JP7499267B2 - Oligonucleotides for modulating ATXN2 expression - Google Patents

Description

The present invention relates to oligonucleotides (oligomers) that are complementary to and target a nucleic acid encoding ataxin 2 (ATXN2), resulting in reduced expression of ATXN2. Reduced expression of ATXN2 is beneficial in a wide range of medical disorders such as neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43 proteinopathy.

Expanded glutamine repeats in ataxin 2 (ATXN2), resulting from 31 or more CAG repeats in the ATXN2 gene, cause the rare neurodegenerative disorder spinocerebellar ataxia type 2 (SCA2). In addition, expanded CAG repeats are a genetic risk factor for amyotrophic lateral sclerosis (ALS) through an RNA-dependent interaction with TAR DNA-binding protein 43 (TDP-43). Other neurodegenerative diseases associated with TDP-43 proteinopathy are, for example, Alzheimer's frontotemporal dementia (FTD) and Parkinsonism. Recently, TDP-43 transgenic mice (TDP-43 ^T/Tg ), an ALS-related mouse model, were crossed with Atxn2-negative mice, which significantly extended the lifespan and improved the motor function of TDP-43 T/ ^Tg Atxn2 ^−/ mice (Becker et al. 2017 Nature 544:367-371). In the same article, treatment of TDP-43 ^T/Tg mice with antisense oligonucleotides targeting ATXN2 was shown to extend the lifespan and improve motor performance.
Antisense oligonucleotides targeting ATXN2 have also been described in U.S. Patent No. 2017/175113, WO 2015/143246, and WO 2017/117496, with WO 2017/117496 specifically relating to the treatment of ALS. Scoles et al. 2017 Nature 544:362 evaluated the ability of antisense oligonucleotides to reduce ATXN2 in the cerebellum, showing localization to Purkinje cells, indicating a potential treatment for SCA2.

OBJECTS OF THEINVENTION The present invention provides antisense oligonucleotides that modulate ATXN2 both in vivo and in vitro. The present invention has identified a specific target sequence present in intron 9 of human ATXN2 pre-mRNA that can be targeted by antisense oligonucleotides to provide effective ATXN2 inhibition. In particular, targeting positions 83118-83146 of SEQ ID NO:1 is advantageous in terms of reducing ATXN2.

The present 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), Alzheimer's frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43 proteinopathies.

The present invention relates to antisense oligonucleotides that target human ataxin 2 (ATXN2) transcripts and can inhibit the expression of ATXN2.

The present invention relates to a compound of the formula TCAcAttttactttaacCTC (SEQ ID NO: 15_4).
where the capital letters are beta-D-oxy LNA nucleosides, the lower case letters are DNA nucleosides, all LNA C's are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages.
The present invention provides an antisense oligonucleotide represented by the formula:

The present invention relates to a compound of formula

or a pharma- ceutically acceptable salt thereof.

In some embodiments, the antisense oligonucleotide is in the form of a pharma- ceutically acceptable salt.

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

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

The present invention provides a conjugate comprising an antisense oligonucleotide according to the present invention and at least one conjugate moiety covalently attached to the oligonucleotide. Stated alternatively, in some embodiments, the antisense oligonucleotide of the present invention is in the form of a conjugated oligonucleotide. In some embodiments, the oligonucleotide is not conjugated.

The present invention provides a pharmaceutical composition comprising an antisense oligonucleotide or conjugate of the present invention and a pharma- ceutically acceptable diluent, solvent, carrier, salt, and/or adjuvant.

In some embodiments, the composition includes a pharma- ceutically acceptable diluent, such as sterile phosphate buffered saline.

In some embodiments, the antisense oligonucleotides are formulated in a pharma- ceutically acceptable diluent at a concentration of 50-300 μM solution. The diluent may be phosphate buffered saline.

In some embodiments, the antisense oligonucleotides are formulated in a pharma- ceutically acceptable diluent at a concentration of 1-100 mg/mL, e.g., 2-30 or 2-50 mg/mL, or e.g., 4-30 mg/mL. The diluent may be phosphate buffered saline.

The present invention provides a method for modulating ATXN2 expression in a target cell expressing ATXN2, comprising administering to the cell an effective amount of an antisense oligonucleotide or conjugate or pharmaceutical composition of the present invention. 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, e.g., a cerebellar cell, e.g., a Purkinje cell, or a cortical cell.

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

The present invention provides an oligonucleotide, conjugate, or pharmaceutical composition of the present invention for use in treating a disease selected from the group consisting of neurodegenerative diseases selected from the group consisting of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43 proteinopathy.

The present invention provides use of an oligonucleotide, conjugate, or pharmaceutical composition of the present invention for preparing 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's disease, frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43.

The present invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide, conjugate, or pharmaceutical composition of the present invention to a subject suffering from or susceptible to a disease, 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's disease, frontotemporal dementia (FTD), Parkinsonism, and conditions associated with TDP-43 proteinopathy.

In some embodiments, the disease is spinocerebellar ataxia type 2 (SCA2).

In some embodiments, the disease is amyotrophic lateral sclerosis (ALS).

In a further aspect, the present invention provides a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharma- ceutically acceptable diluent, carrier, salt, and/or adjuvant.

In a further aspect, the present invention provides a method for in vivo or in vitro methods of modulating ATXN2 expression in a target cell expressing ATXN2 by administering to said cell an effective amount of an oligonucleotide or composition of the present invention.

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

In a further aspect, the oligonucleotide or composition of the invention is used for the treatment or prevention of a neurodegenerative disease, e.g., a neurodegenerative disease selected from the group consisting of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43 proteinopathy.

In a further aspect, the oligonucleotide or composition of the invention is used for the treatment or prevention of spinocerebellar ataxia type 2 (SCA2) or amyotrophic lateral sclerosis (ALS).

Figure 1 shows compound 15_4 (the sequence of the nucleobases is shown in SEQ ID NO: 15). The compound shown in Figure 1 is shown in protonated form, with the S atom on the phosphorothioate linkage being protonated. It will be understood that the presence of protons will vary depending on the acidity of the molecule's environment and the presence of alternative cations (e.g., when the oligonucleotide is in salt form). Protonated phosphorothioates exist in tautomeric forms. Figure 2 shows the screening of over 1500 compounds targeting the entire human Ataxin2 pre-mRNA sequence in human cell lines. Compound 7_1 is shown as an open diamond. Figure 3 shows SEQ ID NO: 6 of the compound-only targeting hotspot region according to Figure 6. Compound 7_1 is shown as an open diamond. FIG. 4 shows an evaluation of the in vitro efficacy of compounds 7_1 and 15_4 compared to compound 37_1 (ASO7). Figure 5 shows in vivo mouse studies - comparison of knockdown (mRNA) of 11 selected compounds, compiled data from 3 experiments: Study 1 = filled circles, Study 2 = open circles, Study 3 = half filled and half open circles. Figure 6 shows in vivo mouse studies - knockdown at protein and mRNA levels and exposure to compound 7_1 in cortical, cerebellar regions. Protein data for cortex is shown. Figure 7 shows in vivo mouse studies - knockdown at protein and mRNA levels and exposure to compound 15_4 in cortical, cerebellar regions. Protein data for cortex is shown. FIG. 8 shows an in vivo study in mice, time course following intracerebroventricular (ICV) administration of 150 μg of Compound 7_1 and Compound 15_4 (measured only on day 7). FIG. 9 shows NHP in vivo PK/PD study—mRNA and protein expression levels in major tissues measured after 14 days of treatment following administration of 4, 8, or 24 mg (compound 7_1), and 8 mg of compound 15_4. 10 shows NHP in vivo PK/PD study--mRNA and protein expression levels in major tissues measured after 14 days of treatment following administration of 4, 8, or 24 mg (compound 7_1), and 8 mg of compound 15_4. Data is presented to show the relative specific activity of the two compounds.

Definitions Oligonucleotides The term "oligonucleotide" as used herein is defined as a molecule that contains two or more covalently linked nucleosides, as is commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually made in the laboratory by solid-phase chemical synthesis, followed by purification and isolation. When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or modifications thereof. The oligonucleotides of the present invention are artificial, chemically synthesized, and usually purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides or nucleotides, such as, for example, 2' sugar modified nucleosides.

Antisense oligonucleotide The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid.Antisense oligonucleotides are not double-stranded in nature, and therefore are not siRNA or shRNA.

Contiguous Nucleotide Sequence The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "contiguous nucleobase sequence" and "oligonucleotide motif sequence." In some embodiments, all nucleotides of an oligonucleotide constitute a contiguous nucleotide sequence. In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence, e.g., an F-G-F' gapmer region, and may optionally include a nucleotide linker region that may be used to attach additional nucleotide(s), e.g., a functional group, to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

Nucleotides Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides). Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers."

Modified Nucleosides The term "modified nucleoside" or "nucleoside modification" as used herein refers to a nucleoside that is modified by the introduction of one or more modifications in the sugar or (nucleic acid) base moiety, compared to the 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 with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA if Watson Crick base pairing is possible.

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

Phosphorothioate linkages can exist in various tautomeric forms, for example as shown below.

The term nucleic acid base includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization.In the context of the present invention, the term nucleic acid base also includes modified nucleic acid bases that may differ from naturally occurring nucleic acid bases, but are functional during nucleic acid hybridization.In this context, "nucleobase" refers to both naturally occurring nucleic acid bases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, and non-natural variants. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Appendix 37 1.4.1.

In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

The nucleobase moieties may be designated by the letter code of each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may optionally include modified nucleobases of equivalent functionality. For example, in exemplary oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, a 5-methylcytosine LNA nucleoside may be used.

Modified Oligonucleotides The term modified oligonucleotide refers to an oligonucleotide containing one or more sugar-modified nucleosides and/or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term used in the literature to refer to oligonucleotides having modified nucleosides.

Complementarity The term "complementarity" refers to the capacity of nucleosides/nucleotides to form Watson-Crick base pairs. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). It will be understood that oligonucleotides may include nucleosides having modified nucleobases, for example, 5-methylcytosine is often used in place of cytosine, and thus the term complementarity encompasses Watson Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Appendix 37 1.4.1).

The term "% complementary" as used herein refers to the percentage of nucleotides of a contiguous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) over the contiguous nucleotide sequence. Thus, the percentage of complementarity is calculated by counting the number of aligned (Watson-Crick base pairs) nucleobases that are complementary between two sequences (when aligned between the target sequence 5'-3' and the oligonucleotide sequence from 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleobases/nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in the calculation of the % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

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

The following are examples of oligonucleotides that are perfectly complementary to the target sequence:

Below is an example of an oligonucleotide (SEQ ID NO:15) that is perfectly complementary to a target sequence (SEQ ID NO:6).
5' ttaaggaggttaaagtaaaatgtgaattt 3' (SEQ ID NO:6)
3' ctccaatttcattttacact 5' (SEQ ID NO: 15)

Identity The term "identity" as used herein refers to the proportion (expressed as a percentage) of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that are identical to a reference sequence (e.g., a sequence motif) over the contiguous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of aligned nucleobases that are identical (matching) between two sequences (in the contiguous nucleotide sequence of the compound of the invention and the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, percentage of identity = (number of matches x 100) / length of aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percentage identity of a contiguous nucleotide sequence. In determining identity, it will be understood that chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson Crick base pairs is retained (e.g., 5-methylcytosine is considered to be identical to cytosine for purposes of calculating % identity).

Hybridization As used herein, the term "hybridizing" or "hybridizing" should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of binding between two nucleic acid strands is the strength of hybridization. This is often represented by the melting temperature (T _m ), defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. In physiological conditions, T _m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° more accurately represents the binding affinity and is related to the dissociation constant (K _d ) of the reaction by ΔG°=-RTln(K _d ), where R is the gas constant and T is the absolute temperature. Thus, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid indicates strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction at 1M concentration in water, pH 7, and temperature 37°C. Hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, in which case ΔG° is less than zero. ΔG° can be experimentally measured, for example, by isothermal titration calorimetry (ITC), as described in Hansen et al., 1965, Chem. Comm. 36-38, and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercially available equipment is available for measuring ΔG°. ΔG° can be measured using the method described in SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465 (using appropriately derived thermodynamic parameters as described in Sugimoto et al., 1995, Biochemistry 34:11211-11216, and McTigue et al., 2004, Biochemistry 43:5388-5405). To ensure the potential for hybridization modulation of its intended nucleic acid target, the oligonucleotides of the present invention hybridize to target nucleic acids with estimated ΔG° values of less than −10 kcal for oligonucleotides 10-30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotides may hybridize to the target nucleic acid with estimated ΔG° values in the range of less than −10 kcal, such as less than −15 kcal, such as less than −20 kcal, and such as less than −25 kcal for oligonucleotides of 8 to 30 nucleotides in length, hi some embodiments, the oligonucleotides hybridize to the target nucleic acid with estimated ΔG° values of −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.

According to the present invention, the target nucleic acid is a nucleic acid that encodes a mammalian ATXN2, and may be, for example, a gene, an RNA, an mRNA, and a pre-mRNA, a mature mRNA, or a cDNA sequence. Thus, the target may be referred to as an ATXN2 target nucleic acid.

Preferably, the target nucleic acid encodes the ATXN2 protein, in particular a mammalian ATXN2, e.g., human ATXN2 (see, e.g., Tables 2 and 3), providing mRNA and pre-mRNA sequences for human, monkey, rat, and porcine ATXN2.

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

When the oligonucleotides of the invention are used for research or diagnostic purposes, the target nucleic acid can be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

For in vivo or in vitro applications, the oligonucleotides of the invention can typically inhibit 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, measured over the length of the oligonucleotide, optionally with one or two mismatches, and optionally with a nucleotide-based linker region or other non-complementary terminal nucleotides (e.g., region D' or D'') that may link the oligonucleotide to any functional group, such as a conjugate. The target nucleic acid can be RNA, such as messenger RNA, such as mature mRNA or pre-mRNA, or DNA, in some embodiments.

In some embodiments, the target nucleic acid is an RNA or DNA encoding a mammalian Ataxin2 protein, such as human ATXN2, e.g., a human ATXN2 mRNA sequence, such as that disclosed in SEQ ID NO: 1. Further information regarding exemplary target nucleic acids is provided in Tables 2 and 3.

Ｆｗｄ＝フォワード鎖 ゲノム座標は、プレｍＲＮＡ配列（ゲノム配列）を提供する。ＮＣＢＩ参照は、ｍＲＮＡ配列（ｃＤＮＡ配列）を提供する。
＊アメリカ国立生物工学情報センター参照配列データベースは、ゲノム、転写物およびタンパク質を含む、包括的で統合された、冗長性のない、十分に注釈が付けられた参照配列のセットである。これはｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｒｅｆｓｅｑ．で主催されている。

Fwd = forward strand Genomic coordinates provide the pre-mRNA sequence (genomic sequence) NCBI reference provides the mRNA sequence (cDNA sequence).
*The National Center for Biotechnology Information Reference Sequence Database is a comprehensive, integrated, non-redundant, well-annotated set of reference sequences, including genomes, transcripts, and proteins. It is hosted at www.ncbi.nlm.nih.gov/refseq.

Target sequence The term "target sequence" as used herein refers to the sequence of nucleotides present in the target nucleic acid, which comprises the nucleobase sequence complementary to the oligonucleotide of the present invention. In some embodiments, the target sequence is comprised of a region on the target nucleic acid that has the nucleobase sequence complementary to the continuous nucleotide sequence of the oligonucleotide of the present invention. This region of the target nucleic acid can be interchangeably referred to as the target nucleotide sequence, the target sequence or the target region. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide, and can represent, for example, a preferred region of the target nucleic acid that can be targeted by several oligonucleotides of the present invention.

In some embodiments, the target sequence is a sequence selected from human ATXN2 mRNA intron 9 (see Table 1 above).

In one embodiment of the present 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 may be 10-50 nucleotides, such as 12-30, such as 14-20, such as 15-18 contiguous nucleotides.

Target Cells As used herein, the term "target cell" refers to a cell expressing a target nucleic acid. In some embodiments, the target cell can 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 cells can be Purkinje neurons, such as Purkinje cells. Other target cells of interest are motor neurons, such as upper and lower motor neurons.

For in vitro evaluation, target cells can be established cell lines, such as A431 or U2-OS cells. Alternatively, motor neurons derived from human induced pluripotent stem cells (iPCS) (see, e.g., Sances et al. 2016 Nat Neurosci. 19(4):542-553) or Purkinje cells derived from iPCS (Wang et al. 2015 Scientific Reports 5:9232) can be used for in vitro screening.

In a preferred embodiment, the target cell expresses ATXN2 mRNA, such as ATXN2 pre-mRNA or ATXN2 mature mRNA. The polyA tail of the ATXN2 mRNA is typically not considered for antisense oligonucleotide targeting.
Naturally occurring variants

The term "naturally occurring variant" refers to variants of the ATXN2 gene or transcript that originate from the same genetic locus as the target nucleic acid but may differ, for example, due to degeneracy of the genetic code resulting in multiple codons encoding the same amino acid, or due to alternative splicing of the pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of a sufficient complementary sequence to the oligonucleotide, the oligonucleotides of the invention can thus target the target nucleic acid and its naturally occurring variants.

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

The term "modulation of expression" as used herein should be understood as a general term for the ability of an oligonucleotide to change the amount of ATXN2 when compared to the amount of ATXN2 before administration of the oligonucleotide. Alternatively, the modulation of expression can be determined by reference to a control experiment. A control is generally understood to be an individual or target cell treated with a saline composition, or an individual or target cell treated with a non-targeting oligonucleotide (mock).

One type of modulation is the ability of the oligonucleotide to inhibit, decrease, reduce, suppress, eliminate, stop, block, prevent, reduce, decrease, avoid, or terminate expression of ATXN2, for example, by degradation of mRNA or blocking transcription. The antisense oligonucleotides of the invention can advantageously inhibit expression of mammalian ATXN2, such as human ATXN2.

High affinity modified nucleosides are modified nucleotides which, when incorporated into an oligonucleotide, increase the affinity of the oligonucleotide for its complementary target, e.g., as measured by melting temperature ( ^Tm ). The high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature of +0.5 to +12 ^°C , more preferably +1.5 to +10 ^°C , and most preferably +3 to +8 ^°C per modified nucleoside. A large number of high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, for example, Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

Sugar Modifications The oligomers of the invention may contain one or more nucleosides having modified sugar moieties, ie, modifications to the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA.

A large number of nucleosides with modifications of the ribose sugar moiety have been created primarily for the purpose of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those in which the ribose ring structure has been modified, for example by replacing it with a hexose ring (HNA) or bicyclic ring (typically having a biradical bridge between the C2 and C4 carbons of the ribose ring (LNA)), or an unlinked ribose ring (e.g., UNA), which typically lacks a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

Sugar modifications also include modifications made via changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group found naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions.

2' Sugar Modified Nucleosides 2' sugar modified nucleosides are nucleosides having a substituent other than H or -OH at the 2' position (2' substituted nucleosides) or nucleosides that contain a 2' linked biradical capable of forming a bridge between the 2' carbon and a second carbon of the ribose ring, e.g., LNA (2'-4' biradical bridged) nucleosides.

Indeed, much attention has been focused on the development of 2' sugar substituted nucleosides, and a number of 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2' modified sugars can provide oligonucleotides with improved binding affinity and/or increased nuclease resistance. 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, for example, Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. See Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are examples of some 2'-substituted modified nucleosides.

In the context of the present invention, 2' substituted sugar modified nucleosides do not include 2' bridged nucleosides such as LNA.
Locked Nucleic Acid Nucleosides (LNA Nucleosides)

"LNA nucleosides" are 2' modified nucleosides that contain a biradical (also referred to as a "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or fixes the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Fixing the conformation of the ribose is associated with improved hybridization affinity (duplex stabilization) when LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules. This can usually be determined by measuring the melting temperature of the oligonucleotide/complementary duplex.

Non-limiting, exemplary LNA nucleosides are described 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, Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1.

Scheme 1:

Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, e.g. (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA.

A particularly preferred LNA is beta-D-oxy-LNA.

The compounds described herein may contain several asymmetric centers and may exist as optically pure enantiomers, mixtures of enantiomers, e.g. racemates, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.

The term "asymmetric carbon atom" means a carbon atom that has four different substituents. According to the Cahn-Ingold-Prelog rules, an asymmetric carbon atom can be of the "R" or "S" configuration.

Pharmaceutically Acceptable Salts The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable.

Protecting Group The term "protecting group", alone or in combination, means a group that selectively blocks a reactive site of a multifunctional compound so that a chemical reaction can be selectively carried out at an otherwise unprotected reactive site. The protecting group can be removed. Exemplary protecting groups are the amino protecting group, the carboxy protecting group or the hydroxy protecting group.

Nuclease-Mediated Degradation Nuclease-mediated degradation refers to oligonucleotides that, when duplexed with a complementary nucleotide sequence, are capable of mediating the degradation of such sequence.

In some embodiments, oligonucleotides may function via nuclease-mediated degradation of target nucleic acids, and the oligonucleotides of the invention may recruit nucleases, particularly endonucleases, preferably endoribonucleases (RNases), such as RNase H. Examples of oligonucleotide designs that function via nuclease-mediated mechanisms are oligonucleotides that typically contain a region of at least 5 or 6 contiguous DNA nucleosides and are flanked on one or both sides by affinity enhancing nucleosides, such as gapmers, headmers and tailmers.

RNase H Activity and Recruitment RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when it forms a duplex with a complementary RNA molecule. WO 01/23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered to be capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate measured in pmol/l/min that is at least 5%, e.g., at least 10% or more than 20% of the initial rate determined when using an oligonucleotide that has the same base sequence as the modified oligonucleotide being tested, but that contains only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Examples 91-95 of WO 01/23613 (herein incorporated by reference). For use in determining RNase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland.

Gapmer The antisense oligonucleotide or its contiguous nucleotide sequence of the present invention may be a gapmer and may also be referred to as a gapmer oligonucleotide or gapmer design. Antisense gapmers are typically used to inhibit target nucleic acids via RNase H mediated degradation. Gapmer type oligonucleotides contain at least three distinct structural regions, 5'-flank, gap and 3'-flank, F-G-F', in the 5'→3' direction. The "gap" region (G) contains a stretch of contiguous DNA nucleotides that allow the oligonucleotide to recruit RNase H. The gap region is flanked by a 5' flanking region (F) that contains one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides, and a 3' flanking region (F') that contains one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides. The one or more sugar modified nucleosides of regions F and F' improve the affinity of the oligonucleotide for the target nucleic acid (i.e., are affinity improving sugar modified nucleosides). In some embodiments, one or more sugar modified nucleosides of regions F and F' are 2' sugar modified nucleosides, such as high affinity 2' sugar modifications, eg, independently selected from LNA and 2'-MOE.

In a gapmer design, the 5' and 3' most nucleosides of the gap region are DNA nucleosides, positioned adjacent to sugar-modified nucleosides in the 5' (F) or 3' (F') regions, respectively. The flanks may be further defined by having at least one sugar-modified nucleoside at the end furthest from the gap region, i.e., at the 5' end of the 5' flank and at the 3' end of the 3' flank.

The regions F-G-F' form a contiguous nucleotide sequence. The antisense oligonucleotide of the present invention or the contiguous nucleotide sequence thereof may comprise a gapmer region of the formula F-G-F'.

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

By way of example, a gapmer-type oligonucleotide of the invention can be represented by the following formula:
F _1-8 -G _6-16 -F' _1-8 , for example F _1-8 -G _8-16 -F' _2-8
With the proviso that the total length of the gapmer region FGF' is at least 12, for example at least 14, nucleotides in length.

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

LNA Gapmer An LNA gapmer is a gapmer in which either or both of regions F and F' comprise or consist of LNA nucleosides. Beta-D-oxy gapmer is a gapmer in which either or both of regions F and F' comprise or consist of beta-D-oxy LNA nucleosides.
In some embodiments, the LNA gapmer is of the formula: [LNA] _1-5 -[region G]-[LNA] _1-5 , where region G is as defined in the definition of gapmer region G.

Alternating Flank Gapmers The flanking regions may comprise both LNA and DNA nucleosides and are referred to as "alternating flanks" because they comprise an alternating motif of LNA-DNA-LNA nucleosides. Gapmers comprising such alternating flanks are referred to as "alternating flank gapmers". An "alternating flank gapmer" is therefore an LNA gapmer oligonucleotide where at least one of the flanks (F or F') comprises DNA in addition to LNA nucleosides. 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, flanking regions F or F', or both F and F', comprise at least three nucleosides and the 5' and 3' most nucleosides of the F and/or F' regions are LNA nucleosides.

The alternating flanking regions can contain up to three consecutive DNA nucleosides, such as one to two, or one, or two, or three consecutive DNA nucleosides.

Region D' or D" in the oligonucleotide
The oligonucleotides disclosed herein can comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, e.g., a gapmer F-G-F', as well as additional 5' and/or 3' nucleosides. The additional 5' and/or 3' nucleosides may or may not be 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" can 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 link a conjugate moiety to a conjugate moiety, it can serve as a biocleavable linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacture.

Regions D' and D" can be attached to the 5' end of region F or the 3' end of region F', respectively, to generate designs of the following formula D'-F-G-F', F-G-F'-D" or D'-F-G-F'-D", where F-G-F' is the gapmer portion of the oligonucleotide and regions D' or D" constitute separate portions of the oligonucleotide.

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 the F or F' region are not sugar-modified nucleotides, but are, for example, DNA or RNA or base-modified versions thereof. The D' or D' region may serve as a nuclease-sensitive biocleavable linker (see definition of linker). In some embodiments, the additional 5' and/or 3' terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biocleavable linkers suitable for use as regions D' or D" are disclosed in WO 2014/076195, including, by way of example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in WO 2015/113922, where they have been used to join multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

In one embodiment, the oligonucleotide of the present invention includes regions D' and/or D" in addition to the contiguous nucleotide sequence that constitutes the gapmer.

An oligonucleotide gapmer may be represented by the formula:
F-G-F'; especially F _1-8 -G _6-16 -F' _2-8
D'-F-G-F', especially D' _1-3 -F _1-8 -G _6-16 -F' _2-8
F-G-F'-D", especially 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 bond between region D' and region F is a phosphodiester bond. In some embodiments, the internucleoside bond between region F' and region D" is a phosphodiester bond.

Conjugate The term conjugate, as used herein, refers to an oligonucleotide covalently attached to a non-nucleotide moiety (the conjugate moiety or region C or a 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 modulates 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 conjugate may target the oligonucleotide to a particular organ, tissue, or cell type, thereby enhancing the efficacy of the oligonucleotide in that organ, tissue, or cell type. At the same time, the conjugate may serve to reduce the activity of the oligonucleotide in non-target cell types, tissues, or organs (e.g., off-target activity or activity in non-target cell types, tissues, or organs).

In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophiles, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

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

Linker A bond or linker is a connection between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds. A conjugate moiety can be attached to an oligonucleotide directly or through a linking moiety (e.g., a linker or tether). A linker functions to covalently attach a third region (region C), such as a conjugate moiety, to a first region (region A), such as an oligonucleotide or a continuous nucleotide sequence that is complementary to a target nucleic acid.

In some embodiments of the invention, the conjugates or oligonucleotide conjugates of the invention may optionally include a linker region (second region or region B and/or region Y) located 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 that includes or consists of a physiologically labile bond that is cleavable under conditions normally found or similar to those found in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidizing or reducing conditions or oxidizing or reducing agents, and salt concentrations found or similar to those found in a mammalian cell. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in mammalian cells, such as from proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In a preferred embodiment, the nuclease-sensitive linker comprises 1-10 nucleosides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferably 2-6 nucleosides, and most preferably 2-4 linked nucleosides, which include at least two consecutive phosphodiester bonds, such as at least three or four or five consecutive phosphodiester bonds. Preferably, the nucleosides are DNA or RNA. Phosphodiester-containing biocleavable linkers are described in more detail in WO 2014/076195, which is incorporated herein by reference - see also region D' or D" herein.

Region Y refers to a linker that is not necessarily biocleavable, but serves primarily to covalently attach the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). Region Y linkers may include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units or aminoalkyl groups. Oligonucleotide conjugates of the invention may be constructed from the following local 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, such as, for example, a C2-C36 aminoalkyl group, including a C6-C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

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

In some embodiments, the treatment is administered to a patient who has been diagnosed with a neurological disorder, such as a neurological disorder selected from the group consisting of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's frontotemporal dementia (FTD), Parkinsonism, and neurodegenerative diseases, including conditions involving TDP-43 proteinopathy.
In some embodiments, the compounds of the invention are for use in the treatment of spinocerebellar ataxia type 2 (SCA2) or amyotrophic lateral sclerosis (ALS).

Oligonucleotides of the Invention The present invention relates to oligonucleotides of the formula TCAcAttttactttaacCTC (SEQ ID NO: 15_4).
where the capital letters are beta-D-oxy LNA nucleosides, the lower case letters are DNA nucleosides, all LNA C's are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages.
The present invention provides an antisense oligonucleotide represented by the formula:

The present invention provides a conjugate comprising an oligonucleotide or 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.

Methods of Production In a further aspect, the invention provides a method of producing an oligonucleotide of the invention, comprising reacting nucleotide units to thereby form covalently linked contiguous nucleotide units contained within the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al., 1987, Methods in Enzymology, Vol. 154, pp. 287-313). In a further embodiment, the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In a further aspect, a method of producing a composition of the invention is provided, comprising mixing an oligonucleotide or a conjugated oligonucleotide of the invention with a pharma- ceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

Pharmaceutical Salts In a further aspect, the present invention provides a pharma- ceutically acceptable salt of the antisense oligonucleotide or conjugate thereof. In a preferred embodiment, the pharma- ceutically acceptable salt is a sodium or potassium salt.

Pharmaceutical Compositions In a further aspect, the present invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof, and a pharma- ceutically acceptable diluent, carrier, salt and/or adjuvant. Pharmaceutically acceptable diluents include phosphate buffered saline (PBS) and pharma- ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharma- ceutical acceptable diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used at a concentration of 50-300 μM solution in the pharma- ceutical acceptable diluent.

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

The oligonucleotides or oligonucleotide conjugates of the invention can be mixed with pharma- ceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for the preparation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

These compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be 3-11, more preferably 5-9, or 6-8, most preferably 7-8, e.g., 7-7.5. The resulting solid form compositions may be packaged in a plurality of single dose units, each containing a fixed amount of the agent or agents, such as a sealed package of tablets or capsules. The solid form compositions may also be packaged in flexible quantity containers, such as squeezable tubes designed for topically applicable creams or ointments.

In some embodiments, the oligonucleotides or oligonucleotide conjugates of the invention are prodrugs. Particularly with respect to oligonucleotide conjugates, when the prodrug is delivered to a site of action, e.g., a target cell, the conjugate moiety is cleaved from the oligonucleotide.

Uses The oligonucleotides of the invention may be utilized, for example, as research reagents for diagnostics, therapeutics and prophylaxis.

In research, such oligonucleotides may be used to specifically regulate synthesis of Ataxin2 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or evaluation of its usefulness as a target for therapeutic intervention. Typically, target regulation is achieved by degrading or inhibiting the mRNA that produces the protein, thereby preventing protein formation, or by degrading or inhibiting a modulator of the gene or mRNA that produces the protein.

When the oligonucleotides of the invention are used for research or diagnostic purposes, the target nucleic acid can be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

The present invention provides an in vivo or in vitro method for modulating ATXN2 expression in a target cell expressing ATXN2, the method comprising administering to the cell an effective amount of an oligonucleotide of the present invention.

In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells may be in vitro cell cultures or in vivo cells that form part of a mammalian tissue. In preferred embodiments, the target cells are present in the brain or central nervous system, including the brainstem and spinal cord. In particular, cells within the cerebellum are relevant target cells, e.g., Purkinje neurons or Purkinje cells, particularly in individuals affected by Spinocerebellar Ataxia Type 2 (SCA2).

Other target cells of interest are motor neurons located in the cortex of the brain and in the spinal cord. Upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord are target cells of the present invention. In particular, motor neurons in individuals affected by amyotrophic lateral sclerosis (ALS) are target cells of interest.

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

For therapeutic purposes, the oligonucleotides may be administered to an animal or human suspected of having a disease or disorder that can be treated by modulating the expression of ATXN2.

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

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

The oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions according to the invention are typically administered in effective amounts.

The present invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the present invention as described above for the manufacture of a medicament for the treatment of a disorder referred to herein or for a method of treating a disorder referred to herein.

The diseases or disorders referred to herein are associated with expression of ATXN2. In some embodiments, the disease or disorder may be associated with a mutation in the ATXN2 gene, such as an expanded CAG repeat region. The disease or disorder may be associated with a gene whose protein product is associated with or interacts with ATXN2. In particular, in diseases associated with TDP-43 proteinopathies, reduction of ATXN2 may have beneficial effects, for example, in amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia (FTD), and Parkinsonism.

The methods of the present invention are preferably used for the treatment or prevention of diseases caused by abnormal levels and/or activity of ATXN2.

The present invention further relates to the use of an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of ATXN2.

In one embodiment, the present invention relates to an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition for use in the treatment of a disease or disorder selected from neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43 proteinopathy. In particular, the use of the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention in the treatment of spinocerebellar ataxia type 2 (SCA2) or amyotrophic lateral sclerosis (ALS) is advantageous.

Administration The oligonucleotides or pharmaceutical compositions of the present invention can be administered parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly, intraocularly, or intrathecally).

In some embodiments, administration is via intrathecal administration.

Advantageously, for example for the treatment of a neurological disorder, the oligonucleotide or pharmaceutical composition of the invention is administered intrathecally or intracranially, for example via intracerebral or intraventricular administration.

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

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

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

In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention is used in a combination treatment with another therapeutic agent. The therapeutic agent may be, for example, a standard treatment for the disease or disorder described above.

EXAMPLES Materials and Methods Oligonucleotide Motif Sequences and Oligonucleotide Compounds

A motif sequence represents a consecutive sequence of nucleobases present in an oligonucleotide.

Design refers to a 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=gap region), followed by the number of modified nucleosides, e.g., 2' modified nucleosides (third number=3' flanking), optionally preceded or followed by further repeated regions of DNA and LNA, which are not necessarily part of the contiguous nucleotide sequence complementary to the target nucleic acid.

The oligonucleotide compounds represent specific designs of motif sequences. Capital letters represent beta-D-oxy LNA nucleosides, lower case letters represent DNA nucleosides, all LNA C's are 5-methylcytosines, all 5-methyl DNA cytosines are represented by "e", and all internucleoside linkages are phosphorothioate internucleoside linkages.

Oligonucleotide synthesis Oligonucleotide synthesis is generally known in the art. Below are applicable protocols. The oligonucleotides of the present invention may be produced by methods that differ slightly in terms of the equipment, support and concentration used.

Oligonucleotides are synthesized on a uridine universal support using the phosphoramidite approach of Oligomaker 48 on a 1 μmol scale. At the end of the synthesis, the oligonucleotides are cleaved from the solid support using aqueous ammonia at 60°C for 5-16 hours. Oligonucleotides are purified by reversed-phase HPLC (RP-HPLC) or solid-phase extraction, characterized by UPLC, and molecular weights are further confirmed by ESI-MS.

Oligonucleotide extension:
Coupling of β-cyanoethyl-phosphoramidites (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) is carried out using 0.1 M of the 5′-O-DMT protected amidite in acetonitrile and a solution of DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. In the final cycle, a phosphoramidite with the desired modification can be used, such as a C6 linker for attaching a conjugate group, or such a conjugate group. Thiolation to introduce a phosphorothioate bond is carried out by using xanthan gum (0.01 M in acetonitrile/pyridine 9:1). A phosphodiester bond can be introduced using 0.02 M iodine in THF/pyridine/water 7:2:1. The remaining reagents are those commonly used in oligonucleotide synthesis.

For conjugation after solid-phase synthesis, commercially available C6 amino linker phosphoramidites 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 conjugate is introduced by activation of the functional group using standard synthetic methods.

Purification by RP-HPLC:
The crude compound is purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μ 150x10mm column. 0.1M ammonium acetate pH 8 and acetonitrile are used as buffers at a flow rate of 5mL/min. Collected fractions are lyophilized to give the purified compound, typically as a white solid.

Abbreviations:
DCI 4,5-dicyanoimidazole DCM: dichloromethane DMF: dimethylformamide DMT: 4,4'-dimethoxytrityl THF: tetrahydrofuran Bz: benzoyl Ibu: isobutyryl RP-HPLC: reversed-phase high-performance liquid chromatography _Tm assay

The oligonucleotide and RNA target (phosphate linked, PO) duplexes are diluted to 3 mM in 500 ml RNase free water and mixed with 500 ml _2xTm buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM phosphate, pH 7.0). The solution is heated to 95°C for 3 minutes and then annealed at room temperature for 30 minutes. The melting temperature ( _Tm ) of the duplex is measured on a Lambda 40 UV/VIS spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature is increased from 20°C to 95°C and then decreased to 25°C and the absorbance is recorded at 260 nm. The first derivative and both the melting and annealing maxima are used to evaluate the duplex _Tm .

Cell lines

Example 1 Testing the in vitro efficacy of LNA oligonucleotides in A431, NCI-H23, and ARPE19 cell lines at 25 and 5 μM An oligonucleotide screen was performed in three human cell lines using the LNA oligonucleotides in Table 4, which target the region of positions 83121-83144 of SEQ ID NO:1. Human cell lines A341, NCI-H23, and ARPE19 were purchased from vendors listed in Table 5 and maintained in a humidified incubator at 37° C. with 5% CO2 as recommended by the supplier. For the screening assay, cells were seeded in 96 multi-well plates in the medium recommended by the supplier (see Table 5 in the Materials and Methods section). The number of cells per well was optimized for each cell line (see Table 5 in the Materials and Methods section).

Prior to addition of oligonucleotides, cells were incubated for 0-24 hours at a concentration of 5 or 25 μM (dissolved in PBS). Cells were harvested 3-4 days after addition of oligonucleotides (incubation times for each cell line are shown in Table 5 in the Materials and Methods section).

RNA was extracted using the 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 using FAM-labeled TaqMan assays from Thermo Fisher Scientific in multiple reactions along with a VIC-labeled GUSB control. TaqMan primer assays for target transcripts of interest ATXN2 (Hs01002833_m1 (FAM-MGB)), and housekeeping gene GUSB (4326320E VIC-MGB probe). A technical duplex setup was used (n=1 biological replicate).

Relative ATXN2 mRNA expression levels are shown in Table 6 as a percentage of control (PBS-treated cells); i.e., the lower the value, the greater the inhibition.

Example 2: In vitro EC50 and efficacy testing of selected compounds from Example 1 in A431, NCI-H23, U251, and U2-OS cell lines.
The EC50 and potency (KD) of the oligonucleotides of Example 1 that exhibited less than 20% residual ATXN2 mRNA at 5 μM in NCI-H23 cells were measured using the assay described in Example 1 at the following oligonucleotide concentrations: 50, 15.81, 5.00, 1.58, 0.50, 0.16, 0.05, and 0.016 μM (half log dilutions, 8 points from 50 μM) and n=1-2 biological replicates.

Table 5 in the Materials and Methods section shows the conditions for two additional cell lines, the TaqMan primer assays used for cell line U2-OS were ATXN2, Hs00268077_m1 (FAM-MGB), housekeeping GAPDH, 4326137E (VIC-MGB). The TaqMan primers for the cell lines used in Example 1 were the same in this example.

EC50 values were calculated using GraphPad Prism 6 and the maximum reduction (Max KD) of ATXN2 mRNA at 50 μM is shown in the table as a % of control (PBS treated cells). Tables 7-10 show the results for each cell line.

Example 3: Comparison of Compounds Targeting "Region 12" with Compounds Targeting Spanning Human AXTN2 Over 1500 LNA gapmer oligonucleotides were designed spanning the ATXN2 pre-mRNA sequence (SEQ ID NO: 1) and their in vitro potency at a low dose of 0.5 μM was evaluated in A431 and U2OS cells. The results are summarized in Figure 2. The data confirm that the hotspot region represented as a solid circle (SEQ ID NO: 6) provides compounds with high potency. Figure 3 shows only selected hotspot region compounds.

Example 4: Testing the in vitro efficacy of LNA oligonucleotides in U2OS and A431 cell lines at 0.5 μM An oligonucleotide screen was performed in three human cell lines using the LNA oligonucleotides in Table 11, which also target the region at positions 83121-83144 of SEQ ID NO:1, as described in the Examples above. The relative ATXN2 mRNA expression levels are shown in Table 11 as a percentage of the control (PBS treated cells), i.e. the lower the value, the greater the inhibition.

For the compounds, capital letters represent beta-D-oxy LNA nucleosides, lower case letters represent DNA nucleosides, all LNA Cs are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages.

Example 5: In Vitro EC50 and Efficacy Testing of Selected Compounds Using the methodology described in Example 2, the EC50 of the compounds tested in Example 3 was determined using 10 mM as the starting concentration. The EC50 values were calculated as follows:

Example 6: Evaluation of selected compounds 7_1 and 15_4 compared to prior art compound ASO7 (compound 37_1) in mouse primary cortical neuronal cells.
Compound 37_1 = gtggg atacaaattc taggc , where the underlined bold letters represent 2'-O-MOE nucleosides, the non-bold letters are DNA nucleosides, and all internucleoside linkages are phosphorothioates (as disclosed in Scholes et al., Nature 544:362-366 (20 Apr. 2017)).

Preparation of mouse primary cortical neuron cell cultures Primary cortical neuron cultures were prepared from 15-day-old mouse embryonic brains according to standard procedures. Briefly, culture dishes were coated with poly-L-lysine (50 μg/ml poly-L-lysine, 10 mM Na tetraborate, pH 8 buffer) for 2-3 hours in a humidified incubator at 37°C and 5% CO2. Dishes were washed with 1×PBS before use. Collected mouse embryonic brains were dissected and homogenized with a razor blade and immersed in 38 ml of dissection medium (HBSS, 0.01 M Hepes, penicillin/streptomycin). 2 ml of trypsin was added and the cells were incubated at 37 ^°C for 30 minutes. After incubation, 4 ml of trypsin stopper was added and the cells were centrifuged.

The cells were dispersed in 20 ml DMEM (+10% FBS) and passed through a syringe with a 13 g needle for further homogenization. This was followed by centrifugation at 500 rpm for 15 min. The supernatant was removed and the cells were dispersed in DMEM (+10% FBS) and seeded in 96-well plates (0.1 x 10^6 cells/well in 100 μl). The neuronal cell cultures were ready to use directly after seeding.

Screening of oligonucleotides in mouse primary cortical neuron cell cultures The next day, medium was changed to growth medium (Gibco Neurobasal medium, B27 supernatant, Glutamax, penicillin-streptomycin) and 5 μM FdU in 96-well plates and incubated with oligonucleotides at the desired concentrations for 6 days. RNA was isolated from total 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_assay1, Mm.PT.58.7178341) run in duplex with either RPL4, Mm.PT.58.17609218, or RPS29, Mm.PT.58.21577577. To each reaction, 4 μL of previously diluted RNA, 0.5 μL water, and 5.5 μL TaqMan MasterMix were added. Plates were centrifuged and heat-closed at 90° C. for 40 seconds, followed by a short incubation on ice, after which samples were analyzed using qPCR (incubation at 50° C. for 15 minutes and 90° C. for 3 minutes, followed by 40 cycles of 95° C. for 5 seconds and 60° C. for 45 seconds).

Data were analyzed using the relative standard curve method, with each sample first normalized to the geometric mean of two housekeeping genes (RPL4 and RPS29) and then expressed as a percentage of untreated control animals.
Compounds used: 7_1, 15_4, and 37_1 (ASO7)
The results are shown in Figure 4.

Example 7: In vivo ICV Mouse Studies Animal Husbandry Compounds were tested for in vivo activity and tolerability in C57BL/6JBomTac female mice (16-23 g, Taconic Biosciences, Ejby, Denmark) housed 5-6 per cage. Animals were kept in a colony room maintained at constant temperature (22±2° C.) and humidity (55±10%) and exposed to 12 h/day light (lights on 0600 h). All animals had free access to food and water during the study period. All mouse protocols were approved by the Danish National Committee for Ethics in Animal Experiments.

Route of administration--intraventricular injection.
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). Intracerebroventricular injections were performed using a Hamilton micro syringe with a FEP catheter fitted with a 23-gauge needle secured to a stand adjusted to penetrate a precise distance (3.9 mm) through the skin and skull into the right lateral ventricle. The mouse to be injected was held at the neck crease with the thumb and index finger of one hand. Gentle but firm pressure was applied, pushing the head up so that the needle penetrated 1-2 mm to the right of the skull midline (mediolateral) and 1-2 mm behind the eye. A bolus of test compound or vehicle was injected for 30 seconds at the previously measured infusion rate. The mouse was held in this position for an additional 5 seconds to avoid reflux, then carefully withdrawn downwards away from the needle. This procedure does not require surgery or incision. The animals were placed under a heat lamp until they recovered from the procedure. Brain tissues (cortex and cerebellum), as well as liver and kidney cortex, were collected on dry ice 2 or 4 weeks after dosing for drug concentration analysis, and ATXN2 mRNA and protein analysis.

Three independent experiments were performed with different groups of compounds as shown in the table below (Table 13).

Compound 36_1 = TCCattaactactCTTT (capital letters represent beta-D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA C's are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages.)

Tolerability results:
Acute toxicity was measured by monitoring animal behavior as described in WO 2016/126995 (see Example 9). Chronic toxicity was measured by monitoring the weight of each animal over the time course of the experiment, with a weight loss of more than 5% indicating chronic toxicity. Animals showing significant signs of toxicity were euthanized, and in some cases the experiment was terminated early (if a high percentage of animals showed signs of toxicity, all animals were euthanized).

Experiment 1
Compound 7_1: None of the animals showed signs of acute toxicity. One mouse showed weight loss during the experiment (27 days).

Compound 14_1: Two of the ten animals showed acute toxicity and had to be euthanized one day after dosing. Five of the remaining eight animals showed weight loss during the study (ended on day eight).

Compound 15_1: One of 10 animals showed acute toxicity and had to be euthanized one day after dosing. Of the remaining eight animals, five showed weight loss during the experiment (ended on day eight).

Experiment 2
Compound 37_1 (ASO7) demonstrated acute toxicity in all 10 animals, with severe convulsions within 30 minutes of dosing, necessitating euthanasia 1 hour after dosing.

Compound 36_1: 3 of 10 animals showed acute toxicity and had to be euthanized 1 day after dosing. 4 of the remaining 7 animals showed weight loss during the study (ended on day 12).

Compound 17_1: None of the animals showed signs of acute toxicity. Two of the ten animals showed weight loss over the course of the experiment (29 days).

Compound 18_1: One of 10 animals showed acute toxicity and was euthanized. Three of the remaining nine animals showed weight loss during the study (29 days).

Experiment 3
Compound 15_3: Two of six animals showed acute toxicity and had to be euthanized one day after dosing. Of the remaining four, two lost weight during the study (ended on day 15).

Compound 15_4: Two of six animals showed acute toxicity and had to be euthanized one day after dosing. None of the remaining four animals showed weight loss during the study (ended on day 14).

Compound 14_3: One of six animals showed acute toxicity and had to be euthanized one day after dosing. Of the remaining five, three lost weight during the study (ended on day 9).

Compound 14_2: Two of six animals showed acute toxicity and had to be euthanized one day after dosing. Of the remaining four, three lost weight during the study (ended on day 15).

Compound 15_2: Two of six animals showed acute toxicity and had to be euthanized one day after dosing. Of the remaining four, three lost weight during the study (ended on day 10).

Compound 15_5: All six animals showed acute toxicity and had to be euthanized one day after dosing.

Tissue homogenization for oligo 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 TissueLyzer II. The homogenates were incubated at room temperature for 30 minutes to ensure complete lysis. After lysis, the homogenates were centrifuged at 13000 rpm for 3 minutes and the supernatant was used for analysis. One half was set aside for biological analysis and the other half was directly continued with RNA extraction.

Oligo content analysis For biological analysis, samples were diluted 10-50 fold for oligo content measurement using a hybridization ELISA method. A biotinylated LNA capture probe and a digoxigenin-conjugated LNA detection probe (both 35 nM in 5x SSCT, each complementary to one end of the LNA oligonucleotide to be detected) were diluted with the diluted homogenate or relevant standard, incubated for 30 min at room temperature and added to a streptavidin-coated ELISA plate (Nunc catalogue no. 436014).

Plates were incubated for 1 hour at room temperature and washed with 2xSSCT (300 mM sodium chloride, 30 mM sodium citrate, and 0.05% v/v Tween-20, pH 7.0). Captured LNA duplexes were detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche Applied Science Catalog No. 11093274910) and alkaline phosphatase substrate system (Blue Phos Substrate, KPL Product Code: 50-88-00). The amount of oligo complex was measured as absorbance at 615 nm in a Biotek reader.

Data were normalized to tissue weight and expressed as nM of oligo.

Depletion of ATXN2 mRNA RNA was purified from 350 μL of supernatant using a MagNA Pure 96 instrument (Roche, Indianapolis, IN) with the kit Cellular RNA Large Volume Kit. RNA samples were normalized to 2 ng/μL in RNase-free water and stored at −20° C. until further use.

For one-step qPCR (cDNA synthesis and qPCR), each sample was run in duplicate with four probe sets (IDT, Leuven, Belgium) run in duplex (RPL4, ATXN2_assay1 in duplex with Mm.PT.58.17609218, and ATXN2_assay2 in duplex with Mm.PT.58.11673123, RPS29). To each reaction, 4 μL of previously diluted RNA, 0.5 μL water, and 5.5 μL TaqMan MasterMix were added. Plates were centrifuged and heat-closed at 90°C for 40 seconds, followed by a short incubation on ice, after which samples were analyzed using qPCR (incubation at 50°C for 15 minutes and 90°C for 3 minutes, followed by 40 cycles of 95°C for 5 seconds and 60°C for 45 seconds).

Data were analyzed using the relative standard curve method, and each sample (geometric mean of two ATXN2 assays) 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 homogenization for ATXN2 protein analysis

Mouse brain samples were homogenized in RIPA buffer with 1% Halt™ Protease and Phosphatase Inhibitor (Thermo Fisher Scientific) using a Qiagen TissueLyzer II. The homogenate was incubated at 4°C for 30 min to ensure complete cell lysis. After cell lysis, the homogenate was centrifuged at 14000rcf for 10 min and the supernatant was aliquoted into small volumes and stored at -20°C until further use.

ATXN2 Protein Reduction Samples were normalized to 0.05mg/ml based on total protein measured using BCA Kit (Thermo Fisher Scientific). ATXN2 protein reduction was measured in duplex (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, CA) and analyzed on a Capillary Western Immunoassay WES instrument (Protein Simple) following the manufacturer's standard procedures.

Data were analyzed in terms of relative abundance. ATXN2 expression for each sample was first normalized to a housekeeping protein (HPRT) and then expressed as a percentage of untreated control animals.

The results are shown in Figures 5 to 7.

Figure 5: Comparison of knockdown (mRNA) of 11 selected compounds, compiled data from 3 experiments, Study 1 = filled circles, Study 2 = open circles, Study 3 = half filled and half open circles.

Figure 6: Knockdown at protein and mRNA levels and exposure to compound 7_1 in cortical and cerebellar regions. Protein data for cortex is shown.

Figure 7: Knockdown at protein and mRNA levels and exposure to compound 15_4 in cortical and cerebellar regions. Protein data for cortex is shown.

Example 8: In vivo ICV mouse study - duration of action A new study was launched to investigate the duration of action of compound 7_1. 15_4 was included at one time point only (7 days). The procedure was as described in Example 7 using the following protocol:

The results of the mRNA knockdown are shown in Figure 8. This shows a robust and potent knockdown of ATXN2 mRNA in both cortical and especially cerebellar tissue for at least 56 days (maximal levels of efficacy were maintained from 7 to 56 days, indicating that the effective duration of action was considerably longer than 56 days). There appear to be some mice for which the treatment was less effective, which may be procedure-related to the mice over time as they are associated with the same individual.

Example 9: In vivo Cynomolgus Monkey Study Subjects Subjects were male and female cynomolgus monkeys weighing 2-4 kg at the start of dosing. Each was implanted with a polyurethane catheter into the lumbar intrathecal space. The proximal end of the catheter was connected to a subcutaneous access port to allow injection into the intrathecal space and withdrawal of CSF samples.

Cynomolgus monkeys were administered either saline, Compound #7_1, or Compound #15_4 (dissolved in saline at 0.33 ml/min in a volume of 1.0 ml followed by 1.5 ml aCSF). Total infusion time was 4.5 minutes. See Table 15 for information on dose, duration, group size, and tissues.

CSF was collected from a lumbar access port by gravity flow with a maximum of 0.8 ml of CSF per sample. CSF was centrifuged and the supernatant was kept at -80°C until analysis. Plasma obtained from an available vein was kept at -80°C until analysis.

Cynomolgus monkeys were euthanized with ketamine and isoflurane while receiving an appropriate volume of a commercially available euthanasia solution. Autopsy tissue was obtained immediately thereafter, and brains were transferred to a cooled surface for dissection. All samples were collected using a clean clearance technique, weighed, and frozen on dry ice for drug concentration and ATXN2 mRNA analysis.

Tolerability No clinical adverse events were reported during the study phase. Histopathology did not indicate any concern for any of the compounds at the doses tested.
Tissue homogenization for oligo content and ATXN2 mRNA analysis

See Example 7 - same procedure was used.

Oligo content analysis For biological analysis, samples were diluted 50-100 fold for oligo content measurement using a hybridization ELISA method. A biotinylated LNA capture probe and a digoxigenin-conjugated LNA detection probe (both 35 nM in 5x SSCT, each complementary to one end of the LNA oligonucleotide to be detected) were diluted with the diluted homogenate or relevant standard, incubated for 30 min at room temperature and added to a streptavidin-coated ELISA plate (Nunc catalogue no. 436014).

Plates were incubated for 1 hour at room temperature and washed with 2xSSCT (300 mM sodium chloride, 30 mM sodium citrate, and 0.05% v/v Tween-20, pH 7.0). Captured LNA duplexes were detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche Applied Science Catalog No. 11093274910) and alkaline phosphatase substrate system (Blue Phos Substrate, KPL Product Code: 50-88-00). The amount of oligo complex was measured as absorbance at 615 nm in a Biotek reader.

Data was normalized to tissue weight and expressed as nM of oligo.
Depletion of ATXN2 mRNA RNA was purified from 350 μL of supernatant using a MagNA Pure 96 instrument (Roche, Indianapolis, IN) with the kit Cellular RNA Large Volume Kit. RNA samples were normalized to 2 ng/μL in RNase-free water and stored at −20° C. until further use.

For one-step qPCR (cDNA synthesis and qPCR), each sample was run in duplicate with four probe sets for ATXN2 (see Table 16) (IDT, Leuven, Belgium) performed in singleplex, and four probe sets for different housekeeping genes (GAPDH, Mf04392546_g1, POLR3F, Mf02860939_m1, YWHAZ, Mf02920410_m1 and UBC, Mf02798368_m1) (Thermo Fisher Scientific).

To each reaction, 4 μL of previously diluted RNA, 0.5 μL water, and 5.5 μL TaqMan MasterMix were added. Plates were centrifuged and heat-closed at 90°C for 40 seconds, followed by a short incubation on ice, after which samples were analyzed using qPCR (incubation at 50°C for 15 minutes and 90°C for 3 minutes, followed by 40 cycles of 95°C for 5 seconds and 60°C for 45 seconds).

Data were analyzed using the relative standard curve method. Each sample (average of four ATXN2 assays) was first normalized to the average of the three best performing housekeeping genes for each tissue as measured by the geNORM analysis described in Vandesompele et al., 2002, Genome Biology 2002, 3(7): research 0034.1-0034.11, and then expressed as a percentage of untreated control animals (see Figure 9).

Tissue homogenization for ATXN2 protein analysis: Same as mouse study. ATXN2 protein reduction: Cerebellar and cortical samples were normalized to 0.2 mg/ml based on total protein measured using the BCA Kit (Thermo Fisher Scientific). ATXN2 protein reduction was measured in duplex (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, CA) and analyzed on a Capillary Western Immunoassay WES instrument (Protein Simple) following the manufacturer's standard procedures.

Data were analyzed in terms of relative abundance. ATXN2 expression for each sample was first normalized to a housekeeping protein (HPRT) and then expressed as a percentage of untreated control animals.

The results are shown in Figures 9 and 10.

Claims (23)

Formula TCAcAttttactttaacCTC (CMP number 15_4)
where the capital letters are beta-D-oxy LNA nucleosides, the lower case letters are DNA nucleosides, all LNA C's are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages.
An antisense oligonucleotide represented by the formula: The antisense oligonucleotide has the formula

2. The antisense oligonucleotide of claim 1, which is an antisense oligonucleotide of the formula: The antisense oligonucleotide according to claim 1 or 2, in the form of a pharma- ceutically acceptable salt. The antisense oligonucleotide according to claim 1 or 2, in the form of a pharma- ceutically acceptable sodium salt. The antisense oligonucleotide according to claim 1 or 2, in the form of a pharma- ceutically acceptable potassium salt. A conjugate comprising the antisense oligonucleotide according to any one of claims 1 to 5 and at least one conjugate moiety covalently bound to the oligonucleotide. A pharmaceutical composition comprising the antisense oligonucleotide according to any one of claims 1 to 5, or the conjugate according to claim 6, and a pharma- ceutically acceptable diluent, solvent, carrier, salt and/or adjuvant. 8. The pharmaceutical composition of claim 7, wherein the pharma- ceutically acceptable diluent is sterile phosphate buffered saline. The pharmaceutical composition according to claim 7 or 8, wherein the antisense oligonucleotide is used in the pharma- ceutically acceptable diluent in a solution having a concentration of 1 to 100 mg/mL. The pharmaceutical composition according to claim 7 or 8, wherein the antisense oligonucleotide is used in the pharma- ceutically acceptable diluent in a solution having a concentration of 2 to 30 mg/mL. An in vitro method for modulating ATXN2 expression in a target cell expressing ATXN2, comprising administering to said cell an effective amount of an antisense oligonucleotide according to any one of claims 1 to 5, or a conjugate according to claim 6, or a pharmaceutical composition according to any one of claims 7 to 10 . A pharmaceutical composition for regulating ATXN2 expression in a target cell expressing ATXN2, comprising an effective amount of an antisense oligonucleotide described in any one of claims 1 to 5 or a conjugate described in claim 6. Use of the antisense oligonucleotide according to any one of claims 1 to 5 or the conjugate according to claim 6 for the preparation of a medicament for modulating ATXN2 expression in a target cell expressing ATXN2 . An oligonucleotide according to any one of claims 1 to 5, or a conjugate according to claim 6, or a pharmaceutical composition according to any one of claims 7 to 10, for use in medicine. An oligonucleotide according to any one of claims 1 to 5, or a conjugate according to claim 6, for use in the treatment of a neurodegenerative disease selected from the group consisting of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43 proteinopathy . Use of an oligonucleotide according to any one of claims 1 to 5, or a conjugate according to claim 6, or a pharmaceutical composition according to claim 7, for the preparation of a medicament for the treatment or prevention of a neurodegenerative disease selected from the group consisting of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP- 43 proteinopathy. 10. A pharmaceutical composition for treating or preventing a neurodegenerative disease comprising a therapeutically or prophylactically effective amount of the antisense oligonucleotide of any one of claims 1 to 5, or the conjugate of claim 6, wherein the neurodegenerative disease is selected from the group consisting of spinocerebellar ataxia type 2 ( SCA2 ), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia (FTD), Parkinsonism, and conditions involving TDP-43 proteinopathy. 16. The oligonucleotide or conjugate of claim 15 , wherein the neurodegenerative disease is spinocerebellar ataxia type 2 (SCA2). 16. The oligonucleotide or conjugate of claim 15 , wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). 17. The use according to claim 16, wherein the neurodegenerative disease is spinocerebellar ataxia type 2 (SCA2). 17. The use according to claim 16, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). 18. The pharmaceutical composition of claim 17, wherein the neurodegenerative disease is spinocerebellar ataxia type 2 (SCA2). 18. The pharmaceutical composition of claim 17, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).