. mu.L of previously diluted RNA, 0.5. mu.L of water and 5.5. mu.L of TaqMan premix. The plates were centrifuged and heat shocked at 90 ℃ for 40 seconds, followed by a brief incubation on ice, and then the samples were analyzed using qPCR (15 minutes incubation at 50 ℃ and 3 minutes incubation at 90 ℃, followed by 40 cycles of 5 seconds incubation at 95 ℃ and 45 seconds incubation at 60 ℃).

The data were analyzed using a relative standard curve method, in which each sample was first normalized to the geometric mean of two housekeeping genes (RPL4 and RPS29) and then expressed as a percentage relative to untreated control animals.

The compounds used were: 7_1, 15_4 and ASO7

The results are shown in FIG. 8.

Example 10: ICV in vivo study in mice

Animal care

Compounds were tested for in vivo activity and tolerability using 5-6C 57BL/6JBomTac female mice (16-23g, Taonic Biosciences, Ejby, Denmark) raised per cage. Animals were kept in a group room maintained at constant temperature (22. + -. 2 ℃ C.) and humidity (55. + -. 10%) and illuminated for 12 hours per day (lights on 0600 hours). Food and water were freely available to all animals throughout the study. All mouse experimental protocols were approved by the danish national ethics committee for animal experiments.

Route of administration-intraventricular injection.

The compounds were administered to mice by Intracerebroventricular (ICV) injection. Prior to ICV administration, mice were weighed and anesthetized with isoflurane or propofol (30 mg/kg). Intraventricular injections were performed using a Hamilton mini-syringe with FEP catheter fitted with a 23 gauge needle, which was fixed to the stent to adjust to the correct distance (3.9mm) through the skin and skull and into the right ventricle. The dorsum of the neck of the mouse to be injected is held with the thumb and forefinger of one hand. A slight but steady pressure is applied and the head is pressed upwards so that the needle penetrates the skull 1-2mm behind the eye 1-2mm to the right of the midline (lateral medial). Test compound or vehicle was injected at a predetermined infusion rate over 30 seconds. To avoid reflux, the mouse was held in this position for an additional 5 seconds and then carefully pulled down the needle. The procedure does not require surgery or incisions. Animals were placed under a heating lamp until they recovered from surgery. At 2 or 4 weeks post-dose, brain tissue (cortex and cerebellum) and the hepatic and renal cortex were collected on dry ice for drug concentration analysis and ATXN2 mRNA and protein analysis.

As shown in the following table (table 19), 3 independent experiments were performed on groups of different compounds.

Compound 906_1 ═ TCCattaactactCTTT, where the capital letters represent β -D-oxy LNA nucleosides, the lowercase letters represent DNA nucleosides, all LNA cs are 5-methylcytosine, and all internucleoside linkages are phosphorothioate nucleoside linkages.

Watch 19

Tolerance results:

acute toxicity was measured by monitoring the behaviour of the animals as described in WO2016/126995 (see example 9 of WO' 995). Chronic toxicity was measured by monitoring the body weight of each animal during the experiment, with a weight loss of > 5% indicating chronic toxicity. Note that euthanization of animals exhibiting signs of toxicity, in some cases led to early termination of the experiment (where a large percentage of animals exhibited signs of toxicity, all were euthanized).

Experiment 1

Compound 7_ 1: no animals showed signs of acute toxicity. During the course of the experiment (27 days), 1 mouse showed weight loss.

Compound 14_ 1: 2 out of 10 animals showed acute toxicity and required euthanasia 1 day after administration. Of the remaining 8 animals, 5 showed weight loss during the course of the experiment (terminated on day 8).

Compound 15_ 1:1 of 10 animals showed acute toxicity and required euthanasia 1 day after administration. Of all the remaining 8 animals, 5 showed weight loss during the course of the experiment (termination on day 8).

Experiment 2

Compound ASO7 was acutely toxic to all 10 animals, with severe convulsions occurring within 30 minutes after administration and required euthanasia 1 hour after administration.

Compound 906_ 1: 3 out of 10 animals showed acute toxicity and were euthanized 1 day after administration. Of the remaining 7 animals, 4 showed weight loss during the course of the experiment (terminated on day 12).

Compound 17_ 1: no animals showed signs of acute toxicity. 2 out of 10 animals showed weight loss during the experiment (29 days).

Compound 18_ 1:1 out of 10 animals showed acute toxicity and were euthanized. Of the remaining 9 animals, 3 showed weight loss during the experimental period (29 days).

Experiment 3

Compound 15_ 3: 2 of 6 animals showed acute toxicity and were euthanized 1 day after administration. Of the remaining 4 animals, 2 showed weight loss during the experiment (terminated on day 15).

Compound 15_ 4: 2 of 6 animals showed acute toxicity and were euthanized 1 day after administration. None of the remaining 4 animals showed weight loss during the course of the experiment (completed on day 14)

Compound 14_ 3: 1 of the six animals showed acute toxicity and required euthanasia 1 day after administration. Of the remaining 5 animals, 3 showed weight loss during the experiment (terminated on day 9).

Compound 14_ 2: 2 of 6 animals showed acute toxicity and were euthanized 1 day after administration. Of the remaining 4 animals, 3 showed weight loss during the experiment (terminated on day 15).

Compound 15_ 2: 2 of 6 animals showed acute toxicity and were euthanized 1 day after administration. Of the remaining 4 animals, 3 showed weight loss during the experiment (terminated on day 10).

Compound 15_ 5: all six animals showed acute toxicity and were euthanized 1 day after administration.

Tissue homogenates for oligonucleotide content and ATXN2 mRNA analysis

Mouse brain, liver and kidney samples were homogenized IN MagNA Pure LC RNA isolation tissue lysis buffer (Roche, Indianapolis, IN) using Qiagen tissue lyzer II. The homogenate was incubated at room temperature for 30 minutes in the absence of light for complete lysis. After lysis, the homogenate was centrifuged at 13000rpm for 3 minutes and the supernatant was taken for analysis. Half was left for biological analysis and the other half was directly subjected to RNA extraction.

Analysis of oligonucleotide content

For bioanalysis, samples were diluted 10-50 fold to measure oligonucleotide content using hybridization ELISA methods. Biotinylated LNA capture probes and digoxigenin-conjugated LNA detection probes (both 35nM solutions in 5xSSCT, each complementary to one end of the LNA oligonucleotide to be detected) were mixed with the diluted homogenate or related standard, incubated at room temperature for 30 minutes, and then added to a streptavidin-coated ELISA plate (Nunc cat No. 436014).

Plates were incubated at room temperature for 1 hour and washed in 2XSSCT (300mM sodium chloride, 30mM sodium citrate, and 0.05% v/v Tween-20, pH 7.0). Captured LNA duplexes were detected using anti-DIG antibodies conjugated to alkaline phosphatase (Roche Applied Science catalog number 11093274910) and alkaline phosphatase substrate system (Blue Phos substrate, KPL product code 50-88-00). The amount of oligonucleotide complex was measured on a Biotek microplate reader with absorbance at 615 nm.

Data were normalized to tissue weight and expressed as nM oligonucleotides.

Reduction of ATXN2 mRNA

RNA was purified from 350 μ L of supernatant using the kit Cellular RNA bulk kit (Roche, Indianapolis, IN) using the MagNA Pure 96 instrument. RNA samples were normalized to 2 ng/. mu.L in RNase-free water and stored at-20 ℃ until further use.

For one-step qPCR (cDNA synthesis and qPCR), each sample was run in duplicate, four sets of probe sets (IDT, Leuven, Belgium) were run in duplicate mode (ATXN2_ assoy 1, mm.pt.58.7178341 and RPL4, mm.pt.58.17609218 duplicate mode; and ATXN2_ assoy 2, mm.pt.58.11673123 and RPS29, m.pt.58.21577577 duplicate mode). To each reaction was added 4. mu.L of previously diluted RNA, 0.5. mu.L of water and 5.5. mu.L of TaqMan premix. The plates were centrifuged and heat shocked at 90 ℃ for 40 seconds, followed by a brief incubation on ice, and then the samples were analyzed using qPCR (15 minutes incubation at 50 ℃ and 3 minutes incubation at 90 ℃, followed by 40 cycles of 5 seconds incubation at 95 ℃ and 45 seconds incubation at 60 ℃).

The data were analyzed using a relative standard curve method, in which each sample (geometric mean of two ATXN2 determinations) was first normalized to the geometric mean of two housekeeping genes (RPL4 and RPS29) and then expressed as a percentage of untreated control animals.

Tissue homogenates for ATXN2 protein analysis

Using Qiagen TissueLyzer II in a cell containing 1% Halt^TMMouse brain samples were homogenized in RIPA buffer with protease and phosphatase inhibitors (Thermo Fisher Scientific). The homogenate was incubated at 4 ℃ for 30 minutes to complete lysis. After lysis, the homogenate was centrifuged for 10 minutes at 14000rcf, the supernatant was aliquoted and stored at-20 ℃ until further processingThe application is as follows.

Reduction of ATXN2 protein

Samples were normalized to 0.05mg/ml based on total protein measured using the BCA kit (Thermo Fisher Scientific). The reduction of ATXN2 Protein was measured in duplexes (primary antibody: mouse anti-Ataxin-2, 1:50, #611378, BD Bioscience and anti-HPRT, 1:100, # ab109021, Abcam, secondary antibody: anti-mouse and anti-rabbit secondary antibodies, Protein Simple, San Jose, Calif.) and analyzed on a capillary Western immunoassay WES instrument (Protein Simple) according to manufacturer's standard protocol.

The data were analyzed in relative amounts, where the ATXN2 expression for each sample was first normalized to Housekeeping Protein (HPRT) and then expressed as a percentage relative to untreated control animals.

The results are shown in FIGS. 9 to 11.

FIG. 9: knockdown (mRNA) comparison of 11 selected compounds, compiled data from three experiments, study 1 as a solid dot, study 2 as a hollow dot, and study 3 as a semi-solid dot.

FIG. 10: knockdown of compound 7_1 at protein and mRNA levels and exposure in cortical, cerebellar regions. Only cortical protein data is shown.

FIG. 11: knockdown of compound 15_4 at protein and mRNA levels and exposure in cortical, cerebellar regions. Only cortical protein data is shown.

Example 11: ICV study in mice-duration of action

A new study was set up to explore the duration of action of compound 7_ 1. 15_4 contains only one time point (7 days). The procedure was as described in example 7, using the following protocol:

watch 20

Study number	Compound ID	Dose, μ g	Point in time	Group size
					4	Saline only	0	1wk	6
4	Saline only	0	8wk	6
					4	7_1	150	24h	6
4	7_1	150	1wk	6
					4	7_1	150	4wk	6
4	7_1	150	6wk	6
					4	7_1	150	8wk	6
4	15_4	150	1wk	6

The results of mRNA knockdown are shown in figure 12, which demonstrates that ATXN2 mRNA is knocked down robustly and efficiently for at least 56 days in the cortex, particularly in the cerebellar tissue (the maximum efficacy level is maintained for 7-56 days, indicating that the effective duration of action is well over 56 days.

Example 12: in vivo study of cynomolgus monkey

Test subject

The subjects were male and female cynomolgus monkeys weighing 2-4kg at the start of dosing. Each was implanted with a polyurethane catheter in the lumbar intrathecal space. The proximal end of the catheter is connected to a subcutaneous inlet to allow injection into the intrathecal space and withdrawal of a CSF sample.

Saline, compound ID 7_1 or compound ID15_4 dissolved in saline, was administered to cynomolgus monkeys at a rate of 0.33ml/min in a volume of 1.0ml, followed by 1.5ml of aCSF. The total infusion time was 4.5 minutes. See table 21 for information on dose, duration, group size and tissue.

TABLE 21

CSF was collected from the lumbar inlet by gravity flow, with a maximum of 0.8ml CSF collected per sample. The CSF was centrifuged and the supernatant was kept at-80 ℃ until analysis. Plasma obtained from available veins was kept at-80 ℃ until analysis.

Appropriate amounts of commercial euthanasia solution were administered to cynomolgus monkeys while anesthetizing with ketamine and isoflurane. Necropsy tissue was obtained immediately thereafter and the brain was transferred to an ice-cold surface for dissection. All samples were collected using a clean removal technique, weighed and frozen with dry ice for drug concentration analysis and ATXN2 mRNA analysis.

Tolerance to stress

In the life stage, no adverse clinical reactions were reported. Histopathology showed that there was no concern with both compounds at the level tested.

Tissue homogenates for oligonucleotide content and ATXN2 mRNA analysis

See example 7-using the same procedure.

Analysis of oligonucleotide content

For bioassays, samples were diluted 50-100 fold to measure oligonucleotide content using hybridization ELISA methods. Biotinylated LNA capture probes and digoxigenin-conjugated LNA detection probes (both 35nM solutions in 5xSSCT, each complementary to one end of the LNA oligonucleotide to be detected) were mixed with the diluted homogenate or related standard, incubated at room temperature for 30 minutes, and then added to a streptavidin-coated ELISA plate (Nunc cat No. 436014).

Data were normalized to tissue weight and expressed as nM oligonucleotides.

Reduction of ATXN2 mRNA

For one-step qPCR (cDNA synthesis and qPCR), each sample was run in duplicate, with four probe sets for ATXN2 (IDT, Leuven, Belgium) (see table 22) and four probe sets for different housekeeping genes (GAPDH, Mf04392546_ g 1; POLR3F, Mf02860939_ m 1; ywtaz, Mf02920410_ m 1; and UBC, Mf02798368_ m1) (Thermo Fisher Scientific) run in singleplex mode.

Table 22: primer and probe sequences for Mf (cynomolgus monkey) ATXN2 assay.

To each reaction was added 4. mu.L of previously diluted RNA, 0.5. mu.L of water and 5.5. mu.L of TaqMan premix. The plates were centrifuged and heat shocked at 90 ℃ for 40 seconds, followed by a brief incubation on ice, and then the samples were analyzed using qPCR (15 minutes incubation at 50 ℃ and 3 minutes incubation at 90 ℃, followed by 40 cycles of 5 seconds incubation at 95 ℃ and 45 seconds incubation at 60 ℃).

The data were analyzed using a relative standard curve method in which each sample (average of four ATXN2 assays) was first normalized to the average of the three best performing housekeeping genes for each tissue-determined by the GENORM analysis described in Vanderampele et al,2002, Genome Biology 2002,3(7): research 0034.1-0034.11-and then expressed as a percentage relative to untreated control animals (see FIG. 13).

Tissue homogenates for ATXN2 protein analysis

Same as the mouse study

Reduction of ATXN2 protein

Cerebellum and cortex samples were normalized to 0.2mg/ml based on total protein measured using BCA kit (Thermo Fisher Scientific). The reduction of ATXN2 Protein was measured in duplexes (primary antibody: mouse anti-Ataxin-2, 1:50, #611378, BD Bioscience and anti-HPRT, 1:50, # ab109021, Abcam, secondary antibody: anti-mouse and anti-rabbit secondary antibodies, Protein Simple, San Jose, Calif.) and analyzed on a capillary Western immunoassay WES instrument (Protein Simple) according to manufacturer's standard protocol.

The results are shown in fig. 13 and 14.

Claims

1. An antisense oligonucleotide 10 to 30 nucleotides in length comprising a contiguous nucleotide sequence of 10 to 22 nucleotides in length having at least 90% complementarity to a sequence selected from the group consisting of SEQ ID NO 1516, SEQ ID NO 6, SEQ ID NO 1505, SEQ ID NO 1506, SEQ ID NO 1507, SEQ ID NO 1508, SEQ ID NO 1509, SEQ ID NO 1510, SEQ ID NO 1511, SEQ ID NO 1512, SEQ ID NO 1513, SEQ ID NO 1514, SEQ ID NO 1515, SEQ ID NO 1517, SEQ ID NO 1518, SEQ ID NO 1519, SEQ ID NO 1520, SEQ ID NO1, SEQ ID NO 1522, SEQ ID NO 1523, SEQ ID NO 1524 and SEQ ID NO 1525, such as 100% complementarity.

2. An antisense oligonucleotide according to claim 1, characterized in that the contiguous nucleotide sequence comprises a sequence selected from the group consisting of: SEQ ID NO 7, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 105, SEQ ID NO 154, SEQ ID NO 161, SEQ ID NO 162, SEQ ID NO 238, SEQ ID NO 385, SEQ ID NO 388, SEQ ID NO 391, SEQ ID NO 398, SEQ ID NO 399, SEQ ID NO 401, SEQ ID NO 423, SEQ ID NO 468, SEQ ID NO 477, SEQ ID NO 534, SEQ ID NO 843, SEQ ID NO 844, SEQ ID NO 845, SEQ ID NO 847, SEQ ID NO 848, SEQ ID NO 849, SEQ ID NO 850, SEQ ID NO 851, SEQ ID NO 852, SEQ ID NO 853, SEQ ID NO 854, 906, 974, 1003, 1004, 1045, 1054, 1180, 1246, 1247, 1248, 1361, 1408 and 1504; or at least 14 contiguous nucleotides thereof.

3. An antisense oligonucleotide according to any of claims 1 to 2, characterized in that the contiguous nucleotide sequence comprises a sequence selected from the group consisting of: SEQ ID NO 7, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 105, SEQ ID NO 385, SEQ ID NO 388, SEQ ID NO 391, SEQ ID NO 1246, SEQ ID NO 1247, SEQ ID NO 1248 and SEQ ID NO 1504; or at least 14 contiguous nucleotides thereof.

4. An antisense oligonucleotide according to claims 1-3, characterized in that one or more nucleotides in the contiguous nucleotide sequence are 2' sugar modified nucleotides.

5. An antisense oligonucleotide according to claim 4, characterized in that the one or more 2' sugar modified nucleosides are independently selected from the group consisting of: 2' -O-alkyl-RNA, 2' -O-methyl-RNA, 2' -alkoxy-RNA, 2' -O-methoxyethyl-RNA, 2' -amino-DNA, 2' -fluoro-DNA, arabinonucleic acid (ANA), 2' -fluoro-ANA, and LNA nucleosides.

6. An antisense oligonucleotide according to any of claims 4 to 5, characterized in that the one or more modified nucleosides are LNA nucleosides.

7. An antisense oligonucleotide according to any of the claims 1-6, characterized in that at least one internucleoside linkage in the contiguous nucleotide sequence is a phosphorothioate internucleoside linkage.

8. An antisense oligonucleotide according to claim 7, characterized in that all internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

9. An antisense oligonucleotide according to claims 1-8, characterized in that the oligonucleotide is capable of recruiting RNase H, such as human RNase H1.

10. An antisense oligonucleotide according to claim 9, characterized in that the antisense oligonucleotide or its contiguous nucleotide sequence consists of or comprises a gapmer of the formula 5' -F-G-F ' -3 '.

11. An antisense oligonucleotide according to claim 10, characterized in that region G consists of 6-16 DNA nucleosides.

12. An antisense oligonucleotide according to claim 10 or 11, characterized in that regions F and F' each comprise at least one LNA nucleoside.

13. An antisense oligonucleotide according to any of claims 1-12, characterized in that it is a compound selected from the group consisting of: CMP ID NO 7_1, CMP ID NO 13_1, CMP ID NO 14_2, CMP ID NO 15_1, CMP ID NO 16_1, CMP ID NO 17_1, CMP ID NO 18_2, CMP ID NO 105_1, CMP ID NO 154_1, CMP ID NO 161_1, CMP ID NO 162_1, CMP ID NO 238_1, CMP ID NO 385_1, CMP ID NO 388_1, CMP ID NO 391_1, CMP ID NO 398_1, CMP ID NO 399_1, CMP ID NO 401_2, CMP ID NO 423_1, CMP ID NO 468_1, CMP ID NO 477_1, CMP ID NO 534_1, CMP ID NO 843_1, CMP ID NO 844_1, CMP ID NO 845_1, CMP ID NO 847_1, CMP ID NO 848_1, CMP ID NO 849_1, CMP ID NO 850_1, CMP ID NO 851_1, CMP ID NO 852_1, CMP ID NO 853_1, CMP ID NO 854_1, CMP ID NO 906_1, CMP ID NO 974_1, CMP ID NO 1003_1, CMP ID NO 1004_1, CMP ID NO 1045_1, CMP ID NO 1054_1, CMP ID NO 1180_1, CMP ID NO 1246_1, CMP ID NO 1247_1, CMP ID NO 1248_1, CMP ID NO 1361_1, CMP ID NO 1408_1 and CMP ID NO 1504_ 1.

14. An antisense oligonucleotide according to any of claims 1-13, characterized in that the antisense oligonucleotide is a compound selected from the group consisting of

ATTTtactttaaccTCC SEQ ID NO:7，CMP ID NO:7_1

TCACattttactttaacCT SEQ ID NO:13，CMP ID NO:13_1

TCACattttactttAACC SEQ ID NO:14，CMP ID NO:14_1

TCAcattttactttAACC SEQ ID NO:14，CMP ID NO:14_2

TCACattttactttaaccTC SEQ ID NO:15，CMP ID NO:15_1

TTCAcattttacttTAAC SEQ ID NO:17，CMP ID NO:17_1

TTCAcattttactttaACC SEQ ID NO:18，CMP ID NO:18_1

TTCacattttactttAACC SEQ ID NO:18，CMP ID NO:18_2

TCACttgacacaacTTC SEQ ID NO:105CMP ID NO:105_1

ACTTtttatacctcatCA SEQ ID NO:385CMP ID NO:385_1

TACTttttatacctcATC SEQ ID NO:388CMP ID NO:388_1

TTActttttataccTCAT SEQ ID NO:391CMP ID NO:391_1

TTCAcattttatactTTAA SEQ ID NO:1246CMP ID NO:1246_1

ATTCacattttatactTTAA SEQ ID NO:1247CMP ID NO:1247_1

ATTCacattttatacTTTA SEQ ID NO:1248CMP ID NO:1248_ 1; and

TTTTattttatattatCTAC SEQ ID NO:1504CMP ID NO:1504_1

15. A conjugate comprising the antisense oligonucleotide of any one of claims 1-14, and at least one conjugate moiety covalently attached to the oligonucleotide.

16. A pharmaceutically acceptable salt of the antisense oligonucleotide according to any one of claims 1-14 or the conjugate according to claim 15.

17. A pharmaceutical composition comprising an antisense oligonucleotide according to claims 1-14 or a conjugate according to claim 15, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

18. An in vivo or in vitro method for modulating the expression of ATXN2 in target cells expressing ATXN2, the method comprising administering to said cells an effective amount of the antisense oligonucleotide according to any one of claims 1-14 or the conjugate according to claim 15 or the pharmaceutical composition according to claim 16.

19. A method of treating or preventing a disease, the method comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of the antisense oligonucleotide of any one of claims 1-14 or the conjugate of claim 15 or the pharmaceutical composition of claim 16.

20. The method of claim 19, wherein the disease is selected from the group consisting of neurodegenerative diseases selected from the group consisting of: spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), Alzheimer frontotemporal dementia (FTD), Parkinson's syndrome, and disorders with TDP-43 proteinopathies.

21. An oligonucleotide according to any one of claims 1-14 or a conjugate according to claim 15 or a pharmaceutical composition according to claim 16 for use in medicine.

22. An oligonucleotide according to any one of claims 1 to 14 or a conjugate according to claim 15 or a pharmaceutical composition according to claim 16 for use in the treatment or prevention of a neurodegenerative disease, such as a disease selected from the group consisting of: spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), Alzheimer frontotemporal dementia (FTD), Parkinson's syndrome, and disorders with TDP-43 proteinopathies.

23. Use of an oligonucleotide according to claims 1-14 or a conjugate according to claim 15 or a pharmaceutical composition according to claim 16 for the preparation of a medicament for the treatment or prevention of a neurodegenerative disease, such as a disease selected from the group consisting of: spinocerebellar ataxia type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), Alzheimer frontotemporal dementia (FTD), Parkinson's syndrome, and disorders with TDP-43 proteinopathies.